Metabolic Consequences of Folate-Induced Reduction of Hyperhomocysteinemia in Uremia

Metabolic Consequences of Folate-Induced Reduction of Hyperhomocysteinemia in Uremia ALSSANDRA F. PRNA,*f DIGO INGROSSO,* NATAL G. D SANTO,t PATRIZIA GALLTTI,* MASSIMILIANO BRUNON,* and VINCNZO ZAPPIA*I *Instjtute of Biochemistry of Macromolecules, Division of Nephrologv/Department of Pediatrics, Sc/tool of Medicine and Surgery, Second University of Naples, Naples, Italy; tlnstitute of Food Sciences and Technology, Avellino, Italy. Abstract. Plasma homocysteine, a well-recognized risk factor for cardiovascular disease, is elevated in uremic patients on hemodialysis. The authors have recently demonstrated that one consequence is the reduction in red cell membrane protein methylation levels, caused by a rise of intracellular adenosylhomocysteine, a potent inhibitor of methyltransferases. Protein methylation is involved in a repair mechanism of damaged membrane proteins, and an impairment in methylation leads to the accumulation of altered proteins. Therapy with folates, cofactors in the transformation of homocysteine to methionine, is effective in lowering plasma homocysteine. This article details a study on the metabolic effects of oral methyltetrahydrofolate, the active form of folic acid, on 14 uremic hemodialysis patients. Two months of therapy led to a significant reduction of plasma homocysteine levels, with a proportional response to pre-folate levels. In five of 1 3 patients with homocysteine levels above 2 pm, plasma homocysteine level was reduced to less than I 5 M. After treatment, levels of adenosylmethionine, the methyl donor in transmethylations. had significantly increased; levels of adenosylhomocysteine had increased to a smaller extent. Therefore, the ratio between the two compounds, an excellent indicator of the presence and the degree of methybation inhibition, was significantly ameliorated. Methionine plasma levels increased after treatment in all patients and were correlated with posttreatment adenosybmethionine levels. It was concluded that treatment with methyltetrahydrofolate brings the plasma homocysteine concentration back to an acceptable level, and the metabolic consequences are in the direction of an increase in the normal flow of transmethybations, as monitored by an increase in the ladenosylmethionine}/[adenosylhomocysteine] ratio. (J Am Soc Nephrol 8: 1899-195, 1997) Recently, homocysteine plasma level has been widely recognized as an independent predictor of an increase in cardiovascular disease accidents (1-5). ven a modest elevation is significantly correlated with an increase in the risk of occurrence of myocardial infarction, stroke, and peripheral artery and deep vein thrombosis (1-5). nd-stage renal disease patients, whose death rate due to cardiovascular accidents is 4% (6), show a significant elevation in homocysteine plasma levels (7-1 1) to be ascribed to a reduction in the metabolic functions of damaged kidneys. rather than to a diminished excretion of this amino acid (12-14). Homocysteinemia increases to levels that in hemodialysis patients are well above the 95% percentile of a healthy reference population; that is, 1 5 p.m (15,16). In erythrocytes from chronic renal failure and hemodialysis patients, we have previously demonstrated that an elevation in plasma homocysteine level beads to an increased level of its precursor, adenosylhomocysteine ( 1 7), a potent natural inhibitor of all adenosylmethionine-dependent transmethylation re- Received March 4. 1997. Accepted May 14, 1997. Correspondence to Dr. Diego Ingrosso, Istituto di Biochimica delle Macromolecole, Facolt#{224} di Medicina e Chirurgia, Seconda Universit#{224}degli Studi di Napoli, via Costantinopoli 16, 8138 Napoli. Italy. 