Influence of nutritional status on plasma and erythrocyte sulphur ...

Nephrol Dial Transplant (2002) 17: 1050–1056

Original Article

Influence of nutritional status on plasma and erythrocyte sulphur amino acids, sulph-hydryls, and inorganic sulphate in end-stage renal disease Mohamed E. Suliman, Peter Ba´ra´ny, Jose´ C. Divino Filho, A. Rashid Qureshi, Peter Stenvinkel, Olof Heimbu¨rger, Bjo¨rn Anderstam, Bengt Lindholm and Jonas Bergstro¨m Divisions of Baxter Novum and Renal Medicine, Department of Clinical Science, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden

Abstract Background. The metabolism of sulphur amino acids and sulph-hydryls is altered in end-stage renal disease (ESRD). Previous studies have focused on the role of vitamin status in the development of hyperhomocysteinaemia in such patients, but little information exists about the influence of global nutritional status and hypoalbuminaemia on sulphur-containing compounds in ESRD. As considerable fractions of sulphhydryls in blood are present in erythrocytes (RBC), which among others participate in intra-organ amino acid transport, the relationship between plasma and RBC levels of several of these compounds and various nutritional parameters were evaluated in the present study. Methods. Thirty-seven ESRD patients (24 males, 13 females) on dialysis treatment (18 haemodialysis, 19 continuous ambulatory peritoneal dialysis) and 21 healthy subjects (seven males, 14 females) were examined. The subjective global nutritional assessment (SGNA) showed that 10 (27%) patients were malnourished and 27 (73%) had normal nutritional status. Results. All the ESRD patients had high plasma total homocysteine (tHcy) levels. The plasma concentrations of methionine (Met) and taurine (Tau) were low, but the levels of the other sulphur-containing compounds were high. In the RBC, the patients had higher levels of tHcy and Tau than in healthy subjects, but no difference was seen in the concentrations of glutathione (GSH), cysteinylglycine (Cys-Gly), Met, and Cys. The plasma inorganic sulphate concentrations were five times higher in the patients

Correspondence and offprint requests to: Dr Bengt Lindholm, Divisions of Baxter Novum and Renal Medicine, K-56, Huddinge University Hospital, Karolinska Institutet, S-141 86, Stockholm, Sweden. Email: [email protected] #

than in healthy subjects, but the levels did not differ significantly between the malnourished patients and those with normal nutritional status. The malnourished patients had lower plasma, but not RBC, levels of tHcy, GSH, and Cys-Gly than those with normal SGNA. Plasma tHcy correlated positively with serum (s)-albumin and anthropometric parameters and negatively with SGNA. RBC and whole blood, but not plasma, GSH concentrations were correlated with haematocrit and were significantly lower in low haematocrit patients (O37%, ns19) than in those with a high haematocrit ()37%, ns18). Conclusions. These results show that nutritional status and s-albumin influence plasma, but not RBC, concentrations of sulph-hydryls in ESRD patients. This should be considered when the relationships between cardiovascular disease and plasma tHcy or other sulphur-containing compounds are assessed. The study also shows that GSH concentrations in RBC and whole blood are related to haematocrit and not to nutritional parameters, indicating that anaemia status rather than nutritional status determines RBC and whole blood GSH levels in ESRD patients. Keywords: anaemia; end-stage renal disease; glutathione; homocysteine; nutritional status; serum albumin; sulphate; sulphur amino acids

Introduction Protein-energy malnutrition in maintenance dialysis patients is common, with signs of malnutrition being seen in 10–70% of haemodialysis (HD) patients and in 18–59% of continuous ambulatory peritoneal dialysis (CAPD) patients [1]. Signs of malnutrition in

