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Hyperhomocystinemia in patients with nonarteritic anterior ischemic optic neuropathy, central retinal artery occlusion, and central retinal vein occlusion1
Hyperhomocystinemia in Patients with Nonarteritic Anterior Ischemic Optic Neuropathy, Central Retinal Artery Occlusion, and Central Retinal Vein Occlusion Pazit Pianka, MD,1 Yehoshua Almog, MD,1 Oran Man, MD,1 Michaela Goldstein, MD,1 Ben-Ami Sela, PhD,2 Anat Loewenstein, MD1 Objective: This study aimed to determine the prevalence of hyperhomocystinemia among patients with nonarteritic anterior ischemic optic neuropathy (NAION), central retinal artery occlusion (CRAO), or central retinal vein occlusion (CRVO). Design: Retrospective, case-control study. Participants: The study cohort consisted of 74 consecutive patients with NAION, CRAO, or CRVO who were examined at the Retina or Neuro-ophthalmological Unit of the Tel-Aviv Sourasky Medical Center from 1998 through 1999. The control group consisted of 81 consecutive patients of similar gender and age with no history of these pathologic conditions. Main Outcome Measures: Plasma homocystine levels of all study participants were obtained. Results: Eighteen of 40 patients (45%) with NAION and eight of 13 patients (61.5%) with CRAO had hyperhomocystinemia compared with three of 21 (14.3%) in the CRVO group (P ⬍ 0.001) and eight (9.8%) in the control group (P ⬍ 0.0001). Hypertension and ischemic heart disease were significantly more prevalent in the NAION patients with elevated plasma homocystine. Conclusions: Our findings suggest that hyperhomocystinemia is a risk factor for NAION and CRAO. Ophthalmology 2000;107:1588 –1592 © 2000 by the American Academy of Ophthalmology. Mild hyperhomocystinemia is an independent risk factor for premature vascular disease.1,2 Elevated homocystine levels are frequently found in patients with arteriosclerosis affecting coronary, cerebral, and peripheral arteries.1,3–7 Homocystine is a highly reactive amino acid.8 High levels of plasma homocystine are toxic to the vascular endothelium, presumably by directly injuring the vessel’s endothelium by the release of free radicals, creating an environment of hypercoagulability, and by modifying the vessel wall.9 –13 Any enzyme deficiency in the process of remethylation (5,10-methylenetetrahydrofolate reductase [MTHFR]) or
Originally received: October 23, 1999. Accepted: March 28, 2000. Manuscript no. 99484. 1 Department of Ophthalmology, Tel-Aviv Sourasky Medical Center, TelAviv, Israel. 2 Institute of Chemical Pathology, Sheba Medical Center, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel. The authors have no proprietary interest in the products mentioned herein. Presented in part at the annual meeting of the American Academy of Ophthalmology, Orlando, Florida, October 1999. Correspondence to Pazit Pianka, MD, Department of Ophthalmology, Tel-Aviv Sourasky Medical Center, 6 Weizman Street, Tel-Aviv, 64239, Israel. E-mail: [email protected].
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© 2000 by the American Academy of Ophthalmology Published by Elsevier Science Inc.
transsulfuration (cystathionine -synthase) of methionine will elevate plasma homocystine. Homozygosity to one of these enzymes results in severe homocystinemia, and, without proper treatment, up to 50% of these patients will experience a thrombotic or occlusive episode at a young age. Heterozygotes, however, rarely develop hyperhomocystinemia. A thermolabile variant of the enzyme, MTHFR, was recently discovered.14,15 In most populations, 5% to 16% of individuals are homozygous to this enzyme and up to 50% are heterozygous. Several studies have shown a correlation between the homozygosity and an increased incidence of thrombosis or arteriosclerosis.16 –21 There are other conditions that may increase plasma homocystine levels: age,22 gender,22 renal failure,23 medications,24 –26 and decrease in uptake of vitamins B6, B12, and folic acid.27 These three vitamins are important cofactors in the processing and conversion of homocystine to either methionine or cysteine. Salomon et al28 have recently shown that nonarteritic anterior ischemic optic neuropathy (NAION) is not associated with certain thrombophilic risk factors (protein C, protein S, antithrombin III, lupus anticoagulant, factor V, factor II, and MTHFR). They did not, however, measure the homocystine levels of these patients. ISSN 0161-6420/00/$–see front matter PII S0161-6420(00)00181-0
Pianka et al 䡠 Homocystine in NAION, Arterial, or Venous Retinal Occlusion Table 1. Comparative Homocystine Levels of Patients with NAION, CRAO, CRVO, and Control Participants Subjects (n)
Age (yrs, mean ⴞ SE)
Homocystine Level (mol/l ⴞ SE)
Hyperhomocystinemia (%)
NAION
40
CRAO
13
CRVO
21
Control
81
66.3 ⫾ 1.96 P ⬎ 0.05 69.38 ⫾ 2.4 P ⬎ 0.05 58.6 ⫾ 2.7 P ⬍ 0.005 66 ⫾ 2
11.68 ⫾ 0.99 P ⫽ 0.0088 12.5 ⫾ 1.03 P ⫽ 0.0034 10.25 ⫾ 1.88 P ⫽ 0.4 8.7 ⫾ 0.5
45 P ⬍ 0.001 61.5 P ⬍ 0.001 14.3 P ⫽ 0.5616 9.8
CRAO ⫽ central retinal artery occlusion; CRVO ⫽ central retinal vein occlusion. NAION ⫽ nonarteritic anterior ischemic optic neuropathy; SE ⫽ standard error of mean.
