Total Plasma Homocysteine and Primary Open-angle Glaucoma

GLORIA WANG, MD, FELIPE A. MEDEIROS, MD, BRUCE A. BARSHOP, MD, AND ROBERT N. WEINREB, MD

To evaluate total plasma homocysteine (tHcy) levels in patients diagnosed with primary openangle glaucoma (POAG) and normal subjects. DESIGN: Case-control study. METHODS: This study involved 55 POAG patients, 16 patients with secondary open-angle glaucoma or angleclosure glaucoma (non-POAG group), and 39 control healthy subjects undergoing ocular surgery. All glaucoma patients had characteristic glaucomatous optic disk damage and visual eld loss. Fasting tHcy concentrations of all study participants were determined using high-performance liquid chromatography. Analysis of variance was used to compare homocysteine levels among the three diagnostic groups, and multivariate analysis was conducted to assess the associations between tHcy and diagnostic group, age, gender, smoking status, systemic hypertension, hyperlipidemia, and cardiovascular or cerebrovascular disease. RESULTS: Mean standard deviation of tHcy levels in POAG individuals, non-POAG patients and control subjects was 14.90 6.45 mol/l, 14.30 4.35 mol/l, and 14.81 4.56 mol/l, respectively (P .93; ANOVA). No statistically signicant difference was found in the proportion of patients with abnormal tHcy levels among the three diagnostic groups. In multivariate analysis, only age and positive smoking status were signicantly correlated with total plasma homocysteine levels. CONCLUSION: No signicant difference was found in plasma homocysteine levels among POAG patients and normal control individuals. (Am J Ophthalmol 2004; 137:401 406. 2004 by Elsevier Inc. All rights reserved.)

PURPOSE:

HE PATHOGENESIS OF OPTIC NERVE DAMAGE IN

Accepted for publication Sept 12, 2003. InternetAdvance publication at ajo.com Sept 16, 2003. From the Departments of Ophthalmology (G.W., F.A.M., R.N.W.) and Pediatrics (B.A.B.), University of California, San Diego, San Diego, California. Inquiries to Robert N. Weinreb, MD, Hamilton Glaucoma Center, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0946
0002-9394/04/$30.00 doi:10.1016/j.ajo.2003.09.041

glaucoma remains poorly understood. Although intraocular pressure is assumed to be the most important risk factor for the disease, there is accumulating evidence that vascular risk factors may also play a role.13 Glaucomatous damage may be caused, or at least facilitated, by inadequate perfusion of the optic nerve. Impaired microcirculation and abnormal perfusion may be linked to anatomical or functional abnormalities of the vessels responsible for the blood supply of the optic nerve head, or both, like arteriosclerosis or vascular dysregulations.1,3 As a result of several epidemiologic studies, increased attention recently has been directed at studying the total plasma level of the amino acid homocysteine (tHcy) as a risk factor for various cardiovascular disorders. Hyperhomocysteinemia has been identied as a possible risk factor for arteriosclerosis and for the development of symptomatic peripheral vascular, cerebrovascular, and coronary heart disease.4 9 Pooled results from retrospective studies indicate that fasting homocysteine concentrations in patients with vascular disease are on average 30% higher than in normal subjects.7 Moreover, hyperhomocysteinemia has also been demonstrated to be a risk factor for retinal vascular disease, including central retinal vein and central retinal artery occlusions10 17 as well as for nonarteritic ischemic optic neuropathy.13,18 In a recent study, Leibovitch and associates19 found higher levels of plasma homocysteine in pseudoexfoliative glaucoma patients compared with age-matched control subjects. However, to date, there has been only one study relating levels of homocysteine in patients with primary open-angle glaucoma (POAG). Using an enzyme-linked immunoassay (EIA), Bleich and coworkers20 reported signicantly higher plasma homocysteine levels in 18 patients with POAG compared with 19 control subjects. The assay of total homocysteine in biological uids is complicated by the ease with which the amino acid forms disulde bonds. Although EIA methods provide simple and rapid assessment of tHcy levels, high-performance liquid chromatography (HPLC) may be more precise.2123 Highperformance liquid chromatography has been shown to
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provide reliable detection of even low concentrations of plasma homocysteine.21,24 The purpose of this study was to evaluate plasma homocysteine levels using the HPLC method in a group of POAG patients and compare them to an age-matched group of healthy control subjects.

