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NDT Advance Access published online on April 8, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn127
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Protein adsorption during LDL-apheresis: proteomic analysis

Hassan Dihazi1,*, Michael J. Koziolek1,*, Tanja Söllner1, Elke Kahler2, Reinhard Klingel3, Rieke Neuhoff1, Frank Strutz1 and Gerhard A. Mueller1

1 Department of Nephrology and Rheumatology 2 Department of Medical Statistics, Georg-August-University Goettingen 3 Apheresis Research Institute, Cologne, Germany

Correspondence and offprint requests to: M. Koziolek, Department of Nephrology and Rheumatology, Georg-August University Göttingen, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany. Tel: +049-551-396331; Fax: +049-551-398906; E-mail: mkoziolek{at}med.uni-goettingen.de



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. The aim of our study was to investigate the clearance of functional proteins by different low-density lipoprotein-apheresis (LDL-A) methods with the help of proteomic analyses.

Methods. Proteins were eluated from the different LDL-A columns and investigated with 2D electrophoresis combined with mass spectrometry methods. In parallel, we quantified the plasma protein loss from patients treated with double-filtration plasmapheresis (DFPP; n = 9), direct adsorption of lipoproteins (DALI; n = 5) or heparin-induced extracorporeal LDL precipitation (HELP; n = 7) with routine laboratory methods and western blots.

Results. Proteomic analyses of the column-bound proteins revealed a column-type-dependent loss with the highest number of protein spots in DALI-treated patients (1001 ± 36), followed by HELP (881 ± 25) and DFPP (535 ± 20). More than 70 functional proteins were identified. These proteins are involved in the coagulation pathway (e.g. kininogen1) and have adhesive (e.g. fibronectin), rheological (e.g. fibrinogen) and immunological/inflammatory properties (e.g. complement components). Quantification with western blot analyses demonstrated a significant depletion (P < 0.01) of these proteins comparing serum samples before and after the column with a systemic lowering in patients’ serum.

Conclusions. These data reveal strong interaction between column and serum proteins during LDL-A. The clearance of proteins with adhesive, rheological, and inflammatory characteristics may have beneficial effects on microcirculation and reduce chronic inflammation but may also concomitantly induce side effects such as an increased bleeding risk.

Keywords: atherosclerosis; chronic inflammation; LDL-apheresis; microcirculation; rheology



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 References
 
The role of cholesterol-bearing lipoproteins in atherogenesis is well established. The accumulation of cholesterol in the intima of arteries seems to be accompanied by endothelial dysfunction [1,2]. The goal of cholesterol-lowering therapy is the primary prevention of end-organ damage or secondary prevention of the recurrence. Low-density lipoprotein-apheresis (LDL-A) can be used as an additional therapeutic tool to decrease LDL-cholesterol levels. In Germany, the method is restricted to a small cohort of patients suffering from severe lipoprotein disorder with an associated end-organ damage and elevated LDL cholesterol despite maximal dietetic and medical therapy, or to patients with homozygous familiar hypercholesterolaemia [3].

In our apheresis centre, the plasma-treating methods double-filtration plasmapheresis (DFPP; Lipidfiltration®) and heparin-induced LDL-precipitation (HELP®), and (as haemoperfusion methods treating whole blood) direct adsorption of lipoproteins (DALI®) are used. In DFPP and HELP®, plasma is separated by a hollow fibre plasma separator. Thereafter, in DFPP, lipoproteins are filtered by a secondary column depending on their size, whereas in HELP® lipoproteins are precipitated by a sodium acetate buffer (pH 4.85) containing heparin. These complexes are subsequently removed by a secondary 0.45 µm polycarbonate filter [1]. In the DALI® system, whole blood is perfused through an adsorber that contains a polyacrylate-coated negatively charged polyacrylamide binding the cationic groups in the apolipoprotein B moiety of LDL and lipoprotein a (Lp(a)) [1]. These therapies are highly effective in lowering pro-atherogenic LDL cholesterol and Lp(a), while high-density lipoprotein (HDL) levels are nearly unaffected [4]. In clinical practice, LDL-A reduces the rate of future cardiovascular events [1,5–8] and has been postulated to have additional effects on potentially pro-atherogenetic factors. In the past, some proteins have been identified with adhesive characteristics to lipoproteins, rheological, immunological and inflammation relevant proteins [6,8,9] that influence microcirculation as well as the inflammatory response.

