General themes of hyperhomocysteinaemia

The Netherlands

JOURNAL OF MEDICINE ELSEVIER

Abstracts I Netherlands Journal of Medicine 52 (1998) SI $6I

General themes of hyperhomocysteinaemia Pathogenesis of hyperhomocysteinaemia Vascular dysfunction at the cellular level. D.W. Jacobsen*, R. Poddar. Department o f Cell Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA. Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Although the causes of hyperhomocysteinemia are multifactorial and include both genetic and acquired components, the consequences of increased circulating levels of homocysteine and its congeners on vascular cell function are poorly understood. Previous in vitro studies using cultured animal or human aortic endothelial cells suggested that homocysteine inhibited anticoagulant activity by decreasing surface expression of thrombomodulin, but promoted procoagulant activity by increasing tissue factor expression and stimulating factor V activity. Other studies found that homocysteine inhibited growth of endothelial cells but stimulated proliferation of smooth muscle cells. Only a limited number of studies have addressed the role of homocysteine in early atherogenesis. We have investigated the effect of homocysteine on chemokine expression in cultured human aortic endothelial cells. Using both Northern blot analyses and RNase protection assays, we have found that relatively low concentrations of homocysteine induce the expression of monocyte chemoattractant protein 1 (MCP-1) and interleukin 8 (IL-8). Maximal expression of MCP-1 mRNA was observed 2-3 h after homocysteine addition which then declined to basal levels within 24 h. Induction of MCP-1 mRNA was achieved with as little as 10/,tM D,[-homocysteine. A three-fold increase of MCP-I mRNA occurred with 50 /tM o,L-homocysteine, but higher concentrations did not further enhance expression of MCP-1. In contrast IL-8 mRNA expression continued to increase with increasing concentrations of D,L-homocysteine up to 10 mM. Homocysteine did not induce expression of TNFa, GM-CSF, IL-lfl or TGF]3 mRNAs over concentrations ranging from 10/IM to 10 mM. L-Cysteine, L-homocystine and L-methionine (50/IM) did not induce expression of MCP-I or IL-8 mRNA. D,L-Homocysteine (50/,tM) stimulated a 10-fold increase in the release of MCP-1 and IL-8 protein into culture medium from human aortic endothelial cells within 60 min. These studies demon-

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strate that low concentrations of homocysteine, but not cysteine, homocystine or methionine, induce expression of MCP1, a potent chemokine for monocytes. We conclude that homocysteine could play a role in atherogenesis by augmenting the recruitment of monocytes to developing arterial lesions (Supported by grant HL52234 from the National Institutes of Health).

Vascular dysfunction in hyperhomocysteinemic animals. S.R. Lentz. Department of Internal Medicine, University of lowa, Iowa City', 1A 52242, USA. Hyperhomocysteinemia is associated with increased risk of thrombotic and atherosclerotic vascular disease. Studies of cultured cells in vitro have led to the hypothesis that homocysteine may predispose to vascular disease by altering the normally antithrombotic and vasoprotective phenotype of vascular endothelium, perhaps through a mechanism that involves generation of peroxides and other reactive oxygen species. Recent findings in animal and human models of hyperhomocysteinemia provide support for this hypothesis. We have observed decreased thrombomodulin-dependent protein C activation and impaired endothelium-dependent vascular relaxation in cynomolgus monkeys with diet-induced mild hyperhomocysteinemia. Similar findings have been subsequently reported in humans with moderate hyperhomocysteinemia. Endothelial dysfunction may contribute to development of atherosclerosis and predispose to complications such as thrombosis and vasospasm. More recently, we have found that monkeys fed high-fat, atherogenic diet develop both hypercholesterolemia (plasma cholesterol>400 mg/dl) and hyperhomocysteinemia (plasma homocyst(e)ine > l0/./M). Supplementation of the atherogenic diet with B vitamins corrected plasma homocysteine to normal levels ( < 4/.tM), but did not restore vascular function to normal or prevent progression of structural lesions in atherosclerotic monkeys with persistent hypercholesterolemia. These observations have implications for the potential clinical utility of dietary intervention to decrease plasma homocysteine, which has been proposed as a strategy to decrease the prevalence of vascular disease. Goals of future studies include determining the relative effects of hyperhomncysteinemia and hypercholesterolernia on progression of atherosclerosis and vascular dysfunction, and defining mechanisms of vascular dysfunction in hyperhomocysteinemia.

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Abstracts / Netherlands Journal q["Medicine 52 (1998) SI-$61

Poster visits Interleukin-8 is correlated with homocysteine in healthy volunteers, but not affected by vitamin supplementation. B.E. Van Aken 1., M. Den Heijer2, P.H. Reitsma ~, G.M.J. Bos 3, H.J. Blom 4, S.J.H. Van Deventer 1. ILab. for Experimental Internal

Medicine, AM(', Amsterdam, 2Dept. of Internal Medicine. Twee Steden Hospital, Tilburg, 3Dept. of Hematology, DDHK, Rotterdam, 4Dept. of Pediatrics, University Hospital St Radboud Nijmegen, Nijmegen, Netherlands. Elevated plasma homocysteine levels are a risk factor for deep-vein thrombosis. The mechanism by which homocysteine increases thrombotic risk is unknown. One possibility is that increased levels of homocysteine induce a chronic inflammatory response in certain cell types (endothelial cells, monocytes) that alters the antithrombotic properties o f the vascular system. Interleukins are expected to play an intermediate role in such an inflammatory process. The aim of the study was to (1) document whether proinflammatory cytokine levels correlate with plasma homocysteine and (2) whether vitamin supplementation not only lowers homocysteine but also cytokine levels. Plasma homocysteine, IL-6, and IL-8 levels were measured in 230 healthy volunteers from the general population, aged 20 90. Samples were obtained from the subjects before and after multivitamin frolic acid, B6, and Bl2) supplementation. The results show that only for detectable IL-8, but not IL-6, a weak correlation with homocysteine was observed both before (r = 0.23, P = 0.001) and after vitamin supplementation. IL-6 levels are detectable in 23% of the volunteers. The percentage of detectable levels in normal compared to hyperhomocysteinemic ( > 16 pM) individuals is equal, the mean of the detectable levels is not significantly different (4.6+ 2.1 versus 4.1 + 1.4 pg/ml). 88%o of the subjects have a detectable level of IL-8, 87% of the normal and 93% o f the hyperhomocysteinemic group have detectable IL-8 levels. The mean detectable IL8 level of the hyperhomocysteinemic group is significantly increased compared to the normal group (6.3+1.1 versus 4.6 + 0.1 (P = 0.01 )). After vitamin supplementation homocysteine was significantly decreased as expected, but no effect on the IL-6 or IL-8 levels could be determined. In conclusion, the weak correlation with IL-8 and the absence of a correlation with IL-6, together with the fact that vitamin supplementation does improve homocysteine but not cytokine levels, do not support the hypothesis that homocysteine increases thrombotic risk through stimulation of proinflammatory cytokine production. Positive effect of cobalamin supplementation on cognitive performance and cerebral function in free-living older subjects with low plasma cobalamin concentrations. D. Van Asselt ~*, J. Pasman 2, D. Vingerhoets2, Y. Kuin j, H. Van Lier3, H.J. Blom 4, D. Hoefnagels l. 1Dept. of Geriatric Medicine, 2Dept.

of Clinical Neurophysiology, 3Dept. of Medical Statistics, 4Lab. of Pediatrics and Neurology, University Hospital Ni/megen. Nijmegen, Netherlands.

Mild cobalamin (Cbl) deficiency is very common in freeliving older subjects. The clinical benefit from Cbl supplementation in these subjects has not been studied before. In a singleblind intervention study with placebo period, we investigated the effect of Cbl supplementation on plasma total homocysteine (tHcy), methylmalonic acid (MMA), cognitive performance, cerebral function in 16 free-living older subjects, aged 64 to 89 y, with low plasma cobalamin concentrations (-<150 pmol/L) and free from significant cognitive impairment. They were examined at baseline, after one month of placebo and after five months of intramuscular hydroxocobalamin injections (total dose 8000 pg). After Cbl supplementation, plasma Cbl levels increased and plasma M M A and tHcy levels decreased significantly. Quantitative electroencephalogram (qEEG) showed significant improvement, i.e. more fast activity and significant less slow activity. The performance on three cognitive tests (Verbal Word Learning Test, Verbal Fluency, Similarities) also improved significantly. Lower concentrations of tHcy were related to increases in fast activity on qEEG on the one hand and to better performances on the Verbal Word Learning Test on the other. Better performances on the Verbal Word Learning Test were related to increases in fast activity on qEEG. These results suggest a positive effect of Cbl supplementation on cognitive performance and cerebral function in free-living older subjects with low plasma Cbl concentrations. The effects on cognitive performance and cerebral functions seem related to homocysteine.

Hoio transcobalamin II concentrations and mild cobalamin deficiency in free-living healthy older subjects. D. Van Asselt I*, C. Thomas:, M. Segers:, L. De Groot s, W. Van Staveren s, R. Wevers 4, H.J. Blom 4, W. Hoefnagels l. IDept. of Geriatric

Medicine, 2Dept. of Obstetrics and Gynecology and Lab. of Endocrinology and Reproduction, 4Lab. of Pediatrics and Neurology, University Hospital Nijmegen. Nijmegen, Netherlands; 3Dept. of Human Nutrition and Epidemioh)gy, Wageningen Agricultural University, Wageningen, Netherlands. It was hypothesized that older subjects with low normal plasma cobalamin (Cbl) and elevated plasma methylmalonic acid (MMA) levels can be Cbl deficient because of impaired delivery of Cbl to the cells due to low holo transcobalamin II (holo TCII) levels. The aim of this study was to determine whether holo TCII levels are reduced in free-living healthy older subjects with low normal plasma Cbl and elevated plasma M M A levels. Plasma Cbl, holo TCII and M M A levels were measured in 102 free-living older subjects, aged 74 to 80 y. Heparin-Conjugated-Sepharose was used for holo TCII absorption. Plasma Cbl was low (-<15¢) pmol/L) in 24 (24%) subjects and low normal (160-250 pmol/L) in 36 (35%). Plasma M M A was elevated ( > 0.32 pmol/L) in 40 (39%) subjects, of whom 15 subjects had low and 10 had low normal plasma Cbl levels. Subjects with low normal Cbl and elevated M M A levels had similar holoTCIl concentrations (median 51 pmol/L (range 2(~95)) to subjects with low normal Cbl and normal M M A concentrations (52 pmol/L (2-114) ( P = 0.8). This finding did not change after exclusion of subjects with increased

Abstracts/Netherlands Journal of Medicine 52 (1998) S1-$61 serum creatinine levels. These results show that decreased holoTCII is not the cause of elevated plasma M M A in subjects with low normal plasma Cbl concentrations. Thus, the delivery of Cbl to cells seems adequate in these subjects. Other causes of these elevated plasma M M A concentrations will probably have to be sought at the cellular level.

Hyperhomocysteinemia and vascular disease in children. I.M. Van Beynum 1., J.A.M. Smeitink 1, C. Thomas 3, M. Den Heijer2, E. Rammeloo 1, M.T.W.B. Te Poele Pothoff1, H.J. Blom 1. 1Dept. of Pediatrics, ZDept. of Internal Medicine,

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with a high resolution lithium column and standard physiological separation. Homocysteine and cysteine peaks are discretely separated. We suggest that tHcy and tCys be routinely analysed by IEC as part of the amino acid profile, and that free Hcy2, Cys2 and Hcy-Cys be regarded as artefacts of no specific diagnostic relevance. This approach surmounts the problem o f protein binding of aminothiols during sample storage, enhances the diagnosis and monitoring o f I M D patients where the value of other amino acids is important, and complements the broad metabolic investigation of patients with neurological disorders who may be at risk of cerebral vascular ,disease.

3Laboratory of Endocrinology and Reproduction, University Hospital St Radboud Nijmegen, Nijmegen, .Netherlands'. Children with classical homocystinuria suffered from arteriosclerotic and thromboembolic events at very young age. Several studies have shown that mild hyperhomocysteinemia (MHH) is associated with arterial vascular disease and thrombosis in adults. Studies on this relation in children have not been published. The aim of our study was to assess a possible relation between M H H and vascular disease in children. We measured plasma total homocysteine levels in 124 patients with vascular disease and in 237 controls (hospital based controls and healthy volunteers), at the age of 0 18 year. Hyperhomocysteinemia was defined as a homocysteine level above the 90th percentile of the controls. The median homocysteine level was 9.1 (range 4.3-20)/tmol/ 1 for the control group and 9.0 (range 2.8 22)/~mol/l for the patients. Homocysteine level increased with age and was not different in boys and girls. Hyperhomocysteinemia was present in 21 (17%) of the 124 patients with vascular disease. The crude odds ratio was 1.8 (95% confidence interval (CI) 1.0.... 3.4). After adjustment for age and sex the odds ratios were 2.7 (95% CI 1.4-5.5) and 2.1 (95% CI 1.0~.7), respectively. This result indicates that M H H is a risk factor for vascular disease in children.

Total homocysteine assay: an argument for ion-exchange chromatography. A. Briddon Clinical Biochemistry, National Hos-

pital for Neurology and Neurosurgery. London, WCIN 3BG, UK. Specific methods for tHcy are convenient for large screening programmes but do not provide the additional useful information available from a complete amino acid profile. Assay of tHcy by ion-exchange chromatography (IEC) following dithiothreitol (DTT) reduction is well established but typically uses a 60 minute, buffered plasma, reduction step. This introduces an analysis delay which also compromises the integrity of other amino acid values, notably glutamine and glutamate. Whilst it is necessary to buffer, or neutralise, aqueous solutions of Hcy/Cys the pH of untreated plasma favours a rapid DTT reduction without the need for additional buffering whilst maintaining the stability of other amino acids. Recovery of added Hcy/Cys is excellent (95 110%) and an inter-assay CV of 8% is attainable at normal physiological concentrations. Analysis was performed on a Biochrom 20 amino acid analyser

Homocysteine response to B-vitamln intervention in women of reproductive age: a meta-analysis of four studies. A. Br6nstrup*, J. Dierkes, M. Kroesen, K. Pietrzik Institute of Nutri-

tional Science, Dept. of Pathophysiology, University of Bonn, Endenicher Allee 11-13, 53115 Bonn, Germany. Several intervention studies have shown that plasma total homocysteine (tHcy) levels can be lowered by vitamin intervention. However, there is still uncertainty as to the combination of vitamins needed as well as to the minimal effective/ optimal doses required. Only few studies have included young women, a group for which Hcy may be relevant because of its possible involvement in the etiology of neural tube defects (NTDs) as well as a risk factor for vascular disease. Design: From 1993 to 1996 we undertook four different studies with a total o f 459 apparently healthy women of age 20-36 years. A total of nine different supplementation regimes each lasting four weeks are now compared in this pooled analysis: placebo; 2 mg vit. B6; 100, 200 or 400/lg folic acid (FA); 400 ]~g FA+2 mg vit. B6; 400 //g FA+2 mg vit. B6+6 /lg vit. B12; 400 /lg FA+6 /lg B~z; 400 /,tg FA+400 /tg vit. BI:. Group size of the different regimes varied between n = 29 and 51. Statistical methods: The primary variable was the change in tHcy level after 4 weeks of treatment. Analysis involved homogeneity checking, paired t-tests and ANOVA models as well as regression analysis. Results. All treatment forms except placebo and vit. B6 alone resulted in a significant decrease of the mean tHcy level (p-<0.04 and lower) after four weeks of treatment. However, there were considerable differences in the extent of tHcy reduction obtained; all treatments including FA supplementation alone (100, 200 or 400 /~g FA) were less effective compared to combined supplementation of two or three vitamins (400 pg F A + 6 / l g vit. Bj2; 400 pg FA+400/~g vit. B~; 400 pg FA+2 mg vit. B6+6 ~tg vit. Bl:). The mean tHcy reductions varied between 5- 9% (FA alone) and 15-18% (combined treatments). Conclusions." In this group of young women with tHcy levels in the lower range, a combined vitamin intervention strategy appears to be most effective to lower tHcy levels. This may be important for example in light of single vitamin, i.e. folate, enrichment of food as mandated in the USA beginning January 1st, 1998.

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Abstracts/Netherlands Journal of Medicine 52 (1998) S1 $61

Determination of total homocysteine in plasma: comparison of a new homocysteine-immunoassay with an established HPLCmethod. A. Br6nstrup 1., K. Sielaff~, D. Janke:, K. Pietrzik1. l lnstitute o f Nutritional Science, Dept. of Pathophysiology, University of Bonn, Endenicher Allee 11-13, 53115 Bonn, Germany; 2Axis Biochemicals ASA, P.O. Box 2123, 0505 Oslo, Norway. Several different methods for measurement of total homocysteine (tHcy) in plasma have been developed. However, use of established methods is often time-consuming and work-intensive. Thus, it may not be suitable to employ these techniques in routine laboratory procedures where expensive equipment is often not available. However, the demand for easy-to-use techniques has increased owing to the attention to tHcy as risk factor for vascular disease and probably neural tube defects. Aim: We aimed to compare the performance of a new, commercially available enzyme-immunoassay (Axis® Homocysteine-EIA) with a reversed-phase HPLC technique including fluorescence detection after derivatization with SBD-F. Design: From a vitamin intervention study with elderly individuals, a subset of samples (n = 149) was analyzed both with HPLC and EIA. Data were compared using the method of Bland-Altman as well as correlation analysis. For both methods, quality of performance was assessed computing withinand between-batch CVs as well as % recovery after dilution and spiking. For HPLC, quality control was also performed via participation in a European quality control-scheme (ERNDIM). Results: The range of tHcy values observed was 3.9 20.2 /zmol/1. When measured with the EIA kit, values were slightly higher by a mean of 0.43 /trnol/l compared to HPLC. The regression coefficient of both methods was r=0.85. For the EIA kit, within- and between-batch CVs were between 4.99.7%, and 4.0-9.9%, respectively. Within- and between-batch CVs for HPLC were 2.8-7.8% and 3.7-5.7%, respectively. Recovery after spiking and dilution ranged between 97.4 110.9% for the EIA kit. The HPLC method gave similar results for recovery after spiking but little lower recovery after dilution. Conclusions: Both methods are comparable over the range of tHcy levels measured. Thus, routine analysis of tHcy is now also accessible for laboratories not equipped with an HPLC but able to perform an EIA. Total plasma homocysteine assays: comparison of liquid chromatography methods. M. Candito 1., F. Parrot:, N. Jacobs, R. Calaf1, P. Charpiot4, M. Sarthou 1, D. Garcon 4, E. Van Obberghen1, P. Kamoun5. Biochemistry: I H6pital Pasteur, BP 69, 06002 Nice Cedex; 2H6pital Pellegrin, 33076 Bordeaux Cedex; 3Hdpital Piti~ Salp~tribre, 75651 Paris Cedex 13; 4Facultb de Pharmacie, 13005 Marseille; 5Hdpital Necker, 75015 Paris Cedex, France. A comparison between common liquid chromatography methods for total plasma homocysteine (Hcy) assay was performed in four laboratories. Two methods were based on HPLC with fluorescence detection using either a pre-chromatographic condensation with ABD-F (Cornwell, 1993) or a postcolumn derivatization with dansyl chlorides. Two laboratories

used an amino acid analyzer (AAA) from Beckman (6300 analyzer) and plasma samples were treated according to Brattstrom et al., 1988. Each laboratory received 20 blindly dedoubled plasma to evaluate exactitude and repeatability; 4 plasma were sampled 6 hours after an oral methionine load (0.1 g/kg). Authentic homocystine or Hcy (Sigma) standards were treated as samples. All the data were compared by variance analysis. Results obtained from the two AAA were highly correlated (r = 0.990). The results from the two distinct HPLC methods were also highly correlated (r = 0.997). Standard deviation calculated on dedoubled plasma was 1.6 for the HPLC methods and 3.3 for the AAA method. A difference in Hcy levels evaluated by the different methods appeared after oral methionine load: levels found with AAA were higher (from 12 to 20%) than those obtained with HPLC (levels on AAA were 24.8; 31.3; 55.9 and 73.0/zmol/1). This discrepancy can be explained by the incomplete separation between homocysteine and methionine when this last amino acid increased greatly after oral loading. Although AAA gave higher results than HPLC in plasma after post methionine load, the liquid chromatography results as a whole were correctly correlated.

HPLC procedure for total and free plasma homocysteine analysis. R. Castro 1., V. Reis ~, M.A.P. Segurado2, 1. Tavares de Almeidat. I Centro de Patogdnese Molecular, Faculdade de Farmhcia, Lisboa, Portugal; 2Dept. of Chemistry, Faculdade de Farmdcia, Lisboa, Portugal. In the last few years, several studies have demonstrated the association of hyperhomocysteinemia versus vascular disease. Increasing attention is being devoted to this risk factor. The need to evaluate the different forms of the circulating homocysteine has prompted us to develop a rapid and sensitive HPLC procedure for the study of the Portuguese population. For total homocysteine evaluation the reduced forms were obtained after incubation with tri-n-butylphosphine 10% (TBP), subsequently proteins were removed by precipitation with trichloroacetic acid. Free Hcys (reduced plus oxidized) determination was achieved taking into account that plasma and protein precipitation should be obtained immediately after blood was drawn. Oxidised form was reduced by incubation with TBP, Cysteamine has been used as internal standard and Hcys has been analysed as a SBD-F derivative on a reverse phase Cls-column (Supelcosil LC-18-S) under isocratic elution (phosphate buffer 0.1 M, pH 2.10/acetonitrile; 95:5), with a flow rate of 1.8 ml/min. The cysteamine peak (3.58+0.320) was clearly resolved from that of Hcys (5.90 +0.091). Plasma calibration curves, corrected for endogenous Hcys, were linear (r = 0.998) over the range 5- 100 Imaol/L. Intra-assay and interassay variation (relative standard deviation) ranged from 0.95% to 1.7% and from 7.1% to 8.3%, respectively, in the range 5 50/lmol/L. Total Hcys (tHcys) and free Hcys (fHcys) basal fasting levels have been studied in 36 healthy controls: 18 males (age: 1825) and 18 females (age: 20--33). tHcys = 6.6/.tmol/L (3.8~.4) and 7.6/lmol/L (5.0 11.6) for males and females, respectively. fl-Icys= 1.1 ktmol/L (0.8-1.5); overall mean. The slightly elevation of total Hcys fasting levels that we found in women com-

Abstracts~Netherlands Journal o[" Medicine 52 (1998) SI-$61 pared with men can be explained by the age difference existing between both groups and the slightly lower values of tHcys when compared with the ones in the literature is probably due to the type of diet, a Mediterranean one. In conclusion the method is suitable for systematically screening of the patients. Further studies are in progress concerning the determination of the different forms of free Hcys.

Effect of homocysteine on cellular oxidation of low density lipoproteins. A. Chango*, F. Barb~, G. Alcindor, J.P. Nicolas. Laboratoire de Biochimie Pbdiatrique et Mbdicinale, INSERM U-308, Facult~ de Mbdicine et Laboratoire de Bioehimie-A, CHUN Brabois, 54511 Vandoeuvre-les-Nano', France. We have evaluated in vitro, the effect of homocysteine (Hey) on low density lipoproteins (LDL) oxidation by human umbilical vein endothelial cells. LDL (d 1.019-1.063) were isolated at 4°C by two sequential ultracentrifugation methods. The isolation by the first method (120 000 × g), was complete within 44 hr of obtaining the plasma. The isolation by the 2nd method (443 000xg) was complete within 4 hr. Isolated LDL were eluted on PD-10 columns (Pharmacia) and sterilized by filtration (0.22 /an). Confluent endothelial cells were incubated in Ham's F-10 medium supplemented with 100/lg LDL protein/ nal. After 30 h incubation, thiobarbituric acid reactive substances (TBARS: a commonly used index of lipid peroxidation), were measured. Incubation with 100, 250 and 500/.tmol/1 of Hey generated 16, 32 and 33 nmol TBARS/mg LDL, respectively, vs. 9 nmol TBARS/mg for control, when LDL isolated by the 44 hr method were used. Hey did not generate TBARS when LDL isolated by the 4 hr method were used. These results suggest that treatment or the state of LDL before the contact with endothelial cells may be a determinant factor in TBARS generation. Aminothiols may enhance the rate of oxidation of LDL preparation that contain peroxides. From a clinical point of view antioxidant deficiencies or pathological states in some subjects may influence in this way LDL, initiating an oxidative process. Homocysteine may only enhance the rate of oxidation of LDL by endothelial cells in subjects with high homocysteine level. Variability and determinants of plasma homocysteine levels in an elderly population. R. Clarke 1., P. Woodhouse:, A. Ulvik3, C. Frost4, P. Sherliker~, H. Refsums, P.M. Uelands, K.-T. Khaw2. I Clinical Trial Service Unit, University of O~[brd. Ox.lord, UK; 2Dept. of Clinical Gerontology, University of Cambridge, Cambridge, UK; 3Dept. of Pharmacology, University q[ Bergen, Bergen. Norway; 4Medical Statistics Unit, London School of Hygiene, London. UK. Epidemiological studies based on single baseline measurements of risk factors may underestimate the strength of associations of the 'usual' (i.e. long-term average) level of the risk factors with disease due to 'regression-dilution' bias. To assess the magnitude of this bias in studies of blood total homocysteine (tHcy), plasma samples were collected from 96 individuals at 2-monthly intervals over a one year period.

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Blood tHcy concentrations varied from 7.1 /maol/L in the bottom quintile to 14.5 ]zmol/L in the top quintile. The mean tHcy was 10.4/~mol/L, the between-person SD was 2.5 gtmol/L and the within-person SD was 0.93 /zmol/L. There was little seasonal variation in tHcy concentrations, and the intra-class correlation coefficient for tHcy was 0.88, which was higher than that for total cholesterol (0.85) and for systolic blood pressure (0.74). Mean tHcy concentrations were inversely related to mean plasma folate (r=-0.36) and vitamin Btz (r =-0.35) concentrations. Median tHcy concentrations were about 1 /lmol/L higher in men than in women, in older (70 to 74 years) than in younger (65 to 69 years) individuals, and in those with the TT and CT genotypes compared with the CC genotype for the MTHFR polymorphism (10.7 and 10.6 vs. 9.6 ~tmol/L). Only a small fraction of the within-person variation in tHcy was due to analytic variability, and a substantial component of the within and between-person variability in tHcy remained unexplained when the factors studied had been allowed for. Epidemiological studies based on single tHcy measurements may underestimate the magnitude of any risk associations with disease by 10 to 15%. The magnitude of this regression dilution bias is less than that observed for both total cholesterol and for systolic blood pressure.

The C677T polymorphism of the MTHFR and the plasma homocysteine concentration in a middle-aged population in East Germany. J. Dierkes*, A. Ambrosch, A. Jeckel, H. Boeing, C. Luley. Institute of Clinical Chemistry', University Hospital, Magdeburg, and German Institute of Nutrition Research, Dept. q[ Medical Epidemiology, Potsdam, Germany. Elevated homocysteine concentrations are an independent risk factor for atherosclerotic disease. The plasma homocysteine concentration can be influenced by both genetic and environmental factors. The present study was undertaken to determine the importance of the C677T polymorphism as a genetic factor in relation to environmentally determined factors for the homocysteine concentration in an apparently healthy, middle-aged, East German population. Design: Study participants (n=350) were recruited from among the participants of the Potsdam cohort of the EPIC study (European prospective investigation into nutrition and cancer). Men and women aged 40-65 years, apparently healthy (especially no known renal or vascular disease) who did not use folate or B12 supplements were eligible to participate. Homocysteine, serum and RBC folate, vitamin BI2, vitamin B6, creatinine and protein were measured in plasma samples. The C677T polymorphism was measured using PCR technique with enzymatic restriction analysis. Anthropometrical and lifestyle data were also obtained. Statistical analysis: Single and multiple regression analysis with plasma homocysteine as the dependent variable. Vitamin users were excluded from the main analysis and analysed separately. Results and interpretation: Homocysteine concentrations between 5 and 45 /lmol/L were measured. The homocysteine concentration correlated inversely with folate (serum and RBC folate), vitamin Bl2, and positively with the creatinine

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Abstracts/Netherlands Journal of Medicine 52 (1998) SI-$61

concentration. There was, however, no correlation between homocysteine and vitamin B6 or protein. Men had higher homocysteine concentrations than women. The frequency of the 677T allele was 0.29, which is slightly lower than the reported frequency in other European populations. Sex, folate status and renal function seem to be more relevant for the homocysteine concentration than the C677T genotype. Excellent correlation between homneysteine concentrations either measured by an HPLC method with fluorescence detection and by a newly developed enzyme immunoassay (EIA). J. Dierkes*, D. Janke, C. Luley. Institute ~f Clinical Chemistry, Uni-

versity Hospital, Magdeburg, German), and Axis Biochemieals, Oslo, Norway. Methods for the determination of the atherogenic amino acid homocysteine are so far based on chromatographic separation (HPLC or GC). Recently, an enzyme immunoassay (EIA) for the determination of homocysteine was introduced into the market. HPLC methods are based on reduction of homocystine, homocysteine-cysteine mixed disulfide and protein-bound homocysteine, protein precipitation and derivatisation with a fluorescence substance. The present assay uses TBP for reduction of the homocysteine moieties in plasma and SDB-F for the derivatisation of homocysteine. Design." Homocysteine was measured in EDTA plasma of patients with non-insulin dependent diabetes mellitus with both methods. HPLC determinations were made during a period of 6 months, while the determinations using the EIA were made within 4 weeks using 2 microtitre plates. Descriptive statistics were calculated for homocysteine measured by both methods. Comparisons were made by traditional correlation analysis and by the method of Bland and Altman (1986). The frequency of hyperhomocysteinemia was calculated using different cut-offvalues for the HPLC method and adjusted cutoff values for the EIA method. Results and interpretation: The correlation between both methods was excellent (r = 0.94, n = 38, p < 0.001 ). The analysis by the method o f Bland and Altman revealed that the HPLC assay measured on average 1.7 = 2.2 ~tmol/L higher than the EIA. There was a trend to lower results by the EIA at homocysteine concentrations by H P L C > 20 /~mol/L. However, if the difference of 1.7/zmol/L was considered for the diagnosis of hyperhomocysteinemia, the diagnosis was correctly done by the EIA in 37 of 39 (95%) patients. These results show that the EIA has an excellent correlation with the HPLC method and can be used for the diagnosis of hyperhomocysteinemia in patients with N I D D M if the cut-off points are adequately chosen. Familial hyperhomocysteinemia and low blood folate. T.B. Domagala, M. Libura*, M. Sanak, R. Czach6r, A. Szczeklik.

Department of Medicine, Jagiellonian Universi0 School ~]' Medicine, 31-066 Cracow, Poland The aim of our study was to assess the relationship between genetic and environmental factors in families of patients with early-onset of atherosclerosis and hyperhomocysteinemia. We

studied 73 members of 15 families; 26 women and 47 men; mean age 34 y. (range, 15-65). In all of them we measured plasma total homocysteine, plasma and erythrocyte folates and plasma vitamin BI2. We also screened for C677T polymorphism in M T H F R gene and performed oral methionine load. Out of 73 subjects, 22 presented with clinical signs of atherosclerosis: 16 coronary artery disease, 1 cerebral artery disease, 5 peripheral occlusive arterial disease. The cut-off for homocysteine was set as 95 percentile o f our control group (14.9 L,mol/1 in the fasting state and 65.2/nnol/1 in the 6th hour o f methionine load). Hyperhomocysteinemia only in the fasting state was observed in 9 persons (12.7%); only after methionine load in one. In 23 subjects (32.4%1 hyperhomocysteinemia was recorded both in the fasting and post-load samples. Thus, to sum up, hyperhomocysteinemia was detected in 45.2% of the studied group. Low folate plasma levels ( < 4 ng/ml) were present in 25.4% of the subjects studied; 51% o f the whole group were below mediana (<6.51 ng/ml) for controls. A similar, significant depression in red blood cell folate was also evident. The thermolabile M T H F R +/+ genotype was shown in 12 (7 o f 15 probands). Our results indicate that: (1) hyperhomocysteinemia is a frequent phenomenon in patients with early onset of atherosclerosis and in their families; (2) low blood folate is common in families of Polish population and significantly increases the phenotypical expression of thermolabile MTHFR. Plasma total cysteine and cardiovascular risk profile: comparison to plasma total homocysteine. L. El-Khairy ~*, P.M. Ueland 2, O. Nyg~trd 1, H. Refsum:, S.E. Vollset SE 1. IDiv.

Jbr Medical Statistics. 2Dept. qf Pharmacology, University of Bergen, Bergen, Norway. Background." Cysteine is formed during the transulfuration of homocysteine. The concentration of this thiol amino acid in plasma is about 20-fold higher than that of homocysteine. We have compared the relations of total cysteine (tCys) and total homocysteine (tHcy) plasma levels to major cardiovascular risk factors. Methods. We studied 7591 men and 8585 women, aged 4067 y, with no history of hypertension, diabetes, or coronary heart disease. The subjects were recruited from the Hordaland Homocysteine study (1992-1993). Results." In the age group 4 0 4 2 y, tCys was higher in men (273/~mol/l, n = 5919), than in women (253 #mol/l, n = 6349), p < 0.001. The levels were significantly higher in the age group 65-67 y, but were similar in the two sexes, men (296/tmol/l, n = 1386), women (296 ~umol/l, n = 1932). At concentrations of tHcy below 20 ~mol/l, tCys showed a consistent increase with increasing tHcy, whereas at higher concentrations of tHcy, tCys decreased with increasing tHcy. Age and sex adjusted correlations between tCys, tHcy and risk factors are as follows: Age: r (tCys) 0.42, r (tHcy) 0.22; BMI: r (tCys) 0.25, r (tHcy) 0.01"; Smoking: r (tCys) -0.01", r (tHcy) 0.19; Physical activity: r (tCys) -0.03, r (tHcy) -0.06; Diastolic blood pressure: r (tCys) 0.12, r (tHcy) 0.06; Cholesterol: r (tCys) 0.14, r (tHcy) 0.07. * Stands for not significant. All other coefficients p < 0.001.

Abstracts/Netherlands Journal of Medicine 52 (1998) S1-S61 tCys shomed no rdation mith smoki~3g in the younger age group, and a negative relation in elderly men (r = - 0 . 0 9 ) and elderly women (r = - 0 . 0 7 ) . Conchus~ons: Oxtr ~a'ta snow ~tha't ~,Cys ~s assoc'~a~ed wi~h several cardio'~ascular risk factors. Except for physical acti~,ity and smc~(ttg, relations were stronger than (or t/-/c%

A simplified method for simultaneous assay of total plasma homocysteine (Hey) and methlonine (Met) using HPLC and pulsed tay, D . ~ . C . Cole. Departments q( Laboratory Medicine and

Pathobk{ogy~ kfeae(e(ne ann' ('aea'&crt~'s, 6'hA'er.s~y o.( i"oronCa~ The Toronto Hospital and the Hospital .[br Sick Children, Toronto, ON, Canada. In clinica'~ studies o'/Ytomocystdme metabo~'~sm, it may b e useful t o he'terrrbne 5 - ~ anla ~ e a concen'~ra'ho'ns m ~'ne same sample, but published methods for simultaneous measurement require ~he:hva'fiza'fion 5or assay "Dr V~C-'t6%, 5~ ,~enera3, 6eflvatizations are labor-intensive and increase the imprecision of the result. Because PIA detection depends only on the presence of a lone pair of electrons in the sulfur species, we have adapted our H P L C - P [ A method for Hqv fClin Chem [995;4l :7573 so that Met is quantified simultaneously. In our modified method, one volume of plasma is mixed with 1/10 volume of 10% tris(2carboxyethyl)phosphine (TCEP) reductant, as described by Gilfix et al. (Clin Chem 1997;43:687), and the protein precipitated with HPLC eluent (100 mM HCIO4, 150 mM NaCIO4, 5% CH3CN) after 30 min at room temperature. After passage through a C~s solid-phase cartridge, the extract is injected directly onto a Dionex DX-500 Ion Chromatograph equipped for heart-cut trapping and PIA, with minor adjt~stment of heart-cut times. Patient Hcy concentrations measured by this technique agree well (rz = 0.96; n = 58 ; range: 5.8 to 126/.tM) with those determined by the original method, based on NaBH4 reduction of Hcy. Moreover, precision of Hcy determination was significantly improved (Interassay CV: 7.4% to 3.1%, F test, p < 0 . 0 5 ) . Met concentrations determined by are comparable (r2=0.96). Recovery o f 6 ./aM reagent-grade L-methi(orhne abBeb "to a ~hasma samy~Se~ b a a nomina5 ~vSea concentra'fio'n ~ '12.'i ~M "wa~ q'ua'ne¢icca'e~ 'i'~'8.'4--.'~.'i'%'). Intra- and inter-assay CV's were 3.4% and 4.3%, respectively. T-tius, fi'FILt%PI~ xs a s~inp~ metlloff tbr sfinuffaneous Met and Hcy assay that could find wide application in clinical and experimental studies of homocysteine metabolism.

Fast anth accurate ~ ' P ~ me~'noh lo puan~+ "tola'lfllasma "nomoeysteine. I. Fermo *~, C. ArceYtoni", G. Mazzo'~a2, A. D'Angelo 2, R. Paroni ~. ILaboratory of Chromatographic and Separative Techniques, 2Coagulation Service, IRCCS H San Raffaele, Via Olgettina 60, Milano. Italy. In relation to the increased interest in measuring total homocysteine (tHcy), we have modified an HPLC procedure based on SBD-F (ammonium 7-fluorobenzo-2-oxa-l,3-diazole-4-sulfonate) pre-column derivatization to obtain an assay useful for clinical routine applications.

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The ri~etheed ~o~sis~ed o f 'the following ste~s: af'~er the addition of an internal standard (mercaptopropionylglycine), the reduction and cleavage of tHcy was performed by treating p'~asma samp'~es wi~h sodium boro~v&i~e 3 M ins~.ea~ o~ ~the more frequent used wi-n-butylphosphine in dimethylformam~de < t~tis ~aadling requires sa(et~ ¢ortdi¢ioas > . After incubation at 50°C for 30 min and HCIO4 deproteinization, supernatant was derivatized with SBD-F at 50°C for 60 min. The apparatus was a simple monopump System Gold Beckman madzu RE 55[ fluorescence detector ~385--525 nmk The chroma~:ogcapt'ffc ana?~sfs was att ~socradc rutt (~6~mfn~ per(armed on a Ultrasphere a D S Beckman column (150:<4.6 mm, 5 pm) w'ith sodium acetate buffer 0.2 M, 2% methanol, pH 4,0. The ~(~h}n- and between-day C ~ % were "~.~/o an(/ ~.~A, respec~Sveby. "ibae ~e~oveD, 05 ~,'ne mea'nt~h was ~'5,b~_5,~ (mean + S.D.) and the lower limit of sensitivity was 0.5 pmol/ "m)ecae6. "S'ne 5"meat r~ress~on anaSygas o] p3asma sampSe supplemented with homocystine (0-80/.tM) yielded r = 0.999. Applying this fast (= 70 samples can be rnn daily) and specific method (SBDF is a thiol labelling reagent) we found as referettce values 7.65+_2.36 .umoI/L and 8.9+_2.[4 .umobrL for women (n =40) and men (n =40), respectively (age 33.0+ 10 yr), in good concordance with ranges reported by literature (Clin Chem 1993;39:1764-1779) Thanks to the use of an open and not expensive HPLC equipment and commercially available reagents this method appears robust and easily handling and therefore suitable for routine use in clinical chemical laboratories.

Higher bomocysteine ]eve|s~ lower cognitive skills? L.P. Van Goor 1 , D.Z.B. Van Asselt2, M.D. Woiski/, A.M. Lagaay 3, A.E. Meinders t, P.P. Tak 4. University Hospital leiden, The Netherlands', I Department of Internal Medicine, 3Department of Geriatrics/Internal Medicine, 4Department of Reumatology, University Hospital St. Radboud, Nijmegen, The Netherlands', 2Department of Geriatrics.

and cognitive skills in the elderly? N%e measuret3 arnon~ o't'ner ~':6~s serum 5~cys 5eveBs a , h living at home. These individuals were part of the 'Leiden 85plhs stUart 'ann' wfilhh tfilg ffamewor~ were vlgfiea" 6y a pfiyslcian who administered the Mini-Mental State Examination (MMSE) and who took a medical history. We analysed 183 individuals with a MMSE-score < 25 and they were matched with 214 individuals with a MMSE-score k>'2~ an6 a compara'D'~e age anh sex ~fisIiS~nfinn, "The mean serum Hcys concentration was significantly higher (p = 0.000) in the group with lower MMSE scores. There were no differences in medical histories of vascular disease between the two groups. Hcys levels were 20% higher in the group with low scores irrespective of their vitamin B12, folate and creatinine levels. It seems that Hcys is a risk factor for cognitive decline. Whether it is a causal factor or an indication of an underlying problem needs to be determined.

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Abstracts~Netherlands Journal of Medicine 52 (1998) SI-$61

~50% of 'healthy' American elderly have low serum transcobalamin, diagnosing reduced vitamin BI2 absorption; 60% also have high serum homocysteine; none have low red cell folate: All elderly should get 25-100/zg oral free crystalline vitamin B~: daily as food fortiflcant or supplement. V. Herbert 1., M.A. Flynne, G.A. Nolph2, G. Krausee. IMount Sinai and Bronx VA Medical Centers, New York City, NY10468, USA, 2 University of Missouri at Columbia, Columbia, MO 65212, USA. Normally, of the total serum vitamin Bl~ level, ~ 20% is on the delivery protein transcobalamin (TC; half-life = 6 min), and 80% is on the circulating storage protein, haptocorrin (halflife=2 weeks); every cell with a metabolic need for B12 has receptors for TC, but only liver and other reticuloendothelial storage cells have receptors for haptocorrin (see citations in Herbert V. Vitamin Bl2. In: Ziegler EE, Filer LJ, Eds. Present Knowledge in Nutrition (7th Ed), Chap. 20. Washington, DC, International Life Sciences Institute (ILSI) Press, 1996:191 205). Serum TC is low within a week of the start of persistent food Bl2 malabsorption of any cause, and therefore is an ideal surrogate Schilling test (op. cit.). Low serum TC (and subtle neuropsychiatric and blood damage) may precede by months to a year or more, high serum HCY, high serum methylmalonate, and low total serum B12 (op. cit.). Our group (Flynn MA, et al. J Am Coll Nutr 1997; 16:258--267) found in 171 (139 men, 32 women) healthy elderly Missouri Caucasians (mean age 65) that all 52 with H C Y > 17.5 nmol/ml had low serum TC ( < 60 pg/ml), but only 7 had total serum BI2 < 200 pg/ml. An additional 31 also had reduced food Bl_~ absorption measured by TC < 60 pg/ml, but did not yet have high HCY. All had normal red cell folate ( > 136 ng/ml) and serum folate > 1.6 ng/ml; however, as expected, there was an inverse relationship between serum HCY and red cell (and serum) folate. Four decades ago, we showed (Ellenbogen L, Herbert V, Williams RR. Proc Soc Exp Biol Med 1958;99:257 259) that 1% of any oral dose (2/.tg or 30/tg) of crystalline vitamin BI2 is absorbed by mass action in gastric atrophy (we used pernicious anemia patients). We petitioned our Government to mandate that a minimum of 25 gg vitamin B12 be added to each portion of US Government-mandated (as of January 1, 1998) folatefortified food, and all folate supplements (Herbert V, Bigaouette J. Am J Clin Nutr 1997;65:572 573). The importance of hyperhomocysteinemia in high age people. W. Herrmann*, S. Quast, H. Schultze, M. Ullrich, J. Geisel. Zentrallabor der Universitiitskliniken des Saarlandes, Homburg, Germany. A disturbed methionine metabolism with moderate elevated homocysteine level has been recognized as an independent atherogenic risk factor. Elevated plasma homocysteine is found in 20-50% of patients with premature CHD, stroke, or peripheral vascular occlusive disease. Homocysteine also has great importance in geriatrics. Here homocysteine is an indicator for an intracellular lack of B-vitamins and/ or folic acid. Therefore we investigated 104 healthy high age people (85-102 years) compared to 100 healthy seniors (age 65 75 years) and 75 younger controls (19-60 years) for serum homocysteine levels. Homocysteine was investigated by HPLC with

fluorescence detector. 58% of the high age people had elevated homocysteine level contrary to 32% of the healthy seniors (6575 years) and none of the younger controls. The healthy seniors only showed moderate homocysteine elevations (15 to 30 /tol/l) while in the high group intermediate homocysteine elevations (30 to 100/tmol/l) occurred in 14% of the subjects. B vitamins were decreased in 55% of high age subjects and in 29°/,, of healthy seniors. The deficiency of vitamin B6 was in the high age group at 43% most frequent compared to the younger seniors at 22%. Vitamin B12 deficiency occurred in high age subjects at 20% and in younger seniors at 8%. Decreased folic acid was found in 1% in both age groups. So the measurement of metabolites like homocysteine tells us more about intracellular vitamin B deficiency than the direct measurement of Bvitamins and folic acid. The very high prevalence of hyperhomocysteinemia in elderly is a strong indicator for a acute intracellular deficiency of B-vitamins and folic acid. Beside vitamin shortage a very common mutation in the tetrahydrofolic acid circle, the mutation of the thermolabile methylene tetrahydrofolate reductase (MTHFR), is seen responsible for moderate homocysteine elevation. The 677-missense mutation in the MTHFR gene is found in 35% of alleles in high age people, in 37% of alleles in younger seniors and in 27% of alleles in controls. There is no significant difference in frequency distributions of the MTHFR gene mutation between all these age groups.