METHODS
STUDY SUBJECTS:

This case-control study involved 55 POAG patients, 16 patients with secondary open-angle glaucoma or angle-closure glaucoma (non-POAG group), and 39 control healthy subjects undergoing ocular surgery at the Hamilton Glaucoma Center, University of California, San Diego. Informed consent was obtained from all participants after detailed explanation of the purpose and methods of the study. The University of California San Diego Human Subjects Committee approved all protocols, and the methods described adhered to the tenets of the Declaration of Helsinki. Each subject underwent a comprehensive ophthalmologic examination including best-corrected visual acuity, slit-lamp biomicroscopy, intraocular pressure (IOP) measurement using Goldmann applanation tonometry, gonioscopy, and dilated fundoscopic examination using a 78diopter lens. A detailed medical history was obtained to identify patients with risk factors for vascular disease such as hypertension, diabetes mellitus, hyperlipidemia, smoking status, and also presence of cardiovascular and cerebrovascular disease, and current drug therapy. Exclusion criteria included major systemic illness (including recent myocardial infarction), evidence of vasculitis, renal or hepatic disease, gastrointestinal malabsorption, cardiomyopathy, pregnancy, psychiatric illness, chronic alcohol abuse, anticonvulsivant therapy, recent exposure (within 3 months) to nitrous oxide, or current use of medications known to be associated with increased plasma levels of homocysteine.25 To be included, POAG subjects had to have openangles on gonioscopy, characteristic glaucomatous optic disk damage (presence of neuroretinal rim thinning, excavation, notching, or characteristic retinal nerve ber layer defects), and repeatable glaucomatous visual eld defects on standard automated perimetry (full-threshold or Swedish Interactive Threshold Algorithm [SITA] strategy, program 24 2, Humphrey Field Analyzer). Glaucomatous visual eld loss was dened as a pattern standard deviation (PSD) outside of the 95% normal condence limits, or a Glaucoma Hemield Test outside 99% normal condence limits. Mean deviation (MD) and PSD of visual elds of POAG patients were 9.2 8.8 dB and 5.2 3.6 dB, respectively. Patients in the non-POAG group had characteristic optic nerve damage and visual eld loss as dened above but with an associated cause for elevated IOP. Four AMERICAN JOURNAL
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patients had pseudoexfoliative glaucoma, three had pigmentary glaucoma, three had neovascular glaucoma, and six had primary angle-closure glaucoma. Mean MD and PSD of non-POAG patients were 6.8 8.7 dB and 4.8 3.8 dB, respectively. Control healthy subjects had no history of ocular disease (except refractive error, strabismus, or cataracts), normal IOP ( 21 mm Hg), and normal eye examination, including open-angles, and normal appearance of the optic disks and retinal nerve ber layer. All patients underwent blood sample collection the day of ocular surgery, ensuring that the patients were fasting. Patients with glaucoma were scheduled to have either cataract extraction by phacoemulsication (32 patients), trabeculectomy with antimetabolite use (31 patients), or a combined procedure (8 patients). All 39 control subjects were scheduled to have cataract extraction by phacoemulsication.
BLOOD SAMPLE COLLECTION AND BIOCHEMICAL