Central retinal artery occlusion (CRAO) is often caused by atherosclerotic-related thrombosis occurring at the level of the lamina cribrosa, whereas emboli are found in approximately 20% of cases.29 The leading cause of death in individuals with CRAO is cardiovascular disease. There are numerous case reports implicating an association between thrombophilic factors (protein S deficiency,30 resistance to activated protein C,31 factor V-Leiden gene mutation, 32,33 MTHFR mutation33) and CRAO. In 1993, Wenzler et al34 described four patients with CRAO and two with central retinal vein occlusion (CRVO) who had elevated homocystine levels after methionine-loading testing. Aging, diabetes, hypertension, cardiovascular risk profile, and glaucoma have all been identified as independent risk factors for branch retinal vein occlusion (BRVO)35 and CRVO.36 Among the biochemical and hemostatic disturbances that have been associated with CRVO are antiphospholipid antibodies syndrome and abnormalities in the anticoagulant system, such as protein C, protein S, and antithrombin-III deficiency.37– 40 Several studies have also shown an association between CRVO and hyperhomocystinemia. Biousse et al41 described a 24-year-old man with bilateral CRVO and increased plasma homocystine. We reported a young patient with bilateral CRVO in association with MTHFR.42 Salomon et al43 investigated the role of genetic polymorphism associated with venous and arterial thrombosis. Their data suggested that homozygosity to MTHFR C677T polymorphism is a risk factor for RVO. We recently documented an incidence of 36% of homozygosity to MTHFR in patients with CRVO.44 The assay of total homocystine in biologic fluids is complicated by the ease with which the amino acid forms disulfide bonds. Therefore, in previous studies, plasma homocystine levels were not quantified directly, but rather were extrapolated from the measurements of methionine loading and enzyme activity tests. Recently developed methods using high-performance liquid chromatography (HPLC) or gas chromatography-mass spectrometry now enable a more precise evaluation of plasma homocystine levels.45,46 High-performance liquid chromatography enables reliable detection of even low concentrations of plasma homocystine.47
The aim of our study was to examine the prevalence of hyperhomocystinemia in the fasting state by the HPLC method among patients with NAION, CRAO, and CRVO.
Patients and Methods All consecutive patients with NAION, CRAO, or CRVO seen at the Retina or Neuro-ophthalmological Unit of the Tel-Aviv Sourasky Medical Center from July 1998 through March 1999 and who agreed to participate in the study were included. Because of the low number of consecutive patients with CRAO seen at our institution during this period (n ⫽ 5), we also retrieved the charts of all the patients with CRAO seen in previous years and invited them to participate in the study (n ⫽ 8). A detailed medical history was obtained to determine those with known or suspected diabetes mellitus, hypertension, cardiovascular disease, cerebrovascular disease, previous thromboembolic events (ocular and nonocular), and current drug therapy. The control group consisted of consecutive patients of similar gender and age with no history of NAION, CRAO, or CRVO. A recent article determined that the intra-individual coefficient of variation of plasma homocystine is relatively low.48 Because a single measurement can be used to characterize the average homocystine concentration, one 2-ml venous blood sample was obtained from each participant. The blood was centrifuged at 3000 g for 6 minutes, and the plasma was removed and analyzed for homocystine by the HPLC method with fluorescent detection.49 A value of plasma homocystine over 11mol/l is considered increased at our laboratory. The study was approved by the institutional review board.
Statistical Analysis The chi-square test was used for comparing differences in proportions among the groups. The two-sample t test was used to examine differences in mean values between the groups. A P value of 0.05 or less was considered as being statistically significant.
