Factors associated with increased plasma homocysteine in patients using an amino acid peritoneal dialysis fluid
Shao-Yu Yang1,2,
Jenq-Wen Huang1,
Kai-Yao Shih1,
Shih-Ping Hsu2,
Pei-Lun Chu1,
Tzong-Shinn Chu1 and
Kwan-Dun Wu1
1 Department of Internal Medicine, National Taiwan University Hospital and, 2 Department of Internal Medicine, Far Eastern Memorial Hospital, Taipei, Taiwan
Correspondence and offprint requests to: Kwan-Dun Wu, MD, PhD, Department of Internal Medicine, Room 1419, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei 100, Taiwan. E-mail: kdw{at}ntumc.org.tw
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Abstract
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Background. Although amino acid peritoneal dialysate (AAPD) substitution is thought to improve protein-energy malnutrition in patients undergoing peritoneal dialysis (PD), it may also increase plasma homocysteine (Hcy) levels due to the methionine load in the dialysate. However, it is still unclear which factors are important for elevating Hcy in patients treated with AAPD.
Methods. Sixteen malnourished PD patients (age 48±18 years) were treated daily with one exchange of 1.1% AAPD for 3 months. The effects of AAPD on nutrition, Hcy, methionine, leptin and insulin resistance were studied. We also analysed factors that influenced plasma Hcy levels.
Results. We found a transient increase in serum albumin (P<0.01) after 1 month treatment, especially in patients with serum albumin
3.5 g/dl. Total plasma Hcy increased markedly after AAPD (the peak at month 2, P<0.001) and returned to baseline after ceasing AAPD, despite no changes in dietary methionine intake and serum methionine levels. Eight patients with Hcy increments >5.65 µM (the median) had lesser dietary intakes of protein (P = 0.01) and methionine (P = 0.028), lower body fat mass (P = 0.05) and lower aspartate transaminase (AST) (P = 0.008) before AAPD treatment than patients with lower increments.
Hcy was inversely correlated with baseline dietary methionine intake (r = 0.61), protein intake (r = 0.54) and AST (r = 0.51) (all P<0.05). There was no change in leptin or insulin resistance. AAPD treatment significantly increased Kt/Vurea (P<0.001), weekly creatinine clearance (P<0.05) and peritoneal glucose transport (P<0.05).
Conclusions. Treatment with 1.1% AAPD transiently increased serum albumin in malnourished PD patients. However, the methionine load from the dialysate in this study significantly elevated plasma Hcy levels, especially in patients with lower protein and methionine intakes, and lower AST levels. Further long-term studies will be needed to clarify potential nutritional benefits and adverse effects of AAPD.
Keywords: amino acid peritoneal dialysate; homocysteine; nutrition; peritoneal dialysis
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Introduction
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Nutritional status provides an independent predictor of clinical outcome in uraemic patients treated with peritoneal dialysis (PD) [1]. Approximately 50% of patients undergoing continuous ambulatory peritoneal dialysis (CAPD) have shown subjective, anthropometric and biochemical evidence of malnutrition [2]. Even though malnutrition has many causes in these patients, the use of amino acid peritoneal dialysate (AAPD) as an osmotic agent and simultaneous nutritional supplement has been shown to transiently or persistently improve nutritional indicators, such as albumin, prealbumin, serum total proteins, transferrin, nitrogen balance and mid-arm muscle circumference (MAMC) [3,4]. In addition, the amino acid substitution may reduce glucose load and, in turn, influence lipid metabolism [4,5], reduce body fat mass [6] and may decrease insulin resistance.
There are, however, adverse metabolic effects of AAPD. Mild metabolic acidaemia is not uncommon, but it can be managed with alkali administration. Increases in urea nitrogen may induce gastrointestinal symptoms and decrease dietary intake, especially when two AAPD exchanges are administered daily [3]. Importantly, increases in plasma homocysteine (Hcy) levels have been reported in patients treated with AAPD. Hyperhomocysteinaemia, commonly encountered in uraemic patients, is an independent risk factor for cardiovascular disease in this population [7]. Elevations in Hcy caused by AAPD are most probably due to the methionine load from the dialysate. Therefore, absorption of amino acids from the peritoneum may be important in this mechanism. However, other factors, including decreased renal clearance and a deranged Hcy metabolic pathway, in uraemic patients, may also explain the hyperhomocysteinaemia in these patients [8]. Malnourished patients are probably deficient in vitamins that play important roles in Hcy metabolism, and are therefore prone to elevations in Hcy when the methionine load is increased. In this context, we speculate that malnutrition per se may influence the absorption of amino acids and aggravate derangements in Hcy metabolism.
