Department of Nephrology, Medizinische Klinik and Medizinische Poliklinik Innenstadt, Universität München, München, Germany
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Abstract |
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Methods. Forty-eight early haemodialysis patients were assigned to either treatment with conventional (potentially microbiologically contaminated) or on-line produced ultrapure dialysis fluid. Study parameters were bacterial growth, markers of systemic inflammation (C-reactive protein (CRP) and interleukin 6), and parameters of nutritional status (estimated dry weight, upper mid-arm muscle circumference, serum albumin concentration, insulin-like growth factor 1, leptin, and protein catabolic rate). Patients were followed for 12 months.
Results. There were no statistically significant differences in demographic and treatment characteristics, degree of bacterial contamination of the dialysate, markers of systemic inflammation, or parameters of nutritional status among the two treatment groups at recruitment. Changing from conventional to ultrapure dialysis fluid reduced significantly the levels of IL-6 (19±3 pg/ml to 13±3 pg/ml) and CRP (1.0± 0.4 mg/dl to 0.5±0.2 mg/dl), and resulted in significant increases in estimated dry body weight, mid-arm muscle circumference, serum albumin concentration, levels of the humoral factors, and in protein catabolic rate after 12 months. Continuous use of conventional dialysis fluid (median 4060 c.f.u./ml) was not associated with significant alterations in markers of inflammation (IL-6 21±4 pg/ml vs 24±6 pg/ml, CRP 0.9±0.3 mg/dl vs 1.1±0.4 mg/dl) or of nutritional status at any time of the study. All differences in systemic inflammation and nutritional parameters observed during the study period (from recruitment to month 12) were significant between the two patient groups.
Conclusions. Cytokine induction by microbiologically contaminated dialysis fluid has a negative impact on nutritional parameters of early haemodialysis patients. The microbiological quality of the dialysis fluid represents an independent determinant of the nutritional status in addition to known factors, such as dose of dialysis and biocompatibility of the dialyser membrane. Ultrapure dialysis fluid adds to the cost of the dialytic treatment, but may improve the nutritional status in long-term haemodialysis patients.
Keywords: biocompatibility; cytokines; dialysis fluid; haemodialysis; nutrition
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Introduction |
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Renal replacement therapy is associated with a systemic inflammatory response syndrome characterized by elevated levels of acute-phase proteins and triggered by enhanced cytokine induction [59]. Uraemia and its metabolic complications as well as bioincompatibility of the components of the dialytic procedure contribute to high cytokine concentrations. Routinely used dialysis fluid prepared by reverse osmosis and bicarbonate concentrate may be microbiologically contaminated. While intact dialyser membranes are impermeable to bacteria, soluble pyrogenic bacterial products penetrate through both low- and high-flux dialyser membranes, cause activation of monocytes, and induce cytokine production. With the introduction of on-line-produced ultrapure dialysis fluid using endotoxin adsorbing synthetic dialysers, a reduction of the cytokine response could be demonstrated [10,11].
We investigated the effects of different microbiological qualities of dialysis fluid on selected nutritional parameters in early dialysis patients in a prospective clinical study.
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Subjects and methods |
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Treatment characteristics
Regular haemodialysis was performed with volumetrically controlled ultrafiltration (MTS 4008, Fresenius, Bad Homburg, Germany). Each treatment session lasted 35 h, with blood flow rates between 200 and 300 ml/min, and dialysate flow rates of 500 ml/min. All patients received single-use biocompatible synthetic high-flux membranes (APS 650, polysulphone, Asahi, Tokyo, Japan). The bicarbonate dialysate was either conventional or ultrafiltered using an additional polysulphone filter (Diasafe, Fresenius, Bad Homburg, Germany) next to the dialyser.
Study design
The study was a prospective comparison with parallel groups. The assignment to conventional or ultrafiltered dialysis fluid was made in alternating order according to the length of time on dialysis (336 months). At recruitment, all patients were on conventional dialysate; after assignment to a treatment group each patient was dialysed during all outpatient treatment sessions with the relevant dialysis fluid for that group.
During the 12-month study period, the attending nephrologist determined the patients dialysis prescription according to the usual clinical standards and estimated the post-dialytic dry weight by clinical acumen (blood pressure using a sphygmomanometer, other signs and symptoms of hyper- or hypovolaemia), X-ray and every 3 months by determination of the diameters of the inferior vena cava (by echography, according to the procedures described by Cheriex et al. [12]; overhydration was defined as a vena cava diameter of 12 mm, underhydration as a vena cava diameter of less than 8 mm). The usual management of long-term dialysis patients was employed, no further restrictions or guidelines were imposed.
Study parameters
Dry weight was recorded at the end of each dialysis session and blood pressure recordings were performed pre- and post-dialysis throughout the study period.
