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Methionine loading test is necessary for detection of hyperhomocysteinemia
Methionine loading test is necessary for detection of hyperhomocysteinemia REN~: VAN DERGRIEND, FRED J. L. M. HAAS, MARINUS DURAN, DOUWE H. BIESMA, OTGER J. A. TH. MEUWISSEN, AND JAN-DIRK BANGA NIEUWEGEIN AND UTRECHT,THE NETHERLANDS
Hyperhomocysteinemia, defined as an elevated concentration of homocysteine 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 (MLT) is, however, controversial. To determine the additional value of the MLT for diagnosis of hyperhomocysteinemia, we prospectively studied 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, cobalamin, pyridoxine, and creatinine concentrations were measured. Hyperhomocysteinemia was defined as a fasting homocysteine concentration and/or an increase in homocysteine concentration after methionine loading exceeding the 95th percentile of a healthy control group. Hyperhomocysteinemia was found in 141 (33%) of the 429 subjects: 15% were diagnosed by fasting homocysteine concentration and 18% by MLT.Seventy-eight (55%) of the 141 hyperhomocysteinemic persons were diagnosed only by the MLT. Folic acid was lower in the group with an elevated fasting homocysteine concentration than in those with only an abnormal MLT result (I I versus 15 nmol/L, p = 0.002). Folic acid was significantly negatively correlated, and creatinine significantly positively correlated, with both fasting homocysteine concentration and increase in homocysteine concentration. Negative correlations of cobalamin and pyridoxine with fasting homocysteine concentration were found. In conclusion, the MLT is necessary for diagnosis of hyperhomocysteinemia, because a considerable number of hyperhomocysteinemic persons (55%) remain undiagnosed with the determination of a fasting homocysteine concentration alone. (J Lab Clin Med 1998;132:67-72)
Abbreviations: HPLC= High performance liquid chromatography; MLT= methionine loading test
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lassic homozygous homocystinuria is associated with premature t h r o m b o e m b o l i c events due to markedly elevated homocysteine concentrations. 1 M o r e than 25 years ago, the MLT was From the Departments of Internal Medicine and Clinical Chemistry Sint Antonius Hospital, Nieuwegein; the Department of Metabolic Disease, University Children's Hospital "Het Wilhelmina Kinderziekenhuis," Utrecht; and the Department of Internal Medicine, University Hospital Utrecht. Submitted for publication Oct. 29, 1997; revision submitted Feb. 4, 1998; accepted March 11, 1998. Reprint requests: Ren6 van der Griend, MD, Department of Internal Medicine, F02.126, UniversityHospital, Post Office Box 85500, 3508 GA, Utrecht, The Netherlands. Copyright © 1998 by Mosby, Inc. 0022-2143/98 $5.00 + 0 5/1/90336
introduced for the detection of heterozygotes for homocystinuria. 2 In 1976, the MLT was used for the first time to detect moderate hyperhomocysteinemia in men with premature cardiovascular disease, 3 Since then, several studies have c o n f i r m e d that h y p e r h o m o c y s t e i n e m i a , detected by elevated fasting and/or post-load homocysteine concentrations, is an independent risk factor for both atherosclerotic disease and venous thrombosis. 41t There is, however, discussion about the role o f the MLT in the detection o f h y p e r h o m o c y s t e i n e m i a . The MLT has logistic limitations. It has been argued that the test is not p h y s i o l o g i c and, moreover, that it adds no information to the fasting homocysteine determination alone. 12 On the other hand, the clinical relevance o f the MLT is supported by observations o f a higher
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Table I. Subject characteristics, plasma total h o m o c y s t e i n e , a n d p r e v a l e n c e of h y p e r h o m o c y s t e i n e m i a as
d e f i n e d by a n e l e v a t e d fasting h o m o c y s t e i n e c o n c e n t r a t i o n a n d an a b n o r m a l MLT in 281 patients w i t h prem a t u r e arterial disease a n d 148 first-degree relatives Parameter
Patients (N = 281)
Relatives (N = 148)
Total (N = 429)
Characteristics
Mean age _+SD (yr) Gender (% male) Creatinine (gmol/L) Folic acid (nmol/L) Pyridoxine (nmol/L) Cobalamin (pmol/L) Plasma total homocysteine (gmol/L) Fasting homocysteine Increase in homocysteine Prevalence of hyperhomocysteinemia (no. [%]) Hyperhomocysteinemia Elevated fasting homocysteine Abnormal MLT
42.7 _+9.1" 57 79 (61-105) 15 (9-36)1 64 (39-160) 300 (180-548)
37.8 _+11.2 48 77 (60-97) 17 (10-42) 70 (42-122) 270 (170-532)
40,9 + 10.2 54 78 (60-102) 16 (9-36) 65 (41-140) 300 (174-532)
12.4 (8.2-24.6)* 33.4 (20.7-70.4)
11.5 (6.8-21.6) 32.3 (21.6-63.2)
12,1 (7.6-23.4) 32,7 (21.4-67.6)
98 (35) 48 (17)1" 50 (18)
43 (29) 15 (10) 28 (19)
141 (33) 63 (15) 78 (18)
Creatinine, vitamin and homocysteine concentrations represent medians (10th to 95th percentile), *p < 0,001 for patients versus relatives, tp : 0,05 for patients versus relatives. ~p : 0,005 for patients versus relatives.
