1 Physiologische Chemie l, Biozentrum, Am Hubland, Würzburg, 2 Nephrologisches Zentrum Niedersachsen, Hannoversch Münden, 3 Kuratorium für Dialyse und Nierentransplantation, Würzburg and 4 Fresenius Medical Care, BioSciences Department, Bad Homburg, Germany
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Abstract |
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Methods. The removal of AGEs by high-flux haemodialysis (HD) using standard and ultrapure dialysis fluid (SDF and UDF), by haemodiafiltration (HDF) and by haemofiltration (HF) was studied by fluorescence spectroscopy and by a carboxymethyllysine (CML)-specific ELISA. In addition, molecular weight distribution of fluorescent AGE products in serum of several patients was analysed by gel filtration.
Results. The highest AGE-typical fluorescence was found in the serum of patients on HD using SDF (114 667±18 967 arbitrary units (AU)), followed by patients on HDF (86 912±24 411 AU, P<0.005), by patients on HD using UDF (74 953±21152 AU, P<0.0001) and by patients on HF (74 039±17 027 AU, P<0.0001). Similar results were found for serum CML levels with the highest values in HD patients on SDF (1609±504 ng/ml), followed by patients on HF (1354±614 ng/ml, P<0.001), then by HD patients on UDF (1310±403 ng/ml, P<0.001) and by patients on HDF (1132±338 ng/ml, P<0.001). The removal rate of AGEs, as evaluated by the determination of the pre-/post-dialysis AGE differences, was comparable across all groups.
Conclusion. These findings suggest that factors other than removal are responsible for the lower pre-dialysis AGE levels found in patients on convective dialysis as well as on HD with UDF. A role of water quality is assumed. This is corroborated by the finding that the high molecular weight AGE-fraction is preferentially lowered in comparison with patients on HD with SDF, as analysed by gel filtration chromatography. These findings could be best explained by a less severe oxidative stress (i.e. resulting in decreased AGE generation) with HF and HDF, as well as with ultrapure HD.
Keywords: advanced glycation end products; end-stage renal disease; haemodiafiltration; haemodialysis; haemofiltration
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Introduction |
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Patients with end-stage renal disease (ESRD) have very high AGE levels [57]. The main factors involved are increased AGE formation by enhanced carbonyl and oxidative stress, and impaired AGE removal by the damaged kidney [8]. In ESRD, dialysis therapy may contribute to the removal of AGEs, particularly AGE peptides [6]. It is therefore of interest to investigate the influence of different modalities of renal replacement therapies. In this study, the removal of AGEs by high-flux haemodialysis (HD) using standard dialysis fluid (SDF) and ultrapure dialysis fluid (UDF), by haemodiafiltration (HDF) and by haemofiltration (HF) was examined by fluorescence spectroscopy and by an enzyme-linked immunosorbent assay (ELISA); in addition, the molecular weight distribution of fluorescent AGE-products in the serum of several patients was analysed by gel filtration chromatography.
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Patients and methods |
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Maintenance HD was performed as follows: blood flow 250300 ml/min, dialysate flow rate 500 ml/min. Dialysers were high-flux polysulfone dialysers (F 60S or HF 80S; Fresenius Medical Care, Bad Homburg, Germany). Standard dialysis fluid was prepared from purified water obtained from reverse osmosis and mixed with the acid electrolyte concentrate (SKF213 with a potassium content of 2.0 mVal/l or SKF313 with a potassium content of 3.0 mVal/l; Fresenius Medical Care) and the liquid bicarbonate solution obtained from NaCO3 powder (Bilog; Fresenius Medical Care).
Microbial contamination of SDF was determined monthly in the dialysate at temperatures of 22°C and 36°C for a prolonged incubation time of 7 days. All bacterial counts were in the range of 070 colony-forming units (CFU)/ml and thus met the standards of the Association for the Advancement of Medical Instrumentation (200 CFU/ml in water, 2000 CFU/ml in dialysate). The endotoxin level in the dialysate was measured by the limulus amoebocyte lysate (LAL) assay. Our data was markedly less than the limit of 0.25 endotoxin units (EU)/ml.