2002 European Renal Association–European Dialysis and Transplant Association

Sulphur compounds and nutritional status in ESRD

end-stage renal disease (ESRD) patients on regular dialysis include weight loss, reduced muscle mass, as estimated by anthropometric methods, low concentrations of albumin and other visceral proteins, as well as abnormal plasma and intracellular amino acid profiles [2,3]. Sulphur amino acids show many abnormalities in chronic renal failure (CRF). Among these, high levels of plasma total homocysteine (tHcy) have attracted special attention because this is a risk factor for atherosclerosis. Hyperhomocysteinaemia in uraemic patients is associated with high levels of plasma total cysteine (tCys), which are correlated with plasma tHcy levels [4]. Taurine (Tau) is one of the most abundant free intracellular amino acids in mammalian tissue. It has been thought to have important cellular functions, including stabilization of membrane potential, modulation of Ca2q transport, a positive inotropic effect on the heart, and antioxidative capacity [5]. In uraemic patients, low Tau levels in muscle, plasma [6], and platelets [7] have been reported in the presence of high Tau concentrations in erythrocytes (RBC) [6]. The concentrations in lymphocytes and granulocytes do not change [7]. Although the consequences of Tau depletion in ESRD patients are unknown, one may surmise that Tau depletion might contribute to muscle fatigue and cardiovascular disease in uraemia. Glutathione (GSH) is a major intracellular antioxidant. It is synthesized mainly in the liver, supplying about 90% of the circulating GSH under physiological conditions [8], and extracellular GSH is transported into the cells of the organs and tissues that do not synthesize GSH de novo [9]. The origin of RBC GSH remains uncertain. It has been speculated that RBC GSH synthesis involves the uptake of precursors and de novo synthesis of GSH in the red cells [10]. Some authors have reported low GSH levels in

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RBC and whole blood [11,12] in dialysis patients. In contrast, normal [13] or high GSH levels in RBC [14] have been reported in dialysed and non-dialysed CRF patients. Very little is known about the effects of nutritional status on sulphur amino acids and sulphhydryls in CRF patients. In almost all studies assessing this relationship, the evaluation focused only on vitamin status, in particular folic acid, vitamin B12, and vitamin B6, but protein-energy malnutrition, which is common in CRF, has received little attention. In a previous study [4], we showed that plasma tHcy is related to nutritional status in HD patients. On the other hand, measurement of amino acids in RBCs gives additional information to that observed from plasma aminograms as RBCs are involved in amino acid transport between various tissues of the body, thus playing an important role in amino acid and protein metabolism in uraemia [6]. In this study, we aimed to explore the relationship of nutritional status not only to tHcy but also to the concentrations of other sulphur-containing compounds measured both in plasma and RBCs in ESRD patients.

Subjects and methods Thirty-seven ESRD patients (24 males, 13 females), having a median age of 55 (range 25–77) years, were enrolled in this study. Eighteen patients were on HD treatment and 19 were on CAPD. The HD patients were dialysed for 4 h three times per week. Dialysers with low-flux, modified cellulose membranes, derivatized cellulose (Hemophan1 (ns14) or cellulose acetate (ns4)) were used. Nineteen patients were treated with CAPD using four daily exchanges of 2 l of glucosebased dialysis fluid (DianealTM, Baxter, Castlebar, Ireland), using different concentrations of glucose as required. Biochemical characteristics of the ESRD patients and healthy subjects are shown in Table 1. Thirty-one patients

Table 1. Clinical, biochemical, anthropometric parameters, plasma-free amino acids, and vitamin concentrations in 37 ESRD patients and 21 healthy subjects

Gender, femaleumale Age, years Urea, mmolul Creatinine, mmolul Albumin, gul Haematocrit % BMI, kgum2 Fat mass, by DXA kg LBM, by DXA kg DBW, % MAMC, % HGS, % nPNA gukg DBWuday Plasma B12, pmolul Plasma folate, nmolul RBC folate, nmolul

Controls (ns21)

Well-nourished (ns27)

Malnourished (ns10)