Results Seventy-four patients (41 males, 33 females) with NAION, CRAO, or CRVO were included in the study group. The control group consisted of 81 healthy adults of similar age and sex (Table 1). The mean age of the NAION and the CRAO groups was not
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Ophthalmology Volume 107, Number 8, August 2000 significantly different from the controls (P ⬎ 0.5), but the patients in the CRVO group were significantly younger than the controls (P ⬍ 0.005). The mean plasma homocystine level was statistically significantly elevated in the NAION (P ⫽ 0.0088) and the CRAO groups (P ⫽ 0.0034), but not in the CRVO group (P ⫽ 0.4) when compared with the control group (Table 1). Eighteen patients (45%) in the NAION group and eight (61.5%) in the CRAO group had hyperhomocystinemia compared with three (14.3%) in the CRVO group (P ⬍ 0.001) and eight (9.8%) in the control group (P ⬍ 0.0001). In the NAION group, 18 patients (45%) had hypertension, 12 (30%) had diabetes, six (15%) had ischemic heart disease, and one patient had stroke. In this group, the patients with hyperhomocystinemia had a significantly higher prevalence of hypertension and ischemic heart disease as compared with the patients with normal homocystine levels (P ⫽ 0.0002 for hypertension and P ⫽ 0.0002 for ischemic heart disease). In the CRAO group, 10 patients (77%) had hypertension, two (15.4%) had diabetes, three (23%) had ischemic heart disease, one patient had stroke and one patient had peripheral vascular disease. The patients with hyperhomocystinemia had an increased prevalence of diabetes mellitus and ischemic heart disease as compared with the patients with normal homocystine levels. However, because of the small sample size, this was not statistically significant (P ⬎ 0.05).
Discussion The results of our study suggest that the prevalence of hyperhomocystinemia in patients with arterial occlusion (NAION or CRAO) is significantly increased. Moreover, in the NAION group, patients with hyperhomocystinemia had a significantly higher incidence of hypertension and ischemic heart disease. A similar trend was seen for the CRAO group, but the sample was too small to be statistically significant. In our previous study,44 we investigated the incidence of homozygosity to MTHFR mutation in patients with CRVO, and homocystine levels were not investigated. It is well known that MTHFR deficiency is associated with hyperhomocystinemia only when there is low folate, and that only one third of patients with homozygosity to MTHFR mutation have hyperhomocystinemia.50 Thus, of the 36% of patients with MTHFR mutation documented in our previous study, an incidence of 12% would be expected, correlating well with the 14.3% of hyperhomocystinemia in patients with CRVO found in our present study. Hyperhomocystinemia is now recognized as an independent risk factor for vascular disease, including coronary and cerebrovascular disease. However, to the best of our knowledge this is the first study that directly evaluated and compared homocystine levels measured by the HPLC method in patients with NAION, CRAO, and CRVO. Our study results suggest that homocystine is a risk factor for NAION and CRAO as well. It has been demonstrated that the risk associated with hyperhomocystinemia appears to be concentration dependent and not attributable to traditional risk factors. The odds ratio for ischemic heart disease has been estimated to be 1.4 for every 5 mol/l increase of total plasma homocystine.51
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Treatment of hyperhomocystinemia is safe, readily available, and affordable. A recent article by Jacques et al52 concluded that fortification by grain products enriched with folic acid is associated with a substantial improvement in folate status in middle-aged and older adults. Treatment with daily vitamin supplementation (vitamin B6, folic acid, and betaine) is now usually recommended in patients with hyperhomocystinemia. Recent studies have shown that doses of folic acid as low as 250 g/day, in addition to usual dietary intakes of folate, significantly decrease total plasma homocystine levels in healthy young women.53 A similar reduction in plasma homocystine level was reported in an elderly population treated with 400 g/day folic acid supplemented with vitamin B6 and B12.54,55 Treatment with these three vitamins can reduce serum homocystine levels by approximately 25%.51 It is assumed that by reducing the toxic effect of homocystine on the blood vessels, the probability of further vascular occlusion, and therefore the resulting morbidity, will be reduced. Recently, de Jong et al56 showed a protective effect of treatment with vitamin B6 and folic acid in patients with premature arterial occlusive disease. This is a preliminary study. Larger scale studies are needed to determine a treatment policy for these patients with NAION and CRAO who are probably at increased risk for thrombosis and arteriosclerosis.