In malnourished patients treated with AAPD, some may experience greater increases in Hcy and should be monitored closely during treatment. The aim of this study was to examine factors associated with elevations in Hcy in patients treated with AAPD. We analysed factors that may affect the absorption of amino acids through the peritoneum and that influence the metabolism of Hcy. The effects of AAPD on leptin levels and insulin resistance were also studied.
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Subjects and methods
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Study design
This was a prospective, open-label, single-group study. The enrolled PD patients received one bag of Nutrineal® (Baxter Healthcare, Singapore) during the second exchange for CAPD or during the daytime exchange for automated peritoneal dialysis (APD), while undergoing treatment lasting 3 months. The dwell volumes and times were unchanged. Biochemistry and anthropometric parameters were assessed either monthly or at the end of the study and 1 month after discontinuing Nutrineal®. A subjective global assessment [9] was made at baseline and at the third month. The patients recorded their daily detailed dietary intakes (including dietary content, weight and how the food was cooked) consecutively every other day, three times, during the first week of each month. The records were reviewed by a dietitian. All medications known to interfere with study parameters, such as lipid-lowering agents, were suspended for 2 weeks before baseline sampling. The supplement of vitamins in patients was not changed during the study.
Patients
We recruited patients having evidence of malnutrition from 135 patients in our hospital-based PD centre. The criteria for recruitment were as follows: (i) ages between 18 and 75 years; (ii) Kt/Vurea>2.0; and (iii) serum albumin
3.9 g/dl, or normalized protein nitrogen appearance (nPNA)
1.0 g/kg/day. Patients were excluded if they had one of the following conditions: (i) blood urea nitrogen (BUN)
100 mg/dl; (ii) significant hepatic dysfunction [aspartate transaminase (AST) or alanine transaminase (ALT) >50 U/l]; (iii) positive anti-HIV test; (iv) malignancy; (v) more than one episode of peritonitis in the past 6 months, or one episode of peritonitis within 1 month; (vi) venous serum bicarbonate
20 mmol/l; and (vii) diseases or medications leading to a catabolic state. All patients provided informed consent and the study was approved by the Ethics Committee of the National Taiwan University Hospital.
Assessments and examinations
Adverse effects of AAPD treatment were recorded from monthly interviews. Blood samples were collected monthly for routine biochemistry after overnight fasting. Plasma total Hcy, insulin, growth hormone, tumour necrosis factor-
, serum folic acid and vitamin B12 were measured by immunoassay with an IMMULITE 2000 analyser and corresponding kits (DPC, Los Angeles, CA). Serum vitamin B6 was measured with high-performance liquid chromatography according to the protocol of Bates et al. [10]. Methionine levels were analysed on an API 2000 LC/MS/MS (Applied Biosystem, Co., Foster City, CA) as previously described [11]. Serum C-reactive protein, prealbumin and transferrin were measured by an ARRAY 360 system (Beckman Coulter, CA). Lactate was measured by a Vitros 950 (Ortho Clinical Diagnosis, Rochester, NY), and other biochemical parameters, including urea nitrogen, creatinine, albumin, AST, ALT, triglyceride and total cholesterol were measured by a TBA-200 FR automatic analyser (Toshiba, Tokyo, Japan). Plasma leptin levels were measured using a human leptin kit (Linco Research, Inc., St Charles, MO).
Anthropometric markers, including triceps, biceps, subscapular, with suprailiac skin fold thickness and mid-upper arm circumference (MUAC) were measured at baseline, the third month and the fourth month. MAMC was derived from MUAC and tricep skin fold thickness, where MAMC = MUAC
x (TST) [2]. Body fat mass and its distribution were assessed using a dual-energy X-ray absorptiometry (DEXA) total body scanner. Subjects were scanned after overnight fasting with an empty bladder and after complete drainage of dialysate.
The amount of total dialysate used monthly was calculated for estimating the average daily dialysate glucose absorption. The glucose absorption ratio, 1 (D/D0 G), was derived from the peritoneal equilibration test (PET); D/D0 G was the ratio of the final dialysate glucose to the initial dialysate glucose level. Dietary intakes of protein, energy and methionine were calculated based on patient dietary records. Normalized protein catabolic rate (nPCR), nPNA and nitrogen balance were calculated according to standard formulae [12]. Weekly Kt/Vurea, weekly creatinine clearance (CCr) and PET were performed to evaluate peritoneal function before and 3 months after Nutrineal® treatment, as previously described [13].