Inhibition of vascular endothelial cell growth by homocysteine: molecular mechanisms. Hong Wang*, N.E.S. Sibinga, M. Yoshizumi, M.A. Pen-ella, E. Haber, Mu-En Lee. Cardiovascular Biology Laboratory, Harvard School of Public Health and Cardiovascular Division, Brigham and Women's Ho.spital, Boston, MA 02115 USA. Elevated blood levels of homocysteine have been recognized as an important risk factor for myocardial infarction and stroke, yet the mechanisms by which homocysteine promotes arteriosclerosis are poorly understood. We found that clinically relevant concentrations of homocysteine (10-50/tM), but not cysteine, arrested the growth of cultured vascular endothelial cells (VEC) at the G1/S junction of the cell cycle and inhibited DNA synthesis. Homocysteine in this range had no effect on the growth of vascular smooth muscle cells or fibroblasts. This difference may result from VEC-specific inhibition of protein methylation, as homocysteine but not cysteine dramatically increased levels of S-adenosylhomocysteine, a potent inhibitor of methyltransferase in VEC. Homocysteine decreased carboxyl methylation of p21 ras (a G1 regulator activated by phenylation and methylation) and reduced levels of membrane-associated p21 ras and mitogen-activated protein (MAP) kinase activity. Moreover, 50 pM homocysteine significantly inhibited expression of the cyclin A gene (a major cell cycle regulator) and cyclin A-associated kinase activity in VEC. Homocysteine decreased transcription of the cyclin A gene in nuclear run on experiments but did not affect the half-life of cyclin A mRNA ( ~ 9 h). By transient transfection experiments in human VEC, homocysteine down-regulated cyclin A promoter activity. These results identify two potentially-related mechanisms, hy-

Abstracts~Netherlands Journal of Medicine 52 (19983 S1 $61 pomethylation-mediated inhibition of p21r~/MAP signalling and decreased transcription of the cyclin A gene, by which homocysteine may inhibit VEC growth. Homocysteine-induced inhibition of VEC growth and regeneration in response to injury may play an important role in pathogenesis of arteriosclerosis.

Hyperhomoeysteinemia is a predictor of overall mortality: The Hoorn Study. E.K. Hoogeveen 1., P.J. Kostense 12, F. de Vegt j, J.M. Dekker 1, H.G. Ruh61, C. Jakobs "~, R.J. Heine r4, L.M. Bouter 12, C.D.A. Stehouwefl '4. UniversiO' Hospital Vrije Universiteit, Amsterdam, The Netherlands. ~Institute Jbr Research in Extramural Medicine, 2Dpt. of Epidemiology and Biostatistics, 3Dpt. of Clinical Chemistry. 4Dpt. q[ Internal Medicine. An elevated serum total homocysteine (tHey) level is a risk factor for cardiovascular disease, but the prognostic value of tHcy for overall mortality has not extensively been defined. We prospectively investigated the relation between serum tHcy level and mortality in a nested case-control study. Between 1989 and 1992 serum was saved from 2484 men and women, aged 50-75 years, randomly selected from the population of Hoorn, a town in the Netherlands. During follow-up to December, 1996, there were 188 incident cases of mortality. Serum tHcy was measured in 188 cases and 633 controls of an age-, sex- and glucose tolerance stratified random subsample. Serum tHey levels were significantly higher in cases than in controls (geometric mean 13.0 [95%CI 10.2 to 15.8] vs. 11.8 [9.0 to 14.6]/tmol/L; P=0.02). After adjustment for age, sex, hypertension, diabetes, hypercholesterolemia and smoking the relative risk of mortality was 1.59 (95% CI; 1.06 to 2.37) for subjects with hyperhomocysteinemia (->18.0 /.tmol/L). Additional adjustment for serum creatinine or creatinine clearance (using the Cockcroft and Gault formula) did not materially attenuate the relation between hyperhomocysteinemia and mortality. This finding suggests that hyperhomocysteinemia is a risk factor for overall mortality independent of classical risk factors. Homocysteine concentrations (Hcy) after aHogeneic hematnpoetic stem cell transplantation (HST). F. Jochum~*, W. Nfirnberger, M. Bogatzki, M. Lehmann, M. Laryea l, U. G6bel, U. Wendel 1. Department for 1Metabolism and J'or Pediatric Hematology and Oncology; Heinrich Heine UniversiO, Medical Center, Diisseldorf Germany. An increased Hey level has been described as risk factor for arterial and venous thromboses. Inhibition of the folinic acid metabolism may result in raised Hcy levels. We performed a prospective study to assess the course of Hyc levels after allogeneic HST. Hcy plasma levels were determined by using high performance liquid chromatography, after labelling with ABDfluorescence. Concentrations were calculated by comparing the areas of retention with a fix standard plasma. Normal range for Hcy was 6.1 -+3.1/trnol/1 (mean -+S.D., n = 43). Male values (6.4_+3.4 /.tmol/l, n=293 were slightly higher than female values (5.5_+ 1.9 pmol/1, n = 14). Children below the age of 2 years (n = 7) had lower levels (4.9_+ 1.7 /trnol/l) compared to adolescents aged > 15 years (8.9 _+3.9/3mol/1, n = 10; p = 0.02).

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Hcy values (n = 31) from 5 children undergoing allogeneic HST were obtained following a prospectively defined time schedule. Hcy values at day -+0 ranged from 3.0-34/_wnol/1 (median: 5.7 /lmol/l). Maximum Hey values after HST ranged from 8.5 102 /lmol/l (median: 23/drool/l) and were reached at day 14 (median). Patients (n = 3) who received methotrexate after HST (15 mg/m 2 body surface area at day +1, 10 mg/m 2 body surface area at days +3 and +6) showed higher Hcy peak values (23, 30 and 102/.tmol/1) compared to those without methotrexate (8.5 and 12.7/Jmol/l). One patient, who had received methotrexate, developed venoocclusive disease (VOD) of the liver with severe capillary leakage (maximum bilirubin 26 mg/dl at day +18 after HST). This patient showed the highest Hcy values before (34/lmol/l) and after (102 /ffnol/l) transplantation. We conclude, that disturbances of the methionine metabolism may lead to increased Hey values after HST. Further analyses are necessary to confirm the association between increased Hcy and thrombotic complications such as VOD. Supported by the Elterninitiative Kinderkrebsklinik e.V., DiJsseldorf

Acetaldehyde, but not alcohol or acetate, inhibits vitamin B~2dependent methionine synthase activity in vitro. S.H. Kenyon l *, A. Nicolaou 2, W.A. Gibbons I. I University-lndustry Centre fi~r Pharmaceutical Research, School of Pharmacy. 2~39 Brunswick Square, London, UK, 2Department of Pharmaceutical Chemistry, School qf Pharmacy, Universit.v of BradJbrd. Richmond Road, Bradford, W. Yorks.. UK. Alcohol has been reported to inhibit vitamin Bjz-dependent methionine synthase in vivo (Sherif F, Gomes C, Orelend L. Pharmacol Toxicol. 1993;73:287-290). This interaction is considered to be important in the pathogenesis of alcoholic cirrhosis and liver cancer (Trimble KC, Molloy AM, Scott JM, Wier DG. Hepatology. 1993; 18:984-9893, thus investigation of this effect may lead to the development of treatment for alcoholics and insights into the pathophysiology of cancer. Using the in vitro methionine synthase assay we investigated the effect of alcohol, and its metabolites: acetaldehyde and acetate using highly purified methionine synthase (Kenyon SH, Nicolaou A, Gibbons WA. Alcohol. 1997;in press). Alcohol, as previously reported (Sherif et al.), had no effect upon enzyme activity, neither did acetate. Acetaldehyde however, inhibited methionine synthase activity, in a dose dependent manner [Acetaldehyde concentration (raM); Mercaptoethanol assay (%inhibition+SD); DTT/cobalamin assay (%inhibition+SD): 0.6; 26.0_+8.0; 1.1_+18.0, 3.6;69.5_+7.3; 23.0_+16.2, 6.0; 79.7_+3.0: 35.3_+18.2, 36.0; 96.5_+0.8; 70.5 + 2.0]. As methionine synthase is associated with two electron donating proteins, and co-purifies with these proteins (Gulati S, Chen Z, Brody LC, Rosenblatt DS, Banerjee R. J Biol Chem. 1997;272:19171 19175), the effect of changing the reducing agent from mercaptoethanol to DTT and hydroxocobalamin was tested. This change increased the apparent IC50 from 2 to 12 mM indicating that acetaldehyde may inhibit methionine synthase by more than one mechanism. SHK is a Maplethorpe Fellow.

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Abstracts / Netherlands Journal o/Medicine 52 (1998) SI $61

The effect of hydrazine on vitamin Ble-dependent methionine synthase activity in isolated rat hepatocytes. S.H. Kenyon 1., C.J. Waterfield2, D.S. Asker:, A. Nicolaou 3, J.A. Timbrell:, W.A. Gibbons 1. 1University-Industry Centre jbr Pharmaceuti-

cal Research, 2Department of Toxicology, School of Pharmacy, 29 39 Brunswick Square, London, UK, ~-Department t~['Pharmaceutical Chemistry, School of Pharmac T, University ~?/"Brad.ford, Richmond Road, BradJbrd, W. Yorks., UK. in rats, hydrazine causes a marked rise in urinary taurine, the end product of sulfur amino acid metabolism (Waterfield CJ, Turton JA, Scales MDC, Timbrell JA. Arch Toxicol. 1993;67:244-254). Methionine synthase (BI2-MS) is one enzyme controlling the recycling of homocysteine. The following study was performed to see if hydrazine affected the activity of methionine synthase. Hepatocytes were isolated (Moldeus P, Hogberg J, Orrenius S. Methods Enzymol. 1978;52:60-71, Chap. 4) and incubated under CO2lO2 (5%195%) for 3 h in Krebs Henseleit buffer containing hydrazine (0, 2, 4, 8 or 12 raM), after which time B12-MS activity was tested using the assay described previously (Nicolaou A, Waterfield C J, Kenyon SH, Gibbons WA. Eur J Biochem. 1997;244:876-882). Toxicity was assessed by LDH leakage and cellular ATP levels. The effect of hydrazine on highly purified methionine synthase was also measured: [Hydrazine (mM); LDH leakage (% total); ATP (nmol/106 cells); Methionine synthase (nmol/h/mg protein): 0; 20.6_+0.7; 7.8_+0.75; 6.68_+1.30, 4; 19.9_+0.5; 4.9_+0.9; 5.56+ 1.50, 8; 21.8_+ 1.8; 2.7_+0.3*; 3.67_+0.8*, 12; 29.1 +5.6; 1.4_+0.28"*; 3.02+0.55", where values are means _+SEM, N = 4 , *p<0.05, **p<0.01, paired t-test, cells from same isolation compared with controls]. Hydrazine (12 mM) caused less than 30% inhibition of purified enzyme, but inhibited B12-MS in isolated hepatocytes in a dose dependent manner, at doses of hydrazine which were not cytotoxic, but which did reduce ATP levels. The results may help to explain the raised levels of taurine seen in vivo after hydrazine treatment. SHK is a Manlethorpe Fellow A rapid and convenient method of measuring betaine and dimethylglycine in blood and urine. M.D. Laryea*, F. Steinhagen, S. Pawliczek, U. Wendel. Metabolic Unit, University Childrens

Hospital, D-40225 Dfisseldorf Germany. Large amounts of betaine are often given orally to patients with inborn errors o f methionine metabolism, in an attempt to decrease elevated plasma homocysteine (Hcy) levels and to increase plasma methionine levels when they are low. However, little is known about its extent of metabolism and regulation in normal subjects and various disease states. Several methods are known for the analysis of betaine and its metabolite dimethylglycine (DMG), however accuracy, sensitivity and simplicity are not enough for routine analysis. We developed a convenient, sensitive, precise and robust HPLC-method for measuring these substances. Betaine and dimethytglycine are derivatised with p-bromophenacyl bromide and the resulting esters are separated on a cation exchange column using acetonitrile-water containing choline as the mobile phase.

With this method we analysed plasma and urine samples of normal subjects and of patients receiving oral therapy with betaine (2 to 8 g/day). Plasma levels ranged from 2-13/zmol/ 1 and from 2(~144/zrnol/l for D M G and betaine, respectively, in the normal subjects. In patients receiving oral therapy with betaine plasma levels ranged from 11-281/.tmol/l and 20---2680 ,umol/l for D M G and betaine, respectively. In urine of normal subjects D M G and betaine ranged 0.8-11.6//mol/mmol creatinine and 6.4-92.7 t,tmol/mmol creatinine. In the urine of betaine treated patients the levels were found to be increased up to 3.6 mmol/mmol creatinine for D M G and up to 20.8 mmol/ mmol creatinine for betaine. The developed method is suitable for monitoring betaine treatment in different forms of homocystinuria. The effect of diet on homocysteine levels in healthy male subjects. N.J. Mann 1., N. Dudman:, X.W. Guo2, D. Li:, A.J. Sinclair/. 1Department of Food Science, RMIT, Melbourne,

3000, Aus'tralia. 2Department ¢~/c Cardiovascular Medicine, Prince Henry Hospital, Sydney, 2036, Australia. Moderate to extreme cases of hyperhomocysteinemia have for some time been associated with genetically determined enzymatic faults of methionine metabolism. More recently, evidence has been accumulating indicating the importance of adequate dietary intake of the three B group vitamins: folate and vitamin Blz (remethylation) and vitamin B6 (transsulfuration), in the prevention of hyperhomocysteinemia. The aim of this study was to determine if habitual diet (omnivorous and vegetarian) resulted in differing plasma levels of homocysteine and whether these levels were related to serum concentrations of folate and vitamin Bl~. We recruited 139 healthy, non-smoking male subjects aged 20--55 years from the Melbourne area. They were divided into four groups based on habitual dietary intake: Vegan in = 18), Ovo-lacto vegetarian (n =43), Moderate meat-eaters (n = 6, < 260 g/day) and High meat-eaters (n = 18, > 280 g/dayl. Plasma total homocysteine concentration was determined by a standard HPLC method and serum folate and vitamin B12 by a competitive immunochemiluminometric assay. Plasma homocysteine levels exhibited a clear and significant step wise increase from the high meat diets through to vegan diets, with the inverse pattern being observed for serum vitamin Bi2 levels. A highly significant correlation was established between these two parameters. No variation in folate levels was detected between groups. Plasma homocysteine was elevated above 16 /~mol/L in 28% of ovolacto vegetarians and 44°A, of vegans, but not in any meat-eaters. Similarly 28'¼, of ovolacto vegetarians and 61% of vegans had serum vitamin B12 levels below 300 ng/L, whereas only 2% of meat-eaters were below this level. This data corresponds with the negligible dietary intake of cobalamin by vegan subjects in western societies. Correlation between plasma total homocysteine and copper. M.A. Mansoor ~*, C. Bergmark e, S.J. Haswell3, l.F. Savage 3, P. Evans 4, A. Demoz 5, R. Berge5, A. Svardal 6. IDivision of

Clinical Chemistry, Central Hospital in Rogaland, 4003 Stavanger, Nor~ay. ~Department ~)[' Vascular Surgery, Karolinska

Abstracts/Netherlands Journal of Medicine 52 (1998) S1-$61 Hospital Stockholm, Sweden. 3Department of Chemistry, University of Hull. 4Department of Public" Health, University of Glasgow, UK. 5Departments of Clinical Biochemistry and SDepartment of Pharmacology, University of Bergen, Norway. Elevated level of plasma total homocysteine (tHcy) or hyperhomocysteinernia is a risk factor for vascular disease. The biochemical mechanism(s) by which increased concentrations of tHcy may induce damage to blood vessels, initiate or cause vascular disease are not established. We measured tHey in 65 patients and 65 sex and age matched healthy control subjects in a case controls study (Mansoor et al. 1995 and Bergmark et al. 1993). In the present study and in the same subjects we explored correlations between t h e y and trace elements, because there were indications that thiols in the presence of copper (Cu) may produce free radicals in vitro. Correlations between tHcy and 17 trace elements including Cu were investigated. Vitamins folate, B12, B6, E, A and fl-carotene, and antibodies against copper oxidised low density lipoprotein and thiobarbituric acid reactive substance were also tested for correlation with tHcy and trace elements. Univariate analysis demonstrated a significant correlation of tHey with copper (Coefficient + SE, 0.337 + 0.18, p = 0.005). In a multivariate regression analysis a positive correlation between tHcy and copper (Coef_+SE 0,437_+0.12, p--0.0004) and a negative correlation between tHcy and folate and vitamin B12 were detected (Coef+ SE -0.351 + 0.097, p = 0.0004, and Coef +_ SE -0.008 + 0.004, p = 0.043). Our findings suggest that the atherogenicity of homocysteine may be related to copper dependent interactions.

Hyperhomocysteinaemia in neurological patients with mild vitamin B12 deficiency. R. McCafferty 1., H. Merlitz 1, A. Briddon:.

Departments of IHaematology and 2Biochemistry, National Hospital for Neurology and Neurosurgery, London WCIN 3BG, UK Serum vitamin Bt2 concentration has been measured using an Abbott IMX Micropartide Enzyme Intrinsic Factor assay in patients presenting with a variety of neurological disorders. A decreased concentration was found in 15% of cases. The assay providers, (Abbott Laboratories, Maidenhead, UK), suggest a reference interval for the assay of 223- 1132 pg/ml with an indeterminate range of 179-223 pg/ml based on the overlap of 95% confidence intervals determined on apparently normal and clinically deficient populations. Correlation studies carried out in our laboratory confirm that mild hyperhomoeysteinaemia occurs at BI2 concentrations in this indeterminate range. Further investigation for vitamin BI2 malabsorption by Schilling Test (Dicopac, Amersham International), showed a mild intestinal B12 malabsorption not corrected by intrinsic factor in 25% of patients with a BI2 value in the range 179-223 pg/ml. This is similar to the 28% incidence of B~2 matabsorption found in patients with overtly low serum B12 concentrations ( < 179 pg/ml). We conclude that mild BI2 deficiency in the range 179-223 pg/ml warrants further investigation and that these patients are candidates for vitamin supplementation in

SI1

view of the risk of cerebral vascular disease associated with hyperhomocysteinaemia.

A randomized controlled study on the effects of 17fl-estradioldydrogesterone therapy on plasma homocysteine in postmenopausal women. V. Mijatovic*, P. Kenemans, C. Jakobs, W.M. van Baal, E.R.A. Peters-Muller, M.J. van der Mooren. Insti-

tute for Cardiovascular Research, Vrije Universiteit, Project 'Ageing Women', Free University Hospital, Amsterdam, The Netherlands. Objective: To investigate the effects of oral 17fl-estradiol (E2)~lydrogesterone therapy on fasting plasma total homocysteine (tHcy) concentrations in healthy postmenopausal women. Methods: We studied 27 postmenopausal women who were randomized into a study group (N= 14) and a control group (N= 13). The study group was treated during the first 12 months with oral E2, 1 mg daily, sequentially combined with dydrogesterone 5 or 10 mg (14 days/28-day treatment cycle). Thereafter these women were treated with oral E2, 2 mg daily, sequentially combined with dydrogesterone, 10 mg daily (14 days/28-day treatment cycle) for a period of 3 months. The control group received no treatment. Fasting plasma tHcy concentrations were determined at baseline, 3, 12 and 15 months after randomization. Results: At baseline, plasma t h e y levels did not differ between the groups. Plasma tHcy did not alter in the control group during the whole study period. As compared to the control group, 15 months of hormone treatment was associated with a significant decrease in plasma tHcy levels (-12.6% vs. baseline; P < 0 . 0 0 1 ; analysis of variance for repeated measures). Significant correlations were found between the absolute changes in plasma tHcy and serum E2 after 3 and 12 months of HRT ( r = - 0 . 5 4 and r =--0.56, resp.; P<0.05). Conclusion: E2-dydrogesterone replacement therapy lowers plasma tHcy levels in postmenopausal women.

The subcutaneous adipose tissue topography (SAT-Top) measured with the optical device Lipometer reflects homocysteine levels in a circadian rhythm. R. M6ller l*, E. Tafeit ~, K.M. Sudi:, K. Vrecko l, R. Horesji l, G. Reibnegger t. Karl-Franzens

University Graz, Austria, Ilnstitute Jor Medical Chemistry and PregI-Laboratory. 2Institute./or Aport Sciences. The aim of the study was to investigate if and to what extent subcutaneous adipose tissue topography (SAT-Top) can reflect homocysteine levels in a male subject throughout a 24-h period. Methods" and subject: The optical device Lipometer analyses the amount of backseattered light in a chosen subcutaneous fatty layer and computes the thickness of this layer in mm. For the measurement of subcutaneous adipose tissue pattern (SATTop) 30 standardized body sites were chosen. The sites were distributed over the whole body symmetrically both on the left and right side from the neck to the calf. A healthy, weight stable male subject 33 years old was assigned to a diet of 11700 kJ of energy per day. Energy intake consisted of approximately 65% carbohydrate, 15';/,, proteins and 20% fat. The measurements of SAT-Top by means of Lipometer were car-

S 12

Abstracts I Netherlands Journal of Medicine 52 (1998) S1-$61

ried out in standing position starting at 0715 am and ending the next at the same time. For the measurement of homocysteine blood samples were taken every 3 hours at the same time when SAT-Top measurements were taken. Results: Most of the SAT-Top body sites showed rhythmic increase and decrease as did the mean value of all body sites with a maximum at 0715 pm. Homocysteine levels showed a time pattern which was not exact the same as for SAT-Top. In a multiple regression analysis we identified the combination of 3 body sites (hip and front thigh at the right body site and neck at the left body site) to describe homocysteine as the dependent variable (r = 0.975; p = 0.0011 ). Discussion: Our study shows that 'simply living a day' is able to change SAT-Top and homocysteine levels in a short time. Due to the observed association between SAT-Top and homocysteine levels we may speculate about the role of body fat distribution and especially SAT-Top as a marker of the endocrine system for at least in the investigated male subject.

In vitro control and regulation of vitamin Bl2 dependent methionine synthase. A. Nicolaou 1., S.H. Kenyon:, C. Waterfield:, W.A. Gibbons:. IDepartment of Pharmaceutical Chemistry, University of Bradford, UK, 2University-Industry Centre jor Pharmaceutical Research, School of Pharmao', University of London, UK, 3Department of Toxicology, School o[" Pharma~T, University of London, UK. Methionine synthase is considered to be important for the maintenance o f normal cell functions. Disturbance of enzyme activity has been linked to megaloblastic anaemias, neuropathy, cancer and atherosclerosis. Control and regulation of methionine synthase under normal physiological conditions, needs to be understood in order to elucidate its role to the pathogenesis of these disease states. In search of such mechanisms, we have performed a series of in vitro experiments, screening a range of compounds and downstream metabolites of methionine synthase. Our experiments have been performed using highly purified rat liver methionine synthase and rat hepatocytes. The polyamines putrescine, cadaverine, spermine and spermidine were found to stimulate methionine synthase activity (Nicolaou A, Kenyon SH, Gibbons JM, Ast T, Gibbons WA. Eur J Clin Invest. 1996;26:167--170) supporting the hypothesis that they may act as feedback regulators of the enzyme. Studies on the nitric oxide (NO) induced inactivation of methionine synthase, performed with NO and NO donors, provided arguments in favour of a mechanism uniting the effects of nitric oxide with elevated levels of homocysteine (Kenyon SH, Nicolaou A, Ast T, Gibbons WA. Biochem J. 1996;316:661-665 and Nicolaou A, Waterfield C J, Kenyon SH, Gibbons WA. Eur J Biochem. 1997;244:876-882). Inactivation of liver methionine synthase by alcohol is considered to be one mechanism resulting in the pathological effects associated with alcohol abuse. Since the inactivation has been shown to occur in vivo but not in vitro, we have performed a series of in vitro studies with alcohol and its metabolites (Kenyon SH, Nicolaou A, Gibbons WA. Alcohol. 1997;in press). The results of this study revealed that acetaldehyde inhibits methionine synthase,

providing thus an attractive and rational explanation for the in vivo findings. SHK is a Maplethorpe Fellow.

Plasma homocysteine, folate, vitamin B12 and B6 status in the oldest old. I.M. Rea 1., D. McMaster 2, G. Doherty s, P. Archibold 4, H. McNulty 5, I.R. Y o u n f , A.S. Whitehead 6. Departments of Geriatric Medicine I . Medicine ~ and Clinical Biochemistry 3 The Queen's University of Belfast, Biochemistry Laboratory, Belfast City Hospital 4, Department of Human Nutrition, University of Ulster5, Coleraine, Department of Genetics and Biotechnology 6 Trinity College, Dublin. There is now accumulating evidence that elevated blood levels of homocysteine increase the risk of vascular disease. Very elderly people have survived early mortality from cardiovascular, cerebrovascular and peripheral vascular disease and might be expected to demonstrate altered homocysteine metabolism. In this preliminary report of a cross-sectional study of community living subjects, homocysteine levels were measured in more than 200 elderly and very elderly subjects who were mentally alert and apparently well. Plasma samples stored at -200°C were analysed by routine methods - approximately 100 ( > 9 0 year olds), 80 (80-90 year olds) and 40 (70-80 year olds) with approximately a 2:1 ratio of women to men. Serum folate, Vitamin B12, urea and creatinine were measured together with dietary folate and B vitamins. Homocysteine was higher than for local younger controls, was lower in females compared to males and fell slightly but significantly with increasing age. Folate, Bt2 and B6 were suboptimal in approximately 25 30% of subjects. Subjects with the lowest percentiles of folate and B6 showed graded increases in homocysteine but there was no change for B12. Homocysteine was not correlated with urea or creatinine in univariate analysis. In a multiple regression model, 25% of the increase in homocysteine was explained by age, urea, protein intake and M T H F R genotype category. These findings show that higher homocysteine and suboptimal folate, vitamin BI2 and B6 co-exist in apparently well, very elderly subjects living in the community but that as age increases plasma homocysteine falls in very old age. We have previously shown that M T H F R genotype frequency is unchanged or undiluted in these elderly subjects. The changes in homocysteine seem only partly explained by presently understood mechanisms but might suggest that an unidentified genetic or environmental 'survivor' effect is present in the oldest old. Funded in part by a Wellcome Summer Student Grant 1995. Are high homocysteine levels a benign phenomenon in the elderly? J.S. Silberberg JS l*, R. Crook R 1, J.L. Fryer ~, B. Nair 1, P.J. Fletcher 1, X.W. Guo 2, N.P.B. Dudman e. IJohn Hunter Hospital & University of Newcastle, Australia, 2Prince Henry Hospital & University of NSW, Australia. Several investigators have reported higher tHcy levels in the elderly, often due to unrecognised Bi2 deficiency. Elderly persons are at high risk of serious vascular events but the contribution of high tHcy levels is unknown.

Abstracts/Netherlands Journal of Medicine 52 (1998) S1-$61 We studied the frequency of vascular risk factors in 53 'elite seniors' who were actively competing in lawn bowls at least one day per week, were free of self-reported vascular disease, had physical examination findings which were unremarkable except for vascular bruits, had normal resting ECG and no regional wall motion abnormality on echocardiography. The salient findings are summarised below as mean (S.D.); values in 89 younger persons with early familial CHD are shown for comparison [Healthy elderly, HE; Young familial CHD, YF. N (% male): HE, 53 (81%); YF, 89 (63%). Age: HE, 79 (4); YF, 51 (7). Total cholesterol (mmol/L): HE, 5.9 {1); YF, 5.9 (1). hdl cholesterol (mmol/L): HE, 1.3 (0.4); YF, 1.1 (0.4). Triglycerides (mmol/L): HE, 1.4 (0.6); YF, 1.9 (1). lp(a) >300 g/L: HE, 20%; YF, 39%. Creatinine ~mol/L): HE, 106 (21); YF, 97 (21). tHcy Cumol/L): HE, 17.2 (8); YE, 12.6 (4). Serum B12 (pmol/L): HE, 298 (235); YE, 355, (294). Plasma folate (/~g/L): HE, 19.3 (11); YF, 18.4 (6). Red cell folate (/.tg/L): HE, 891 (370); YF, 1002 (332). MTHFR ll genotype: HE, 3/53 (6%); YF, 2/24 (8%). ACE dd genotype: HE, 8/53 (15%); YF, 17/53 (32%)]. All putative risk factors tended to be favourable in these healthy elderly except for tHcy levels. We suggest that high tHey in the elderly may not increase the risk of vascular events. Natural history studies of vascular events in patients with pernicious anaemia would be worthwhile. Hyperhomocysteinemia and race: different causes. S.P. Stabler SW*, R.H. Alle1, L.P. Fried:, M.S. Pahors, J. Kittner4, J.M. Guralnik 5. 1U of CO Health Sciences Center, Denver, CO,

zJohn Hopkins U and U of Maryland, Baltimore. MD. 3U ol" Tennessee, Memphis, TN and 4Epid, Demog and Biometry, NIH, Bethesda, MD, USA. Elderly (mostly white) cohorts have been found to have a high prevalence of cobalamin (Cbl) deficiency. Most subjects with hyperhomocysteinemia in these cohorts also had elevated methylmalonic acid (MMA). We measured MMA, total homocysteine (tHcy) and serum vitamin levels in 762 elderly disabled women from the Women's Health and Aging Study, aged 65-99 yr, 28% African-American (Afr-Am). The mean serum tHcy was significantly lower in whites as compared to the AFr-Am, 11.2 vs. 12.7/IM (p=0.005) and the number with high tHcy ( > 13.9/zM) was lower in the whites, 19% vs. 31°/o. In contrast, the mean serum MMA was higher in the whites as compared to the Afr-Am, 301 vs. 220 nM (p=0.0001) with a significantly greater number having a high (>271 nM) level 39°/,, vs. 20%. Serum creatinine (Cr) was lower in white subjects as compared to the Afr-Am, 1.0 vs. 1.2 mg/dl (p = 0.001). Cbl deficiency (serum Cbl < 350 pg/ml with high MMA) was found in 21% of the whites as compared to 8% of the Afr-Am (p < 0.001). Folate deficiency (serum folate < 4 ng/ml and elevated they with normal MMA level) was found in 2% of the whites as compared to 6% of the Afr-Am (p = 0.02). Whites were almost twice as likely to take multivitamins (MVI) as the Afr-Am, 23% vs. 12% (p<0.001). No user of MVI had folate deficiency and the mean tHey in subjects with Cr < 1.2 mg/dl was 8.9 ItM for users as compared to 11.2/tM for nonusers (p < 0.05). There was also a significant effect of MVI use

SI 3

with higher serum Cr ( > 1.2 mg/dl); users 11.6/tM vs. nonusers 15.9 ¢tM. MVI use did not significantly decrease the serum MMA. Linear regression showed that age, black race, Cr > 1.0 mg/dl, serum Cbl and folate levels, serum MMA and MVI use were determinants of tHey. For MMA, older age, white race, Cr, Cbl, folate and tHcy were determinants of the serum MMA. We conclude that in elderly disabled women the mean tHcy and folate deficiency rates are higher in Afr-Am and mean MMA and Cbl deficiency rates are higher in whites. Influence of H.E.L.P. therapy on homocysteine in human plasma. K. Vrecko I*, M. Walzi2, G. Schied2, B. Walzt:, G. Reibnegger1. 1Institute Jbr Medical Chemistry, Karl-Franzens

University of Graz, Austria, 2Department of Electrobiology/ H.E.L.P. -Center, University Clinic of' Neurology, Graz, Austria. Recent investigations have shown that increased homocysteine concentration in plasma, caused either by malabsorption and/or vitamines B6, Bl2 and folic acid deficits, or by genetic defects concerning homocysteine methyltransferase and cystathionine fl-synthetase, is an independent risk factor for neural tube defects and atherosclerosis. The biochemical role of homocysteine in vascular diseases remains unclear. Endothelial cells toxicity, increased attachment of blood platelets and changes of coagulation factors are discussed as reaction to high homocysteine levels. H.E.L.P. (Heparin-induced Extracorporal LDL Precipitation) therapy is applied to patients with different kinds of atherosclerosis (coronary heart disease CHD, cerebrovascular insufficiency CVI, hyperlipemia HL, peripheral occlusive vessels disease POVD) showing very good positive reactions. During this therapy fibrinogen, cholesterol, LDL, lipoprotein (a) and triglycerides are precipitated and washed out extracorporally from approximately three liters of plasma within two hours. The question raised whether homocysteine concentration in blood can also be lowered by this therapy. Therefore we measured first homocysteine concentrations in plasma from 27 patients with CHD (homocysteine= 12.68 /tmol/1; SD=4.79), 20 patients with POVD (14.69 ¢tmol/l; SD=3.30), 22 with CVI (18.97 ~tmol/1; SD=12.57) and 47 with untreated HL (11.58 /lmol/l; SD=3.651. We found significantly increased homocysteine levels (Wilcoxon test) in patients with POVD in comparison to those with KHK (P<0.02), POVD in comparison to HL (P<0.03), CVI in comparison to KHK (P<0.05) and CVI in comparison to HL (P < 0.004). Afterwards we measured in 67 patients, suffering from the above mentioned diseases, homocysteine before and after H.E.L.P. therapy, and we found a highly significant homocysteine reduction by 14.4% (P < 0.005). These results are a first indication that elevated homocysteine can be lowered by H.E.L.P. therapy and that patients with homocysteinemia might be prevented from the consequences of this disorder. Stabilisation of blood samples for total homocysteine with acidic citrate. H.P.I. Willems j*, G.M.J. Bos2, W.B.J. Gerrits 1, H.J. Blom s. 1Dept. of Haematology, Leyenburg Hospital, The

Hague, 2Dept. ()f Haematology, Dr Daniel den Hoed Clbffc.

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Abstracts I Netherlands Journal of Medicine 52 (1998) S1 $6I

Rotterdam, 3Dept. of Paediatrics, University Hospital St Radboud, Nijmegen, The Netherlands. Background Production o f homocysteine continues in whole blood after collection. To avoid falsely elevated total homocysteine (tHcy), blood is usually sampled in tubes with EDTA, immediately put on ice and the plasma is separated as soon as possible. When blood is collected in non-laboratory conditions, this method can be problematic. We therefore investigated if tHcy remains stable in other collection media. Methods Blood was drawn from healthy laboratory workers (n = 30). Blood was drawn in vacuum tubes with acidic citrate (Biopool, Stabilyte~ ) and with EDTA (Vacutainer, Becton and Dickinson). The tubes with EDTA were immediately put on ice after collection or kept at room temperature. Tubes with acidic citrate were kept at room temperature or at 37°C. The blood was centrifuged 0, 2, 4 and 6 hours after collection. Plasma was stored at - 3 0 ° C until determination of tHcy. Results Six hours after collection, tHcy did not rise significantly in the tubes containing EDTA which were stored at 0°C (0.3/.tmolfl [95% CI with paired samples T-test -0.1 to 0.7]) as well as in the tubes with the acidic citrate which were stored at 21°C (0.3 pmol/1 [95% CI - 0 . 2 to 0.6]). tHcy concentrations rose significantly in the tubes with EDTA when they were stored at room temperature: 2.0 /~nol/1 after 2 hours up to 4.7 pmol]l after 6 hours (95% CI 1.6 to 2.4 and 4.1 to 5.3, resp.). In the tubes with acidic citrate which were kept at 37°C tHcy concentrations remained stable in the first two hours but after 4 hours there was an increase of 0.9 pmol/1 (95% CI 0.5 to 1.3). Mean baseline tHcy concentrations were lower in the samples taken in tubes with EDTA than in the samples with acidic citrate (12.7 and 14.0 pmol/l, resp.). Conclusion Acidic citrate seems to be a good collection medium for determination of tHcy in blood sampled in non-laboratory conditions.

Free communications Folic acid supplements lower plasma homocysteine and improve flow-mediated endothelial function in healthy adults. M.F. Bellamy*, I.F.W. McDowell, M.W. Ramsey, M. Brownlee, M.J. Lewis. Cardiovascular Sciences Research Group, University ~f" Wales College of Medicine, Cardiff, UK. Endothelial injury with loss o f nitric oxide bioavailability may be a mechanism whereby homocysteine (Hcy) promotes arteriosclerotic vascular disease. Mild to moderate elevation of Hcy has been shown to be associated with endothelial dysfunction in man. Improvement of endothelial function by lowering Hcy would provide evidence for a causal role. We studied 18 healthy adults (age 35 + 6[SD] years, 12 male) with plasma Hcy concentrations in the upper part of the normal range from a screening population of 890 volunteer blood donors. The study design was a double-blind, placebo-controlled, crossover trial of oral folic acid (5 mg daily for 6 weeks) with a 6 week interval between treatments. Using ultrasonic vessel wall-track-

ing, Doppler ultrasound and photoplethysmography, we measured brachial artery (BA) diameter responses to hand hyperaemia (flow-mediated, endothelium-dependent vasodilatation [FMD]) and to sublingual glyceryl trinitrate (3 min after 400 pg [GTN]). Oral folic acid lowered plasma Hcy cf. placebo (8.7 _+2.5 p M cf. 12.0 -+ 3.7 pM, n = 18, p < 0.05) and improved flow-mediated endothelial responses. There was a carryover effect between treatments such that subjects receiving placebo 6 weeks after stopping folic acid showed a residual effect from the first treatment. This will lessen the magnitude of a positive result. In spite of this, a significant result was obtained using a standard crossover analysis. Folic acid improved F M D to +0.08 + 0.05 mm cf. +0.04 + 0.04 mm placebo. (SE = 0.014, 95% CI +0.009 to +0.069, p = 0.015). GTN responses were unchanged in both groups. Basal BA diameter, heart rate and systolic blood pressure were similar following treatment with folate compared with placebo as were increases in hyperaemic blood flow (A50_+ 30 cf. A60 + 53 ml s/min). Healthy subjects with Hcy levels in the upper range of normal respond to high dose folic acid supplementation by lowering Hcy and enhancing flow-mediated, endothelium-dependent vasodilatation.

Inactivation of the folate binding protein genes: implications for the role of homocysteine in neural tube defects. R.H. Finnell t*, G.D. Bennett 1, S.W. Lacey2, B.A. Kamen 2, J. Van Waes ~, J.A. Piedrahita I . 1Texas A & M University, College Station, Texas, USA and 2 University ~/' Texas Southwestern Medical Center, Dallas, Texas, USA. Neural tube defects (NTDs) are common human congenital malformations whose etiological basis remain poorly understood. In an effort to learn more about environmental risk factors and genetic determinants of susceptibility to NTDs, a unique transgenic mouse model system was developed. The two murine folate binding protein genes, FBP-I and FBP-2, were inactivated by homologous recombination in embryonic stem cells to develop mice that had impaired ability to internalize folic acid. This vitamin co-factor has been shown to significantly reduce the occurrence of NTDs when provided to the mother during the periconceptional period. The mechanism underlying this beneficial effect is unknown, as is the issue of whether the observed NTDs are the result of insufficient folic acid concentrations or due to elevated levels of homocysteine. The development of these knockout mice demonstrate that adequate folic acid concentrations are essential for the embryos to progress normally through neural tube closure. Among the progeny of FBP-1 heterozygote incrosses, approximately one-quarter of the embryos had NTDs. The NTDaffected embryos were homozygous lbr the null allele ( - / - ) , as confirmed by Southern Blot analysis. In addition to NTDS, the embryos had lethal malformations of the cardiovascular system and were significantly growth and developmentally retarded. No such defects were observed with the FBP-2 nullizygotes. Folic acid concentrations for animals maintained on either a standard rodent diet, or one that was 90% depleted of folic

Abstracts~Netherlands Journal o[' Medicine 52 (1998) S1--$61 acid were determined. The FBP-1 heterozygous and the FBP-2 nullizygous dams had significantly reduced levels of folic acid when compared to their wildtype controls under normal dietary conditions. Irrespective of FBP genotype, all of the dams were significantly folate depleted under the conditions of folate dietary restriction. In terms of homocysteine levels, dams of all genotypes had highly significant elevations in homocysteine levels when dietary folic acid was restricted. The relative role of folic acid deficiency and elevated homocysteine levels in terms of NTD risk will be discussed. Supported by NIH grants HD/ES 35396 and ES 07165, and a grant from the March of Dimes Birth Defects Foundation

Disturbed ratio of plasma and erythrocyte S-adenosylmethionine/S-adenosylhomocysteinein peripheral arterial occlusive disease. P. Litynski1., M. Tsch6pl2, F.M.T. Loehrer 1, C. Angst l, K. Jfiger2, W.E. Haefeli3, B. Fowlerj. University Children's Hospital Basel1; Div. of Angiology 2 and Div. ~[' Clinical Pharmacology 3, University Hospital Basel, Switzerland Elevated homocysteine (tHcy) concentrations are associated with peripheral arterial occlusive disease and with impairment of key methylation reactions through accumulation of S-adenosylhomocysteine (AdoHcy). We determined tHcy, S-adenosylmethionine (AdoMet), AdoHcy and 5-methyltetrahydrofolate (MeTHF) in plasma as well as AdoMet and Ado-Hcy in erythrocytes (RBC) in 61 patients with peripheral arterial occlusive disease (age 75+ 1.8; 29 men, 32 women) and in 53 healthy controls (age 63+2.4, p<0.01 versus patients; 23 men, 30 women). Analysis was performed by various HPLC methods [Plasma values: tHcy (~mol/l)**, patient 18.6_+ 1.3, control 10.8 + 0.5; AdoMet (nmol/1)**, patient 138 + 16, control 55.5 +2.5; AdoHcy (nmol/1)**, patient 81.1 + 14, control 23.3 + 1.2; AdoMet/AdoHcy*, patients 2.3 +0.2, control 2.7 + 0.2. Erythrocytes: AdoMet (lamol/l)*, patient 3.(I+_0.1. control 3.8 + 0.1 ; AdoHcy (nmol/l)*, patient 862 +_384, control 22.6+ 15; AdoMet/AdoHcy*, patient 11.5+ 1.0, control 18.8+1.5. Values: means+SEM; **p<0.001; *p<0.01 (Mann-Whitney U-test)]. MeTHF values were similar for patients (24.6 + 4.7 nmol/l) and controls (26.8 + 4.2; p = NS). Adjustment for age and creatinine clearance did not change the mean values significantly (multifactorial analysis, MANOVA). Beside the expected alteration of tHcy in patients, both AdoMet and AdoHcy in plasma increased, together with a moderate decrease in the ratio which might be due to the increase in AdoHcy. In RBC AdoHcy increased to the same extent, AdoMet however decreased expressed by the marked decrease in the ratio. This might be due to the lack of remethylation in RBC additionally to the inhibition of transmethylation reactions by elevation of AdoHcy. Since the ratio of AdoMet/ AdoHcy is closely linked with the activity of numerous enzymatic methylation reactions, these results suggest that methylation may be impaired in these patients, which needs to be considered in pathophysiological studies in such patients. Hyperhomocysteinemia following oral methionine load impairs endothelium-dependent arterial dilatation in atherosclerotic sub-

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jects but not in healthy controls. M. Krzanowski*, T.B. Domagala, A. Motyl, Frolow and Szczeklik A. Department of Medicine, Jagiellonian University School of Medicine, 31-066 Cracow. Poland. The aim of our study was to find whether hyperhomocysteinemia induced by an oral methionine load (0.1 g/kg) impairs endothelial function in man. The loading test was performed in 32 symptomatic patients suffering from coronary, peripheral, or extracranial arterial occlusive disease and in 11 healthy controls. Flow mediated dilatation (FMD) of brachial artery to forearm hyperaemia, following release of a forearm cuff" inflated to suprasystolic pressure for 5 min, was recorded. Patients with any degree of upper limb ischemia were excluded from the study. The homocysteine levels and FMD were measured before and at 4, 6 and 24 hours after the methionine load; the patients remained fasting up to 6th hour of the test. Both in patients and controls plasma total homocysteine rose significantly and peak values at 6 hours were 56.0_+ 20 /1tool/1 and 43.6 + 9.2 ~mol/l, respectively. The FMD decreased in patients (9.5_+6.7% before the load, 7+6.3')'o at 4 hours (NS), 6.3 + 6.2°/,, at 6 hours (p = 0.05) and 8 + 8.8% at 24 hours (NS)) but not in healthy controls (7.9 + 4% before the toad, 9.4+5.5% at 4 hours (NS), 9.8+4.1% at 6 hours (NS) and 7.4+ 2.3% at 24 hours (NS) (mean_+ SD)). We were unable to show any significant diurnal changes in FMD when we measured it over 24 hours in 13 healthy volunteers not loaded with methionine. In conclusion, transient hyperhomocysteinemia induced by methionine load, impairs endothelial function in patients with symptomatic atherosclerosis, but not in healthy controls. It could be partly attributed to greater increase in homocysteine in the patients than in controls. Perhaps, endothelium of patients with atherosclerosis is more sensitive to plasma homocysteine rise, a phenomenon that may be relevant to development of atherosclerosis. Methionine load test combined with brachial artery vasodilatory response measurement may prove useful in detecting a subgroup of healthy people prone to develop atherosclerosis. Homocysteine remethylation in mammalian cells. L.-S. Chen, K. Peng, J.-C. Hsu, Y.-J. Choi, B. Shane*. Department q/' Nutritional Sciences, University q/Cal~/'ornia, Berkeley, Cal!/ornia, USA. At physiological homocysteine and folate levels, most cells, and most tissues, appear to be limited in their abilities to remethylate homocysteine, which may explain why homocysteine is exported by most cells. The major sites of homocysteine remethylation may be limited to tissues such as liver and kidney. We have investigated the control of the metabolic one carbon flux into the homocysteine remethylation cycle in model mammalian cells. CHO and HepG2 cells have a limited capacity to grow under methionine-deficient conditions and growth requires high levels of homocysteine and folate. Modelling of kinetic parameters for methionine synthase (MS) and methylenetetrahydrofolate reductase (MTHFR) suggests that both enzymes may be partly rate-limiting in homocysteine reinethylation in these cells and that their activities, even with high folate, are insufficient to support methionine synthesis

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Abstracts~Netherlands Journal o[' Medicine 52 (1998) S1 $61

rates needed for optimal cell growth. CHO and HepG2 cells were transfected with full length human MS and M T H F R cDNAs and cells expressing elevated levels of these enzymes were obtained. Preliminary studies indicate that increased expression of M T H F R activity improves growth and reduces the folate requirement under methionine-free conditions, demonstrating that M T H F R is partly rate-limiting in the methionine resynthesis cycle. Homocysteine remethylation is promoted by increased cellular folate. High folate increases MS activity in CHO cells but not in HepG2 cells. MS m R N A levels are unaffected by medium folate although a small, but reproducible effect of medium methionine was observed. Folate accumulation by mammalian cells is proportional to FPGS activity when cells are cultured in medium containing high folate levels. We have investigated the flux through the cytosolic and mitochondrial serine hydroxymethyltransferase (SHMT) reactions by measuring the interconversion of labeled carbons of serine and glycine, their incorporation into serine, glycine and methionine in protein and their secretion into the medium. The net flux through the cytoSHMT reaction is normally in the direction o f serine synthesis under steady state conditions and cytoSHMT catalyzes the removal of one carbons from the cytosolic folate pool and redirection away from methionine synthesis. Increased expression of cytoSHMT increases the rate of serine synthesis and excretion into the medium. Supported in part by NIH grant number DK42033. Hyperhomocysteinemia: an independent risk factor for histopathologically-confirmed Aizheimer's disease. A.D. Smith/*, R. Clarke 2, K.A. Jobst j, H. Refsum s, L. Sutton/, P.M. Urland s.