Upper mid-arm muscle circumference (UMAMC) was measured at recruitment and after 3, 6 and 12 months. UMAMC was derived from the mid-arm circumference (MA) between the midpoint of the acromial and olecranon process of the ulna of the patient's upper limb on the non-access arm as well as from the triceps skinfold thickness (TSF). For TSF, the thickness of the subcutaneous tissue was measured with a skinfold calliper. UMAMC was calculated according to the equation [13]: UMAMC=MA-0.134xTSF.
Blood samples were taken pre-dialysis for haematological parameters (blood count, ferritin), for routine biochemical parameters (urea), for serum albumin and for serum CRP levels and interleukin 6 (IL-6) levels at baseline and after 3, 6 and 12 months. Insulin-like growth factor and leptin were measured from serum samples at the initiation and termination of the study. Post-dialysis blood samples were taken for determination of urea concentration.
Serum albumin was measured by the bromocresol green method (ALB plus; Roche Diagnostics, Mannheim, Germany) using a Hitachi autoanalyser. The sensitivity of this method is 0.2 g/dl. The intra-assay coefficient of variation at a mean concentration of 2.7 g/dl is 0.39% and the inter-assay coefficient at a mean concentration of 3.2 g/dl is 1.71%. CRP was measured using particle-enhanced immunoturbidimetry (COBAS Integra 700, Roche, Mannheim, Germany). This assay detects values as low as 0.025 mg/dl. The intra-assay coefficient of variation was 1.8% at a level of 0.62 mg/dl and 1.5% at a level of 14.2 mg/dl. The expected values for adults of serum CRP are less than 0.5 mg/dl. Serum IL-6 was measured using a commercially obtained immunoassay (IL-6 Quantikine assay, R&D Systems, Abingdon, UK) with a sensitivity of 0.7 pg/ml and an inter-assay coefficient of variation of less than 5%. Using this assay the upper limit of normal for human serum IL-6 concentrations is 12.5 pg/ml. Serum insulin-like growth factor 1 (IGF-1) concentration was measured by radioimmunoassay (Medgenix, Fleurus, Belgium) as previously described by Niebauer et al. [14]. The IGF-1 RIA has a sensitivity of 0.25 ng/ml, the within assay coefficient of variation at concentrations around 200 ng/ml was 4%, the between-assay coefficient of variation was 9% at a concentration of 250 ng/ml. The normal values of serum IGF-1 of adults are 88240 ng/ml.
Serum leptin levels were measured in duplicate at each time point by radioimmunoassay using commercial kits (Human leptin RIA, Linco Research, St Charles, MO, USA). Sensitivity was 0.5 µg/l, the inter- and intra-assay coefficients of variation were 6 and 7% respectively [15].
Dialysate bacteriology
Samples of standard dialysis fluid (1 ml) were analysed by the pour-plate method using thioglycolate agar and trypticase soy agar (TSA) at 20°C and 37°C for at least 5 days. One litre of ultrafiltered dialysis fluid was filtered through a bacteria filter (0.22 µm) and the entire filter was placed on the same types of agar plate.
Determinations of the endotoxin concentration of the dialysis fluids used were not made routinely. However, measurements were performed in filtered dialysis fluid at the beginning and at the end of the study. Endotoxin concentrations were measured with the chromogenic Limulus amoebocyte lysate (LAL) assay (Coatest Endotoxin Chromogenix, Mölndal, Sweden) using microtitre plates, which were measured in an automatic reader. Commercially available LPS from E. coli was used for the calibration curve. The test system is calibrated in endotoxin units (EU). The assay was validated according to the European Pharmacopoeia and performed in duplicate. The detection limit was 0.03 EU/ml.
All filtered dialysis fluid samples contained less than 0.1 c.f.u./ml and 0.03 EU and met the definition of ultrapure dialysate [16,17]. These findings are in accordance with previous reports, showing that the use of one or two additional filters reduces bacterial contamination at least to ultrapure levels [1821].
Calculations
To calculate the dialysis dose the second-generation Daugirdas formula [22] was used: Kt/V=-ln(R-0.008 t)+(4-3.5R) UF/W, where R=BUN post/BUN pre, while t, UF, W, BUN post, BUN pre respectively mean length of dialysis session (h), weight loss (kg), dry weight (kg), and post-/pre-dialysis blood urea nitrogen (mg/dl).
The protein catabolic rate was determined by the formula published by Garred et al. [23]: PCR=0.0076 (Kt/V) (Urea pre+urea post)+0.17. This formula relates PCR (g protein/kg/day) to Kt/V and pre- and post-dialysis blood urea nitrogen measurements (mg urea nitrogen/dl) for the midweek session.