References 1. Clarke R, Daly L, Robinson K, et al. Hyperhomocystinemia: an independent risk factor for vascular disease. N Engl J Med 1991:324:1149 –55. 2. den Heijer M, Koster T, Blom HJ, et al. Hyperhomocystinemia as a risk factor for deep-vein thrombosis. N Engl J Med 1996;334:759 – 62. 3. Scott J, Weir D. Homocystine and cardiovascular disease [editorial]. QJM 1996;89:561–3. Comment on: QJM 1996;89: 571–7. 4. Boushey CJ, Beresford SA, Omenn GS, Motulsky AG. A quantitative assessment of plasma homocystine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. JAMA 1995;274:1049 –57. 5. D’Angelo A, Selhub J. Homocystine and thrombotic disease [review]. Blood 1997;90:1–11. 6. Nygard O, Nordrehaug JE, Refsum H, et al. Plasma homocystine levels and mortality in patients with coronary artery disease. N Engl J Med 1997;337:230 – 6. 7. Graham IM, Daly LE, Refsum HM, et al. Plasma homocystine as a risk factor for vascular disease. The European Concerted Action Project. JAMA 1997;277:1775– 81. 8. Du Vigneaud V. A Trial of Research in Sulfur Chemistry and Metabolism, and Related Fields. Ithaca, NY: Cornell University Press, 1952;213. 9. McCully KS. Vascular pathology of homocystinemia: implications for the pathogenesis of arteriosclerosis. Am J Pathol 1969;56:111–28 10. Upchurch GR Jr, Welch GN, Fabian AJ, et al. Homocystine decreases bioavailable nitric oxide by a mechanism involving glutathione peroxidase. J Biol Chem 1997;272:17012–7. 11. Stamler JS, Osborne JA, Jaraki O, et al. Adverse vascular effects of homocystine are modulated by endothelium-derived
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relaxing factor and related oxides of nitrogen. J Clin Invest 1993;91:308 –18. Jakubowski H. Metabolism of homocystine thiolactone in human cell cultures. Possible mechanism for pathological consequences of elevated homocystine levels. J Biol Chem 1997;272:1935– 42. Harpel PC, Zhang X, Borth W. Homocystine and hemostasis: pathogenic mechanisms predisposing to thrombosis [review]. J Nutr 1996;126(4 Suppl):1285S–9S. Kang SS, Wong PW, Susmano A, et al. Thermolabile methylenetetrahydrofolate reductase: an inherited risk factor for coronary artery disease. Am J Hum Genet 1991;48:536 – 45. Frosst P, Blom HJ, Milos R, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase [letter]. Nat Genet 1995;10: 111–3. Margaglione M, D’Andrea G, d’Addedda M, et al. The methylenetetrahydrofolate reductase TT677 genotype is associated with venous thrombosis independently of the coexistence of the FV Leiden and the prothrombin A20210 mutation. Thromb Haemost 1998;79:907–11. Arruda BR, von Zuben PM, Chaparini LC, et al. The mutation Ala677-⬎Val in the methylene tetrahydrofolate reductase gene: a risk factor for arterial disease and venous thrombosis. Thromb Haemost 1997;77:818 –21. Morita H, Kurihara H, Tsubaki S, et al. Methylenetetrahydrofolate reductase gene polymorphism and ischemic stroke in Japanese. Arterioscler Thromb Vasc Biol 1998;18:1465–9. Gallagher PM, Meleady R, Shields DC, et al. Homocystine and risk of premature coronary heart disease. Evidence for a common gene mutation. Circulation 1996;94:2154 – 8. Kluijtmans LA, Kastelein JJ, Lindemans J, et al. Thermolabile methylenetetrahydrofolate reductase in coronary artery disease. Circulation 1997;96:2573–7. Boushey CJ, Beresford SA, Omenn GS, Motulsky AG. A quantitative assessment of plasma homocystine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. JAMA 1995;274:1049 –57. Andersson A, Brattstrom L, Israelsson B, et al. Plasma homocystine before and after methionine loading with regard to age, gender, and menopausal status. Eur J Clin Invest 1992;22:79 – 87. Bostom AG, Shemin D, Lapane KL, et al. Hyperhomocystinemia and traditional cardiovascular disease risk factor in endstage renal disease patients on dialysis: a case-control study. Atherosclerosis 1995;114:93–103. Broxson EH Jr, Stork LC, Allen RH, et al. Changes in plasma methionine and total homocystine levels in patients receiving methotrexate infusions. Cancer Res 1989;49:5879 – 83. Ermens AA, Refsum H, Rupreht J, et al. Monitoring cobalamin inactivation during nitrous oxide anesthesia by determination of homocystine and folate in plasma and urine. Clin Pharmacol Ther 1991;49:385–93. Blankenhorn DH, Malinow MR, Mack WJ. Colestipol plus niacin therapy elevates plasma homocystine levels. Coron Artery Dis 1991;2:357– 60. Selhub J, Jacques PF, Wilson PW, et al. Vitamin status and intake as primary determinates of homocystinemia in an elderly population. JAMA 1993;270:2693– 8. Salomon O, Huna-Baron R, Kurtz S, et al. Analysis of prothrombotic and vascular risk factors in patients with nonarteritic anterior ischemic optic neuropathy. Ophthalmology 1999;106:739 – 42. Ahuja RM, Chaturvedi S, Eliott D, et al. Mechanisms of retinal arterial occlusive disease in African American and Caucasian patients. Stroke 1999;30:1506 –9.