UDF was achieved by placing an additional ultrafilter (polysulfone, 0.1 µm) close to the dialyser. UDF is characterized by a bacterial contamination of <0.1 CFU/ml and unmeasurable endotoxin levels (<0.03 EU/ml) [9], which is in line with our data.
Post-dilution haemodiafiltration (HDF) was performed using AK 200 machines (Gambro, Hechingen) as well as a specially equipped Fresenius 4008 machine. Haemodiafilters were HF60 and HF80, depending on the body surface. Ultrapure water was used both for the dialysate and for online production of the highly purified substitution fluid. Blood flow averaged 300350 ml/min, total dialysate flow 650 ml/min, dialysate flow 500550 ml/min, infusate flow 100 ml/min. Total filtrate volume was aimed at one-third of the body weight, corresponding to 2025 l per session.
Online HDF (post-dilutional method) was performed by use of AK 200 ULTRA machines (Gambro), as well as Online PLUS 4008 machines from Fresenius. Haemodiafilters were HF60 and HF80, depending on the body surface. Ultrapure water was used both for the dialysate and for online production of the highly purified substitution fluid. Blood flow averaged 300350 ml/min, total dialysate flow 650 ml/min, dialysate flow 500550 ml/min, infusate flow 100 ml/min. Total filtrate volume was aimed at one-third of the body weight, corresponding to 2327 l per session.
In a similar manner, post-dilutional haemofiltration (HF) was performed using AK 200 ULTRA machines, and HF60 and HF80 haemofilters. Total fluid exchange was identical to that of HDF.
Blood sampling
After receiving informed consent, blood samples of the patients were collected at midweek and endweek sessions before (prior to heparin administration) and after the therapy session (<5 min). Samples were collected into Sarstedt monovettes, centrifuged for 10 min and the serum stored at -20°C before determination.
Fluorescence spectroscopy
The fluorescence measurement of the 50-fold diluted serum samples (corrected for background) was performed in triplicate on a FluoroMax spectrometer (Spex Instruments, Edison, NJ, USA) at a wavelength of 440 nm (excitation 370 nm) as described previously [10]. Haemolytic sera were excluded. No significant shift of the fluorescence maximum was observed after dialysis.
Measuring the content of N-(carboxymethyl)lysine (CML) by competitive ELISA
Serum CML concentrations were measured in triplicate by a competitive ELISA developed by ROCHE Diagnostics (Penzberg, Germany) using the anti-CML monoclonal antibody 4G9 (Alteon Inc., New York, NY, USA) as described previously [11]. Briefly, Proteinase-K-digested serum (to make hidden epitopes accessible) was incubated on AGEBSA-coated, BSA-blocked microtitre plates in the presence of peroxidase-conjugated anti-CML monoclonal antibody for 1 h. After three washing steps, colour reaction was induced with 2,2'-azino-di-3-ethylbenzthiazoline-sulfonic acid (Roche Diagnostics) and 0.01% H2O2 in 0.01% glycine/citrate buffer and absorbance was read in a microtitre ELISA plate reader (Multiskan Ascent; Labsystems, Helsinki, Finland) at 405 nm. All steps were carried out at room temperature. N-(carboxymethyl-)amino-caproic acid (Alteon Inc.) served as standard.
Gel filtration chromatography
Molecular weight distribution of serum AGEs was determined by gel filtration chromatography. The separation was performed by fast protein liquid chromatography (Biologic System BioRad; BioRad, Hercules, CA, USA) on a Superdex 75 HR 10/30 column (Pharmacia, Freiburg, Germany) equilibrated with PBS, the flow rate being 1 ml/min. Fluorescence was measured at an emission wavelength of 440 nm (excitation 370 nm) with a Merck-Hitachi F-1080 fluorospectrometer (Hitachi, Tokyo, Japan).
