I n the past several years, a large number of invest!-

Homocysteine and Folate Concentrations in Blood from Patients Treated ith Hemodialysi& Tsunenobu Tamura,2 Kelley E. Johnston, and Suzanne M. Bergman 1. Tamura, K.E. Johnston, Department of Nutrition Sciences, Universily of Alabama at Birmingham, Birmingham, AL SM. Bergman, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL (J. Am. Soc. Nephrol. 1996; 7:2414-2418) ABSTRACT Plasma homocysteine and plasma and erythrocyte folate concentrations before and after hemodialysis ere measured in 31 patients ith ESRD. Homocystelne and folate ere measured by HPLC-fluorometric and microbiological methods, respectively. The mean plasma homocysteine level declined from 36.8 to 24.2 mol/l during hemodialysis, Indicating that homocysteine can be partly removed by hemodialysis (P <.1). Mean plasma folate concentration before hemodialysis as 46.4 nmol/l and decreased to 25.9 nmol/l after hemodialysis (P <.1), hereas mean erythrocyte folate concentration did not change (1295 and 1385 nmol/l before and after hemodialysis, respectively). Plasma folate concentrations shoed a significant negative correlation ith homocystelne concentrations before and after hemodialysis (r = -.53, P <.3, and r = -.59, P <.1, respectively). Furthermore, there ere significant negative correlations beteen plasma homocysteine and erythrocyte folate concentrations both before (r = -.6, P <.5) and after hemodlalysis (r = -.49, P <.5). All patients had homocysteine concentrations over 12. mol/l before hemodlalysis, and only three had homocystelne concentrations loer than 12. moi/l after hemodlalysis. Although significant correlations existed beteen homocysteine and folate concentrations, the majority of the patients In this study appeared to have adequate folate nutriture as assessed by blood folate concentratlons. It remains to be determined hether patients ith ESRD have an altered homocystelne metabolism. Key Words: ESRD. foilc acid supplementation, cardiovascular disease 1 Received January 1, 1996. Accepted April 16, 1996. 2 Correspondence to Dr. T. Tamura, Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham. AL 35294-336. 146.6673/711-241413./ Journal of the American SocIety of Nephrology CopyrIght C 1996 by the AmerIcan society of Nephrology I n the past several years, a large number of invest!- gators have shon that increased plasma/serum homocysteine concentration (hyperhomocysteinemla) is an independent risk factor for development of atherosclerosis ( 1,2). It is ell knon that there is a high incidence of cardiovascular disease in patients ith ESRD, and many researchers have determined the concentrations of plasma/serum homocysteine in these patients. It Is no ell established that these patients have abnormally high concentrations of homocysteine in circulation (3-2). There are several reports indicating an association beteen hyperhomocysteinemia and folate nutritional status by either determining the concentrations of plasma/serum folate or evaluating the effect of folic acid (pteroylglutamic acid) supplementation on plasma/serum homocysteine levels in patients ith ESRD (5,8.11,14,17, 18,2). There appears to be a consensus that folate nutriture is one of the most important factors regulating the concentrations of homocysteine in circulation. We undertook the study presented here to evaluate the relationship beteen plasma homocysteine and plasma and erythrocyte folate concentrations in patients treated ith hemodialysis. MATERIALS AND METHODS In this study, e used plasma samples that ere originally obtained from 3 1 patients (23 omen and eight men; age range, 36 to 7 1 yr) ith ESRD ho ere treated ith hemodialysis. They participated in a study to measure plasma folate conjugase activities before and after hemodialysis beteen February