30. Greven CM, Weaver RG, Owen J, Slusher MM. Protein S deficiency and bilateral branch retinal artery occlusion. Ophthalmology 1991;98:33– 4. 31. Vignes S, Wechsler B, Elmaleh, C et al. Retinal arterial occlusion associated with resistance to activated protein C [letter]. Br J Ophthalmol 1996;80:1111. 32. Tayyanipour R, Pulido JS, Postel EA, et al. Arterial vascular occlusion associated with factor V Leiden gene mutation. Retina 1998;18:376 –7. 33. Talmon T, Scharf J, Mayer E, et al. Retinal arterial occlusion in a child with factor V Leiden and thermolabile methylene tetrahydrofolate reductase mutations. Am J Ophthalmol 1997; 124:689 –91. 34. Wenzler EM, Rademakers AJ, Boers GF, et al. Hyperhomocystinemia in retinal artery and retinal vein occlusion. Am J Ophthalmol 1993;115:162–7. 35. Risk factors for branch retinal vein occlusion. The Eye Disease Case-control Study Group. Am J Ophthalmol 1993;116: 286 –96. 36. Baseline and early natural history report. The Central Vein Occlusion Study. Central Vein Occlusion Study Group. Arch Ophthalmol 1993;111:1087–95. 37. Glacet-Bernard A, Bayani N, Chretien P. Antiphospholipid antibodies in retinal vascular occlusions. A prospective study of 75 patients. Arch Ophthalmol 1994;112:790 –5. 38. Glacet-Bernard A, Chabanel A, Lelong F, et al. Elevated erythrocyte aggregation in patients with central retinal vein occlusion and without conventional risk factors. Ophthalmology 1994;101:1483–7. 39. Cassels-Brown A, Minford AM, Chatfield SL, Bradbury JA. Ophthalmic manifestations of neonatal protein C deficiency. Br J Ophthalmol 1994;78:486 –7. 40. Ciardella AP, Yannuzzi LA, Freund KB, et al. Factor V Leiden, activated protein C resistance, and retinal vein occlusion. Retina 1998;18:308 –15. 41. Biousse V, Newman NJ, Sternberg P Jr. Retinal vein occlusion and transient monocular visual loss associated with hyperhomocystinemia. Am J Ophthalmol 1997;124:257– 60. 42. Loewenstein A, Winder A, Goldstein M, et al. Bilateral central retinal vein occlusion associated with 5,10-methylenetetrahydrofolate reductase mutation. Am J Ophthalmol 1997;124:840 –1. 43. Salomon O, Moisseiev J, Rosenberg N, et al. Analysis of genetic polymorphisms related to thrombosis and other risk factors in patients with retinal vein occlusion. Blood Coagul Fibrinolysis 1998;9:617–22. 44. Loewenstein A, Goldstein M, Winder A, et al. Retinal vein occlusion associated with methylenetetrahydrofolate reductase mutation. Ophthalmology 1999;106:1817–20. 45. Jacobsen DW, Gatautis VJ, Green R. Determination of plasma homocystine by high-performance liquid chromatography with fluorescence detection. Anal Biochem 1989;178:208 –14. 46. Stabler SP, Marcell PD, Podell ET, et al. Quantitation of total homocystine, total cysteine, and methionine in normal serum and urine using capillary gas chromatography-mass spectrometry. Anal Biochem 1987;162:185–96. 47. Moat SJ, Bonham JR, Tanner MS, et al. Recommended approaches for the laboratory measurement of homocystine in the diagnosis and monitoring of patients with hyperhomocysteinaemia. Ann Clin Biochem 1999;36(Pt 3):372–9. 48. Rossi E, Beilby JP, McQuillan BM, Hung J. Biological variability and reference intervals for total plasma homocystine. Ann Clin Biochem 1999;36(Pt 1):56 – 61. 49. Fiskerstrand T, Refsum H, Kvalheim G, Veland PM. Homocystine and other thiols in plasma and urine: automated determination and sample stability. Clin Chem 1993;39:263–71.
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Ophthalmology Volume 107, Number 8, August 2000 50. Selhub J. Homocystine metabolism [review]. Annu Rev Nutr 1999;19:217– 46. 51. Taylor LM Jr, Moneta GL, Sexton GJ, et al. Prospective blinded study of the relationship between plasma homocystine and progression of symptomatic peripheral arterial disease. J Vasc Surg 1999;29:8 –19; discussion 19 –21. 52. Jacques PF, Selhub J, Bostom AG, et al. The effect of folic acid fortification on plasma folate and total homocystine concentrations. N Engl J Med 1999;340:1449 –54. 53. Brouwer IA, van Dusseldorp M, Thomas CM, et al. Low-dose folic acid supplementation decreases plasma homocystine concentrations: a randomized trial. Am J Clin Nutr 1999;69:99 –104.
54. Bronstrup A, Hages M, Pietrzik K. Lowering of homocystine concentrations in elderly men and women. Int J Vitam Nutr Res 1999;69:187–93. 55. Lobo A, Naso A, Arheart K, et al. Reduction of homocystine levels in coronary artery disease by low-dose folic acid combined with vitamins B6 and B12. Am J Cardiol 1999;83: 821–5. 56. de Jong SC, Stehouwer CD, van den Berg M, et al. Normohomocystinaemia and vitamin-treated hyperhomocystinaemia are associated with similar risks of cardiovascular events in patients with premature peripheral arterial occlusive disease. A prospective cohort study. J Intern Med 1999;246:87–96.