Oxford Project to Investigate Memory and Ageing (OPTIMA), University of Oxford, Oxford, England, ~Department of' Pharmacology, University of Oxford, 2Clinieal Trial Service Unit, University of OxJbrd, 3Department ~['Pharmaeology, University of Bergen, Norway. In relation to the idea that vascular factors may be involved in the development o f Alzheimer's disease, we examined the serum levels of total homocysteine (tHcy), vitamin BI: and folate in 164 cases with a clinical diagnosis of dementia of Alzheimer's type (DAT), including 76 who had histopathological confirmation of the diagnosis (AD), and in 108 cognilively-screened controls. The cases and controls were followed on a yearly basis. Serum tHcy concentrations at presentation (mean of 29 months prior to death in confirmed AD cases) were significantly higher, and serum folate and vitamin B12 levels were significantly lower, in both DAT and AD cases than in controls. The association of serum tHcy with DAT and with AD was independent of possible confounders such as age, sex, social class, smoking and the apoE e4 genotype. The odds ratio of confirmed AD for tHcy concentrations in the upper tertile of controls was 4.5 (95% CI 2.2- 9.2) after adjustment for these confounders. The adjusted odds ratios of AD for folate and vitamin B12 concentrations in the lower tertiles of controls were 3.3 (95% CI 1.8-6.3) and 4.4 (95'7,, CI 2.1 8.8), respectively, but these were no longer significant after adjust-

ment for tHcy, suggesting that the vitamin associations were mediated through tHcy levels. Notably, individuals with higher tHcy concentrations at presentation showed a more rapid progression of their disease over a three year period, which could reflect a causal relationship. Clinical trials with folic acid and vitamin Blz intervention are needed to confirm this hypothesis.

Pathology of hyperhomocysteinaemia Obstetrical and gynecological complications of hyperhomocysteinaemia Hyperhomocysteinemia in obstetrics and gynecology. An unifying concept. T.K.A.B. Eskes*, E.A.P. Steegers, H.M.W.M. Merkus. Department of Obstetries and Gynecology, University

Hospital Nijmegen, Netherlands. Homocysteine is an amino acid with toxic properties for blood vessels and the embryo. Hyperhomocysteinemia can be present in patients with vascular disease [1], in patients with birth defects in their offspring like neural tube defects-NTD [2], recurrent early pregnancy loss [3], congenital heart anomalies [4], schisis [5] and vascular pathology like placental abruption [6] and preeclampsia [7]. Hyperhomocysteinemia can be caused by lack of B-vitamins frolic acid) or by genetic mutation of the genes that regulate the activity of 5,10-methylenetetrahydrofolate reductase (MTHFR) or both. These genes are located on chromosome one: C677T and A1298C and involved in NTD [8,9], recurrent early pregnancy loss [10] and vascular disease [11]. These mutations reduce the activity of M T H F R and cause thermolability. M T H F R catalyses the reduction of 5,10-methylene THF to 5-methyl THF, the last being necessary for the remethylation of homocysteine to methionine. Thereafter the major methyldonor S-adenosylmethionine is available tbr DNA synthesis and necessary for fast growing tissues. Folic acid can overcome this genetic blockade and can also prevent the clinical consequences of these mutations like neural tube defects [12,13]. Hyperhomocysteinemia is also present in the postmenopausal state [14], can be corrected with estrogens, and can be considered at least in part as a cause for the susceptibility for cardiovascular disease at this particular phase of life. It is challenging to synthesize the above mentioned data in a unifying clinical concept in which the obstetric (familial) performance is linked to the cardiovascular events of life that might happen in the postmenopause. Such research could also unify Obstetrics and Gynecology into a core activity that could lead to life-long guidance of women with emphasis on prevention. It is postulated that these cardiovascular complications will occur especially in women who carry genetic mutations (nature) without knowing. It is also postulated that folic acid (nurture) can prevent part of these disasters. ReJerences: [1] Graham et al. J A M A 1997;277:1775-1781. [2] Steegers-Theunissen et al. Metabolism 1994;43:1475-1480.

Abstracts/Netherlands Journal of Medicine 52 (1998) S1-$61 [3] Wouters et al. Fertil. Steril. 1993:60:820 825. [4] Haagmans et al., 2nd Int. Conf. Homocysteine Metabolism Nijmegen, April 26-29, 1998. [5] Steegers-Theunissen et al., Am. J. Hum. Genet. 1997:$61, A396, 2319. [6] Goddijn-Wessel et al. Eur. J. Obstet. Gynecol. Reprod. Biol. 1996; 66:23-29. [7] Sohda et al. Med. Genet. 1997:34:525-526. [8] Van der Put et al. Lancet 1995:346:1070-1071. [9] Van der Put et al. 2nd Int. Conf. on Homocysteine Metabolism Nijmegen April 2(~ 29, 1998. [10] Nelen et al. Lancet 1997:350:861. [11] Van der Molen et al. 2nd Int. Conf. on Homocysteine Metabolism Nijmegen April 26-29, 1998. [12] M R C Vitamin Study Group Lancet 1991:338:131-137. [13] Czeizel and Dudas New Engl. J. Med. 1992:327:1832-1835. [14] Wouters et al. Eur. J. Clin. Invest. 1995:25:801-805. [15] Van der Mooren et al. Eur. J. Clin. Invest. 1994:24:733-736. Grants: CDI; Preventie fonds; Neth. Heart Foundation; Princess Beatrix Foundation. Disciplines: Ob and Gyn, Lab Endocrinology and Reproduction, Paediatrics, Lab Paediatrics and Neurology, Internal Medicine, Clinical Genetics, Epidemiology and Toxicology at Nijmegen University and Department of Genetics, Montreal, Canada. Homocysteine and Congenital Defects: a Role for the NMDA Receptor. T.H. Rosenquist l, V.J. Andaloro:, D.T. Monaghan e, J. Selhub 3. Departments of Cell Biology I and Pharmacology2, University of Nebraska Medical Center, 600 S. 42nd St., Omaha, N E USA 68198-6395; USDA-Tu[ts University Human Nutrition Center on Aging at Tufts Universio,3, 711 Washington Street, Boston, MA USA 02111, USA. The relationship between folic acid deficiency and the occurrence of neural tube and neural crest defects has been demonstrated repeatedly in both experimental and epidemiologic studies. It had been hypothesized by others that the teratogenic agent in folic acid insufficiency was homocysteine (e.g., Steegers-Theunissen et al. Am. J. Ob. Gyn. 172:1436, 1994). In testing this hypothesis, we found that homocysteine induced neural tube and neural crest defects in an avian model (Rosenquist et al. PNAS USA 93:15227, 1996); this study did not show the teratogenic mechanism of homocysteine. Because homocysteine may act as an antagonist of the N-methyl-D-aspartate (NMDA) type of glutamate (Lipton et al. PNAS USA 94:5923, 1997); and because the N M D A receptor is highly significant in the regulation of adhesion and cytoskeleton genes, it was hypothesized that homocysteine could induce congenital defects by disrupting the function of the N M D A receptor. To test this hypothesis, we showed first that the ability to induce neural tube and neural crest defects was a general property of well-defined N M D A receptor antagonists (Andaloro et al. Ped. Res. 43:1). Next we showed that the frequency of occurrence and severity o f homocysteine-induced defects, as well as embryonic death, may be reduced or prevented by activation of the N M D A receptor. Of the agonists tested, the most effective protection against homocysteine-induced congenital defects was given by l-aminocyclopropane-l-carboxylic acid (ACPC). We also found that homocysteine may act addi-

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tively or synergistically with other N M D A receptor blockers to increase the frequency of occurrence or severity of neural crest and neural tube defects, e.g. a non-teratogenic concentration of the N M D A receptor calcium channel blocker MK801 increases the teratogenicity of homocysteine by 1/3. Supported by NIH ROI HL-55940.

Poster visits Mild hyperhomocysteinemia is a graduate risk factor for arterial and venous thrombosis disease. A. Ankri*, G. Montalescot, B. Chadefaux, J. Blacher, M.C. Couty, D. Thomas, P. Kamoun. CHU Pitib-Salpbtrikre." H6pital Necker, Paris, France. Mild hyperhomocysteinemia is considered as a risk factor for both arterial and venous thrombotic disease. Study: Plasma homocysteine (HCY) level was measured in 419 consecutive patients referred to our coagulation Department from 1991 to september 1996, 3 to 6 months after single or recurrent arterial orivenous thrombotic events and in 208 healthy volunteers (control group), thrombotic disease free. Among the 422 patients, 172 presented an arterial thrombotic disease (ATD) and 250 a venous thrombotic disease (VTD). Fasting total HCY (radio-isotopic assay), folates and vitamin 812 levels were measured in patients and in controls. All patients had: ATDIII, PC, PS, plasminogen measurements, detection of antiphospholipid antibodies and dysfibrinogemia. APC resistance and presence or absence of f.V Leiden were assayed in 156 patients. Results, Control group: Mean HCY level, m + SD was significantly higher in men (M, n = 75) 9.9 + 3.9 /lmol/L than in women (W, n = 133), 7.3+2.1 pmol/L (p < 0.0001). The cut off value was defined as M + 2 SD, 17.7 pmol/L in men and 11.5 /.tmol/L in women. Results, Patients: Mean HCY level was significantly higher (p < 0.0001) in patients with ATD than in controls: 15.1 + 8.7 /.tmol/L in men (n=100) and 13.2+4.6 /tmol/L in women (n = 72). In patients with VTD, mean HCY level was significantly higher (p < 0.0001) than in controls: 14.8 _ 5.1 pmol/L in men (n=78) and 12.1_+6.7/lmol/L in women (n= 171). In the 2 groups of patients, plasma HCY levels were significantly (p<0.0001) higher in men than in women. Prevalence of mild hyperHCY in patients with ATD, was significantly higher (p < 0.0001) in women than in men : 62.3% in women, 21% in men. Prevalence in VTD was also significantly higher (p = 0.04) in women: 41.2°/,, (71/172) than in men: 28% (22/78). inherited or acquired predisposing factors for thrombophilia were found in 35 patients with ATD and 48 patients with VTD. 30 patients (11 ATD and 19 VTB) among the 156 tested presented FV Leiden. Mean plasma HCY and prevalence of mild hyperHCY was not different in the group with or without congenital or acquired thrombophilia. Recurrent thrombotic events were associated with significantly higher HCY levels: we established a score system: 0 = n o thrombosis, l = o n e thrombotic event, 2 = t w o thrombotic events, 3 for more than 2 thrombotic events. In ATB and VTB, there was a sig-

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Abstracts/Netherlands" Journal of Medicine 52 (1998) S1-$61

nificantly positive correlation between the score of thrombosis and the level of homocysteinemia: r=0.334, p<0.0001 in ATB, r =0.458, p < 0.0001 in VTB. Although increasing age is a risk factor for recurrent thrombosis, the level of HCY appears as an independent risk factor for recurrent thrombosis. Conclusion: Our results (1) suggest that different normal values must be used in male and female to evaluate homocysteinemia and (2) confirm the high prevalence of hyperhomocysteinernia in thrombotic disease and show that the thrombotic risk grows linearly with the homocysteine level. Homocysteine concentration in pregnant and nonpregnant women on folate controlled diets. L.B. Bailey*, G.P.A. Kauwell GPA. Food Science and Human Nutrition Department, Universi O, of Florida, Gainesville, Florida. USA. The total homocysteine (tHey) concentrations in response to an 84 d folate controlled diet were determined in pregnant (n=12, 14-25 wks gestation) and nonpregnant controls (n = 12). These women were fed a diet approximating the current (400 #g/d) and previous (800/1g/d) Recommended Dietary Allowance (RDA) for pregnant women. Folate was provided as both food folate (120/.tg/d), and either 330 or 730 pg/d of supplemental folic acid. Plasma tHey concentration was significantly lower in pregnant women in the second trimester of pregnancy than in non-pregnant controls, independent ot" folate treatment group. The overall mean plasma t h e y concentration of the pregnant subjects (5.4_+ 1.4 /zmol/L) was 62% lower than that observed in the nonpregnant control group (8.7 _+ 1.7/tmol/L) (p<0.05). This difference in tHcy concentrations remained constant throughout the I2 wk investigation. No effect of dietary folate intake (450 or 850/,tg/d) on plasma t h e y concentration was observed within either pregnant or nonpregnant groups. Consequently, these folate intakes may either meet or exceed the amount required to maintain normal plasma tHey concentrations in pregnant and nonpregnant women. The lower plasma tHcy observed in pregnant subjects compared to nonpregnant controls appears to be a normal physiological response to pregnancy. The data suggest that plasma tHey concentration may not be a useful indicator of folate status in pregnant women. It may be necessary to establish different 'normal' reference ranges for plasma tHey in pregnant women for use in the assessment of folate status. Homocysteine as determinant of arterial stiffness in hypertensive subjects with end-stage renal disease. J. Blacher*, K. Demuth, A. Guerin, N. Moatti, G. London, M. Safar. Department (~[ Internal Medicine, I N S E R M U 337. Broussais HosT~ital, Paris. France. To test the hypothesis that increased plasma homocysteine levels in end-stage renal disease (ESRD) could be associated with structural and functional alterations of large arteries, and to assess the principal determinants of arterial stiffness in this population, we measured aortic (carotid-femoral), brachial (carotid-radial) and lower limb (femoral-tibial) pulse wave velocity in 74 patients undergoing hemodialysis (mean age _+SD: 51.6_+ 15.6 years).

We determined arterial blood pressure by sphygrnomanometry, aortic calcifications by echography, routine biochemical parameters, total plasma homocysteine (fluorimetric high performance liquid chromatography) and plasma endothelin (Elisa). Multiple stepwise regression analysis was used to assess the correlations between pulse wave velocity and determinants of biochemical and arterial parameters. Arterial blood pressure was 150 + 30/80 + 14 mmHg, aortic calcifications were found in 59 patients (80%), nine (12%) patients were insulin-dependent diabetic, total plasma homocysteine was 36 + 13 (normal < 15) ~tmol/1 and plasma endothelin was 4.6_+3.8 ( n o r m a l < 2 ) pg/ml. In this population, aortic pulse wave velocity was positively and independently correlated with systolic blood pressure (p < 0.0001), age (p < 0.0001), the prevalence of aortic calcifications (p = 0.0004) and the prevalence of diabetes mellitus (p =0.0043). The only determinant of brachial pulse wave velocity was mean blood pressure (p < 0.0001). Lower limb pulse wave velocity was positively and independently correlated with plasma total homocysteine (p = 0.0001), age (p = 0.011) and plasma endothelin (p = 0.017). Pulse wave velocity on any vascular site was not independently correlated with tobacco use, cholesterol, triglyceride, fibrinogen, hemoglobin, body mass index, or heart rate. In conclusion, the determinants of arterial stiffness will differ depending on whether the aortic, brachial or lower limb vascular site is considered. This study suggests that increased homocysteine concentrations in end-stage renal disease may be of pathophysiological significance in the process of lower limb vascular remodeling. Plasma homocysteine levels are associated with arterial stiffness in hypertensive subjects. J. Blacher*, S. Djane, E. Billaud, C. Lacroix, M. Safar. Department ~/" internal medicine. INSERM U 337, Broussais hospital. Paris. France. It has been widely reported that hyperhomocysteinemia was associated with both vascular structure and vascular clinical endpoints. Homocysteine has also been shown to enhance smooth muscle cell proliferation and even alter elastic tissue. In order to assess the relation between homocysteine and the structure and function of large arteries, we investigated a population of 135 hypertensive patients. We estimated arterial stiffness by measuring the carotid-femoral pulse wave velocity (PWV), arterial blood pressure was measured by sphygmomanometry, and total plasma homocysteine was determined by fluorimetric high performance liquid chromatography. Multiple stepwise regression analysis was used to assess the correlations between pulse wave velocity and determinants of biochemical and arterial parameters. In this population, PWV was positively correlated with plasma homocysteine (p=0.016), even after adjustments on systolic blood pressure (p < 0.0001), age (p = 0.0002), plasma glucose (p=0.018), and the duration of hypertension (p = 0.044); (R-squared = 0.523, F ratio = 15.34, p < 0.00001). PWV was not independently correlated with tobacco use, cholesterol, triglyceride, body mass index, or heart rate. In conclusion, this study suggests that increased homocysteine concentrations in hypertensive subjects may be of path-

Abstracts~Netherlands Journal of Medicine 52 (1998) S1 $61 ophysiological significance in the process of vascular remodeling of large arteries in hypertensive subjects. Since vitamin supplements are known to decrease plasma homocysteine concentrations, their effects on arterial stiffness needs to be evaluated. Can an elevated endogenous thrombin potential explain the elevated risk for venous thrombosis in case of hyperhomocysteinemia? G.M.J. Bos 1., D.T.S. Rijkers 3, H. Willems2, M. den Heijer2, W. Gerrits 2, H.C. Hemker ~. Daniel den Hoed Cancer

Center, University Rotterdam, Rotterdam; 2Leyenburg Hospital The Hague," 3University Maastricht, Maastricht, The Netherlands. Hyperhomocysteinemia is now known to be a risk factor for venous thrombosis. However, the pathogenesis of this clinical observation is not known. The endogenous thrombin potential (ETP) has been introduced as an overall function test of the blood coagulation system that reflects the combined action of thrombin formation and thrombin breakdown processes. The ETP is defined as the area under the thrombin generation curve. The ETP is decreased in hypocoagulable states and increased in subjects hypercoagulability (oral contraceptives, antithrombine III deficiency etc). Addition of activated protein-C (APC) or thrombomodulin (TM) both inhibit the thrombin generation, thus decrease the ETP. To test if there is a relationship between the concentration of homocysteine (tHcy) in a normal reference population and the ETP, we determined the ETP under standard conditions and in the presence of exogenous APC or TM. The addition of thrombomoduine was performed since an interaction of homocysteine with thrombomoduline has been suggested in the literature. 60 samples were selected out of samples available from a Dutch reference group (no history of venous thrombosis): 30 with elevated tHcy levels > 18 ~tmol/1; Mean = 22.7/tmol/l) and 30 with normal tHcy levels (M = 14.2/tmol/l) and matched for gender and age. We determined the extrinsic and intrinsic ETP in the 60 samples and were not able to find a relationship between the concentration of tHcy and the ETP or a difference of the ETP in both groups (extrinsic: elevated tHcy: 395 = 55.6 nM rain (100% of normal); normal tHcy: 419 + 64.6 nM min (106'% of normal); intrinsic: elevated tHcy: 412= 65.5 nM min (99% of normal); normal tHcy: 412+72 nM min (102% of normal). Furthermore, the anticoagulant effect of APC (elevated tHcy: 70% of pool plasma: normal tHcy: 73% of pool plasma) or M (elevated tHcy: 92% of pool plasma; normal tHcy: 90% of pool plasma) did not show a correlation with the concentration o f tHcy. These data indicate that in a normal reference group hyperhomocysteinemia is not reflected by an elevated ETP or a reduced anticoagulant activity of APC or TM. These data suggest that hyperhomocysteine does not induce a hypercoagualable state at the level of clotting factors [High homocysteine (M=22.7): ETP extrinsic M = 3 9 5 ; ETP intrinsic M =412 ( P > 0 . 5 ) . Normal homocysteine ( M = 14.2): ETP extrinsic M =419; ETP intrinsic M = 412 (P > 0.5)]. Hyperhomocysteinemia and resistance to activated protein C in patients with history of venous thromboembolism. M. Bo2iU*,

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I. Fermo 2, A. Ritonja 3, A. D'Angelo 2, P. Peternel l, M. Stegnat l 1Dept. Angiol., Univ. Med Ctr., Ljubljana, Slovenia.

2Scient. Inst. H. San Rafaelle, Milan, Italy, 3Dept. Biochem. and Mol. Biol., J. Stefan Inst., Ljubljana, Slovenia. Background and aim: There is growing evidence that mild hyperhomocysteinemia is a risk factor for venous thromboembolism. The aim was to establish prevalence of hyperhomocysteinemia in patients with history of venous thromboembolic disease, and its possible association with resistance to activated protein C (APC), the major inherited risk factor for venous thromboembolism. Subjects and methods: Fasting total plasma homocysteine (tHcys) was measured by high-performance liquid chromatography in 157 patients (61 females/96 males; mean age 43 years, range 19-60 years) with history of objectively confirmed venous thromboembolic disease and in 138 apparently healthy controls (80 females/58 males) of comparable age. Resistance to APC was determined by a modified activated partial thromboplastin time and defined as APC ratio < 2.0. Results: In patients tHcys was significantly higher than in controls (8.2/tmol/L vs. 7.2/~mol/L, p < 0.001). 22/157 patients (14%) had tHcys levels above the 95th percentile ( > 11.8/~mol/ L) of controls. In these patients odds ratio as a relative risk of venous thromboembolism was 3.59 (1.32-10.2: 2~-test=6.9, p<0.01). In patients folate and B12 concentrations (22.1 = 6.6 nmol/L and 400_+ 1185 pmol/L, respectively) were not lower than in controls (19.4+7.1 nmol/L and 242+329 pmol/L, respectively). None of the measured vitamins correlated with tHcys levels. In multiple regression model age, total serum cholesterol and low-density lipoprotein cholesterol explained 11% of tHcys variability. 11/62 patients (18%) without oral anticoagulant treatment were found to be APC resistant. Only 1/11 APC resistant patients had tHcys above the cut-off point. Conclusions: Our study suggests that fasting hyperhomocysteinemia is an important risk factor for venous thromboembolic disease. Low dose B-vitamin intervention in elderly individuals: Extent of homocysteine plasma reduction and association with vitamin and genotype status for thermolabile methylenetetrahydrofolate reductase (MTHFR). A. Br6nstrup A*, K. Pietrzik. Institute o/'

Nutritional Science, Dept. o/ Pathophysiology, University of Bonn, Endenicher Allee 11-13, 53115 Bonn, German),. Total homocysteine (tHcy) in plasma increases with age, probably as a function of decreased enzyme activi.ty in the elderly in conjunction with impaired vitamin status. Thus, the risk of vascular diseases in elderly individuals may be particularly increased. The tHcy concentration is a function of the vitamins folate, Bl2 and probably B6 acting as cofactors in its metabolism. Another determinant may be a newly recognized mutation in the enzyme M T H F R leading to thermolability of the enzyme which is involved in cofactor formation for Hcy remethylation. Aim: To investigate to what extent the plasma level of tHcy is determined by and can be influenced through an improved status of its vitamin cofactors in a group of apparently healthy

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Abstracts/,Netherlands Journal of Medicine 52 (1998) S1-$61

elderly individuals of both sexes. The genotype status for the mutation leading to thermolabile M T H F R is also assessed. Design: Ninety-nine men and women (65 88 years) were enrolled in an intervention study lasting for 4 weeks Blood for tHey and vitamin parameters was taken after an overnight fast at the beginning of the study and 2 and 4 weeks later. At onset of the study, individuals were randomly assigned to a treatment group receiving daily supplements of folic acid (400 /~g)+vitamin BI~ (4 gg)+vitamin B6 (2 mg) or to a placebo group. Results: At onset of the study, tHcy was significantly associated with plasma folate as well as erythrocytic folate, but not with plasma vitamin B12 and B6. After 2 weeks of supplementation, tHcy decreased significantly by a mean of 15%. Continuation o f the supplementation for another 2 weeks only slightly extended the mean reduction to 19%. The extent of tHey decrease was dependent on the tHcy level at start of the study resulting in greater reduction with higher initial tHcy. It also depended on baseline plasma folate levels as well as erythrocytic folate, but not on baseline vitamin B12 and B6 levels. No association was observed between homozygosity for the C677T mutation in the allele for M T H F R and tHey and the vitamin parameters at baseline as well as the extent of decrease after vitamin treatment. Postmethionine load homocysteine values, MTHFR polymorphism and another common mutation on CBS in vascular diseases. M. Candito 1., R. Defranchis:, P. Bedoucha3; P. Gibelin 4, J.L. Sadoul 5, P. V6nembre l, M. Chatel 3. E.Van Obberghen ~. IBioehemistry, 3Neurology, 4Cardiology, 5Diabet-

ology H6pital Pasteur, CHU Nice, BP 69, 06002 Nice Cedex, France. 2pediatrics, University Federico H, Naples, Italy. The frequency of the C677T homozygous mutation on methylenetetrahydrofolate reductase (MTHFR) is 11 to 16% in both cardiovascular patients and healthy controls. However, the mutation is associated with higher fasting homocysteine levels (HCY). A methionine loading test (MLT) can identify patients at increased risk for vascular disease (despite normal fasting plasma HCY levels). We thus investigated the relation with M T H F R polymorphism and HCY level 6 hours after MLT (0.1 g/kg) in 54 cardiovascular patients (age 18 to 70 yr, mean 45.8 yr). Eleven +/+ (homozygous mutants) had a significantly higher mean fasting HCY level (20.2 _+ 18.0 retool/ l) than 23 - / - patients (10.1 + 3.8). Moreover, after MLT. in the +/+ patients, HCY mean level (72.4+35.1) was significantly higher than in - / patients (47.1+20.3). This prompted us to search in the 13 first patients who were - / + for the C677T mutation on the M T H F R gene, for another common mutation on cystathionine 6-synthase (CBS), an enzyme activated by dietary methionine intake: the 844 ins 68 mutation (15% of healthy controls are heterozygous + / - ) . We found this last mutation in 3 patients: 2 patients have suffered from stroke and 1 from multiple venous thrombosis. The C677T mutation on the M T H F R gene is known to be aggravated by a drop in folates. Other contributory genetic factors such as a common mutation on the CBS gene might be necessary for the development of thrombosis.

Hyperhomocysteinemia and dissection of vertebral artery: could they be related? A case report. G. Cardaioli 1., L. Parnetti 1, A. Alberti 1, M. Venti ~, T. Bottiglieri:, V. Gallai 1. Neurological

Clinicl, Perugia University, Italy and 2Baylor University Medical Center, Institute of Metabolic Disease, Dallas Texas, USA. A 43 year-old man complained of a sudden pain in the hack of the neck followed by cephalalgia, left palpebral ptosis, hiccup and vomiting. At neurological examination left lateropulsion on the stand position, left eye miosis and left anhydrosis of the upper part of the face were observed. Brain Magnetic Resonance Imaging was negative for focal lesions. Duplex color-flow imaging and angiography showed the presence of left vertebral artery dissection. Neither classical vascular risk factors nor previous head trauma were documented. The only positive abnormal findings was a high total homocysteine level (38 /zM/L), which was confirmed twice. Folate levels (2.5 ng/ ml; normal range 3-20 ng/ml), as well vitamin B12 levels (172 pg/ml; normal range 200-900 pg/ml) were slightly below the normal range. In view of the existence o f a possible correlation between hyperhomocysteinemia and dissection of cervical arteries, the whole patient's family is under investigation for plasma total homocysteine and 5,10-methylentetrahydrofolate reductase (MTHFR) genotyping. Hyperhomocysteinaemia in abruptio placentae. A.A. Carolissen ~*, H.J. Odendaal ~, D.W. Steyn:, E.P. Owen:, L.E. Human:, E.H. Harley:. 1Department of Obstetrics and Gynaecol-

ogy, University of" Stellenbosch and MRC Perinatal Mortality Research Unit, Tygerberg Hospital, Tygerberg, South AJrica. 2Department of Chemical Pathology, University o[' Cape Town Medical School, Groote Schuur Hospital, Cape Town, South Ajriea. Objective: To determine the prevalence of hyperhomocysteinaemia in patients who had abruptio placentae. Study design: A case-control study set in a clinical academic environment. Twenty-one patients where the pregnancy was complicated by abruptio placentae at a gestation of ---28 weeks were recruited as the study group, with seventeen control patients between March 1995 and September 1995. The main measures were baseline total homocysteine levels and peak total homocysteine levels after an oral methionine loading test. Statistical analyses were performed with ANOVA for normally distributed data. For non-parametric data the Kruskal-Wallie H-test was used for calculation of p-values. Results: Baseline mean total homocysteine levels were significantly higher in the study group namely 8.0/,nnol/l (range 3.4-16.5 pmol/l; SD=3.7) compared to the 4.8 /nnol/l of the control group (range 2.8-8.2 pmol/l SD = 1.6), p = 0.0013. Peak mean total homocysteine levels were also significantly higher in the study group namely 30.8 pmol/l (range 15.4-106.1 /lmol/1 ; SD = 20.4) compared to the 20.0 pmol of the control group (range 12.2-44.6; SD = 9.0), p = 0.012. Hyperhomocysteinaemia occurred in 43% (9/21) of the study group and 6°/, (1/17) of the control group patients. Conclusion : Hyperhomocysteinaemia has been identified as a possible risk factor for abruptio placentae in our study population.

Abstracts I Netherlands Journal of Medicine 52 (1998) SI-$61 Fasting plasma homocysteine levels do not predict the occurrence of deep vein thrombosis after elective HIP replacement surgery. M. Cattaneo*, M.L. Zighetti, R.M. Turner, S.G. Thompson, G.D.O. Lowe, F. Haverkate, R.M. Bertina, A.G.G. Turpie, P.M. Mannucci, on behalf of the ECAT DVT Study Group. A. Bianchi Bonomi Hemophilia and Thrombosis Center. IRCCS Ospedale Maggiore and University of Milano, Italy; Department of Medical Statistics and Evaluation, Imperial College School of Medicine, London, UK; Department of Medicine, University of Glasgow, UK; TNO Prevention and Health, Gabius Laboratory, Leiden, The Netherlands; Haemostasis and Thrombosis Research Center, Academisch Ziekenhuis, Leiden, The Netherlands; HGH McMaster Clinic, Hamilton, Ontario, Canada. Background. Case-control studies have shown that mild hyperhomocysteinemia is associated with an increased risk for venous thromboembolism. Objective. To evaluate whether or not homocysteine plasma levels predict the risk of post-operative deep vein thrombosis (DVT) after elective hip replacement. Methods. The European Concerted Action on Thrombosis (ECAT) DVT Study was a multicenter study of preoperative hemostatic tests in prediction of DVT (diagnosed by routine bilateral venography) after elective hip replacement. Most patients received at least one method of thromboprophylaxis, which was chosen by each participating center. 480 patients were recruited in 11 centers across Europe. Informative bilateral (n = 370) or monolateral (n = 5) venography was available for 375 of them. On the day before surgery or on the day of surgery, blood samples were obtained in ice-cold 0.109 mol/] trisodium citrate from fasting patients. Blood samples were mixed, immediately placed in melting ice and centrifuged as soon as possible at 0~°C, 3000×g for 20 rain. The supernatant platelet-poor plasma was aliquoted, snap-frozen and stored at -70°C. The plasma concentration of total homocysteine was retrospectively measured in 371 adequate samples from patients with informative venography. Results. Of the 371, 119 patients (32%) had venography proven DVT. The geometric means (approx. SD) of pre-operative plasma levels of total homocysteine were 18.2 (5.86) pmol/l for patients with DVT, and 17.1 (5.53) pmol/l for patients without DVT. The percentage difference ( D V T - N o DVT) after adjustment for age, sex, current smoking, systolic blood pressure, pulse rate, BMI, presence/absence of varicose veins, prophylactic use of stockings, center and assay batch was 4 (95% CI: - 4 to 12, p=0.34). Higher levels of plasma homocysteine were associated with age, male sex, smoking, blood pressure, pulse rate and serum creatinine. Conclusion. DVT is common after elective hip replacement despite thromboprophylaxis. The fasting plasma levels of homocysteine are not clinically useful in prediction of such events. Vitamin E and Probucol-sensitive mechanisms in the abnormally high thromboxane biosynthesis in homocystinuria due to homozygous cystathionine-/~-synthase deficiency. A. Coppola*, A.M. Cerbone, G. Guiotto, U. Piemontino, P. Della Valle 1. G.

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Andria2, A. D'Angelo1, G. Davi3, G. Di Minno. Departments

of Clinical and Experimental Medicine and 2Pediatrics, Universit), "Federico H" of Naples; 3Hematology, University 'G. D'Annunzio', Chieti, and I Coagulation Service, I. 1~ C. C.S. 'Ospedale S. Raffaele', Milan, Italy. In patients homozygotes for cystathionine-fl-synthase deficiency (CBSD) we have previously shown an abnormally high in vivo platelet biosynthesis of thromboxane (TX) A2. We have now tested potential mechanisms involved in such phenomenon. Platelet biosynthesis of TXA2 was evaluated by monitoring the urinary excretion of its major metabolite, 11-dehydro-TXB2 (TXM). Protein C and S, thrombomodulin, thrombin-antithrombin complexes, prothrombin fragments 1+2 and activated protein C served to detect a hypercoagulable state. In 9 homozygous patients (5 males, 4 females; 7 ~ 4 yrs. old) the plasma levels of these hemostatic variables were consistent with a hypercoagulable state. However, none of the variables correlated with TXM excretion and a bolus infusion of the thrombin inhibitor r-hirudin (0.3 mg/kg over a 15 min period), that prolonged the aPTT 1.5-3 fold normal and impaired platelet aggregation > 4 hrs., did not affect TXM excretion (1175+236 pg/mg creatinine, pre-infusion samples; 926 + 131 and 973 + 152 samples obtained 8 and 24 hrs. after, p > 0.01 vs. pre-infusion). In contrast a short-course trial of probucol (500 mg/d for 3 wks.) significantly lowered TXM excretion of 8 CBSD patients (1152 _+241 pg/mg creatinine in pre-treatment samples, 772_+ 184 and 762_+ 192 at withdrawal and 3 wks. after withdrawing probucol, p = 0.007 vs. pre-treatment), without affecting the hemostatic variables and TXM excretion in matched control subjects. A short-course vit. E administration behaved similarly with respect to TXM excretion, a 30% lowering (p < 0.05) of its urinary levels being detectable in 6 CBSD patients who ingested 600 mg/d vit. E for 2 wks. Our data argue for enhanced in vivo TXA2 biosynthesis in this setting as being largely independent of a hypercoagulable state and for its partial dependence on probucol and vitamin E-sensitive oxidative mechanisms.

Monitoring Homocysteine in stroke patients, acute or chronic phase? R. Deulofeu*, N. Vilz, E. Mas, A. Chamorro l, M.J. Medrano:, A.M. Ballesta, and the Spanish study group on Homocysteine. Hospital Clinic Universitari, Villarroel I70, Barcelona 08036; Servei de Bioquimica Clinica, 1Servei de Neurologia, 2Instituto de Salud Carlos II1. Madrid, Spain. The timing of plasma homocysteine measurements relative to stroke onset is a matter of controversy. To ascertain this question, we studied 61 patients, 26 from our hospital and 35 patients from the Spanish Study Group on Homocysteine. Total plasma homocysteine was measured by HPLC and fluorescence detection, and albumin by standard methods. Based upon the results on albumin at both phases and acute phase homocysteine we computed a theoretic chronic phase homocysteine using the formula: theoretic homocysteine= acute phase homocysteine × chronic albumin/acute phase albumin. During acute phase mean total plasma homocysteine levels were not statistically different from those found during chronic

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Abstracts/Netherlands Journal of Medicine 52 (1998) S1-$61

phase (10.4 _+4.8/.tmol/L vs. 9.6 _+4.1 pmol/L, p = ns.). By contrast albumin was significantly different between acute and chronic phase 43.2_+ 5.6 g/L and 41.9 + 6.1 g/L, respectively p=0.034. The mean theoretic chronic phase homocysteine was 9.9_+ 4.9 that was very similar to that measured and correlated closely with measured chronic phase homocysteine (r =0.6712, p < 0.0001). We also found a good correlation between measured acute and chronic phase homocysteine (r = 0.6301, p < 0.0001) but not between homocysteine and albumin in neither phases. The good correlation between the measured and theoretic chronic phase homocysteine suggests that this change could correctly be attributed to changes in vascular volume alterations between both phases. Furthermore this results allow monitoring homocysteine in any phase of the patients disease and reduces the need of repeating homocysteine measurements during the chronic phase. Is hyperhomocysteinemia a risk factor for neuropathy in patients with non-insulin dependent diabetes mellitus (NIDDM)? A. Ambrosch, J. Dierkes*, lR. Lobmann, JW. Ktihne, tH. Lehnert, C. Luley. Institute of Clinical Chemistry and I Dept. of Internal Medicine, Clinic of Endocrinology, UniversiO, Hospital Magdeburg, FRG. It is known from in-vitro studies that homocysteine exhibits toxic effects on neurogenic cells. Since neuropathy is a common complication of NIDDM, we investigate the role of homocysteinemia in NIDDM patients with and without neurogenic complications. Design: NIDDM patients (n = 59) were consecutively enrolled into the study. Neuropathy was diagnosed by standardized questionnaire, by autonomic function test (AFT) and by testing peripheral sensomotoric neuropathy including vibratory perception, temperature perception, discrimination perception and muscular force. Neuropathy was diagnosed when two of the three subtests were positive. Besides neuropathy testing, demographic data (age, sex, duration of NIDDM), renal function (creatinine, glomerular filtration rate (GPR)t and metabolic parameters of glucose metabolism (HbAlc) were investigated. Homocysteine, serum folate, RBC folate and vitamin Bl2 were measured by standard procedures. Results: NIDDM patients without (n= 19) and with diabetic neuropathy (n=40) were comparable with regard to demographic data. Duration of NIDDM was significantly longer in patients with diabetic neuropathy. There were no significant differences in folate, vitamin BI:, creatinine, GFR or HbAlc measured. The homocysteine concentration was higher in patients with neuropathy (p=0.05). In a multivariate logistic regression with diabetic neuropathy as dependent variable, homocysteine (adjusted for age, renal function, folate and vitamin BI2, duration of NIDDM, HbAlc) was the only variable which was significantly associated with neuropathy. A 5 /,tmol/L increase of homocysteine was associated with an odds ratio of 2.32 t95% CI 1.0-5.4) for diabetic neuropathy. These results suggest that hyperhomocysteinemia may contribute to the neuropathic pathogenesis in NIDDM patients.

Hyperhomocyst(e)inemia as an independent risk factor for cardiovascular disease in renal transplant recipients. D. Duclou×*, C. Ruedin, J.M. Rebibou, R. Gibey, J.M. Chalopin. Department of Nephrology and Renal Transplantation." Laboratory oJ Biochemistry. Hopital St. Jacques; Besancon; France. Limited data are available on the prevalence, determinants and significance of hyperhomocysteinemia in renal transplant recipients. In the present study, plasma homocyst(e)ine concentrations and factors known to influence homocysteine metabolism were analyzed in 224 renal transplant recipients with and without cyclosporin A (CsA). Atherosclerotic complications were evaluated with respect to plasma homocysteine concentrations and other known risk factors for cardiovascular disease (CVD). Plasma tHcy was measured by high-performance liquid chromatography. Plasma folic acid, vitamin BI2 and creatinine concentrations were also measured. Mean plasma tHcy was 21.3 _+9.6 pmol/1. We observed significant correlations between on one hand tHcy and serum creatinine concentration (r=0.213; p <0.0001) and, on the other hand tHcy and folic acid concentration (r=0.053; p < 0.005) both in patients with and without CsA. Plasma tHcy were higher in patients with a past history of CVD (25.2_+ 11 pmol/l vs. 20.5_+9 pmol/1; p<0.005). Moreover we found a persistent association between plasma tHcy and CVD after multiple logistic regression analyses adjusted for age, transplant duration, hyperlipidemia, diabetes, hypertension, smoking and hemodialysis duration (p < 0.02). Despite methodologic limitations, hyperhomocyst(e)inemia seems to be an independent risk factor for CVD in renal transplant recipients. Placebo-controlled clinical trials of effective tHcy-lowering treatment are required to confirm this hypothesis. Homocysteine production in chronic renal failure. N.P.B. Dudman *1, W. Fu j, K, Yang z. J.S. Silberbergz, R. Crooks:. I Centre for Thrombosis and Vascular Research. Prince Henry Ho.wital, Universi O, of New Sauth Wales, Sydney, Australia, 2 Univ. o1"Newcastle, Australia. Patients with chronic renal failure (CRF) have raised erythrocyte S-adenosylhomocysteine (SAH), which was thought to arise from their elevated plasma homocysteine (Perna A, et al. Enzymatic methyl esterification of erythrocyte membrane proteins is impaired in chronic renal failure. J Clin Invest. 91;1993:2497-2503t. Our aim was to study relations between levels of these two metabolites. Standard HPLC methods were used to measure levels of plasma homocysteine and methionine, and erythrocyte SAH and S-adenosylmethionine (SAM). A standardised oral methionine load (4 gm/m2) was given to 36 fasted adults with normal plasma homocysteine values. We found that, whilst oral methionine led to greatly increased plasma methionine (22.0_+8.3 fold, mean_+ SD, n = 32) and homocysteine (3.0_+0.8 fold, n=31), these changes had no significant effect on levels of erythrocyte SAM and SAH. We confirmed that fasted erythrocyte SAH in 10 CRF patients was higher than in 13 controls by 2.8 fold, p = 0.003. We hypothesised that this increased SAH resulted from accelerated SAM-dependent

Abstracts/Netherlands Journal of Medicine 52 (1998) S1 $61 methylation, and predicted that homocysteine would be released into plasma more quickly in pre-dialysis whole blood drawn from CRF patients, than from either post-dialysis CRF blood, or from healthy controls. This was supported by measurement. A likely methylation target in CRF erythrocytes is the membrane protein (Perna et al.). Since the protein methylation is followed by peptide rearrangement and demethylation, we predicted that whole blood incubated with [methyl-3H]methionine should release a volatile [3H]product (MeOH), and that this should be increased in pre-dialysis compared with post-dialysis blood drawn from CRF patients. Both these predictions were also supported by experiment. Thus control of erythrocyte SAH and SAM levels is highly resistant to elevations of plasma homocysteine and methionine. Increased erythrocyte SAH levels in CRF patients may reflect enhanced repair methylation of the plasma membrane proteins, which could lead to accelerated release of homocysteine into the plasma. The distribution and correlates of elevated total homocysteine: the stroke prevention in young women study. W.H. Giles WH / *, S.J. Kittner2, J.B. Croft 1, M.A. Wozniak:, R.J. Wityk~, B.J. Stern4, M.A. Sloan5, T.R. Price:, R.J. McCarter:, R.F. Macko:, C.J. Johnson3, B.R. Feeser:. C.J. Earley 3, D.W. Buchholz3, P.D. Stolley:. 1Centers for Disease Control and

Prevention, Atlanta, GA; 2University of Maryland, Baltimore. MD ; 3Johns Hopkins University, Balt#nore, MD : 4Emoo, University, Atlanta, GA; 5Harbin Clinic, Rome, GA, USA. Background: Elevated total homocysteine (tHcy) is an independent risk factor for cardiovascular disease. Limited information is available regarding the distribution and correlates of elevated tHcy in healthy young adults. Methods: Data from the Stroke Prevention in Young Women Study, a population-based case-control study, were used to examine the distribution and correlates of elevated tHcy levels (->10.0 pmol/L) in 304 young healthy women (34% black) free of vascular disease. Multivariate linear and logistic regression analyses were used to examine these relationships. Results: The mean tHcy level for the population was 6.58 pmol/L (range 2.89-26.5 pmol/L). Mean tHcy levels increased with age, cholesterol level, alcohol intake, and number of cigarettes smoked (all; p < 0.05). There were no race differences. Regular use of multivitamins and increasing education were associated with significantly lower mean tHcy concentrations. Approximately 13% of the samples had elevated tHcy levels. Multivariate adjusted correlates of elevated tHcy included education > 12 vs. -<12 years (odds ratio (OR)=0.4, 95% confidence interval (CI)= 0.2-0.8); smoking ->20 cigarettes/day vs. non-smokers (OR = 2.8, 95% CI = 1.1-7.3); and regular use of multivitamins (OR = 0.4, 95% CI = 0.2 0.9). Conclusions: A substantial proportion of healthy young women have tHcy levels that increase their risk for vascular disease. A number of potentially modifiable behavioral and environmental factors appear to be significantly related to elevated tHcy levels, suggesting additional strategies for the reduction of elevated tHcy levels in young women.