Statistical analysis
Prior to statistical analysis, all variables were tested for normality using a KolmogorovSmirnov test. All data were found to be normally distributed. Although most results were expressed as means±SD, some variables with a large SD were reported as medians and interquartile ranges (IQRs). Paired t-tests were performed to compare differences in parameters during and after the study with the baseline. The MannWhitney U-test was used to compare the variables in subgroups categorized by Hcy increments (
Hcy) after AAPD. A P-value <0.05 was considered statistically significant. All computations were performed by SPSS 10.0.1 for Windows (SPSS Inc., Chicago, IL).
Results
Eighteen patients were recruited into the study, and two were dropped because one was diagnosed with pulmonary tuberculosis in the first month and the other did not follow the study protocol precisely. The remaining 16 patients (six males, 10 females; age 48±18 years) underwent PD treatment (11 CAPD, five APD) for >3 months (median 28, IQR 1181.25 months). Two patients experienced nausea and poor appetite during AAPD treatment. These symptoms subsided after increased dialysis exchange. Transient hypotension occurred in one APD patient and this was alleviated by increasing fluid intake and reducing the daily dwell volume of Nutrineal® by 300 ml. The other patients showed good tolerance of Nutrineal® during the study, and no peritonitis was noted during the protocol. Two patients took 5 mg of folic acid per day before the study, and the dosage was not altered during the study. The patients were not given vitamin B6 or vitamin B12 supplements during the study.
Nutritional and anthropometric parameters
At 1 month after Nutrineal® treatment, serum albumin levels increased significantly from 3.60±0.21 to 3.77± 0.21 g/dl (P = 0.002). However, the effect was not sustained to the end of the treatment. The increment in serum albumin was slightly although not significantly greater in patients with baseline albumin
3.5 g/dl (n = 6) than in patients with higher albumin levels (0.28±0.17 vs 0.10±0.16 g/dl, respectively, P = 0.06). There was no significant change in prealbumin or transferrin levels. Although one exchange with AAPD did not change dietary protein intake of the patients (P = 0.16), the dietary energy intake increased (25.2±5.2 to 27.8±4.1 kcal/kg/day, P = 0.04) at month 1. Nutrineal® also caused significant beneficial effects in nPNA, nPCR and nitrogen balance (Table 1). The levels of serum folic acid, vitamin B6 and vitamin B12 did not change significantly during treatment.
Nutrineal® elevated BUN (from 51.0±17.6 to 61.3± 8.1 mg/dl, P<0.01) and chloride (from 91.6±4.4 to 93.1± 4.6 mmol/l, P<0.05), but reduced serum bicarbonate (from 29.4±2.3 to 26.3±2.5 mmol/l, P<0.01). Serum creatinine, uric acid, lactate and electrolytes remained unchanged (data not shown), except for a decrease in high-density lipoprotein cholesterol at month 3 (54±23 vs 45±16 mg/dl, P<0.01). MAMC was significantly lower only at month 1 (19.7±2.3 vs 19.2±2.1 cm, P<0.01). There was a decrease in body fat mass, but this was not significant.
Effects on homocysteine metabolism
Dietary methionine intake did not change during the treatment. Despite no changes in serum methionine levels, plasma Hcy increased significantly by 22% (P<0.05) after 3 months of Nutrineal® treatment, but returned to baseline levels at 1 month after discontinuing AAPD (Table 2). The median increase in Hcy (
Hcy) at month 1 was 5.65 µM. There was no significant correlation between baseline total Hcy and
Hcy.
Eight patients with
Hcy
5.65 µM had fewer dietary intakes of protein (P = 0.01) and methionine (P = 0.028) compared with patients with smaller increases (Table 3). They also had lower baseline body fat mass (P = 0.05) and AST (P = 0.05) than patients with
Hcy<5.65 µM. The two subgroups were not different in peritoneal function, plasma Hcy, serum methionine, albumin, increased albumin in month 1 (
Alb), Kt/V, weekly CCr, inflammation markers or homeostasis model assessment (HOMA-IR) [14]. Serum folic acid, vitamin B6 and vitamin B12 were not different between the two groups, although patients with less
Hcy had higher levels of these vitamins.
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Table 3. Comparison of baseline parameters for homocysteine metabolism and peritoneal function between patients having plasma homocysteine changes greater than or less than 5.65 µM
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The
Hcy was inversely correlated with baseline dietary methionine intake (r = 0.61, P<0.05), protein intake (r = 0.54, P<0.05) and AST (r = 0.51, P<0.05) (Figure 1). There were no correlations between
Hcy and the measured vitamins (data not shown).

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Fig. 1. Relationships between increments in Hcy ( Hcy) and baseline intake of protein and methionine (Met), plasma Hcy and AST levels.
