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Protein carbonylation is an irreversible and not reparable reaction which is caused by the introduction into proteins of carbonyl derivatives such as ketones and aldehydes, generated from direct oxidation processes or from secondary protein reaction with reactive carbonyl compounds. Several studies have demonstrated significantly increased levels of reactive carbonyl compounds, a general increase in plasma protein carbonyls and carbonyl formation on major plasma proteins in blood from uremic patients, particularly those undergoing chronic haemodialysis.
In the present preliminary study, we first assessed by an in vitro filtration apparatus the possible effects of different materials used for haemodialysis membranes on protein retention and carbonylation. We employed hollow fiber minidialyzers of identical structural characteristics composed of either polymethylmethacrylate, ethylenevinyl alcohol, or cellulose diacetate materials. Protein Western Blot and SDS-PAGE coupled to mass spectrometry analysis were applied to highlight the carbonylated protein-binding characteristics of the different materials. We also investigated in vivo protein carbonylation and carboxy methyl lisine-modification in plasma obtained before and after a haemodialysis session.
Our data underline a different capability on protein adsorption associated with the different properties of the filter materials, highlighting the central buffering and protective role of serum albumin. In particular, polymethylmethacrylate and cellulose diacetate showed, in vitro, the highest capacity of binding plasma proteins on the surface of the hollow fiber minidialyzers.
The present study suggests that biomaterials used for fabrication of haemodialysis membrane may affect the carbonyl balance in chronic uremic patients.
Protein carbonylation is an irreversible and not reparable reaction which is caused by the introduction into proteins of carbonyl derivatives such as ketones and aldehydes, generated from direct oxidation processes or from secondary protein reaction with reactive carbonyl compounds, eventually forming advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs)1. Loss of protein function usually stem from the carbonylation process and may lead to cellular dysfunction and tissue damage2. Carbonyl stress-modified proteins can also exhibit several biological activities initiating a range of inflammatory responses (rev. in 3). Coupled with increased levels of protein carbonyls observed in several human pathological conditions, these findings suggest “carbonyl stress” (carbonyl overload) a potential causative role in disease onset/progression2,4.
The uremic syndrome is related to a complex set of biochemical and pathophysiological disturbances, resulting in a state of malaise and dysfunction. In uremic patients, particularly those undergoing maintenance haemodialysis, proteins can be subjected to a variety of modifications including carbonylation5. Several studies have demonstrated significantly increased levels of reactive carbonyl compounds, a general increase in plasma protein carbonyls, and carbonyl formation on major plasma proteins in blood from uremics6–10. Thus, uremia is considered to be a state of carbonyl stress with potentially damaging proteins.
While it appears that carbonyl stress in haemodialysis patients may result from uremia per se10,11, haemodialysis therapy, and in particular the dialyzer membrane, may have a relevant impact on carbonyl balance. During haemodialysis procedure, biochemical reactivity following the contact of blood with the membrane and the loss of antioxidant substances, may in fact promote carbonyl formation via an increase of oxidative stress. On the other hand, membranes for haemodialysis therapy might lessen the carbonyl overload by removing low molecular weight reactive carbonyl compounds.
Furthermore, membrane contained in the haemodialyzer might remove carbonylated proteins. This would occur mainly via the protein adsorptive properties of the membrane biomaterial, since diffusion and convection, the principal mechanisms for solute removal during dialysis, have a poor ability to remove high molecular weight solutes. Protein deposition and adsorption occurs almost immediately when blood is exposed to artificial membrane surfaces. Detailed analysis of protein adsorption onto dialysis membrane materials, however, has been generally limited by the absence of adequate protein separation and identification techniques.
Proteomic methods have been developed to investigate a large number of proteins simultaneously12, and allow separation and identification of single proteins from a complex mixture without requiring assumptions as to which protein may be present13. We have recently shown that proteomic techniques are a promising approach for the investigation of proteins surface-adsorbed onto haemodialysis membrane14. In the present preliminary study, we assessed by proteomic and immunochemical investigation the carbonylated protein-binding characteristics of different materials used for haemodialysis membranes (cellulose diacetate, ethylenevinyl alcohol, and polymethylmethacrylate), an issue not yet investigated.
We pursued further investigation to detect carbonylation levels and carboxy methyl lysine (CML) modification of proteins in uremic plasma. Such analyses were carried out on blood obtained before and after the haemodialytic session carried out with ethylenevinyl alcohol (EVAL) and cellulose diacetate (CDA) membranes; during the study period there was no patient treated with polymethylmethacrylate (PMMA) membrane.
The commercially available dialysis membranes tested included CDA (Hospal, Italy), EVAL (Asahikasei Kuraray Medical Co., Japan), and PMMA (Toray, Japan). The hollow fiber minidialyzers (kindly provided by Kuraray Analytical Technology Centre, Japan) used in the experimental model were developed as a small scale model of a standard hollow fiber dialyzer. Their structural characteristics were identical in terms of effective length (80 mm), number of fibers (300), effective surface area (130 cm2), and sterilization (g-ray).