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Combination of low-dose folio acid and pyridoxine for treatment of hyperhomocysteinaemia in patients with premature arterial disease and their relatives. R. van der Griend 1., F.J.L.M. Haas:, D.H. Biesma1, M. Duran 3, O.J.A.Th. Meuwissen1, J.D. Banga4. Sint Antonius Hospital, Nieuwegein, The Nether-

lands', l dept. of Internal Medicine, 2 dept. o f Clinical Chemistry, University Children's Hospital "Het Wilhelmina Kinderziekenhuis', Utrecht, 4 University Hospital, Utrecht, The Netherlands'. Background: Hyperhomocysteinaemia is an independent risk factor for atherosclerotic disease and venous thrombosis. The optimal homocysteine-lowering vitamin dose and target homocysteine concentration is currently unknown. The optimal effective and safe dose should be established from a pharmacological point of view, because neuropathy could be induced, and for recommendations in food supplements. Methods: We prospectively studied the homocysteine-|owering effect of a low-dose combination of folic acid (0.5 mg) and pyridoxine (100 mg) in 49 hyperhomocysteinaemic persons (33 patients with documented premature arterial disease and 16 of their first-degree relatives). A methionine loading test and vitamin analysis was repeated after 8 weeks of therapy. Results: Low-dose vitamin therapy significantly reduced fasting homocysteine concentration (median 13.9 to 9.3 pmol/L, reduction 32% [95 percent confidence interval, 27 37%]) and post-load homocysteine concentration (median 55.2 to 36.5 /zmol/L, reduction 30% [95 percent confidence interval, 25 35%]). Fasting homocysteine reduction was similar in women and men, as well as in patients and relatives. Postload homocysteine reduction was significantly less in men compared to women (p=0.04) and in relatives compared to patients (p= 0.03). Of all vitamin concentrations baseline folic acid significantly correlated with the reduction both in fasting and post-load homocysteine concentration. Conelusion: Treatment with low-dose combination of folic acid (0.5 rag) and pyridoxine (100 rag) daily appears effective in reducing homocysteine concentrations in subjects with hyperhomocysteinaemia. Assessment of the contribution of the thermolabile M T H F R allele to hyperhomocysteinemia and cardiovascular disease in a spanish population. N. Garcia-Girald, I. Ferrer2, M.A. Vilaseca 2, X. Pint6 3, S. Balcellsj, D. Grinberg1.. IDpt. Genbti-

ca, Facultat de Biologia, Universitat de Barcelona, 2Scrvei de Bioquimiea, Hospital Sant Joan de Dbu. 3 Unitat d'Arteriosclerosi, Ciutat Sanithria Universit?tria de Bellvitge. Barcelona, Spain. Mild hyperhomocysteinaemia is accepted as an independent risk factor for premature coronary artery disease (CAD). Genetic contributions to this biochemical parameter may be found in the genes for CBS and for MTHFR. The common mutation 677 C-->T in MTHFR (MTHFRts), that produces a thermolabile protein, has been studied in relation to CAD in different populations, giving controversial results. We present the genotype analyses of 80 patients with premature CAD ( < 50 years) and 100 controls matched by age and sex, representative of a Spanish population. Total plasma

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Abstracts / Netherlands Journal of' Medicine 52 (1998) SI $61

homocysteine (plHcy) and folate was measured for all individuals, and genotype-phenotype correlations were studied. The frequencies o f the three M T H F R genotypes in controls' were: 39% wt/wt, 45% wt/ts and 16% ts/ts; and in patients were: 40% wt/wt, 41.2% wt/ts and 18.8% ts/ts. Comparison of the genotype distributions in patients and controls by a Chi-square test gave a non significant result. However, in the control sample significant associations were found between the genotypes and the levels o f plHcy or folate: individuals with the ts/ts genotype have higher levels of plHcy (~ = 15.8/tM) and lower levels of folate (~ = 12.1 nM), than individuals wt/wt or wt/ts, whose means for plHcy and folate are 11.3/tM and 15.3 nM, respectively (t-test associated probabilities of 0.015 and 0.009, respectively). Because plHcy and folate are dependent variables, we assayed the association between genotypes and the ratio plHcy/folate and found that it is still significant (t-test associated probability of 0.024). We also subdivided the samples in two groups according to folate levels below or above 15.4 nM. While in the first group the association was significant, no association was detected in the second. This data points out to the protecting effect of high folate levels. Our data clearly associate the M T H F R ts/ts genotype to hyperhomocysteinaemia. On the other hand, when establishing the plHcy value of the 95% percentile in controls, we observe that 20% of the patients have plHcy levels above it. The fact that we cannot associate the ts/ts genotype to CAD may be due to the fact that its effect is small and is masked by other factors. To quantify this effect on CAD in terms of relative risk should be of aid to the clinicians. Vitamin B profiles are not suitable for identifying women at risk for congenital heart defects. M.L.M. Haagmans MLM*, L. Kapusta ~, E.A.P. Steegers2, T.K.A.B. Eskes 2, M. van Hoven2 , N. Hanael-van Bruggen 2, M.H.M. Cuypers/, O. Daniels 1. University Hospital St Radboud, Nijmegen, The Nether-

lands," IChildren's Heart Centre, 2Dept. of Gyn./Obst. Deficiencies of folate, vitamin B~2 and vitamin B6 can be a cause of raised plasma homocysteine concentration. Hyperhomocysteinemia, a very sensitive marker for folate metabolism, is a known risk factor for neural tube defects (NTD). Periconceptional multivitamin use is reported to reduce the prevalence of NTD and of conotruncal heart defects. Aim: The aim of the study was to investigate whether low maternal folic acid, vitamin B12 and vitamin B6 concentrations are risk factors for congenital heart defects (CHD). Methods: A case-control study was performed in 27 mothers of infants born with CHD and 56 controls. Fasting serum and red cell folate, as well as vitamin Bl2 and vitamin B~ were measured. Results: There was no statistical significant difference in concentrations of serum and red cell folate, vitamin BI2 and vitamin B6 between both groups. On the contrary, fasting homocysteine concentrations were significantly higher in the study group as compared to controls (p = 0.0001). Conclusion: This preliminary study suggests vitamin B profiles do not identify women at risk for CHD. The presence of hyperhomocysteinemia deserves further investigation.

South Australian experience with hyperhomocysteinaemia as a risk factor in obstetrics. Time for a trial? B. Hague 1., M. Whiting2, G. Tallis 2. IDepartment of Obstetrics, Women's

& Children's Hospital North Adelaide, South Australia 5006; 2Department of Biochemistry and Chemical Pathology, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia. In recent years, hyperhomocysteinaemia has associated with early onset pre-eclampsia and placental abruption. We have therefore studied 41 consecutive women with a history of early-onset ( < 35 weeks) pre-eclampsia/eclampsia in the recent or distant past, 5 women with known antiphospholipid syndrome and previous fetal death, and 18 consecutive pregnant or puerperal women with a history of recent or previous thromboembolism, measuring non-fasting plasma homocysteine. In a control group of 24 unselected women at 28 weeks gestation, mean plasma homocysteine was 5.8 (SD 1.7) pmol/L (range 3.5 10.9 pmol/L), the reference range for non-pregnant persons being 3-.13 Imaol/L. 15/41 (36.6%) women with earlyonset pre-eclampsia (OR compared with controls 9.2 95% CI 1.1 to 78.7), 6/18 (33.3%} women with thromboembolism (OR compared with controls 11.5 95"/o CI 1.24 to 10.7) and 1/5 (20%) women with antiphospholipid syndrome had values of plasma homocysteine > 11.0 pmol/L. Identification of women with hyperhomocysteinaemia will provide the opportunity for testing an intervention with folic acid in an appropriately designed randomised controlled clinical trial. For example, to detect a 33% reduction in the risk of recurrent pre-eclampsia from 30% to 20% in women with previous early onset pre-eclampsia, 626 women would need to be recruited ( a 0.05, 80% power). Prevention of recurrent pre-eclampsia by folic acid supplementation in women with hyperhomocysteinaemia: proposal for a randomised controlled trial. B. Hague I *, C. Crowther t, J. Robinson 1, G. Dekker z, H. Odendaal s. 1Department of Obstetrics,

Women's and Children's Hospital, North Adelaide, South Australia 5006; 2Department of Obstetrics, Free University Hospital Amsterdam, Netherlands; 3Department of Obstetrics. Tygerberg Hospital University of Stellenbosch, Cape Town. South ~{/'riea. 14~pothesis: Folic acid supplements (5 mg/day) will reduce the frequency of recurrent pre-eclampsia in women with hyperhomocysteinaemia. Study design: A multi-centred randomised controlled clinical trial. Entry criteria: Women with a past history of early onset ( < 3 5 weeks gestation at delivery) pre-eclampsia/eclampsia and found to have hyperhomocysteinaemia will be recruited to the trial either prior to attempting a further pregnancy or in the first or second trimester of the at risk pregnancy up to 20 weeks gestation. Exclusion criteria : Gestation > 20 completed weeks, women with known vitamin Bi2 deficiency, women with epilepsy taking anticonvulsant medication, women with a previous infant/ fetus affected by neural tube defect enrolled prior to 12 weeks gestation, women with homocystinuria.

Abstracts~Netherlands Journal of Medicine 52 (1998) S1 $61 Following informed consent, women will be randomly allocated to either folic acid 5 mg daily or to placebo with stratification by centre and by time of gestation at entry. All women recruited prior to 12 weeks gestation will be asked to be responsible for their own low dose folate for prophylaxis of neural tube defect. The primary endpoint of the trial will be the frequency of recurrent pre-eclampsia. Secondary endpoints will include incidence of early miscarriage, presence of proteinuria, severity of pre-eclampsia, gestational age at onset of pre-eclampsia, need for early delivery for pre-eclampsia, method of delivery, gestational age at delivery, incidence of placental abruption, incidence of venous thrombo-embolism, birthweight standardized for gestational age, Apgar scores, cord blood gases and pH, and time spent in neonatal nursery. Power calculations: A minimum of 375 women in each arm will need to be recruited to detect a 33'~ reduction in the risk of pre-eclampsia from 30% to 20% (~ 0.05,/3 80%). This will need to be increased to allow for those who do not become pregnant in the study period. Homocysteine, serum folate, mutated methylenetetrahydrofolate reductase (mthfr) and factor V Leiden and the risk of venous thrombosis. M. den Heijer 1., H.P.J. Willems:, F.R. Rosendaal s, G.M.J. Bos 4, N. van der Put s, W.B.J. Gerrits:, H.J. Blom5. l Dept. of Internal Medicine, TweeSteden Hospital, Til-

burg," 2Leyenburg Hospital, The Hague; 3Dept. of Clinical Epidemiology and Dept. of Hematology, University Hospital Leiden; 4Department of Hematology, Daniel den Hoed Clinic; 5Lab. of Paediatrics and Neurology, Univ. Haapital N!'imegen," The Netherlands'. Hyperhomocysteinemia is associated with venous thrombosis. Whether determinants of homocysteine such as low serum folate and mutated M T H F R are also risk factors for venous thrombosis is unknown. We evaluated the possible risk of these factors and the interaction with factor V Leiden in 185 patients with a history of venous thrombosis and 500 healthy volunteers. Hyperhomocysteinemia was defined at the 90th percentile and low serum folate at the 10th percentile of the controlgroup. In this study the risk for venous thrombosis was 3.7 (2.45.7) for fasting homocysteine; 0.8 (0.4 1.5) for low serum folate; 0.9 (0.5-1.8) for mutated M T H F R and 5.8 (3.2 10.4) for factor V Leiden. The combined risk for hyperhomocystenmia and factor V Leiden was 6.9 (2.4-20) and for mutated M T H F R and factor V Leiden 13.9 (1.6~116). Hyperhomocysteinemia and factor V Leiden are risk factors for venous thrombosis. Low folate concentration and mutated M T H F R are important determinants of homocysteine levels but seem not to be risk factors for venous thrombosis. There might be some positive interaction between hyperhomocysteinemia, mutated M T H F R and factor V Leiden, although confidence limits are rather wide to allow definite conclusions.

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Hyperhomocysteinemia is associated with microaibuminuria. E.K. Hoogeveen 1,, p.j. Kostense~,:, A. Jager 1, C. Jakobs s, 74 ~ R.J. Heine~' , L.M. Bouter 1 % C.D.A. Stehouwerj,4. University •

Hospital Vrije Universiteit, Amsterdam, The Netherlands, llnstitute jor Research in Extramural Medicine. 2Dpt. of Epidemiologv and Biostaristics 3Dpt. of Clinical Chemistry. 4Dpt. of Internal Medieine. Microalbuminuria (MA), a slightly elevated urinary albumin excretion rate, is a strong predictor of cardiovascular disease. The increased risk of cardiovascular disease in individuals with MA is only partly due to a higher prevalence of classical risk factors• Hyperhomocysteinemia is a recently recognized risk factor for cardiovascular disease independent of classical risk factors. Since it is not known whether hyperhomocysteinemia is associated with microalbuminuria we studied the association in an age-, sex- and glucose-tolerance stratified random sample of a 50-.75 year-old general Caucasian population (n = 680}. The urinary albumin-to-creatinine ratio (ACR) was measured in an early morning spot urine sample. M A was defined as ACR > 2.0 mg/mmol. The prevalence of MA was 8.6% (26/ 304) in subjects with normal glucose tolerance, 12.4% (23/185) in impaired glucose tolerance and 26.2% (431164) in non-insulin-dependent diabetes mellitus (NIDDM); it was 7.2% (29/ 402) in subjects without hypertension and 25.1% (631251) in those with hypertension. After adjustment for age, sex, NIDDM, hypertension, dyslipidemia and smoking, the odds ratio (OR; 95% confidence interval (95% CI) per 5 /zmol/L total homocysteine increment for MA was 1.27 (1.04 to 1.54)). Additional adjustment for HbAlc, waist-hip ratio, animal protein intake and serum creatinine did not attenuate the association between M A and total homocysteine (OR (95% CI); 1.24 (1.01 to 1.53)). We conclude that hyperhomocysteinemia is associated with microalbuminuria independent of non-insulin-dependent diabetes mellitus, hypertension and animal protein intake. Mild hyperhomocysteinemia detected in offsprings of parents operated on for aortocoronary or peripheral artery bypasses depends on age. J. Hyfinek*, J. Stribrny, J. Dvorfikovfi, V. Martinikovfi. Dept. Clin. Biochemistry-Metabolic Unit, Hospital Na

Homolee, Prague, Czech Republic. The total homocysteine (tHcy) in plasma was followed in 30 children born from parents operated on for aortocoronary (ACB) or peripheral artery (PAB) bypasses. For the estimation of tHcy the HPLC method of Araki and Sako with fluorescence detection was used. As expected in operated parents the significant increase of tHcy levels was observed. In ACB 16 _+5.3 pmol/l for men and 14.4_+4.9 ,umol/1 for women (healthy men: 12.5_+ 1.7 pmol/l and healthy women: 10.3 _+ 1.9 pmol/1. In a group of children born from operated parents the significant increase of tHcy was also observed: 8.8_+ 2.30 pmol/l (healthy children 5.8_+ 1.34 pmol/l). In older children at risk (over 12 yrs) the increase of tHcy was more evident. The dependence of mild hyperhomocysteinemia on the age of children is further discussed.

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Abstracts / Netherlands Journal of Medicine 52 (1998) SI-$61

Severe post-thrombotic syndrome in a young patient with Hyperhomocysteinemia and Factor V Leiden. D.N. Kolbach 1., J.C.J.M. Veraart I, J. Boer:, L.J.M. Spaapen~, K. Hamulyrik 4, H.A.M. Neumann I. 1Dept. of Dermatology University Hospital Maastricht, The Netherlands, 2 Dept. of Dermatology Deventer Hospital, The Netherlands, 3 Clinical Genetics Liraburg, The Netherlands, 4 Dept. of Hematology, UniversiO' Hospital, Maastricht, The Netherlands. A 24 years-old male was referred to our institute with a history of three documented episodes of proximal spontaneous deep venous thrombosis of both legs, the first event when he was 17 years old; at age 21 he developed severe post-thrombotic syndrome with bilateral ulcers, that did not improve on adequate compression therapy. At time of referral he did not use oral anticoagulant therapy. The family history was negative for venous thromboembolism and there was no evidence for underlying acquired disorders. Additional investigations revealed that he was heterozygous carrier of the factor V Leiden mutation in combination with hyperhomocysteinemia, based on clearly elevated values of homocysteine, basal as well as post methionine loading in the presence of normal values for folic acid, vitamin B6 and vitamin Bi2. Oral anticoagulant therapy, in principle life long, was restarted, folic acid 5 mg once daily was added and the compression therapy continued; a control methionine loading test after 3 months folic acid was normal, in 3.5 years follow-up a slow improvement of his leg ulcers was observed, with complete healing in one leg and no recurrent thromboembolic events. Post-thrombotic syndrome (PTS) is rare complication at this age. PTS is a long-term sequelae of acute deep-vein thrombosis and varies from mild edema to incapacitating swelling with pain and ulceration. PTS is a serious problem in terms of prevalence, complications and treatment with considerable socio-economic consequences. Young patients with symptomatic deep venous thrombosis ( < 50 years), also when transient risk factors for deep venous thrombosis are present, should have a good thrombophilia work-up to detect genetically determined tendency to venous thrombosis. Synergism of factor V Leiden and hyperhomocysteinemia, with regards to the thrombotic risk, has been postulated. Treatment with folic acid in combination with oral anticoagulant therapy and adequate compression therapy has led to slow improvement of his leg ulcers in our case, however if metabolic correction of hyperhomocysteinemia leads to improvement of long term prognosis remains to be proven. Homocysteine in elderly patients with stroke and vascular dementia. M.A. Mansoor 1., D.J. Stott2, A. Rumley3, G.D.O. Lowe3, P.H. Evans4, O. Kristensen/. 1Division q/ Clinical Chemistry, Central Hospital in Rogaland Stavanger. Norwm,, 2Academic Section of Geriatric Medicine, ~University Department of Medicine, 4University Department ~[ Public Health, University of Glasgow, UK. Epidemiological surveys and case control studies have shown that elevation of plasma total homocysteine (tHcy) is a risk factor for premature vascular disease. Patient populations investigated in most of these studies include individuals

aged -<60 years. In the present study we investigated tHcy concentrations in elderly patients with vascular dementia (n--42) age 74.4+8.7 years, stroke (n=82) age 73.4+10.1 years, and healthy control subjects (n = 40) age 71.8 + 7.4 years. Patients with vascular dementia had significantly higher concentrations of tHcy than control subjects (mean+SD, 19.9+7.6 /amol/L vs. 16.9_+6.1 /lmol/L (p=0.05). There was a trend for tHey to be elevated in ischaemic stroke however this was not statistically significant. We detected significant positive correlations between tHcy and fibrin degradation products (r = 0.19, p < 0.05), protrombin fragment 1+2 (PF 1+2) (r =0.24, p =0.005) and tissue plasminogen activator (r = 0.21, p = 0.02), and negative correlations with vitamin B12 (r = 0.2, p < 0.05) and erythrocyte folate (r = 0.18, p < 0.05). The findings of this study suggest that increased concentrations of tHcy may be a risk factor for vascular dementia. Increased levels of tHcy were associated with evidence of activated thrombosis and endothelial disturbance, supporting the hypothesis that these mechanisms play a role in causing atherothrombotic disease due to elevated tHey.

Heteroduplex analysis for C677T genotyping of methylene tetrahydrofolate reductase: a case-Control study in men with angina. I.F.W. McDowell*, Z.E. Clark, D.L Bowen, M.F. Bellamy, M.L. Burr. Cardiovascular Sciences Research Group, c/o Dept. of Medical Biochemistry, University of Wales College of Medicine, Health Park, Cardiff, CF4 4XN. UK. Individuals who possess the C677T variant of methylene tetrahydrofolate reduetase (MTHFR) can develop hyperhomoo cysteinaemia and may be at increased risk of cardiovascular disease. A technique for C677T genotyping has been developed using heteroduplex analysis. This is a modified version of the polymerase chain reaction (PCR) in which a 147-bp region of the MTHFR gene containing the C677T mutation is amplified from genomic DNA in a PCR which also includes a 144-bp fragment of DNA known as the heteroduplex generator. This is identical to the normal PCR product except for a 3-bp deletion one nucleotide upstream from the mutation site. Following PCR heteroduplexes forn~ between the heteroduplex generator and the 147-bp PCR product amplified from the genomic DNA. The 3-bp deletion in the heteroduplex generator causes a 3-bp loop to protrude from the PCR product whose shape determines the electrophoretic mobility of the heteroduplex when analysed on a polyacrylamide gel. The presence of the C677T point mutation produces a larger protruding loop (5 bp) such that the mobility of the mutant heteroduplex is different from that of the wild-type heteroduplex. Hence the gel profile is characteristic of each genotype. Heteroduplex analysis requires only three steps (DNA extraction, PCR, electrophoresis) and produces results that are easy to interpret. DNA extracted from blood and mouthwash are both suitable samples for genotyping. Men with angina (n = 624) recruited for the Diet and Angina Randomised Trial (DART2) were genotyped and compared with 605 healthy subjects (blood donors) recruited from the same region (South Wales). Genotype frequencies (+/+

Abstracts/Netherlands Journal of Medicine 52 (19983 S1-$61 12.5%; + / - 43.6%; - / - 43.9% (patients); +/+ 12.1%; + / 40.0%; - / - 47.9% (controls)) were not significantly different. Heteroduplex analysis is a rapid and robust technique for C677T genotyping. The population frequency in South Wales is similar to that reported for populations from northern Europe. Possession of the C677T allele was not associated with ischaemic heart disease in this study.

The cardioprotective effect of HDL is not modified by elevated tHcy. R. Mclcady1., L. Daly2, I. Graham I and the EC concerted action project Hornocysteine and Vascular Disease. 1Adelaide Hospital and 2Department of Epidemiology and Public Health Medicine, University College, Dublin, Ireland Elevated plasma total homocysteine(tHcy) is now considered a strong and independent risk factor for vascular disease but the mechanism by which this occurs remains unclear. An oxidising effect on LDL cholesterol has been proposed. Using data from a multicentre European case-control study of 750 cases of vascular disease and 800 controls, we have undertaken an analysis of the contribution to vascular risk from total cholesterol(TCHOL), LDE, HDL and the HDL/LDL ratio. In addition, we have examined the independence of the risk estimate for TCHOL and for each subfraction in relation to elevated fasting plasma tHey level. Odds ratios (OR) and associated 95% confidence intervals were calculated using logistic regression analysis treating cholesterol and subfractions as continuous variables and comparing risk in subjects at the top quintile to that in subjects at the bottom quintile of each of the control distributions [Data for males: total Chol., unadjusted 1.4 (1.1.-1.7), adj. smok./BP 1.3 (1.1-1.73, adj. smok./BP/ftHcy 1.3 (1.1-1.73; LDL chol., 1.3 (1.0-1.6), 1.3 (1.1-1.7), 1.3 (1.1-1.73; HDL chol., 0.4 (0.3 0.5), 0.4 (0.3 0.6), 0.4 (0.3-0.6); HDL/LDL ratio, (L5 (0.40.6), 0.5 (0.4-0.7), 0.5(0.4-0.7)]. These values show the effect of elevated TCHOL and of each subfraction in males. In females (data not shown), the OR are somewhat greater. In both sexes, the independence of TCHOL, of each subfraction, and HDL/LDL ratio to vascular risk is demonstrated. Adjustment for the presence of elevated fasting tHey (ftHcy) did not alter the risk estimate. The present data do not support the hypothesis that elevated plasma tHey levels adversely affect risk due to elevated serum cholesterol levels. Homocysteine and restenosis post-PTCA. S.E.S. Miner*, H.S. Banijamali, J. Teitel, H.K. Lau, K.A. Bowman, R.J. Chisholm, B.H. Strauss. Saint Michael's Hospital, Toronto, Ontario, Canada. Introduction: Although hyperhomocysteinemia is an independent risk factor for occlusive arterial and venous disease, there have been no published studies documenting its role in restenosis post-percutaneous transluminal coronary angioplasty (PTCA). Objective: To investigate the potential association between hyperhomocysteinemia and restenosis post-PTCA. Method~: In 147 patients undergoing PTCA. fasting homocysteine levels were determined by HPLC with pulsed inte-

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grated amperometry. Mandatory repeat angiography was performed at 6 months, or earlier if clinically indicated. At the time of angiography, a clinical determination of restenosis was made by operators blinded to homocysteine levels. In addition, quantitative coronary angiography (QCA) was performed and analyzed at a later date to quantitate the degree of restenosis in all patients. Results: For the entire group, the mean homocysteine levels was 10.1 pmol/L. Only 11 patients (7%) were found to have homocysteine levels above 15 ,umol/L. In the 37 patients with clinical restenosis, the mean homocysteine level was 9.6 ~mol/L vs. 10.2/gnol/L in those free of restenosis (p = NS). Similarly, no correlation was noted between homocysteine levels and QCA-determined restenosis. Conclusions: The findings of this study do not support the hypothesis that plasma homocysteine levels predict restenosis post-PTCA.

High dose vitamin therapy to treat hyperhomocysteinemia in chronic renal failure: preliminary correlation with myocardial function. S.E.S. Miner*, H.S. Banijamali, M. Freeman, M. Goldstein. Saint Michael's Hospital, Toronto, Canada. Introduction: Hyperhomocysteinemia is an independent risk factor for occlusive arterial and venous disease, and is a common finding in the hemodialysis population. The mechanisms underlying this observation are unclear, but may relate to the induction and perpetuation of endothelial dysfunction. Transient (silent) ischemia due to endothelial dysfunction is postulated to underlie the frequent, transient decreases in ejection fraction seen on nuclear vest studies (NVS) in the hemodialysis population. Objective: To document the efficacy of high dose vitamin therapy to reduce elevated homocysteine (Hey) levels, and to evaluate the impact of this therapy on silent ischemia in the hemodialysis population. Methods: NVS provide a continuous measure of ejection fraction, with transient reductions felt to be due to transient ischemia. Results are presented as minutes of decreased ejection fraction ( > 7% fall) per hour, and are divided into predialysis (pre-HD) and dialysis (HD) periods. Eight patients receiving chronic hemodialysis therapy had previously been studied at this institution with NVS. In these patients, nonfasting Hey levels were determined by HPLC with pulsed integrated amperometry. In an unblinded fashion, treatment was initiated with high dose vitamin therapy frolic acid 15 mg/d, pyridoxine 100 rag/d, cobalamin 25 microgram/d). After 4 to 6 weeks of treatment, non-fasting Hey and NVS were repeated. Results: The average Hey level fell from 29.3 ,umol/L to 19.8 flmol/L with treatment (p=0.009). All eight patients underwent repeat NVS, but one was disqualified due to uninterpretable data. Of these 7 patients, a mean drop of 10.8 ~mol/L was associated with mean decreases of 12.6 min/hr pre-HD (13.3 to 0.7) and 4.6 min/hr HD (14.0 to 9.4) on NVS. These results did not achieve statistical significance. Conclusions: Non-fasting homocysteine levels are significantly decreased by high dose vitamin therapy. This treatment effect is associated with a trend toward decreased myo-

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Abstracts/Netherlands Journal o["Medicine 52 (1998) SI-$61

cardial ischemia. Larger trials will be required to verify this finding. Hyperhomocysteinemia is one of independent risk factors for coronary artery disease in Japanese. H. Mokuno*, H. Daida, Y. Watanabe, Y. lwama, H. Yokoi, H. Yamaguchi. Department of Cardiology, Juntendo University, Tokyo, Japan. Moderate elevated levels of total plasma homocysteine are independently associated with coronary artery disease (CAD) and might be attributed to its direct vascular damage and thrombogenic effects. To examine this association between homocysteine levels and angiographically defined CAD in Japanese, we measured plasma homocysteine by high pressure liquid chromatography and evaluated other risk factors in 313 subjects aged 22 to 79 years who underwent coronary angiography examination. CAD(+) was defined as diseased vessel of > 5 0 % stenosis in any major coronary artery (n = 238). Homocysteine levels tended to be higher in CAD(+) than C A D ( - ) ( m e a n + S D : 17.6+9.97 vs. 15.8_+5.16/2mol/l, p = 0.1). Especially, subjects with multiple diseased vessels in CAD(+) had significantly higher levels of homocysteine compared to C A D ( - ) (18.3_+011 vs. 15.8_+5.16 pmol/l, p < 0 . 0 5 ) . Prevalence of CAD were higher in hyperhomocysteinemia subjects with highest 5% of homocysteine levels ( > 30 pmol/l) than in the rest subjects (94% vs. 75%~ p=0.1). In other conventional risk factors, age and lipoprotein(a) were higher (61.6_+7.9 vs. 55.8+12.0 years, p < 0 . 0 1 . 28.0=24.5 vs. 22.4 + 17.2 mg/dl, p < 0.01 ) and high density lipoprotein cholesterol was lower (39.7 _+ 12.1 vs. 48.2 _+24.4 mg/dl, p < 0.01 ) in CAD(+) than in C A D ( - ) significantly. However, total cholesterol and triglycerides were not different between CAD(+) and C A D ( - ) . In correlation analysis, homocysteine levels were not correlated with these any other risk factors. These results suggest that hyperhomocysteinemia is not prevalent, but independent of other conventional risk factors for coronary artery disease in Japanese. ]'he mutation 677C --,T of the methylenetetrahydrofolate reductase (MTHFR) gene as a possible risk factor for placenta vasculopathy. E.F. van der Molen j*, W.L.D.M. Nelen2, S.G. Heil I, T.K.A.B. Eskes2 and H.J. Biota 1. Laboratory of Paediatries and Neurology, and Department o f Obstetrics/ Gynaecology 2, University Hospital St Radhoud Nijmegen, The Netherlands. Hyperhomocysteinemia as a risk factor for placental vasculopathy (placental abruption and placental infarcts with fetal growth retardation), may result from genetic changes or nutrient deficiencies. Homozygosity of the 677C ~ T conversion in the M T H F R gene is associated with hyperhomocysteinemia and was investigated as a risk factor for placental vasculopathy in 165 women (study group) and 139 matched control women and 1250 population based controls [Results for CC: study group (n=165) 74 (45%), controls (n= 139) 63 (45%), Dutch population (n= 1250) 617 (49%); for TC: 72 (44%), 69 (50%), 527 (42%); for TT: 19 (12%), 7 (5%,), 106 (8.5"/,,); OR: controls 2.45, Dutch 1.40; 95% CI: controls (1.00-6.02), Dutch (0.84~2.36). Odds ratio (OR) and 95% confidence inter-

val (CI) were calculated for prevalence of homozygosity in both groups]. Conclusion : Homozygosity for 677C ~ T mutation in M T H F R gene is likely a risk factor for placental pathology. Are the effects of low-dose folic acid on homocysteine (tHcy) and folate concentrations methylenetetrahydrofolate reductase (MTHFR)-genotype related? W.L.D.M. Nelen 1., H.J. Blom2, C.M.G. Thomas ~, G.H.J. Boers3, E.A.P. Steegers1, T.K.A.B. Eskes 1. Departments o/" Obstetrics and Gynaecology j , Paediatrics2 and General Internal Medicine3, University Ho~pital St Radboud N!]megen, The Netherlands. Purpose: Hyperhomocysteinaemia and homozygosity for the 6 7 7 C ~ T mutation in the MTHFR-gene are known as risk factors for unexplained recurrent early pregnancy loss (REPL). The aim of this study was to investigate in these women the effect of folic acid supplementation (0.5 mg daily) on tHcy and folate concentrations after subdivision into the three genotypes of the 677 C ~ T mutation in the M T H F R gene. Methods: Homocysteine metabolism was investigated in 49 women with a history of REPL by methionine loading test before and after two months of folic acid supplementation. Per genotype (homozygous T/T n = 8 ; heterozygous T/C n = 23; wild type C/C n = 18) the effect on tHey and concerning vitamin concentrations was observed. Results: Before folic acid supplementation we determined a difference in the median fasting tHcy concentration between the T/T (14.9 ~tmol/L) and the other genotypes (12.8 pmol/L) (P < 0.05). Furthermore, the median serum folate concentration was lower in T/T (7.3 nmol/L) compared with TIC (12 nmol/L) and C/C (13 nmol/L) women (P<0.01). After two months of supplementation T/T women showed the strongest decline in median fasting tHcy concentration ( - 4 1 % ; P < 0.01 ) and after-load ( - 3 7 % ; P < 0.05), but the lowest absolute increase in folate serum concentrations (+26 nmol/L; P < 0.05). Conelusion: Folic acid supplementation (0.5 mg daily) for two months in women with REPL results in substantially reduced tHcy concentrations. This effect was most obvious in women homozygous for the 677 C ~ T mutation of the M T H F R gene. Therefore improving folate metabolism in these women by folic acid supplementation may reduce the incidence of REPL. The relationship between folate and homocysteine (tHcy) concentrations and dietary intake pattern in women during their reproductive ages. W.L.D.M. Nelen ~*, M.C.J.G. Nelen2, H.J. Blom2, E.A.P. Steegers j, W.M.M. Verschuren4, T.K.A.B. Eskes I. Dept. o1" Obstetrics and Gynaecology 1 and Paediatries3, University Hospital St Radboud Nijmegen, Dept. Nutrition2, Ignatius Hospital Breda, Dept. Epidemiology4 National Institute of Public Health and the Environment, Bilthoven, The Netherlands. Introduction: Folate deficiency and hyperhomocysteinaemia have been proposed as risk factors for recurrent early pregnancy loss (REPL). Purpose: To investigate the relationship between the blood

Abstracts/Netherlands Journal of Medicine 52 11998) S1 $61 concentragcons o~ v'aam'm ~ ~'~Z~)t, vi~am'm ~ , fdta~e, respectively homocysteine (tHcy) and the dietary intake o f these vitamin¢~, ~'W~e¢;~,5 ~ q~e~;~tboT~;~e~. Study design: In a case-control study we examined 18 women with REPL and 19 unrelated controls which were acquaintances of the patients and who had at least one liveborn child and no spontaneous miscarriages. At the same day homocysteine metabolism was investigated by a standardized oral methionine loading test and dietary data were collected by a dietary h/story regarding the past fourteen days. Results: Between women with REPL and their controls were no significant differences in the dietary intake pattern and the measured blood concentrations. Folate intake was correlated with the ~oJa~e concemra't'aons measure6 ~n serum ~P='6.5~; rs = 0.41]~ and red cells (t° = 11.113; rs = (1.36~. ~'olate and methionine intake seemed to correlate with fasting tHcy concentrations but this reached no statistical significance &=0.0e~; rs = - 0 . 2 8 ) . For all other dietary data no significant correlations with the measured concentrations were observed. Conctlusion: X~xese res,a~'ts s ~ a e s t 'ay~e~'aomoq-~ste'maemia in REPL is not due to inadequate folate dietary intake, but may be genetically mediated. However, consumption of folate-rich food increases serum and red ce~l folate concentrations and may lower fasting tHcy concentrations.

Homocysteine plasma levels in patients with coronary artery disease: influence on restenosis after per-cutaneous transluminal coronary reva~ca~a~zml'ton, ~ . ~'tk~ar~'lara~, 3. ~orn)~ewycz, G. Zasmeta, G. Zom, G. Maurer, M. Gottsauner-Wolf, K. Huber. Department of Cardiology, University qf Vienna. Austria. risk factor fo~ coronary a~t~-y disease {CAD}. The majo-~ longterm complication of PTCR is the development of restenosis in 30-50%. The influence of plasma homocysteine level on anglographic restenosis rate after PTCR is unclear. Methods: Plasma homocysteine levels were measured in 74 patients with CAD (median age 58 years, range 38 to 82 years; m = 60) by use of an ELISA test (Axis, Norway). All patients revascularisation). 27 patients received angioplasty and 47 patients additional stent implantation. Restenosis was evaluated by quantitative angiography (defined as a late lumen loss of

Results: Thirty one per cent (23174) patients developed restenosis. Homocysteine plasma levels were defined in quartiles (range between 4.9 to 34.4 umol/[). In the Ist quartile (4.9 12.3 ,umol/l) 10 patients had restenosis, in the 2nd quartile 12.4-19.7 /tmol/l) 10 patients in the 3rd quartile (19.8-27.0 ,umol/l) 2 patients and in the last quartile (27.1-34.4 /,tmol/l) 1 patient, Conclusion: There was no association between basal homocysteine plasma levels and the development of late anglographic restenosis in patients with elective PTCR. Impact of homocysteine plasma Ie~e~s on coronary artery disease in patients o~ young age. ~v~.~'t'ttfar~am~, %. ~ornykewvcz. C~.

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Zorn, "oh_.Gortsauner-'NoYi, Q. 'bhaurer, It(. "t-iuber. ~eeartmen~

&~Cardiology, University of Vienna, Austria. known risk factors for coronary artery disease (CAD) and associated with mortality. We investigated possible associations between plasma Hcy levels and clinical parameters in a cohort of young patients with CAD (age < 50 years). Methods: Plasma Hcy levels were measured in 94 consecutive young patients (median age 38 years, range 19 to 50 years: m/f= 73/21) with angiographically proven stable CAD by use of a commercially available ELISA test (Axis, Norway: arbitrary nomal range < 9 mmol/l). Hcy levels were related to age, sex, anatomical extent o f disease, previous myocardial infarcYaon ~NSt'S), as ~e55 as p~esence o~ a~osence o*i risk ~ac~.ors (smoking, hypedigidemia, hypertettsion ) by u~/,~ariate (logistic regression) or multivariate analysis (forward stepwise logistic regression). Results: In 45% of our patients no significant coronary artery sclerosis (narrowing of > 70% of vessel diameter) could be demonstrated, 35% sb,omed a "~-'¢esseX-~isease~-'qV)) and 20% a ->2-VD, respectively. In univariate analysis elevated plasma Hcy was associated with smoking (p = 0.0005), PMi{p
Conclusions: Our data support the view of plasma Hcy being primarily an independent thrombogenic than atherosclerotic

Plasma total homocysteine: a risk factor for mortaIity in aortic stenosis patients. O. NygArd t*, P.M. Ueland 2, H. Refsum:, J.E, Nordrehaug~, S.E. Vollset I. 1Division for Medical Statistics; 2Department of Pharmacology; University of' Bergen; and the t'3Department of Heart Disease, Haukeland University Hospital; Bergen. Norway. are at increased risk of mortality when the levels of plasma total homocysteine (tHcy) are elevated. Methods: In a prospective study, we have evaluated plasma "~5-5cy be'~ebs m reSa'bc,n ao mor'~a'i~'~ ~m ,r,a'fieJ)'~s #~'~ aoy'bc stenosis confirmed at cardiac catheterization in 1991-1992. A total of 93 consecutive patients (44 women) with mean + SD age 68+ 10 yrs were included. 72 of the patients had subsequent valve replacement (AVR) and 32 had additional CABG. The causes for no AVR were: 14 had high risk, 6 had borderline valve area or mild symptoms, and 1 refused surgery. Results." After a median follow up of 5 years, 26 patients (28%) had died. Overall, plasma tHcy was 11.3 /.tmol/L in survivors and 14.6 ,umol/L in patients who died (p=0.001). In a sex and age adjusted Cox regression, AVR, ejection fraction IF,F), plasma tl-tcy, serum crealinine and age were al) strone pred't~tors o~ mortff(tty UJ < ~.~051k "~'nen fft't (nese ~ac-

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Abstracts~Netherlands Journal of Medicine 52 (1998) SI-$61

tars were adjusted for, only AVR and EF remained significantly related to mortality. The tHcy-mortality relation was in particular attenuated by adjustment for creatinine and AVR, but the relation remained significant until both factors were included. Moreover, the sex and age adjusted relation between they and mortality was significant both with cardiovascular (n= 15) and non-cardiovascular deaths (based on death certificate information) as endpoint. Conclusion: Plasma tHey is a strong predictor of mortality in aortic stenosis patients. The present data also underlines the importance of creatinine as a determinant of the tHey level. Hyperhomocysteinemia as a risk factor for premature coronary artery disease. Relation with vitamin status and the T677 mutation of the MTHFR gene. X. Pint6*, M.A. Vilaseca, I. Ferrer, C. Mainou, M. Palfi, J.F. Meco, S. Balcells, R. Pujol, D. Grinberg. Bellvitge and Sant Joan de Deu Hospitals and

Department of Molecular Biology, University of Barcehma. Spain. Patients and methods: We studied 76 male outpatients with early MI history (age < 55 years) and 99 age-matched male healthy controls with a mean age of 45.8 + 7 and 45.6_+ 10 years, respectively. Cardiovascular risk factors, lipid profile, basal plasma homocysteine, vitamin B6, vitamin Bl2, folate and the presence of the T677 mutation of the MTHFR gene were evaluated in all subjects. Hyperhomocysteinemia was defined as a plasma concentration of homocysteine in the patients greater than the 95 percentile (16.8/zmol/1) of the healthy control group. The MTHFR genotype was analyzed by PCR followed by Hint digestion. Results: Smoking, hypertension, and diabetes were more frequent in patients than in healthy subjects. Patients had higher mean levels of creatinine, urate, glucose, triglycerides, apo B, Lp(a), and fibrinogen concentrations, and lower HDLc and apo AI than control subjects. There were no differences in the other parameters studied. The prevalence of T677 mutation of the MTHFR gene was similar in cases and controls. Moderate hyperhomocysteinemia ( > 16.8 /,tmol/l) was more frequent in case patients than in controls (21.4% vs. 5%, respectively; p = 0.008). In both groups, a significant correlation was noted between homocysteine levels and folate and vitamin BI2 levels (p < 0.001), but not with vitamin B6. The results of the discriminant function analysis, the odds ratios (OR) and the 95% confidence intervals (CI) are presented below for several variables: Age (years), OR 1.0250, CI 0.874-1.078: Smoking status, OR 8.1151, CI 2.366-27.831; Homocysteine ~mol/1), OR 4.8914, CI 1.074-22.277; Creatinine (/zmol/l), OR 0.9464, CI 0.923-0.970; Total cholesterol (mmol/l), OR 3.1782, CI 1.315 7.679; Lipoprotein (a) (mg/dl), OR 3.4483, CI 1.397-8.510; HDLc (mmol/1), OR 0.2240, CI 0.079 0.631; Triglycerides (mmol/1), OR 1.1676, CI 0.333~.096. Conclusions: Mild hyperhomocysteinemia, but not the T677 mutation of the MTHFR gene, is associated with premature coronary artery disease in patients from the Baix Llobregat area. Mild hyperhomocysteinemia is significantly related with low concentrations of folate and vitamin B~2 but not of vitamin B6.