risk for arterial disease and venous thrombosis in patients with hyperhomocysteinemia as determined by an abnormal MLT result only, compared with persons without hyperhomocysteinemia.6J3-15 To assess the contribution of the MLT to the detection of hyperhomocysteinemia, a large group of patients with premature arterial disease and their first-degree relatives was studied. METHODS Subjects. From January 1992 to August 1996, 429 persons
were screened for hyperhomocysteinemia; there were 281 patients with documented premature peripheral, cerebral, or coronary artery disease (before 50 years of age) and 148 unaffected first-degree relatives. All patients were referred by vascular surgeons, neurologists, or cardiologists after assessment of the vascular problem. First-degree relatives were screened on their own request after detection of the index patient. Laboratory investigations. All persons underwent an oral MLT (0.1 grrdkg body weight of 1-methionine) after an overnight fast. L-Methionine was dissolved in orange juice. During the test only low-methionine nutrients were allowed. Women were tested on the 20th through the 22nd day of a menstrual cycle. 16 Blood was sampled before and 6 hours after ingestion, collected in tubes containing lithium-heparin, and immediately centrifuged; plasma was frozen at -20 ° C and stored until assayed. Plasma total homocysteine was measured by HPLC with fluorescence detection as described by Araki and Sako. 17 This method has an interassay coefficient of variation of <7%. Determination of plasma folic acid and cobalamin was performed by a dual-count radioassay (Diagnostic Products Corporation, Los Angeles, Calif.), pyridoxine by fluorescence
HPLC technique, and creatinine according to a modified Jaffe reaction (Dimension, Du Pont, Wilmington, Del.). Definition of hyperhomocysteinemia. Hyperhomocysteinemia was defined as fasting homocysteine concentration and/or an increase in homocysteine concentration (concentration after methionine loading minus fasting concentration) exceeding the 95th percentile of controls. The normal range was derived from 68 healthy volunteers (38 women and 30 men); their mean age was 35 years (range, 18 to 50 years). Their homocysteine values showed a non-normal distribution with a positive skew. The 95th percentile for fasting homocysteine concentration was 16.3 gmol/L in women and 18.8 gmol/L in men; the 95th percentile for increase in homocysteine concentration after loading was 42.3 gmol/L for both sexes. Our reference values are similar to those from two other Dutch reference populations.6J8 Statistical methods. Homocysteine, creatinine, and vitamin concentrations are expressed as median (10th to 95th percentile). The Wilcoxon rank-sum test was used to analyze differences for these variables between patients and relatives and between the different groups stratified for methionine loading outcomes. The Student's t-test was used to analyze differences in mean age. The Pearson X2 test was used to compare frequency measurements between patients and relatives. Spearman correlation coefficients were calculated to study the relations between the variables. All reported probability values are two-tailed, and a value of <0.05 was considered significant. For the analysis, SPSS software was used. RESULTS
Table I lists the characteristics, homocysteine concentrations, and prevalence of hyperhomocysteinemia as defined by an elevated fasting homocysteine concen-
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Table II. Characteristics of subjects stratified for t h e h o m o c y s t e i n e o u t c o m e s d u r i n g m e t h i o n i n e l o a d i n g
Characteristic
Elevated fasting homocysteine (N = 63)
Gender (% male) Mean age _+SD (yr) Patients (%) Creatinine (/.tmol/L) Felic acid (nmol/L) Pyridoxine (nmol/L) Cobalamin (pmol/L)
44 43.7 + 8.6 76 80 (60-136) 11 (7-23)t 56 (37-249) 270 (164-504)§
Abnormal MLT only (N = 78)
50 43.5 _+9.6 64 80 (63-105) 15 (9-30)2 65 (41-107) 280 (160-585)
Normal homocysteine (N = 288)
57 39.4 _+10.5* 64 77 (60-99) 17 (10-41) 66 (41-144) 310 (190-540)
Creatinine and vitamin values represent medians (10th to 95th percentile). *p = 0.004 for normal homocysteine versus elevated fasting homocysteine and p = 0.002 for normal homocysteine versus abnormal MLT. t p = 0.002 for elevated fasting homocysteine versus abnormal MLT and p < 0,001 for elevated fasting homocysteine versus normal homocysteine, ~p = 0.008 for abnormal MLT versus normal homocysteine. §p = 0.005 for elevated fasting homocysteine versus normal homocysteine.