The blood samples used for the preparation of serum were collected into an evacuated tube containing heparin and immediately placed on ice. The samples were then centrifugated at 3500 rpm for six minutes, usually within one hour and after a maximum of two hours of collection. After centrifugation, the plasma supernatant was placed into plastic Eppendorf tubes and stored at 10 C. Within a few days the samples were delivered on ice to UCSD Biochemical Genetics Laboratory where they were stored at 20 C until tHcy analysis was performed. The duration of storage was from a few days up to one month. Total homocysteine (free and initially proteinbound) was measured using sodium borohydride reduction of disulde bonds and derivatization of sulfhydryl groups with monobromobimane followed by deproteinization and HPLC with uorescence detection.26 All measurements were performed in duplicate. Standard curves and quality control samples were run with each batch. The normal range for plasma tHcy was between 5 and 15 mol/l in fasting subjects.
ANALYSIS:

Analysis of variance was used to detect differences in continuous variables (tHcy and age) among the three diagnostic groups. The TukeyKramer honestly signicant difference (HSD) test was used to evaluate pair wise differences between groups. For categorical variables, 2 tests were used to assess differences between groups. General linear regression models were used to evaluate the associations between total plasma homocysteine and diagnostic group, age, gender, smoking status, systemic hypertension, hyperlipidemia, and cardiovascular or cerebrovascular disease. A P value less than .05 was considered statistically signicant. Statistical analyses were performed using software SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). OPHTHALMOLOGY MARCH 2004

STATISTICAL ANALYSIS:

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TABLE 1. Demographic and Clinical Data for Primary Open-angle Glaucoma (POAG) Patients, Non-POAG Patients and Control Healthy Subjects*
POAG (n 55) Non-POAG (n 16) Control (n 39)

Variable

Age, years ( SD) Female gender Systemic hypertension Hyperlipidemia Diabetes Mellitus Cardiovascular/ Cerebrovascular disease Smoking

75 9 34 (62) 29 (53) 9 (16) 6 (11) 12 (22)

75 6 14 (88) 8 (50) 6 (38) 2 (13) 5 (31)

73 7 22 (56) 22 (56) 14 (36) 6 (15) 10 (26)

.51 .09 .90 .05 .81 .75

2 (4)

4 (10)

.22

SD standard deviation. *Values are expressed as number (percentage) unless otherwise specied.

of 5 mol/l, statistical power was above 90% to detect a signicant difference among the diagnostic groups at 0.05. Assuming a tHcy level higher than 15 mol/l as abnormal, 26 POAG patients (47%), 7 non-POAG patients (43%), and 14 control subjects (36%) had abnormal tHcy levels. No statistically signicant difference was found in the proportion of patients with abnormal tHcy levels among the three diagnostic groups (P .56). The correlation between severity of visual eld damage, as assessed by the visual eld indices MD and PSD, and tHcy levels in glaucoma patients was also evaluated. No statistically signicant correlation was found between MD and tHcy levels in POAG (r 0.234; P .13) as well as in non-POAG patients (r 0.348; P .27). Also, no signicant correlation was found between PSD and tHcy levels in POAG (r 0.163; P .30) and non-POAG (r 0.384; P .22) groups. In the multivariate model, only age (P .048) and positive smoking status (P .003) were signicantly correlated with total plasma homocysteine levels.