14u7 38 (9) 4.8 (1.2) 87 (9) 43 (4) 39 (3) 24 (4) – – – – –

8u19 53 (12)*** 23 (6)*** 827 (194)*** 37 (5)** 37 (4) 26 (4) 24 (11) 52 (11) 112 (19) 90 (10) 84 (19) 1.18 (0.29) 475 (200)** 12 (5) 737 (214)*

5u5 64 (13)***,q 20 (8)***,q 574 (197)***,qq 33 (5)***,q 36 (5)* 21 (4)*,qq 15 (7)q 41 (7)qq 91 (16)qq 81 (8)qq 56 (13)qqq 1.14 (0.30) 427 (149)** 13 (6) 872 (160)**

266 (107) 11 (5) 671 (200)

The patients were divided into two groups on the basis of SGNA. Values are given as mean (SD). *P-0.05, **P-0.01, and ***P-0.001, compared with control subjects. qP-0.05 and qqP-0.01, and qqqP-0.001 compared with well-nourished patients.

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received recombinant human erythropoietin (Epo) 2000– 12 000 U a week (mean (SD) 5730 (2420) U) and six did not. Iron supplements were given to nine patients, in combination with Epo. All patients were routinely given supplements of water-soluble vitamins, including L-pyridoxine chloride 10 mg daily, but not folic acid or B12. The daily protein intake was estimated by calculating the daily protein equivalent of total nitrogen appearance (PNA) based on urea kinetic modelling and normalized (nPNA) for desirable body weight (DBW). The dialysis dose, expressed as KtuVurea, was calculated based on urea kinetic modelling. Twenty-one healthy subjects (seven males, 14 females), having a median age of 36 (range 24 –54) years were recruited from the hospital and clinical research centre employees for comparative analyses. The nature and purpose of the study were carefully explained to all participants before they agreed to take part. The local Ethics Committee of Karolinska Institutet at Huddinge University Hospital approved the study protocol. Venous blood samples from the patients and healthy subjects were collected in the morning, after an overnight fast, in cooled EDTA tubes. The tubes were centrifuged immediately at 48C and then the plasma and RBC were separated and treated, as described previously [13]. Another aliquot of blood was kept on crushed ice and then the whole blood was diluted with an equal amount of distilled water and sonicated in tubes placed on ice. Thereafter, the diluted whole blood, plasma, and RBC were stored at 708C, pending determination of GSH. The blood samples from the HD patients were collected on a mid-week dialysis-free day. The plasma, RBC and whole blood sulph-hydryls (Hcy, Cys, GSH, cysteinylglycine (Cys-Gly) and c-glutamylcysteine (c-Glu-Cys)) were determined with high-performance liquid chromatography (HPLC) using fluorescence detection with only a minor modification as described elsewhere [15]. The plasma and RBC-free amino acid (methionine (Met), cysteinesulphinic acid (CSA), and Tau) concentrations were determined with HPLC, as described elsewhere [15]. Sulphate in the plasma was determined in an isocratic anion HPLC system (using Waters method of application # A 101), with an IC-Pak Anion, 4.6 3 50 mm column (Waters Corporation, Milford, MA, USA), borateugluconate eluent, and a 432 conductivity detector (Waters Corporation). Folate and vitamin B12 concentrations were determined with the Dualcount SPNB (solid phase no boil) radioimmunoassay kit from DPC (Diagnostic Product Corporation, Los Angeles, CA, USA), serum albumin (s-albumin) with the bromcresol purple method and serum creatinine (s-creatinine) and blood urea concentrations with routine laboratory methods, using the Hitachi 737 Automatic Analyser (Naka Works, Hitachi Ltd, Tokyo, Japan).

