Riboflavin Is a Determinant of Total Homocysteine Plasma Concentrations in End-Stage Renal Disease Patients

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J Am Soc Nephrol 13: 1331–1337, 2002 Riboflavin Is a Determinant of Total Homocysteine Plasma Concentrations in End-Stage Renal Disease Patients

SONJA SKOUPY,* MANUELA FO¨ DINGER,† MARIO VEITL,† AGNES PERSCHL,* HEIDI PUTTINGER,* CLAUDIA RO¨ HRER,† KARIN SCHINDLER,* ANDREAS VYCHYTIL,* WALTER H. HO¨ RL,* and GERE SUNDER-PLASSMANN* *Division of Nephrology and Dialysis, Department of Medicine III, and †Division of Endocrinology and Metabolism, Department of Laboratory Medicine, University of Vienna, Vienna, Austria.

Abstract. The effect of thiamine (vitamin B1) or riboflavin tHcy plasma levels within the fourth quartile (plasma tHcy Ͼ ␮ ␣ Ͼ (vitamin B2) availability on fasting total homocysteine (tHcy) 38.3 mol/L) was increased by an -EGR median (odds plasma levels in end-stage renal disease patients is unknown. A ratio, 4.706; 95% confidence interval, 1.124 to 19.704; P ϭ cross-sectional study was performed in a population of non– 0.026). By way of contrast, ␣-ETK had no effect in these vitamin supplemented patients maintained on continuous am- analyses. Independent predictors of tHcy plasma levels were bulatory peritoneal dialysis. Red blood cell availability of serum albumin, ␣-EGR, red blood cell folate, and certain thiamine (␣-ETK) and of riboflavin (␣-EGR), along with other MTHFR genotypes. A logistic regression analysis showed that predictors of tHcy plasma levels, was considered in the anal- the MTHFR genotype is a predictor for having a tHcy plasma ysis. There was a linear association of ␣-EGR with tHcy concentration within the fourth quartile. In summary, ribofla- plasma concentrations (P ϭ 0.009), which was not observed vin availability, as measured by ␣-EGR, is a determinant of for ␣-ETK. Among red blood cell vitamins, ␣-EGR was the fasting tHcy plasma levels in peritoneal dialysis patients. This only predictor of tHcy levels (P ϭ 0.035), whereas ␣-ETK, red finding may have implications for tHcy lowering therapy in blood cell pyridoxal-5-phosphate supply (␣-EGOT) and red individuals with end-stage renal disease. blood cell folate levels had no effect. The risk for having a high

The majority of patients with impaired renal function present far from clear, although vitamin B1 (thiamine pyrophosphate) elevated total homocysteine (tHcy) plasma levels (1). Estab- and vitamin B2 (riboflavin) are involved in the metabolism of lished predictors of tHcy plasma levels in the renal failure methionine and homocysteine. population include serum albumin and serum creatinine levels, We assumed that thiamine or riboflavin availability is a creatinine clearance, folate status, vitamin B12 and vitamin B6 predictor of fasting tHcy plasma levels in end-stage renal levels, as well as genetic variants in enzymes involved in the disease (ESRD). To test this hypothesis, we performed a cross- folate cycle or in the remethylation of homocysteine (2–7). An sectional study among a population of non–vitamin supple- elevated tHcy plasma level can indicate folate and/or vitamin mented patients maintained on continuous ambulatory perito-

B12 deficiency (8) and is associated with a variety of patho- neal dialysis (CAPD). Red blood cell availability of thiamine logic conditions such as vascular disease (9–11) or birth de- and of riboflavin, along with other important predictors of tHcy fects (12). Although genetic and nongenetic factors have been plasma levels, was considered in the analysis. shown to determine tHcy concentrations of patients with renal insufficiency, the cause of hyperhomocysteinemia among these patients is not completely understood (13). Materials and Methods Study Design The role of B-group vitamins other than vitamin B6 or vitamin B as determinants of hyperhomocysteinemia in the In a cross-sectional study, the association of red blood cell avail- 12 ␣ ␣ general population and in the setting of renal insufficiency is ability of thiamine ( -ETK) and riboflavin ( -EGR) with tHcy levels was evaluated in stable peritoneal dialysis patients. Erythrocyte pyridoxal-5-phosphate availability (␣-EGOT), red blood cell folate,