Statistical methods
Results are presented as mean±SD. To prevent problems associated with multiple testing, statistical analyses were restricted to time point 0 (baseline) and 12 months. Comparison between the groups at baseline and 12 months was done by an unpaired t-test. Comparison of changes in parameters within a group between baseline and 12 months were analysed by a paired t-test. Comparison of changes in parameters from baseline to 12 months between the groups the Wilcoxon rank sum test was used. Simple linear regression analysis was used to investigate the relationship between serum IL-6 levels and corresponding serum CRP levels. All data analyses were performed using the SAS statistical software packages. Significance was accepted at a P<0.05 level.
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Results |
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There were no statistically significant differences in estimated dry weight, upper arm muscle circumference, and serum albumin concentrations between the groups at recruitment (Table 3). Both groups of patients had comparable serum IGF-1 and leptin concentrations (Table 4
). There were no statistically significant differences in mean protein catabolic rate or Kt/V between the two study groups at baseline (Table 5
). The blood pressure recordings and the post-dialysis vena cava diameter did not reveal over- or underhydration at baseline (Table 6
).
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Ultrafiltration of dialysis fluid resulted in a microbiological quality that was in accordance with the definition of ultrapure dialysis fluid. Haemodialysis patients treated with ultrapure dialysis fluid had a significant and sustained decrease in mean serum CRP levels and IL-6 concentrations compared to their baseline values (Table 2). At the end of the study period, 11 of 24 (46%) of the patients had CRP levels below the expected normal value and 12 of 24 patients (50%) had IL-6 concentrations below the upper limit of the normal range. There was a highly significant linear correlation between serum IL-6 and corresponding CRP concentrations obtained at termination of the study (y=0.05575x0.20516, r=0.974, P<0.001). In contrast, no statistically significant changes from baseline were observed in the mean CRP levels and IL-6 concentrations of the patients who were treated with mildly contaminated conventional bicarbonate dialysis fluid.
Changes in nutritional parameters induced by different microbiological qualities of dialysis fluid during the study period
Estimated dry weight
Haemodialysis patients who were switched from conventional to ultrapure dialysis fluid had a clinically relevant increase in their estimated dry weight compared to their baseline post-dialysis weights. Table 3 shows the changes in dry weight for the study months 3, 6 and 12. As can be seen, the increase in dry weight in the ultrapure group was observed throughout the study period and became statistically significant compared to baseline at month 3. The mean increase in weight was 1.4 kg after 3 months of treatment with ultrapure dialysis fluid, 2.6 kg after 6 months, and 4.2 kg after 12 months. Each patient treated with ultrapure dialysis fluid had a weight gain of at least 2 kg at the termination of the study. Sixteen of 24 patients treated with ultrapure dialysis fluid (66%) had weight gains over 4 kg. There was no centre effect regarding weight gain (or other inflammatory markers or nutritional parameters). Sub-analysis of the patients treated with ultrapure dialysis fluid revealed that patients with weight gains of less than 4 kg or over 4 kg had differed significantly in their serum IL-6 levels at recruitment (17.3±2.2 vs 23.3±0.9 pg/ml).
In contrast, no statistically significant change from baseline dry weight was observed in the mean dry weights measured at subsequent times in patients treated exclusively with conventional dialysis fluid (Table 3). The changes in dry weight from baseline to end of the study among the groups were statistically significant (0.6±3.1 vs 4.2±0.9, P<0.01).
Upper mid-arm muscle circumference
Patients dialysed with ultrapure dialysis fluid had a significant increase in their mid-arm muscle circumference at month 12 compared to baseline. There was no statistically significant change from baseline in UMAMC at subsequent time points in patients who were treated with conventional dialysis fluid (Table 3). The changes in UMAMC from baseline to the end of the study among the groups were statistically significant (-0.4±0.6 vs 1.2±0.6, P<0.01).
Serum albumin concentration
Serum albumin levels increased above baseline values in both study groups after initiation of the study. However, this effect was continuous throughout the entire study period only for the ultrapure group. The increase in serum albumin concentration in patients treated with ultrapure dialysis fluid was statistically significant compared to baseline at 12 months. In contrast mean serum albumin concentrations of the patients who remained on conventional dialysis fluid did not differ statistically from baseline (Table 3). The changes in serum albumin from baseline to the end of the study among the groups were statistically significant (0.1±0.1 vs 0.3±0.2, P<0.01).
Humoral factors indicative of nutritional status
Patients switched from conventional to ultrapure dialysis fluid showed a significant increase in both serum IGF-1 and serum leptin concentrations compared to baseline at month 12. Continuous use of conventional dialysis fluid did not affect IGF-1 and leptin serum levels. The differences in circulating IGF-1 concentrations between the groups reached statistical significance after 12 months (Table 4). The changes in IGF-1 and leptin from baseline to the end of the study among the groups were statistically significant (IGF-1,-13±13 vs 45±26, P<0.01; leptin,-0.3±0.5 vs 0.8±0.5, P<0.01).