Subjective global nutritional assessment Subjective global nutritional assessment (SGNA) was used to evaluate the overall protein-energy nutritional status [16]. This SGNA includes six subjective assessments, three based on the patient’s history of weight loss, incidence of anorexia, and incidence of vomiting, and three based on the physician’s grading of muscle wasting, presence of oedema, and loss of subcutaneous fat. These variables were graded as 1snone, 2smild, 3smoderate, and 4ssevere. The patients were divided into two groups, according to the SGNA score: group 1, normal nutritional status (score -8) and group II, mild to severe malnutrition (score P9).

M. E. Suliman et al.

Anthropometric measurements Anthropometric measurements were made in the morning after taking the blood samples. Hand-grip strength (HGS) was measured with a Harpenden dynamometer, triceps skin fold thickness (TSF) with a Harpenden calliper on the nondominant arm in the CAPD patients and on the arm without vascular access in the HD patients. The mid-arm muscle circumference (MAMC) was derived from the TSF and mid-arm circumference (MAC) as follows: MAMCsMAC (p 3 TSF). The DBW was corrected for the patient’s height, sex, and frame-size match, using Metropolitan height and weight tables. Lean body mass (LBM) was evaluated by dual-energy X-ray absorptiometry (DXA). The individual values of anthropometric variables (TSF, MAMC, and HGS) were normalized by converting them to per cent of the mean values in healthy subjects (23 males, 12 females) of the same sex [2].

Statistical analyses All values are expressed as mean (SD), unless otherwise indicated. A P-0.05 was considered significant. Comparisons between two groups were assessed for continuous variables with the Student’s unpaired t-test, the Mann–Whitney test when the distribution was skewed and for nominal variables with the x2-test. Spearman’s rank correlation was used to determine correlations between variables and stepwise multiple regression analysis to identify Hcy predictors.

Results Patients vs healthy subjects On the basis of SGNA, 10 ESRD patients (27%) were classified as malnourished and 27 (73%) as normally nourished. The data on anthropometric and biochemical variables are given in Table 1. The ESRD patients were older and had significantly lower s-albumin levels (Table 1). The BMI and haematocrit per cent were significantly lower in the malnourished patients than in healthy subjects, but the values of the well-nourished patients did not differ significantly from those in healthy subjects (Table 1). The plasma concentrations of tHcy, tCys, CSA, c-Glu-Cys, GSH, Cys-Gly, and plasma sulphate were significantly higher while those of Met, Tau, and TauuCSA ratio were significantly lower in the patients than in the healthy subjects (Table 2). Table 3 shows that the RBC concentrations of tHcy and Tau were significantly higher in the patients, but those of GSH, tCys, Cys-Gly, and Met did not differ significantly from those in healthy subjects. The whole blood GSH concentrations were significantly lower in the two patient groups than in healthy subjects (Table 3). Table 1 shows that the RBC folate and vitamin B12 concentrations were significantly higher in the two patient groups than in healthy subjects, but the plasma folate concentrations were similar in the patient groups and healthy subjects.


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Table 2. Plasma sulphur amino acids, sulph-hydryls, and sulphate concentrations (mmolul; mean (SD)) in 37 ESRD patients, divided into two groups as in Table 1, and 21 healthy subjects

Met Hcya Cys CSA Tau TauuCSA ratio c-Glutamylcysteine GSH Cys-Gly Inorganic sulphate

Controls (ns21)


Malnourished (ns10)

26 (4) 10 (3) 228 (34) 0.89 (0.22) 55 (6) 67 (26) 3.3 (0.7) 3.3 (1.0) 28 (5) 317 (86)

19 (5)** 43 (13)** 406 (90)** 5.2 (1.7)** 39 (13)** 9 (6)** 6.7 (2.5)** 6.4 (1.5)** 69 (21)** 1441 (660)**

17 (6)** 31 (13)**,qq 390 (84)** 5.2 (1.6)** 40 (16)** 9 (4)** 5.8 (2.7)* 4.7 (1.5)*,qq 54 (12)**,q 1414 (825)**

Values are given as mean (SD). Amino acid concentrations in mmolul. *P-0.01 and **P-0.001, compared with control subjects. qP-0.05 and qqP-0.01, compared with well-nourished patients. a The total number of patients was 35 because we excluded one outlier tHcy level in each patient group (see Results).