Discussion by William E. Benson, MD Both nonarteritic ischemic optic neuropathy and central retinal artery obstruction sometimes can affect the fellow eye. Therefore, when a patient presents with either, it behooves us to identify treatable risk factors. The authors, using high performance liquid chromatography, a technique that reliably detects low concentrations of homocystine, have found that hyperhomocystinemia is statistically significantly associated with both of these conditions. They may well be right, because hyperhomocystinemia has already From the Wills Eye Hospital, Philadelphia, Pennsylvania. Address correspondence to William E. Benson, MD, 1051 Indian Creek Road, Wynnewood, PA, 19096.
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been associated with coronary and cerebral vascular disease. Further, we should hope that they are right, because treatment with nontoxic doses of folic acid, vitamin B6, and vitamin B12 can reduce serum homocystine levels by 25%. The authors’ study is an excellent start. However, their numbers are small and one must be very careful before implicating any risk factor with such small numbers, even if they are statistically significant. Before we routinely recommend vitamin supplements to patients with ischemic optic neuropathy and central retinal artery occlusion, two conditions must be fulfilled. First, larger studies must confirm their results. Second, studies must be conducted to show that lowering the homocystine levels will actually prevent involvement of the fellow eye.
Originally received: October 23, 1999. Accepted: March 28, 2000. Manuscript no. 99484. 1 Department of Ophthalmology, Tel-Aviv Sourasky Medical Center, TelAviv, Israel. 2 Institute of Chemical Pathology, Sheba Medical Center, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel. The authors have no proprietary interest in the products mentioned herein. Presented in part at the annual meeting of the American Academy of Ophthalmology, Orlando, Florida, October 1999. Correspondence to Pazit Pianka, MD, Department of Ophthalmology, Tel-Aviv Sourasky Medical Center, 6 Weizman Street, Tel-Aviv, 64239, Israel. E-mail: [email protected].
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© 2000 by the American Academy of Ophthalmology Published by Elsevier Science Inc.
transsulfuration (cystathionine -synthase) of methionine will elevate plasma homocystine. Homozygosity to one of these enzymes results in severe homocystinemia, and, without proper treatment, up to 50% of these patients will experience a thrombotic or occlusive episode at a young age. Heterozygotes, however, rarely develop hyperhomocystinemia. A thermolabile variant of the enzyme, MTHFR, was recently discovered.14,15 In most populations, 5% to 16% of individuals are homozygous to this enzyme and up to 50% are heterozygous. Several studies have shown a correlation between the homozygosity and an increased incidence of thrombosis or arteriosclerosis.16 –21 There are other conditions that may increase plasma homocystine levels: age,22 gender,22 renal failure,23 medications,24 –26 and decrease in uptake of vitamins B6, B12, and folic acid.27 These three vitamins are important cofactors in the processing and conversion of homocystine to either methionine or cysteine. Salomon et al28 have recently shown that nonarteritic anterior ischemic optic neuropathy (NAION) is not associated with certain thrombophilic risk factors (protein C, protein S, antithrombin III, lupus anticoagulant, factor V, factor II, and MTHFR). They did not, however, measure the homocystine levels of these patients. ISSN 0161-6420/00/$–see front matter PII S0161-6420(00)00181-0
Pianka et al 䡠 Homocystine in NAION, Arterial, or Venous Retinal Occlusion Table 1. Comparative Homocystine Levels of Patients with NAION, CRAO, CRVO, and Control Participants Subjects (n)
Age (yrs, mean ⴞ SE)
Homocystine Level (mol/l ⴞ SE)
Hyperhomocystinemia (%)
NAION
40
CRAO
13
CRVO
21
Control
81
66.3 ⫾ 1.96 P ⬎ 0.05 69.38 ⫾ 2.4 P ⬎ 0.05 58.6 ⫾ 2.7 P ⬍ 0.005 66 ⫾ 2
11.68 ⫾ 0.99 P ⫽ 0.0088 12.5 ⫾ 1.03 P ⫽ 0.0034 10.25 ⫾ 1.88 P ⫽ 0.4 8.7 ⫾ 0.5
45 P ⬍ 0.001 61.5 P ⬍ 0.001 14.3 P ⫽ 0.5616 9.8
CRAO ⫽ central retinal artery occlusion; CRVO ⫽ central retinal vein occlusion. NAION ⫽ nonarteritic anterior ischemic optic neuropathy; SE ⫽ standard error of mean.