plasma folate, plasma vitamin B6, plasma vitamin B12, and MTHFR Received November 4, 2001. Accepted December 26, 2001. genotypes were also determined. We included all patients in our study Correspondence to: Dr. Gere Sunder-Plassmann, Klinische Abteilung fu¨r who were on peritoneal dialysis treatment at the Division of Nephrol- Nephrologie und Dialyse, Universita¨tsklinik fu¨r Innere Medizin III, A-1090 ogy and Dialysis, Department of Medicine III, University of Vienna Wien, Wa¨hringer Gu¨rtel 18-20. Phone: ϩ43-1-40400-4391; Fax: ϩ43-1- for at least 3 mo. Exclusion criteria comprised time on peritoneal 40400-4392; E-mail: [email protected] dialysis of less than 3 mo, acute illness, current vitamin supplemen- 1046-6673/1305-1331 tation, or participation in other clinical studies. Journal of the American Society of Nephrology The ethical review board at the University of Vienna approved the Copyright © 2002 by the American Society of Nephrology study. All patients gave written informed consent according to the DOI: 10.1097/01.ASN.0000013299.11876.F6 Declaration of Helsinki and the Austrian Law on Gene Technology. 1332 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 1331–1337, 2002

Biochemical Methods levels are given across the quartiles of thiamine, riboflavin, pyridoxal- Blood was drawn for all analyses after an overnight fast. Whole 5-phosphate, plasma vitamin B12, and whole blood folate. Differences blood counts and blood and dialysate chemistry were performed by were examined by ANOVA. Correlations were assessed by Pearson standard methods. correlation coefficient. A four-way ANOVA model was built to For determination of red blood cell folate, plasma tHcy, plasma examine the association of red blood cell vitamin supply (covariables: ␣-ETK, ␣-EGR, ␣-EGOT, and red blood cell folate) with tHcy plasma folate, plasma vitamin B6, and plasma vitamin B12, blood anticoagulated with ethylenediaminetetraacetic acid (EDTA) was drawn, im- levels. The proportion of patients with poor vitamin supply (vitamin mediately placed on crushed ice, and protected from light. Blood level Յ median or ␣-ratio Ͼ median) among subjects presenting with aliquots were snap frozen at Ϫ70°C for extraction of DNA. Blood tHcy plasma levels in the fourth quartile was compared with the aliquots for determination of red blood cell folate concentrations were proportion of patients with tHcy plasma levels in the first three 2 hemolysed with 0.2% ascorbic acid (1:21) for 2 h and frozen at quartiles showing poor vitamin supply by ␹ test. The risk (odds ratio Ϫ70°C. Red blood cell folate was determined using a radioassay and 95% confidence interval) for a tHcy level within the fourth (SimulTRAC-SNB; ICN Pharmaceuticals Inc., Costa Mesa, CA) ac- quartile (Ͼ38.3 ␮mol/L) was calculated for poor supply of thiamine, cording to the instructions of the manufacturer (red blood cell folate riboflavin, pyridoxal-5-phosphate, plasma vitamin B12, and red blood [nmol/L of packed red blood cells] ϭ [(hemolysate folate concentra- cell folate. tion) ϫ 21]/[hematocrit (in decimal notation)]; normal, Ͼ272 nmol/ A multiple linear regression model was built to examine indepen- L). The remaining EDTA-anticoagulated blood samples were centri- dent predictors of tHcy plasma concentrations. tHcy measurements fuged within 30 min at 2.000 ϫ g at 4°C (20 min). Plasma aliquots were positively skewed; therefore, natural logarithmic transformation were snap frozen and stored at Ϫ70°C. was used to normalize the distribution. This model included all Fasting tHcy plasma concentrations were determined by a fluores- variables that showed a linear association with tHcy plasma concen- cence polarization immunoassay (IMx analyzer; Abbott Laboratories, trations (serum creatinine, serum albumin, red blood cell folate, and Abbott Park, IL). Hyperhomocysteinemia was defined as tHcy plasma ␣-EGR) as well as a MTHFR (677/1298) TT/AA, CT/AC genotype levels Ͼ15 ␮mol/L (14). Folate (normal, Ͼ3.4 nmol/L) and vitamin yes/no term. Ͼ A logistic regression analysis was performed to identify indepen- B12 (normal, 118 pmol/L) plasma levels were measured with a radioassay (SimulTRAC-SNB, ICN Pharmaceuticals Inc.). Vitamin dent predictors of tHcy plasma levels in the upper quartile as com- Ͼ pared with the first three quartiles. ␣-ETK, ␣-EGR, ␣-EGOT, red B6 plasma levels (normal, 20 nmol/L) were determined with a radioenzymatic assay (Bu¨hlmann Laboratories AG, Allschwil, blood cell folate, plasma vitamin B12 levels, as well as albumin, Switzerland). creatinine, and a MTHFR (677/1298) TT/AA, CT/AC genotype For examination of thiamine-, riboflavin-, and pyridoxal-5-phos- yes/no term were included as independent variables. All analyses were phate status of red blood cells, blood anticoagulated with heparin was performed using SPSS for Windows (Version 10.0.7; SPSS, Chicago, drawn and incubated in melting ice for 15 min. After centrifugation at IL). 