SSCP analysis of the human methylenetetrahydrofolate dehydrogenase gene: relevance to neural tube defects. N.M.J. van der Put 1., F.A. Hole, S.G. Hell 1, M. Geurts:, J.M.F. Trijbels 1, E.M.C. Mariman:, B. Hamel:, H.J. Blom 1. Departments o[

I Pediatrics and 2Human genetics, University Hospital Nijmegen, The Netherlands. No more than 25% of the folic acid preventable neural tube defects (NTD) cases can be explained by the observed 677C-*T mutation in the MTHFR gene. In order to look for possible other mutations present in the folate dependent homocysteine (Hcy) metabolism, we now studied the methylenetetrahydrofolate dehydrogenase (MTHFD) gene on chromosome 14q24 by SSCP analysis. This gene encodes a single protein with three catalytic properties; the 5,10-methylenetetrahydrofolate dehydrogenase; the 5A0-methylenetetrahydrofolate cyclohydrolase and 10-formyltetrahydrofolate synthetase activity. Here we report on the mutation analysis of the MTHFD cDNA in patients with NTD. Results: The cDNA of the MTHFD gene of 38 familial and 79 sporadic patients was screened for the presence of mutations. Two amino acid substitutions were identified. The first one a 9 3 1 G ~ A mutation was detected in a patient with familial NTD and was not detected in 300 controls. The second mutation was a 2011G~A substitution. This mutation was fairly prevalent and in similar frequencies present among NTD patients and controls. An AA-genotype was present in 11.4% of the familial NTD patients, 17.9% of the sporadic NTD patients and 18,8% of the controls. Furthermore, there was no association observed between the prevalence of this mutation and Hcy levels. Conclusion : We have no indications for a major involvement of genetic abberations in the MTHFD gene in the etiology of NTD. A major role for the homozygous C677--*T mutation of the MTHFR gene in the association between hyperhomocysteinemia and early-onset ischemic stroke. L. Soriente*, A. Coppola, A. D'Alessio1, G. Guiotto, P. Madonna, V. Palmieri, R. Albisinni, A.M. Cerbone, G. Orefice ~, G. Di Minno. Departments o/

Clinical and Experimental Medicine and 1Neurological Sciences, University o/" Naples "Federico H', Naples, Italy. Hyperhomocysteinemia is a risk factor for ischemic stroke. Conflicting data are available concerning the association between the risk of ischemic stroke and the homozygous C677-~T mutation of the MTHFR gene, a common cause of mild hyperhomocysteinemia. To elucidate the role of this mutation in patients with early-onset ischemic stroke, we have evaluated its prevalence and relationship with hyperhomocysteinemia in a sample of 59 patients (31 males, 28 females, mean age 38 yrs., mean age at the event 34 yrs.) and compared them with a population of 182 matched healthy subjects. A significantly higher prevalence of homozygous C677~T MTHFR mutation (35.6% vs. 21.4%, p < 0.05) and of hyperhomocysteinemia (22% vs. 9.8%, p < 0.05) was found in patients when compared to controls, patient mean fasting total homocysteine levels (tHey) being significantly higher than that of controls (15.9+ 14.5 vs. 12.5+7.8//M; p < 0.05). tHey and the number

Abstracts/Netherlands Journal of Medicine 52 (1998) S1-$61 of hyperhomocysteinemic individuals were higher in patients with the homozygous mutation than in non-homozygotes (23.3±22.2 vs. 11.8±4.9 and 9/21, 42.8% vs. 4/38, 10.5%, respectively, p always < 0.005). A logistic regression analysis showed a significant association between the homozygous MTHFR mutation and the risk of early-onset ischemic stroke (OR=2.1, 95% CI 1.1~,.0, p=0.02). The association of the event with tHey was statistically significant only when the genetic marker was excluded from the analysis. Accordingly, the homozygous MTHFR mutation was the major determinant of tHcy in a multiple regression analysis Ip < 0.000). In conclusion our data show an association of the homozygous MTHFR C 6 7 7 ~ T mutation with juvenile stroke and confirm the relevance of this genetic marker in the regulation of tHcy. Nutritional modulation of homocysteine metabolism in dialysisdependent end-stage renal disease patients: a pilot study in Spain. M.J. Jimeno ~, B. Malla 2, G. Torres 2, R. Deulofeu l, E. Alonso-Aperte4, G. Varela-Moreiras 4.. 1Universidad de Burgos; 2Hospital Gral. Yagiie, Burgos; 3Hospital Clinic i Provincial, Barcelona; 4Universidad San Pablo-CEU, Madrid. Spain. Spain shows a much lower prevalence of Cardiovascular Disease (CVD) compared to central and northern european countries, although CVD remains the leading cause of mortality in end-stage renal disease (ESRD) patients on maintenance hemodialysis. Conventional risk factors as hypertension, smoking, dyslipidemia, etc., are not good predictors of incident CVD in dialysis patients. Many patients with renal disease have elevated homocysteine (Hey), possibly due to uremia. However, there are no data on the determinants of homocysteinemia in Spain where a mediterranean dietary pattern is usually followed. The aim was to analyze how Hcy metabolism may be regulated through several dietary factors in ESRD patients. For this purpose 61 (39 rrd22 f, 62+ 13.9 yr~ ESRD patients on dialysis were selected corresponding to the total of dialysed patients in the province of Burgos. Time on dialysis ranged from 0-2 yr (n--17) to > 1 0 yr (n=8). Diet study consisted of a semiquantitative food frequency questionnaire (150 items, 1 yr) including a vitamin supplements questionnaire, that was validated with a '24 h dietary recall'. Several biomarkers of Hcy/methionine metabolism and 'conventional' CVD risk factors were also evaluated. Plasma Hey was much higher in ESRD (16.4 ± 5.6 ~nol/L) compared to a subsample of a healthy spanish population (8.0 + 3.16 /~moI/L, n = 48), although lower compared to previous results for Hcy in ESRD from other countries. Folate+Btz+B6 supplementation resulted in a significant reduction of Hcy (11.9 + 3.51 /zmol/L). Plasma folic acid (FA) in the ESRD group was 8.7 + 6.0 ng/ml, and vitamin B12 values found were 787 _+355 pg/ml. Mean folic acid (122 gg/d) and vitamin B6 (0.8 rag/d) intakes were much below the actual spanish RDA's (200/~g/d FA; 1.8 mg/d B6), whereas vitamin Bl2 (6.1 /,tg/d) was much higher compared to RDA's (2 /tg/d). The effect of Cho1.(183 mg/dl), HDL-chol. (44.1 mg/dl), LDL-chol. (111.7 mg/dl), triglycerides (147.7 mg/dl), BMI, and others were also evaluated. Results suggest that Hey, and FA, B12 intakes/plasma levels should be consid-

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ered as independent factors, and that dietary pattern in our ESRD sample seems to be protective and useful on the basis to understand if Hcy may be considered as a biological marker for CVD in a mediterranean area as Spain. Hyperhomocysteinemia in children with stroke. M.A. Vilaseca~*, E. Cardo2, R. Artuch 1, J. Campistol 2, M. Pineda:. Serveis de Bioquimica I i Neuropediatria 2, Hospital Universitari Sant Joan de Dbu, Barcelona, Spain. Objective: To screen for hyperhomocysteinemia in children with stroke (ischaemic infarct, thrombosis or haemorrhage; N = 55): (a) to discover individual patients with primary hyperhomocysteinemia and (b) to compare plasma total-homocysteine (tHey) concentrations of this group of children with those of age-matched reference values. Method: tHcy was determined by HPLC with fluorescence detection of the SBDF derivatives. Results: We found a 6-month-old male with fatal haemorrhagic infarct and homocystinuria (plasma tHey: 202/zmol/L) caused by cystathionine fl-synthase deficiency (Dr. Blom. Nijmegen). Moderate hyperhomocysteinemia was found in three patients (tHey: 15, 16.5 and 25.2/~mol/L), one of them homozygous for the G677T mutation of the MTHFR gene (Drs. Grinberg and Balcells, Barcelona), and the others under investigation. Significant differences were found in children with stroke compared with age-matched reference values (RV): 2 months-10 years (N=33): mean: 6.7 (2.6-15) (RV:5.8 (3.3 8.3) (P<0.0001); 11 15 years(N=14): mean: 8.7 (5.5 25.5) (RV:6.6 (4.4~10.8) (P<0.001) and 16-18 years (N=8): mean: 10.8 (6.1--16.5) (RV: 8.1 (4.6-11.3) (P<0.05). tHcy values in patients with antiepileptic treatment was similar to those of untreated patients. Conclusions: (1) The diagnosis of one patient with CBS deficiency and three with mild hyperhomocysteinemia confirms the need of including tHey determination in the differential diagnosis of stroke, independently of the age of presentation. (2) Considered as a group, the patients with stroke showed significantly higher tHey values compared with age-matched controls. (3) The diagnostic protocol for stroke should also include the determination of vitamins (B12, B6 and folates) as well as other determinants of plasma tHey concentration, so as to be able to interpret the etiology of moderate hyperhomocysteinemia in childhood. Pregnancy outcome in hyperhomocysteinaemic women without vitamin supplementation. M.E. Van Leerdam l, S.C. De Jong:, J.A. Rauwerda 2, G.A. Dekker 1, J.I.P. de Vries l*. IDept of G YN/Obstet., Free University Hospital, Amsterdam, The Netherlandv, 2Dept of Surgery, Free University Hospital, Amsterdam. The Netherlands. In this study we had the unique opportunity to study hyperhomocysteinaemic (HHC, fasting and/or postloading total homocysteine > 97.5 percentile) women who received no vitamin supplementation during their pregnancies. The women participated in an ongoing study of the division of vascular surgery being either vascular patient with/without HHC or sibling of vascular patient with/without HHC. By means of a question-

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Abstracts~Netherlands Journal of Medicine 52 (1998) S1-$61

naire the subjects (95% response) described each pregnancy and the following two items are presented here: spontaneous abortions (SP Ab, pregnancy loss < 16 weeks) and pregnancy induced hypertensive disorder (PIHD). Statistical analysis with chi-square test [Women: Vascular patients, HHC pos 26, HHC neg 41; Siblings, HHC pos 49, HHC neg 18. Pregnancies: Vascular, HHC pos 82, H H C neg 103; Siblings, HHC pos 138, H H C neg 58. SP Ab: Vascular, HHC pos 11 (13.4%), HHC neg 13 (12.6%); Siblings, HHC pos 18 (13.0%), HHC neg 13 (22.4%). PIHD: Vascular, HHC pos 27 (32.9%), HHC neg 19 (18.4%); Siblings, HHC pos 42 (30.4%), HHC neg 10 (17.2'7o)]. Conclusion: HHC positive women showed the same percentage Sp Ab and an increased percentage PIHD compared with the general population 12-15% and 10%, respectively. HHC negative siblings demonstrated a remarkable high percentage Sp Ab and PIHD compared to a general population. PIHD occurred significantly more in pregnancies of HHC pos vascular women versus HHC neg vasc women (p < 0.04) and more in pregnancies of HHC pos vascular and sibling women versus HHC neg vascular and sibling women (p < 0.05). The high occurrence of PIHD in the 4 studied groups may open the discussion about prospective investigation randomizing placebo controlled vitamin supplementation to prove therapeutical benefit for treatment during pregnancy in HHC positive and negative siblings of vascular patients despite the current advise to use periconceptionally folic acid. Homocysteine as a risk factor for venous thrombosis in the elderly. H.P.J. Willemfl*, M. Havekes l, H. Berenschot:, S. Vloet 3, W.B.J. Gerrits, H.J. Blom 3, G.M.J. Bos:. 1Dept. ~['

Haematology, Leyenburg Ho.~pital, The Hague,'2 Dept. qf' Haematology, Dr. Daniel den Hoed Clinic, Rotterdam; 3Dept. ~[' Paediatrics, University Hospital St. Radboud, N(imegen, The Netherlands. Baekground: Homocysteine is a risk factor for deep venous thrombosis. In the elderly higher homocysteine levels are observed more frequently. It is not known whether homocysteine is also a risk factor for deep venous thrombosis in the elderly. Therefore we performed a case-control study with approx. 250 cases and 300 controls in Rotterdam and the Hague. Currently the results from The Hague are available. Cases: Case subjects were 68 consecutive patients of 65 years and older suffering from a first and objectivated episode of idiopathic deep venous thrombosis. They were selected by the anticoagulation clinic in The Hague, The Netherlands and screened for total homocysteine (tHcy) levels. Controls: Control subjects were selected from the files of a general practice in the same area. All the elderly (545) were invited to participate as control subjects. Blood was obtained from the first 152 patients who were interested to participate. Blood: Blood for tHcy measurement was drawn in tubes containing acidic citrate (Biopool, Stabilyte~). Values were corrected for the acidic citrate present in the tubes before blood sampling. Results: Mean tHcy of the cases was slightly higher in the cases than in the controls (18.7/tmol/l (SD 7.8) vs. 16.6/,tmol/l

(SD 5.5), resp.). Mean age was comparable: 74.2 yrs. (SD 6.0) vs. 73.4 (SD 5.7). Crude odds ratio's are 1.6 (95% CI 0.7 to 3.9) for tHcy above the P90 (22.6/,tmol/l) and 2.4 (95% CI 0.8 to 6.9) for tHcy above the P95 (24.5 /,tmol/l). Subgroup analysis will be performed after completion of the entire study population. Interpretation: Although the numbers of both cases and controls are very small and therefore the confidence intervals are very wide, the results indicate that homocysteine might be a risk factor for venous thromboembolism in the elderly. Interrelationship between folate status and methylenetetrahydrofolate reductase genotype and effects on plasma homocysteine levels of healthy adults. B. Wilson j*, H. McNulty l, McPartlin 2, j.j. Strain/, D.G. Weir2, J.M. Scott 3. t The ,Vorth-

ern Ireland Centre .]For Diet and Health, University of Uster. Coleraine, U.K. and Departments o[' Clinical Medicine and Biochemistry, Trinity College Dublin, h'eland. Mild hyperhomocysteinemia has been identified as an independent risk factor for vascular disease. Both sub-optimal B vitamin status and genetic factors are implicated in the elevation of plasma homocysteine (hcy). Amongst the latter, the presence of a thermolabile variant of methylenetetrahydrofolare reductase (MTHFR) with impaired ability to catalyze the reduction of 5,10-methylene THF to 5-methylTHF has been identified and is reported to occur in up to 15% of normal healthy populations. However, there is some controversy as to the association between folate status and M T H F R genotype. Some studies (Van der Put NMJ, et al. Lancet 1995; 346: 1070-1071) report significantly higher red cell folate (RCF) levels in individuals who are homozygous for thermolabile MTHFR, while others (Molloy AM, et al. Lancet 1997; 349:1591 1593) show significantly lower RCF levels in nonpregnant homozygous women relative to those showing no mutation. The aim of this pilot study is to examine the association between M T H F R genotype, RCF status and hcy levels. One hundred healthy adults (21-59 years; M = 4 0 , F = 6 0 ) were recruited as part of a larger ongoing study being carried out at this centre. RCF and hcy levels were compared (Student's ttest) between subjects who were homozygous for thermolabile M T H F R (+/+) relative to heterozygotes ( + / - ) or those showing no mutation ( - / - ) . Mean (SD) RCF and fasting plasma hcy levels are as follows: M T H F R genotype +/+. number of subjects 14, red cell folate (/,tg/l) 253 (136), plasma hcy (/,tmol/l) 13.7 (10.3); M T H F R genotype +/ , number of subjects 44, red cell folate (/.t~l) 248 (94), plasma hcy (,umol/1) 9.8 (5.9); M T H F R genotype - / - , number of subjects 42, red cell folate (/2g/l) 275 (128), plasma hcy (,umol/1) 8.1 (2.8)** (**Significantly different from the + / - group: p < 0.01i. While these results show no significant difference in RCF levels between the genotype groups, the trend towards lower RCF in the +/+ group is consistent with Molloy et al., possibly suggesting that these individuals, because of reduced M T H F R activity, may require higher folate intakes to maintain equivalent folate status. The significant difference between hcy levels found in the - / + and - l - group demonstrate the link be-

Abstracts/Netherlands" Journal of Medicine 52 (1998) S1-Srl tween hcy and genotype. Further investigation of a larger sample, including measurements of serum folate will confirm the relationship between genotype, RCF and hcy.

Free communications Maternal plasma homoeysteine, placenta status and docosahexaenoic acid concentration in erythrocyte phospholipids of the newborn. H. B6hles*, S. Arndt, U. Ohlenschl/iger, S. Rill, A.C. Sewell. Dept. Pediatrics, Johann Wolfgang Goethe Universit),, Frankfurt, Germany. The enhanced transport of long chain polyunsaturated fatty acids, especially docosahexaenoic acid (22:6 w-3), to the fetus is a placental function, important for an adequate membrane phospholipid formation and herewith decisive for the quality of the fetal CNS-myelination. A compromised placental function is correlated with signs of vascular pathology. As elevated plasma homocysteine (HCY) concentrations are considered an independent risk for premature occlusive vascular disease, the influence of maternal plasma HCY concentrations on placental function was indirectly studied, determining docosahexaenoic acid (DHA) content in erythrocyte membrane phospholipids of the newborn. 60 unselected pregnant women (age range: 21 39 years) were investigated at delivery. Gestational age ranged from 2&41 weeks. Prior to delivery a placental ultrasound scan was performed. Complete sets of data could be obtained from 43 mothers and their offspring. HCY concentrations were determined in the plasma of cord- and maternal blood. The fatty acid pattern o1" erythrocyte membrane phospholipids was determined by GC in the mothers and their newborns. Zscores of the birth weights ranged from -3.4 to 2.1 and of the placental weights from -3.8 to 4.7. The mean maternal plasma HCY concentration was 6.29_+ 3.34 ~moles/1 ranging from below our limit of detection up to 15 /lmoles/l. These maternal concentrations were correlated with those of their infants (r = 0.71; p < 0.0001). The HCY concentrations were significantly higher in mothers with pregnancies complicated by gestosis or placental calcifications. The Z-scores of birth weights as well as placental weights showed a significant negative correlation with the maternal plasma HCY concentrations. The mean percentage of DHA in erythrocyte phospholipids was 3.2+2.2% in the mothers and 3.4+2.3% in their newborns. Most importantly the maternal plasma HCY levels and the erythrocyte phospholipid DHA concentrations of their offspring were significantly correlated ( r = - 0 . 5 1 ; p < 0.0003). We conclude from these data that maternal HCY influences the integrity of placental vasculature and herewith placental function as for instance represented by DHA accumulation in fetal membrane phospholipids. As the plasma HCY concentration reflects the status of folic acid, vitamin Bt_, and vitamin B6 availability, the nutritional status of the pregnant mother has to be followed more thoroughly. Blood homocysteine lowering with folic acid based supplements: A systematic overview of the randomised trials. Homocysteine

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Lowering Trialists' Collaboration. Cl&ical Trial Service Unit and Epidemiologieal Studies Unit, University of Oxford, Oxford, UK Blood tHcy levels are inversely related to blood levels of folate, vitamin BI2 and vitamin B6, but there is substantial uncertainty about the optimal treatment regimen to lower tHcy levels. A meta-analysis was conducted of individual data on 1114 people in 12 randomised controlled trials to determine the effects on basal blood tHcy levels of dietary supplements of folic acid and of vitamins B12 and B6. Multivariate regression analysis was used. The proportional and absolute reductions in blood tHcy produced by dietary folic acid were greater in those with higher pre-treatment blood tHcy levels (p < 0.1301) and lower pretreatment blood folate levels (p < 0.001). After standardisation to pre-treatment blood tHcy levels of 12 pmol/L and folate levels of 12 nmol/L (i.e. approximate averages for Western populations), folic acid reduced blood tHcy levels by 25% (95% CI: 23% to 28%), with similar effects in the dose range of 0.5 to 5 mg folic acid daily. Dietary vitamin BI2 (mean dose 0.7 mg) produced an additional 7% (95% CI: 3% to 10%) reduction in blood tHcy, whereas dietary vitamin B6 (mean: 16.5 mg daily) did not have any significant effect, at least on basal levels. Hence, daily supplementation with 0.5-5 mg folic acid and about 0.5 mg vitamin BL2 in Western populations would be expected to reduce blood tHcy levels by about one-quarter to one-third (e.g. from about 12/~mol/L to about 8-9/tmol/L). Large-scale randomised trials of such regimens in high risk populations are now needed to determine whether lowering blood tHcy levels reduces the risk of vascular disease.

Renal homocysteine uptake in rats during acute and chronic hyperhomocysteinemia. J.D. House*, M.E. Brosnan, J.T. Brosnan. Dept. Of Bioehemisto'. Memorial University, St. John's, NF, Canada. Elevated plasma total homocysteine (tHCY), an independent risk factor for cardiovascular disease, is commonly observed in renal patients. We have previously shown that, in the rat, the kidney is a major site for the removal of plasma tHCY (Bostom et al. Atherosclerosis 1995;116:59-62). To further characterize renal HCY metabolism, we investigated renal HCY uptake during acute and chronic hyperhomocysteinemic conditions, in a rat model of renal clearance. Acute hyperhomocysteinemic conditions were produced by the primed (5 /zmoles/100 g body wt)-continuous (25 nmoles/(min.100 g body wt)) infusion of [-HCY (+HCY), while control rats ( - H C Y ) received an osmotic equivalent of saline. Renal haemodynamics were determined by [14C]inulin clearance. Arterial plasma tHCY, arterio-renal vein ( A - V ) difference, and arterial plasma free HCY (fHCY) (pM), as well as renal HCY uptake and filtered load (nmoles/min.100 g body wt) are reported below (means=SE; n=8/group), with significant differences (17< 0.05) denoted by '*'. HCY infusion had no significant effect on renal haemodynamics. The urinary excretion of HCY was negligible ( < 2% of filtered load) in - H C Y rats. - H C Y : tHCY 7.9~0.6, A - V 1.4-T-0.3, fHCY 5.9-T-0.5,

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Abstracts/Netherlands Journal of Medicine 52 (1998) S1 $61

HCY uptake 4.l-T- 1.6, Filtered load 5.4w 1.1 ; +HCY: tHCY 47.7-T-5.3", A--V 5.3~1.2", fHCY 36.5~4.8', HCY 18.0-T-3.1", Filtered load 30.8w4.9". During acute hyperhomocysteinemia, renal HCY uptake was increased 4 fold and was equivalent to 50% of the infused dose, while urinary excretion remained negligible. The results obtained provide strong evidence that the kidney has a substantial capacity for metabolizing acute elevations in plasma tHCY. We subsequently initiated studies to examine renal HCY metabolism during chronic hyperhomocysteinemia, induced by the 4 week feeding of a folate-devoid, semi-purified diet. Arterial plasma tHCY was increased in folate deficient vs. folate replete rats (27.5~ 1.7 vs. 12.8~ 1.4/,tM, p < 0.05, respectively (n = 6/group)). Renal A--V differences were 3.5w-0.4 vs. 1.3~0.4 /tM (p < 0.05), for folate-deficient and folate-replete animals, respectively. These data again support a substantial reserve capacity for the renal clearance of HCY, even in the face of a chronic hyperhomocysteinemia induced by folate deficiency. Further, they support a major role for the folate-independent transulfuration pathway in the renal metabolism of HCY in rats. Funding: Kidney Foundation and Medical Research Council of Canada. Cystathionine fl-synthase and mild hyperhomocysteinemia: an association study. L.A.J. Kluijtmans ~, P. Verhoef2, G.J.J. Boers 3, M. Den Heijer 4, L.P.W.J. Van den Heuvel 1, J.M.F. Trijbels ~, H.J. Blom s. Depts. of 1Pediatrics and 3Internal Med-

icine, University Hospital Nijmegen, the Netherlands'. 2Division of Human Nutrition and Epidemiology, Agricultural University Wageningen, the Netherlands. 4Dept. of Internal Medicine, TweeSteden Hospital Tilburg, the Netherlands. Introduction: Molecular defects in genes encoding enzymes involved in homocysteine (Hcy) metabolism may account for mild hyperhomocysteinemia (mHH), an independent and graded risk factor for cardiovascular disease (CVD). Although heterozygosity for cystathionine fl-synthase (CBS) deficiency seems to be excluded as origin of mHH in vascular disease, mutations in (non)-coding DNA sequences leading to mildly affected CBS expression have never been investigated as risk factors for CVD. In the present study, we investigated the possible involvement of CBS in CVD. Methods: We analyzed three silent polymorphisms in the CBS gene (i.e. 6 9 9 C ~ T ; 1080T-~C; 844ins68) as markers in linkage disequilibrium with a possible pathogenic mutation, and assessed their association with fasting, post-methionine load, and delta (increase upon methionine loading) Hcy in 191 patients with arterial occlusive disease and in 109 controls. Results: The 699 TT and 1080 CC genotypes appeared to be associated with significantly elevated Hcy levels compared with 699 CC ( P = 0.02) and 1080 TT ( P = 0.03) genotypes, respectively, however only after a methionine loading test. The 844ins68 insertion variant in either heterozygous or homozygous state was associated with significantly decreased Hcy concentrations. Noteworthily, those CBS genotypes associated with elevated Hcy concentrations were, although nonsignificantly, positively related to the risk of CVD (OR: 1.2

[95%CI: 0.6-2.5] and 1.3 [95% CI: 0.6-2.8] for 699 TT and 1080 CC genotypes, respectively). Conclusion: Our results suggest the existence of molecular defects in the CBS gene predisposing to elevated plasma Hcy concentrations after methionine loading. Whether these defects contribute to the risk of CVD needs further investigation. A second common mutation in the methylenetetrahydrofolate reductase gene; a risk factor for neural tube defects? N.M.J. van der Put 1., F. Gabre~ls:, E. Stevens ~, J.A.M. Smeitink l, F.J.M. Trijbels 1, T.K.A.B. Eskes 3, L.P. van den Heuvel 1, Henk J. Blom 1"4. Departments of JPediatrics, 2Pediatric Neu-

rology, 3Obstetrics and Gynecology, University Hospital N(]megen, The Netherlands. 4Correspondence should be addressed to H.J. Blom. Recently, we showed that a common 677C ~ T mutation in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, causing thermolability of the enzyme, is a risk factor for spina bifida. We now report on another mutation in the same gene, a 1298A ~ C transition, which alters a glutamate into an alanine residue. This mutation destroys a MboII recognition site. We examined the effect of the 1 2 9 8 A ~ C mutation on the M T H F R activity, homocysteine (Hcy) and folate levels. The total study group consisted of a 122 families with mostly sporadic NTD offspring and 403 controls. Enzymatic analysis shows that homozygosity for the 1 2 9 8 A ~ C mutation causes decreased M T H F R activity (Anova, p < 0.0001). Homozygosity for this mutation has no effect on Hcy or folate levels and no thermolability of the enzyme. The combined effect of the 1298A ~ C and 677C ~ T mutations was analysed on Hcy, folate levels and enzyme activity showing that compound heterozygosity for both the 1298 A ~ C and 6 7 7 C ~ T mutations in the coding region of MTHFR, results in reduced M T H F R activity and an impaired Hcy and folate metabolism and thus may be a genetic risk factor for NTD (Anova, p < 0.03). If so these two common mutations in the M T H F R gene may account for a significant proportion of the observed protective effect of folate supplementation. Conclusion: We identified a second common mutation in the M T H F R gene affecting its enzyme function, which may be a risk factor for NTD. Plasma total homocysteine and previous pregnancies: the Hordaland homocysteine study. S.E. Vollset j,2*, A.L. Bjorke-Monsen 3, L.M. Irgens 1"2, H.K. Gjessing 1, B.M. Emblem ~, P.M. Ueland 3, H. Refsum 3. University of Bergen, Bergen, Norway.

JDept of Public Health and Primam" Health Care, ~-Medieal Birth Registry, aDept, of Pharmacology. Background. Plasma total homocysteine (tHcy) measured in serum or plasma is a marker of folate deficiency and has been linked with pregnancy complications and adverse pregnancy outcomes. Methods. Among 5883 40~2-year-old women who participated in a cardiovascular health screening and had their plasma tHcy determined in 1992 or 1993, 14492 pregnancies were reported to the Medical Birth Registry of Norway during the

Abstracts/Netherlands Journal of Medicine 52 (1998) Sl-$61 years 1967 1996, with about 80% of the births occurring ten years or more before the tHcy measurement. Unless otherwise noted quartile-categories of tHey were used, and we report odds ratios (OR) comparing the upper versus the lower tHcy quartile. All p-values refer to trend tests. Findings. After adjustment for age at birth, parity and smoking, tHcy was significantly associated with increased risk of preeclampsia (n = 451, OR = 1.33, p = 0.0l) and all malformations (n = 191, OR = 1.55, p = 0.049). Among malformations, neural tube defects and foot deformities showed the strongest associations with tHcy. No overall relation was observed for still births, but a strong association was seen between tHcy and stillbirths with a reported birth weight of 1500 g or less (n= 112, OR=2.11, p=0.002). Elevated tHcy was also associated with prematurity (gestational age of less than 37 weeks, n = 770, O R = 1.41, p=0.001), birth weight less than 2500 g (n=708, O R = 1.46, p=0.001) and small for gestational age (SGA) births (n = 1727, OR = 1.21, p-- 0.006). The relation of tHcy to SGA was weak, and the latter condition was more strongly associated with smoking habits. Risk of placental abruption did not change significantly over tHcy-quartiles, but was strongly associated with tHcy above 15 ,umol/L (n = 73, OR = 3.03, p = 0.001). Conclusions. This study implicates elevated tHcy as an important biologic marker of several common pregnancy complications and adverse pregnancy outcomes.

Workshops on homocysteine Epidemiology High prevalence of hyperhomocysteinemia in patients with inflammatory bowel disease. M. Cattaneo*, M. Vecchi, M.L. Zighetti, P. Omodei, I. Martinelli, S. Saibeni, P.M. Mannucci, R. de Franchis. A. Bianchi Bonomi Hemophilia and Thrombosis

Center, and Institute of Internal Medicine, IRCCS Ospedale Maggiore. University of Milano, Milan, Italy. Background. Why patients with inflammatory bowel disease are at increased risk for arterial and venous thrombosis is unknown. Since they may have impaired absorption of vitamins that regulate the metabolism of homocysteine, we tested the hypothesis that they have hyperhomocysteinemia, an established risk factor for arterial and venous thrombosis. Methods'. The concentrations of total homocysteine, folate and cobalamin were measured in blood samples from 61 consecutive patients with inflammatory bowel disease and 183 ageand sex-matched healthy controls. Results. The mean concentration of plasma total homocysteine was higher in patients (12.2+7.7) than in controls ( 10.5 _+4.6). Eight patients (13%) had concentrations of homocysteine higher than the 95th percentile of distribution among healthy controls, as compared with 9 healthy controls (5%, OR 2.9, 1.1 7.9). The prevalences of folate or cobalamin deficiencies were higher in patients (15% and 11.5%) than in healthy

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controls (5%). Oral administration of folate, cobalamin and pyridoxine for 30 days to 15 patients decreased their mean ( + SD) homocysteine levels from 20.3 + 9.9 to 9.5 _+3.4/tmol/ L (p<0.001). Two additional non consecutive patients with inflammatory bowel disease, who had had thrombotic episodes at a young age were studied. They had markedly high homocysteine levels (61.9 and 38.8 ~-nol/L), which were corrected by the administration of vitamins. Conclusions: In patients with inflammatory bowel disease there is an increased prevalence of hyperhomocysteinemia, which can account for their high risk for thrombosis and can be corrected by the administration of folate, cobalamin and pyridoxine. Whether or not this therapeutic approach will decrease the thrombotic risk should be tested in properly designed clinical trials. Hyperhomocysteinemia and abdominal aortic aneurysm. M. Wullink *'l, A.A.J. van Landeghem 2, S.E. Kranendonk ~, S.J. Graafsma 3, M. den Heijer3. 1Department of Surgery and

3Department of Internal Medicine, Twee-Steden Hospital, Tilburg," 2Department of Clinical Chemistry and Haematology, St. Elisabeth Hospital, Tilburg, The Netherlands. Hyperhomocysteinemia is an established risk factor for arterial vascular disease and venous thrombosis. Although there are some reports on abdominal aortic aneurysm in patients with homocystinuria, no systematic study has been performed on the relation between hyperhomocysteinemia and aneurysm of the abdominal aorta, which was the aim of our study. Patients which abdominal aortic aneurysm (diameter ->30 mm) or patients who underwent surgery for their aneurysm were recruited from our out-patient clinic. Each patient was asked to bring a friend or neighbour as a control subject. Homocysteine concentrations were measured before and six hours after oral methionine loading. Hyperhomocysteinemia was defined at the 90th percentile of the control group (n = 60). Out of 89 patients 22 (25"/0) had hyperhomocysteinemia at the fasting state (odds ratio 3.6 [95%CI 1.3 to 10.2]) and 15 (17%) after methionine loading (odds ratio 1.8 (95%CI 0.7 to 5.1]). After adjustment for age, sex and creatinine concentration the odds ratio were 1.9 (95%CI 0.6 to 6.0) for the fasting level and 1.2 (95%CI 0.3 to 4.0) for the postmethionine level. Our results suggests that hyperhomocysteinemia is a risk factor for abdominal aortic aneurysm. This work was supported by the 'Stichting Voorzieningen Wetenschappelijk onderzoek', Tilburg, The Netherlands. Plasma homocyst(e)ine concentrations in preeclamptic and normotensive Zimbabwean women. A. Rajkovic *,1, M.R. Malinow, T.K. Sorensen, K. Mahomed, G.B. Woelk, S. Mudzamiri, S. Madzimi, I. King, B. Upson, E. Graf, M.A. Williams.

1Department of Obstetrics and Gynecology, Case Western Reserve University, Metrohealth Medical Center, Cleveland, Ohio 44106-3105, USA. Background: Preeclampsia is a major cause of maternal and infant mortality and morbidity in developed and developing countries. We examined if homocyst(e)ine, a potentially treatable risk factor for premature vascular disease, is elevated in

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Abstracts I Netherlands Journal of Medicine 52 (19983 S1-$61

preeclamptic women from Zimbabwe. In addition, we examined statistically interactions among homocyst(e)ine, obesity and nulliparity. Methods: We studied 382 Zimbabwean women from Harare Maternity Hospital: 191 cases (154 with preeclampsia and 37 with eclampsia) and 191 normotensive controls in this casecontrol study. Plasma was collected from cases and controls 12 72 hours postpartum and homocyst(e)ine concentrations measured. In addition, maternal mid-arm circumference was measured at the time of the delivery and used as primary measure of maternal pre-pregnancy adiposity. Findings: Homocyst(e)ine concentrations in 191 cases with eclampsia and preeclampsia were significantly higher than 191 normotensive controls (mean 12.73 + 5.37 versus 9.93_+ 3.69 /,tmol/L, p<0.0001). The relative risk of preeclampsia increased significantly across increasing quartile of concentrations of maternal plasma homocyst(e)ine (p for linear trend in risk across quartiles < 0.0001). Women in the highest quartile, after adjusting for potential confounders, had a 4.74 fold excess risk o f preeclampsia as compared to women in the lowest quartile. Women in the highest decile as compared to women in the lowest quartile had a relative risk of 7.58. Analysis of interaction of maternal adiposity and increased homocyst(e)ine concentrations produced a 7.54 fold relative risk of preeclampsia. Similarly, nulliparous women with elevated homocyst(e)ine had a 15.4 fold increased risk o f preeclampsia and the excess risk was greater than the sum o f the each factor considered independently. Interpretation: Homocyst(e)ine levels are significantly higher in Zimbabwean women with preeclampsia as compared to normotensive subjects. The relative risk of preeclampsia increased across quartiles of maternal plasma homocyst(e)ine concentrations and synergistic interactions with maternal adiposity and nulliparity were statistically significant. Hyperhomocysteinemia and lower normal levels of the involved vitamins as a risk factor for placental pathology (PV). E.F. van der Molen 1., W.L.D.M. Nelen2, C.M.G. Thomas 3, T.K.A.B. Eskes2, L.A.H. Monnens 4, H.J. Blom -¢. Laboratories of Paediatrics & Neurology and Endocrinology~, Departments of Obstetri~w/Gynaecology 2 and Paediatrics4, University HosT)ital St Radboud Nijmegen, The Netherlands. Hyperhomocysteinemia is a risk factor for placental vasculopathy (PV; placental abruption and placental infarcts with fetal growth retardation). Low levels of the vitamins folic acid, vitamin BI2 and B6 are determinants of hyperhomocysteinemie and therefore may be risk factors for PV. We studied levels of plasma homocysteine (tHcy) and vitamins of 175 women with a history of PV and 141 matched control women [Results. tHey (lamol/l): PV (n = 175 ; median, range) 12.2 ( ~ 49.8), Controls (n=141; median, range) 11.6 (6.4-27.13, OR (95%CI) 3.3 (1.4-7.9); Folio acid (sl; nmol/ll: PV 12 (3.4~42), Contr. 14 (4.1-32), Correlation with tHcy p-value -0.44 (<0.0001), OR 2.4 (1.2~.2); Folie acid (e2; nmol/l): PV 530 (150-1300), Contr. 530 (16(~t100), Correlation with tHcy pvalue --0.38 ( < 0.0001 ), OR 2.1 (1.1- 4.0) ; Vitamin Bi2 (pmol/ 1): PV 210 (69-16003, Contr. 250 (77 720), Correlation with

tHcy p-value - 0 . 3 5 ( < 0.0001), OR 2.6 (1.3-4.9); Vitamin B6 (nmol/l): PV 41 (18-120), Contr. 45 (27-140), Correlation with tHcy p-value -0.15 (0.04), OR 2.9 (1.5-5.5). *Wilcoxon ranksum, Spearman Rank correlation tests and Odds ratio (OR) with 95% confidence interval (CI) homocysteine above 96th and vitamins below 10th percentiles of controls. ISerum, 2erythrocytes]. Conclusions: Significant differences in homocysteine concentrations between women with placental vascolopathy and their controls were found. Low normal levels of the vitamins are also strong risk factors and vitamin B6 may be an independent risk factor for placental vasculopathy. Serum total homocysteine and coronary heart disease: prospective study in middle aged men. P.H. Whincup i*, H. Refsum:, I.J. Perry 3, R. Morris t, L. Lennon j, M. Walker t, A. Thomson j, P.M. Ueland 2, S. Ebrahim 1. 1Royal Free Hospital School oJ Medicine, Lomton, UK; 2 University o f Bergen, Norway: 3 University College, Cork, Ireland. Objective: To examine the prospective relationship between serum total homocysteine (tHcy) and risk of coronary heart disease, taking potential confounding factors into account. Methods: Nested case control study within a prospective study of cardiovascular disease in men aged 40-59 years at baseline (British Regional Heart Study), based on 386 cases of myocardial infarction (MI) and 454 controls, frequency matched by age and town. Stored baseline samples for these subjects were used for retrospective blind analysis of tHcy concentration. Results: Geometric mean tHcy levels were slightly higher in cases (14.2 micromol/L) than controls (13.5 micromol/L) (mean difference 5.5%, 95% CI -0.02 to 10.8%, p=0.06). Age-adjusted risk of MI increased with increasing log t h e y concentration. For a one SD increase in log t h e y (equivalent to a 48"/o rise in tHey), the odds of MI rose by 1.15 (95% CI 1.00 to 1.32; p=0.053. Within the control group, tHey was positively related to haematocrit (r = 0.12, p = 0.01) and inversely related to forced expiratory volume in 1 s ( r = - 0 . 1 3 , p=0.006). However, adjustment for these factors and for blood pressure, cigarette smoking, total cholesterol, social class and creatinine had little effect on the odds ratio observed (1.17, 95% CI 0.99 to 1.38; p = 0.07). In an analysis based on tenths of t h e y concentration, the increased risk of MI was concentrated in the top three groups (tHey > 15.7 micromol/L) which had a combined odds ratio of 1.75 (95% CI 1.29 to 2.36) when compared with the other 7 groups. Elevated tHcy levels ( t H c y > 15.7 micromol/L) accounted for 18% of the attributable risk of MI in this study population. Conclusion: The results provide further prospective evidence that marked elevation of tHey levels is related to increased coronary risk and strengthen further the case for trials of tHey reduction with folate. Hyperhomocysteinemia rosis. F.F. Willems l*, A.H. Zwinderman2, Verheugt 1. UniversiO'

and progression of coronary atheroscleW.R.M. Aengevaeren 1, J.W. Jukema:, G.H. Boers-c, H. Blom 4, F.W.A. Ho57)ital St Radboud Nijmegen. The

Abstracts/Netherland~ Journal q[ Medicine 52 (1998) S1 $61 Netherkan(~ t~3em. ~ ~nr6io]~X'~, abe m. ]merna'~ ~et~,, 4LaboraWry of Pediafrics and Neurology, 2 UnA,ersiry Hospim~ Leiden, The Netherlands'. Hyperhomocysteinemia (HHCY) is an independent riskfactor for the development of coronary artery disaese (CAD) although the mechanisms responsible for the atherogenic effects of homocysteine (HCY) have not yet been determined. Aim of this study was to evaluate the effect of HHCY on angiographic progression of coronary atberosclerosis. Methods: To assess the effects of HHCY on progression of coronary atherosclerosis, HCY was determined after an overnight fast in 295 male patients. These patients had normal Io nary arteriog~aph'£ T-~r~ "~ea~s "aP.e~ en~olrnen't a sec~md coronary arteriography was performed. Progression of coronary atherosCE'a-,y~N" ~ ~ a e e g 4L9 ~4umt&t,~e~ ~e,",~mwO "megiog~ raphy (¢OU)~). Y'yo~res~o~ was tlebneO as a Becre,~se)n ;~vera,ge Mean Segment D/omelet (MSD) and average Mean Obstruction Diameter {'MOO) o~ a~'~a;'a~kab'~e coronary, segmems. Results: Of the 295 patients 218 patients had an evaluable final coronary angiography. The median of the HCY concentration was 13.4 (7.7-56.91. [Correlation coefficient values. HCY: change MSD 0.14 p < 0 . 0 5 , change M O D 0.09 p < 0 . 1 0 . Change MSD: H C Y < 15 prnol/1 (n=157), -0.117 (SD 0.22) ram; H C Y > 15 pmol/1 (n=61), -0.133 (SD 0.18)p=0.38. Change MOD: H C Y < 1 5 /.tmol/l (n=157), -0.116 (SD 0.22) ram; H C Y > 15 pmol/1 (n=61), -0.167 (SD 0.34)p=0.25]. Conclusion: Patients with hyperhomocysteinema show a stronger, though not significant, decrease of MSD and MOD compared to patients with normal HCY levels. Homocysteine levels are significantly correlated with the change in average Mean Segcaent O&meter e~xggestiag ~a effect o( h~;qecho~ocysteinemia on progression o f coronary atherosclerosis.

Nonsyndromie orofacial clefts: association with maternal hyperhomocysteinemia. W.Y. Wong*, T.K.A.B. Eskes ], A. KuijpersJagtman:, P.H.M. Spauwen ~, E.A.P. Steegers ~, C.M.G. Thomas ], BJ.C. Hamel ~, H.J. Blom s, R.P.M. SteegersTheunisser~:'~. UniversiD, Hospital St Rad6oud, No~egen, T~e Netherlands, ~Dept. of Gyn./Obsr, 2Dept. 01" Orthodontics, 3Dept. of Plastic and Reconstructive Surgery, 41)epl. qf Human Genetics, SDepr of Pediatrio, 6Dept. of Epidemiology. Background: Nons),ndromic orofacia~ c~ef~s, i.e.~ c~eft ~ip with or without cleft palate, are serious congenital malformations with a multifactorial aetiology. Folate has been suggested to play a role in the prevention of orofacial clefts (OFC). Therefore, we investigated the folate-dependeut homocysteiue metabolism in mothers with O F C offspring. Methods: A case-control study has been performed in thirty-five women who had given birth to a child with OFC and in fifty-six controls. The folate-dependent homocysteine metabolism was studied by performing an oral methionine loading test. In addition serum and red cell folate, vitamin BI2 and vitamin B6 as well as liver and renal functions were determined. Findi~gs" g o t h ~asCt~ as we't't as a£ter'toad p'tasma totak go-

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s~udy group (n=35) as compared to the controls (n=56) (p < 0.01 and p < 0.05, respectively). Hyperhomocysteinemia was present in 23% of the study group and 9% of the controls. The odds ratio was 3.0 (CI 95%: 0.95-9.6). The median concentrations o f the serum and red cell folate were significantly higher in women of the study group as compared to the controls (p < 0.01 and p < 0.05, respectively). Vitamin B6 concentrations were significantly lower in ~he study group (p < 0.05) while no difference was observed for vitamin BI2. Interpretation: This preliminary study suggests that maternal byperfiomocystNnemia may be a risk factor for nonsyndromic

Comparison between the 7.-4mur and the 4~our methioffme loading test for the identification of subjects with methionine intolerance. M. Cattaneo*, B. Agati, A. Lecchi, R. Lombardi, M.L. Zighetti, E. Taioli, P.M. Mannucci. A. Bianchi Bonomi Hemophilia and Thrombosis" Center and the Epidemiology Unit, IRCCS Ospeda& Maggiore, University of Milano. Milan, Italy Background. High plasma levels of homocysteine 4 to 8 h after an oral methionine loading identify subjects with methionine intolerance, which is a risk factor for arterial and venous thrombosis. In 1995, B o s t o n el al. demonstrated the equivalence of homocysteine determinations 2 h and 4 h postmethionine loading (PML) for identification of these subjects. Using the 2-hour protocol would facilitate expanded application of

Aim of our study was to validate the 2-hour protocol for the methionine loading test in our laboratory. Subjects and methods: Total plasma homocysteine levels were measured by HPLC with fluorescence detection before, 2 h and 4 h after an oral methionine loading (3.8 g/m z b.s.a.) in 187 patients with previous episodes of arterial and/or venous ~hrombosis (median age: 40.5 "y, range: ~3 ~ l y; M" F = 9(}:97) and in 84 healthy controls (median age: 41 y, 20-72 y; M : F =42:42). Both the PML plasma homocysteine absolute levels and their increments above fasting levels were calculated and considered for comparison of the 2-hour and the 4-hour protocols. Results: (1) PML absolute levels of homocysteine. The median 2-hour and 4-hour values (umol/L) for the 271 subjects stud(ed were 18.5 (range: 10.3-87.1~ and 22.5 (12.1-9(I.0). Linear (Pearson r) and rank order (Spearman rho) correlations between the 2-hour and the 4-hour values were 0.97 and 0.94 (p < 0.0001 for both). The 2-hour PML homocysteine levels were higher than the 95th percentile of distribution among controls in 13 patients (7%). In contrast, the 4-hour PML levels were higher than the 95th percentile in 17 patients (9.I%). The sensitivity and specificity o f the 2-hour versus the 4-hour protocol were 70.6% and 99.4%. (2) PML homocysteme increments abo'~e ~'asting ~eve_t,~. 3"he median 2-.h~x

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Abstracts / Netherlands Journal of Medicine 52 (1998) S1-$61

and 4-hour values for the 271 subjects studied were 9.7 (range: 2.9~2.9) and 14.0 (5.4-54.3). Linear and rank order correlations between the 2-hour and the 4-hour values were 0.90 and 0.88 (p<0.0001 for both). The 2-hour PML homocysteine levels were higher than the 95th percentile of distribution among controls in 19 patients (10.2%). In contrast, the 4hour P M L levels were higher than the 95th percentile in 25 patients (13.4%). The sensitivity and specificity of the 2-hour versus the 4-hour protocol were 76% and 100%. Conclusions: The results of our study suggest that the 2-hour protocol of the methionine loading test should not replace the 4-hour protocol, since it is less sensitive to methionine intolerance. Isolated methionine intolerance is a risk factor fur early-onset venous thromboembolic disease. A. D'Angelo 1., I. Fermo:, G. Mazzola j, R. Paroni2, G. Di Minno 3. S. Vigano'D'Angelo:.

I Coagulation Service and 2Dept. of Laboratory Mealie'me, 1RCCS H. S. Raffaele, Milan, Italy, and 3Dept. of Clinical and Experimental Medicine, University 'Federico H', Naples, Italy. Moderate fasting hyperhomocysteinemia (HHcy) has been recognized as an independent risk factor for venous thromboembolic diseases (VTE). Whether isolated methionine intolerance (= abnormal elevation in t-Hcy following an oral methionine load) is also a risk factor for VTE remains to be proven. Protein C, protein S, antithrombin, plasminogen, heparin cofactor II, APC resistance (APC-r) and fasting t-Hcy were measured in a consecutive series of 429 patients (F/M 233/196, mean age 44.1 + 16.7 yrs, mean age at first event 41.5+17.2 yrs, 109 patients with recurrent VTE) with early-onset deep vein thrombosis of the inferior limbs and/or pulmonary embolism (n = 233) or VTE occurring at unusual sites (central retinal vein occlusions, superficial vein thrombosis, thrombosis of the cerebral veins, thrombosis of the internal veins, n = 196), in the absence of acquired coagulation abnormalities, overt cancer, liver and renal diseases. The prevalence of abnormalities was 39.5%, with APC-r and fasting HHcy accounting for 70.8% of the defects detected. The odds ratio (OR) for recurrent VTE in patients with defects was 2.00 ( p < 0.005). Familial history of thrombosis was present in 79.3% of patients with deficiencies of natural anticoagulants, but it was similar in patients with APC-r (50.0%), fasting HHcy (35.4%) and no defects (44.3%). Postmethionine load t-Hcy (A PML at 8 hrs) was measured in a subgroup of 230 patients. In this group of patients, 14.4% had isolated fasting HHcy+methionine intolerance (OR: 3.18, p < 0.005), 12.2% had isolated methionine intolerance (OR:2.63, p < 0 . 0 1 / and 6.5% had isolated APC-r (OR: 2.25, p < 0.05). Combinations of two defects were observed in 6.9% of the patients, who had a higher risk of recurrent VTE than patients with no abnormalities detected (OR:4.13, p < 0.02). APC-r and HHcy (any form) were associated in 5.7% of the patients, a figure 2.9-fold greater (p<0.001) than expected based on the prevalence of the isolated abnormalities. These results show that isolated methionine intolerance is a risk fac-

tor for early-onset VTE, and confirm the increased thrombotic risk of patients with association of APC-r and moderate HHcy. The methionine loading test is necessary for detection of hyperhomocysteinaemia. R. van der Griend 1., F.J.L.M. Haas:, M. Duran 3, D.H. Biesma 1, O.J.A.Th. Meuwissen 1, J.D. Banga 4.

Sint Antonius Hospital, Nieuwegein, The Netherlands dept. of Internal Medicine, 2dept. of Clinical Chemistry 3University Children's Hospital 'Het Wilhelmina Kimterziekenhuis', Utrecht, 4 University Hospital, Utrecht, The Netherlands'. Background: Hyperhomocysteinaemia, defined by an elevated homocysteine concentration in the fasting state or after methionine loading, is an independent risk factor for premature atherosclerosis and venous thrombosis. The role of the methionine loading test is, however, controversial. We have, therefore, determined the additional value of a methionine loading test for diagnosing hyperhomocysteinaemia. Methods: We, prospectively, studied 429 persons (281 patients with premature arterial disease and 148 of their first degree relatives) in the outpatient clinic of a general hospital. Total plasma homocysteine (fasting and 6 hours after methionine loading), folic acid, cobalamm, pyridoxine and creatinine concentrations were measured. Hyperhomocysteinaemia was defined as fasting homocysteine concentration and/or increase in homocysteine concentration after methionine loading exceeding the 95th percentile of a healthy control group. Results: Hyperhomocysteinaemia was found in 141 (33°/,,) of 429 persons: 15% diagnosed by an elevated fasting homocysteine concentration and 18% diagnosed by an abnormal methionine loading test. Of the 141 hyperhomocysteinemic persons, 78 (55%) were diagnosed only after methionine loading. Folic acid was lower in the group with an elevated fasting homocysteine concentration (t 1 nmol/L) as compared to those with only an abnormal methionine loading test (15 nmol/L, P = 0.002). Folic acid and creatinine were significantly correlated, negative and positive, respectively, to both fasting and increase in homocysteine concentration. Negative correlations of cobalamin and pyridoxine with fasting homocysteine concentrations were found. Conclusion: The methionine loading test is necessary for diagnosing hyperhomocysteinaemia, because a considerable number of hyperhomocysteinemic persons (55%) remains undiagnosed with the determination of a fasting homocysteine concentration alone. Results of methionine loading tests and vitamin status in young patients with vascular problems in the region of West-Brabant. T. Rammeloo*, T. Noordzij, L.J.M. Spaapen, A. Augustijn.