146-6673/81 2-1899$3./ Journal of the American Society of Nephrology Copyright 1997 by the American Society of Nephrology actions ( 18-22). According to this notion. it has been shown that an increased adenosylhomocysteine level, in uremic patients, significantly inhibits the adenosylmethionine-dependent repair of molecular fatigue damage in membrane proteins (17,23-2S). Based on the mechanism of the thioether inhibitory effect upon transmethylation reactions, it has been shown that the [adenosylmethionine]/[adenosylhomocystei ne] ratio can be used as a key metabolic parameter to evaluate the degree of such inhibition (17-23). It has been shown, in fact. that the reduction of this ratio reflects the chronic derangement of protein repair in uremia (17,25). Folic acid, and its active circulating derivative 5-methyltetrahydrofolate (MTHF), functions as the one-carbon donor in the remethylation pathway from homocysteine to methionine. the in vito adenosylmethionine precursor (26,27). Folate therapy is known to reduce homocysteine plasma levels in hyperhomocysteinemic patients (except in cases of homozygous cystathionine -synthase-deficient homocystinunia, in which vitamin B6 is usually sufficient), such as patients with increased risk of cardiovascular accidents, including those affected by chronic renal failure (28-3). We have therefore started this study to assess the effects of MTHF on the methionine-homocysteine cycle and to clarify whether a reduction in homocysteine plasma levels induced by MTHF administration affects the ladenosylmethioninel/lad-

19 Journal of the American Society of Nephrology enosylhomocysteine] ratio, which is the crucial indicator of the presence and degree of methylation inhibition (18-22). Materials and Methods Patients and Treatment Sixteen end-stage renal disease patients were selected, provided that they were not affected by systemic diseases such as lupus erythematosus. arterial hypertension antecedent to renal failure, diabetes mellitus. cardiac disease, etc. Patients were being treated by regular bicarbonate hemodialysis treatment. thrice weekly, using hollow-fiber no-reuse dialyzers. Kt/V was routinely checked with monthly intervals and was always above 1.5 during the study and in the previous 6 mo. Patients, for a washout period of 2 mo, did not receive any group B vitamins or phosphodiesterase inhibitors (31). MTHF (Prefolic, Knoll. Germany) was administered for 2 mo at a dose of 15 mg/day per os. Compliance with treatment was assessed by pill count. A report of symptoms after the start of therapy was requested from each patient. Two patients dropped out from the study: one left to undergo a kidney transplant, and the other died of respiratory complications. Blood was drawn by venipuncture while the patient was in a fasting state at the start and at the end of the study immediately before the dialysis session by using ethylenediaminetetraacetate ( 1 mg/ml of blood). Whole blood was immediately centrifuged to separate plasma from cells. Plasma aliquots were stored at -8#{176}Cbefore homocysteine determination. Packed red cells were obtained according to the procedure of Perna ci al. ( I 7). Routine blood biochemical tests (using the Hitachi 91 1 Automatic Analyzer. Hitachi. Tokyo, Japan) were performed before and after treatment. Plasma Ho,nocysteine Plasma homocysteine concentration was determined according to the method of Ubbink et al. (32), as modified by Perna et al. (33). Homocysteine represents total. protein-bound. and non-protein-bound homocysteine. In brief, the procedure involved a preliminary step of reduction and release from plasma proteins. using tni-n-butyl-phosphine in dimethylformamide. followed by precolumn derivatization with ammonium 7-fluorobenzo-2-oxa- I.3-diazole-4-sulphonate (SBD-F. a thiol-specific fluorogenic probe). The mobile phase was.1 M KH2PO4. ph 2. 