The differences between these established LDL-A methods are related to their different physicochemical principles resulting in differences in selectivity and specification of the different plasmaproteins. The aims of this study were the following: to achieve the mapping of the plasma proteins that interact with the column during LDL-A treatment with the help of proteomic methods; to investigate the differences in protein capture between the different column types and finally to quantify the clearances of functional proteins.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 References
 
Patients and treatment
Twenty-one patients undergoing LDL-A therapy routinely were included in this study. All patients received an individual risk-adapted drug therapy (Table 1). The study protocol had been approved by the local ethics committee prior to the study commencement (no. 10/11/03). All patients or their parents, respectively, gave their written informed consent.


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  		Table 1 Demographic, clinical and apheresis data. Demographic data, the underlying diseases, relevant laboratory values, lipid reduction rates and lipid-lowering therapy are listed.

 
For the extracorporeal LDL-A therapy, DFPP, HELP® or DALI® were used. For DFPP the Octonova® (Diamed, Cologne, Germany) using the EC50® column (Asahi Kasei Medical, Tokyo, Japan), for HELP® the Plasmat Futura® using the precipitate column® (both Braun, Melsungen, Germany) and for DALI the 4008ADS® system using the DALI500 or 750 column® (both Fresenius Medical Care, St. Wendel, Germany) were applied. The LDL-A method was chosen according to individual history and not changed during the study.

Sample collection and protein eluation
Plasma and serum samples were taken immediately before and after the end of LDL-A session from a cubital vein or arteriovenous fistula, respectively. Additional samples were collected from the extracorporeal circulating blood immediately before and after the apheresis-relevant column (in DFPP the EC50® column, in HELP® the precipitation column® and the DALI500 or 750® column), each exactly 5 and 60 min after the start of therapy. After the therapy, proteins bound to DFPP and HELP® secondary columns (EC50® and precipitation column®) as well as DALI500® columns were eluated at 4°C with different protocols depending on the column criteria. Elution of proteins from the apheresis columns was carried out with different protocols depending on the column criteria. Prior to elution all columns were washed with a PBS buffer and according to the column type we applied the following protocols to elute (each step with 50 mL) the proteins: in the EC50® column the PBS-washing step was followed by a three-step elution protocol using solution A (100 mM sodium acetate, 1 M NaCl pH 5), solution B (20 mM Tris–HCl, 1 M NaCl pH 8.5) and solution C (20% acetonitril in ddH2O). In the case of DALI500 or 750® columns, protein elution was achieved by washing the column with solution A, solution D (100 mM sodium acetate, 1 M NaCl pH 4) and solution E (100 mM sodium acetate 1 M NaCl pH 3). In the case of the precipitation columns® (HELP®) the protein elution was performed with solution A, followed by solution F (20 mM Tris–HCl 1 M NaCl pH 7) and solution G (20 mM Tris–HCl buffer 1 M NaCl pH 8.5). For all three types of columns the proteins eluated from all three steps were pooled together and aliquots of 10 mL were used for protein precipitation, protein estimation and 2D gel electrophoresis.

Protein precipitation and estimation
Precipitation of the total protein content was performed prior to 2D gel electrophoresis as following: 10 mL of the eluated solution were concentrated to 2 mL with an Amicon column cut-off 5000 (Beverly, MA, USA). Subsequently, protein precipitation was carried out by mixing the samples with an ice-cold precipitation solution (20% trichloroacetic acid in acetone) with one volume of the sample and three volumes of precipitation solution [10]. Thereafter, protein pellets were washed twice with ice-cold acetone and dried for 5 min. The resulting pellets were dissolved in a 2D gel buffer [urea 9.5 M, CHAPS 2% (w/v), ampholytes 2% (w/v), DTT 1%, and CompleteTM protease 1 tablet/25 mL solution (Roche, Germany)] for 2D gel electrophoresis or in a Laemmli buffer [50 mM Tris–HCl pH 6.8; 2% (w/v) SDS; 0,1% bromphenol blue; 10% (v/v) glycerine and 5% (v/v) ß-mercaptoethanol] for western blot analysis. The protein concentrations were measured according to the Bradford method [11].