tration and an abnormal MLT in patients and relatives. Patients had a higher mean age, a lower folic acid concentration, and a higher median fasting homocysteine concentration than relatives. The prevalence of hyperhomocysteinemia in the total group was 33% (141 persons). In 15% the condition was detected by an elevated fasting homocysteine concentration (6% had a normal increase in homocysteine after methionine loading and 9% had elevated concentrations in both instances); in 18%, hyperhomocysteinemia was detected by an abnormal MLT result only. Those who had elevated homocysteine concentrations with both fasting and methionine loading were included in the former group, since the MLT was not necessary for the detection of their hyperhomocysteinemia. Among the 141 persons defined as having hyperhomocysteinemia, 63 (45%) had an elevated fasting homocysteine concentration and 78 (55%) were diagnosed after MLT. A significantly higher percentage of patients had an elevated fasting homocysteine concentration, compared with relatives. No differences were found in the percentages of hyperhomocysteinemic persons diagnosed only after MLT between men and women (19% versus 24%, p = 0.29) or between patients and relatives (both 21%). Table II summarizes the characteristics of the study subjects stratified for the homocysteine outcomes during methionine loading. All vitamin concentrations were in the reference range. No differences between the two hyperhomocysteinemic subgroups (i.e., those detected by an elevated fasting homocysteine concentration and those diagnosed only by an abnormal MLT result) were found with respect to gender, age, category (patient or relative), creatinine, pyridoxine, or cobalamin. Only folic acid was significantly lower in hyperhomocysteinemic persons diagnosed by an elevated
fasting homocysteine concentration compared with persons diagnosed by an abnormal MLT result. Folic acid was significantly lower in both hyperhomocysteinemic subgroups, compared with persons without hyperhomocysteinemia. Cobalamin was significantly lower in the subgroup with elevated fasting homocysteine, compared with persons without hyperhomocysteinemia. Seventy-eight (21%) of 366 persons with a normal fasting homocysteine concentration had an abnormal MLT result. In Fig. 1, hyperhomocysteinemic persons whose fasting homocysteine concentration was normal are represented in the left upper quadrant. Data points for normal subjects are found in the left lower quadrant. Reducing the threshold for a normal fasting homocysteine concentration to 15 gmol/L and then to 12 gmol/L resulted in diagnosis by an abnormal MLT in 62 (20%) of 314 persons and 30 (14%) of 213 persons, respectively. There was a strong negative correlation of folic acid with both the fasting concentration and the increase in homocysteine concentration (r = -0.41 and -0.22, respectively; p < 0.001); there was also a strong positive correlation of creatinine concentration with the same parameters (r = 0.32 and 0.18, respectively; p < 0.001). A negative correlation with fasting homocysteine concentration was found for cobalamin (r = -0.17; p = 0.001) and for pyridoxine (r = -0.11; p = 0.05). However, after calculation of a partial correlation coefficient, with adjustment for the two most important variables (folic acid and creatinine), the correlation with fasting homocysteine of cobalamin appeared much lower (r = -0.11; p = 0.05) and that for pyridoxine disappeared. DISCUSSION Hyperhomocysteinemia is now recognized as a serious risk factor for premature atherosclerosis and venous thrombosis. This study demonstrates the essential role
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Fig. 1. A graphical presentation of the individual values for fasting homocysteine concentration and increase in homocysteine concentration after MLT for 230 men and 199 women. The reference lines represent the 95th percentile of the control group (fasting homocysteine, 18.8 gmol/L for men and 16.3 gmol/L for women; increase in homocysteine, 42.3 gmol/L for both sexes).
of the MLT in the detection of hyperhomocysteinemia: 55% of all hyperhomocysteinemic persons were identified only after methionine loading. Our findings are in accordance with previous observations regarding value of the MLT. 7,14,19 In patients with early-onset venous or arterial thromboembolic disease, the prevalence of hyperhomocysteinemia was almost twice as high after methionine loading. 7 In a population-based investigation, more than 40% of the hyperhomocysteinemic volunteers had a normal fasting homocysteine concentration, 19 Both smaller studies used different postload homocysteine measurements (8 and 4 hours, respectively), and the vitamin status was not given. Very recently, the largest study so far demonstrated that 101 (27%) of 375 hyperhomocysteinemic arterial disease patients were identified only by MLT.14 This percent-
age was smaller than in our study, probably because a relatively low cutoff point for the fasting homocysteine concentration was defined (12 gmol/L). Our results refute recent correspondence stating that methionine loading has little additional value. 12 Both the elevated fasting homocysteine concentration and the concentration after methionine loading are reported to represent independent risk factors for arterial disease and venous thrombosis. 6,13-15 Only the elevated postload concentration indicated an increased risk for a first episode of deep-vein thrombosis. 20 Furthermore, from pooled data of more than 500 patients with arterial disease and more than 300 control subjects, an odds ratio of 13.0 (95% confidence interval, 5.9 to 28.1) was calculated for persons with an abnormal MLT result compared with normal responders.13 Among young patients