1. containing 4% acetonitrile, with a flow rate of 2. mi/mm. Micromolar concentrations of homocysteine are referred to 1 liter of plasma. Detection conditions were optimized for homocysteine. Fluorescence intensities were measured with excitation at 385 nm and emission at 5 15 nm using a Shimadzu RF-535 fluorescence detector (Shimadzu Co.. Kyoto, Japan), equipped with a Shimadzu Chromatopac C-R6A data processor. Pre-folate and after-folate homocysteine level determinations were performed at the same time for each patient. Methionine Determination Plasma methionine was assayed according to the method of Jones et al. (34). In brief, plasma samples were deproteinized by perchloric acid precipitation and neutralized by KOH. The insoluble KCIO4 was removed by centrifugation. Pre-column derivatization was then performed by adding 15 jsl of o-phtalaldehyde solution (5 mg in I.5 ml methanol, plus 5 p1 -niercaptoethanol and 1.2 ml of a.4 mm potassium borate solution. ph 1.4) to 1 pj of each sample just before injection. A C- I 8 15 mm X 5 mm reverse-phase column was used (Supelco: Du Pont, Boston. MA). The mobile phase components used were the killowing: buffer A, 5 mm sodium phosphate/so mm sodium acetate, ph 7.4. 2% tetrahydrofurane. and 2% methanol; buffer B. 65% methanol. 45% Milli-Q Millipore water. Buffer B increased from 1% to 1% in 35 mm, then S mm of isocratic elution at 1% buffer B followed. Reequilibration of the system to the initial conditions required S mm. The flow rate was 1.5 mllmin. The fluorometric detector was set at 335 nm excitation and 445 nm emission. Adenosylmethion me and Adenosylhomocysteine Determinations Adenosylmethionine and adenosylhomocysteine were determined chromatographically in red blood cells following the method modified by Perna et al. (17). Statistical Analyses Statistical analyses were performed using the paired a test. Linear regression analysis was done to assess the independent effects of the different variables (35). All calculations were performed using the software package StatWorks (Cricket Software. Philadelphia, PA), running on an Apple Macintosh IIfx (Cupertino. CA) personal computer. All results are presented as means ± SM. Results MTHF ffects on Homocvsteine Levels in Hemodialysis Patients The effect of 2 mo of treatment with MTHF are reported in Table 1, whereas data from individual patients are shown in Figure 1. The mean plasma homocysteine concentration decreased significantly after 2 mo of therapy. It can be noted that the majority of patients had levels above 2 M before MTHF treatment, and in five of these 13 patients there was a reduction to less than 15 p.m, which is considered an acceptable ( normative ) range (3). The higher the plasma homocysteine pretreatment levels were, the greater the decrease after therapy (coefficient of correlation r =.98, P = <.1). Table 1. Key patient characteristics before and after Parameter treatment with MTHF. Medium Before MTHF (n 14) After MTHF (ii = 14) Homocysteine (p.m) P 67.99 ± 14.89 18.87 ± 237b Methionine (M) P 3.78 ± 3.93 4.38 ± 5.36c Urea(mg/dl) P 153.7± 11.46 158.1 ± 13.13 Creatinine (mg/dl) P 9.31 ±.72 9.33 ±.69 AST (Un) P 15.36 ± 1.56 16.5 ± 1.78 ALT (U/I) P 8. ±.79 6.86 ±.55 AdoMet (p.m) 3.531 ±.354 6.164 ± 658b AdoHcy (p.m) 2.68 ±.28 4.1 ± 784d AdoMet/AdoHcy 1.498 ±.2 1 2 2.9 1 6 ± 737d a MTHF, 5-methyltetrahydrofolate; AST, aspartate aminotransferase; ALT, alanine aminotransferase; AdoMet, adenosylmethionine; AdoHcy, adenosylhomocysteine: P, plasma;, erythrocyte. hp <.3. C p <.6. d p < o.os.