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Effects on leptin, glucose metabolism and inflammatory markers
Similar to a previous study [15], leptin levels were correlated with body fat mass (r = 0.66, 0.55, 0.60 at baseline, month 3 and month 4, respectively, all P<0.05). The leptin levels, although higher at month 3, were not significantly different from the baseline. Compared with baseline values, growth hormone, TNF-
and CRP did not change significantly (Table 4). AAPD did not change fasting glucose, fasting insulin levels or the insulin resistance index determined by HOMA-IR.
Peritoneal membrane function, weekly Kt/Vurea and clearance of creatinine
Nutrineal® treatment produced a tendency towards increased peritoneal solute transport. The ratio of dialysate glucose at 4 h to that at time zero (D/D0 G) decreased after 3 months of treatment with AAPD, but the ratio of dialysate to plasma for creatinine did not change (Table 5). The weekly Kt/Vurea and CCr increased significantly. Two patients received an increased dialysis dose due to uraemic symptoms during Nutrineal® treatment. However, the overall Kt/V (2.43± 0.44) and weekly CCr (61.10±16.73 l/week) were still greater than baseline (P = 0.0014 and 0.037, respectively), even after removal of these two patients from the analysis.
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Discussion
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As observed by others [3], we found that Nutrineal®-induced increases in serum albumin were more prominent in malnourished patients (serum albumin
3.5 g/dl). However, even in these patients, this benefit was only transient. Consistent with these findings, the anthropometric markers did not change, although nPNA, nPCR and nitrogen balance significantly increased.
AAPD treatment also caused elevations in plasma tHcy levels. This elevation may have resulted from an increase in methionine intake, increased Hcy synthesis, decreased Hcy degradation or increased albumin levels. However, our patients did not change their dietary methionine intake during AAPD treatment, and previous studies have shown that
80% of amino acids given in AAPD are absorbed through the peritoneum [16]. Given this assumption, the daily methionine load from AAPD would be 1.36 g, a value
1.2 times the dietary intake. Nevertheless, the serum methionine levels were not increased in these patients, in spite of a significant elevation in Hcy. This suggests that there was increased synthesis of Hcy from methionine in these patients, resulting in no change in serum methionine. As a second mechanism to explain the increased Hcy, there is a strong correlation between changes in tHcy levels and changes in serum albumin levels, which is possibly related to the high albumin binding of Hcy [17]. However, Hcy levels increased moderately (
25%) during the 3 months, whereas albumin levels increased only slightly during month 1. This finding suggests that the albumin change was not the main cause of Hcy increase in our study.
We also observed correlations between Hcy elevation and baseline levels of protein and methionine intake. Several mechanisms may account for this observation. First, the absorption of amino acids (methionine) was greater in malnourished patients. Our patients with greater
Hcy had similar peritoneal function test results to those with less
Hcy. However, it is not known whether peritoneal function tests based on glucose dialysates can be applied to the absorption of amino acids. Secondly, the vitamins needed for Hcy metabolism, including folic acid, vitamin B6 and B12, are deficient in these patients. Although not statistically significant, patients with greater
Hcy had lower levels of these vitamins. Even though this correlation could become significant after inclusion of more patients, this finding is not sufficient to indicate that routine vitamin supplementation is needed in patients receiving AAPD. Thirdly, the transmethylation pathway of methionine may be up-regulated in these patients, which in turn may increase Hcy. Despite varying aetiologies, patients with lower protein and methionine intakes may have greater increases in plasma Hcy after a methionine load. However, hyperhomocysteinaemia does not result in poor outcomes in end-stage renal disease patients [18], and AAPD may improve nutritional status.
In the present study, we also found an inverse correlation between
Hcy and baseline AST. We previously had demonstrated that in haemodialysis patients there is an inverse correlation between plasma Hcy levels and AST [11], indicating that the transamination of methionine may play an important role in the development of hyperhomocysteinaemia in uraemic patients in whom transmethylation is deranged. The present work further supports speculation that patients with lower transamination activity have greater increments in plasma Hcy during AAPD substitutions, because more methionine is catabolized through the transmethylation pathway. Even though the transamination of methionine does not play an important role in normal subjects [19], it may be more important in uraemic patients who receive an additional methionine load. A limitation of the present study was the lack of change in methionine or Hcy in the PD fluid.
Even though patients with higher
Hcy had lower folic acid and reduced vitamin B6 and B12 levels than patients with less pronounced
Hcy, these findings were not significant in our study.
In conclusion, 1.1% AAPD (Nutrineal®) elevated plasma Hcy in PD patients, especially in those with lower dietary protein/methionine intakes and with low AST levels. Further investigation will be required to elucidate the long-term effects of AAPD as well as the underlying mechanisms of the correlations revealed in this study.
Conflict of interest statement. The study was financially supported by Baxter Healthcare Corporation.
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Received for publication: 26. 1.04
Accepted in revised form: 10. 8.04