2.000 ϫ g for 20 min at 4°C, red blood cells were washed thrice with 0.9% saline (centrifugation for 10 min; 2.000 ϫ g;4°C). For analysis of thiamine status, 400 ␮l of the pellet were diluted with 360 ␮lof Results saline and stored at Ϫ70°C. For analysis of riboflavin and pyridoxal- Patients 5-phosphate supply, 200 ␮l of pelleted red blood cells were diluted Fifty-four (28 female patients and 26 male patients) of 62 with 900 ␮l of saline and stored at Ϫ70°C. eligible peritoneal dialysis patients were included in this study Thiamine status was assessed by determination of erythrocyte (three patients participated in other clinical studies, four patients transketolase activity (ETK) without (ETKo) and with addition of had high-dose vitamin supplementation, and one patient refused to thiamine diphosphate (ETKϩ) (15). The ratio of enzyme activity after participate). Recruitment of patients was performed between No- and before addition of thiamine is given as ␣-ratio (␣-ETK; adequate vember 2000 and March 2001. The mean age of these patients Ͻ thiamine status, 1.18) (16). was 54.4 Ϯ 15.1 yr, and the duration of dialysis treatment was 1.8 Riboflavin supply was evaluated by measuring erythrocyte gluta- Ϯ 1.5 yr. The mean weekly Kt/V was 2.43 Ϯ 0.49, the mean thione-reductase (EGR) activity without (EGRo) and after addition of Ϯ ϩ protein catabolic rate was 0.92 0.19 g/kg per d, and the mean flavin adenine dinucleotide (EGR ) (17–19). The ratio of enzyme Ϯ activity after and before addition of the vitamin is indicated as ␣-ratio dialysate/plasma ratio of creatinine was 0.63 0.16 (32 low-low (␣-EGR; adequate riboflavin status, Ͻ1.52) (16). average transporters and 22 high-high average transporters). The For estimation of pyridoxal-5-phosphate supply, erythrocyte glu- mean serum albumin was 33.7 Ϯ 3.8 g/L, the mean serum tamic-oxaloacetic transaminase activity (EGOT) was determined (20). creatinine was 7.9 Ϯ 2.3 mg/dl, and the mean hemoglobin con- The ␣-ratio (␣-EGOT; adequate pyridoxal-5-phosphate status, Ͻ1.8) centration was 11.5 Ϯ 1.4 g/dl. The mean body mass index was (16) was calculated from enzyme activity after (EGOTϩ) and before 23.7 Ϯ 3.6 kg/m2. There were 20 smokers, and 10 patients (EGOTo) addition of pyridoxal-5-phosphate. presented with diabetes mellitus. Dialysis treatment was initiated Identification of the 677C3T transition and of the 1298A3C because of ESRD due to shrunken kidneys of unknown etiology transversion in MTHFR was performed by restriction fragment length (n ϭ 24), diabetic nephropathy (n ϭ 10), polycystic kidney polymorphism analysis (21,22). disease (n ϭ 8), chronic glomerulonephritis (n ϭ 7), chronic Weekly Kt/V, residual renal clearance, and characterization of the interstitial nephritis (n ϭ 2), hemolytic uremic syndrome (n ϭ 1), peritoneal transport type was performed as previously reported (4). juvenile nephronophthisis (n ϭ 1), and Alport syndrome in one case. Thirty patients were maintained on CAPD, and 24 were Statistical Analyses treated with automated peritoneal dialysis (APD), including 16 Continuous data are given as mean Ϯ SD. Categorical data are patients on continuous cyclic peritoneal dialysis (CCPD) and 8 given as absolute counts and percentages. Nonadjusted tHcy plasma patients on nightly intermittent peritoneal dialysis (NIPD). None J Am Soc Nephrol 13: 1331–1337, 2002 Vitamin B2 Determines tHcy Plasma Levels in ESRD 1333 of the patients included in this study received folic acid or B Ϫ0.338; P ϭ 0.013), and of folate plasma levels (r ϭϪ0.284; vitamin supplementation. Fifty patients were on regular therapy P ϭ 0.037) with tHcy plasma levels. The association of tHcy with recombinant human erythropoietin and intravenous iron su- plasma levels with ␣-EGR is shown in Figure 1 (excluding one crose. Two patients were on erythropoietin therapy alone, and outlier). There was also a positive association of creatinine and another two patients were on iron therapy without erythropoietin. albumin levels with tHcy plasma concentrations. Red blood cell thiamine supply, red blood cell pyridoxal phosphate sup-