Folates and Hyperhomocysteinemia in Uremia 19()l a) C a) (1) > C.) I Ce U) Ce 2 19 18 17-16- 11 1-9- 8-7- 5 4 3 2 1 enzymatic methylations is donated by adenosylmethionine. We measured methionine plasma levels in hemodialysis patients before and after MTHF treatment. We found that methionine levels significantly increased after treatment (Table 1). Methionine is, in turn, the in vivo precursor of the methyl donor adenosylmethionine (23.27). Red cell adenosylmethionine concentration increased significantly after 2 mo of therapy with MTHF in hemodialysis patients (Figure 3. panel A, and Table I). In our study, we found that the difference between pretreatment and posttreatment methionine levels significantly correbated with posttreatment adenosybmethionine levels (r =.76, P <.1). These results substantiate the interpretation that treatment of hemodialysis metabolism with folates leads to an improvement of homocysteine metabolism through the remethylation pathway, which ultimately leads to a significant increase of the intracellular pool of the methyl donor adenosylmethionine. The large majority of the adenosylmethionine formed is consumed in transmethylation reactions, yielding adenosylhomocysteine as the demethylated product (27). We measured the concentration of this thioether in red cells of hemodialysis patients before and after 2 mo of MTHF treatment. Adenosylhomocysteine concentration increased, but to a smaller extent with respect to adenosylmethionine (Figure 3, panel B, and Table I). The [adenosylmethionine]/[adenosylhomocysteinej ratio, a crucial indicator of the presence and degree of methylation inhibition, increased significantly after therapy (Table I ). These results prompted us to conclude that treatment with MTHF critically relieves transmethylation inhibition in hemodialysis patients. The mechanism consists in the facilitated removal of excess homocysteine, which induces an increase of the biosynthesis of the methyl donor adenosylmethionine. and, at the same time, favors the hydrolysis of adenosylhornocysteine, the common product of transmethylations. - #{182} Before MTHF After MTHF Figure 1. Plasma homocysteine concentration of individual patients before and after 2 mo of treatment with oral 5-methyltetrahydrofolate (MTHF). After 2 mo of treatment with MTHF, plasma homocysteine levels decreased in all 14 patients. In 13 patients with pretreatment levels above 2 M, homocysteine levels decreased to values less than 15,.M in five patients. valuation of the intermediates of Methionine- Homocysteine Cycle in Hemodialysis Patients The metabolism of methionine can be envisioned as a true metabolic cycle because the carbon backbone of this amino acid is sequentially converted to adenosybmethionine, then to adenosylhomocysteine, and finally to homocysteine (Figure 2). Methionine is the product of homocysteine metabolism through homocysteine remethylation. This reaction crucially depends on MTHF, which acts as the methyl donor (27). It is worth noting that this is the only methyl transfer reaction in which MTHF donates the methyl group, which in all other Biochemical and Clinical Parameters The main biochemical parameters-urea, creatinine, aspartate aminotransferase, alanine aminotransferase- of patients before and after therapy are reported in Table 1. There was no significant difference in these parameters after therapy with MTHF. Moreover, our patients did not report any side effects or particular complaints during folate administration, cornpared with the previous period. Compliance was excellent because the patients were closely followed and counseled in relation to the importance of strict adherence to therapy. Discussion In the work presented here, we demonstrate that lowering plasma homocysteine levels in hemodialysis patients by using MTHF, the methyl donor in homocysteine conversion to methionine, leads to a significant reduction of homocysteinemia and to an increase of the intracellular concentration of adenosylmethionine. adenosylhomocysteine, and their ratio. Plasma homocysteine levels are a recognized predictor of an individual s risk of cardiovascular complications. Investigated explanations of homocysteine toxicity are various. including

