1Department of Nephrology, Medical Centre Alkmaar, Alkmaar, 2Department of Nephrology, VU Medical Centre, Amsterdam, 3Department of Clinical Chemistry, Medical Centre Alkmaar, Alkmaarand 4Institute of Professional Education Hogeschool Alkmaar, Alkmaar, The Netherlands
Correspondence and offprint requests to: Anne van Tellingen, Department of Nephrology, Medical Centre Alkmaar, Wilhelminalaan 12, 1815 JD Alkmaar, The Netherlands. Email: avantellingen{at}worldmail.nl
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Abstract |
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Methods. To assess the influence of HD on plasma leptin, 10 CHD patients were crossover randomized to low-flux polysulfone (PS: F 6HPS), high-flux PS (F 60S), super-flux PS (F 500S) or super-flux cellulose-tri-acetate (CTA: Tricea 150G) for 12 weeks each. Blood samples were collected at the start of the study and each 12-week period. In addition, the relationship between patient characteristics, inflammation and leptin was analysed.
Results. At baseline, all groups showed similar leptin concentrations (mean 33.6±21.7 ng/ml). After a single HD session, a significant (P<0.01) decrease was observed with all three high permeable devices (Tricea 150G 52.7±6.4%; F 60S 63.1±5.7%; F 500S 68.7±8.2%), whereas leptin remained stable with low-flux PS. After 12 weeks, a marked increase was observed with low-flux PS (week 1, 30.4±23.0; week 12, 40.5±5.4 ng/ml, P = 0.05), no change with super-flux CTA and high-flux PS (Tricea 150G week 1, 29.4±23.7; week 12, 32.0±27.9 ng/ml, P = ns; F 60S week 1, 36.0±31.8; week 12, 33.0±31.2 ng/ml, P = ns), and a significant decrease with super-flux PS (week 1, 38.3±33.0; week 12, 29.5±31.9 ng/ml, P = 0.02). The change in leptin after 12 weeks was significantly different between super-flux PS, and both low-flux PS (P = 0.009) and super-flux CTA (P = 0.01). Besides interleukin-6 (IL-6) at the start of the study (P = 0.006), no correlations were observed between patient characteristics, parameters of inflammation and plasma leptin levels.
Conclusions. Apart from low-flux PS, plasma leptin decreased considerably with all three high permeable dialysers after a single HD session. In the long run, leptin levels were lower with high-flux PS than with low-flux PS. Moreover, after switching from high-flux PS to super-flux PS (but not super-flux CTA), an additional decrease in leptin was observed. Apart from IL-6 at the start of the study, neither patient characteristics nor inflammatory parameters correlated with plasma leptin levels in this patient group.
Keywords: CRP; dialyser; haemodialysis; IL-6; leptin; polysulfone; super-flux
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Introduction |
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The identification of leptin (molecular weight 16 kDa), encoded by the ob gene [3], has markedly increased the understanding of the complex physiological system that regulates satiety and eating behaviour. A major action of leptin on the hypothalamus is to inhibit the production of neuropeptide Y, resulting in a decreased appetite and increased energy expenditure [4]. Recent studies have demonstrated that end-stage renal disease (ESRD) patients have inappropriately high leptin levels [5] and it has been speculated that leptin may be a uraemic toxin that mediates anorexia and weight loss, which are commonly observed in chronic HD (CHD) patients [6,7]. The pathophysiological mechanism of hyperleptinaemia in ESRD is not well understood. As the kidney eliminates a variety of polypeptide hormones, leptin might accumulate in ESRD due to reduced renal clearance. In humans, the kidney appeared to be a major site of leptin removal [5]. Moreover, an inverse correlation between leptin and glomerular filtration rate has been demonstrated in patients with various degrees of renal failure [8]. The important role of the kidney in leptin metabolism is further underscored by the fact that leptin levels are reduced after transplantation [9]. In line with the data in ESRD patients, elevated leptin levels have been described, although low values have been reported as well [10]. In this respect, various factors might play a role, including removal by splanchnic organs, gender, body fat mass and insulin levels [11,12]. Finally, it has been suggested that chronic inflammation plays an important role in the elevation of plasma leptin concentrations. Indeed, an association between pro-inflammatory cytokines and leptin synthesis in adipocytes has been demonstrated recently [13]. Therefore, it is tempting to speculate that the chronic micro-inflammatory state, commonly observed in ESRD patients, may contribute to the relatively high leptin levels in this patient group.