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with l o w e r - e x t r e m i t y arterial disease, the severity of atherosclerosis appeared to be even more closely related to the h o m o c y s t e i n e concentration after MLT than to the fasting homocysteine concentration. 21 Moreover, in a large European multicenter study including 750 subjects with atherosclerotic vascular disease, the relative risk for vascular disease in patients with hyperhomocysteinemia increased from 1.6 in those with elevated fasting h o m o c y s t e i n e only to 2.5 in patients with both determinants elevated. 14 Interestingly, elevated fasting homocysteine concentrations occurred significantly less often among the younger and asymptomatic relatives than among the patients. Furthermore, Franken et al. 22 found that if the index patient had an elevated fasting h o m o c y s t e i n e concentration, 29% o f the h y p e r h o m o c y s t e i n e m i c relatives showed a normal fasting value. Both are arguments in favor of performing the MLT as part of the screening for familial hyperhomocysteinemia. Several investigators have reported an increased risk o f vascular disease at h o m o c y s t e i n e concentrations b e l o w the 95th percentile cutoff as used in this study. Reducing the cutoff point for fasting h o m o c y s t e i n e increases the n u m b e r o f h y p e r h o m o c y s t e i n e m i c persons d i a g n o s e d by a r a n d o m b l o o d sample. We observed that the percentage of persons with hyperhom o c y s t e i n e m i a who could be detected only b y an abnormal MLT result does not decrease until the cutoff point for fasting homocysteine is reduced to as low as 12 g m o l / L or less. In this study, folic acid and creatinine concentrations were important determinants for both the fasting homocysteine concentration and the increase with methionine loading. This is in accordance with earlier results indicating an inverse correlation of folic acid and cobalamin and a positive correlation of creatinine concentration with fasting homocysteine. 23 Only very recently have the determinants o f h o m o c y s t e i n e concentration both before and after methionine loading been studied in the general population. 24 A low folic acid concentration and a c o m m o n mutation (677C-+T) in the methy l e n e t e t r a h y d r o f o l a t e reductase gene 18 were strong determinants o f h y p e r h o m o c y s t e i n e m i a in the fasting state and after methionine loading, again demonstrating the key role of folic acid. The creatinine concentration influenced only the fasting homocysteine concentration. Our results indicate that the MLT m a y identify more than 50% o f subjects with h y p e r h o m o c y s t e i n e m i a . These persons w o u l d not have been d i a g n o s e d b y screening with a fasting plasma homocysteine determination alone. A l t h o u g h our study was p e r f o r m e d in patients with p r e m a t u r e arterial vascular disease and their healthy relatives, it seems reasonable to extend the
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findings of the value of the MLT to patients with venous thrombosis and arterial disease as a whole. Together with the observations that postload hyperhomocysteinemia is an independent risk factor for both atherosclerosis and venous thrombosis, we conclude that the MLT remains essential in the detection o f h y p e r h o m o c y s teinemia. The authors thank Marijke van Kooten, medical secretary, for her excellent assistance in performing the MLTs and Helma Scheepers and Barbara van Sterkenburg for collecting the blood samples. REFERENCES
1. Mudd SH, Levy HL, Skovby F. Disorders of transsulfuration. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The metabolic and molecular bases of inherited disease. 7th ed. New York: McGraw-Hill, 1995:1279-327. 2. Fowler B, Sardharwalla IB, Robins AJ. The detection of heterozygotes for homocystinuria by oral loading with Lmethionine. Biochem J 1971;122:23P-4R 3. Wilcken DEL, Wilcken B. The pathogenesis of coronary artery disease: a possible role for methionine metabolism. J Clin Invest 1976;57:1079-82. 4. Boers GHJ, Smals AGH, Trijbels FJM, Fowler B, Bakkeren JAJM, Schoonderwaldt HC, et al. Heterozygosity for homocystinuria in premature peripheral and cerebral occlusive arterial disease. N Engl J Med 1985;313:709-15. 5. Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B, et al. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med 1991;324:1149-55. 6. den Heijer M, Blom HJ, Gerrits WBJ, Rosendaal FR, Haak HL, Wijermans PW, et al. Is hyperhomocysteinemia a risk factor for recurrent venous thrombosis? Lancet 1995; 345:882-5. 7. Fermo I, Vigano' D'Angelo S, Paroni R, Mazzola G, Calori G, D'Angelo A. Prevalence of moderate hyperhomocysteinemia in patients with early-onset venous and arterial occlusive disease. Ann Intern Med 1995;123:747-53. 8. den Heijer M, Koster T, Blom HJ, Bos GMJ, Briet E, Reitsma PH, et al. Hyperhomocysteinaemia as a risk factor for deep-vein thrombosis. N Engl J Med 1996;334:759-62. 9. Stampfer MJ, Malinow MR, Willett WC, Newcomer LM, Upson B, Ullman D, et al. A prospective study of plasma homocyst(e)ine and risk of myocardial infarction in US physicians. JAMA 1992;268:877-81. 10. Perry IJ, Refsum H, Morris RW, Ebrahim SB, Ueland PM, Shaper AG. Prospective study of serum total homocysteine concentration and risk of stroke in middle-aged British men. Lancet 1995;346:1395-8. 11. Arnesen E, Refsum H, BCnaa KH, Ueland PM, F#rde OV, Nordrehaug JE. Serum total homocysteine and coronary heart disease. Int J Epidemiol 1995;24:704-9. 12. den Heijer M, Blom HJ, Rosendaal FR. Hyperhomocysteinemia as a risk factor for deep-vein thrombosis [correspondence]. N Engl J Med 1996;335:975-6. 13. Ueland PM, Refsum H, Brattstrtm L. Plasma homocysteine and cardiovascular disease. In: Francis RB Jr, editor. Atherosclerotic cardiovascular disease, hemostasis, and endothelial function. New York: Marcel Dekker; 1992:183-236. 14. Graham IM, Daly LE, Refsum HM, Robinson K, Brattstrtm LE, Ueland PM, et al. Plasma homocysteine as a risk factor for vascular disease. The European Concerted Action Project. JAMA 1997;277:1775-81.