and April 1993 (21). The patients ere maintained by hemodialysis using a high-flux polysulfone membrane (3.5 to 4. h three times per k). Of the 31 patients, 2 received a prescription of daily multivitamin tablets containing folic acid (.68 to 2.27 mol/day), and 29 received erythropoletln administration at dosages ranging from 2 to 8 U three times per k. The study as approved by the Institutional Revie Board of the University of Alabama at Birmingham and each patient signed an informed consent form. Blood samples ere obtained from the arterial line immediately before and after the procedure and ere transferred to evacuated tubes containing heparin. Blood samples ere stored at 4#{176}C until centrifugation to avoid falsely high values resulting from homocystelne leaching out from the erythrocytes (22). After separation, plasma samples ere stored at -7#{176}C until analysis (a condition under hich plasma homocysteine is knon to be stable for a long period of time) (23). Plasma homocysteine levels ere determined using an HPLC-fluorometric method after samples ere treated ith 7-flourobenzo-2-oxa- 1,3-diazole-4-sulfonamlde to convert homocysteine to a fluorescent compound (24). Homocystelne thiolactone hydrochloride (Sigma Chemical, St. Louis, MO) as used as a standard for the assay. The concentration of the standard as verified spectrophotometrically using the absorption maximum at 238 nm of A = 4.9 per mol/l at 2414 Volume 7 Number 1 1. 1996

Tamura et al ph 1. (Baggott JE, personal communication). In our laboratory, the coefficient ofvariation for the homocysteine assay is approximately 9%, using repeated determinations of pooled plasma samples. Plasma and erythrocyte folate concentratlons ere determined by a microbiological method using Lactobactilus casel as the assay organism (25); folate values ere previously reported (2 1). Statistical analyses ere performed using the linear regression analysis and paired t test after all values ere logarithmically transformed. P values less than.5 ere considered significant. RESULTS As shon in Table 1, the mean concentration of plasma homocysteine immediately before hemodialysis as 36.8 tmol/l, hich declined to 24.2 pmol/l after hemodialysis (P <. 1). SimIlarly, plasma folate concentrations decreased during hemodialysis from 46.4 to 25.9 nmol/l (P <.1), hereas erythrocyte folate concentrations remained unchanged (1295 and 1385 nmol/l before and after hemodialysis, respectively) (2 1). As shon in Figures 1A and B, plasma folate concentrations shoed a significantly negative correlation ith plasma homocysteine concentrations before (r = -.53, P <.3) and after hemodialysis (r = -.59, P <.1). There as a significantly negative correlation beteen plasma homocysteine and erythrocyte folate concentrations before (r = -.6, P <.5) and after hemodialysis (r = -.49, P <.5) as shon in Figure 2A and B. Of the 3 1 patients, none had homocystelne concentrations belo 12. mol/l before hemodialysis, hich is generally accepted as a cutoff point for normal range (26; and Tamura et at., unpublished), and only three had concentrations belo this value after hemodialysis. Only four patients had erythrocyte folate concentrations belo the loer limit of normal range (<454 nmol/l) (27). The mean plasma homocysteine level in the 2 patients ith folic acid supplementation before hemodialysis as 3.6 mol/l, hich as significanfly loer than that in the 1 1 patients ithout supplementation (47.9 mol/l, P <.3); hoever, this difference as not significant after hemodialysis. Plasma and erythrocyte folate levels ere not significantly -J E z I- U) >- C-) I Cl) I 1 1 1 o A..iI,. 