Table 3. Erythrocyte sulphur amino acids, sulph-hydryl, and whole blood GSH concentrations in 37 ESRD patients, divided into two groups as in Table 1, and 21 healthy subjects

Met Hcy Cys Tau GSH in RBC GSH in whole blood Cys-Gly

Controls (ns21)


Malnourished (ns10)

23 (6) 3.4 (0.9) 12 (5) 150 (59) 2840 (467) 1100 (147)

22 (5) 6.1 (3.6)* 12 (8) 273 (121)** 2690 (493) 920 (192)*

19 (7)*,qq 5.7 (2.5)** 14 (10) 249 (233)* 2698 (725) 830 (210)**

13 (3)

11 (3)

16 (11)q

Values are given as mean (SD). The erythrocyte sulphur amino acids and sulph-hydryl concentrations are in mmolul intracellular water, but the whole blood GSH concentrations are in mmolul. *P-0.01 and **P-0.001, compared with control subjects. q P-0.05 and qqP-0.01, compared with well-nourished patients.

Malnourished patients compared with those with normal nutritional status As expected, anthropometric parameters were significantly higher in patients with normal nutritional status than in malnourished ones. The latter were older, had low s-albumin, s-creatinine and blood urea levels. The haematocrit percentages were similar in both patient groups. Protein intake was similar in the well-nourished and malnourished patients (Table 1). There were more males than females among the well-nourished patient group, as compared with the malnourished group, but x2 analysis showed no significant difference (Ps0.13). Plasma sulphur amino acids and sulph-hydryl compounds in well-nourished and malnourished ESRD patients are compared in Table 2. Two patients, one in each group, with extremely high plasma tHcy concentrations (134 and 170 mmolul, respectively, one patient with heterozygous A1298C mutation of the MTHFR gene), were excluded as outliers [17]. The lowest plasma tHcy level in this patient material was 16 mmolul. The plasma concentrations of tHcy, GSH,

and Cys-Gly were significantly lower in the malnourished group than in those with normal nutritional status, but there were no significant differences in the plasma concentrations of tCys, c-Glu-Cys, Met, CSA, and Tau between the two patient groups (Table 2). In the RBC (Table 3), the malnourished patients had lower Met concentrations and marginally higher Cys-Gly concentrations than the well-nourished patients. However, the RBC Cys-Gly concentrations in the malnourished patients were similar to those in the well-nourished group if one outlier (40.6 mmolul) was excluded. No significant differences between the two patient groups were found in other RBC sulphur amino acids and sulph-hydryl concentrations. The plasma sulphate concentration showed no significant difference between the malnourished and wellnourished patients (Table 2). In patients, the plasma sulphate was significantly higher in men than in women (1593 (649) vs 1110 (672) mmolul, P-0.05), but the concentrations in healthy subjects were similar in both sexes. Table 1 shows no significant differences in plasma folate, RBC folate, and plasma vitamin B12 concentrations between the well-nourished and malnourished patients. The plasma tHcy was significantly and negatively correlated with RBC folate. No significant correlation was found between the RBC tHcy concentration and plasma or RBC folate. SGNA was positively correlated with RBC folate (rs0.54; P-0.01), but not with plasma folate (rs0.07; PsNS). The numbers of patients on CAPD and HD treatment were similar (19 and 18, respectively). Apart from a higher concentration of plasma Met and lower plasma sulphate level in CAPD than in HD patients, no differences were found in sulphur amino acids and sulph-hydryls levels, vitamin concentrations, subjective and objective nutritional parameters, age, and sex between the two patient groups. Protein intake, expressed as nPNA, did not significantly differ between the HD and CAPD patients (1.25 (0.31) vs 1.16 (0.22) gukg DBWuday, PsNS). In addition, KtuVurea did not differ significantly