Two-dimensional gelectrophoresis, protein visualization and image analysis
Each sample was processed as described in detail previously [12]. Briefly the sample was diluted in a rehydration buffer (8 M Urea, 1% (w/v) CHAPS, 0.2% ampholytes, pH 3–10, 15 mM DTT and a trace of bromphenol blue) to a final volume of 350 µL. The mixture containing 400 µg (for Coomassie staining) or 150 µg (for silver staining) protein was used for the hydration of IPG strips. The strips (pH 3–10, 17 cm) were allowed to rehydrate for 1 h before adding mineral oil. The passive hydration of the gels was carried out overnight for at least 12 h at room temperature in a focusing chamber. Isoelectric focusing with a Protean IEF cell was performed at 20°C using the following multistep protocol: 500 V for 1 h, 1000 V for 1 h and 8000 V for 5 h. After the first dimension, the individual strips were equilibrated in 6 M urea, 30% (w/v) glycerol, 2% (w/v) SDS, 0.05 M Tris–HCl, pH 8.8, and 15 mM DTT for 20 min. An additional incubation in the same buffer supplemented with iodoacetamide (40 mg/mL) was carried out for another 20 min. The second dimension was performed overnight at 120 V using a homogenous acrylamide gel (12% T, 200 x 230 x 1.5 mm) applying a continuous Laemmli buffer system. Image analysis was performed using the PDQuest system according to the protocols provided by the manufacturer. To account for experimental variation, three gels were prepared for each experiment. The gel maps from the different columns were compared to yield information about differences related to the different LDL-A methods.

In-gel digestion, MALDI-TOF MS identification of protein spots and database search
In-gel digestion experiments and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) identification of protein spots were performed according to previously published data [13]. Briefly, silver- or Coomassie-stained spots were manually excised from the gels. After three washes with distilled water for 5 min each, in the case of silver-stained spots, a destaining procedure was carried out with the destaining kit according to the manufacture's protocol (Invitrogen, Karlsruhe, Germany). After dehydrating the spots with acetonitril (ACN) for 15 min, they were dried in a vacuum centrifuge for ~15 min. Thereafter the proteins in gel pieces were digested overnight at 37°C with a 40 µL Trypsin solution (10 ng/µL, in 100 mM ammonium bicarbonate). The peptide extraction was performed with different concentrations of ACN and trifluoroacetic acid (TFA) and the resulting tryptic digests were co-crystallized with the matrix ({alpha}-cyano-4-hydroxycinnamic acid) on a stainless steel target using a 1 µL matrix and 1 µL sample. The mass to charge ratios of the tryptic-digested peptides were acquired on a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems, Darmstadt, Germany) operating in delayed reflector mode with an accelerated voltage of 20 kV. The mass spectra were obtained by averaging 50 individual laser shots. All samples were externally calibrated with a peptide mix of des-Arg-bradykinin ([M + H]+ 904.46), angiotensin I ([M + H]+ 1296.68), Glu1-fibrinopeptide B ([M + H]+ 1570.67), ACTH (1–17) ([M + H]+ 2093.08), ACTH (18–39) ([M + H]+ 2465.19). Internal calibration of the mass spectra was performed with trypsin autolysis products (m/z 842.50 and m/z 2211.10).

A database search with the detected peptide masses was performed against the Mass Spectrometry Protein Sequences Database (MSDB) or the National Center for Biotechnology non-redundant (NCBInr) database using the Mascot peptide mass fingerprint software provided by Matrix Science (Oxford, UK, www.matrixscience.com/search_form_select.html) [14]. Carboxamidomethylation and methionine oxidation were considered as variable modifications. A database search was performed so that after identification each hit was inspected visually to match as much spectral information as possible. The quality criteria encompassed optimized mass accuracy (~50 ppm), minimal mass deviation (in the mDa range), maximized sequence coverage, and the highest possible probability score had to be assigned to the identified protein.