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15. Verhoef R Kok FJ, Kruyssen DACM, Schouten EG, Witteman JCM, Grobbee DE, et al. Plasma total homocysteine, Bvitamins and risk of coronary atherosclerosis. Arterioscler Thromb Vasc Biol 1997;17:989-95. 16. Steegers-Theunissen RPM, Boers GHJ, Steegers EAR Trijbels FJM, Thomas CMG, Eskes TKAB. Effects of sub-50 oral contraceptives on homocysteine metabolism: a preliminary study. Contraception 1992;45:129-39. 17. Araki A, Sako Y. Determination of free and total homocysteine in human plasma by high performance liquid chromatography with fluoresence detection. J Chromatogr 1987;422:43-52. 18. Kluijtmans LAJ, van den Heuvel LPWJ, Boers GHJ, Frosst P, Stevens EMB, van Oost BA, et al. Molecular genetic analysis in mild hyperhomocysteinemia: a common mutation in the methylenetetrahydrofolate reductase gene is a genetic risk factor for cardiovascular disease. Am J Hum Genet 1996;58:35-41. 19. Bostom AG, Jacques PF, Nadeau MR, Williams RR, Ellison RC, Selhub J. Post-metbionine load hyperhomocysteinemia in persons with normal fasting total plasma homocysteine: initial results from the NHLBI Family Heart Study. Atherosclerosis 1995;116:147-51.
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20. Cattaneo M, Martinelli I, Mannucci PM. Hyperhomocysteinemia as a risk factor for deep-vein thrombosis [letter]. N EnglJ Med 1996;335:974-5. 21. van den Berg M, Stehouwer CDA, Bierdrager E, Ranwerda JA. Plasma homocysteine and severity of atherosclerosis in young patients with lower extremity atherosclerotic disease. Arterioscler Thromb Vasc Biol 1996; 16:165-71. 22. Franken DG, Boers GHJ, Blom HJ, Cruysberg JRM, Trijbels FJM, Hamel BCJ. Prevalence of familial mild hyperhomocysteinemia. Atherosclerosis 1996; 125:71-80. 23. Brattstr6m L, Lindgren A, Israelsson B, Andersson A, Hultberg B. Homocysteine and cysteine: determinants of plasma levels in middle-aged and elderly subjects. J Intern Med 1994;236:633-41. 24. den Heijer M, Bos GMJ, van der Boor RPGM, van Geel AACM, Lamers H-J, van der Put N, et al. Mutated methylenetetrahydrofolate reductase and other determinants of plasma homocysteine before and after methionine loading in a general Dutch population. In: den Heijer M. Hyperhomocysteinemia and venous thrombosis [dissertation]. Leiden: Leiden University; 1997:37-55.
Hyperhomocysteinemia, defined as an elevated concentration of homocysteine 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 (MLT) is, however, controversial. To determine the additional value of the MLT for diagnosis of hyperhomocysteinemia, we prospectively studied 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, cobalamin, pyridoxine, and creatinine concentrations were measured. Hyperhomocysteinemia was defined as a fasting homocysteine concentration and/or an increase in homocysteine concentration after methionine loading exceeding the 95th percentile of a healthy control group. Hyperhomocysteinemia was found in 141 (33%) of the 429 subjects: 15% were diagnosed by fasting homocysteine concentration and 18% by MLT.Seventy-eight (55%) of the 141 hyperhomocysteinemic persons were diagnosed only by the MLT. Folic acid was lower in the group with an elevated fasting homocysteine concentration than in those with only an abnormal MLT result (I I versus 15 nmol/L, p = 0.002). Folic acid was significantly negatively correlated, and creatinine significantly positively correlated, with both fasting homocysteine concentration and increase in homocysteine concentration. Negative correlations of cobalamin and pyridoxine with fasting homocysteine concentration were found. In conclusion, the MLT is necessary for diagnosis of hyperhomocysteinemia, because a considerable number of hyperhomocysteinemic persons (55%) remain undiagnosed with the determination of a fasting homocysteine concentration alone. (J Lab Clin Med 1998;132:67-72)
Abbreviations: HPLC= High performance liquid chromatography; MLT= methionine loading test
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lassic homozygous homocystinuria is associated with premature t h r o m b o e m b o l i c events due to markedly elevated homocysteine concentrations. 1 M o r e than 25 years ago, the MLT was From the Departments of Internal Medicine and Clinical Chemistry Sint Antonius Hospital, Nieuwegein; the Department of Metabolic Disease, University Children's Hospital "Het Wilhelmina Kinderziekenhuis," Utrecht; and the Department of Internal Medicine, University Hospital Utrecht. Submitted for publication Oct. 29, 1997; revision submitted Feb. 4, 1998; accepted March 11, 1998. Reprint requests: Ren6 van der Griend, MD, Department of Internal Medicine, F02.126, UniversityHospital, Post Office Box 85500, 3508 GA, Utrecht, The Netherlands. Copyright © 1998 by Mosby, Inc. 0022-2143/98 $5.00 + 0 5/1/90336