Human blood from healthy donors (n=3) was allowed to flow into the minidialyzer by a peristaltic pump (flow rate 1 mL/min) in a closed loop system, under meticulous experimental technique (details are provided in ref. # 14). After 30 minutes blood recirculation, proteins adsorbed onto the minidialyzer were eluted by a strong chaotropic solubilization buffer (6 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 65 mM dithiotheitol) and after appropriate steps, quantified by the bicinchoninic acid assay, according to the protocol supplied by the vendor (BCA, SIGMA). Proteins adsorbed onto the minidialyzer were then derivatized of carbonyl groups by 2,4-dinitrophenyldrazine (DNPH) 10 mM in Cloridric Acid (HCl) 2N, in order to get carbonylated proteins, which were precipitated over night with an ice-cold solution containing 50% ethanol, 25% methanol, and 25% acetone at −20 °C, and washed twice with the same ice-cold solution. The final pellet was dried at room temperature and redissolved in Laemli sample buffer. Proteins were separated by SDS-PAGE in triplicate gels and then stained with silver nitrate without glutaraldehyde15 (protein stain), or used for western blot analysis to evaluate carbonylated proteins adsorbed onto each minidialyzer.
After SDS-PAGE separation, proteins were electroblotted onto nitrocellulose membranes, according to Towbin16. Protein transfer was carried out in an Amersham Bioscence transblot semidry transfer cell at 0.8 mA/cm2 constant current for 2 hours. Immunodetection was performed using anti-2,4-dinitrophenyl-keyhole limpet haemocyanin (KLH) rabbit immunoglobulin G (Invitrogen, Molecular Probes; dilution 1:1,000). The immunoreactive bands were detected using goat peroxidase-conjugated antirabbit immunoglobulin G (Santa Cruz Biotechnology; dilution 1:20,000) and a chemiluminescence detection method (Santa Cruz Biotechnology).
For mass spectrometry (MS) analysis, protein bands were excised from the gel, in-gel reduced, thiol-alkylated, and digested with sequence grade porcine trypsin (SIGMA)17 in 50 mM ammonium bicarbonate (SIGMA) at 37 °C for 16–18 h; the reaction was stopped by addition of 0.1% trifluoroacetic acid (TFA) (Fluka). Carbonyl proteins were then identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)/MS mass fingerprinting14. A microcrystalline matrix surface was made by spotting a saturated solution of α-cyano-4-hydroxycinnamic acid in ethanol (SIGMA) onto ground steel MALDI plate. Tryptic peptides were extracted by ZipTip C18 (Millipore) reverse phase material and directly eluted and crystallised in an acetonitrile/water 1:1 (v/v) saturated solution of α-cyano-4- hydroxycinnamic acid, onto the first matrix surface. The solvent was allowed to evaporate at room temperature.
MALDI mass spectra were recorded in the positive ion mode with delayed extraction on a Reflex IV time-of-flight instrument equipped with an MTP multiprobe inlet and a 337 nm nitrogen laser. A 50 pmol/μL standard peptide mix solution of Angiotensin I (1296.68 Da), Adrenocorticotropic hormone (ACTH) 18–39 (2465.19 Da), Bradikynin (1060.57 Da), [Glu1]-Fibrino peptide B (1570.68 Da) and Renin Substrate (1758.93 Da) was used for external calibration. Internal spectrum calibration was performed by a three-point linear fit using the autolysis products of trypsin at m/z 842.50, m/z 1045.56 and m/z 2211.10. A database search with the mono-isotopic peptide masses was performed against the National Center for Biotechnology Information (NCBI) non-redundant database using the peptide search algorithm MASCOT (Matrix Science, http://www.matrixscience.com). The parameters employed in the database search were: mass tolerance of 100 ppm, single miss cleavage site per peptide fragment, carboamidomethyl modification of cysteine residues and optional presence of methionine oxidation. Masses corresponding to keratin tryptic fragments or evaluated as environmental contaminants by specific blank controls were excluded.
Finally, plasma protein targets of carbonylation and presence of plasma CML-modified proteins were evaluated in blood from uremic patients (n=2) pre and post a haemodialytic session carried out with EVAL or CDA membranes, by proteomic and immunochemical investigations previously described. Immunodetection was performed using anti-CML rabbit (Novus Biologicals; dilution 1:5000). The immunoreactive bands were detected using goat peroxidase-conjugated antirabbit immunoglobulin G (Santa Cruz Biotechnology; dilution 1:20000) and a chemiluminescence detection method (Santa Cruz Biotechnology).
Whole blood samples from uremic patients were collected in K3EDTA vials (3 mL) and centrifuged at 4000 rpm for 20 minutes at 4 °C. Plasma samples were stored at −80 °C until analyses.