RESULTS
TABLE 1 SHOWS THE DEMOGRAPHIC AND CLINICAL DATA

for the three diagnostic groups. No signicant difference was found in age, gender, prevalence of systemic hypertension, diabetes mellitus, cardiovascular or cerebrovascular diseases, and smoking status among the three diagnostic groups. The POAG group had a signicantly lower proportion of patients being treated for hyperlipidemia than the control group (16.4% vs 35.9%; P .03; square test). Mean SD of tHcy levels in POAG individuals, nonPOAG patients and control subjects was 14.90 6.45 mol/l, 14.30 4.35 mol/l, and 14.81 4.56 mol/l, respectively (P .93; ANOVA). For non-POAG patients, mean tHcy levels according to diagnostic category were 13.21 2.81 mol/l (angle-closure glaucoma), 14.31 5.91 mol/l (pseudoexfoliative glaucoma), 16.40 7.02 mol/l (neovascular glaucoma), and 14.36 3.15 mol/l (pigmentary glaucoma). Considering all 71 glaucoma patients (POAG and non-POAG) together, the mean tHcy level was 14.80 6.01 mol/l. There was no signicant difference between mean tHcy levels in glaucoma patients and control subjects (P .97; Student t test). Figure 1 shows the distribution of values of tHcy in the three diagnostic groups using a box plot chart. The Box extends from the 25th to 75th percentile and the whiskers extend to largest and smallest observed values within 1.5 box lengths. The range dened by the inner fences (end of the whisker) contains approximately 95% of the data points, and points outside this range are called outliers.27 Two subjects in the control group and three in the POAG group were considered outliers. The exclusion of these ve subjects from the analysis did not change the signicance of the results (P .85; ANOVA). Assuming a minimum detectable difference in homocysteine levels of 4 mol/l and a common standard deviation VOL. 137, NO. 3 HOMOCYSTEINE

DISCUSSION
IN THE CURRENT STUDY, NO SIGNIFICANT DIFFERENCES

were found between mean total plasma homocysteine levels among POAG individuals, non-POAG patients, and control subjects. Also, the distribution of tHcy values was similar among the subgroups, and no signicant difference was found in the proportion of patients diagnosed with hyperhomocysteinemia among the three diagnostic groups. These results contrast with those of Bleich and associates,20 who found a signicantly higher mean tHcy level in 18 POAG patients (12.52 mol/l) compared with 19 control subjects (8.40 mol/l). Different characteristics of the included populations such as age, severity of glaucoma, nutritional status, and presence of associated illnesses may account for the conicting results. Also, different methodologies used to assess plasma tHcy levels may explain at least part of the different results. Bleich and associates20 used an EIA method to assess plasma homocysteine levels, whereas in the current study, an HPLC method was used. Several investigators have evaluated the different available analytical methods to assess plasma homocysteine concentrations. Nexo and coworkers22 found that measurements of tHcy with EIA have a higher inaccuracy and imprecision compared with other standard methods. In another study, Ducros and associates21 concluded that EIA methods are more suitable for large screening programs of hyperhomocysteinemia detection, but their limitations require that high values be checked using reference methods in specialized laboratories. Using an HPLC method, Leibovitch and associates19 found higher tHcy levels in 30 pseudoexfoliative glaucoma patients compared with 30 age-matched control subjects.
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FIGURE 1. Distribution of total plasma homocysteine values in primary open-angle glaucoma (POAG) patients, non-POAG patients, and normal control subjects.