Vitamin Status, tHcy Plasma Levels, and MTHFR ply, vitamin B12 plasma levels, and vitamin B6 plasma levels Genotypes showed no association with fasting tHcy plasma concentrations Vitamin dependent enzyme activities in red blood cells in this analysis. (␣-ETK, ␣-EGR, ␣-EGOT), red blood cell folate, vitamin The tHcy plasma levels according to quartiles of vitamin plasma levels, and tHcy plasma levels are given in Table 1. supply are given in Table 2. Poor red blood cell folate, plasma ␣ Ն Vitamin B1 deficiency ( -ETK 1.18) was present in five vitamin B12, and red blood cell vitamin B2 supply was asso- ␣ Ն patients, vitamin B2 deficiency ( -EGR 1.52) in 10 patients, ciated with increased tHcy plasma concentrations. ␣ Ն and vitamin B6 deficiency ( -EGOT 1.8) in 46 patients. Low A four-way ANOVA showed that riboflavin supply, as Ͻ ␣ plasma levels of vitamin B6 ( 20 nmol/L) were observed in 25 determined by -EGR, is the most important predictor of tHcy patients. Plasma folate and red blood cell folate was normal levels among red blood cell vitamins (Table 3). The other (Ͼ3.4 nmol/L and Ͼ272 nmol/L, respectively) in all patients, variables, namely ␣-ETK, ␣-EGOT, and red blood cell folate Ͻ and vitamin B12 plasma levels were low ( 118 pmol/L) in showed no effect on tHcy concentrations in that model. three patients. Normal tHcy plasma levels were present in six There were significantly more patients with an ␣-EGR Ͼ ␮ Ͻ patients. Moderate (16 to 30 mol/L), intermediate (31 to 100 median, with a vitamin B12 plasma level median, or with a ␮mol/L), and severe (Ͼ 100 ␮mol/L) hyperhomocysteinemia red blood cell folate Ͻ median among individuals with tHcy was detected in 31, in 14, and in three patients, respectively. plasma levels in the upper quartile (Table 4). The MTHFR 677/1298 TT/AA genotype was identified in five A multiple linear regression model showed that serum albu- patients, and the CT/AC genotype in 18 patients. min levels (P ϭ 0.001), ␣-EGR (P ϭ 0.043), red blood cell folate (P ϭ 0.001), and the MTHFR genotype (P ϭ 0.007) are Riboflavin Supply Is a Determinant of tHcy Plasma independent predictors of tHcy plasma concentrations. Serum Concentrations creatinine had no major effect on tHcy concentrations in this Regression analysis showed a linear association of riboflavin model (P ϭ 0.091). supply (r ϭ 0.351; P ϭ 0.009), of red blood cell folate (r ϭ Logistic regression analysis demonstrated that the MTHFR