192 Journal of the American Society of Nephrology CYSTIN HOMOCYSTIN MTHF THF ADNOSYLHOMOCYSTIN MTHIONIN MTHYLATD MTHYL ACCPTORS MTHYL ACCPTORS ADNOSYLMTHIONIN Figure 2. The methionine-homocysteine cycle. Adenosylmethionine, enzymatically synthesized from methionine and ATP (step 1), is the common methyl donor for transmethylation reactions catalyzed by methyltransferases, which transfer the methyl groups to methyl acceptors; adenosylhomocysteine is the common demethylated product of these reactions, as well as the competitive inhibitor of adenosylmethioninedependent methyltransferases (step 2). Adenosylhomocysteine is hydrolyzed to adenosine and homocysteine in a reversible reaction catalyzed by adenosylhomocysteine hydrolase (step 3). Homocysteine is utilized for methionine resynthesis in a reaction catalyzed by methionine synthase (step 4). The methyl donor for this reaction is MTHF. Homocysteine is also metabolized through the side-pathway of transsulfuration (step 5). This process involves the sequential action of cystathionine--synthase and cystathioninase, beading to cysteine (23,26,27). 14 ri- 12 8 >1 6 U 4 V 4 4 2 Before MTHF After MTHF Before MTHF After MTHF Figure 3. Adenosylmethionine (panel A) and adenosylhomocysteine (panel B) concentrations of individual patients before and after 2 mo of treatment with oral MTHF. Adenosylmethionine and adenosylhomocysteine concentrations were measured in the acid soluble fraction of erythrocyte cytosol by means of high-performance liquid chromatography ( 17). AdoMet, adenosylmethionine; AdoHcy, adenosylhomocysteine. stimulation of muscle cell proliferation; increased binding of dying of cardiovascular accidents (6). Their average plasma Lp(a) to fibnin. a significant prooxidative activity; and activa- homocysteine concentration is well above the top 5% of a tion of factor V of coagulation or of thrombomodulin expres- normal reference population, even if supplemented with twosion (36-4). to threefold physiologic doses of B vitamins (15,16). More- nd-stage renal disease patients have an increased risk of over, hemodialysis lowers plasma homocysteine levels only

Folates and Hyperhomocysteinemia in Uremia 193 partially (7,41). Thus uremia represents a suitable model to study the metabolic effects of hyperhomocysteinemia and its management. Our study was specifically undertaken to elucidate the mechanism of the effect of folate upon elevated homocysteinemia in end-stage renal disease. For this purpose we used MTHF, the readily available form of folate, as a direct precursor of the methyl moiety for methionine resynthesis from homocysteine. Before MTHF administration, a vitamin B washout period was performed in our study to obtain a baseline situation with high homocysteine levels, reflecting those present in standard hemodialysis patients, in whom either no folate supplementation or a low-dose supplementation is prescribed (7-1 1, 15,29,3,41-43). Diet was not modified, therefore the usual dietary folate intake was maintained. However, the washout period cannot be expected to induce an unusual state of folate deficiency because it has been described that after a three- (42) or four (29)-month washout period, blood folate concentrations were still significantly above the upper reference limit. This is consistent with the report that end-stage renal disease patients have folate plasma levels higher than those in a comparable population of healthy individuals (15). Nevertheless, folate levels are, in renal disease patients. inversely correlated with the degree of hyperhomocysteinemia (15). These data have been interpreted in the light of a relative resistance to folate in kidney failure (16,3). Although folate levels were not determined in our study, the mean (Table 1) or the median (45 M) homocysteine prefolate plasma levels compare with values obtained in hemodialysis patients (7-1 1,15,29,3,41,43), as well as other key parameters given in Table 1. We can conclude therefore that our patient population reflected a typical hemodialysis patient population. We have previously demonstrated that as a consequence