Although in the present study tHcy levels in the nonPOAG patients were not signicantly different from the control group, only four subjects had a diagnosis of pseudoexfoliative syndrome, making any comparison of the present results to those found by Leibovitch and associates difcult. An elevated plasma homocysteine level may occur as the result of inherited disorders of the enzymes of its metabolism but also as the result of nutritional deciencies of the vitamin co-factors (B6, B12, and folates); the presence of systemic diseases like chronic renal failure, systemic hypertension, diabetes mellitus, malignancies; physiological factors (that is, age, gender); use of medications; or lifestyle determinants such as smoking, coffee consumption, alcoholism, and physical activity.25,28 In the current study, an attempt was made to evaluate and match the patients by possible confounding factors associated with plasma homocysteine levels. Accordingly, no signicant difference was found in any of the evaluated factors among the three diagnostic groups, except for hyperlipidemia. In multivariate analysis, only older age and positive smoking status were signicantly associated with higher tHcy levels. One limitation of the present study is that it did not evaluate measurements of nutritional status or other lifestyle determinants possibly associated with plasma homocysteine concentrations. The mean tHcy level for the normal subjects included in the present analysis was higher than those reported in previous studies evaluating homocysteine as a risk factor for glaucoma or other ocular diseases.11,19,20 However, the 404 AMERICAN JOURNAL
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presence of a large number of factors inuencing plasma levels of homocysteine makes the comparisons among different studies difcult. Further, the intermethod and interlaboratory variations of tHcy measurements have been reported to be high.21 Preliminary data from the Centers for Disease Control and Prevention on 14 laboratories performing tHcy assay on reference materials indicate a high variation between laboratories.29 For one reference material with a mean concentration of 11.1 mol/l, the reported values ranged from 8.3 to 14 mol/l. Nonetheless, the reproducibility of the method used in the present investigation allows signicant conclusions based on comparisons between groups. Elevated plasma homocysteine is generally dened as tHcy 15 mol/l.30 Using this cutoff, a relatively high number of subjects in each of the subgroups would carry a diagnosis of hyperhomocysteinemia. This observation may be related to several factors. It is well known that tHcy increases with age, so that as for individuals aged 65 or older tHcy levels up to 20 mol/l are usually considered normal.25 Ninety percent of the subjects included in the present study were older than age 65. Using 20 mol/l as a cutoff value, six (11%) POAG patients, two (13%) non-POAG patients, and three (8%) controls would be diagnosed as having hyperhomocysteinemia, with no signicant difference among the diagnostic groups. Systemic hypertension has also been associated with higher levels of tHcy.28,31 Plasma homocysteine concentration has been demonstrated to be positively correlated with levels of systolic blood pressure.31 In the present investigation, OPHTHALMOLOGY MARCH 2004

about half of the subjects had systemic hypertension, which may be associated with the relatively high tHcy levels found in each of the diagnostic groups. The exact mechanisms of action of homocysteine remains to be elucidated. Current evidence suggests that an increase in homocysteine concentration may induce atherosclerosis by a combination of endothelial injury,32 smooth muscle proliferation,33,34 platelet activation and thrombogenesis.35,36 These vascular effects could lead to changes in the optic nerve head microvasculature and consequent impairment of optic nerve blood ow. Some evidence has also suggested that excess levels of homocysteine may induce neuronal cell death.3739 Moore and associates37 demonstrated that homocysteine induces apoptotic cell death in retinal ganglion cells by overstimulation of n-methyl-D-aspartate receptors and caspase-3 activation. However, the homocysteine concentrations used in many of these experiments were in the pharmacologic range and may not be relevant to the levels encountered in clinical practice. The results of these studies must, therefore, be interpreted with caution. In accordance, conicting evidence largely exists among the clinical studies evaluating tHcy as a risk factor for cardiovascular disease.40 43 In a comprehensive meta-analysis, Ford and coworkers44 found that the evidence available from prospective studies suggests only a weak association between homocysteine concentration and coronary heart disease. Thus, it is still not clear whether homocysteine is a causative factor or only a marker of vascular diseases. In conclusion, no signicant difference was found in plasma homocysteine levels among POAG patients and normal control individuals in the present analysis. Although these results provide evidence against the role of homocysteine as a risk factor for POAG, larger-scale prospective studies will be needed to resolve conicting results in the literature.

REFERENCES
1. Hayreh SS. Blood ow in the optic nerve head and factors that may inuence it. Prog Retin Eye Res 2001;20:595624. 2. Weinreb RN, Ciof GA, Harris A. Optic nerve blood ow. In: Van Buskirk EM, Shields B, editors. 100 Years of progress in glaucoma. Philadelphia: Lippincott-Raven Healthcare, 1997:59 78. 3. Flammer J, Orgul S, Costa VP, et al. The impact of ocular blood ow in glaucoma. Prog Retin Eye Res 2002;21:359 93. 4. Nygard O, Nordrehaug JE, Refsum H, Ueland PM, Farstad M, Vollset SE. Plasma homocysteine levels and mortality in patients with coronary artery disease. N Engl J Med 1997; 337:230 236. 5. McCully KS. Vascular pathology of homocysteinemia: Implications for the pathogenesis of arteriosclerosis. Am J Pathol 1969;56:111128. 6. 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;346:13951398.