So far, the effect of HD on plasma leptin concentrations has only been investigated in a small number of studies, generally showing a marked intra-dialytical reduction during HD with high-flux devices [14,15]. Only one sequential analysis demonstrated a long-term benefit in eight CHD patients with exceptionally high leptin (>70 mg/l) levels, who were treated with biocompatible high-flux dialysers [16]. In the present prospective randomized crossover study, we compared the effectiveness of four types of dialysers, differing in pore size and/or membrane material, on the elimination of plasma leptin, both during a single dialysis session and in the long-term, in 10 CHD patients.
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Subjects and methods |
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Dialysis procedure and materials
The dialysis sessions lasted 35 h, depending on the individual prescription of the patient. Only first-use dialysers were used. Characteristics of the four dialysers used in this study (PS: F 6HPS, F 60S and F 500S, Fresenius, Bad Homburg, Germany; CTA: Tricea 150G, Baxter, Osaka, Japan) are depicted in Table 2.
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Analytical methods
Total leptin
Plasma samples were stored at 25°C until required for testing. Total leptin concentrations were analysed with a commercially available enzyme-linked immunosorbent (ELISA) kit (Diagnostic Systems Laboratories, Inc., TX, USA) based on anti-human leptin antibodies. The lower limit of detection was 0.05 ng/ml. The intra- and inter-assay coefficients of variation were, respectively, 4.4% at a standard concentration of 4.9 ng/ml and 4.9% at a standard concentration of 4.7 ng/ml. The reference value was <16 ng/ml.
IL-6
IL-6 was determined in K3-EDTA plasma by a sandwich-type immunoassay, according to the manufacturers procedures (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands) [17]. After centrifugation (10 min, 1500 g), the plasma samples were stored immediately at 70°C until required for testing. The lower limit of detection for IL-6 was 0.3 pg/ml. The IL-6 reference value of healthy individuals was below 10 pg/ml.
C-reactive protein (CRP)
Serum CRP concentrations were determined by nephelometry (BN II, Dade Behring B.V., Leusden, The Netherlands). The lower limit of detection of CRP was 2 mg/l. CRP reference values for healthy individuals were below 5 mg/l.
Correction for haematocrit (Ht)
Plasma leptin concentrations at t180 were corrected for changes in Ht: corrected value t180 = (Htt0/Htt180) x valuet180.
Body mass index (BMI)
The BMI was defined as the weight in kilograms divided by the square of the height in meters.
Adequacy of dialysis
Kt/Vurea was measured using a urea monitor (Biostat, Baxter, Utrecht, The Netherlands). This measurement was performed by means of a membrane bound urease and an ammonium-ion-selective electrode, which measures the ammonium generated by the reaction: urea(urease) ammonia (upper and lower detection levels urea: 1.028.5 mmol/l and precision 1.8%). Correction for the two-compartment model was performed by the formula of Daugirdas and Maduell: eKt/V = Kt/V [1 (0.06/T)] + 0.03.
Endotoxin assay
Dialysate samples for the determination of lipopolysaccharide (LPS) concentrations were collected at t180 in pyrogen-sterile FALCON 2063 polypropylene tubes (Becton Dickinson, Franklin Lakes, USA). LPS activity in dialysate was quantified by a kinetic chromogenic method based on the LAL-assay (Bio Whittaker, Wakersville, USA). Standard series of purified Escherichia coli 055:B5 LPS were made in the range 0.00550.0 EU/ml. Inhibition and interference testing was performed on each sample by an endotoxin spike. To overcome inhibition/enhancement, all dialysate samples were diluted 10 times. All determinations were performed in duplicate and recoveries of spikes between 50 and 150% were accepted. Limit of determination was 0.05 EU/ml.
Statistical analysis
Data are expressed as mean ± SD, or median and range when appropriate. Analysis was performed with the Statistical Package for Social Sciences/PC+ software system using paired T-tests. Correlation coefficients were calculated with the Pearson method. Differences were considered statistically significant at P<0.05.
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Results |
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LPS content of dialysate
During the study period, mean LPS concentration in unfiltered dialysate was 0.051±0.005 EU/ml [18].