1 1 O.. OO 1 1 PLASMA FOLATE (nmol/l) I. Figure 1. RelationshIp beteen plasma homocysteine and plasma folate concentrations before and after hemodlalysis. (A) Plasma homocysteine and folate concentrations before hemodialysis (r = -.53, P <.3). (B) Plasma homocysteine and folate concentrations after hemodialysis (r = -.59, P <.1). Closed circles represent patients ho ere given a prescription of folic acid and open circles represent those ho ere not. different beteen patients ith and ithout folic acid supplementation. Furthermore, there ere no significant differences in plasma and erythrocyte folate and B TABLE 1. Changes in homocysteine and folate concentrations before and after hemodialysis in 31 patients ith ESRD Parameter (normal values in our laboratory) Before (Mean ± SD (Range)) Geometric Mean (1th, 9th percentile) After (Mean ± SD (Range)) Geometric Mean (1th, 9th percentile) P Value Plasma Homocysteine (jmol/l) 36.8 ± 18.3 32.8 24.2 ± 1.1 22.2.1 (<12,.mol/L)a (13.1 to 83.1) (16.3, 69.2) (9.6 to 47.4) (15.3, 4.9) Plasma Folate (nmol/l)b 46.4 ± 72.3 24.3 25.9 ± 3.7 15.3.1 (>1 1.3 mol/l) (3.9 to 362.1) (8.7, 95.5) (3.6 to 1 12.) (4.9, 89.7) Erythrocyfe Folate (nmol/l) 1295 ± 1239 863 1385 ± 1276 992 NSC (>454 mol/l) (16 to 427) (3, 324) (23 to 53) (44, 36) a Unpublished data. b Some of these values ere published previously (21). C Not significantly different before and after hemodialysis. Journal of the American Society of Nephrology 2415

Homocysteine and Folate in ESPD Patients -J E z I- U) >- C.) I Cl) a- 1 1 1 1 o c9 o A : O 1 1 1 o 1 1 B ERYTHROCYTE FOLATE (nmol/l) Figure 2. Relationship beteen plasma homocysteine and erythrocyte folate concentrations. (A) Plasma homocysteine and erythrocyte folate concentrations before hemodialysis (1 = -.6, P <.5). (B) Plasma homocysteine and erythrocyte folate concentrations after hemodialysis (r = -.49, P <.5). Closed circles represent patients ho ere given a prescription of folic acid and open circles represent those ho ere not. plasma homocysteine levels beteen patients ho ere administered erythropoletin (N = 29) and those ho ere not (N = 2). DISCUSSION We found that hemodialysis reduced the levels of plasma homocysteine; hoever, this reduction as not sufficient to bring the concentrations to less than 12 mol/l, hich is the upper limit of normal range. The mean posthemodlalysis homocysteine concentration as 24.2 mol/l. There ere significant negative correlations beteen plasma homocysteine and erythrocyte and/or plasma folate concentrations, suggesting that folate nutriture Is one of the important factors for controlling plasma homocysteine concentrations. In the study presented here, both plasma and erythrocyte folate levels In pre- and posthemodialysis samples ere not significantly different beteen patients ho received a prescription of folic acid and those ho did not. Posthemodialysis plasma homocysteine levels ere similar in these to groups of patients, hereas prehemodialysis levels ere higher In the folic acidsupplemented group than In the nonsupplemented group. These findings indicate poor compliance by our patient population in taking supplemental vitamin tablets, and self-medication ith over-the-counter supplements containing folic acid by those ho did not get a prescription. Furthermore, it is not clear hether extra care should be taken ith the patients ith ESRD ho receive erythropoietin, In terms of folic acid supplementation (28,29). We could not address this question In the study presented here. Because only to of the 3 1 patients In this study did not receive erythropoietln, none of the differences reached statistical significance. The decline in plasma homocysteine concentrations during hemodialysis in our patients is consistent ith findings by other investigators (6-8, 1 1 ). Although Bostom et at. ( 1 8) reported that a negative correlation existed beteen homocysteine and folate concentrations in prehemodialysis plasma samples, to our knoledge it has never been