Table 1. Vitamin status and tHcy plasma levels of 54 peritoneal dialysis patientsa

Mean Ϯ SD Median (Range)

Vitamin B1 ETKo 0.24 Ϯ 0.05 0.25 (0.12 to 0.36) ETKϩ 0.26 Ϯ 0.05 0.26 (0.14 to 0.38) ␣-ETK 1.08 Ϯ 0.08 1.06 (1.00 to 1.29)

Vitamin B2 EGRo 0.0263 Ϯ 0.0068 0.0272 (0.0138 to 0.0396) EGRϩ 0.0321 Ϯ 0.0050 0.0322 (0.0155 to 0.0444) ␣-EGR 1.28 Ϯ 0.30 1.19 (1.00 to 2.68)

Vitamin B6 EGOTo 0.0143 Ϯ 0.0047 0.0138 (0.0071 to 0.0354) EGOTϩ 0.0318 Ϯ 0.0062 0.0317 (0.0174 to 0.0473) ␣-EGOT 2.33 Ϯ 0.47 2.28 (1.34 to 3.79) plasma (nmol/L) 27.3 Ϯ 26.4 23.7 (1.6 to 141.9)

Vitamin B12 plasma (pmol/L) 304 Ϯ 181 268 (49 to 1200) Folate RBC (nmol/L) 1367 Ϯ 874 1108 (435 to 5706) plasma (nmol/L) 16.1 Ϯ 7.2 14.5 (5.4 to 40.0) tHcy plasma (␮mol/L) 33.0 Ϯ 24.5 25.7 (8.4 to 127.5)

a ETK, erythrocyte transkelotase activity; EGR, erythrocyte glutathione-reductase activity; EGOT, erythrocyte glutamic-oxaloacetic transaminase activity; o, enzyme activity without cofactor; ϩ, enzyme activity following addition of cofactor; RBC, red blood cells; tHcy, total homocysteine. 1334 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 1331–1337, 2002

sensitive to impaired riboflavin status (26). Thus, riboflavin availability may be of importance in renal failure, where tHcy plasma levels are sensitive to mutations in MTHFR and to low folate levels. A few studies indicated that a low dietary intake of thiamine and riboflavin is associated with an elevation of tHcy plasma levels in subjects without renal failure (27–29). In the physi-

cian health study, a negative correlation of vitamin B1 and vitamin B2 intake with tHcy plasma levels was observed in healthy subjects (27). An inverse association of plasma tHcy was also found with thiamine intake and riboflavin intake among participants of the Atherosclerosis Risk in Communities (ARIC) Study (28). The fifth examination cycle of the Fra- Figure 1. Linear association of red blood cell riboflavin supply mingham Offspring Study suggested that riboflavin intake is a (␣-EGR) and plasma homocysteine (tHcy) concentration in 53 peritoneal dialysis patients (one extreme outlier was omitted). predictor of tHcy in non–vitamin supplemented people without renal failure (29). Most recently, the riboflavin plasma level was shown to be an independent predictor of tHcy plasma genotype is an important predictor of high tHcy plasma levels levels in healthy blood donors with the MTHFR 677TT geno- in the upper quartile (regression coefficient, 3.228; P ϭ 0.027), type (30). These findings are in some contrast to results from whereas all other variables in this analysis showed no effect on the Health Professionals Follow-up Study that showed that high tHcy plasma levels. there is no association of the MTHFR 677TT genotype and riboflavin intake with respect to tHcy levels among generally Discussion well-nourished men (31).