Data for quantitative adsorption of proteins onto membrane contained in minidialyzers are reported as mean ± standard deviations. Data were analyzed by one-way analysis of variance (ANOVA) test, followed by Least Significance Difference (LSD) pairwise comparison. A p value <0.05 was considered as statistically significant.
Results for adsorption of whole blood proteins from healthy subjects onto PMMA, EVAL, and CDA membranes contained in minidialyzers are shown in Figure 1, panel A. Total protein adsorption was significantly higher for PMMA and CDA (both p-value <0.01) as compared to EVAL membrane.
Monodimensional SDS-PAGE and DNPH-immunoblot analysis of eluted proteins are shown in Figure 1, panel B. Both silver stained gel and western blot visibly showed the lower contents of adsorbed and carbonylated proteins onto EVAL vs. PMMA and CDA membranes, according to quantification assay.
After derivatization with 2,4-DNPH of adsorbed proteins, all membranes displayed the ability to adsorb and carbonylate proteins, albumin being the predominant type. Particularly with PMMA and CDA, carbonylated proteins other than albumin were also detected in the eluate obtained after experimental in vitro dialysis (Figure 1, panel B). Proteins identified by MALDI-TOF mass fingerprinting (Table I) were α2-macroglobulin, transferrin, albumin, chain A α1-antitrypsin, immunoglobulin G1, fibrinogen gamma and proapolipoprotein.
We also examined plasma obtained from chronic haemodialysis patients for evidence of protein carbonylation. Figure 2, panel A shows SDS-PAGE and western blot experiments of total plasma proteins before and after in vivo haemodialytic treatment with EVAL or with CDA membranes. The immunoblot shows that plasma DNPH reactive carbonyl derivates are apparently increased after the haemodialysis session. Identification by MS analysis (Table II, A) confirmed in vivo the presence of the same carbonylated proteins previously identified after the in vitro experiments.
In Figure 2, panel B, CML immunoblot shows that in both pre- and post- haemodialysis blood samples, albumin is the major plasma protein target of oxidative stress in chronic haemodialysis patients (Table II, B).
Uremia has been described as a state of “carbonyl stress” resulting from either increased oxidation of carbohydrates and ligands (oxidative stress) or inadequate detoxification or inactivation of reactive carbonyl compounds derived from both carbohydrates and ligands by oxidative and non oxidative chemistry3.
The present study shows that biomaterials used for fabrication of haemodialysis membrane may have the capacity in vitro to adsorb proteins. Then we evaluated the possible effects on protein carbonylation using PMMA, EVAL, and CDA membranes. Statistically significant differences concerning the capability for protein adsorption onto minidialyzer membranes were found (Figure 1, A), though the findings should be somewhat cautiously interpreted because of the small sample size. The pattern of eluted carbonylated protein, however, showed some differences between the 3 tested membranes. For EVAL membrane, MS analysis of protein eluate demonstrated mostly albumin, which is one of the main target of carbonylation in uremic patients11. With the use of PMMA and CDA materials, other adsorbed proteins were also identified: transferrin, immunoglobulin G1, fibrinogen gamma, which beside albumin are the major plasma proteins target of carbonyl modification, α2-macroglobulin, chain A α 1-antitrypsin, and proapolipoprotein.
The in vivo analysis on plasma from uremic patients before and after dialytic treatment with EVAL and CDA membrane confirmed the identification of the same carbonylated proteins. The DNPH immunoblot analysis shows that carbonylated protein levels after haemodialysis session are apparently higher than before. Moreover, our in vivo results showing the presence in uremic blood of CML-modified proteins are in keeping with previous observations that albumin is the major plasma protein target of oxidative stress in chronic haemodialysis patients11.
Protein adsorption onto artificial membrane surfaces depends to a large extent on membrane surface characteristics such as hydrophilicity, roughness, charge, and chemistry18. The lower the hydrophilicity, the greater the adsorption of proteins during use19. PMMA is a hydrophobic membrane, whereas EVAL is more hydrophilic than CDA14. These different structural characteristics may at least partly explain the different adsorptive behaviour for carbonylated plasma proteins of tested biomaterials. This is also supported by the different quantitative ability of the 3 membranes to adsorb proteins from healthy human blood. In this regard, it should be born in mind that highly adsorptive capacity may have untoward effects, for example by limiting the diffusive and convective capacity of the membrane and hence its therapeutic usefulness. Thus, a moderated degree of protein adsorption coupled with the ability to bind carbonylated proteins appears to be advisable for a safe membrane.
Carbonyl stress has been proposed as a new uremic toxicity, which may contribute to long-term complications associated with chronic renal failure and haemodialysis such as dialysis-related amyloidosis and accelerated atherosclerosis1,20. Our study suggests that haemodialytic membranes may affect the carbonyl balance in chronic uremic patients, though the study design does not allow us to draw any definitive conclusion on the issue.
At present, however, these assumptions are only speculative and require further investigation in vivo with the aim to verify that adsorption onto the haemodialysis membrane surface might represent a mechanism to antagonize protein carbonylation in uremia.