shon that the concentrations of plasma homocysteine negatively correlated ith erythrocyte folate concentrations both before and after hemodlalysis. Erythrocyte folate concentrations are considered to be a more reliable indicator of long-term folate nutriture, unlike plasma folate, 1 hich is readily Influenced by dietary folate Intakes. Judged by erythrocyte folate concentrations, four of the 3 1 patients ( 1 3%) In the study presented here had subnormal erythrocyte folate concentrations (27), Indicating that the majority of our patients had adequate folate nutriture. On the basis of these findings, homocysteine concentrations should, In theory, have been normal in our patients. Therefore, there appears to be another unknon factor(s) involved In homocysteine metabolism in these patients. Thus far, the literature has uniformly pointed out that folic acid supplementation reduces homocysteine in circulation (5,8, 1 1, 1 4, 1 7, 1 8,2). By revieing these studies, hoever, e became aare that folic acid supplementation, even a large dose such as 11.3 pmol (5. mg) per day, does not necessarily normalize plasma/serum homocysteine concentrations in all patients ith ESRD. In a recent placebo-controlled study, Bostom et at. (2) administered a large daily dose of folic acid ( 15 mg = 34 j.mol), vitamin B6 (1 mg), and vitamin 12 ( 1 mg), in addition to the regularly prescribed supplementation of these vitamins ithout any adverse effects. They observed a 26% decline in plasma homocysteine after 8 k of administration. Hoever, the mean concentration of homocysteine as reduced only to 2 1.9 jimol/l and only five of 15 patients ho ere supplemented ith vitamins had homocysteine levels belo 15.tmo1/L. These investigators suggested that long-term controlled studies In patients ith ESRD be carried out to evaluate the effect of folic acid supplementation at higher than 34 mol per day on plasma homocysteine levels (2). 2416 Volume 7. Number 1 1 1996

Tamura et al It has been ell established that homocysteine 1evels in circulation are controlled by the nutriture of folate, vitamin B12, and vitamin B6 (26). Based on the results of increased concentrations of these vitamins in circulation reported by Bostom et a!. ( 18), it is unlikely that patients ith ESRD ho are supplemented ith multivitamins ill be deficient in these B vitamins. Other factors may also have an important role in regulating homocysteine metabolism in these patients. Perna et at. ( 15) suggested several possible mechanisms of increased plasma homocystelne, including decreased renal excretion, altered transsulfuration pathay, Impaired transmethylation reactions, and a combination of these. Hoever, it is unlikely that the altered transmethylatlon is the major reason for hyperhomocysteinemia among patients ith ESRD, based on the observation by Guttormsen et at. ( 19), ho found the increase In plasma methionine after homocystelne load and concluded that the methylation of homocysteine is Intact in these patients. They suggested that hyperhomocysteinemia is the result of reduced renal elimination of homocysteine from circulation, and that the kidney is an important site of homocysteine metabolism. Furthermore, using an in vivo rat model, Bostom et at. (3) shoed that homocystelne is metabolized in the kidney, and its urinary excretion is minimal. They extrapolated these findings to patients ith ESRD, and suggested that chronic loss of renal metabolism contributes to a high prevalence ofhyperhomocysteinemia among these patients. Serine is essential for the transsulfuration pathay in homocysteine metabolism ( 1 8). Wilcken et a!. (5) reported that plasma serine concentrations are lo in renal transplant recipients as compared to healthy control subjects. They also reported that plasma serine concentrations further declined