introduced for the detection of heterozygotes for homocystinuria. 2 In 1976, the MLT was used for the first time to detect moderate hyperhomocysteinemia in men with premature cardiovascular disease, 3 Since then, several studies have c o n f i r m e d that h y p e r h o m o c y s t e i n e m i a , detected by elevated fasting and/or post-load homocysteine concentrations, is an independent risk factor for both atherosclerotic disease and venous thrombosis. 41t There is, however, discussion about the role o f the MLT in the detection o f h y p e r h o m o c y s t e i n e m i a . The MLT has logistic limitations. It has been argued that the test is not p h y s i o l o g i c and, moreover, that it adds no information to the fasting homocysteine determination alone. 12 On the other hand, the clinical relevance o f the MLT is supported by observations o f a higher
67
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Table I. Subject characteristics, plasma total h o m o c y s t e i n e , a n d p r e v a l e n c e of h y p e r h o m o c y s t e i n e m i a as
d e f i n e d by a n e l e v a t e d fasting h o m o c y s t e i n e c o n c e n t r a t i o n a n d an a b n o r m a l MLT in 281 patients w i t h prem a t u r e arterial disease a n d 148 first-degree relatives Parameter
Patients (N = 281)
Relatives (N = 148)
Total (N = 429)
Characteristics
Mean age _+SD (yr) Gender (% male) Creatinine (gmol/L) Folic acid (nmol/L) Pyridoxine (nmol/L) Cobalamin (pmol/L) Plasma total homocysteine (gmol/L) Fasting homocysteine Increase in homocysteine Prevalence of hyperhomocysteinemia (no. [%]) Hyperhomocysteinemia Elevated fasting homocysteine Abnormal MLT
42.7 _+9.1" 57 79 (61-105) 15 (9-36)1 64 (39-160) 300 (180-548)
37.8 _+11.2 48 77 (60-97) 17 (10-42) 70 (42-122) 270 (170-532)
40,9 + 10.2 54 78 (60-102) 16 (9-36) 65 (41-140) 300 (174-532)
12.4 (8.2-24.6)* 33.4 (20.7-70.4)
11.5 (6.8-21.6) 32.3 (21.6-63.2)
12,1 (7.6-23.4) 32,7 (21.4-67.6)
98 (35) 48 (17)1" 50 (18)
43 (29) 15 (10) 28 (19)
141 (33) 63 (15) 78 (18)
Creatinine, vitamin and homocysteine concentrations represent medians (10th to 95th percentile), *p < 0,001 for patients versus relatives, tp : 0,05 for patients versus relatives. ~p : 0,005 for patients versus relatives.
risk for arterial disease and venous thrombosis in patients with hyperhomocysteinemia as determined by an abnormal MLT result only, compared with persons without hyperhomocysteinemia.6J3-15 To assess the contribution of the MLT to the detection of hyperhomocysteinemia, a large group of patients with premature arterial disease and their first-degree relatives was studied. METHODS Subjects. From January 1992 to August 1996, 429 persons
were screened for hyperhomocysteinemia; there were 281 patients with documented premature peripheral, cerebral, or coronary artery disease (before 50 years of age) and 148 unaffected first-degree relatives. All patients were referred by vascular surgeons, neurologists, or cardiologists after assessment of the vascular problem. First-degree relatives were screened on their own request after detection of the index patient. Laboratory investigations. All persons underwent an oral MLT (0.1 grrdkg body weight of 1-methionine) after an overnight fast. L-Methionine was dissolved in orange juice. During the test only low-methionine nutrients were allowed. Women were tested on the 20th through the 22nd day of a menstrual cycle. 16 Blood was sampled before and 6 hours after ingestion, collected in tubes containing lithium-heparin, and immediately centrifuged; plasma was frozen at -20 ° C and stored until assayed. Plasma total homocysteine was measured by HPLC with fluorescence detection as described by Araki and Sako. 17 This method has an interassay coefficient of variation of <7%. Determination of plasma folic acid and cobalamin was performed by a dual-count radioassay (Diagnostic Products Corporation, Los Angeles, Calif.), pyridoxine by fluorescence
HPLC technique, and creatinine according to a modified Jaffe reaction (Dimension, Du Pont, Wilmington, Del.). Definition of hyperhomocysteinemia. Hyperhomocysteinemia was defined as fasting homocysteine concentration and/or an increase in homocysteine concentration (concentration after methionine loading minus fasting concentration) exceeding the 95th percentile of controls. The normal range was derived from 68 healthy volunteers (38 women and 30 men); their mean age was 35 years (range, 18 to 50 years). Their homocysteine values showed a non-normal distribution with a positive skew. The 95th percentile for fasting homocysteine concentration was 16.3 gmol/L in women and 18.8 gmol/L in men; the 95th percentile for increase in homocysteine concentration after loading was 42.3 gmol/L for both sexes. Our reference values are similar to those from two other Dutch reference populations.6J8 Statistical methods. Homocysteine, creatinine, and vitamin concentrations are expressed as median (10th to 95th percentile). The Wilcoxon rank-sum test was used to analyze differences for these variables between patients and relatives and between the different groups stratified for methionine loading outcomes. The Student's t-test was used to analyze differences in mean age. The Pearson X2 test was used to compare frequency measurements between patients and relatives. Spearman correlation coefficients were calculated to study the relations between the variables. All reported probability values are two-tailed, and a value of <0.05 was considered significant. For the analysis, SPSS software was used. RESULTS