Dialysis dose and protein catabolic rate
The dose of dialysis remained unchanged during the whole study period in both groups. However, PCR increased significantly (months 3, 6 and 12) only in patients switched from conventional to ultrapure dialysis fluid. By comparison, there was no significant increase in PCR in patients treated exclusively with conventional dialysis fluid (Table 5). The changes in PCR from baseline to the end of the study among the groups were statistically significant (0.1±0.1 vs 0.3±0.1, P<0.01).
Clinical course of the patients participating in the investigations
All 48 patients completed the study. Four patients needed hospitalization. Two patients had failure of their vascular access (one patient in each group), one patient (ultrafiltered dialysis fluid) had a bleeding gastric ulcer, and another patient (conventional dialysis fluid) had pneumonia. Neither the intercurrent illness nor its treatment permanently affected the inflammatory markers or the parameters of nutritional status. Switch from conventional to ultrapure dialysis fluid resulted in a significant and sustained reduction of rHuEpo dose in these patients at months 3, 6 and 12 compared to baseline values (rHuEpo dose at month 12, 66±18 vs 96±22 U/kg/week at baseline; Hb 10.5±0.3 vs 10.4±0.4 g/dl). In contrast, patients exclusively treated with conventional dialysis fluid showed no significant improvement of their response to rHuEpo during the study period (rHuEpo dose at month 12, 90±18 vs 92±15 U/kg/week at baseline; Hb 10.3±0.2 vs 10.4±0.3 g/dl), and needed significantly higher rHuEpo doses at all times during the study. Predialysis blood pressure was well controlled in both groups throughout the study period. The percentage of patients taking antihypertensive drugs did not change significantly during the study period. Postdialysis measurements of the diameter of the inferior vena cava disclosed intravascular hypervolaemia and did not reveal differences among patient groups (Table 6).
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Discussion |
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According to the design of the study, the only difference in the therapy of the two groups of early dialysis patients was the microbiological quality of the dialysis fluid. Conventional bicarbonate dialysate showed mild to moderate bacterial growth (<200 c.f.u./ml) and was well within the accepted standard limits. No bacterial growth was detected in the ultrafiltered dialysate. Compelling evidence has been accumulated that small-molecular-weight pyrogens derived from bacterial contamination of the dialysis fluid are able to penetrate both low- and high-flux dialyser membranes and that these endo- or exotoxins induce cytokine production in circulating mononuclear cells even in the absence of complement activation [10]. The changes in circulating IL-6 concentrations and CRP levels after a switch from conventional to ultrapure fluid observed in our present and previous investigations [9,24] are principally reversible, as shown by studies utilizing a cross-over design [24]. They support the concept that repetitive cytokine response to microbiologically contaminated dialysis fluid contributes to raised levels of pro-inflammatory cytokines and induce the inflammatory syndrome associated with uraemia and its therapy.
Inflammation and malnutrition not only coexist in a significant number of maintenance dialysis patients, but there may be also a cause-and-effect relationship. The adverse effects of pro-inflammatory cytokines, particularly of IL-6, may be caused by enhanced protein catabolism, increased energy expenditure, and inhibited appetite [25]. The observation of lower body weight and serum albumin concentrations in our patients with higher IL-6 levels is in accordance with data of recent studies [26,27], indicating that serum albumin levels and/or albumin generation as well as changes in body weight were negatively correlated with IL-6 levels. Furthermore, elevated IL-6 levels have been associated with increased mortality in dialysis patients [27]. The mechanisms underlying the beneficial long-term effects of ultrapure dialysis on nutritional parameters are not fully elucidated. This is true for the nutritional parameters in our investigation or high serum albumin levels in patients receiving ultrapure dialysis fluid prepared by a batch type machine [28] or in patients on on-line haemodiafiltration [29]. The changes in nutritional parameters of our patients reflect higher protein intake (by improved appetite), less protein catabolism, and probably an anabolic effect of higher growth hormone levels or improved action of this hormone.
In conclusion, inflammation mediated by the cytokine response to bacterial contamination of the dialysis fluid appears to be an independent factor (in addition to underdialysis and to the properties of the dialyser membrane) affecting the nutritional status of haemodialysis patients. Ultrapure dialysis fluid is desirable, but adds to the cost of dialysis therapy. Further studies are needed to clarify the question as to whether sterile and pyrogen-free dialysis fluid or substitution fluid can prevent the development of other dialysis-related inflammatory diseases and reduce morbidity and mortality in end-stage renal disease patients to more acceptable levels. This may be true, particularly for dialysis patients who are not eligible for early transplantation.
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Notes |
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References |
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