Peptide sequence analysis
To confirm and accomplish the data obtained from mass finger print analysis, all samples were subjected to peptide sequence analysis. After in-gel digestion, a part of the extracted peptides was dissolved in 0.1% formic acid. One microlitre of sample was introduced using a CapLC auto sampler (Micromass, Manchester, UK) onto µ-precolumnTM Cartridge a C18 pepMap (300 µm x 5 mm; 5 µm partical size) and further separated through the C18 pepMap100 nano SeriesTM (75 µm x 15 cm; 3 µm particle size) analytical column (LC Packings, Amsterdam, The Netherlands). The mobile phase consisted of solution A (5% ACN in 0.1% formic acid (FA)) and solution B (95% ACN in 0.1% FA). The total sample run time was 60 min. In the first step, samples were injected onto a precolumn and washed for 5 min with 0.1% formic acid (30 µL). The washing step was followed by an elution step with an exponential gradient starting with 10% B and ending with 95% B. The nanospray needle was held at 2 kV and the source temperature at 40°C. After chromatographic separation, peptide sequencing was performed on a Q-TOF Ultima Global (Micromass, Manchester, UK) mass spectrometer equipped with a nanoflow ESI Z-spray source in positive ion mode. Multiple charged peptide parent ions were automated, marked and selected in the quadrupole, fragmented in the hexapole collision cell and their fragment patterns were analysed by TOF. The data acquisition was performed using MassLynx (v 4.0) software on a Windows NT PC, while the data were further processed on a Protein-Lynx-Global-Server (v 2.1) (Micromass, Manchester, UK). The raw data files were deconvoluted and deisotoped using the Max EntTM lite 3 algorithm and a peak list was generated. Processed data were searched against MSDB and Swissprot data bases through Mascot search engine using a peptide mass tolerance of 50 ppm and fragment tolerance of 100 mmu. Protein identification with at least two peptides sequenced or a PMF minimal score of 70 were considered significant.

Western blot analysis
Western blot analyses were performed according to previously published data [15] with 20 µg from plasma proteins collected from the extracorporeal circulating blood as described above and 20 µg eluated proteins from every type of the column. For immunodetection of proteins the following antibodies were used: rabbit polyclonal antibodies to fibrinogen, complement factor B, coeruloplasmin, plasminogen or transferrin (all Dako, Hamburg, Germany). Goat anti-alpha-antitrypsin polyclonal antibody, rabbit anti-alpha-2 macroglobulin polyclonal antibody, rabbit anti-C1q complement antiserum and mouse anti-ß2-microglobulin monoclonal antibody were from Sigma-Aldrich (Missouri, USA). Horseradish peroxidase-linked donkey anti-rabbit antibody, horseradish peroxidase-linked rabbit anti-goat antibody, horseradish peroxidase-linked sheep anti-mouse antibody were from Amersham Biosciences (Freiburg, Germany).

Clinical chemistry
All clinical chemical parameters were measured by standard routine methods. Fibrinogen was measured in citrate plasma using the method of Clauss [16]. The complement components C3c and C4 were investigated nephelometrically. Complement activity CH50 was tested by haemolysis of erythrocytes at a wavelength of 541 nm. Total-(Total-C), LDL-(LDL-C), HDL cholesterol (HDL-C), albumin, fibrinogen, protein S, apoB, haptoglobin and transferrin were determined with standardized test kits from routine analyses.

Statistics
Mass spectrometry identified proteins were described qualitatively. The other results are expressed as mean values ± standard deviation (SD). Comparative statistical analyses of within-subject within-treatment changes were assessed using t-test for paired samples and intergroup comparisons were assessed using ANOVA with subsequent pairwise comparison using the t-test for independent samples. In order to correct for multiple comparisons, the closure principle was used. The analysis was performed using Sigma-StatTM software 2.03 (Jandel Scientific, San Rafael, CA, USA). A P-value of <0.05 was considered to be statistically significant.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 References
 
All investigated LDL-A methods led to a significant reduction of pro-atherogenic LDL cholesterol and Lp(a). The relevant data of patient characteristics as well as the apheresis data did not differ between the different groups and are shown in Table 1.

2D-electrophoresis mapping of the column-bound proteins
In our study, we investigated whether differences in physicochemical mode of action of the different LDL-A methods are associated with differences in the clearance of proteins. For this purpose, adapted protocols for each column were developed for the elution of binding proteins. The amount and number of adsorped proteins were different from column to column. The highest spot number was detected in DALI® gel (1001 ± 36) whereas the DFPP and HELP® gels displayed lower spot numbers (535 ± 20 or 881 ± 25, respectively) in the pH range of 3–10 when 20 000 pixels were used as the filter limit of the automated image analysis (Figure 1).


Figure 1
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  		Fig. 1 Protein patterns of eluate from the three different columns (DFPP, DALI500® and HELP®) analysed by 2D gel electrophoresis. The protein spots were visualized by silver staining.