of the increased plasma levels of homocysteine in uremic patients, a rise in the intracellular concentration of adenosylhomocysteine emerges (17). This thioether is the precursor of homocysteine and the natural inhibitor of all adenosylmethionine-dependent transmethylation reactions (1 8-22). The ratio [adenosylmethionine]/[adenosylhomocysteine] is the most reliable indicator of the normal flow of methyl groups transferred from the methyl donor to methyl acceptors within the cell (18-22). In this respect, we would like to quote,... it has become well established that modulation of adenosylmethionine/adenosylhomocysteine ratios can be utilized to probe for the involvement of methylation reactions in a variety of physiological systems since changes in this ratio brought about by pharmacological manipulations can be associated with striking biological effects (22). We previously found that the reduction of the [adenosylmethionine]/[adenosylhomocysteine] 1evels, measured by straightforward analytical procedures ( 17), is in good agreement with the degree of impairment of membrane protein repair (25). We have demonstrated that the rise of adenosylhomocysteine concentration in uremia, which is not paralleled by any rise of adenosylmethionine concentration, gives way to a significant reduction of the [adenosylmethionine]/[adenosylhomocysteine] ratio (17). The finding that a reduction of homocysteine plasma levels, as it is induced by the oral administration of MTHF for 2 mo to hemodialysis patients. is important with respect to the metabolic repercussions of MTHF administration, because MTHF is able to increase the [adenosylmethionine]/[adenosylhomocysteine] ratio to levels not significantly different from those detected in healthy individuals (17,33). In fact, the MTHFinduced increase in methionine is handled by the cell through an increase in adenosylmethionine, an ATP-dependent reaction (erythrocyte ATP concentration is normal, if not elevated in uremic cells, 44). Treatment with MTHF in hemodialysis patients increases the [adenosylmethionine]/[adenosylhomocysteine] ratio, indicating that the blockage in the normal flow of transmethylations is eased, if not dispelled, by treatment with MTHF. An increase in transmethylations should therefore induce a diminished accumulation of altered proteins. with the relevant consequences in terms of protein function. Our findings involve other considerations as well, because several crucial transmethylation-dependent processes, in addition to protein repair in cells different from erythrocytes. can be affected by a reduction in the [adenosylmethioninel/ladenosylhomocysteine] ratio (23). In conclusion, our findings clarify the mechanism and the metabolic consequences of the reduction of plasma homocysteine levels induced in hemodiabysis patients by the administration of pharmacological doses of folates. as advocated by concerns related to the increase in the cardiovascular risk associated with hyperhomocysteinemia. We conclude that the benefits are not only in the direction of reducing hyperhomocysteinemia. whose reflections on cardiovascular risk remain to be established, but also in preventing the metabolic alterations induced at a cellular level by the decreased fadenosylmethionine]/[adenosylhomocysteine] Acknowledgments ratio. This work was partially supported by grants from the Ministry of University and Scientific and Technological Research. from Targeted Project Prevention and Control of Disease Factors, and from the National Research Council to the Institute of Biocheniistry of Macromolecules and the Chair of Nephrology. References I. Clarke R, Daly L. Robinson K. Naughten. Cahalane S. Fowler B, Graham I: Hyperhomocysteinemia: An independent risk factor for vascular disease. N ngi J Med 324: 1 149-1 155. 1991 2. Stampfer Mi, Malinow MR. Willett WC. Newcomer LM. Upson B. Ullmann D. Tishler PV, Hennekens CH: A prospective study of plasma homocyst(e)ine and risk of myocardial infarction in US physicians. JAMA 268: 877-881. 1992 3. Malinow MR. Nieto FJ, Szklo M, Chambless L, Bond G: Carotid artery intimal-medial wall thickening and plasma homocyst(e)ine in asymptomatic adults: The atherosclerosis risk in communities study. Circulation 87: 1 17-1 1 13, 1993 4. Nygard. Vollset S. Refsum H, Stensvold I. Tverdal A. Nordrehaug J, Ueland PM. Kvale G: Total plasma homocysteine and cardiovascular risk profile: The Hordaland homocysteine study. JAMA 274: 1526-1533. 1995 5. Robinson K, Mayer L, Miller DP, Green R, van Lente F. Gupta