7. Ueland PM, Refsum H, Brattstrom L. Plasma homocysteine and cardiovascular disease. In: Francis RBJ, editor. Atherosclerotic cardiovascular disease, hemostasis, and endothelial function. New York: Marcel Dekker, 1992:183236. 8. Clarke R, Daly L, Robinson K, et al. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med 1991;324:1149 355. 9. Selhub J, Jacques PF, Bostom AG, et al. Association between plasma homocysteine concentrations and extracranial carotid-artery stenosis. N Engl J Med 1995;332:286 291. 10. Biousse V, Newman NJ, Sternberg P Jr. Retinal vein occlusion and transient monocular visual loss associated with hyperhomocystinemia. Am J Ophthalmol 1997;124:257 260. 11. Cahill M, Karabatzaki M, Meleady R, et al. Raised plasma homocysteine as a risk factor for retinal vascular occlusive disease. Br J Ophthalmol 2000;84:154 157. 12. 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 Fibrinol 1998;9:617622. 13. Pianka P, Almog Y, Man O, Goldstein M, Sela BA, Loewenstein A. Hyperhomocystinemia in patients with nonarteritic anterior ischemic optic neuropathy, central retinal artery occlusion, and central retinal vein occlusion. Ophthalmology 2000;107:1588 1592. 14. Loewenstein A, Goldstein M, Winder A, Lazar M, Eldor A. Retinal vein occlusion associated with methylenetetrahydrofolate reductase mutation. Ophthalmology 1999;106:1817 1820. 15. Loewenstein A, Winder A, Goldstein M, Lazar M, Eldor A. Bilateral retinal vein occlusion associated with 5,10-methylenetetrahydrofolate reductase mutation. Am J Ophthalmol 1997;124:840 841. 16. Weger M, Stanger O, Deutschmann H, et al. The role of hyperhomocysteinemia and methylenetetrahydrofolate reductase (MTHFR) C677T mutation in patients with retinal artery occlusion. Am J Ophthalmol 2002;134:5761. 17. Brown BA, Marx JL, Ward TP, et al. Homocysteine: a risk factor for retinal venous occlusive disease. Ophthalmology 2002;109:287290. 18. Biousse V, Kerrison JB, Newman NJ. Is non-arteritic anterior ischaemic optic neuropathy related to homocysteine? Br J Ophthalmol 2000;84:555. 19. Leibovitch I, Kurtz S, Shemesh G, et al. Hyperhomocystinemia in pseudoexfoliation glaucoma. J Glaucoma 2003;12:36 39. 20. Bleich S, Junemann A, Von Ahsen N, et al. Homocysteine and risk of open-angle glaucoma. J Neural Transm 2002;109: 1499 1504. 21. Ducros V, Demuth K, Sauvant MP, et al. Methods for homocysteine analysis and biological relevance of the results. J Chromatogr B Analyt Technol Biomed Life Sci 2002;781: 207226. 22. Nexo E, Engbaek F, Ueland PM, et al. Evaluation of novel assays in clinical chemistry: quantication of plasma total homocysteine. Clin Chem 2000;46:1150 1156. 23. Ducros V, Candito M, Causse E, et al. Comparison of plasma total homocysteine determinations in 9 French hospital laboratories by using 6 different methods. Ann Biol Clin (Paris) 2002;60:421428. 24. Jacobsen DW, Gatautis VJ, Green R. Determination of plasma homocysteine by high-performance liquid chromatography with uorescence detection. Anal Biochem 1989; 178:208 214. 25. Clarke R, Stansbie D. Assessment of homocysteine as a
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HOMOCYSTEINE

POAG

405

26.

27. 28. 29. 30.

31.

32.

33.