after folic acid supplementation in patients ith ESRD, although plasma glycine concentrations increased (8). Hoever, the exact mechanism(s) of these phenomena Is Unknon. A similar observation as reported by Bostom et at. ( 18), ho hypothesized that a limited amount of serine is a cause of hyperhomocysteinemia In patients ith ESRD and gave oral serine supplementation (29 to 38 mmol [3 to 4 gi/day for 7 days) to these patients. Hoever, this supplementation did not affect plasma homocysteine concentrations. Furthermore, these investigators supplemented betaine in combination ith folic acid in patients ith ESRD ( 1 7). This therapy is knon to reduce the increased homocysteine concentrations found in patients ith homocystinuria, a congenital deficiency ofcystathionlne f3-synthase (31). Hoever, this supplementation did not change plasma homocysteine in patients ith ESRD (17). An additional cause of hyperhomocysteinemia may be increased sulfate levels in patients ith ESRD. We shoed that the activities of plasma folate conjugase, the enzyme that hydrolyzes polyglutamyl forms of folate to monoglutamyl folate, is sensitive to sulfate (2 1 ). The activity of folate conjugase is lo in prehemodialysis plasma samples but increases after hemodialysis. We postulated that the increased level of sulfate inhibits folate conjugase. Therefore, It Is possible that in addition to the reduced metabolism of homocystelne In the nonfunctioning kidney, a similar inhibition of sulfate on homocystelne metabolism occurs in extrarenal tissues such as the liver, hich presumably is one of the major sites of metabolism. Further investigation of the mechanisms of hyperhomocysteinemia In patients ith ESRD Is arranted. ACKNOWLEDGMENTS The authors thank Ms. Jane Forehand and Ms. Judy Waitha for their help in obtaining the blood samples used for this investigation. REFERENCES 1. Clarke R, Daly L, Robinson K, et at. : Hyperhomocysteinemla: An Independent risk factor for vascular disease. N Engl J Med 1991;324:l 149-1155. 2. Seihub J, Jacques PF, Bostom AG, et at.: Association beteen plasma homocysteine concentrations and extracranial carotid-artery stenosis. N Engl J Med 1995; 332:286-291. 3. Robins AJ, Mileczyk BK, Booth EM, Mallick NP: Plasma amino acid abnormalities in chronic renal fallure. Chin Chim Acta 1972;42:215-217. 4. Wilcken DEL, Gupta VJ: Sulphur containing amino acids in chronic renal failure ith particular reference to homocystine and cystelne-homocysteine mixed disulphide. Eur J Clin Invest 1979:9:31-37. 5. Wilcken DEL, Gupta VJ, Betts AK: Homocysteine In the plasma ofrenal transplant receipients: Effect of cofactors for methionine metabolism. Chin Sci 1981:61:743-749. 6. Kang S-S, Wong PWK, Bidani A, Milanez 5: Plasma protein-bound homocyst(e)ine In patients requiring chronic haemodlalysis. Clin Sd 1983;65:335-336. 7. Laidla SA, Smolin LA, Davidson WD, Kopple JD: Sulfur amino acids in maintenance hemodialysis patients. Kidney mt 1987;32lSuppl 22J:S191-S196. 8. Wilcken DEL, Dudman NPB, Tyrrell PA, Robertson MR: Folic acid loers elevated plasma homocysteine in chronic renal insufficiency: Possible implications for prevention of vascular disease. Metab Clin Exp Ther 1988: 37:697-71. 9. Sona C, Chadefaux B, Coude M, Gaillard, Kamoun P: Concentrations of total homocystelne In plasma in chronic renal failure. Clin Chem 199:36:2137-2138. 1. Chauveau P, Chadefaux B, Cloude M, et at.: Increased plasma homocystelne concentration in patients ith chronic renal failure. Miner Electrolyte Metab 1992; 18: 196-198. 1 1. Chauveau P, Chadefaux B, Cloude M, et at.: Hyperhomocysteinemia, a risk factor for atherosclerosis in chronic uremic patients. Kidney mt 1993;43ISuppl 411: 572-577. 