IL-6
At baseline, mean values were within the reference range. IL-6 levels remained stable after 12 weeks of HD with all four modalities (F 6HPS week 1, 2.4±0.9 pg/ml and week 12, 2.5±1.1 pg/ml; F 60S week 1, 2.6±0.9 pg/ml and week 12, 2.6±1.0 pg/ml; F 500S week 1, 2.5±1.3 pg/ml and week 12, 2.3±1.2 pg/ml; Tricea 150G week 1, 2.8±1.4 pg/ml and week 12, 3.0±1.3 pg/ml).
CRP
Baseline CRP levels were slightly elevated when compared with the reference value of healthy individuals (<5 mg/l). Significant differences were not observed between the four modalities at the start of the study (F 6HPS: 5.3±4.3 mg/l; F 60S: 6.3±3.8 mg/l; F 500S: 6.4±6.2 mg/l; Tricea 150G: 5.9±5.8 mg/l; mean 6.0±4.8 mg/l). Marked fluctuations were not observed after 12 weeks of HD.
Relationship between patient-related characteristics, inflammation and plasma leptin concentrations
Significant correlations were not found between age, sex, BMI, time on dialysis, residual kidney function, dialysis dose (Kt/V) and plasma leptin concentrations. At the start of the study, a strong correlation was observed between IL-6 and leptin levels (r = 0.59, P = 0.006), whereas CRP and leptin concentrations were not significantly correlated. As mentioned before, neither IL-6 nor CRP changed over time in any of the four modalities. In contrast, during HD with super-flux PS plasma leptin concentrations decreased significantly. Consequently, any correlation between the change in plasma leptin and fluctuations in the two inflammatory parameters was absent (data not shown).
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Discussion |
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Recent findings suggest that leptin levels are influenced by inflammation. In animal studies elevated leptin levels appeared to be responsible for the anorexia that was observed during inflammatory conditions [20]. Data regarding the effect of inflammation on plasma leptin concentrations in humans, however, are not unanimous [8,13]. In healthy controls, the administration of cytokines, such as tumour necrosis factor (TNF
) and IL-1
, led to an increase in plasma leptin levels [21]. In ESRD patients, both negative [22] and positive [6] correlations with inflammation have been described.
From a prior long-term sequential analysis on the influence of low-flux cellulose, low-flux PS and high-flux PS on plasma leptin levels in eight CHD patients with exceptional high leptin (>70 mg/l) levels [16], it appeared that high-flux PS reduced plasma leptin levels significantly over a period of 8 weeks, when compared with the two low-flux devices. Of note, in the latter analysis no correlations were found between TNF and leptin concentrations, despite lower TNF
levels in both PS groups. In the present study a highly significant correlation was found between plasma leptin concentrations and IL-6, but remarkably, only at the start. As mentioned, leptin levels decreased after 12 weeks of HD with super-flux devices, whereas parameters of inflammation did not. In a concurrent study [23], intercurrent clinical events were predictive of changes in both plasma IL-6 and CRP, whereas neither the bacteriological quality of the dialysate nor the type of dialyser had any influence on these parameters. Hence, our combined data suggest that in a group of patients with only mild inflammation (mean CRP: 6.0±4.8 mg/l) baseline leptin levels can be modified by HD, depending on the flux characteristics of the dialysers used. Whether HD with high permeable dialysers can lower leptin values in more serious inflammatory states, warrants further investigation.
With respect to the present study, at baseline none of the patients had plasma leptin concentrations within the reference range, in contrast to 60% of the patients after 12 weeks of HD with super-flux PS. As elevated leptin concentrations have been associated with a low protein intake and loss of lean body mass in peritoneal dialysis [6] and HD patients [7], and positive correlations have been described in HD patients between leptin levels and markers of malnutrition, including hypo-albuminaemia [2] and the protein catabolic rate, our findings may have important consequences for clinical practice.
To summarize, with the exception of low-flux PS, plasma leptin was effectively reduced during a single HD session with high-flux PS, super-flux PS and super-flux CTA dialysers, suggesting enhanced convective removal. However, marked differences were observed in the long-term, super-flux PS being more effective than both high-flux PS and super-flux CTA, whereas an increase was observed with low-flux PD. Whether a decline in plasma leptin concentrations results in an improvement of the nutritional state and a reduced cardiovascular risk in CHD patients deserves further study.
Conflict of interest statement. None declared.
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References |
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