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M. E. Suliman et al.

when the patients were divided into malnourished and well-nourished groups in HD (KtuVurea per HD session: 1.5 (0.36) vs 1.8 (0.39), PsNS) as well as in CAPD (weekly KtuVurea: 2.0 (0.32) vs 2.2 (0.46), PsNS) patients. Correlations In ESRD patients, Spearman’s rank correlation coefficients (r) showed that the plasma tHcy concentration was negatively correlated with SGNA and positively with DBW%, s-albumin, s-creatinine, plasma tCys, BMI (rs0.44; P-0.01), LBM (rs0.33; P-0.05), fat mass (rs0.33; P-0.05), MAMC% (rs0.42; P-0.01), and HGS% (rs0.37; P-0.05). In these patients, plasma GSH was negatively correlated with SGNA (rs–0.43; Ps0.007), positively with HGS% (rs0.40; Ps0.02), and tended to be correlated with s-albumin (rs0.30, Ps0.06). However, no relationship was found between nutritional parameters and the concentrations of GSH in RBC and whole blood. In the patients, the plasma Tau concentration was negatively correlated with the plasma CSA level (rs 0.50, Ps0.001) (Figure 1). The concentration of plasma c-Glu-Cys showed a positive correlation with plasma tCys (rs0.50, Ps0.001) and plasma GSH levels (rs0.34, Ps0.04). The plasma sulphate concentration in ESRD patients was significantly correlated with plasma tCys (rs0.44, Ps0.005), s-creatinine (rs0.38, Ps0.02), and blood urea (rs0.42, Ps0.01) levels, but no significant relationship was found with other sulphur compounds or nutritional parameters. In this study, nPNA was significantly correlated with plasma tCys (rs0.41, Ps0.01), sulfate (rs0.41, Ps0.01), and Tau (rs0.38, Ps0.02), but nPNA did not correlate with plasma Met and tHcy. Using stepwise multiple regression analysis to find the predictors of plasma tHcy in the patients, in a model including plasma folate, SGNA, BMI, s-albumin, plasma tCys, and age, we found that SGNA was a significant predictor (r2s0.11, P-0.05) of plasma tHcy followed by plasma folate (r2s0.09, Ps0.05). However, when we excluded plasma folate from the model and replaced it with RBC folate, we found that BMI was the only parameter that could be entered into the model (r2s0.21). GSH and haematocrit In ESRD patients, the RBC GSH concentrations (Table 3) were similar to those in healthy subjects (Ps0.14), but the whole blood concentrations were significantly lower (Table 3) and the plasma concentrations significantly higher (Table 2) than in healthy subjects. The mean haematocrit per cent was significantly lower in the ESRD patients than in healthy subjects (Table 1). Table 4 shows that when the patients were divided into one group with low haematocrit per cent

Fig. 1. Relationship between plasma Tau and plasma CSA concentrations in 37 ESRD patients. Spearman rank correlation coefficient rs–0.50, Ps0.001.

Table 4. The GSH concentrations in plasma, whole blood, and RBC in 37 ESRD patients when the patients were divided into two groups on the basis of the median haematocrit per cent

Number Malnourished Plasma GSH Whole blood GSH Erythrocyte GSH

Haematocrit )37%

Haematocrit O37%

18 4 6.4 (1.5) 995 (216) 2865 (631)

19 6 5.6 (1.7) 802 (172) 2528 (424)

P value

NS -0.01 -0.05

Values are given as mean (SD). The plasma and whole blood concentrations are in mmolul, but the erythrocyte concentrations are in mmolul intracellular water.

and another with high haematocrit per cent, not only the whole blood GSH concentrations, but also the RBC concentrations were significantly lower in the patient group with the low haematocrit per cent, but the plasma GSH did not differ significantly (Ps0.09) between the two groups. As mentioned above, no difference was found in GSH concentrations in RBC or whole blood when the patients were divided according to SGNA, but the whole blood concentrations were significantly lower in the ESRD patients than in healthy subjects, and the RBC GSH concentrations in both groups were similar to those in healthy subjects (Table 3). The haematocrit was significantly correlated with the GSH concentrations in whole blood (rs0.47; P-0.01) and RBCs (rs0.34; P-0.05), but not with the plasma levels (rs0.30; PsNS).