Dept. ~?/'Clinical Chemistry and Internal medicme, Franciscus Hospital. Roosendaal, Netherlands' and Foundation of Clinical Genetics Limburg. Maastrieht, Netherlands'. Hyperhomocysteinemia (HHcy) is an independent risk factor for premature atherosclerosis. Diagnosis either via basal homocysteine values or by means of a methionine loading test (MLT) is under discussion. Since 1993 standardized methionine loading tests were carried out in 205 patients living in

Abstracts~Netherlands Journal of Medicine 52 (1998) SI $61 Roosendaal and the surrounding area (region West-Brabant). Plasma total homocysteine (tHCy) was measured by a HPLC method equipped with a fluorescence detector using pre-column derivatisation with SBD-F. Plasmas were pretreated with tri-n-butylphosphine in order to release plasma protein-bound homocysteine. Cut-off points of fasting tHcy are: 16.6 and 18.0 /maol/l; those of post-load tHcy: 54.6 and 56.3 ¢tmol/l for women and men, respectively. All patients presented with vascular disease of coronary, cerebral or peripheral origin. In about 17% of the patients HHcy was detected. Of this group the percentage of patients with normal fasting tHcy and increased post-load tHcy was 37%. HHcy was found in 11% of patients with acute myocardial infarction, in 18% of patients with young stroke and in 35% of patients with peripheral vascular disease. Moreover, 4 patients with deep-venous thrombosis and 1 patient with recurrent abortion showed increased tHcy values after methionine loading. In 32 patients with HHcy treated with folic acid (5 mg/day) pre- and post-load tHcy concentrations normalized. The vitamin status in 1530% of these patients revealed decreased folate, vitamin B6 or BI2 values, whereas 15% of these patients had decreased concentrations of 2 or 3 of these vitamins. From our study it is concluded that by assaying only fasting plasma tHcy levels in vascular patients a significant number of hyperhomocysteinemic patients would be missed. In patients with arterial occlusive disease HHcy is observed frequently, sometimes accompanied by low plasma vitamin levels. Treatment of these patients with folic acid appeared to be effective in lowering pre- and post-load plasma tHcy concentrations. Hyperhomocysteinemia is associated with elevation of exitotoxic sulfur amino acids in human CSF. T. Bottiglieri ~'2., K. Hyland:,:, J. Griener3, C. Quinn 3, B. Maddux 2, E. Frohman:, B. Kamen3. Baylor University Medical Center, Institute oJ"Met-

abolic Disease 1, Departments of Neurology 2 and Pediatrics 3, University of Texas South Western Medical Center, Dallas, Texas, USA. Hyperhomocysteinemia may be due to one or more genetic or non-genetic disorders and is a strong independent risk factor for vascular disease including stroke and carotid artery stenosis. In addition, Homocysteine (HCY) may be neurotoxic by way of its metabolism to the excitotoxic sulfur amino acids, homocysteine sulfinic acid (HCSA), homocysteic acid (HCA), cysteine sulfinic acid (CSA) and cysteic acid (CA), that are endogenous agonists of the NMDA receptor. We here report levels of plasma and CSF homocysteine and CSF excitotoxic sulfur amino acids in a 13 year old female (KN) with T-cell lymphoblastic leukemia prior to any antineoplastic chemotherapy, and a 58 year old female (PY) with isolated CNS vasculitis. Both had elevated total plasma HCY concentrations (KN, 22.5 `aM; PY, 52.8 ~M; control range 3 14 `aM), elevated CSF HCY (KN, 1.9 ,aM; PY, 1.8 ,aM; control range 0.28 0.66 ,uM) and decreased CSF methyltetrahydrofolate (KN, 12 ,aM; PY, 6 ,aM; control range 40-150 ,aM). Excitotoxic sulfur amino acids were undetectable in CSF from 16 control subjects but present in large amounts in CSF from the patients KN and PY, particularly HCA (154 and 172

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`aM, respectively) and CSA (36 and 122 `aM, respectively). These findings are in keeping with our previous observations of increased CSF HCY and excitotoxic sulfur amino acids in patients with acute lymphoblastic leukemia treated with the antifolate methotrexate. Increased excitotoxic sulfur amino acids in the central nervous system may play a role in the neurological complications associated with hyperhomocysteinemia. Molecular beacons: a new approach for the semi-automated detection of the C677T mutation in the MTHFR gene. B.A.J. Giesendorf1., J.A.M. Vet2, S. Tyagi 2, J.M.F. Trijbels j, HJ. Blom1. 1University Hospital Nijmegen, dept. of Pediatrics,

P.O. Box 9101, 6500 Nijmegen, The Netherlands," 2public Health Research Laboratory, New York University, dept. o[ Molecular Genetics, New York, USA. Introduction: Mild hyperhomocysteinaemia is a risk factor for vascular disease. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of methylene-THF to methyI-THF, the methyl-group donor for remethylation of homocysteine to methionine. A mutation (C677T) in the MTHFR gene has been identified which alters a highly-conserved amino acid and correlates with reduced enzyme activity and increased homocysteine levels. Current techniques to investigate this mutation are based on the PCR, restriction enzyme analysis and electrophoresis. The method presented here consists of a PCR in which allele-specific fluorescent probes, molecular beacons, are included. The probes undergo a fluorogenic conformational change by hybridisation to their targets and only under this condition emit a fluorescent signal. Methods: Two molecular beacons, differing in only one basepair, were designed to discriminate between wildtypeand mutant target. Both probes were added to DNA amplification samples and fluorescence was monitored by the ABI Prism 7700. Samples were also analysed by conventional techniques. Results: A PCR-probe assay for the detection of the C677T mutation in the MTHFR gene was developed and 45 samples were analysed. Results obtained by this method were in complete agreement with conventional procedures. Conclusion: Molecular beacons are excellently suited for single basepair mutation detection. The method presented here is a fast, automated single-tube procedure for the diagnosis of the MTHFR mutation with a high sample throughput and a reduced risk for cross-contamination. This work is supported by the EU Commission Demonstration Project Contract No. BMH4-CT95-0505 and United States National Institutes of Health grant HL-43521. Quantitation of total homocysteine, total cysteine and cystathionine in human plasma and serum by gas chromatography-mass spectrometry (GC-MS). A.B. Guttormsen*, E. Solheim, P.M. Ueland, H. Refsum. Department of Pharmacology. University

of Bergen, Armauer Hansens Hus. 5021 Bergen, Norway. We have developed a sensitive GC-MS method for measurement of total homocysteine, total cysteine and cystathionine in human plasma and serum. Amino-. carboxyl and sulfhydryl

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Abstracts / Netherlands Journal of Medicine 52 (1998) S1-$61

groups are rapidly derivatized with ethylchloroformate (ECF) and ethanol in an aqueous medium, and the N-ethoxycarbonyl ethyl esters are measured in the S1M mode by GC MS. Deuterated internal standards are used for the three analytes. The assay is linear for cystathionine between 0.05 300/zmol/ L. for total homocysteine and total cysteine in the range 0.3 1000/.tmol/L and 3-1000/zmol/L, respectively. The coefficients of variation (within day and between day) are between 3 and 15% for all metabolites, and recovery about 90%. The sample requirement is 100/tL, total run time about 10 minutes, and the sample throughput 100 samples/24 h. Measurement of plasma S-adenosylmethionine and S-adenosylhomocysteine by a novel fluorescent method. C. Wagner j':*, A. Capdevila:. 1Veterans AdminL~tration Medical Center, Nashville, TN~ USA; 2 Vanderbilt Univ. School of Medicine, Nashville, TN, USA. The levels of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in human plasma are in the nM range which makes their measurement by standard HPLC techniques using ultraviolet absorbance very difficult. We have developed a fluorescent method using naphthalene dialdehyde (NDA) to form the isoindole derivatives of the free amino group of these compounds. The method is sensitive to 5 pmol of SAM and SAH. Both SAM and SAH give identical standard curves. The method consists of deproteinization of plasma with trichloroacetic acid (TCA) followed by extraction of the remaining TCA with trioctylamine and l,l,3-triehlorotrifluoroethane. The sample is first chromatographed by HPLC on a strong cation exchange column for SAM or a C-8 column for SAH in order to remove the majority of interfering compounds. The fractions containing SAM or SAH are concentrated and desalted on an Oasis cartridge (Waters). Samples are derivatized with N D A and CN at pH 8.0 for 10 min at room temperature. The reaction goes to 85% completion. The reaction mixture is then chromatographed on a C-18 column and monitored by fluorescence (Ex 420 nm, Em 483 nm). Specificity of the method was shown by heating plasma at 100 ° for 10 min to destroy SAM. No other fluorescent peak eluted in the same position. The overall recovery is about 65%. The S.E.M. of replicate analyses is about + 8%. We have used this method to obtain normal values for plasma SAM while values for SAH are still in development. Using this method we have obtained mean values of 79.1_+ 3.7 nM for plasma SAM in normal subjects. Values reported by Bottiglieri and Hyland (Acta Scand Supp 1994;154:19 26) range from about 70 to 85 nM obtained using double isotope enzymatic methods. Loehrer et al. (Clin Sci 1996;91:79 86) reported values o f 25 to 40 nM using a method that employs an 8 hr reaction of chloracetaldehyde with SAM at 37°C to form a fluorescent derivative that is then separated by HPLC. The overall time needed to carry out the measurement of SAM by our new method is about 3 hr. (This work was supported by grants DK-15289 and DK-46788 from the U.S. Public Health Service and by the Department of Veterans Affairs).

Treatment and intervention Homocysteine and postmenopausal HRT: is progestagen the determining reducing factor? W.M. van Baal*, P. Kenemans, T. Teerlink, H. Kessel, E.R.A. Peters-Muller, C.D.A. Stehouwer, M.J. van der Mooren. hTstitute Jbr Cardiovascular Research, Vr(]e Universiteit, Project 'Ageing Women', Free University Hospital, Amsterdam, The Netherlands. Introduction: Recent data have shown a reducing effect of hormone replacement therapy (HRT) on homocysteine levels in postmenopausal women. However, these studies lack a placebo-controlled design. Methods: In healthy postmenopausal women aged 45 60 years, we performed a prospective, randomised, double-blind three-months study to investigate the influence on plasma homocysteine levels of: (1) unopposed micronised oestradiol 2 mg daily (n=18) and (2) micronised oestradiol 2 mg daily sequentially combined with either dydrogesterone or trimegestone (total n = 28) versus (3) placebo (n = 15). Results: Four weeks of combined oestradiol/progestagen therapy reduced homocysteine levels by almost 10% (P<0.01), which decrease sustained during the following 8 weeks of treatment. Homocysteine levels did not changes significantly, neither in the placebo group, nor in the oestradiol group. Homocysteine levels decreased significantly in the combined HRT group compared to placebo ( P < 0.01, ANOVA). Conclusion: Combined oestradiol/progestagen reduces plasma homocysteine concentrations in healthy postmenopausal women already after 4 weeks of treatment. Our results indicate a significant role of progestagens in the reduction of homocysteine. Effects of folate supplementation on prevention of neural tube defects and cardiovascular diseases in France: a medico-economic analysis. J. Blacher*, G. Potier De Courcy, C. Silberztein, G. Strauch, G. Duru, M. Safar. Department of Internal Medicine, 1NSERM U 337, Broussais Hospital, Paris, France. Increasing periconceptional folate intakes to 400 ~g per woman and per day leads to an important reduction in the incidence of spina bifida and other neural tube defects, with a high level of proof. In France, this supplementation could avoid nearly 500 neural tube defect births ~ a great proportion of these pathologic pregnancies are detected and prematurely interrupted, with a cost evaluated at French Francs (FF) 13 million. Although public health decisions of supplementing food with folio acid have been taken in several countries, such a decision has not yet been taken in France. Homocysteine is a well-known independent and graded cardiovascular risk factor, the plasma concentration of which decreases with folate treatment. Although no interventional trial proves that this decrease is associated with a reduction in cardiovascular morbidity and mortality, we assessed this hypothesis in our work. The modelisation of homocysteine-attributable cardiovascular risk, on the basis of the less optimistic evaluation, shows that increasing folate intakes up to 500 /.tg 1 mg per day of the entire French population could avoid

Abstracts / Netherlands' Journal cff Medicine 52 (1998) SI-$61 more than 7000 coronary heart disease and cerebrovascular deaths. The gain on the expenses for cardiovascular disease management could reach F F 2.8 billion. In France, increasing folate intakes could be reached, at best, by food supplementation, which could concern flour without any technical, legal or operational diffÉculties. The total cost of this supplementation has been evaluated at F F 20.8 million. By a cost-effectiveness analysis, integrating only the neonatal benefits, each neural tube defect birth avoided would cost F F 16000. If we integrate cardiovascular benefits in this analysis, although they are not as proven as the neonatal ones, we obtain a net benefit that could be more than F F 2.5 billion. In this analysis, as part of future costs and benefits would probably be delayed, the use of discounting has not significantly modified the results. Performed sensitivity analysis only increased the level of confidence of this public health decision which has already been much delayed in France. High daily intake of dietary folate decreases homocysteine and improves folate status: a controlled dietary trial in young healthy volunteers. I.A. Brouwer j,2*, M. van Dusseldorp 1, C.E. West ¢, S. Meyboom, C.M.G. Thomas 24, M. Duran 1, K. van het Hoff, T.K.A.B. Eskes 1, J.G.A.J. Hautvast l, R.P.M. SteegersTheunissen 2~ I Div. o f Hum. Nutr. and Epid.. Wageningen Agrieuhural University, 2Dept. of Obst. and Gyn. and Epid. 3 and the Lab. or Endocrin. and Reprod. 4, Universi O, Hospital St. Radboud, N(]megen, 5Lab. of Metab. Diseases, Wilhelmina Children's Hospital, Utrecht, 6 Unilever Research Laboratory, Vlaardingen, The Netherlands'. We investigated the effects of dietary folate versus folic acid on homocysteine concentrations and the folate status in healthy volunteers (45 women and 21 men, 18~5 yrs), in a 4-week randomised controlled dietary intervention trial with parallel design. At lunch time, all subjects ate their hot meal at our department. Foods for the rest of the day and for the weekend were supplied and taken home. All subjects daily received a basal diet with a similar nutrient composition for all groups. On top of the basal diet, we daily supplied the dietary folate group with natural food rotate, from vegetables (+ 350 g) and citrus fruits (1 piece+200 mL juice), and a placebo tablet (additional folate: 375/.tg/d). The folic acid group daily received extra foods that were naturally low in folate and a folic acid tablet (additional folate: 25 pg/d; additional folic acid: 250 pg/d). The placebo group received the same low folate foods and a placebo tablet (additional folate: 20 pg/ d). At baseline, the geometric mean + SD of the total plasma homocysteine concentration was 10.2 + 1.3 pmol/L. After four weeks of intervention, homocysteine decreased in the dietary folate group (mean 16.8%; 95% CI, 9.8% to 23.8%) and in the folic acid group (20.7%; 13.7% to 27.7%) more than in the placebo group. In the dietary folate group plasma folate concentrations increased by 51.8% (38.6"/, to 65.1%) and in the folic acid group by 48.1% (34.8% to 61.3~/,) more than in the placebo group. Red blood cell folate concentrations increased in the dietary folate group (mean 16.5%; 95('/O C1 7.6% to 25.5%) and in the folic acid group (14.0%: 5.1% to 22.9%) more than in the placebo group. We conclude that additional

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dietary folate, from vegetables and citrus fruit, decreases homocysteine concentrations and increases the folate status in young healthy volunteers. Our data suggest a bioavailability of 65 75% of food folate relative to folic acid. Diagnosis and treatment of homocysteine disease using recombinant homocysteinase. R.M. Hoffman*, M. Lenz, A.W. Perry, Y. Tan. AntiCancer, 7917 Ostrow Street, San Diego, CA 92111, USA. Elevated levels of homocysteine in the urine and blood have been correlated with the development of atherosclerosis and with mortality from existing cardiovascular disease. Even moderate homocysteinemia is now regarded as a risk factor for cardiac and vascular disease. However until now there has been no high throughput assay developed for homocysteine. To develop a widely-available homocysteine diagnostic specifically for determining the amount of homocysteine that is present in biological samples such as urine and blood, a large series of homocysteine-selective recombinant homocysteinases (rHYase) have been cloned in our laboratory. The amount of homocysteine in the sample is measured colorimetricaUy by the amount of hydrogen sulfide produced by rHYase. The total concentration of homocysteine present in biological samples may include homocysteine molecules that are not present in free form, being instead covalently coupled to other molecules. rHYase is active under conditions which allow the reductive cleavage of homocysteine in disulfide linkage releasing bound homocysteine to be measured. Injection o f rHYase in highly homocysteinemic cystathionine-fl-synthase knockout mice reduced the circulating levels of homocysteine by over 90% and in homocysteine-normal cancer patients reduced homocysteine levels by over 50%. Thus, rHYase allows the possibility of a rapid, simple, widely-available diagnostic for homocysteine disease and of a therapeutic for the large numbers of patients with homocysteine disease that do not respond to vitamin intake. Normohomocysteinemia and vitamin-treated hyperhomocysteinemia are associated with similar risks of cardiovascular events in patients with premature peripheral arterial occlusive disease. A prospective study. S.C. de Jong 1':*, C.D.A. Stehouwer 1,3, M. van den Berg 1"2, T.W. Geurts 2, J.A. Rauwerda j,-~. Academiseh Ziekenhuis Vrije Universiteit, Amsterdam, The Netherlands'. l lnstituteJbr Cardiovascular Researeh, 2Dept. of Surgery, Division q]' Vascular Surgery, 3Dept. of Internal Medicine. Mild hyperhomocysteinemia (HHC), fasting or after methionine loading, is associated with an increased risk and severity of atherosclerotic vascular disease. Post-methionine and fasting HHC are responsive to treatment with vitamin B6 and folic acid. We investigated the clinical efficacy with regard to the incidence of cardiovascular events of treatment of mild HHC with vitamin B6 (250 mg) and folic acid (5 mg) in patients with premature peripheral arterial occlusive disease and post-methionine HHC. We studied 273 consecutive patients with clinically manifest peripheral arterial occlusive disease with onset before the age of 56, 79 (28.9%) of whom had post-methionine HHC. Follow-up

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Abstracts/Netherhmds Journal of Medicine 52 (1998) S1-$61

was obtained in 232 (85%) patients. At baseline, 70 (30%) were hyperhomocysteinemic after methionine loading and started treatment with vitamin B6 and folic acid; 162 (70%) were normohomocysteinemic (untreated; reference group). During the follow-up period (median 20; range 1 to 63 months), 48 (29.6%) and 23 (32.9%) of the normo- and the hyperhomocysteinemic patients, respectively, had a new cardiovascular event. Most (75%) involved the peripheral arterial system. The crude incidence rate for any cardiovascular event was 0.16 (95% CI, 0.12 to 0.21) per person-year in the normoand 0.16 (95% CI, 0.09 to 0.22) per person-year in the hyperhomocysteinemic group. Multivariate Cox-regression analyses showed that higher plasma homocysteine levels were associated with an increased risk of new cardiovascular events in the normohomocysteinemic patients (relative risk [RR] per 1 pmollL, 1.17 (CI, 1.05 to 1.30) for fasting and 1.06 (CI, 1.01 to 1.12) for post-methionine levels), but not in the hyperhomocysteinemic (vitamintreated) patients. The adjusted RR for new cardiovascular events in the hyper- as compared to the normohomocysteinemic patients was 0.76 (CI, 0.33 to 1.74). These data are consistent with a protective effect of treatment with vitamin B6 and folic acid in patients with premature peripheral arterial occlusive disease and post-methionine HHC. Double-blind randomized trials are necessary to confirm this. Effect of low dose and high dose riboflavin (RBF) supplementation on plasma homocysteine (hcy) levels: a pilot study. M.C. McKinley*, H. McNulty, J. McPartlin t, J.J. Strain, S. Madigan, D.G. Weir ~, J.M. Scotte. Northern Ireland Centre for

Research into Diet and Health, University of Ulster, Coleraine. UK, and Departments of 1Clinical Medicine and 2Biochemistry, Trinity College Dublin, Ireland. It is probable that elevated plasma hcy is an independent risk factor for coronary vascular disease and stroke. Studies have shown that the status of pyridoxal phosphate (PLP), vitamin Bl2, and folate are inversely related to plasma hcy level and supplementation with folic acid, either alone, or in combination with vitamins B6 and Biz, can significantly lower plasma hey. The active coenzyme forms of RBF, flavin mononudeotide (FMN) and flavin adenine dinucleotide (FAD), are also essential in hey metabolism. FMN is required for the formation of PLP, which in turn is required for cystathionine-fl-synthase activity. [n addition, RBF in the form of FAD acts as a prosthetic group for methylenetetrahydrofolate reductase (MTHFR). Despite this, to date, little attention has been devoted to the role of RBF as a potential hcy-lowering agent. The aim of this pilot study was to determine the effect of RBF supplementation on plasma hcy levels. Subjects (aged 65 years and over) were randomly assigned to one of three intervention groups to receive either 1.6 mg RBF or 25 mg RBF, or placebo, daily for a period of 12 weeks. Available baseline and post-intervention blood samples (n = 29) were compared (paired I test) to examine responses of both hey and RBF to supplementation. For the sample as a whole, only the high dose intervention group showed a sig-

nificant increase (P = 0.049, n = 8) in RBF status (i.e. a decrease in erythrocyte glutathione reductase activation coefficient (EGRAC)), with no corresponding change in plasma hcy (not shown). Individuals who were classified as biochemically deficient in RBF (EGRAC> 1.20, n= 16) at baseline were examined separately (see table). In these subjects there was a significant improvement in RBF status at both levels of supplementation, but no corresponding change in the plasma hcy level. Plasma hcy levels in those who were biochemically deficient in RBF were not significantly different from RBF replete individuals (independent t-test, 15.04+ 8.01, 12.74 + 3.03, respectively, P = 0.203). EGRAC values. Placebo (n=6): Pre 1.29 (0.08), Post 1.23 (0.10) P value 0.106; Supplementation (n = 10): Pre 1.26 (0.05), Post 1.11 (0.05), P value 0.000; Supplementation 1.6 mg/d ( = 7): Pre 1.27 (0.05), Post 1.13 (0.04), P value 0.003; Supplementation 25 mg/d (n = 3): Pre 1.24 (0.05), Post 1.07 (0.05), P value 0.051. H C Y values (Imml/1). Placebo (n = 6): Pre 18.70 (12.2), Post 23.81 ( 1 2 . 0 ) P value 0.051; Supplementation (n=10): Pre 12.84 (3.15), Post 12.68 (1.99), P value 0.413; Supplementation 1.6 mg/d (=7): Pre 12.66 (3.25), Post 12.86 (2.36), P value 0.396; Supplementation 25 mg/d (n = 3): Pre 13.26 (3.54), Post 12.26 (8.05), P value 0.313. Results of this pilot study show that plasma hcy levels do not appear to be raised in association with suboptimal RBF status nor lowered when RBF status is improved by supplementation. This suggests that RBF is not a limiting nutrient in homocysteine metabolism in the elderly, but confirmation of this result is under further investigation. Hyperhomocysteinemia in vascular disease; detection and consecutive treatment. L.J.M. Spaapen*, K. Hamulyzik. Founda-

tion oJ Clinical Genetics, and Dept. of Internal Medicine, Academic Hospital Maastricht. P.O. Box 1475, 6201 BL Maastricht, The Netherlands. During the last decades numerous studies have established that mild or moderate hyperhomocysteinemia (HHcy) is associated with premature occlusive vascular disease. Investigation of the homocysteine metabolism is part of the work-up of patients with occlusive vascular disease. From 1989 to 1997 more than 2000 patients were investigated by means of a standardized methionine loading test (100 mg/kg body weight). Blood sampling occurred in fasting state and 6 hours post methionine loading. Plasma total homocysteine (tHcy) was analysed by HPLC equipped with a fuorescence detector using pre-column derivatization with SBD-F. HHcy was present in about 27%, of the patients tested, independent of the clinical presentation. Three categories of HHcy patients were found: (1) patients with increased fasting tHey and normal post-load tHcy values (+20%), (2) patients with normal fasting tHey and elevated post-load tHcy levels (+ 39%), (3) patients with both fasting and post-load tHcy levels elevated (+41%). Treatment of HHcy patients occurred in a consecutive way independent of the type of hyperhomocysteinemia and was started with 5 mg folic acid daily. In case of no or insufficient normalization of the tHcy levels pyridoxine (10-50 mg/day) was

Abstracts~Netherlands Journal of Medicine 52 (1998) $1 $61 added to the medication. A few patients with a post-load HHcy did not respond to either folate or folate+pyridoxine. These patients who had normal vitamin B12 levels, were successfully treated with betaine (6 g/day). In categories (1) and (3) of HHcy patients more than 70% appeared to be responsive to folate monotherapy. Of the patients with only a post-load hyperhomocysteinemia more than 40% was able to normalize the post-methionine load tHcy level upon folate monotherapy. Conclusions: (a) The vitamin status of vascular patients is a main determinant of HHcy. (b) Three types of HHcy are equally distributed among patients presenting with vascular disease of coronary, cerebral or peripheral origin. (c) Postload HHcy is not merely determined by the vitamin B6 related transsulfuration pathway, involvement of the homocysteine remethylation pathway may be important in the aetiology of post-load HHcy. The VITRO trial: study design. H.P.I. Willems 1., W.B.J. Gerrits1, F.R. Rosendaal:, M. den Heijev~, H.J. Blom4, G.M.J. Bos5. 1Dept. of Haematology, Leyenburg Hospital,

The Hague, 2Dept. of Epidemiology, University Hospital Leiden, University Hospital St Radboud, Nijmegen, The Netherlands. 3Dept. of Internal Medicine, 4Dept. of Paediatrics SDept. ~" Haematology, Dr. Daniel den Hoed Clinic, Rotterdam, The Netherlands. The 'Vitamins and Thrombosis' study (VITRO) is a doubleblind, randomized and placebo-controlled trial in which the effect of 5 mg folic acid, 400 /tg hydroxocobalamin and 50 mg pyridoxine daily as secondary prevention of deep vein thrombosis and pulmonary embolism is studied. Patients are selected by the anticoagulation services of 5 major cities in the Netherlands. From every patient with a first episode of venous thromboembolism blood is taken for determination of the total homocysteine concentration. 300 patients with a normal (i.e. < 17.7/trnol/1) and 300 patients with an elevated homocysteine level will be recruited. Inclusion criteria: (1) an objectivated venous thrombosis (compression ultrasonography, venography in DVT, high probability VQ scanning or angiography in pulmonary embolism), (2) a spontaneous venous thrombosis (no major trauma or surgery, no malignancy, no pregnancy and no immobilisation prior to the thrombosis), (3) age between 20 and 80 years. Exclusion: use of vitamin B or medication which influences homocysteine metabolism. After informed consent, the trial medication is started at least 4 weeks before the oral anticoagulants are stopped. The follow up period is 2.5 years. End-points are an objectivated recurrent deep-vein thrombosis or recurrent pulmonary embolism. The trial started in 1996. Interim data of the screening will be presented at the congress.

Regulation of metabolism Is homocysteine metabolism hormonally regulated? Studies in the streptozotocin-induced diabetic rat. R.L. Jacobs*, L.M.

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Stead, J.D. House, M.E. Brosnan, J.T. Brosnan. Dept. of Bio-

chemistry, Memorial University, St. John's, NF, Canada. Elevated concentrations of total plasma homocysteine (Hcy) are known to be an independent risk factor for the development of vascular disease. Patients with diabetes mellitus have also been reported to have alterations in Hcy metabolism. Diabetes patients who have clinical signs of kidney dysfunction exhibit elevated total plasma Hcy levels, whereas type 1 diabetic patients with no clinical features of kidney dysfunction have a tendency for decreased plasma Hcy concentrations. The purpose of this study was to investigate Hcy metabolism in a type 1 diabetic animal model, to determine whether insulin is involved in its regulation and if so to elucidate the mechanism of hormonal regulation. Diabetes was induced by a single dose of 100 mg/kg streptozotocin (iv.) to Sprague Dawley rats. One group of diabetic rats received insulin for 5 days followed by saline for 5 days (untreated) while the second diabetic group received insulin injections for 10 days (treated); in addition we examined a saline treated control group. We observed a 30% decrease in plasma Hcy in the untreated-diabetic rats. This reduction in Hcy was prevented when diabetic rats received insulin. We also observed an increase in the activity of the hepatic transsulfuration enzymes (cystathionine fl-synthase and cystathionine ~,qyase) in the untreated-diabetic rats. Insulin treatment normalized the activities of those enzymes in the diabetic rat. There was a significant correlation between plasma Hcy and the hepatic activity of both enzymes of the transsulfuration pathway. In separate experiments we observed that the normalization of enzymatic activity was achieved after 1 day of insulin treatment. Congruent studies have also shown that glucagon decreased homocysteine export in an in vitro hepatocyte preparation. These results suggest that insulin and/or its counterregulatory hormones are involved in the regulation of plasma homocysteine concentrations by affecting the hepatic transsulfuration pathway, which is involved in the catabolism of homocysteine. Supported by: The Canadian Diabetes Association and the Medical Research Counsel of Canada Decrease of Plasma Total Homocysteine after Oral Contraception. J. Dvorfikovh 1., E. Bocanovfi e, J. Hyfinek 1, J. Stribrny~.

Dept. Clin. Biochemistry -- Metabolic Unit 1, Dept. o/ Gynaecology2 Hospital Na Homolce, Prague, Czech Republic. Premenopausal women have lower levels of total plasma homocysteine (tHCy) than postmenopausal women or men - in the fasting state as well as after methionine loading. In pregnant women the average concentrations of homocysteine were detected lower than in non pregnant same aged women. In 32 young, healthy women (age: 19.6 _+2.6 years) the total plasma homocysteine, vitamin B12 and folate before use and after 3 months of using oral contraceptives (OC) of third generation have been followed. Total homocysteine levels in plasma (before use: 9.52_+ 2.66/.tmol/1, after 3 months: 7.25 _+2.04 pmol/l) were significantly lower after 3 months of using OC (p < 0.0005). There was no significant difference in the levels of folate. The levels of vitamin B12 have significantly fallen from 355.1 + 130.3 to 303.9+ 113.97 pmol/1 (p< 0.05).

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Abstracts / Netherlands Journal q[' Medicine 52 (1998) SI-$61

Our results do not fit with the findings of Steegers-Theunissen (1992), and do not support the hypothesis that mild hyperhomocysteinemia explains cardiovascular risk in women using oral contraceptives. Reference: Steegers-Theunissen RPM, Boers GHJ, Steegers EAP, et al. Effects of sub-50 oral contraceptives on homocysteine metabolism: a preliminary study, Contraception 1992 ;45:129-139. Homocysteine: influences of menopausal status and postmenopausal hormone replacement therapy. M.J. Van der Mooren. Department o f Obstetrics and Gynaecology, Project 'Ageing Women', Academic Hospital Vrije Universiteit, Amsterdam. The Netherlands. Introduction: Mild hyperhomocysteinaemia is an independent risk factor for cardiovascular disease. The relation with menopause and hormone replacement therapy (HRT) is still poorly understood. A summary will be given of the latest data obtained from studies that investigated differences in plasma homocysteine concentrations between pre- and postmenopausal women, and changes in homocysteine concentrations during several regimens of postmenopausal HRT. Methods: Homocysteine metabolism was tested in healthy premenopausal (N= 46) and postmenopausal (N---26) women by methionine loading test. Further, in healthy postmenopausal women fasting homocysteiue concentrations were measured during three different HRT-regimen: oral sequential oestradiol/dydrogesterone therapy given for two years (N= 21), oral sequential conjugated oestrogen/medrogestone therapy given for 6 months (N = 39), and transdermal oestradiol-only therapy in two dosages (50/2g: N= 17; 80 /,tg: N= 15) given for 7 months. Results: Postmenopausal women showed 20% higher fasting {P < 0.05), and 44°/,, higher after-load (P < 0.05) mean homocysteine concentrations than premenopausal women. Fasting homocysteine decreased during HRT, especially in those women with high homocysteine levels. Plasma homocysteine concentrations were reduced by 8.0% (P < 0.01) during oestradiol/ dydrogesterone and by 9.5% (P < 0.01) during conjugated oestrogen/medrogestone. Probably due to small numbers, reductions in plasma homocysteine during transdermal oestradiol replacement did not reach statistical significance. Conclusions: The observed differences in homocysteine concentrations between pre- and postmenopausal women, and their reduction during HRT may well in part explain the reported high incidence of cardiovascular disease in postmenopausal women who do not use hormone replacement. Abnormal sulfur-amino acid metabolism in the curly-tail (ct) mouse, a model of neural tube defects (NTD). P. Tran 1.. W. Wang ~, N. Sabbaghian 1, T. Bottiglieri -~, J. Selhub4, J. Trasler 12, R. Rozen*. McGill University. Montreal. Canada, 1DtTts. of Human Geneticw/Paediatrics, 2Dept. o['Pharmacology and Therapeutics, 3Bavlor University Medical Centre, Dallas, TX, USA, 4Human Nutrition Research Centre, Tt4/Ls"University, Boston, MA, USA.

Approximately 70% of NTD are preventable by periconceptional folate supplementation. A combination of low folate status with a homozygous 677C---,T mutation in methylenetetrahydrofolate reductase (MTHFR), which converts 5,10methylenetetrahydrofolate to 5-methyltetrahydrofolate, is a risk factor for NTD. Since this mutation accounts for only a portion of folate-preventable NTD, the search for other folatedependent mechanisms continues. Among the animal models of NTD, the ct mouse most resembles the human defect. Sixty percent of embryos are affected phenotypically with variable expressivity. Previous work has shown that a folate-/methyldeficient diet increases the incidence of tail flexion defects in ct mice. The objective of this study was to examine the biochemical effects of inadequate dietary folate/methyl groups on folate metabolism in ct mice and in control C57BI/6J mice. Following eight weeks of administration of a moderately folate-/methyldeficient diet to adult (male) mice, we assayed plasma and tissue levels of folate and related metabolites (homocysteine, SAM/SAH ratios, glutathione, etc.), MTHFR activity, methionine synthase (MS) activity, and methylation status. Metabolic differences between the two strains were found on the control diet and in response to the folate-lmethyl-deficientdiet. On the control diet, ct mice had higher levels of plasma homocysteine (p =0.07), liver MTHFR activity (p =0.05), liver MS activity (p = 0.009), but lower levels of plasma glutathione (p = 0.04). In response to a moderately folate-/methyl-deficient diet, both strains showed increased plasma homocysteine levels, but ct mice maintained higher levels of homocysteine (p = 0.03), liver MTHFR activity (p=0.0l), liver MS activity (p=0.09) and lower levels of glutathione (p = 0.0007). In addition, ct mice showed significantly lower levels of liver SAM/SAH ratios (p=0.008). Since the sulfur atom of glutathione is derived from methionine, we propose that the lower glutathione levels in ct mice result in upregulation of homocysteine remethylation (MTHFR and MS activity). This aberrant methionine/ glutathione metabolism may contribute to the pathogenetic mechanism for NTD in ct mice. Methionine concentration in blood is not affected by mild remethylation defects or vitamin supplementation. D.F.D. van de Gevel ~, A.A.J. van Landeghem:, H.J. Bloms, M. den Heijer/*. JDept. of Internal Medicine, TweeSteden Hospital, Tilburg; ZDept. of Clin. Chemistry and Haematology, St. Elisabeth Hospital, Tilburg and 3Lab. of Paediatrics and Neurology, University Hospital Nijmegen, The Netherlands. Low folate levels, low vitamin Bi2 levels and mutated methylenetetrahydrofolate reductase (MTHFR) are associated with hyperhomocysteinemia, which is a risk factor for cardiovascular disease and neural tube defects. Data from in-vitro experiments suggest that low methionine levels are associated with neural tube defects. We studied the relation between low folate or low vitamin B~2 levels, mutated MTHFR and methionine concentrations in blood and the influence of supplementation of several vitamins on methionine levels. A total of 230 healthy volunteers were randomised to placebo, 0.5 mg folic acid, 5 mg folic acid, 0.4 mg vitamin B12 or multivitamin (containing 5 mg folic acid, 0.4 mg hydroxo-

Abstracts / Netherlands Journal of Medicine 52 (1998) S1-$61 cobalamin and 50 mg pyridoxine). Before and after supplementation methionine concentrations were measured. The mean methionine level before intervention was 28.0 (95% CI 26,0 to 30.0)/.tmol/L in subjects with low folate levels, 27.6 (95% CI 25.6 to 29.5)/zmol/L in subjects with low vitamin Bl2 levels, 29.4 (95% CI 27.3 to 31.5)/~mol/L in subjects with mutated MTHFR and 28.7 (95% C! 28.1 to 29.3)/,tmol/L in the whole group. After supplementation methionine was 0.4 (95% CI -0.7 to 1.5) /lmol/L increased in the multivitamin group and 0.3 (95% CI -1.3 to 2.0)/.tmol/L decreased in the group receiving 5 mg folic acid. We concluded that mild remethylation defects (such as mutated MTHFR and low folate levels) and vitamin supplementation do not affect the methionine concentration in healthy volunteers. Human serum proteins contain bomocysteine bound via amide bonds. H. Jakubowski. Department o f Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark. NJ 07103, USA. Pathological consequences of elevated levels of homocyst:in: (Hey) can be due to incorporation of Hcy into protein by translational and/or post-translational mechanisms (Jakubowski, H. J Biol Chem 1997;272:1935). Here, the levels of translational and post translational incorporation of Hcy were determined in samples of dithiothreitol-reduced serum proteins (to remove Hcy bound via disulfide bonds) by acid hydrolysis under reducing conditions followed by HPLC and by automated Edman degradation. Acid hydrolysis would yield total Hey (incorporated translationally and post-translationally) whereas Edman degradation would yield posttranslationally incorporated Hcy, if present. Up to 8 /tM and < 1 /zM translationally incorporated Hey was found in serum proteins from subjects with homocysteinuria and controls, respectively. Serum proteins from all subjects contained < 1 ,uM post-translationally incorporated Hey. Control experiments have shown that proteins containing post-translationally incorporated Hey (prepared by in vitro homocysteinylation of the amino groups of protein lysine residues with Hcy thiolactone) quantitatively yield Hey in the first cycle of Edman degradation. These data suggest that serum proteins from homocysteinurics contain elevated levels of translationally incorporated Hcy. (Supported by the NSF grants MCB9600522 and MCB-9533127.) Pyridoxine hydrochloride (vitamin B6) administration decreases the concentration of serum folate in healthy subjects. M.A. Mansoor1., C.J. Bates:, K.D. PentievaS, O. Kristensenj. IDivision o f Cliniea/ Chemistry, Central Hospital in Rogaland, 4003 Stavanger Norway. 2Dunn Nutrition Centre, Cambridge, UK. Hyperhomocysteinemia is a risk factor for vascular disease and is usually treated with folic acid solely or in combination with vitamin B6 or vitamin BI2. Recently, we observed that vitamin B6, 120 mg given orally to healthy individuals for 5 weeks caused a significant decrease in the concentration of serum folate (p = 0.02).

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In the present study we gave 400 mg vitamin B6 orally to 13 healthy subjects (6 males and 7 females) and collected blood samples before and 30 min, 1 hour, 2, 4 and 6 hours after vitamin B6 administration. The mean concentrations of serum folate were altered (p= 0,051, Friedman test) and there was a significant decrease in the mean concentration of serum folate after 6 hours (12.9 nmol/L vs. 11.8 nmol/L, p = 0.02, Wilcoxon signed rank test). The concentrations of erythrocyte folate peaked after one hour and decreased later on, the change was significant (p = 0.04, Friedman test). There was not a significant decrease in the mean concentration of erythrocyte folate after six hours ( P > 0.05) however the mean concentration of erythrocyte folate after six hours was significantly different than the peak concentrations after one hour (p=0.02, Wilcoxon signed rank test). The findings of this study indicate that concentrations of serum folate decrease and concentrations of erythrocyte folate alter in healthy subjects during oral doses of 400 mg vitamin B6. Plasma and red cell folate, homocysteine and coronary disease. J.S. Silberberg1., R. Crooks 1, J.L. Fryer l, X.W. Guo:, L. Xie2, N.P.B. DudmanS. 1John Hunter Hospital and University of Newcastle, Australia: 2Prince Henry Hospital & University of NSW, Australia. Homocysteine levels are higher in those with the MTHFR 677tt genotype and fall with folate supplementation but most studies have found no association between the tt polymorphism and CHD. We examined the relation between plasma and red cell folate and total homocysteine (tHcy) in a population-based case--control study of CHD. We enrolled 277 cases with a MONICA diagnosis of MI, 118 population controls and also 51 clinic cases and 155 sundry volunteers. Overall, tHcy levels were higher in cases but in stratified analysis the difference was confined to women, tHcy was correlated with plasma folate (r = 0.23, p < 0.0001 ) and to a lesser extent with red cell folate (r = 0.15, p < 0.001 ). Plasma folate was associated with CHD independent of tHcy (OR 0.95//:g/L, p <0.005), but red cell folate was not (p = 0.8). In a subgroup of 190 subjects (upper and lowermost deciles of tHcy distribution) we examined the relation between the MTHFR c677t polymorphism and tHcy. tt Subjects had higher tHcy (21(5)/:tool/L, compared with 13(7I) for cc) and lower plasma folate (15(6)/.tg/L vs. 21(I l) for cc) but red cell folate was not lower (tt: 1083(480)/:g/L, cc: 972(345)). The ratio of red cell to plasma folate was higher in those with tt genotype. Similar findings have been reported by Molloy et al. (Lancet 1997;349:1591) and v.d. Put et al. (Lancet 1995;346:1070). We caution that there may be differences between red cell and plasma folate in regard to association with CHD. The link between genetic susceptibility to low folate, raised they and CHD is not consistent and it remains possible that low plasma folate is a marker of otherwise poor diet. Controlled trials of folate treatment in those with CHD and raised tHey are required before embarking on folate fortification of foodstuffs for the general population.

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Cystathionine [3 synthase Risk factors for neural tube defects: analysis of common genetic variants of methylenetetrahydrofolate reductase and cystathionine ~-synthase. De Franchis R 1., A. Buoninconti 1, C. Mandato 1, G. Sebastio 1, M.P. Sperandeo ~, V. Capra:, P. De Marco:, R. Ricci~, E. Salvaggio 3, A. Iolascon 4, R. Del Gado s p. Mastroiacovo s, G. Andria 1. 1Dept. o f Pediatrics. Federico

H University, via S. Pansini, 80131 Naples, Italy; 2Dept. of Neurosurgery, G. Gaslini Inst., Genoa; 3Dept. ~ Pediatrics, Catholic University, Rome," 4University oJ Bari; 5Second University, Naples. Italy. Moderate hyperhomocysteinemia (HHC) is considered a risk factor for Spina Bifida (SB). The association between moderate HHC and the thermolabile variant of the methylenetetrahydrofolate reductase (MTHFR), due to the 677C ~ T mutation of the gene, has been established as well. We studied the frequency of the 6 7 7 C - , T mutation in 216 SB Italian patients and in 552 suitable controls, representative of the entire country. In Italy, the frequency of homozygosity (+/+) for the 6 7 7 C ~ T mutation in controls (15.8%) is one of the highest in the world so far reported. Among the 216 SB patients, 56 were homozygotes (25.9%) thus suggesting that in Italy the M T H F R mutation is a risk factor for SB (OR = 1.87; CI = 1.26-2.79), as already found in other populations. Cystathionine ~-synthase (CBS) deficiency is another main cause of HHC. Heterozygosity for the CBS 844ins68 (Ins+), found in 8.6% of the 552 Italian controls, is not significantly higher in our SB patients (9.2%; O R = 1.10; 95% CI=0.61 1.96). However, the co-existence of the two mutations ( 6 7 7 C - , T and 844ins 68) in the Italian SB patients might increase the risk for SB (see below). This result is in agreement with the re-analysis of the data observed in Ireland by Ramsbottom et al. (OR = 5.2; 95% CI 1.4-21.2, as re-calculated). Results: M T H F R +/% Ins +, Cases (n=216) 7, Controls (n=552) 6, OR 3.37, CI 195%) 0.95 12.30; M T H F R +/+, Ins --, Cases 49, Controls 81, OR 1.74, CI 1.15 2.66; M T H F R +/ - ; - / - , Ins +, Cases 13, Controls 41, OR 0.91, CI 0.45 1.82; M T H F R + / - ; - / - , Ins - , Cases 147, Controls 424, OR Ref. The financial support of Telethon-Italy (Grant No. E. 439) is gratefully acknowledged. Unstable CBS mRNA resulting from two mutations in cis: direct sequencing of the entire coding region in genomic DNA samples. Janogik M r*, Sokolovfi jl, Mugkovfi B l, Kraus jp2, Ko2ich

V I . Ilnstitute fiJr Inherited Metabolic Diseases, 1st Faculty of Medicine, Prague, Czech Republic; 2Dept. Pediatrics, Universit), of Colorado School o/Medicine, Denver, CO, USA. Methods: Eighteen CBS exons with at least 80 bp of flanking intronic sequences were PCR amplified using 15 primer pairs with universal T7/RP overhangs. Purified PCR products were subjected to cycle sequencing with FS polymerase and run on ALFExpress automated sequencer. The ratio of both alleles of fibroblasts cytosolic and nuclear R N A was quantified by radioactive RT PCR, PAGE and PhosphorImager analysis.