34. 35.

cardiovascular risk factor in clinical practice. Ann Clin Biochem 2001;38:624 632. Jacobsen DW, Gatautis VJ, Green R, et al. Rapid HPLC determination of total homocysteine and other thiols in serum and plasma: sex differences and correlation with cobalamin and folate concentrations in healthy subjects. Clin Chem 1994;40:873881. Frigge M, Hoaglin DC, Iglewicz B. Some implementations of the boxplot. The Am Statist 1989;43:50 54. Refsum H, Ueland PM, Nygard O, Vollset SE. Homocysteine and cardiovascular disease. Annu Rev Med 1998;49:3162. Centers for Disease Control and Prevention. Assessment of laboratory tests for plasma homocysteineselected laboratories, JulySeptember 1998. MMWR 1999;48:10131015. Malinow MR, Bostom AG, Krauss RM. Homocyst(e)ine, diet, and cardiovascular diseases: a statement for healthcare professionals from the Nutrition Committee, American Heart Association. Circulation 1999;99:178 182. Sutton-Tyrrell K, Bostom A, Selhub J, Zeigler-Johnson C. High homocysteine levels are independently related to isolated systolic hypertension in older adults. Circulation 1997; 96:17451749. Stamler JS, Osborne JA, Jaraki O, et al. Adverse vascular effects of homocysteine are modulated by endotheliumderived relaxing factor and related oxides of nitrogen. J Clin Invest 1993;91:308 318. Tsai JC, Perrella MA, Yoshizumi M, et al. Promotion of vascular smooth muscle cell growth by homocysteine: a link to atherosclerosis. Proc Natl Acad Sci USA 1994;91:6369 6373. Tang L, Mamotte CD, Van Bockxmeer FM, Taylor RR. The effect of homocysteine on DNA synthesis in cultured human vascular smooth muscle. Atherosclerosis 1998;136:169 173. Rodgers GM, Conn MT. Homocysteine, an atherogenic stimulus, reduces protein C activation by arterial and venous endothelial cells. Blood 1990;75:895901.

36. Rodgers GM, Kane WH. Activation of endogenous factor V by a homocysteine-induced vascular endothelial cell activator. J Clin Invest 1986;77:1909 1916. 37. Moore P, El-sherbeny A, Roon P, Schoenlein PV, Ganapathy V, Smith SB. Apoptotic cell death in the mouse retinal ganglion cell layer is induced in vivo by the excitatory amino acid homocysteine. Exp Eye Res 2001;73:4557. 38. Lipton SA, Kim WK, Choi YB, et al. Neurotoxicity associated with dual actions of homocysteine at the N-methyl-Daspartate receptor. Proc Natl Acad Sci USA 1997;94:5923 5928. 39. Kim WK, Pae YS. Involvement of N-methyl-d-aspartate receptor and free radical in homocysteine-mediated toxicity on rat cerebellar granule cells in culture. Neurosci Lett 1996;216:117120. 40. Ridker PM, Stampfer MJ, Rifai N. Novel risk factors for systemic atherosclerosis: a comparison of C-reactive protein, brinogen, homocysteine, lipoprotein(a), and standard cholesterol screening as predictors of peripheral arterial disease. JAMA 2001;285:24812485. 41. Chasan-Taber L, Selhub J, Rosenberg IH, et al. A prospective study of folate and vitamin B6 and risk of myocardial infarction in US physicians. J Am Coll Nutr 1996;15:136 143. 42. OGrady H, Kelly C, Bouchier-Hayes D, Leahy A. Homocysteine and occlusive arterial disease. Br J Surg 2002;89: 838 844. 43. Verhoef P, Hennekens CH, Allen RH, Stabler SP, Willett WC, Stampfer MJ. Plasma total homocysteine and risk of angina pectoris with subsequent coronary artery bypass surgery. Am J Cardiol 1997;79:799 801. 44. Ford ES, Smith SJ, Stroup DF, Steinberg KK, Mueller PW, Thacker SB. Homocyst(e)ine and cardiovascular disease: a systematic review of the evidence with special emphasis on case-control studies and nested case-control studies. Int J Epidemiol 2002;31:59 70.

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