194 Journal of the American Society of Nephrology A. Kottke-Marchant K. Savon SR. Selhub I. Nissen S. Kutner M. Topol l. Jacobsen DW: 1-lyperhomocysteinemia and low pyridoxal phosphate: Common and independent reversible risk factors for coronary artery disease. Circulation 92: 2825-283. I992 6. Parfrey PS: Cardiac and cerebrovascular disease in chronic uremia. Am J Kidney I)is 21: 77-8, 1993 7. Wilcken DL. Gupta Vi. Reddy SG: Accumulation of sulfurcontaining amino acids including cysteine-homocysteine in patients on maintenance haemodialysis. C/in Sci 58: 427-43, 198 8. Chauveau P. Chadefaux B. Coud#{233}M. Aupetit J, Hannedouche T. Kamoun P. iungers P: Increased plasma homocysteine concentration in patients with chronic renal failure. Miner lectrolyte Metab 1992:18:196-198. 9. Chauveau P. Chadefaux B, Coud#{232}M, Aupetit I. Hannedouche T, Kanioun P. iungers P: Hyperhomocysteinemia. a risk factor for atherosclerosis in chronic uremic patients. Kidney hit 43: S72- S77, 1993 1. Hultberg B. Andersson A, Sterner G: Plasma homocysteine in renal failure. C/in Nephrol 4: 23-235, 1993 I I. Bachmann J. Tepel M. Raidt H. Riezler R. Graefe U, Langer K, Zidek W: Hyperhomocysteinemia and the risk for vascular disease in hemodialysis patients. J Am Soc Nephrol 6: 121-125, I 995 I 2. Guttormsen AB, Svarstad, Ueland PM, Refsum H: limination of hoinocysteine from plasma in subjects with end stage renal failure. Jr J Med Sd 164: (S)8-(S)9, 1995 13. Bostom AG. Brosnan IT, Hall B, Nadeau MR. Selhub I: Homocysteine metabolism by the rat kidney in vivo. JrfMedSci 164: (S)2-(S)3. 1995 14. Bostom AG. Brosnan it. Hall B. Nadeau MR, Selhub I: Net uptake of plasma homocysteine by the rat kidney in vivo. Atheroselerosis 1 16: 59-62, 1995 I 5. Robinson K. Gupta A. Dennis V. Arheart D. Chaudhary D. Green R, Vigo P. Mayer L. Selhub I. Kutner M, iacobsen DW: Hyperhomocysteinemia confers an independent increased risk of atherosclerosis in end-stage renal disease and is closely linked to plasma folate and pinidoxine concentrations. Circulation 94: 2743-2748. 1996 16. Dennis VW, Robinson K: Homocysteinemia and vascular disease in end stage renal disease. Kidney In! 5(57): 51 1-5 17. I 996 17. Perna AF. Ingrosso D. Zappia V. Galletti P. Capasso G, De Santo NG: nzymatic methyl esterification of erythrocyte membrane proteins is impaired in chronic renal failure: vidence for high levels of the natural inhibitor 5-adenosylhomocysteine. J Cliii Invest 9 1: 2497-253, 1993 I 8. Zappia V. Zydek-Cwick CR, Schlenk F: The specificity of 5- adenosylmethionine derivatives in methyl transfer reactions. J Biol Cheni 244: 4499-455. 1969 19. Cantoni GL, Richards HH, Chiang PK: Inhibitors of 5-adenosylhomocysteine hydrolase and their role in the regulation of biological methylation. In: Transmethvlations. edited by Usdin. Borchard RT. Creveling CR. Amsterdam. lsevier/north Holland, 1979, pp 155-164 2. Cantoni GL, Chiang PK: The role of S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase in the control of biologic methylations. In: Natural Sulfur Compounds: Novel Biochemical and Structural Aspects. edited by Cavalbini D. Gaull G, Zappia V. New York. Plenum Press, 198, pp 67-89 2 1. Barber JR. Clarke 5: Inhibition of protein carboxyb methylation by S-adenosyl-L-homocysteine in intact erythrocytes: Physiological consequences. J Biol Chem 259: 71 15-7122. 