Fifty-four stable HD patients treated at the Hemodialysis Service of our hospital (Ospedale Policlinico, Verona, Italy) during the period June 2008–July 2009 were included in the study. Patients were on maintenance with intravenous erythropoiesis-stimulating agents (ESAs) epoetin or darbepoetin and chronically treated with intravenous iron, which was stopped 10 weeks before the study. All subjects were dialyzed against standard, not ultrapure, dialysate. Different protocols were used. Thirty-nine patients were on bicarbonate HD (BHD), 23 with low-flux synthetic (11 polysulfone, Fresenius, Germany and 12 polyamide, Gambro, Sweden), and 16 with high-flux membranes (6 cellulose triacetate, Nipro, Japan, 5 each polysulfone, Fresenius, Germany, and polymethylmethacrylate, Toray, Japan). Eleven patients were on acetate-free biofiltration (AFB), a low-volume hemodiafiltration technique based on buffer-free dialysate, a biocompatible high-flux AN69 membrane, and sterile hypertonic bicarbonate infusion in post-dilution mode (Hospal, France) [20
]. Finally 4 patients were on HFR (double-chamber hemodiafiltration with reinfusion of regenerate ultrafiltrate), a technique that utilizes convection, diffusion, and adsorption, using a 0.7
high permeability polyphenylene membrane as a convective dialyzer, a 1.70
low-flux polyphenylene membrane as a diffusive dialyzer, and a regenerating adsorbent cartridge containing undissolvable macroporous-structured styrenic resin as adsorbent material (Bellco, Italy) [21
]. Dialyzers were not reused. In all patients the length of the dialysis session was set at 240 minutes, and the dialysate flow rate at 500
mL/min, the dialysis blood pump flow rate at 300
mL/min, and in the patients on AFB, the 164
mM bicarbonate reinfusion fluid rate was at 2
Blood samples for laboratory testing were obtained prior to the first-of-the-week hemodialysis sessions. Blood samples were collected for detection of C-reactive protein (CRP), ferritin, and interleukin-6 (IL-6) before the dialysis session. Serum ferritin was measured by routine laboratory methods, IL-6 by enzyme-linked immunoadsorbent assay (by Human IL-6 ELISA BMS213/2CE Bender MedSystems GmbH, Vienna, Austria), and CRP by commercially available automated PENIA assays (Dade Behring, Germany).
The effect of the dialysis session on serum hepcidin-25 and hepcidin-20 was evaluated in 39 patients (14 on low-flux BHD, 12 on high-flux BHD, 9 on AFB, and 4 on HFR) by measuring hepcidins also at the end of the dialysis sessions and by calculating the reduction ratio (RR) as follows: RR = (Cpre − Cpost/Cpre) × 100, where Cpre is the concentration at the start of dialysis and Cpost the concentration at the end of dialysis. Cpost was corrected for hemoconcentration due to ultrafiltration by multiplying the uncorrected Cpost for a correction factor computed as the ratio predialysis Hb/postdialysis Hb.
Fifty-seven controls were enrolled among healthy volunteers participating in a phase II trial at the Centre for Clinical Research of the Azienda Ospedaliera-Universitaria di Verona, as described previously in detail elsewhere [12
]. Briefly, at enrollment they completed a questionnaire with specific items relevant to iron metabolism (i.e., any history of blood donations, previous pregnancy, menstrual losses, etc.) and were evaluated by laboratory studies including ferritin, CRP, liver function tests, and creatinine. To be considered as appropriate “normal controls” for the serum hepcidin assay, all these parameters were required to be normal.
The main clinical features of the subjects included in the study are shown in .
Clinical features and baseline laboratory data.
Each patient gave written informed consent. The study was conducted according to the principles contained in the Declaration of Helsinki. The protocol was approved by the Institutional Review Board of the Azienda Ospedaliera-Universitaria di Verona.
2.1. Serum Hepcidin-25 and Hepcidin-20 Assay
We used a protocol based on PBSCIIc mass spectrometer and copper-loaded immobilized metal-affinity capture ProteinChip arrays (IMAC30-Cu2+
L of serum were applied to an IMAC30- Cu2+
surface that binds hepcidin based on its affinity to Cu2+
ions. The binding surface was equilibrated and washed with appropriate buffers according to the manufacturs's instrunctions (Bio-rad, Hercules, CA). Subsequent work-up, SELDI-TOF MS instrumental settings, read-out, and data analysis are described elsewhere [12
], with the addition that protein chip handling was performed in a nitrogen atmosphere to prevent methionine oxidation [9
Briefly, we used synthetic hepcidin-25 (Peptides International, Louisville, KY) for external calibration and a synthetic hepcidin analogue (Hepdicin-24, Peptides International, Louisville, KY) as an internal standard [9
]. Spectra were collected in duplicate for each sample, with or without the internal standard spiked in at a concentration of 10
nM. Concentrations of both serum hepcidin-25 and hepcidin-20 were expressed as nM and were the results of the following equations.
- Hepcidin 25 concentration: (sample 2789m/z peak intensity) × 10nM/(hepc24 spiked sample 2673m/z peak intensity—nonspiked sample 2673m/z peak intensity).
- Hepcidin 20 concentration: (sample 2192m/z peak intensity) × 10nM/(hepc24 spiked sample 2673m/z peak intensity—nonspiked sample 2673m/z peak intensity).
Standard curves of the internal standard were constructed by serial dilutions of hepcidin-24 (0–20
nM) in tubes with blank serum to an end volume of 500μ
L. These were immediately applied to IMAC-Cu2+
Chips, and processed according to protocol and measured by MS. Linear standard curves were obtained for hepcidin-24 blank serum (y
+ 3.97; R2
= 0.994). Based on the measured background noise in each MS spectrum for serum samples, the lower limit of detection (LLOD) ranged from 0.55 to 1.55
nM. Since this method is based on the level of hepcidin-25 peak intensity relative to that of hepcidin-24, we determined hepcidin-24/hepcidin-25 intensity ratios in blank serum samples spiked with both synthetic compounds in duplicate of 8 different concentrations. As compared to our original description of the method [
, recent technical improvements allowed us to increase the mean peak ratios hepcidin-24/hepcidin-25 from 0.71 to 0.93.
Protocols for identification of peaks by immunological and mass spectrometry approaches have been described in detail elsewhere [22
]. Hepcidin-25 and hepcidin-20 concentrations of 0.55
nM were arbitrarily assigned to samples with undetectable serum levels of hepcidin isoforms. The intra- and inter-assay coefficient of variations of this method ranged from 6.1 to 7.3 percent, and from 5.7 to 11.7 (mean 7.7) percent, respectively, [9
2.2. Statistical Analysis
All calculations were performed using the SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). As some of the continuous variables of interest, including serum hepcidin and ferritin, showed a non-Gaussian distribution, their values were log-transformed and expressed as geometric means with 95% Confidence Intervals (CIs). For other variables, results were expressed also as means ± SD unless otherwise indicated. Correlations between quantitative variables were calculated by Pearson r test. Results were considered significant when P was <.05 (two tailed).