Table I lists the characteristics, homocysteine concentrations, and prevalence of hyperhomocysteinemia as defined by an elevated fasting homocysteine concen-
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Table II. Characteristics of subjects stratified for t h e h o m o c y s t e i n e o u t c o m e s d u r i n g m e t h i o n i n e l o a d i n g
Characteristic
Elevated fasting homocysteine (N = 63)
Gender (% male) Mean age _+SD (yr) Patients (%) Creatinine (/.tmol/L) Felic acid (nmol/L) Pyridoxine (nmol/L) Cobalamin (pmol/L)
44 43.7 + 8.6 76 80 (60-136) 11 (7-23)t 56 (37-249) 270 (164-504)§
Abnormal MLT only (N = 78)
50 43.5 _+9.6 64 80 (63-105) 15 (9-30)2 65 (41-107) 280 (160-585)
Normal homocysteine (N = 288)
57 39.4 _+10.5* 64 77 (60-99) 17 (10-41) 66 (41-144) 310 (190-540)
Creatinine and vitamin values represent medians (10th to 95th percentile). *p = 0.004 for normal homocysteine versus elevated fasting homocysteine and p = 0.002 for normal homocysteine versus abnormal MLT. t p = 0.002 for elevated fasting homocysteine versus abnormal MLT and p < 0,001 for elevated fasting homocysteine versus normal homocysteine, ~p = 0.008 for abnormal MLT versus normal homocysteine. §p = 0.005 for elevated fasting homocysteine versus normal homocysteine.
tration and an abnormal MLT in patients and relatives. Patients had a higher mean age, a lower folic acid concentration, and a higher median fasting homocysteine concentration than relatives. The prevalence of hyperhomocysteinemia in the total group was 33% (141 persons). In 15% the condition was detected by an elevated fasting homocysteine concentration (6% had a normal increase in homocysteine after methionine loading and 9% had elevated concentrations in both instances); in 18%, hyperhomocysteinemia was detected by an abnormal MLT result only. Those who had elevated homocysteine concentrations with both fasting and methionine loading were included in the former group, since the MLT was not necessary for the detection of their hyperhomocysteinemia. Among the 141 persons defined as having hyperhomocysteinemia, 63 (45%) had an elevated fasting homocysteine concentration and 78 (55%) were diagnosed after MLT. A significantly higher percentage of patients had an elevated fasting homocysteine concentration, compared with relatives. No differences were found in the percentages of hyperhomocysteinemic persons diagnosed only after MLT between men and women (19% versus 24%, p = 0.29) or between patients and relatives (both 21%). Table II summarizes the characteristics of the study subjects stratified for the homocysteine outcomes during methionine loading. All vitamin concentrations were in the reference range. No differences between the two hyperhomocysteinemic subgroups (i.e., those detected by an elevated fasting homocysteine concentration and those diagnosed only by an abnormal MLT result) were found with respect to gender, age, category (patient or relative), creatinine, pyridoxine, or cobalamin. Only folic acid was significantly lower in hyperhomocysteinemic persons diagnosed by an elevated
fasting homocysteine concentration compared with persons diagnosed by an abnormal MLT result. Folic acid was significantly lower in both hyperhomocysteinemic subgroups, compared with persons without hyperhomocysteinemia. Cobalamin was significantly lower in the subgroup with elevated fasting homocysteine, compared with persons without hyperhomocysteinemia. Seventy-eight (21%) of 366 persons with a normal fasting homocysteine concentration had an abnormal MLT result. In Fig. 1, hyperhomocysteinemic persons whose fasting homocysteine concentration was normal are represented in the left upper quadrant. Data points for normal subjects are found in the left lower quadrant. Reducing the threshold for a normal fasting homocysteine concentration to 15 gmol/L and then to 12 gmol/L resulted in diagnosis by an abnormal MLT in 62 (20%) of 314 persons and 30 (14%) of 213 persons, respectively. There was a strong negative correlation of folic acid with both the fasting concentration and the increase in homocysteine concentration (r = -0.41 and -0.22, respectively; p < 0.001); there was also a strong positive correlation of creatinine concentration with the same parameters (r = 0.32 and 0.18, respectively; p < 0.001). A negative correlation with fasting homocysteine concentration was found for cobalamin (r = -0.17; p = 0.001) and for pyridoxine (r = -0.11; p = 0.05). However, after calculation of a partial correlation coefficient, with adjustment for the two most important variables (folic acid and creatinine), the correlation with fasting homocysteine of cobalamin appeared much lower (r = -0.11; p = 0.05) and that for pyridoxine disappeared. DISCUSSION Hyperhomocysteinemia is now recognized as a serious risk factor for premature atherosclerosis and venous thrombosis. This study demonstrates the essential role
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van der Griend et al.