Central retinal artery occlusion (CRAO) is often caused by atherosclerotic-related thrombosis occurring at the level of the lamina cribrosa, whereas emboli are found in approximately 20% of cases.29 The leading cause of death in individuals with CRAO is cardiovascular disease. There are numerous case reports implicating an association between thrombophilic factors (protein S deficiency,30 resistance to activated protein C,31 factor V-Leiden gene mutation, 32,33 MTHFR mutation33) and CRAO. In 1993, Wenzler et al34 described four patients with CRAO and two with central retinal vein occlusion (CRVO) who had elevated homocystine levels after methionine-loading testing. Aging, diabetes, hypertension, cardiovascular risk profile, and glaucoma have all been identified as independent risk factors for branch retinal vein occlusion (BRVO)35 and CRVO.36 Among the biochemical and hemostatic disturbances that have been associated with CRVO are antiphospholipid antibodies syndrome and abnormalities in the anticoagulant system, such as protein C, protein S, and antithrombin-III deficiency.37– 40 Several studies have also shown an association between CRVO and hyperhomocystinemia. Biousse et al41 described a 24-year-old man with bilateral CRVO and increased plasma homocystine. We reported a young patient with bilateral CRVO in association with MTHFR.42 Salomon et al43 investigated the role of genetic polymorphism associated with venous and arterial thrombosis. Their data suggested that homozygosity to MTHFR C677T polymorphism is a risk factor for RVO. We recently documented an incidence of 36% of homozygosity to MTHFR in patients with CRVO.44 The assay of total homocystine in biologic fluids is complicated by the ease with which the amino acid forms disulfide bonds. Therefore, in previous studies, plasma homocystine levels were not quantified directly, but rather were extrapolated from the measurements of methionine loading and enzyme activity tests. Recently developed methods using high-performance liquid chromatography (HPLC) or gas chromatography-mass spectrometry now enable a more precise evaluation of plasma homocystine levels.45,46 High-performance liquid chromatography enables reliable detection of even low concentrations of plasma homocystine.47
The aim of our study was to examine the prevalence of hyperhomocystinemia in the fasting state by the HPLC method among patients with NAION, CRAO, and CRVO.
Patients and Methods All consecutive patients with NAION, CRAO, or CRVO seen at the Retina or Neuro-ophthalmological Unit of the Tel-Aviv Sourasky Medical Center from July 1998 through March 1999 and who agreed to participate in the study were included. Because of the low number of consecutive patients with CRAO seen at our institution during this period (n ⫽ 5), we also retrieved the charts of all the patients with CRAO seen in previous years and invited them to participate in the study (n ⫽ 8). A detailed medical history was obtained to determine those with known or suspected diabetes mellitus, hypertension, cardiovascular disease, cerebrovascular disease, previous thromboembolic events (ocular and nonocular), and current drug therapy. The control group consisted of consecutive patients of similar gender and age with no history of NAION, CRAO, or CRVO. A recent article determined that the intra-individual coefficient of variation of plasma homocystine is relatively low.48 Because a single measurement can be used to characterize the average homocystine concentration, one 2-ml venous blood sample was obtained from each participant. The blood was centrifuged at 3000 g for 6 minutes, and the plasma was removed and analyzed for homocystine by the HPLC method with fluorescent detection.49 A value of plasma homocystine over 11mol/l is considered increased at our laboratory. The study was approved by the institutional review board.
Statistical Analysis The chi-square test was used for comparing differences in proportions among the groups. The two-sample t test was used to examine differences in mean values between the groups. A P value of 0.05 or less was considered as being statistically significant.
Results Seventy-four patients (41 males, 33 females) with NAION, CRAO, or CRVO were included in the study group. The control group consisted of 81 healthy adults of similar age and sex (Table 1). The mean age of the NAION and the CRAO groups was not
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Ophthalmology Volume 107, Number 8, August 2000 significantly different from the controls (P ⬎ 0.5), but the patients in the CRVO group were significantly younger than the controls (P ⬍ 0.005). The mean plasma homocystine level was statistically significantly elevated in the NAION (P ⫽ 0.0088) and the CRAO groups (P ⫽ 0.0034), but not in the CRVO group (P ⫽ 0.4) when compared with the control group (Table 1). Eighteen patients (45%) in the NAION group and eight (61.5%) in the CRAO group had hyperhomocystinemia compared with three (14.3%) in the CRVO group (P ⬍ 0.001) and eight (9.8%) in the control group (P ⬍ 0.0001). In the NAION group, 18 patients (45%) had hypertension, 12 (30%) had diabetes, six (15%) had ischemic heart disease, and one patient had stroke. In this group, the patients with hyperhomocystinemia had a significantly higher prevalence of hypertension and ischemic heart disease as compared with the patients with normal homocystine levels (P ⫽ 0.0002 for hypertension and P ⫽ 0.0002 for ischemic heart disease). In the CRAO group, 10 patients (77%) had hypertension, two (15.4%) had diabetes, three (23%) had ischemic heart disease, one patient had stroke and one patient had peripheral vascular disease. The patients with hyperhomocystinemia had an increased prevalence of diabetes mellitus and ischemic heart disease as compared with the patients with normal homocystine levels. However, because of the small sample size, this was not statistically significant (P ⬎ 0.05).