Patient: The patient is a 13 year old Czech male with severe B6 non-responsive CBS deficiency, who exhibits very low methionine tolerance (total Hcy 60-100 ,umol/1 at intake of 7 mg Metlkg/d). Results: (A) System for PCR amplification and DNA sequencing has been developed for 18 CBS exons and flanking intronic regions, with average readout of 350M00 bp. (B) Mutations: Maternal allele (MA): carries a common splicing mutation AI224-2C which leads to exon 12 skipping, Paternal allele (PA): contains two mutations in cis- a transition in intron 7 splice donor (Gszs+IA), and a deletion of 99 bp affecting three tandem repeats in intron 11. We have demonstrated, exclusively in the nuclear RNA, that the Gszs+IA mutation activates a cryptic donor site 104 bp downstream from the exon 7/intron 7 junction and leads to a frameshift with premature stop codon (1277fs, 296X). The product of the PA is abnormally processed already in the nucleus (PA/MA ratio is 0.4/1) and this disturbance results in complete absence of the paternal allele in the cytosolic mRNA. Conclusions: (A) We established a system for direct sequencing of all exons of the CBS gene which is capable of detecting mutations in virtually any tissue, with high speed and accuracy. (B) We describe two mutations linked in cis (Gs28+IA and 1223+37de199, respectively) which result in abnormally processed or unstable CBS mRNA. This illustrates the necessity of analyzing both DNA and RNA in patients with homocystinuria. Homozygous cystathionine-[~-synthase deficiency, combined with factor V Leiden or thermolabile methylenetetrahydrofolate reductase in the risk of venous thrombosis. L.AJ. Kluijtmans 1., G.H.J. Boers2, B. Verbruggen -~,4, J.M.F. Trijbels l, I.R.O. Novfikovfi3, H.J. Blom. Depts. of Pediatrics j, Internal Medi-

cine 2, and Haematology 3, Central Laboratory .[br Haematology 4, University Hospital St Radboud Nijmegen, the Netherlands'. Introduction: Severe hyperhomocysteinemia in its most frequent form, is caused by a homozygous deficiency o f cystathionine fl-synthase (CBS). A major complication in CBS deficiency is deep venous thrombosis or pulmonary embolism. A report by Mandel et al. iN Engl J Med. 1996;334:763 768) postulated factor V Leiden (FVL) to be an absolute prerequisite for the development of thromboembolism in patients with severe hyperhomocysteinemia. Methods: We studied 24 patients with homocystinuria due to homozygous CBS deficiency from 18 unrelated kindreds for FVL and for the 677-~T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, and investigated their possible interaction in the risk of venous thrombosis. Results: FVL, in heterozygous state, was detected in three individuals all belonging to the same kindred. The homozygous 677C---,T mutation was observed in five homocystinuria patients. Thrombotic complications were diagnosed in six patients, of whom only one was carrier of FVL. On the contrary, thermolabile M T H F R caused by the 677C--* T mutation, was observed in three out of the six homocystinuria patients who had suffered from a thromboembolic event.

Abstracts/Netherlands Journal of Medicine 52 (1998) S1-$61

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Conclusion: These data indicate that FVL is not an absolute prerequisite and probably not even a major determinant of venous thrombosis in homocystinuria, but, interestingly, thermolabile M T H F R may constitute a concomitant significant risk factor for thromboembolic complications in this inborn error of methionine metabolism.

and fragment libraries of both clones constructed. D N A sequencing of the entire gene has been completed and exon/ intron junctions determined. The sequence comprises 27 938 nucleotides. The CBS gene sequence will be helpful in studies of normal and mutant CBS and in accurate detection of carriers.

Functional suppression of disease causing human cystathionine~synthase mutations in yeast. W.D. Kruger*, Xiaoyin Shah. Division of Population Science, Fox Chase Cancer Center, Philadelphia, PA 19111, USA. Mutations in cystathionine fl-synthase (CBS) are known to cause homocystinuria, a recessive disorder which results in extremely elevated plasma homocysteine levels that mainly affect ocular, skeletal, vascular, and central nervous systems. Using a previously developed yeast system, we have identified a new mutation in human CBS which can restore the enzyme activity of several different CBS mutations. This suppressor mutation, GI385A (W 409 OPA), results in the truncation of 145 amino acids at the C-terminus o f CBS. In vitro enzyme activity data demonstrate that both wild type and mutant CBS protein lacking in this region are significantly more active, indicating that the C-terminus serves as a negative regulatory domain. Furthermore, our data show that the C-terminus is also necessary for regulation by S-adenosylmethionine (SAM), an allosteric affecter. These results not only suggest that most mutations found in homocystinurics inhibit the activation of the enzyme without dysfunction of the catalytic domain, but also that the mutant forms of enzymes can be activated by C-terminal truncation. These findings may have important implications in the treatment o f homocystinuria.

Mutation C677T in the MTHFR gene and polymorphism 844ins68bp in the CBS gene: risk factors for peripheral arterial occlusive disease? O r e n d ~ M ~*, Mu~kovfi B l, Richterovfi F 1, Zv~irovfi J:, Stefek M 2, Zaykov~. E 3, Stfibn~' js, Hy~inek j3, Kraus jp4, Ko2ich V ~. tlnstituteJor Inherited Metabolic Diseases, 1st Faculty of Medicine, Prague, Czech Republic'," 2EuroMISE Center, Charles University and Academy of Sciences, Prague, Czech Republic; 3Dept. Clinical Biochemistry, Hospital Na Homoh'e, Prague, C2ech Republic; 4Dept. Pediatric% University of Colorado School ¢~["Medicine, Denver, CO, USA. Aim: Analysis of the role of C677T M T H F R mutation and 844ins68bp CBS polymorphism in the development o f peripheral arterial occlusive disease (PAOD) in Czech patients who did not exhibit major conventional risk factors for atherosclerosis. Methods: We performed an unmatched case-control study; patients (n = 162) and controls (n=400) were genotyped by PCR/RFLP. The data were evaluated statistically at 5% level of significance. Results: (A) M T H F R : We found slightly higher prevalence of T/T genotype in patients (11.8%) compared to controls (10.2%); this difference is non-significant (p < 0.67). Similarly to other observations, higher mean plasma fasting homocysteine levels were found in patients with the T/T genotype compared to patients with either C/C or C/T genotypes (p < 0.08). (B) CBS: Prevalence of wt/ins genotype in patients (16.8%) was higher than in controls (11.0%), and was close to the 5% level of significance (p < 0.07). There was no significant difference in plasma fasting homocysteine and cysteine levels between patients with wt/wt genotype and those with wt/ins genotype (p<0.72). (C) MTHFR/CBS genotype combinations were analyzed by Fisher's exact test, and significant difference in prevalence of individual genotype combinations in patients and controls was observed (p<0.03). This was attributable especially to the decrease in number of controls with wt/ins CBS and C/T M T H F R genotype combination, and its increase in patients. Conclusions: (1) The data suggest that the T/T M T H F R genotype is not a risk factor for PAOD (OR=I.17, C.I. 0.66-2.09). (2) The 844ins68bp/wt CBS genotype may be an additional risk factor for PAOD (OR = 1.63, C.I. 0.97 2.74). (3) The study suggests that certain MTHFR/CBS genotype combinations may be more prevalent in patients with PAOD compared to healthy controls.

Gene organisation of human cystathionine ~-synthase. J. Oliveriusovfi I'2., E. Kraus 1, J. Sokolovfi:, (~. Vl6ek s, R. de Franchis l, G. Bukovskfi 1, D. Patterson 4, V. Pa6ess, V. Ko2ich 2, J.P. Kraus 1. I Departments of' Pediatrics and Cellular and Structural Biology, University of Colorado School of Medicine, Denver, CO 80262, U.S.A., elnstitute of Inherited Metabolie Disorders, Charles University, Prague, Czech Republic, 31nstitute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic, 4Eleanor Roosevelt Institute, Denver, CO 80206, USA. Cystathionine fl-synthase (CBS) catalyzes the condensation of serine and homocysteine to form cystathionine. It is the first enzyme in the transsulfuration pathway of eukaryotes. CBS deficiency is the most common cause of homocystinuria and may be a significant risk factor for cardiovascular disease. The CBS gene is located on chromosome 21, at q22.3. It is about 30 kbp long and contains 23 exons. The 5'UTR contains five alternatively used exons, - l a to - l e , and an invariable exon 0. The coding region consists of sixteen exons. Exon 15 is alternatively spliced and found only in a minor m R N A species. The 3'UTR is located in exons 16 and 17. Intron 16 is retained in the majority of all mRNAs. In order to determine the complete structure of the CBS gene, we have isolated two genomic clones, one from a P1 and one from a cosmid library. Restriction fragment maps were established

Characterization of human recombinant cystathinnine ]3-synthase in E. coli. S. Taoka*, D. McMurtry, R. Banerjee. Deparmwnt ~[" Biochemistry, UniversiO, of Nebraska-Lincoln, The Beadle ('enter. P.O. Box. 880664, NE 68588-0664 USA.

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We have isolated human recombinant cystathionine fl-synthase (CBS). The purified protein shows a 63 KDa band on a denaturing polyacrylamide gel, and has a specific activity of ~ 2 1 0 unit/mg (/maole/mg h) at 37°C at pH 8.6. Pyridoxal 5Jphosphate (PLP) binds tightly during the purification; and the enzyme activity is not dependent on exogenous PLP in the assay mixture. Metal analysis of the isolated CBS indicates that it contains ~ 2 iron atoms per tetramer. The specific activity increases ~ 1.5-fold under anaerobic conditions. This activity decreases ~2-fold when CBS is reduced by addition of sodium dithionite or Ti(III) citrate; however, the reduced CBS recovers its original activity upon re-oxidation by addition of ferricyanide. This indicates that the CBS-catalyzed reaction is redox regulated. There is no inhibition of the CBS activity by heine ligands, CN , S C N - , F , by the PLP blocked agent, nicotinic acid, and the thiol agent, HgCI2 even at high concentrations. We gratefully acknowledge the recombinant plasmid expressing human CBS from Dr. Warren Kruger at FCCC. This research was supported by a grant from American Heart Association. Genetic causes of mild hyperhomocysteinemia in patients with premature coronary artery diseases. M.Y. Tsai*, M.K. Bignell, K. Schwichtenberg, F. Yang, N.Q. Hanson. University of Min-

nesota Medical School, Minneapolis, MN, USA. Elevated plasma homocysteine is increasingly being recognized as a risk factor for coronary artery disease (CAD). Although there is general agreement on the importance of micronutrient and genetic predisposition to elevated plasma homocysteine, the exact influence of the known prevalent mutations in genes which regulate homocysteine metabolism is not clear. We studied 376 cases of individuals with premature CAD with respect to their fasting and post-methionine load (PML) total homocysteine (tHey), plasma B vitamin and serum creatinine concentrations. We also genotyped the T~33C and G919A mutations and the 68 bp insertion in the cystathionine-/3-synthase (CBS) gene, the C677T mutation in the methylene tetrahydrofolate reductase (MTHFR) gene, and the A2756G transition in the B12 dependent methionine synthase (MS) gene. Approximately 13% of the CAD patients had fasting hyperhomocysteinemia, 8.5% had PML hyperhomocysteinemia, and 4.8% had combined hyperhomocysteinemia. The Ts33C and G919A mutations in the CBS gene and thermolabile M T H F R mutation play an important role only in the subset of individuals with combined hyperhomocysteinemia. The 68 bp insertion in the CBS gene and the A2756G transition in the MS gene may protect against the development of elevated plasma tHey. After accounting for possible impaired renal function as a cause of hyperhomocysteinemia, many cases (25-40%) of hyperhomocysteinemia still could not be explained by either nutritional or currently known genetic mutations. Further work is needed to study whether unknown mutations, particularly those residing in the introns of the genes involved in homocysteine metabolism, and/or other environmental factors may predispose individuals to mild hyperhomocysteinemia.

Molecular genetic aspects of MTHFR Mutated methylenetetrahydrofolate reductase and the distribution of folate metabolites. M. den Heijer 1., N. van der Put 2, M.F.G. Segers3, I.A. Brouwer 4, H.J. Blom2, C.M.J. Thomas 3.

I Dept. of Internal Medicine, TweeSteden Hospital, Tilburg; 2Lab. of Paediatrics and Neurology, and Lab. of Endocrinology and Reproduction, Univ. Hospital Nijmegen, 4Dept. of Nutrition, Univ. of Agriculture, Wageningen, The Netherlands. Mutated M T H F R (677C--9T} is associated with elevated homocysteine concentrations and decreased total folate levels in serum. To investigate a possible shift in the distribution of folate metabolites, due to impaired M T H F R activity, we measured total folate and 5-methyltetrahydrofolate (MeTHF) in plasma and erythrocytes from 20 subjects with 677TT and 20 subjects with 677CC genotype. We calculated a MeTHF/total folate ratio for plasma and red blood cells and compared the mean ratio in both groups before and after 8 weeks daily administration of 5 mg folic acid. The mean total folate concentration in plasma was in subjects with 677TT and in subjects with 677CC. The mean MeTHF concentration in plasma was in subjects with 677TT and in subjects with 677CC. The MeTHF/tota] folate ratio did not differ in both groups. However in red blood cell the mean ratio was 0.5 in subjects with 677TT and 1.0 in subjects with 677TT. A ratio below 0.3 in red cells was seen in 9 out of 20 homozygotes, compared to 1 in subjects with 677CC. This decreased MeTHF/total folate ratio in red cells remains after folic acid supplementation. Our study shows that subjects with mutated M T H F R have a decreased ratio of MeTHF/total folate in red blood cells, independent of folic acid intake. Hyperhomocysteinaemia in chronic inflammatory bowel disease and its implications. N. Mahmud*, A. Molloy, J. McPartin, J.M. Scott I, D.G. Weir. Departments of Clinical Medicine and Biochemistry 1, Trinity College and St James's Hospital,

Dublin, Ireland. The pathogenetic mechanisms underlying the increased incidence of thrombo-embolic events in inflammatory bowel disease (IBD) is still poorly understood. Recently, hyperhomocysteinaemia (tHcy) has been described in IBD patients, which is reducible with folic acid therapy. Since hyperhomocysteinaemia is now known to be associated with an increased incidence of cardiovascular and thromboembolic events, raised tHcy could be the cause of such complications in patients with IBD. Hence, we studied plasma homocysteine levels and its relationship with serum and red cell folate, serum vitamin B12 in patients with IBD and to the presence or absence of the 5 10 thermolabile methylenetetrahydrofolate reductase (MTHFR C677T) genotype. Materials and methods: We prospectively studied 87 patients with established IBD [ulcerative colitis (n =43); Crohn's disease (n--44)] and also enrolled 74 healthy controls. Plasma homocysteine levels were measured by HPLC in these groups. Plasma and red cell folates were measured by a microbiological

Abstracts/Netherlands Journal of Medicine 52 (1998) SI-$61 method and vitamin BI2 levels were evaluated by RIA method. DNA samples were genotyped for the MTHFR (C677T) mutation by PCR and restriction analysis. Two groups were then categorised according to whether they were homozygous wildtype (CC), heterozygous for wild-type and thermolabile (CT), or homozygous for the thermolabile (TT) variant. Results: Patients with inflammatory bowel disease had a higher proportion of TT homozygous 16 (18.4%) compared to healthy controls 7 (9.2%); Odds Ratio=2.09: 95% C1 0.80 to 5.41; p = NS. Plasma homocysteine levels were significantly higher in IBD patients who were TT homozygous compared to CT heterozygous [mean _+SE (pmol/L); 14.86 _+2.17 vs. 9.99+0.48; p<0.002] and CC wild-type homozygous (10.02 + 0.95; p < 0.05). The mean red-cell folate and serum vitamin B12 levels in IBD patients were lower in TT homozygous variant comCT heterozygous or CC wild-type. The homozygous state of the C677T variant and hyperhomocysteinaemia was not associated with the type of inflammatory bowel disease, severity, extent or longevity of the disease. Conclusions: The C677T variant of MTHFR in our patients with chronic inflammatory bowel disease is associated with significantly higher plasma homocysteine and lower folate levels in the homozygous carriers. This suggests that homozygous carriers would be at an increased risk of the complications of high tHcy. The raised tHcy found in this study would appear to be an excellent candidate to explain the presence of these complications in chronic inflammatory bowel disease. It may also be a factor in the pathogenesis of the vasculitis associated with the disease. We believe that all IBD patients should receive low dose folic acid therapy to protect them from the thrombo-embolic complication of raised tHcy.

The relation between hyperhomocysteinemia, coagulation/librinolysis and 6 7 7 C - , T mutation in methylenetetrahydrofolate reductase (MTHFR) gene in healthy persons. E.F. van der Molen ~*, M. den Heijer:, J. Postma3, C. Kluft4, H.J. BlomI. Laboratory o f Paediatrics and Neurology, University Hospital St Radboud Nijmegenl ; Departments of Haematology 2 and Clinical Chemistry 3, Municipal Hospital Leyenburg, The Hague; Gaubius Laboratory, Leiden 2, The Netherlands. Hyperhomocysteinemia is a risk factor for vascular disease. Homozygosity of the 677C-*T genotype is associated with hyperhomocysteinemia. Elevated concentrations of coagulation/fibrinolysis factors: (1) Von Willebrand Factor (vWF), (2) tissue Plasminogen Activator (tPA), and (3) Plasminogen Activator Inhibitor (PAI-1) are associated with vascular disease. The relationship between all these parameters were studied in plasma of 114 healthy volunteers. Results: tHcy (/.tmol/l): Homocysteine > 16 ktmol/1 (n= 30, A) 19.8 (16A-49.8), Homocysteine < 16 lumol/l (n = 64, B) 11.3 (6.4-16), Genotype TT (n = 14) 17.1 (9~,9.8), MTHFR CT and CC (n= 100) 13.3 (6.4-34.8); vWF (%): Horn. (A) 81 (26-213), Horn. (B) 71 (27-167), TT 60.91 (38-128) CT,CC 73.7 (25.8213); tPA (ng/ml): Horn. (A) 9 (2.5-17.9), Horn. (B) 8.6 (3.2 21), TT 9 (3.1-17.7) CT,CC 8.9 (2.5-21); PAI-I (ng/ml): Horn. (A) 30.8 (2.4-267.4), Horn. (B) 41.1 (3.2-304), TT 42.7 (2.4250) CT,CC 39.3 (3.2-304); tPMPAI: Horn. (A) 0.282 (0.06-

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1.3), Horn. (B) 0.18 (0.04-1.1), TT 0.20 (0.04-1.3) CT,CC 0.21 (0.03-1.1). lp = 0.03 in vWF between TT and CT,CC. 2p = 0.02 between group A and B. Conclusions: The significant higher tPA/PAI-1 ratio in persons with hyperhomocysteinemia suggests a disbalance between tPA and PAI-1 resulting in a more activated fibrinolytic system. The significant lower vWF concentration (lower risk vascular disease) in persons with the TT genotype suggests a beneficial effect on endothelial cells, despite that TT genotype is related to elevated homocysteine. This may explain why the TT genotype is not strongly associated with an increased risk for vascular disease.

Infant C677T mutation in MTHFR, maternal periconceptional vitamin use, and cleft lip. G.M. Shaw 1., g. Rozen2, R.R. Finneil3, K. Todorofft, E.J. Lammer4. JCalifornia Birth DeJects Monitoring Program, 1900 Powell Street, Suite 1050 Emeryville, CA, 94608, 2McGill University-Montreal Children's Hospital, 3Texas A & M UniversiO,. 4Children's ttospital, Oakland, CA, USA. Several studies have reported an association between fetal homozygosity for a variant form (C677T/C677T genotype) of the 5,10 MTHFR gene and risk for neural tube defects (NTDs) in individuals. It has been hypothesized that maternal folic acid supplementation prevents NTDs by partially correcting the lower MTHFR activity linked with the variant form of the enzyme. Evidence has emerged to suggest that maternal use of a multivitamin with folic acid in early pregnancy results in reduced risk for cleft lip with or without cleft painte (CLP). Because of MTHFR's involvement with the metabolism of folate, we hypothesized that infants homozygous for the C677T genotype would be at increased risk for CLP due to lower MTHFR enzymatic activity. We further hypothesized that elevations in maternal serum folate levels resulting from periconceptional supplementation of folic acid could improve the activity of the poorly functioning MTHFR enzyme and that CLP risk among infants homozygous for C677T would be lower among those whose mothers used vitamins containing folic acid, compared to the group whose mothers did not. Data were derived from a large population-based case--control study of fetuses and liveborn infants among a cohort of 1987--1989 California births (n = 548,844). Analyses were restricted to: (1) infants diagnosed with isolated CLP whose mothers completed a telephone interview (348 of 412 eligible) and whose DNA was available from stored newborn screening blood specimens (310 of 348); and (2) infants (controls) without a congenital anomaly, selected randomly from all infants born alive in the same geographic area and time period as cases, whose mothers completed a telephone interview (734 of 972 eligible) and whose DNA was available (652 of 734). To minimize genotyping, the 652 controls were further randomly reduced to 400 (383 were genotyped). Genotyping was performed blinded to subjects' case or control status and to maternal vitamin use status. Cases and controls were genotyped TT if homozygous for the C677T allele, CT if heterozygous for the C677T allele, and CC if homozygous for the C677 (wildtype) allele. The odds ratios for CLP were 0.89 (0.55-1.4) and 0.78 (0.56-1.1)

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for infants with TT vs. CC and infants with TC vs. CC genotypes, respectively. Compared to the CC genotype, the odds ratios for CLP among infants with the TT genotype were 0.74 (0.39 1.4) for those infants whose mothers were users, and 1.4 (0.54-3.6) for those infants whose mothers were not users of vitamins containing folic acid in the period 1 month before to 2 months after conception. Although the difference between the risk estimates is in the hypothesized direction, the two estimates were not statistically heterogeneous (p = 0.30). Our results do not indicate an increased risk for CLP among infants homozygous for the C677T genotype, nor do they indicate an interaction between infant. C677T genotype and maternal supplemental vitamin use on the occurrence of CLP. It is possible that the reduced risk associated with vitamins relates to correction of a maternal metabolic defect, rather than that of the fetus. The mechanism underlying the risk reduction associated with maternal folic acid supplementation remains an important question for the etiology of CLP and other congenital anomalies.

Pre- and post-load hyperhomocysteinemia in vascular patients with MTHFR 677 C - , T mutation; Response on vitamin suppletion. L.J.M. Spaapen*, K. Hamuly~.k. Foundation of Clinical Genetics, and dept. of Internal Medicine, Academic Hospital Maastricht, P.O. Box 1475, 6201 BL Maastrieht. The Netherlands. During the last decades numerous studies have established that mild or moderate hyperhomocysteinemia (HHcy) is associated with premature occlusive vascular disease. Investigation of the homocysteine metabolism is part of the work-up of patients with occlusive vascular disease. From 1989 to 1997 more than 2000 patients were investigated by means of a standardized methionine loading test (100 mg/kg body weight). Pre- and post-load total plasma homocysteine (tHcy) was analysed by HPLC equipped with a fluorescence detector using pre-column derivatization with SBD-F. 5,10-Methylenetetrahydrofolate reductase (MTHFR) is an important folate-dependent enzyme in the remethylation pathway of homocysteine. The MTHFR 677 C ~ T mutation, leading to a thermolabile enzyme with diminished activity has been associated with mild or moderate HHcy. In a random sample of 127 patients the MTHFR 677 C-~T (alanine~valine) mutation was investigated. Homozygosity for this mutation (+/+) was found in 15%, heterozygosity ( + / - ) in 43% of the patients. HHcy was detected in 68% of the +/+ patients, in 33'I/;, of the + / - patients and in 26% of the patients with the - / genotype. In the patients with the homozygote (+/+) genotype combined fasting and post-load HHcy was found mostly. Treatment of HHcy patients occurred in a consecutive way independent of the type of hyperhomocysteinemia and was started with 5 mg folic acid daily. In case of no or insufficient normalization of the tHcy levels pyridoxine (10 50 rag/day) was added to the medication. In about 50% of the +/+ and + / - subjects with HHcy folate monotherapy led to normalization of pre- and post-load plasma homocysteine levels. Omelusion: (1) Homozygosity or heterozygosity for the MTHFR 677 C--+T mutation appears not merely be related

to increased fasting tHcy levels; post-load HHcy is found as well. (2) In this group of patients with HHcy folate suppletion is not always sufficient to normalize post-load tHcy levels.

Plasma total homocysteine, vitamin status and the 5,10-methylenetetrahydrofolate rednctase polymorphism in children. A.L. Bjorke-Monsen1., S.E. Vollset:, P.M. Ueland1, H. Refsum l. University of Bergen, Norway," l Dept. o f Pharmacology and 2Div. jor Medical Statistics. In adults, the C677T mutation in the gene coding for the enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) has been associated with elevated levels of total homocysteine (tHcy), especially under conditions of low folate status. In the present study, we investigated tHcy and vitamin status in relation to the MTHFR polymorphism in children. Methods: Fasting tHcy and the MTHFR genotype was determined in 112 healthy children aged 2 15.5 y (median 5.3 y). They were recruited among children admitted to an outpatient clinic for minor ear, nose and throat surgery. Results: Median plasma tHcy tended to be higher in older children; 4.7 pM (2 8.99 y) vs. 5.6 pM (9-15.5 y). The distribution between homozygosity for the C677T substitution (TY), heterozygosity (CT) and the genotype without the mutation were 8%, 39% and 53%, respectively, Children with the TT genotype had higher red cell folate and tended to have higher plasma tHcy values compared to children with the CT or the CC genotype (see below). The difference in red cell folate was significant (TT vs. CT, p=0.0002; TT vs. CC, p=0.001, Student's t-test). Conclusion: tHcy tends to increase when the child reaches prepubertal age. Red cell folate and tHcy are higher in children with the TT genotype. Data are given as median and interquartile range. RC folate (nM): CC 342 (264-422), CT 314 (237 399), "IT 564 (365624); S folate (nM): CC 12.3 (10.3 15), CT 10.4 (8.4--14.3), TT 11.6 (9 15.7); S cobalamin (pM): CC 528 (460-6371, CT 487 {441-639), TT 528 (438-67(I); P tHcy (tiM): CC 4.8 (4.3 5.5), CT 4.8 (4-5.6), TT 5.5 (4.8-5.8). Persistent total homocysteine reduction and folate accumulation following one month course of methyltetrahydrofolate (Prefolic®) administration in thrombophilic patients with moderate hyperhomocysteinemia and/or homozygosity for the thermolabile variant of MTHFR. A. D'Angelo*, A. Coppola l, I. Fermo2, A. Pagano l, G. Mazzola, G. Guiotto 1, R. Paroni2, A.M. Cerbone t, L. Crippa, G. Di Minno ~. Coagulation Service and 2Department of Laboratory Medicine, IRCCS H. S. Raffiwle, Milan, and JDepartment o f Clinical and Experimental Medicine, University of Naples 'Federico H', Naples, Italy. Folate deficiency is a major determinant of hyperhomocysteinemia (HHcy) and folate administration is known to lower fasting total plasma homocysteine levels (tHcy). We have tested the effects of a short course of methyltetrahydrofolate (Prefolic®) administration to thrombophilic patients with moderate HHcy and/or homozygosity for the thermolabile variant of MTHFR (C677-~T mutation of the MTHFR gene, MTHFRmut). Twenty-eight patients [9 F, 19 M, mean age

Abstracts/Netherlands Journal of Medicine 52 (1998) S1 $61

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43 + 14 yrs; with a history of arterial (n = 11), venous thrombosis (n= 14) or both (n=2)] were studied. According to the 95th percentile of the tHcy distribution in 143 healthy subjects without homozygosity for M T H F R m u t (18.6 and 13.2/tM for males and females), 20 patients had HHcy; of these 16 were homozygous (tHey: 34.3__.23.9 /.tM) and 4 fieterozygous (tHey: 25.6 + 15.3 ¢tM) for M T H F R m u t ; the remaining 8 patients, homozygous for MTHFRmut, had normal tHcy (13.5+8.5 ItM). Patient plasma folate and cobalamin levels were 4.43_+2.3 ng/ml and 428_+ 191 pg/ml, respectively. After a 30 day course of Prefolic® ingestion (15 mg/day) tHcy was significantly lowered (10.7_+4.3 ,uM vs. 26.0+20 ],tM, p=0.0001) in all patients and it was normalized in 19 of the 20 patients with HHcy. Plasma folate was > 21 ng/ml after treatment (p <0.00001) and cobalamin levels were not significantly affected (346_+ 112 pg/ml, p=0.062). The reduction of tHcy was positively correlated to baseline tHcy (r = 0.98), with a 86% lowering for each tHey/.tmole being observed for tHcy concentrations ->7 /IM. In a subgroup of 8 HHcy patients (fiomozygous for MTHFRmut), plasma folate was still high one month after the end of treatment (14.0_+5.7 ng/ml, p = 0.014 vs. baseline levels) and tHcy levels were still normal in 6 patients (13.4_+6.6/IM vs. 11.9+3.5/.tM, p=0.044). A one-month course of methyltetrahydrofolate (15 mg/day) leads to normalization of tHcy in HHcy due to the M T H F R m u t with effects still detectable 30 days after the end of treatment.

r =0.175, respectively, Pearson's test). A significantly higher number of hyperhomocysteinemic patients was found among those with folate or vit. B12 below (11 nmol/L and 426 pg/ml, respectively) the median values (34°,4. vs. 18% for folate, 35% vs. 16°,4, for vit. Bi2; p always <0.05). In parallel, tHey was also significantly higher among patients with folate below the median value (17 + 8 vs. 14+ 8, p =0.036, after correction for age, sex and vit. Bt2 levels, analysis of covariance). On the other hand tHcy was significantly higher among patients with BI2 below the median value (19+13.2 vs. 13+5.5, p--0.002 after correction for age, sex and folates, analysis of covariance); this relationship was confirmed only in the + / subgroup (p = 0.0002, t-test). In a multiple regression model, in addition to sex, vitamin status and homozygosity for the M T H F R mutation, the interaction between +/+ genotype of M T H F R and vit. Bi2 (r = -0.32, p = 0.0000) was a determinant of tHcy. In conclusion, in this sample of patients with earlyonset thrombotic events, the influence of vit. B12 on tHcy seems to be genotype-dependent and this interaction is a major determinant of tHcy.

Fasting plasma homocysteine in patients with early-onset thrombotic events. Genotype-dependent interaction between vitamin BI: and the 677C ~ T mutation of MTHFR gene. G Di Minno, A. Pagano*, I. Fermo l, V. Palmieri, L. Soriente, F. Cirillo, A. D'Angelo ~. Department of Clinical and Experimental Medicine,

sit)" of Nebraska, Lincoln, NE 68588-0664, USA.

University of Naples 'Federico H', Naples and I Coagulation Service, 1.R. C. C.S. "Ospedale S. Raffaele', Milan, Italy. In 170 consecutive patients (89 m, 81 f; mean age 41 + 12 yrs) with documented early-onset thrombosis (89 venous, 69 arterial, 12 both; mean age at first episode 36+ 11 yrs), we have evaluated homozygosity (+/+) for the 677C ~ T mutation of the M T H F R gene, vitamin Blz and folate plasma levels. One hundred eighty-two matched healthy subjects served as controls. Total homocysteine plasma levels (tHcy) were evaluated by HPLC and fluorescence; the 6 7 7 C ~ T mutation of the M T H F R gene by PCR; B12 and folates by chemiluminometric immunoassays. All parameters were determined 3 11 too. after the thrombotic event. Elevated tHcy was detected in 44/170 patients (25.8%) and in 18/182 controls (9.8%) (p < 0.0005, chi squared), patient mean tHcy being significantly higher than that of controls (16+10.6 vs. 12.5+7.8 p M ; p < 0 . 0 0 5 , t-test). The + / - 677C--,T mutation of M T H F R gene was detected in 471170 patients (27.6%) and in 39/182 controls (21.4%) (p = 0.217, chi-squared). Among the hyperhomocysteinemic patients 25/44 (56.8%) were homozygotes for the mutation; tHey was higher in the +/+ subgroup (22_+ 16 vs. 14 + 6 in + / - and 13 + 4 in - / - patients, p < 0.001, Scheffe post-hoe test). An inverse correlation was found between tHcy and folate or B12 levels (p<0.005, r = - 0 . 2 3 7 and p < 0 . 0 3 ,

Methionine synthase Identification and purification of the redox proteins for the activation of mammalian methionine synthase. Zhiqiang Chen*, R Banerjee. Department of Biochemistry, Beadle Center, UniverMethionine synthase (MS) is one of the two known mammalian enzymes which uses vitamin B~2 as cofactor. It catalyzes a methyl transfer reaction from methyltetrahydrofolate to homocysteine to generate methionine and tetrahydrofolate. In humans, dysfunction of methionine synthase is associated with megaloblastic anemia, hypomethioninemia and homocystinuria. It may also be associated with heart diseases, because it directly relates to the cellular metabolism of homocysteine, which is an independent risk factor of cardiovascular diseases. One of the active forms of the cofactor in the catalytic cycle is cob(I)alamin which is a supernucleophilic and is oxidized occasionally to cob(II)alamin during catalysis. The oxidized form of enzyme is inactive. To reenter the catalytic cycle, a reductive activation system is required. The physiological reductive activation system in mammals remains unknown. We will present our results on the identification and purification of the pig liver redox proteins involved in MS activation. We have recently reported that the porcine liver MS activation needs at least two protein components (J Biol Chem 1997;272:19171 19175). Our data demonstrate that one of the components (component I) is microsome associated and the other (component If) is a cytosolic protein. By using a fixed amount of purified microsome in the anaerobic N A D P H assay, we can monitor component II activity. After DEAEcellulose, hydroxyaptite, Mono Q and gel filtration chromatography, we identified a small protein which has component II activity. Addition of microsome and the partially purified component lI, activates MS more efficiently than does DTT and

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B12 used in the standard assay. Further characterization of this protein will be reported. Methionine synthase polymorphism and risk of coronary heart disease in the health professionals follow-up study. C.J. van de Poll*, E.B. Rimm, J. Chen, P. Verhoef, W.C. Willett, K. Kelsey, M.J. Stampfer, D.J. Hunter. From the Departments of

Nutrition (Drs. Rimm, Willett and Stampfi'r), Epidemiology (Drs. Rimm, (?.hen, Hunter, Willett and Stampfer), Cancer Biology' (Dr. Kelsey), and Environmental Health (Dr. Kelsev), Harvard School of Public Health, Boston, Mass: The Channing Laboratory, Department of Medicine. Harvard Medical School and Brigham and Women's Hospital, Boston, Mass (Ms. van de Poll and Drs. Rimm, Chen. Willett, Hunter, Kelsey, and Stampfer); and the Division of Human Nutrition and Epidemiology, Agricultural University Wageningen, the Netherlands (Dr. Verhoef). Methionine synthase ([MS) is a vitamin BI2 dependent enzyme responsible for the remethylation of homocysteine to methionine. We examined the risk of nonfatal coronary heart disease (CHD) associated with homozygosity for the D919G polymorphism of MS in a case-control study conducted within the Health Professionals Follow-up Study. From the cohort of 44940 US male health professionals, 18025 members provided blood samples. We compared 499 men with nonfatal CVD and 499 age-matched controls. Genotyping was carried out using polymerase chain reaction followed by restriction fragment length polymorphism. Frequencies of homozygosity (+/+) and heterozygosity (+/ - ) for the mutation were 4% and 32% in cases and 4% and 30% in controls. With subjects homozygous or heterozygous for the wildtype allele as a reference, the odds ratio (OR) of CHD was 1.12 (95"/0 CI: 0.58 2.15) for +/+ subjects. The +/+ genotype was also not associated with risk of CHD among men with low intake o f vitamin B12, B6, B2 and folate, or high intake of alcohol or methionine. In these men, no evidence was found for an association between the D919G polymorphism of MS and CHD. However, since no data are available on plasma homocysteine, further research is needed to determine functionality of this polymorphism. Sequence analysis of the human Methionine Synthase: relevance to hyperhomocysteinemia in neural-tube defects and vascular disease. N.M.J. van der Put 1., E.F. van der Molen l, L.A.J. Kluijtmans 1, S.G. Heil 1, J.M.F. Trijbels/, T.K.A.B. Eskes2, D. van Oppenraaij-Emmerzaal I, R. Banerjee s, H.J. Blom t.

I Departments ~ Pediatrics, University Hospital Nijmegen, The Netherlands, 2Departments of Gynaecology & Ohstetrkw, University Hospital Nijmegen, The Netherlands, 3Departments of Biochemistry, University of Nebraska, Lincoln, USA. Two apparently unrelated diseases, i.e. neural-tube defects (NTDs) and premature vascular disease, are both associated with moderately elevated homocysteine (Hcy) levels. Recently the human methionine synthase (MS) cDNA-sequence has been cloned and mapped to chromosome lq43. A defective MS could result in elevated Hcy levels.

Results: By sequencing analysis of the coding region of MS of 8 hyperhomocysteinemic patients (4 NTD patients and 4 patients who suffered from pregnancies complicated with spiral arterial disease (SAD)), we examined if a mutated MS gene is involved in these Hcy related diseases. We identified an A to G transition at bp 2756, converting an aspartic acid (D919) into a glycine (G). No other mutations resulting in an amino acid substitution were observed. To examine whether the observed 2756A--*G mutation is a risk factor for NTD or vascular disease we screened on genomic DNA for its prevalence among 56 NTD patients, 69 mothers of a child with NTD, 108 SAD patients and 364 controls. We observed no increased prevalence of the GG- and AG-genotype in N T D patients, their mothers and SAD patients, indicating that the D919G mutation is not a risk factor for NTD or vascular disease. Furthermore, we observed no association between the presence of the 2756A--, G mutation in MS and mild hyperhomocysteinemia. Conclusion: The D919G mutation is a fairly prevalent, and probably benign polymorphism. This study provides no evidence for a major involvement of MS in the etiology of homocysteine-related diseases like NTD or vascular disease. The A2756G mutation in the gene coding for methionine synthase: associations with plasma homocysteine and coronary atberosclerosis. P. Verhoeff, L.A.J. Kluijtmans 2, H.J. Blom 2, F.J. Kok 1 ~Division of Human Nutrition and Epidemiology, Agricul-

tural University, l~21geningen, The Netherlands 2Department of Pediatrics, University Hospital, Nijmegen, The Netherlands. Introduction: The vitamin B~2 dependent enzyme methione synthase (MS) remethylates homocysteine to methionine. Recently, a common A2756G mutation was discovered, which causes an aspartic acid to glycine substitution (D919G) in a region believed to bind cobalamin. We assessed whether the mutation has functional significance, i.e. whether it affects plasma total homocysteine (tHcy) levels, and whether homozygosity for the mutation is more common among patients with coronary atherosclerosis than among normal control subjects. Methods: In 1992 1994, a case-control study was conducted in the Rotterdam area, the Netherlands. Information on MS A2756G genotype and tHcy values (fasting and in response to methionine load) were available for 123 subjects with severe coronary atherosclerosis and 99 population-based control subjects. Participants were males and females, aged 25 to 65 years. Results: Frequencies of homozygosity (+/+) and heterozygosity ( + / - ) for the mutation and of the homozygous normal genotype ( - / - ) were 7.3%, 26.8% and 65.9% among the patients and 3.0%, 38.4%, and 58.6% among population-based controls, respectively (Pz 2= 0.09). The odds ratio (OR) of coronary atherosclerosis for the +/+ genotype, taking the other two genotypes as a reference, was 2.5 (95% confidence interval 0.7-9.6). The +/+ subjects did not have elevated plasma tHcy levels (fasting or in response to methionine load) compared to subjects with - / - or + / - genotypes. In a subgroup of subjects with low serum B]2 levels (i.e. lower than the population median of 242 pmol/L), there was a tendency of higher fasting

Abstracts~Netherlands Journal of Medicine 52 (1998) S1-$61 tHcy levels among subjects with the +/+ genotype: geometric mean fasting tHcy levels were 15.3 ~nol/L and 13.2 /tmol/L among +/+ subjects and subjects with other genotypes, respectively (P= 0.66). Conclusion: Homozygosity for the A2756G mutation does not appear to affect plasma tHcy levels. The higher frequency among cases of coronary atherosclerosis relative to normal controls may have been due to chance. Functional significance of this mutation should preferably be evaluated in a large sample of subjects with low vitamin Bi2 status.

Changes in amniotic fluid folate and Bj2 levels (15-20 weeks) associated with neural tube defects. E.B. Dawson*, D R . Evans, J.W. VanHook. The University of Texas Medical Branch, Dept.

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protein synthesis inhibitor. This is consistent with irreversible damage of MS and CH3Cbl, and new synthesis of both enzyme and co-factor is required to restore enzyme activity. In contrast, restoration of the MCM system is only dependent on repletion of the AdoCbl cofactor. We also observed a synchronous fluctuation in AdoCbl and the much larger OHCbl pool during inactivation and recovery phase, suggesting that the loss and regain of AdoCbl reflects changes in the overall Cbl homeostasis. Since total MCM seems to increase during negative Cbl balance (induced by nitrous oxide), the most responsive indicator, however, was the ratio between MCM in the absence vs. in the presence of added AdoCbl to the enzyme assay. This parameter, denoted the activity ratio, should be evaluated as a clinical indicator of cellular Cbl status.

OB-GYN, Galveston, TX 77555-0587, USA. Folate and B12 were measured in amniotic fluid (AF) from ten non-neural tube defect {non-NTD) pregnancies each week between ]5 and 20 weeks gestation age (GA) and ten with a NTD fetus. The group mean _+SD was determined for each week of the non-NTD and the NTD pregnancies. There was a progressive decrease in the AF levels of both folate and BI2 between 15 and 20 weeks of the non-NTD pregnancies. The folate decreased from 6.1 + 1.9 pg/ml at 15 weeks to 2.3_+0.9 /lg/ml at 20 weeks. The AF BI2 levels decreased from 695 _+200 pg/ml at 15 weeks to 228_+94 pg/mg at 20 weeks. The AF samples from the NTD pregnancies were obtained between 16 and 19 weeks GA. The AF folate and B12 levels of the NTD samples were significantly below the range of the nonNTD samples at the same GA in 7 of the 10 NTD pregnancies. The remaining 3 NTD samples apparently were not associated with deficiencies of either folate or BI2, but some other factor, perhaps the homocysteine substrate.

The co-ordinate variations in methionine synthase, methyhnalonyl CoA mutase, and the cobalamin cofactors during nitrous oxide exposure and the subsequent recovery. B. Riedel*, T. Fiskerstrand, H. Refsum, P.M. Ueland. Department of Pharmacol-

ogy, University of Bergen, Armauer Hansens Hus, 5021 Bergen, Norway. The two cobalamin (Cbl)-dependent enzymes, methionine synthase (MS) and methylmalonyl CoA mutase (MCM), and the levels of their respective cofactors, methylcobalamin (CH3Cbl) and adenosylcobalamin (AdoCbl), were measured in cultured human glioma cells during nitrous oxide exposure and a subsequent recovery period of culture in a nitrous oxidefree atmosphere. MS as the primary target of nitrous oxide was rapidly inactivated (initial rate of 0.058 h -1) during exposure, followed by reduction of CH3Cbl, AdoCbl and MCM levels, in that order. After 48 hours, there were small residual activities of MS and CH3Cbl ( < 20%) whereas about half of AdoCbl and MCM remained. For nitrous oxide treated cells, MS and CH3Cbl recovered rapidly up to half of the control values, while AdoCbl and MCM increased more slowly during 24 hours of culture in an atmosphere without nitrous oxide. Notably, the recovery of MS and CH3Cbl was completely blocked by cycloheximide, whereas the increase in AdoCbl and to a lesser extent MCM was actually enhanced by the

Diagnosing cobalamin deficiency in general practice: A rationale for requesting methylmalonic acid. J. SchneedeI *, G. Holleland:, P.M. Ueland 1, H.M. Refsuml, P.K. LundJ, S. Sandberg4. University Hospital Haukeland Sykehus, Bergen,

Norway, IDept. of Clin. Pharmacol., 2Dept. of Internal Medicine, 3Fiirst Medical Laboratory, Oslo, Norway, 4Dept. of Clinical Chemistry. Determination of s-cobalamin still is the most important tool to diagnose cobalamin deficiency in general practice. However, the final decision to supplement a patient or not is normally made on the basis of a total clinical evaluation which includes clinical findings, other laboratory tests, and the age and sex of the patient. In many cases, the clinical diagnosis and decision to treat the patient is characterized by considerable uncertainty. Recently, homocysteine and methylmalonic acid have been introduced as markers of functional cobalamin deficiency. By the help of cost/benefit analysis the present study intends to give a rationale for when s-methylmalonic acid should be requested. Patients with s-cobalamin levels < 300 pmol/l were included. Six weeks after s-cobalamin was requested the general practitioners (GPs) received a questionnaire concerning their patient. The questionnaire sought information on the indications for requesting s-cobalamin, additional analyses ordered, as well as the final conclusion whether the patient was deficient or not. In the remaining sera from the primary cobalamin analysis s-creatinine, s-folate, s-homocysteine and s-methylmalonic acid were analysed. The GPs did not know the results of these analyses when they answered the questionnaire. Cost/benefit analysis indicates that the diagnostic profit of additional analysis of s-methylmalonic acid in terms of expected increase in correct diagnoses is greatest at s-cobalamin levels between 100 and 250 pmol/1. In patients with cobalamin levels in this interval it seems justified to request s-methytmaIonic acid.

Folate and homocysteine metabolism in copper-deficient rats. T Tamura/*, K.H. Hong2, Y. Mizuno ~, K.E. Johnston 1, C.L. Keen2. I University of Alabama at Birmingham, Birmingham,

AL, USA," 2University of California, Davis, CA, USA To investigate the effect of copper (Cu) deficiency on folate and homocysteine (Hcy), we measured plasma, red-cell, and

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hepatic folate (L. casei assay, HPLC-L. casei), plasma Hcy (HPLC-fluorescent detection), and hepatic methionine synthase (MS) activities (using 5-[14C]methyl-tetrahydrofolate [CH3HaPteGIu] as substrate). The values in Cu-deficient (CuD group) male Sprague-Dawley rats were compared to those in Cu-sufficient control rats (control group). The two groups of rats (n = 11 in each group) were fed a semi-purified diet containing either 0.1 or 9.2 mg of Cu/kg of diet. At 6 weeks, the mean body weights were 358 and 411 g in the CuD and control groups, respectively (P < 0.001). Results. CuD: Plasma Cu (lamol/l) 1.0, Hepatic folate (lag/g) 6.13, CH3H4PteGIu (%) 75.0, MS activity (nmol/g/h) 1057, Plasma Hcy (lamol/1) 14.9; Control: Plasma Cu 18.4, Hep. fol. 6.45, CH3H4PteGIu 50.0, MS 1434, Plasma Hcy 11.7; P: Plasma Cu 0.001, Hep. fol. NS, CH3H4PteGIu 0.01, MS 0.01, Plasma Hcy 0.05. Plasma folate levels were similar in both groups, whereas red-cell folate levels were slightly higher in CuD rats (2583 nmol/L) than in controls (2205 nmol/l). Our data indicate that Cu deficiency resulted in a 26% reduction in hepatic MS activity which caused an increase in hepatic 5-CH3H4PteGlu and plasma Hey levels. In summary, our results suggest that hepatic MS is a Cu metalloenzyme, and plasma Hcy levels can be influenced by Cu nutriture in rats. These data support the concept that Cu deficiency can be a risk factor for cardiovascular disease.