Vitamin B1 and vitamin B2 are involved in the metabolism So far the potential association of thiamine or riboflavin of methionine and homocysteine and may therefore influence status with tHcy plasma levels has not been examined in renal tHcy plasma concentrations. Methionine can be catabolized via failure patients. This is the first study to investigate the effect the transamination pathway by oxidative decarboxylation to of thiamine and riboflavin availability on fasting tHcy plasma 3-methylthiopropionate (23). Thiamine pyrophosphate, the ac- concentrations in ESRD patients treated by peritoneal dialysis. tive form of thiamine, is a cofactor of this supposed rate- We provide evidence that riboflavin (vitamin B2) supply, as limiting oxidative decarboxylation and thus may contribute to measured by red blood cell glutathione reductase activity, is a lowering of tHcy plasma levels by lowering of methionine determinant of fasting plasma tHcy concentration in these (24). Furthermore, suboptimal supply of riboflavin, a precursor patients. In contrast, availability of thiamine (vitamin B1)as of flavin adenine dinucleotide (FAD), may also enhance the measured by red blood cell transketolase activity, had no effect risk for hyperhomocysteinemia. Flavin adenine dinucleotide is on fasting tHcy concentrations. a cofactor of the enzyme 5,10-methylenetetrahydrofolate re- In general, thiamine (32–35) and riboflavin (16,34–37) sup- ductase (MTHFR), which produces the active form of folate, ply is considered to be normal in the majority of renal failure 5-methyltetrahydrofolate, required for remethylation of homo- patients. However, some studies suggest the presence of thia- cysteine to methionine. Interestingly, mutation in bacterial mine (37–39) or riboflavin deficiency in renal failure patients MTHFR was shown to affect FAD dissociation kinetics (25). (40), thus providing the rational basis for an association of

In addition, MTHFR is among the flavoenzymes that are most vitamin B1 or B2 with hyperhomocysteinemia in ESRD pa-

Table 2. Total homocysteine plasma levels (mean Ϯ SD; ␮mol/L) by quartiles of vitamin availability (␣-ratios) or of vitamin levels of 54 peritoneal dialysis patientsa

Quartiles of Vitamins

1st 2nd 3rd 4th

Patients (n) 14131314 ␣ Ϯ Ϯ Ϯ Ϯ Vitamin B1 ( -ETK) 35.8 27.8 29.7 13.0 26.3 13.3 39.5 34.7 ␣ b Ϯ Ϯ Ϯ Ϯ Vitamin B2 ( -EGR) 24.9 9.9 30.0 24.1 33.5 28.5 43.5 29.5 ␣ Ϯ Ϯ Ϯ Ϯ Vitamin B6 ( -EGOT) 35.9 23.2 24.0 13.2 30.7 16.8 40.6 36.5 c Ϯ Ϯ Ϯ Ϯ Vitamin B12 (P) 49.1 34.7 27.3 14.4 31.8 23.8 23.2 11.0 Folate (RBC)c 49.8 Ϯ 34.2 32.7 Ϯ 12.3 27.5 Ϯ 24.6 21.6 Ϯ 10.6

a P, plasma; RBC, red blood cells. b ␣ The lower the -EGR, the better is the vitamin B2 supply, resulting in low tHcy plasma levels. c ϭ Suboptimal folate and suboptimal vitamin B12 levels are associated with higher tHcy plasma levels (P 0.002 for RBC folate). J Am Soc Nephrol 13: 1331–1337, 2002 Vitamin B2 Determines tHcy Plasma Levels in ESRD 1335