 
Further comparative examination showed significant differences between the 2D protein maps from the three different columns (Figure 2). Close-up comparisons between protein maps of 2D gels of the three columns revealed prominent differences in both low- (Figure 2A) and high-molecular-mass proteins (Figure 2B). In the EC50-filter eluates (DFPP), we found an agglomeration of protein spots below 15 kDa, around 25 kDa and between 37 and 100 kDa. In the range between 100 and 250 kDa there were only negligible amounts of protein spots detectable. Comparing the distribution according to the pH value, most protein spots were detected above pH 5. In the precipitate-filter eluates (HELP®) most protein spots were detectable between 75 and 150 kDa in the pH range 3–10 and about 37 kDa in the pH range above 10. In the other range there was only a scattered protein spot profile detectable. In eluate of the DALI filter, an accumulation of protein spots between 15 and 150 kDa in the pH range between 5 and 10 was observed.


Figure 2
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  		Fig. 2 Close-up of gels showing differences in proteins bound to the different columns. (A) Close-up of gels showing differences in proteins identified in lower molecular weight ranges (MW < 37 kDa). (B) Close-up of gels showing proteins identified after in-gel digestion and mass spectrometric analysis in the high molecular weight ranges (MW > 37 kDa). The names of the proteins are given directly near their positions on the gels.

 
After in-gel digestion, mass spectrometric analysis and database searches, 74 different proteins could be identified from all the three columns: 28 of the identified proteins were detected in the precipitate (HELP®), 46 in the DALI500® and 42 in EC50 (DFPP) column eluates. When the mass spectrometric data were interpreted, post-translational modifications such as carboxyamidomethylation and methionine oxidation were considered as variable identification parameters. Each peptide hit of a database search was inspected visually to match as much spectral information as possible. The identified proteins could be referred to five different functional protein groups: apoliproproteins, rheological relevant proteins, inflammatory proteins, adhesive proteins and others (Table 2). Only 13 proteins were found to bind ubiquitously to all three LDL-A systems: alpha-1-antitrypsin, alpha-1-antiplasmin, complement component C3, complement component C4B, complement factor H precursor, fibrinogen A, fibrinogen B, fibrinogen gamma, fibronectin 1, haptoglobin, kininogen 1 and transthyretin.


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  		Table 2 List of identified proteins (pH 3–10) in eluate from LDL-A columns using mass spectrometry microsequencing and database comparisons.

 
Western blot quantification of protein clearance during LDL-A
In order to confirm and validate the 2D gel data, and to quantify the protein lost during LDL-A, western blot analyses were performed for prominent proteins identified after 2D gel (Figure 3). Immunoblotting with specific antibodies after 1D gel separation confirmed the presence of plasma proteins such as pre-albumin, collagen 1, plasminogen, transferrin, alpha-2-macroglobulin, alpha-1-antitrypsin, alpha-2-antiplasmin, fibronectin, TGF-ß and complement factor B in the column eluates (Figure 3A). As confirmation for 2D gel data, western blot analysis showed a loss in plasma proteins after passing the LDL-A column. The analysed plasma samples showed a clear decrease in content of the proteins after column in comparison to those taken prior to the column. For almost all analysed proteins the reduction rate tended to be higher using the DALI® column. For selected proteins such as TGFβ, collagen 1 and complement factor B, western blot analyses showed a total removal after the column (Figure 3B).


Figure 3
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  		Fig. 3 Western blot analysis of selected plasma proteins identified in LDL-A column eluates. Immunoblots of column eluates and of plasma samples were compared. Serum proteins were taken from the extracorporeal circulating blood before (1) and after (2) the LDL-apheresis relevant column exactly 5 min after the start of therapy. Specific mass spectrometrically identified proteins were confirmed to bind to the appropriate column by immunoblotting with specific antibodies. (A) The right part of the figure shows the results from serum analysis and the left part shows the eluate results. (B) The western blot quantification is presented as a grouped bar chart with SD (black bar = before the column; grey bar = after the column). Each bar represents the intensity means ± SD of blots from three independent experiments.

 
Laboratory analysis of selected proteins
Some of the detected proteins were analysed from blood samples taken from the vein or arteriovenous fistula before starting LDL-A and after the end of therapy. We found a significant decrease of total protein (–18.0 ± 5.9%, P < 0.01) in all investigated LDL-A methods, reaching from –13.5 ± 5.7% in DALI® to –20.8 ± 5.4% in HELP®. A significantly higher decrease, compared to total protein, was detected for fibrinogen, haptoglobin, complement C3c, C4 and CH50 (Tables 3 and 4). Exemplarily, we analysed the kinetics of complement and demonstrated a significant decrease in all investigated LDL-A methods (Table 4).