Discussion In this study, we assessed for the first time the relation of nutritional status to both the plasma and RBC concentrations of sulphur-containing compounds in CRF patients. All the patients had hyperhomocysteinaemia, which was associated with higher levels


of plasma tCys, CSA, inorganic sulphate, c-Glu-Cys, GSH, Cys-Gly, lower levels of plasma Met and Tau, and higher levels of RBC tHcy and RBC Tau than in the healthy subjects, confirming our previous findings in a smaller group of HD patients [13]. The relationship between plasma tHcy and nutritional parameters was supported by the univariate analysis and the stepwise multivariate regression model which showed that malnutrition and hypoalbuminaemia are associated with lower plasma tHcy levels. In contrast, no relationship was found between the RBC tHcy concentration and nutritional status. The levels of vitamin B12 and folic acid were similar in the malnourished and well-nourished ESRD patients suggesting that the difference in plasma tHcy between the two patients groups was not due to B vitamins. Previous studies in the general population have shown that plasma tHcy increases with age and is higher in males. Our malnourished patients were, on average, older than the well-nourished patients, and it cannot be excluded that this may have contributed to differences between the groups. The influence of age and sex on tHcy levels in CRF patients may be much less important than that observed in the general population because of the remarkable increase induced by uraemia per se. In this study, plasma tHcy levels were in fact lower in the malnourished patients who were older, and also had lower s-albumin, than the younger patients. We had found previously that plasma Met in HD patients was correlated with SGNA and protein intake, estimated from nPNA [4]. However, in the present study, nPNA was not correlated with plasma Met or tHcy, but we found that protein intake was significantly correlated with plasma tCys, sulfate, and Tau. In addition, the RBC Met level was significantly lower and the plasma Met level tended to be lower in the malnourished than in well-nourished patients. A correlation between plasma Met and plasma tHcy has been reported by us [4] and others [18]. In this study, we found that the plasma Met level was correlated with the RBC tHcy concentration; again suggesting that intake of Met (protein) is related to Hcy synthesis. The plasma and RBC concentrations of tCys, Tau, and CSA in ESRD patients did not differ significantly between malnourished patients and those with normal nutritional status, and were not related to nutritional parameters. However, in a previous larger study of HD patients, we found that plasma tCys levels were associated with nutritional status [4]. Interestingly, in the presence of low plasma Tau and high plasma CSA concentrations, we saw an inverse correlation between plasma Tau and plasma CSA in ESRD patients (Figure 1) as well as a reduction in the TauuCSA ratio (Table 2). These findings support the hypothesis that the metabolic conversion of CSA to Tau may be impaired in CRF patients [15]. Among the other sulph-hydryls measured in this study, plasma concentrations of GSH and Cys-Gly were significantly lower in malnourished patients