1984 22. Cantoni G L: The centrality of S-adenosylhomocysteinase in the regulation of the biological utilization of S-adenosylmethionine. In: Biological Met/ivlation and Drug Design:.vperimenta/ and Clinical Roles ofs-adenosv/methionine, edited by Borchardt RT, Creveling CR. Ueland PM, Clifton Ni, Humana Press, 1986, pp 227-238 23. Perna AF, Ingrosso D, Galletti P. Zappia V. De Santo NG: Membrane protein damage and methylation reactions in chronic renal failure. Kidney mt SO: 358-366, 1996 24. Galletti P. Ingrosso D. Manna C, Clemente G, Zappia V: Protein damage and methylation mediated repair in the erythrocyte. Biochem J 36: 313-325, 1995 25. Perna AF, D Aniello A, Lowenson ID, Clarke 5, De Santo NG, Ingrosso D: D-aspartate content of erythrocyte membrane pro- teins is decreased in uremia: implications for the repair of damaged proteins. J Am Soc Nephrol 8: 95-14, 1997 26. Mudd SH, Levy HL. Skovby F: Disorders of transsulfuration. In: The Metabolic Basis ofinherited Disease, edited by Scniver CR, Beaudet AL, Sly WS. Valle D. New York. McGraw Hill, 1989, pp 693-737 27. Selhub I, Miller 1W: The pathogenesis of homocysteinemia: Interruption of the coordinate regulation by 5-adenosylmethionine of the remethylation and transsulfuration of homocysteine. Ant J CliiiNutr 55: 131-138, 1992 28. Wilcken DL, Dudman NPB, Tyrrell PA, Robertson MR: Folic acid lowers elevated plasma homocysteine in chronic renal insufficiency: Possible implications for prevention of vascular disease. Metabolism 37: 697-71, 1988 29. Arnadottir M, Brattstrom LA, Simonsen, Thysell H, Hultberg B, Andersson A, Nibsson-hle P: The effect of high-dose pyridoxine and folic acid supplementation on serum lipid and plasma homocysteine concentrations in dialysis patients. C/in Nephrol 4: 236-24. 1993 3. Bostom AG, Shemin D, Lapane KL, Hume AL. Yoburn D, Nadeau MR. Bendich A, Selhub J, Rosenberg IH: High dose B-vitamin treatment of hyperhomocysteinemia in dialysis patients. Kidney mt 49: 147-152, 1996 3 1. Macfarlane D: Inhibitors of cyclic nucleotide phosphodiesterases inhibit protein carboxyl methylation in intact blood platelets. J Biol Cheiii 259: 1357-1362, 1984 32. Ubbink JB, Vermaak WJH, Bissbort S: Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum. J Chromatogr 565: 441-446, 1991 33. Perna AF. Ingrosso D. De Santo NG, Galletti P, Zappia V: Mechanism of erythrocyte accumulation of methylation inhibitor S-adenosylhomocysteine in uremia. Kidney Jut 47: 247-253. I 995 34. Jones BN, Paabo 5, Stein 5: Amino acid analysis and enzymatic sequence determination of peptides by an improved o-phtalabdehyde precolumn labeling procedure. J Liq Chromatogr 4: 565-586, 1981 35. Dawson-Saunders B. Trapp RG: Basic and Clinical Biostatistics, Norwalk, CT, Appleton & Lange, I 99 36. Tsai C, Perrella MA, Yoshizumi M, Hsien C-M, Habe, Schbagel R, Lee M-: Promotion of vascular smooth muscle cell growth by homocysteine: A link to atherosclerosis. Proc Natl AcadSci USA 91: 6369-6373, 1994 37. Harpel PC, Chang VT. Bort W: Homocysteine and other sulf-

Folates and Hyperhomocysteinemia in Uremia 195 hydryl compounds enhance the binding of lipoprotein(a) to fibrim: A potential biochemical link between thrombosis. atherogenesis. and sulfhydryl compound metabolism. Proc Natl Acad Sci USA 89: 1193-1197, 1992 38. Preibisch G, Kuffner C, lstner F: Biochemical model reactions on the prooxidative activity of homocysteine. J Biosci 48: 58-62, 1993 39. Rodgers GM, Kane WH: Activation of endogenous factor V by a homocysteine-induced vascular endothelial cell activator. J C/in Invest 77: 199-1916, 1986 4. Hayashi 1, Honda G. Suzuki K: An atherogenic stimulus homocysteine inhibits cofactor activity of thrombomodulin and enhances thrombomodulin expression in human umbilical vein endotheliab cells. Blood 79: 293-2936, 1992 41. Laidlaw SA, Smolin LA, Davidson WD, Kopple JD: Sulfur amino acids in maintenance hemodialysis patients. Kidney Jut 32: Sl91-S196, 1987 42. Westhuyzen J, Matherson K, Tracey R. Fleming Si: ffect of withdrawal of folic acid supplementation in maintenance hemodialysis patients. C/in Nephrol 4: 96-99, 1993 43. Janssen MJFM, van den Berg M. van Guldener C. Boers GHJ, Stehouwer CDA: Withdrawal of folic acid supplementation in maintenance hemodialysis patients. C/in Nephrol 42: 136-137, 1994 44. Toigo G, Situlin R. Vasile A, Guerra UP, Tamaro G, Pagoni G, Sergiani G, Russo M. Guarnieri G: ffects of erythropoietin administration on nutritional state and erythrocyte metabolism in maintenance hemodialysis patients. In: Metabolic and Nutritional Abnormalities in Kidney Disease. Contrib Nephro/ 98: 79-88, 1992