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Fig. 1. A graphical presentation of the individual values for fasting homocysteine concentration and increase in homocysteine concentration after MLT for 230 men and 199 women. The reference lines represent the 95th percentile of the control group (fasting homocysteine, 18.8 gmol/L for men and 16.3 gmol/L for women; increase in homocysteine, 42.3 gmol/L for both sexes).
of the MLT in the detection of hyperhomocysteinemia: 55% of all hyperhomocysteinemic persons were identified only after methionine loading. Our findings are in accordance with previous observations regarding value of the MLT. 7,14,19 In patients with early-onset venous or arterial thromboembolic disease, the prevalence of hyperhomocysteinemia was almost twice as high after methionine loading. 7 In a population-based investigation, more than 40% of the hyperhomocysteinemic volunteers had a normal fasting homocysteine concentration, 19 Both smaller studies used different postload homocysteine measurements (8 and 4 hours, respectively), and the vitamin status was not given. Very recently, the largest study so far demonstrated that 101 (27%) of 375 hyperhomocysteinemic arterial disease patients were identified only by MLT.14 This percent-
age was smaller than in our study, probably because a relatively low cutoff point for the fasting homocysteine concentration was defined (12 gmol/L). Our results refute recent correspondence stating that methionine loading has little additional value. 12 Both the elevated fasting homocysteine concentration and the concentration after methionine loading are reported to represent independent risk factors for arterial disease and venous thrombosis. 6,13-15 Only the elevated postload concentration indicated an increased risk for a first episode of deep-vein thrombosis. 20 Furthermore, from pooled data of more than 500 patients with arterial disease and more than 300 control subjects, an odds ratio of 13.0 (95% confidence interval, 5.9 to 28.1) was calculated for persons with an abnormal MLT result compared with normal responders.13 Among young patients
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with l o w e r - e x t r e m i t y arterial disease, the severity of atherosclerosis appeared to be even more closely related to the h o m o c y s t e i n e concentration after MLT than to the fasting homocysteine concentration. 21 Moreover, in a large European multicenter study including 750 subjects with atherosclerotic vascular disease, the relative risk for vascular disease in patients with hyperhomocysteinemia increased from 1.6 in those with elevated fasting h o m o c y s t e i n e only to 2.5 in patients with both determinants elevated. 14 Interestingly, elevated fasting homocysteine concentrations occurred significantly less often among the younger and asymptomatic relatives than among the patients. Furthermore, Franken et al. 22 found that if the index patient had an elevated fasting h o m o c y s t e i n e concentration, 29% o f the h y p e r h o m o c y s t e i n e m i c relatives showed a normal fasting value. Both are arguments in favor of performing the MLT as part of the screening for familial hyperhomocysteinemia. Several investigators have reported an increased risk o f vascular disease at h o m o c y s t e i n e concentrations b e l o w the 95th percentile cutoff as used in this study. Reducing the cutoff point for fasting h o m o c y s t e i n e increases the n u m b e r o f h y p e r h o m o c y s t e i n e m i c persons d i a g n o s e d by a r a n d o m b l o o d sample. We observed that the percentage of persons with hyperhom o c y s t e i n e m i a who could be detected only b y an abnormal MLT result does not decrease until the cutoff point for fasting homocysteine is reduced to as low as 12 g m o l / L or less. In this study, folic acid and creatinine concentrations were important determinants for both the fasting homocysteine concentration and the increase with methionine loading. This is in accordance with earlier results indicating an inverse correlation of folic acid and cobalamin and a positive correlation of creatinine concentration with fasting homocysteine. 23 Only very recently have the determinants o f h o m o c y s t e i n e concentration both before and after methionine loading been studied in the general population. 24 A low folic acid concentration and a c o m m o n mutation (677C-+T) in the methy l e n e t e t r a h y d r o f o l a t e reductase gene 18 were strong determinants o f h y p e r h o m o c y s t e i n e m i a in the fasting state and after methionine loading, again demonstrating the key role of folic acid. The creatinine concentration influenced only the fasting homocysteine concentration. Our results indicate that the MLT m a y identify more than 50% o f subjects with h y p e r h o m o c y s t e i n e m i a . These persons w o u l d not have been d i a g n o s e d b y screening with a fasting plasma homocysteine determination alone. A l t h o u g h our study was p e r f o r m e d in patients with p r e m a t u r e arterial vascular disease and their healthy relatives, it seems reasonable to extend the
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findings of the value of the MLT to patients with venous thrombosis and arterial disease as a whole. Together with the observations that postload hyperhomocysteinemia is an independent risk factor for both atherosclerosis and venous thrombosis, we conclude that the MLT remains essential in the detection o f h y p e r h o m o c y s teinemia. The authors thank Marijke van Kooten, medical secretary, for her excellent assistance in performing the MLTs and Helma Scheepers and Barbara van Sterkenburg for collecting the blood samples. REFERENCES
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