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than in well-nourished ones and were correlated with nutritional parameters, but the concentrations in RBC and whole blood were not, suggesting that plasma but not RBC concentrations of sulph-hydryls are influenced by nutritional status in CRF patients. GSH concentrations are reported to be high in plasma [11,13], low in whole blood [11,12], and low [11,12], normal [13,19], or high [14] in RBCs in CRF patients. However, in the studies reporting low RBC levels, the GSH concentrations were not determined directly in RBCs but in whole blood corrected for haematocrit [11] or haemoglobin [12]. In the present study, the RBC concentrations of GSH were measured simultaneously with the concentrations in whole blood and plasma. There was no significant difference in RBC GSH concentrations between the patients and healthy subjects in the presence of low whole blood and high plasma levels in the ESRD patients. Although the relationship between plasma GSH and total body content of GSH is not clearly established in the human, findings of high plasma and normal RBC concentrations of GSH in the ESRD patients argue against GSH depletion in the tissues of ESRD patients. As noted above, no relationship was found between nutritional status and GSH concentrations in RBC and whole blood in contrast to findings in plasma. The ESRD patients had a lower haematocrit than the healthy subjects, and the haematocrit was significantly correlated with RBC and whole blood GSH levels. Both the RBC and whole blood GSH concentrations differed significantly when the patients were divided into two groups, based on the median haematocrit (Table 4). This accords with a previous observation that treatment of anaemia in HD patients with recombinant Epo increases GSH levels in whole blood. The slight increase in intracellular RBC GSH content in the high haematocrit group may be due to a higher rate of erythropoiesis and a higher proportion of young RBC than in the anaemic patients. Thus, low concentrations of GSH in whole blood in ESRD patients, in the presence of normal RBC GSH levels, are mainly related to a low haematocrit and may not be due to a real GSH deficiency in the body. A substantial fraction of plasma sulph-hydryls is bound to protein and this may explain the significant relationship between plasma tHcy and s-albumin. Previous studies have shown lower plasma tHcy concentrations in CAPD than in HD patients [20,21] and this difference was attributed to higher folate levels in CAPD patients [21]. Another explanation could be that the levels of s-albumin are usually lower in CAPD than in HD patients [1]. However, s-albumin levels were not reported in the previous studies [20,21]. In the current study, the vitamins, protein intake, and nutritional parameters, including s-albumin, were similar in the CAPD and HD patients, and this may explain the absence of a difference in sulph-hydryl concentrations between the two patient groups. The plasma sulphate concentration was five times higher in the ESRD patients, as reported elsewhere

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[22], and was significantly correlated with plasma tCys, but not with the other sulphur-containing compounds or nutritional parameters. Moreover, the plasma sulphate concentrations were similar in the malnourished and well-nourished ESRD patients. The difference in plasma sulphate levels between the HD patients and CAPD patients is probably due to intermittent treatment with HD with accumulation of sulphate during the interdialytic period. Earlier studies have shown that plasma sulphate is efficiently removed by HD [22] and CAPD [23] treatment. Thus, the high plasma sulphate levels in ESRD patients in the presence of low plasma Tau levels suggests that a large proportion of sulphur-containing compounds are metabolized to inorganic sulphate instead of being metabolized to Tau, a reaction which appears to be inhibited in uraemia [15]. In summary, our study shows that ESRD patients with malnutrition have lower plasma tHcy, GSH, and Cys-Gly concentrations than patients with normal nutritional status, suggesting that plasma (but not RBC) concentrations of thiols appear to be influenced by nutritional status and s-albumin in CRF patients. This should be taken into consideration when evaluating plasma tHcy, or other sulphur-containing compounds, as cardiovascular risk factors in CRF patients. As all ESRD patients had hyperhomocysteinaemia, the findings of lower plasma tHcy levels (but still abnormally high levels) in patients with malnutrition and hypoalbuminaemia do not necessarily alter the assumption that hyperhomocysteinaemia is a risk factor for cardiovascular disease in renal failure patients [24]. In contrast, GSH concentrations in RBCs are related to haematocrit, not to nutritional parameters, i.e. anaemia status rather than nutritional status influences RBC GSH levels in CRF patients. Further studies will be required to confirm the interactions between hyperhomocysteinaemia, nutritional status, and cardiovascular disease and to improve our understanding of these relationships in CRF patients. Acknowledgements. This study was supported by Karolinska Institutet, Stockholm, Sweden and by Baxter Healthcare Corporation, Deerfield, IL, USA.

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