Statistics
Results are expressed as mean±SD. Comparison between different groups was performed by one-way analysis of variance (ANOVA) using the SPSS program package (SPSS Inc., Chicago, IL, USA). Statistical significance was defined at P<0.05.
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Results |
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AGE levels in serum measured by competitive ELISA
In accordance with the fluorescence measurements, the ELISA of CML showed a significant difference between dialysis patients and controls (P<0.0001) for all three renal replacement therapies (Figure 1). Also, significantly lower CML levels were observed for patients on HDF and HF (P<0.001) when compared with HD patients with SDF. CML levels of patients on HD with UDF were found to be intermediate, being significantly lower than levels of patients on HD with SDF, but having slightly higher levels of CML compared with HDF (P>0.1, not significant) (Table 2
). The differences between HD with UDF and HF as well as HDF vs HF were not significant. Serum CML values of HF patients were slightly higher (1354±614 ng/ml) than those of HDF patients (1132±338 ng/ml) (P<0.1). The average reduction of CML within one dialysis session was 28% (P<0.001) for HD with SDF, 28% (P<0.001) for HD with UDF, 32% (P<0.001) for HDF and 31% (P<0.001) for HF. There was no significant difference between the different dialysis modalities with respect to CML reduction (Table 3
).
Gel filtration chromatography
Molecular weight distribution of serum AGEs was determined by gel filtration chromatography on a Superdex column (separation range 370 kDa). Serum samples of five patients of each subgroup (SDF HD, UDF HD, HDF, HF) were analysed and compared with controls (Figure 2). While differences in serum fluorescence between HD patients and controls are due to considerably higher levels of both the high- (MW >12 kDa) and low-molecular weight (MW <12 kDa) fluorescent AGEs, the dissimilarity in overall fluorescent AGE concentrations of the patients on different dialysis modalities appears to be mainly due to a lower AGE modification of proteins, in particular albumin, as judged from the quantification of the gel filtration chromatograms (Table 4
).
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Discussion |
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An important factor to be discussed as a possible promotor of oxidative stress is the quality of dialysis fluid. Water containing pyrogens may stimulate pro-inflammatory processes, as seen by increased levels of pro-inflammatory cytokines and acute phase proteins [9]. Even a low-grade bacterial contamination of the dialysate (<200 CFU/ml) may exert toxic effects with increased release of radicals and cytokine formation from the mononuclear cells (the so-called cytokine-inducing substances) [9]. Naturally, for haemofiltration and haemodiafiltration, only sterile and double-filtered pyrogen-free substitution fluid is used. Apart from convective transport, another important difference between haemodiafiltration and haemodialysis with SDF is the use of ultrapure dialysis fluid as well as highly purified sterile infusions. To test the role of water quality, AGE levels of haemodialysis patients using standard or ultrapure dialysis fluids were compared. The results suggest an influence of water quality on AGE levels, although the bacterial counts in the HD patients on standard dialysis met with criteria of the Association for the Advancement of Medical Instrumentation (Figures 1 and 2
). Also, the LAL test was within normal limits.
According to our studies, we assume that in the case of AGE-typical fluorescence, water purity of the dialysate and/or of the infusion fluid may account at least partially for the observed reduction in pre-dialysis AGE levels of patients on haemofiltration or haemodiafiltration. Our results indicate that, besides many other potential factors (e.g. residual renal function, membrane properties, oxygen radical formation and nutritional factors), water quality appears to have an impact on serum AGE levels of dialysis patients as well.
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Notes |
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Correspondence and offprint requests to: Reinhard Schinzel, Physiologische Chemie I, Theodor-Boveri-Institut, University of Würzburg, Josef-Schneider Strasse 2, D-97080 Würzburg, Germany. Email: schinzel{at}biozentrum.uni-wuerzburg.de
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References |
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