12. Arnadottir M, Brattstr#{246}m L, Simonsen, et at.: The effect of high-dose pyridoxine and folic acid supplementation on serum lipid and plasma homocysteine concentrations In dialysis patients. Chin Nephrol 1993;4:236-24. 13. Massy ZA, Chadefaux-Vekemans B, Chevalier A, et at.: Hyperhomocysteinaemia: A significant risk factor for cardiovascular disease in renal transplant recipients. Nephrol Dial Transplant 1994:9:113-118. 14. Janssen MJFM, van den Berg M, van Guldener C, Boers GHJ, Stehouer CDA: Withdraal of folic acid supplementation in maintenance hemodialysis patients. Chin Nephrol 1994:42:136-137. 15. Perna AF, Ingrosso D, Dc Santo NG, Galletti P, Zappla V: Mechanism of erythrocyte accumulation of methylation Inhibitor S-adenocylhomocysteine In uremia. Kidney mt 1995;47:247-253. 16. Hultberg B, Andersson A, Arnadottir M: Reduced, free Journal of the American Society of Nephrology 2417

Homocysteine and Folate in ESRD Patients and total fractions of homocystelne and other thiol cornpounds In plasma from patients ith renal failure. Nephron 1995;7:62-67. 17. Bostom AG, Shemin D, Nadeau MR, et at.: Short term betaine therapy fails to loer elevated fasting total plasma hornocystelne concentrations in hemodialysis patients maintained on chronic folic acid supplementation. Atherosclerosis 1995; 1 13:129-132. 18. Bostom AG, Shemin D, Lapane KL, et al.: Hyperhornocystelnemia and traditional cardiovascular disease risk factors In end-stage renal disease patients on dialysis: A case-control study. Atherosclerosis 1995; 1 14:93-13. 19. Guttormsen AR, Svarstad E, Ueland PM, Refsum H: Elimination of homocysteine from plasma In subjects ith endstage renal failure. Irish J Med Sci 1995; l64lsuppl 11:8-9. 2. Bostom AG, Shemin D. Lapane KL, et at.: High dose B-vitamin treatment of hyperhornocystelnernia in dialysis patients. Kidney mt 1996:49:147-152. 2 1. Livant EJ, Tamura T, Johnston KE, et at.: Plasma folate conjugase activities and folate concentrations in patients receiving hemodialysis. J Nutr Biochern 1994;5: 54-58. 22. Stabler SP, Marcell PD, Podehl ER, Alien RH: Quantitation of total homocysteine, total cystelne, and methionine in normal serum and urine using capillary gas chromatography-mass spectrometry. Anal Blochern 1987:162:185-196. 23. Ueland PM, Refsum H, Stabler SP, Malino MR. Andersson A, Allen RH: Total hornocystelne in plasma or serum: Methods and clinical applications. Chin Chern 1993;39: 1764-1779. 24. Cornell BAR, Morgan SL, Vaughn WH: Modification of a high-performance liquid chromatographic method for assay of homocystelne In human plasma. J Chromat 1993;617: 136-139. 25. Tamura T. Microbiological assay of folates. In: Picciano MF, Stokstad ELR, Gregory JF III, eds. Fohic Acid Metabohlsrn in Health and Disease. Contemporary Issues in Clinical Nutrition, Vol. 13. Ne York: Wiley-Liss; 199: 12 1-137. 26. Seihub J, Jacques PF, Wilson PWF, Rush D, Rosenberg 111: VitamIn status and intake as primary determinants of homocysteinernia in an elderly population. JAMA 1993:27:2693-2698. 27. Tamura T, Soong S-J, Sauberlich HE, Hatch KD, Cole P, Butterorth CE Jr: Evaluation of deoxyuridine suppresslon test by using hole blood samples from folic acidsupplemented subjects. Am J Clin Nutr 199:51:8-86. 28. Ono K, Hisasue Y: Is folate supplementation necessary in hernodialysis patients on erythropoietin therapy? Clin Nephrol 1992;38:29-292. 29. Westhuyzen J, Matherson K, Tracey R, Fleming SJ: Effect of ithdraal of folic acid supplementation in maintenance hemodialysis patients. Chin Nephrol 1993: 4:96-99. 3. Bostom A, Brosnan JT, Hall B, Nadeau MR, Seihub J: Net uptake of plasma homocysteine by the rat kidney in vivo. Atherosclerosis 1995; 116:59-62. 3 1. Wilcken DEL, Wilcken B, Dudman NPB, Tyrrell PA: Hornocystinuria-The effects of betaine In the treatment of patients not responsive to pyridoxine. N Engl J Med 1983:39:448-453. 2418 Volume 7 - Number 1 1 1996