Discussion The results of our study suggest that the prevalence of hyperhomocystinemia in patients with arterial occlusion (NAION or CRAO) is significantly increased. Moreover, in the NAION group, patients with hyperhomocystinemia had a significantly higher incidence of hypertension and ischemic heart disease. A similar trend was seen for the CRAO group, but the sample was too small to be statistically significant. In our previous study,44 we investigated the incidence of homozygosity to MTHFR mutation in patients with CRVO, and homocystine levels were not investigated. It is well known that MTHFR deficiency is associated with hyperhomocystinemia only when there is low folate, and that only one third of patients with homozygosity to MTHFR mutation have hyperhomocystinemia.50 Thus, of the 36% of patients with MTHFR mutation documented in our previous study, an incidence of 12% would be expected, correlating well with the 14.3% of hyperhomocystinemia in patients with CRVO found in our present study. Hyperhomocystinemia is now recognized as an independent risk factor for vascular disease, including coronary and cerebrovascular disease. However, to the best of our knowledge this is the first study that directly evaluated and compared homocystine levels measured by the HPLC method in patients with NAION, CRAO, and CRVO. Our study results suggest that homocystine is a risk factor for NAION and CRAO as well. It has been demonstrated that the risk associated with hyperhomocystinemia appears to be concentration dependent and not attributable to traditional risk factors. The odds ratio for ischemic heart disease has been estimated to be 1.4 for every 5 mol/l increase of total plasma homocystine.51
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Treatment of hyperhomocystinemia is safe, readily available, and affordable. A recent article by Jacques et al52 concluded that fortification by grain products enriched with folic acid is associated with a substantial improvement in folate status in middle-aged and older adults. Treatment with daily vitamin supplementation (vitamin B6, folic acid, and betaine) is now usually recommended in patients with hyperhomocystinemia. Recent studies have shown that doses of folic acid as low as 250 g/day, in addition to usual dietary intakes of folate, significantly decrease total plasma homocystine levels in healthy young women.53 A similar reduction in plasma homocystine level was reported in an elderly population treated with 400 g/day folic acid supplemented with vitamin B6 and B12.54,55 Treatment with these three vitamins can reduce serum homocystine levels by approximately 25%.51 It is assumed that by reducing the toxic effect of homocystine on the blood vessels, the probability of further vascular occlusion, and therefore the resulting morbidity, will be reduced. Recently, de Jong et al56 showed a protective effect of treatment with vitamin B6 and folic acid in patients with premature arterial occlusive disease. This is a preliminary study. Larger scale studies are needed to determine a treatment policy for these patients with NAION and CRAO who are probably at increased risk for thrombosis and arteriosclerosis.
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Ophthalmology Volume 107, Number 8, August 2000 50. Selhub J. Homocystine metabolism [review]. Annu Rev Nutr 1999;19:217– 46. 51. Taylor LM Jr, Moneta GL, Sexton GJ, et al. Prospective blinded study of the relationship between plasma homocystine and progression of symptomatic peripheral arterial disease. J Vasc Surg 1999;29:8 –19; discussion 19 –21. 52. Jacques PF, Selhub J, Bostom AG, et al. The effect of folic acid fortification on plasma folate and total homocystine concentrations. N Engl J Med 1999;340:1449 –54. 53. Brouwer IA, van Dusseldorp M, Thomas CM, et al. Low-dose folic acid supplementation decreases plasma homocystine concentrations: a randomized trial. Am J Clin Nutr 1999;69:99 –104.
54. Bronstrup A, Hages M, Pietrzik K. Lowering of homocystine concentrations in elderly men and women. Int J Vitam Nutr Res 1999;69:187–93. 55. Lobo A, Naso A, Arheart K, et al. Reduction of homocystine levels in coronary artery disease by low-dose folic acid combined with vitamins B6 and B12. Am J Cardiol 1999;83: 821–5. 56. de Jong SC, Stehouwer CD, van den Berg M, et al. Normohomocystinaemia and vitamin-treated hyperhomocystinaemia are associated with similar risks of cardiovascular events in patients with premature peripheral arterial occlusive disease. A prospective cohort study. J Intern Med 1999;246:87–96.
Discussion by William E. Benson, MD Both nonarteritic ischemic optic neuropathy and central retinal artery obstruction sometimes can affect the fellow eye. Therefore, when a patient presents with either, it behooves us to identify treatable risk factors. The authors, using high performance liquid chromatography, a technique that reliably detects low concentrations of homocystine, have found that hyperhomocystinemia is statistically significantly associated with both of these conditions. They may well be right, because hyperhomocystinemia has already From the Wills Eye Hospital, Philadelphia, Pennsylvania. Address correspondence to William E. Benson, MD, 1051 Indian Creek Road, Wynnewood, PA, 19096.
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been associated with coronary and cerebral vascular disease. Further, we should hope that they are right, because treatment with nontoxic doses of folic acid, vitamin B6, and vitamin B12 can reduce serum homocystine levels by 25%. The authors’ study is an excellent start. However, their numbers are small and one must be very careful before implicating any risk factor with such small numbers, even if they are statistically significant. Before we routinely recommend vitamin supplements to patients with ischemic optic neuropathy and central retinal artery occlusion, two conditions must be fulfilled. First, larger studies must confirm their results. Second, studies must be conducted to show that lowering the homocystine levels will actually prevent involvement of the fellow eye.