Table 3. Tissue vitamin supply as determinant of plasma more, the risk for hyperhomocysteinemia, presenting with tHcy tHcy levels of 54 peritoneal dialysis patients plasma levels within the fourth quartile, was substantially increased by suboptimal red blood cell riboflavin supply. This Tissue Vitamin Supply Pa finding supports the concept of Hustad et al. (30), who dem- ␣ onstrated an association of riboflavin plasma levels with tHcy Vitamin B1 ( -ETK) 0.319 ␣ plasma concentrations in healthy individuals. Suboptimal red Vitamin B2 ( -EGR) 0.035 ␣ Vitamin B6 ( -EGOT) 0.265 blood cell folate content and low vitamin B12 plasma levels of Folate (RBC) 0.102 peritoneal dialysis patients were also associated with an increased risk for fasting tHcy levels within the fourth quartile. A a ϭ ϭ Results from four-way ANOVA: F 3.558; P 0.013. multivariate analysis including all variables showing associa- tions with tHcy plasma concentrations disclosed that red blood tients. Furthermore, an independent graded influence within cell riboflavin supply is an independent predictor of tHcy the reference interval of both vitamins on tHcy levels may be plasma concentrations. possible as was previously shown for folate concentrations or The majority of our patients presented poor red blood cell response to folic acid therapy (41,42). vitamin B6 availability. Not unexpectedly, this was not asso- The results of our study, demonstrating no association of red ciated with fasting tHcy plasma levels. There was also no association of red blood cell vitamin B6 availability and plasma blood cell vitamin B1 supply with hyperhomocysteinemia are in line with the observation of Franken et al. (24). In this study, levels of vitamin B6, which may be due to uremia-related disturbances of vitamin B6 metabolism (45). A multivariate no effect of vitamin B1 supplementation on tHcy levels was shown in patients with homocystinuria due to cystathionine-␤ analysis showed that the MTHFR genotype is an important synthase deficiency. Lack of effect of thiamine supply on tHcy predictor of very high tHcy plasma concentrations, which is in levels in renal failure may be related to the impairment of line with previous observations (2,4,6,7). neural tissue transketolase (43) or erythrocyte transketolase The findings of this study may have therapeutic implica- activity (44), a potential indicator of thiamine deficiency, tions. Supplementation of water-soluble vitamins resulted in a which was ascribed to low molecular weight uremic toxins in substantial improvement of vitamin B1 and/or vitamin B2 sup- renal failure patients. In this context, Descombes et al. (16) ply in virtually all renal failure patients (46–48). Therefore, described an impaired basal erythrocyte transketolase activity some recommendations (49–51) included the advice to add despite normal thiamine plasma levels without improvement of vitamin B1 and B2 supplementation to the dietary regimen of transketolase activity after addition of thiamine in vitro in the dialysis patients, whereas others did not (52,53). On the basis majority of hemodialysis patients. This finding supports the of the results of this study, the effect of vitamin B2 supple- assumption of a thiamine-independent enzyme inhibition in mentation on tHcy plasma concentrations is worth examination uremia. Nevertheless, a significant proportion of patients in renal failure patients. Importantly, there may be a permissive showed improvement of enzyme activity after addition of this effect of riboflavin therapy on the tHcy-lowering effect of cofactor in our study. folate supplementation that may overcome some of the so- There was a positive association of ␣-EGR with tHcy called folate resistance in renal failure. This assumption is plasma concentration (Figure 1), which resembles a negative based on the fact that riboflavin is the precursor of flavin association of riboflavin supply with tHcy plasma status be- adenin dinucleotide that facilitates the conversion of 5,10- cause the lower ␣-EGR indicated a better tissue riboflavin methylenetetrahydrofolate to 5-methyltetrahydrofolate, which supply. Thus, riboflavin supply was negatively associated with is necessary for the remethylation of homocysteine to tHcy plasma concentrations of peritoneal dialysis patients. methionine. Interestingly, among red blood cell vitamins, riboflavin supply A potential limitation to our study includes that serum levels was the only predictor of tHcy levels in a multivariate model of thiamine and riboflavin were not analyzed. We have mea- that included red blood cell vitamin B1 supply, red blood cell sured supply of thiamine, of riboflavin, and of pyridoxal-5- vitamin B6 supply, and red blood cell folate levels. Further- phosphate by quantifying vitamin-dependent erythrocyte en-

Table 4. Risk for having a tHcy plasma level within the fourth quartile by poor vitamin supply of 54 peritoneal dialysis patientsa

Vitamin Odds Ratio (95% CI) P ␣ Vitamin B1 ( -ETK) 0.816 (0.234 to 2.851) NS ␣ Vitamin B2 ( -EGR) 4.706 (1.124 to 19.704) 0.026 ␣ Vitamin B6 ( -EGOT) 1.225 (0.351 to 4.278) NS Vitamin B12 (P) 4.706 (1.124 to 19.704) 0.026 Folate (RBC) 8.594 (1.680 to 43.953) 0.004

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