Renal disease Homocysteine concentration in renal transplant recipients: change after transplantation and influence on graft function. M. Arnadottir*, B. Hultberg, J. Wahlberg, B. Fellstr6m, E. Dim6ny. Dept. of Medicine, National University Hospital,

Reykjavik, Iceland, Dept. of Clinical Chemistry, University Hospital, Lund, Sweden, Dept. o[ Transplant Surgery, University Host~ital, Uppsala, Sweden, Dept. of Medicine, University Hospital, Uppsala, Sweden, Dept. (~f Medicine, University Hospital, Umed, Sweden. The aim of the present investigation was to study the change in plasma total homocysteine concentration (tHcy) associated with renal transplantation and the influence of tHey on renal graft outcome. Eighty-one consecutive renal transplant recipients were followed up for five years regarding patient and graft survival, graft function and histopathology. Before and at six months after transplantation blood samples were drawn for analysis of tHey. Mean tHcy before transplantation was 33.2 + 19.2 /lmol/L. In 57 patients, tHcy was 36.6_+20.9 /tmol/L before and 27.7_+ 14.6 /.tmol/L at six months after transplantation. The postransplant changes in tHcy varied widely and in 17 patients, tHcy actually increased. The mean change was a reduction of 13% (p < 0.001 ). There was a direct correlation between pre- and posttransplant tHcy (r = 0.40, p < 0.01 ). Serum creatinine concentrations at six months (mean 148_+ 57 /tmol/L) correlated directly with t h e y (r = 0.41, p < 0.011. The preva-

lence of hyperhomocysteinemia was 94% before and 88% after transplantation. Pretransplant tHey did not predict patient survival, graft survival, graft function or chronic graft damage score at six months or at five years. Neither did posttransplant tHcy predict the outcome at five years. Considering the marked improvement in renal function after transplantation the change in tHey was surprisingly small. This suggests the introduction of a factor that counteracts the tHeylowering effect of improved renal function, possibly cyclosporin. The results did not support the hypothesis that hyperhomocysteinemia adversely influences renal graft outcome.

Association between plasma homocysteine concentrations and cardiac hypertrophy in end-stage renal disease. J. Blacher*, K. Demuth, A. Guerin, C. Vadez, N. Moatti, M. Safar, G. London. Department of internal mediehze, INSERM U 337, Brous-

sais hospital, Paris, France. In patients with end stage renal disease (ESRD), plasma homocysteine and cardiac mass are both increased and considered as independent risk predictors for cardiovascular-specific morbidity and mortality. However, to date, no relationship between these two parameters has ever been established. We determined cardiac mass (echocardiography) and plasma homocysteine (fluorimetric high performance liquid chromatography), in a population of 74 patients with ESRD undergoing chronic hemodialysis. Plasma folates and vitamin Bt2 were also measured together with plasma lipids, serum albumin, fibrinogen, urea, creatinine, hemoglobin, calcium and phosphates. LV mass was increased (280 + 101 g) and the prevalence of LV hypertrophy was 78%. Homocysteine was increased (35.9+ 12.8 /lmol/1) and was negatively correlated with plasma vitamin Blz but not with plasma folic acid. The odds ratio for LV hypertrophy was 6.1 (95% confident interval : 1.6,23.9) for subjects with the highest plasma homocysteine concentrations ( > 33.8 /~mol/l) as compared with those with the lowest concentrations ( < 33.8 /~mol/1) (p < 0.05). We observed a statistically significant positive association between plasma homocysteine and cardiac mass (r= 0.31, p < 0.01) or either of its components (interventricular septal thickness, left ventricular posterior wall thickness and left ventricular end diastolic diameter). This finding was observed even after adjustment for age, gender, systolic blood pressure, body surface area and hematocrit (p=0.0022). Plasma lipids, glucose and serum albumin did not significantly enter into the multivariate analysis. This cross sectional study is the first to provide a statistical link between plasma homocysteine and cardiac structure, independently of mechanical factors. High plasma homocysteine concentrations are associated with an increased risk of LV hypertrophy in ESRD patients.

Vitamin B6 treatment independently reduces the post-methionine load increase in total homocysteine levels: results of a randomized, placebo-controlled, 2 x 2 factorial study in renal transplant recipients. A.G. Bostom 1., R.Y. Gohh:, A. Beaulieu:, M.R. Nadeau 4, A.L. Hume 3, P.F. Jacques 4, J. Selhub 4, I.H. Rosen-

Abstracts/Netherlands" Journal of Medicine 52 (1998) SI-$61 berg4. Brown University School of Medicine, Providence, RL USA, IDivision of Cardiology, 2Division of Renal Diseases, 3Family Medicine, 4Jean Mayer USDA Human Nutrition Research Center, Boston, MA, USA. Earlier we demonstrated that there is an excess prevalence of both fasting and post-methionine loading (PML) hyperhomocysteinemia among stable renal transplant recipients (RTRs). These data suggested that folate and vitamin B12 plasma status were inversely related to fasting plasma total homocysteine (tHcy) levels, while vitamin B6 status was inversely associated with the PML increase in tHcy levels, in follow-up, using a block randomized, placebo-controlled, 2 ×2 factorial design, 29 clinically stable RTRs were assigned to treatment for 6weeks with: vitamin B6 (50 mg/day); folic acid (5 rag/day) and vitamin B12 (0.4 rag/day); vitamin B6 (50 rag/day), folic acid (5 rag/day), and vitamin Bl2 (0.4 rag/day); matched placebo. Fasting and the 2-hour PML increase in tHcy levels were determined twice prior to treatment, and twice during the last week of treatment, and the results averaged. Results: There was no evidence of interaction between the two treatment arms. Analysis of covariance adjusted for age, sex, and pre-treatment levels of PML or fasting tHcy, plasma B-vitamins, and serum creatinine, revealed that B6 treatment resulted in a 22.1% reduction in the geometric mean PML increase in tHcy levels (p = 0.042), while combined folic acidvitamin BI2 treatment caused a 26.2% reduction in geometric mean fasting tHcy levels (p = 0.027). Conclusions: These data provide the first placebo-controlled evidence that vitamin B6 treatment independently lowers the PML increase in tHcy levels. Vitamin B6 should be added to the combination of folic acid and vitamin BI2 for effective reduction of both PML and fasting tHcy levels in RTRs, and possibly other patient populations. Increased homocysteine as a risk factor for complications in renal allograft recipients. G.P. C a n d f * , J.B. Ubbink:, L. Margolius s, J.S. Galpin 4. Departments ~d' Nuclear Medicine 1, Medicine 2, and Actuarial Science 4, University o[" the Witwatersrand, Johannesburg 2000 and Chemical Pathology 3, University of Pretoria, Pretoria 0001, South AJHca. Elevated concentrations of plasma homocystine represent an independent risk factor for premature occlusive vascular disease. Vascular disease is a major cause of renal graft loss and subsequent morbidity and mortality in these recipients. Aim: To correlate plasma homocysteine concentrations with post-transplant complications in renal allograft recipients. Methods': Total plasma homocysteine was determined by HPLC in 72 consecutive renal allograft recipients for up to 14 days in the immediate post-transplant period. Plasma concentrations were correlated with complications including acute rejection episodes. Results: Plasma homocysteine. Average and days post transplant (mean=std; pM). Patients, uncomplicated (n=14), Average 15+9, Day 1 8+1, Day 5 13+2, Day 9 11_+6; Patients with ARE I (n=26), Average 24_+ 11; ARE 2 (n=9126), Day 1 15_+2", Day 5 37_+16, Day 9 31_+14". tARE=acute

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rejection episodes; 2All patients with ARE; SPatients (9/26) with ARE at day 11 post transplant. Discussion and Conclusions: Plasma homocysteine is elevated in most recipients who experienced ARE. Routine homocysteine determinations may prove a useful additional test in monitoring the post-transplant progress of renal transplant recipients. Total plasma homocysteine (Hcy) concentrations are strongly correlated with whole blood cyclosporin levels in heart transplant patients. D.E.C. Cole*, H.J. Ross, L.J. Langman, J. Evrovski, S.E.S. Miner, B.Y.L. Wong, P.A. Daly. Depts. of Lab Medicine and Pathology, Medicine, and Genetics, Univ. of Toronto, and Div. o[" Cardiology, The Toronto Hospital, Toronto ON, Canada. Circulating total homocysteine (Hcy) concentrations are generally elevated in transplant patients but the pathogenesis and benefits of treatment have not yet been established. However, transplant coronary artery disease is an important cause of late allograft failure. We examined Hcy in a cohort of 72 cardiac transplant recipients (3.98 + 0.37 yr after transplantation) to identify clinical, biochemical and genetic factors that influence the extent of the problem. We found that all 72 patients had Hcy levels above our upper limit of normal ( > 15 pM), the mean in the transplant group (25.4 + 0.8 pM) was significantly greater than in controls (8.3 + 2.1/aM, n = i4, p < 0.001), or in patients attending a high-risk thrombosis clinic (16.8+ 1.3 pM, n=94, p<0.05). In the transplant group, parameters examined in our analysis included: age, gender, time since transplant, serum folate and B~2 levels, total protein, urate, creatinine, albumin, and trough whole blood cyclosporin concentrations. The prevalent methylenetetrahydrofolate reductase (MTHFR) polymorphism, C677T, was analyzed as a covariate. In our patients, serum creatinine was elevated (144+6 /zM) and serum folate decreased (6.75+0.22 nMI. Multiple linear regression showed that creatinine (p = 0.021) and cyclosporin levels (191 + 19 pg/L, p=0.015) were independent positive predictors of Hcy, while serum folate (p = 0.018) and time since transplant (p = 0.049) were significant negative predictors. There was no significant association of Hcy with A677V MTHFR status, even when interaction effects with serum folate were included in the regression model. We conclude that hyperhomocysteinemia is a significant problem in cardiac transplant recipients. Our analysis suggests that folate and renal glomerular dysfunction are important contributory factors, but cyclosporin appears to have an independent adverse effect on Hcy. Because folate levels are relatively low, supplementation with high-dose folic acid may be a simple means of reducing the impact of transplant coronary artery disease on long-term graft survival. Early effects of peritoneal dialysis on plasma homocyst(e)ine concentrations. D. Ducloux*, L. Lecornu-Heuze, V. Fournier, C. Bresson-Vautrin, R. Gibey, J.M. Chalopin. Department of Nephrology and Renal Transplantation; Laboratory q/' Biochemistry. Hopital St Jacques; Besam,'on, France.

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The end-stage renal disease (ESRD) population experiences an excess morbidity and mortality due to arteriosclerotic cardiovascular disease (CVD). Recently, controlled evidence has been provided that hyperhomoeyst(e)inemia occurs more commonly than any of the traditional CVD risk factors in ESRD patients on maintenance dialysis. There was no evidence that dialysis adequacy has any significant effect on chronic tHcy levels in maintenance dialysis patients. Hemodialysis results in a transient reduction of plasma tHey, followed by a rapid return to predialysis tHey levels within 24 hours. Limited data are available on the reduction o f plasma tHcy provided by PD. Plasma tHey was measured by high-performance liquid chromatography, immediately before and one month after the initiation of PD in 16 consecutive patients. Plasma folic acid, vitamin B12 and albumin were also measured. Weekly PD creatinine clearance was assessed (ADEQUEST). Plasma tHey were 34.9 + 9.4 #mol/l before PD and 22.7 + 6.9 ,umol/1 one month later (p < 0.0005). Both folic acid and vitamin BI2 concentrations remained stable. Serum albumin decreased from 34.5+6 to 29.6_+8.1 g/l (p<0.01). There was no relationship between the decrease in tHcy and the decrease in serum albumin (R 2 = 0.072; p = NS). By contrast there was a significant relationship between the decrease in tHcy and dialysis adequacy (R 2 = 0.41 ; p < 0.02). Our study demonstrates that PD allows a significant decrease of tHey in ESRD patients. The variation in plasma tHcy is correlated with the measure of dialysis adequacy. Further studies with long follow-up period are required to confirm this result and determine its relevance in the prevention of CVD. No net renal homocysteine extraction in fasting man. C. van Guldener 1., A.J.M. Donker 1,:, C. Jakobs s, T. Teerlink 3, K. de Meet ~,4, C.D.A. Stehouwer:. Depts. o[ Nephrology I, b~ternal Medicine 2 and Clinical Chemistry 3, Academisch Ziekenhuis Vr(je Universiteit and Institute for Cardiovasular Research, Amsterdam, and University Children's Hospital 4, Utrecht, The Netherlands. Hyperthomoeysteinaemia occurs in end-stage renal disease (ESRD) patients and may contribute to their propensity to atherothrombotic disease. The pathophysiological mechanism of elevated plasma homocysteine levels in ESRD is obscure. Loss of urinary homocysteine clearance is unlikely as the amount of homocysteine in urine of healthy subjects is negligible. Loss of active homocysteine uptake, which has been demonstrated in the normal rat kidney by the finding of a substantial (20%) difference between arterial and renal venous plasma homocysteine, seems an attractive alternative explanation. We measured plasma total (i.e. free and protein-bound) homocysteine (tHcy) and free homocysteine (fHcy) sampled from the aorta (A) and renal vein (V) of 20 fasting patients with normal renal function (serum creatinine 90.5+SD, 15.6 Ftmol/I), who underwent elective cardiac catheterisation. Arterial tHcy and fHey were 10.8 + 3.1 and 3.2 + 1.6/Jmol/l, respectively. Renal extraction, calculated as (A-- Vj× 100%/A, was 0.9 (SD 5.8; 95% CI - 1 . 8 to 3.6)% for tHcy, and - 0 . 2 ( 1 1 . 0 : - 5 . 4 to 4.9) % for fHcy.

We conclude that no significant net renal uptake of homocysteine occurs in fasting humans with normal renal function and that elevated fasting homocysteine levels in patients with renal failure thus cannot be explained by the loss of such extraction. Prevalence of tMTHFR in a chronic renal failure population: lack of correlation with homocysteine levels. S.E.S. Miner*, D.E.C. Cole, K. Nanthakumar, M. Goldstein. Saint Michael's Hospital, Toronto, Canada. Introduction: A common, thermolabile variant of methylene tetrahydrofolate reductase (tMTHFR) has been associated with elevated homocysteine levels in the general population. To date, there have been limited published data pertaining to the prevalence and relevance of this polymorphism in the chronic renal failure population. Ot?jectives: To investigate the prevalence and association with hyperhomocysteinemia of t M T H F R in the chronic renal failure population. Methods: In a hemodialysis population, non-fasting plasma was obtained for determination of homoeysteine levels via HPLC with pulsed integrated amperometry. Whole blood was obtained concurrently with genotype determination by standard techniques. Results: 112 patients were screened, with a mean, non-fasting homocysteine level of 22.3 ,umol/L. 37% of this population was heterozygous for the thermolabile polymorphism, while 7% were homozygous. No differences in mean homocysteine levels were noted between the 'normal', heterozygous, or homozygous individuals (22.0, 23.4, 19.3). Conclusion: The common thermolabile variant of M T H F R does not appear to significantly affect non-fasting homocysteine levels in a chronic renal failure population.

Endothelial dysfunction Homocysteine-induced elastolysis in arterial media: activation of MMP2. A. Bescond, T. Augier, C. Chareyre, P. Charpiot, D. Garcon*. Laboratoire de Biochimie, Facult~ de Pharmacie, Marseille, France. We have previously shown that hyperhomocysteinemia induces elastolysis in arterial media of minipigs. This elastolysis resulted in increased elastic laminae-fenestration and was associated with smooth muscle cell (SMC) migration, suggesting the involvement of enzymes produced by SMC. Moreover, we demonstrated an increase in metalloproteinase elastase-like activities in hyperhomocysteinemic-atherosclerotic minipig arteries. Based on these observations, we investigated the activation of the arterial elastolytic matrix metalloproteinases (MMPs) by homocysteine (Hcy). Segments of pig abdominal aortas were cultured in serumfree NCTC 135 medium, with 0, 10 or 50/~M Hcy for 17 and 65 hours. Transverse histological sections (6/.tm) of paraffinembedded arterial segments were stained with (+)-catechin for elastic component investigation. As shown by morphodensito-

Abstracts/Netherlands Journal oJ Medicine 52 (1998) S1-S61 metric analysis, the elastin content was decreased in arteries cultured with Hcy where the elastic structure alterations were similar to those observed in hyperhomocysteinemic animals. SDS-PAGE zymography showed an increase of active MMP2 (gelatinase A), an elastolytic MMP. In vitro, the isolated inactive zymogene pro-MMP2 (72 kDa) was incubated with homocysteine or methionine (Met) at different molar ratios (37°C, 3 h). Pro-MMP2 was activated by Hcy at molar ratio 10:1 (Hcy:pro-MMP2). Met did not activate pro-MMP2 at any molar ratio from 1:1 to 1000:1, showing that the presence of the free reactive thiol group of Hcy is required for activation. This activation occurred without generating lower molecular weight derivatives in accordance with the hypothesis of a 'cysteine-switch' mechanism. These results suggest that hyperhomocysteinemia induces elastic structure alterations in the arterial wall at least by direct activation of the elastolytic MMP2. Upregnlation of collagen accumulation by homocysteine and its abrogation by heparin(s). A.K. Majors;*, D.W. Jacobsen:, R.E. Pyeritz1. 1Allegheny University of the Health Sciences,

Pittsburgh, USA.

USA; 2Cleveland Clinie Foundation, Cleveland,

Collagen plays an important role in the pathogenesis of atherosclerosis by contributing substantially to the mass of connective tissue responsible for arterial stenosis. We cultured rabbit arterial smooth muscle ceils (SMC) for 3 weeks with 300 /~M L-Hcy, L-homocystine, L-Cys or penicillamine (fl,fl-dimethylcysteine). Hcy-treated cultures accumulated approximately twice as much collagen, and up to 43% more noncollagenous protein as control cultures. The increase in noncollagenous protein was primarily due to increased cell numbers (10-30%), while the increase in collagen was primarily due to a specific increase in collagen synthesis per cell. Limited pepsin digestion and polyacrylamide gel electrophoresis of the cell layers indicated that collagen types 1, III, and V were all elevated. In contrast, Cys and homocystine did not increase SMC collagen, and penicillamine inhibited collagen accumulation by up to 40%. Five ltg/ml of heparin, a potent inhibitor of vascular SMC, blocked the increase in collagen accumulation induced by Hcy by 87%. Low molecular weight heparin, which has therapeutic advantages over unfractionated heparin, was as effective in reducing collagen accumulation. Analysis of noncollagenous protein demonstrated that heparin was not toxic to the SMC, and that the inhibition was specific for collagen. Pentosan polysulfate, a semisynthetic drug with actions similar to heparin, but which can be administered orally, was also effective in blocking the Hcy-induced collagen accumulation. These results indicate that Hcy might contribute to atherogenesis by upregulating the accumulation of collagen and that the inhibition of collagen accumulation by heparin(s) and pentosan polysulfate may provide insight into potential therapeutic applications. Association between chorionic villous vascularization and homocysteine concentrations in women known with unexplained recurrent early pregnancy loss? W.L.D.M. Nelen;*, J. Bulten:, H.J.

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Bloms, E.A.P. Steegers 1, A.G.J.M. Hanselaar2, T.K.A.B. Eskes/. Departments of Obstetrics and Gynaecology I, Patholog))

and Paediatrics 3, University Hospital St Radboud Nijmegen, The Netherlands'. Introduction: Hyperhomocysteinaemia is known as a risk factor for unexplained recurrent early pregnancy loss (REPL), but pathogenesis remains unknown. Theoretically, maternal elevated total plasma homocysteine (tHcy) concentrations may cause vascular damage and lead to defective chorionic vasculogenesis. Purpose: To investigate chorionic villous vascularisation in collected miscarriage tissue of 20 women known with REPL and relate it to the tHcy and folate concentrations measured in these women at least six weeks after the miscarriage concerned. Methods: In 4 / t m coupes (formalin fixed and paraffin embedded) CD34 (Q-Bend) was used to stain vascular endothelial cells. By Vidasplus image analysis system seven vascularization parameters were obtained: total median vessel area, vessel circumference, median number of vessels per mm 2 chorionic stroma and, as measured per vessel, the median area, median circumference, minimal diameter and maximum diameter. Results: No correlations were observed between tHcy or folate concentrations and the parameters concerning overall chorionic villous vascularisation. However, measured per blood vessel the fasting tHey was negatively correlated with the circumference (P=0.004; rs=-0.61) and the maximum diameter (P=0.02; rs=-0.53). Higher after-load tHcy inclined to be associated with smaller vessel circumferences, but this reached no statistical significance. No significant correlations were determined between folate concentrations and blood vessel parameters. Conclusion: Higher fasting tHcy is associated with a normal number of chorionic blood vessels but decreased vessel diameter and circumference. One may argue that this may lead to disturbed embryogenesis by less diffusion of nutrients from the mother to the embryo. Homocysteine attenuates haemodynamic responses to nitric oxide-related agnnists in anaesthetised rats. W. Fu r, N.P.B. Dudman;*, M.A. Perry". 1Centre Jbr Thrombosis and Vascular

Research, and 2School of Physioh)gy and Pharmacology, University of New South Wales, Sydney, Australia. Nitric oxide (NO) contributes to blood flow by inducing vascular relaxation and inhibiting platelet aggregation. We hypothesised that elevated homocysteine in vivo could antagonise these effects by sequestering NO to form S-nitrosohomocysteine. Our aim was to test whether homocysteine infusion in anaesthetised rats would influence haemodynamic responses to various injected NO-related vasorelaxants. Adult male Sprague-Dawley rats were anaesthetised with inactin. Femoral artery blood pressure was monitored, and blood samples were drawn from, and injections and infusions made through, a jugular catheter. NO-related compounds which were injected included sodium nitroprusside, acetylcholine, S-nitrosocysteine and S-nitrosohomocysteine. We also injected Verapamfl and Nicardipine as NO-unrelated vasodilator controls. Rat plasma homocysteine levels increased approximately 1l-fold during 90

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min infusion with low dose homocysteine, and 33-fold with high dose homocysteine. No change in rat blood pressure resuited. Treatment of rats with each NO-related compound led to a rapid and substantial lowering of mean arterial blood pressure. With each compound, concurrent homocysteine infusion decreased this fall in blood pressure (p<0.01), and substantially diminished the time required for the return of blood pressure to baseline (p<0.01). Homocysteine had no effect on either the magnitude of the blood pressure decrease or its recovery time, when Nicardipine or Verapamil were injected. Thus homocysteine, at plasma concentrations comparable with those found in human homocystinuria, selectively attenuated the haemodynamic effects of four NO-related vasorelaxant compounds administered intravenously in anaesthetised rats. The possibility that homocysteine-dependent attenuation of normal NO-related vascular functions could contribute to the development of vascular disease in human patients with hyperhomocysteinemia may be considered.

Homocysteine alters interactions between human leukocystes and vascular endothelial cells in vitro. X.W. Guo, N.P.B. Dudman*. Centre for Thrombosis and Vascular Research, Prince Henry Hospital, University of New South Wales, Sydney, Australia. Homocysteine treatment of human neutrophils (PMNs) increases their adhesion to, and damage of cultured human umbilical vein endothelial cells (HUVECs) (Guo XW, Dudman NPB. Homocysteine-treated human neutrophils damage endothelial cells in vitro. Proc. Aust. Soc. Med. Res. 1996;35:P119). Our goals were to test in vitro whether exposure of HUVECs to homocysteine might influence their interactions with monocytes, and whether homocysteine treatment of purified human monocytes might increase their damage of HUVECs. Monocytes from healthy human peripheral blood were purified by elutriation. We assessed the adhesion of SlCrO4-1oaded monocytes to, and migration through, HUVEC monolayers growing on 0.5/,tm filters. We also studied the detachment of SlCrO4.1oaded HUVECs co-cultured with homocysteinetreated monocytes, e-Homocysteine (50 ~tmol/L) pre-treatment of HUVECs clearly enhanced monocyte adhesion, p < 0.05, and migration, p < 0.05. Pre-treatment of monocytes with Lhomocysteine (50/,tmol/L) substantially increased HUVEC detachment, p<0.05. Detachment was inhibited by catalase (p <0.01), signalling a role for H202 Antibody studies indicated the involvement of monocyte surface antigens CDIlb, CD 18, and CD14 in homocysteine-induced monocyte adhesion (p<0.05 in each case), migration (p<0.05, CDllb/CDI8 only), and HUVEC detachment (p < 0.00l for each antigen). Homocysteine concentrations (10-50 /zmol/l), such as are found in patients with homocysteine-associated vascular disease, consistently induced changes of cell behaviour in HUVECs, monocytes, and in PMNs (p<0.05). These changes were concentration-dependent, and significantly selective for homocysteine over cysteine. The selective sensitivity of monocytes, PMNs and HUVECs to low concentrations of homocysteine, and the responses of these cell types, hint that homocysteine could play a physiological role in directing vascular leukocyte traffic.

Homocysteine inhibits cGMP accumulation in cultured porcine aortic endothelial cells via a mechanism involving superoxide anion production. M.B. Kredan*, D. Lang, C. Rix, M.J. Lewis. Cardiovascular Sciences Research Group, University of Wales College of Medicine, Cardiff CF4 4XN, UK. Elevated plasma homocysteine (HCS) levels are associated with premature cardiovascular disease and endothelial dysfunction. The mechanism of this HCS-induced endothelial dysfunction is however unknown. The present study investigated the effects of HCS on cultured porcine aortic endothelial cell (PAE) cGMP levels (as an index of nitric oxide (NO) release) and superoxide anion ( 0 2 - ) production. Cultured (lst passage) PAE were incubated for 24h at 37°C with HCS (lmM) or with culture medium alone. After this time, some cells were exposed to either of the NO-releasing agents, bradykinin (BK, 1 /3M for 90 s) or the calcium ionophore A23187 (1/.tM for 90 s). The cGMP content of these cells was measured by radioimmunoassay and normalised to cell protein content. The remaining cells were trypsin (0.05% w/v) digested and then harvested for measurement of 02- production by lucigenin (500 ~tM) chemiluminescence in the presence of 1% triton X-100. All data is expressed as mean + s.e.m, and n->4. Basal levels of cGMP were 1.17+0.14 fmol//tg protein and remained unaltered following incubation with HCS (0.98 + 0.13 fmol/gtg protein). Incubation with HCS did however cause a highly significant (P < 0.001) inhibition of the cGMP response to BK and A23187 (3.29+0.35 and 4.90+1.34 fmol//zg protein cf. 8.68+ 1.30 and 12.58+2.86 fmol//zg protein for BK and A23187 alone, respectively. In cells that were incubated with culture medium alone, basal 02- production was 35.85+2.47 mV.s/106 cells. This level was significantly (p < 0.001) increased following incubation with HCS (62.29 + 6.97 mV s/106 cells). Incubation of the cells with the intracellular 02 scavenger tiron (10 mM) at the same time as HCS, resulted in a significant (p < 0.001) inhibition of the HCS-induced increase in O~- production (32.13 + 2.45 mV s/106 cells). These data therefore demonstrate that exposure to HCS for 24 h inhibits the cultured PAE cGMP response to BK and A23187 via a mechanism which involves an increase in the intracellular production of 02-. These observations provide a possible mechanism for the endothelial dysfunction associated with hyperhomocysteinaemia. Circulating enzyme activity is influenced by plasma total homocysteine (tHey). S.J. Moat t*, J.R. Bonham2, H.J. PowersI. ~University Department of Paediatrics, and 2Department of Paediatric Chemical Pathology, Sheffield Children's Hospital, Sheffield. UK. Disturbance to endothelial cell function by elevated plasma homocysteine may be mediated by oxidative mechanisms, including the generation of hydrogen peroxide and superoxide. A study was conducted to explore the activity of antioxidant enzymes in subjects with cystathionine ,8-synthase (CBS) deficiency compared with their obligate heterozygous parents. Forty one fasting blood samples were collected from 10 CBS patients during routine monitoring. Single fasting blood sam-

Abstracts~Netherlands Journal of Medicine 52 (1998) S1 $61 pies were collected from 13 of their parents. Plasma tHcy was measured by HPLC, and red blood cell superoxide dismutase (SOD) and catalase activity, and red blood cell and plasma glutathione peroxidase (GSHPx) activity were measured by spectrophotometric methods automated for a centrifugal analyser. For data analysis, samples were classified as those with plasma tHcy < 20/~mol/l or -20~tmol/. Results: values are mean (SEM). Plasma tHcy (pmol/l) < 20 in = 17) 12.7 (0.91); Red blood cell enzyme activity (U/glib): SOD 5098 (122.7), Catalase 194 (5.7), GSHPx 34 (2.7); Plasma GSHPx (U/l) 781 (45.1). Plasma tHcy (grnol/l) _>20 (n=37) 112.8 (11.02); Red blood cell enzyme activity (U/glib): SOD 5699** (90.0), Catalase 204 (4.2), GSHPx 35 (1.2); Plasma GSHPx (U/l) 906* (31.4). Significantly higher than group with tHcy <20 /tmol/1 (Student's t-test). **P<0.001, *P < 0.02. Neither RBC catalase nor GSHPx activity showed any association with plasma tHcy. In contrast, red blood cell SOD and plasma GSHPx activity were significantly higher in subjects with tHcy ---20/tmol/l. Furthermore, plasma GSHPx activity showed a significant positive association with plasma tHcy (r = 0.626, P < 0.001). Results suggest an enzymic response to oxidative stress imposed by elevated plasma tHcy in vivo, possibly at the level of protein synthesis.

Lessons from homocystinuria Coexistence of methylenetetrahydrofolate reductase polymorphism and factor V Leiden in vascular disease. M. Candito 1., D. Jambou2, P. Gibelin3, P. Bedoucha4, J.L. Sadoul 5, F. Fisher:, M. Chatel 4, E. Van Obberghen1. 1Biochemistry,

2Hematology, 3Cardiology, 4Neurology, 5Diabetology, CHU Nice, Hrpital Pasteur, BP 69, 06002 Nice Cedex, France. Mild hyperhomocysteinemia has been described in vascular disease. Among genetic causes, the C677T mutation on the methylenetetrahydrofolate reductase gene (MTHFR) is correlated with moderately increased homocysteine levels (HCY), but the frequency of the homozygous mutant (+/+) is similar in both patients and controls (approx. 12%). No association has been reported in the literature between factor V Leiden (the most frequent cause of familial thrombosis) and increased risk of coronary heart disease or cerebrovascular disease; However, this factor might increase arterial and venous thrombosis in patients with hereditary homocystinuria. We first searched for the C677T mutation on the MTHFR gene in 70 consecutive cardiovascular patients; we then sought for factor V Leiden in the 14 patients homozygous mutants (20%). Coexistence of the homozygous C677T mutation on MTHFR and factor V Leiden was found in 3 patients: one suffered from multiple venous thrombosis, as expected, and 2 suffered from coronary heart disease. In cardiovascular disease, factor V Leiden might aggravate the pathological vascular effect caused by the C677T mutation on the MTHFR gene.

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Raised in vivo thromboxane biosynthesis in heterozygotes for cystathionine-~-synthase deficiency. Similarities and differences with the homozygous state. G. Davi t, A. Coppola, P. Madonna*, U. Piemontino, R. Albisinni, P. Strisciuglio 2, C. Perricone3, G. Andria2, G. Di Minno. Departments of Clinical

and Experimental Medicine and 2pediatrics, University of Naples "Federico H' and 3Division of Hematology, Pausillipon Hospital Naples; t Hematology, University 'G. D'Annunzio', ChietL Italy. Homocystinuria due to cystathionine-fl-synthase deficiency (CBSD) is characterized by a tendency to thrombosis and premature atherosclerosis. In homozygotes for CBSD, we have previously shown an abnormally high in vivo biosynthesis of thromboxane A2 (TXA2), as reflected by the urinary excretion of its major metabolite, 11-dehydro-thromboxane B2 (TXM). We have now evaluated TXA2 biosynthesis in 16 heterozygotes for CBSD (7 M, 9 F, 8-60 yrs old). Ex vivo patient platelet function was within control values. In contrast, TXM excretion was > 2 SD of controls in 11 out of the 16 subjects (943+649 pg/mg creatinine, mean+SD, in patients vs. 345 + 136 in matched controls, p<0.05). Similar to homozygous subjects, the administration to heterozygotes (n=41 of low-dose aspirin (50 mg/d for 1 wk) reduced TXM excretion by =80% (145+71 pg/mg creatinine, p<0.001 vs. pre-aspirin), its level slowly recovering to basal values over the 10 d that followed aspirin withdrawal (302 _+143 and 730 + 257 pg/ mg creatinine, respectively, 4 and 10 d after aspirin cessation). However, at variance with bomozygous patients, no significant reduction in the abnormally high TXM excretion was observed in heterozygotes (n = 7) who ingested 500 mg/d for 3 wks of the antioxidant drug probucol (755 + 450 pg/mg creatinine in pretreatment samples, 681 + 441 and 569 +_295 at withdrawal and 3 wks after withdrawing probucol, p > 0.05 vs. pre-treatment). Moreover several hemostatic indices (antithrombin III, protein C, Protein S, thrombomodulin, t-PA, PAl-l) that reflect a hypercoagulable state were within normal values in this setting, and did not correlate with TXM excretion. Our results extend to heterozygous CBSD the evidence of enhanced TXM excretion and argue against similarities between the two settings in the mechanisms leading to the elevated TXA2 biosynthesis.

Carotid and femoral artery thickness and stiffness in patients at risk for cardiovascular disease, with special emphasis on hyperhomoeysteinemia. T.J. Smilde:*, F.W.M.F. ,,'an den BerkmorteP, G.H.J. Boers 1, H. Wollersheim~, H. van Langen2, A.F.H. Stalenhod. ~Dept. of Internal Medicine,

2C\lin. Vascular Laboratory, University Hospital St Radboud Nijmegen, The Netherlamts. Background: The effect of homocysteine on the arterial wall is still unclear: reports on intima-media thickness (IMT) yield conflicting results, whereas data on vessel wall stiffness are lacking. As several cardiovascular risk factors result in an increased IMT or stiffness, different groups at risk for atherosclerotic disease, with special emphasis on hypcrhomocysteinemia, were studied.

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Abstracts / Netherlands Journal oJ Medicine 52 (1998) S1-$61

Methods: 19 patients homozygous and 14 subjects heterozygous for Cystathionine-fl-Synthase (CS) deficiency, 21 patients with familial hypercholesterolemia (FH), 15 patients with essential hypertension, 20 smokers and 28 control subjects, respectively, ~¢ere studied. T'ae IMT's (both right arid ?eft'¢ of the common carotid artery (CCA, far and near wall), bulb (BUL, far and near wa'l'l~t,interna'~ carotid artery ~'ICA, ?ar wa~) and common femoral artery (CFA, far wall) were measured in mm by high resolution ultrasound (Biosound). The distensibility (DC in 10 ~ kPa -~) and compliance (CC in mm 2 kPa 1) of the CCA (right and left) and C F A (right side) were determined by a wall track system (Pie Medical). Results: The groups were comparable regarding sex and age. The mean IMT in the CCA was 0.73 + 0.09 mm in healthy controls. For patients with FH, hypertension and smokers the mean CCA-IMT was significantly increased, 0.99+0.23 mm, 1.02+_0.17 m m and 0.92+_0.16 mm, respectively. The IMT in the CCA, BUL, and ICA was not significantly different either in homo- or heterozygotes for CS deficiency when compared to healthy subjects. The DC and CC in the right CCA were 23.5+_6.9 (10 -3 kPa - l ) and 0.9+0.3 (mm 2 kPa 1) in healthy subjects, 14.8+6.7 and 0.6+0.3 in patients with FH, 9.9+5.2 and 0.4=0.2 in hypertensives, and 17.7_+5.7 and 0.7+_0.3 in smokers, respectively, all significantly decreased. The DC of the right CCA was significantly decreased in homozygotes for CS deficiency and also a significant difference in ~ g a~c) D C )n ~ e UJa)~ ~vas Yozm~) belwee,9 )~oxoogvgotes and heterozygotes for CS deficiency versus controls. However, no positive correlation was found between plasma homocysteine level and either IMT, CC and DC. In a stepwise regression analysis plasma homocysteine concentration only explained a small proportion of the variation in decrease in DC and CC. Conclusions: In this group sample no relationship was found between the homocysteine level and thickness of the arterial wall and only a marginal one with stiffness. Factor V Leiden (AS06G) and thrombosis in homozygotes and obligate heterozygotes for eystathionine ~-synthase deficiency, S. Yap I*, K.A. O'Donnell 2, C. O'Neill 2, P.D. Mayne 2, P. Thornton 1, E. Naughten j . National CentreJbr Inborn Metabolic Disorders I and the Dept of Pathology 2, The Children's Hospital, Temple Street, Dublin 1, Ireland. Homozygosity and heterozygosity for Factor V Leiden (A506G; FVL) are associated with an increased risk of thrombosis, particularly with additional risk factors. Thrombosis is a recognise-~ q,~tpli',:tt;~i'o~. ' ~ 'i'b~'n~)~ht a'~h, ,('~C~~', -e.~a'~?o nine fl-synthase deficiency). Patients with HCU and FVL have been shcwn to flare an increase
programme and were treated at diagnosis. No individual demonstrated any clinical evidence of thrombosis at the time of investigation. D N A was analysed for FVL using PCR amplification followed by endonuclease digestion (Mnll). Two independent H C U patients (ma~e ~4.8 yrs; femalre ir8.2 yrs) were heterozygous for FVL (allelic frequency 3.8%) and two H C U oNigate heterozygotes {ma~e 45.~ yrs; fem-~e 45.6 yrs) were also heterozygous for FVL (allelic frequency 4.3%). Two H C U patients heterozygous for FVL and treated from birth demonstrated no clinical evidence o f thrombosis (mean age 16.5 yrs), in contrast to the findings of Mandel et al., where 4 of 4 HCU patients, homo- or heterozygous for FVL developed thrombosis by the mean age of 7,5 yrs. This study in a small group suggests that treatment of HCU not only reduces the thrombotic risk in patients with isolated HCU but also in those with FVL heterozygosity. The implication of detecting FVL in HCU cases, however, may indicate a more rigorous anticoagulation therapy.

Are spina bifida occulta and mild hyperhomocysteinemia related? D.G. Franssen-Franken 1., G.H.J. Boers2, A. Lemmens 1. I Department of Radiology, 2Department of Internal Medicine, University Hospital St Radboud Nijmegen, The Netherlands. Mild hyperhomocysteinemia, a risk factor for premature arteriosclerosis and thrombosis, can also frequently be established in mothers with a neural tube defect child. In line with culta, a minor neural tube defect, is also related to mild hyperhomocysteinemia. To e×9lore this hypothesis, in 138 out o f 727 vascular patients younger than 55 years of age, who were screened for mild hyperhomocysteinemia in the period January 1980 until June 1993, plain abdominal or lumbar spine X-ray film had been made for various reasons. A spina bifida occulta at lumbal 5, sacral 1 and sacral 2 was detected in 25 out of 55 patients (45%) with, and in 18 out of 83 (22'7,,) patients without mild hyperhomocysteinemia, which implies a relative risk (RR) of 1.84 (95% confidence interval (CI) van 1,25 tot 2.72), In two-third of the premenopausal women (18 out of 27) with spina bifida occulta mild hyperhomocysteinemia was detected. Moreover, 25 (45%) and 27 (32%) patients with and without mild hyperhomocysteinemia, respectively, showed arteriosclerosis also in the abdominal aorta or lilac arteries on the X-ray film (RR 1.38; 95% CI 0.92 to 2.06). In conclusion, an association between mild hyperhomocysteinemia and spina bifida occulta is indicated.

Detection of four novel mutations in the cystathionine beta-synCBS-defieiency. M. Gaustadnes 1., N. Riidiger ~, O. Kusk 2, K. R a s m ~ s e n ~ F. SkovbyS~ H. Biota ¢, J~ Krans s, J. I~3ersJev-'. 1Department of Clinical Biochemistry, Sktjby University Hospital, Aarhus, Denmark: 2Centre jbr Haemophilia and Thrombosis, Skejby University Hospital, Aarhus, Denmark: 3 5Department of Pediatrics." 3Rigshospitalet, Copenhagen, Denmark: 4 University Hospital Niflnegen, The Netherlands: 5 University oJ Colorado, Denver, CO, USA.

Abstracts~Netherlands Journal of Medicine 52 (1998) S I $61 Mutations in the cystathionine beta-synthase (CBS) gene are known to cause homocystinuria, a risk factor for premature atherosclerosis. At present, we know of four Danish patients with homocystinuria due to CBS-deficiency. We sequenced the entire coding region of the CBS gene in these four patients to detect disease causing mutations. All patients were compound heterozygous. Patient 1 is a 37 y old man with epilepsy and mental retardation without any thromboembolic episodes. On one allele a splice mutation that causes skipping of exon 8 was detected. The mutation was detected by sequencing of genomic DNA and cDNA. On the other allele a missense mutation resulting in the amino acid substitution I ~ T in exon 12 was detected. The fibroblast CBS-activity of the patient is virtually zero. Patient 2 is a 45 y old woman who had no thromboembolic episodes. She is heterozygous for the D444N mutation. On the other allele we detected a missense mutation resulting in a G ~ A substitution in exon 2. Patient 3 is a man of 34 y and patient 4 is his sister of 22 y. Both patients experienced thrombosis before the age of 20 and the brother is mentally affected. Both patients are heterozygous for the 1278T mutation. On the other allele we identified a 22 bp deletion of exon 4 followed by a stop codon. This defect was verified by sequencing of genomic DNA. Sequencing of cDNA from these patients showed lack of transcription from the deleted allele. We are currently undertaking the molecular characterization of these novel mutations. Our findings of private mutations in the four Danish patients are consistent with the results of other groups that CBS-deficiency is caused by a wide heterogeneity of mutations in the CBS gene. The molecular basis of homocystinuria due to cystathionine [3-synthase (CBS) deficiency in the Netherlands. Effect of CBS genotype on biochemical and clinical phenotype in patients and on response upon pyridoxine treatment. L.A.J. Kluijtmans:*, G.H.J. Boers:, M.C.M. De Visser1, J.P. Kraus 3, E.M.B. Stevens 1, L.P.W.J. Van den Heuvel:, J.M.F. Trijbels 1, H.J. Blom:. Depts. ~f tPediatrics. 2Internal Medicine, University

Hospital Nijmegen. the Netherlands'. 3Dept. ~f Pediatrics, University of Colorado, Denver, CO, USA. Introduction: Homocystinuria due to homozygous cystathionine/3-synthase (CBS) deficiency is the most common inborn error of methionine metabolism. Biochemically, patients are characterized by severe hyperhomocysteinemia, hypermethioninemia, and hypocysteinemia. CBS activities in extracts of cultured fibroblasts are less than 2% of the control mean in most patients. Methods: We investigated the molecular basis of CBS deficiency in 29 Dutch homocystinuria patients from 22 unrelated pedigrees. CBS genotypes were associated to clinical and biochemical phenotype at diagnosis, and clinical effect of pyridoxine treatment. Results: Myopia/Ectopia lentis, Homozygotes TB33C (n = 12) 100%/75%, Other genotypes (n = 17) 71°A,/76%; Arteriosclerosis/Thrombosis, H 33%/33%, O 18%/24%; Osteopo-

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rosis, H 50%, O 53%; Scoliosis, H 67%, O 82%; Marfanoid features, H 67%, O 59%; Mental retardation, H 33%, O 47%; Convulsions, H 8%, O 35%; Free Hcy (median [range]), H 106 [47-266], O 102 [42~04]; B6-responsiveness ( > 7 5 % reduction), H 100%, O 59%. On a total of 412 patient-years of treatment, 2 vascular events occurred, whereas, without an effect of treatment, 16 accidents would have been expected (Mudd et al. Am J Hum Genet. 1985;37:1-31; Z ~= 9 .2, P < 0.005). Conclusions: Homozygosity for the T833C (1278T) mutation, which is the most frequently observed genotype in Dutch patients, was associated with pyridoxine responsiveness. Homocysteine lowering therapy markedly reduced cardiovascular risk in CBS deficient patients. Homocysteine influences the activation of human polymorphonuclear leukocytes in vitro and in vivo. U. Till 1., H. V61ksch 1, M. G6tzrath 1, H.S. Fink I, (3. Briitsch:, R. Riezler2, K. Bellstedt3.

1Centre ~[ Vascular Biology and Medicine, University of Jena, Erfi~rt, 2Severi-Med. Miinster, 3Clinic of Pediatrics, University of Jena, Jena, Germany. Investigations on the underlying mechanisms of the atherogenic and thrombogenic actions of homocysteine (HC) suggest endothelial cell damage, increased platelet reactivity, oxidative modification of LDL, and enhanced affinity of Lp(a) to fibrin. To our knowledge no results are published on the influence of HC on polymorphonuclear leukocytes (PMNL) although these cells are deeply involved in pathological events within the vasculature. Different functional parameters of human P M N L were followed (1) in vitro under incubation with 60, 300, and 600 ¢tM DL-HC in isolated fractions or whole blood, respectively, and (2) in vivo in whole blood samples from healthy volunteers after methionine loading. Ad (1): Ca2+-ionophore induced exposition of C D I l b at the membrane (flowcytometric evaluation) is enhanced by all HC concentrations but mostly with 60/zM. Spontaneous mobility of PMNL, measured as migration distance into micropore filters in a modified Boyden-chamber, is significantly enhanced by the two smaller HC concentrations. F M L P induced chemiluminescence (Auto Lumat LB953, Berthold) is most increased by the medium HC concentration. Phagocytosis induced by zymosan (microscopic evaluation) as well as by opsonized E. coli (flowcytometric evaluation) is significantly increased by the two higher HC concentrations. Ad (2): Temporary and moderate increase of plasma HC (between 25 and 40 /3M) is accompanied by significant enhancement of P M N L count and phagocytosis. CD1 lb exposition tends to be increased. Chemiluminescence shows deviations in both directions dependent on the donor. Influence of cysteine and other amino acids could be excluded. Conclusion: The activation of human PMNL is enhanced with respect to most of the investigated stimuli by HC in concentrations reached under pathophysiological conditions.