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  		Table 3 Reduction rates of several proteins dependent on the LDL-apheresis method. Mean reduction rate ± SD of ApoB, total protein, albumin, fibrinogen, protein S, complement CH50, C3c and C4, haptoglobin and transferrin are shown.

 

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  		Table 4 Time course of total protein, complement factors C3c, C4 and complement activity CH50. Blood samples were taken before (AP) and after the end of apheresis therapy (EP), 5 (5') and 60 min (60') before (BC) and after the LDL-A relevant column (AC). Mean values ± SD are shown.

 


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
References
 
Improvement of myocardial ischaemia as a result of LDL-A was reported in the LDL-apheresis atherosclerosis regression study and other trials [17–21]. These effects were not only attributed to the reduction of LDL cholesterol [2,22,23], but also to additional effects such as vasodilatation and improvement of microcirculation [24–27]. This is the first study investigating the clearance of functional proteins by different LDL-A methods with the help of proteomics. The main results of our study are as follows: (i) we identified some captured proteins which had so far not been described and (ii) our results suggested interactions between plasma proteins and LDL-A columns independent of so far known physicochemical principles. However, our study does have some limitations: (i) the number of patients undergoing the particular LDL-A method was limited, but within the range of other published LDL-A studies [18–20], (ii) patients could not be randomized to treatment options due to historic individual side effects following treatment with one particular LDL-A method in the history of some subjects, (iii) patients received individual drug therapy and (iv) differences in filters and elution protocols could result in differences in 2D gel patterns. However, selected proteins detected by 2D gel and MALDI-TOF-MS were validated by western blot and clinical chemistry analyses.

We found that a number of different proteins were captured by the different LDL-A columns. Comparing the proteins, it was conspicuous that they can be divided into functional protein classes consisting of lipoproteins, adhesive, rheological and inflammation relevant proteins.

Haemorheological relevant proteins showed a strong interaction with LDL-A columns. In addition to known proteins, e.g. fibrinogen, we identified some other determinants of plasma viscosity and coagulation, i.e. albumin, transthyretin (pre-albumin), kininogen 1, protein S, thrombin, isoforms of fibrinogen or alpha1-antitrypsin, that had partially not been described before to have been captured by the particular LDL-A method. The lowering of the plasma viscosity by LDL-A is a well-known phenomenon [28], which was related to a significant improvement of myocardial, cerebral, and peripheral flow even after a single LDL-A [26,28–30]. However, the functional relevance of the clearance of these newly detected proteins beyond lowering the plasma viscosity remains to be determined.

Among adhesive proteins that had been adsorped, ficolin and fibulin were identified for the first time to interact with LDL-A columns. Previous studies have identified fibulin as a predominant binding protein for extracellular superoxide dismutase that is known to play an important role in atherosclerosis and endothelial function by modulating levels of the superoxide anion in the extracellular space [31]. Ficoliin initiates the lectin pathway of complement activation through attached serine proteases [32], but its role in atherosclerosis or lipoprotein metabolism was so far undefined.

Among inflammatory and acute phase proteins, we identified a series of different components of the complement system to be removed by all investigated LDL-A methods, but mostly in HELP®. One study has shown a minimal complement activation with a negligible increase in C3a and C5a after DALI® therapy [8], but another study described an adsorption of the complement component factor D by dextran sulphate adsorption [33]. However, although we demonstrated a removal of complement components by the different LDL-A methods, we could not exclude a local activation during extracorporeal circulation. Our data were specific as we demonstrated a decrease of complement components C3c and C4 serologically as well as a reduced activation of the classical pathway of the complement system CH50 comparing AP and EP values along with compatible kinetics after 5 and 60 min treatment. Complement activation contributes to myocardial damage through various pathways, including activation of leucocytes and endothelial cells, upregulation of apoptosis, cytokine production and NO-synthase activity [34,35]. In hyperlipoproteinaemic animal models, inhibition of the complement system has reduced the progression of atherosclerotic lesions although data are not clear so far [35]. In humans there exists so far no data on complement inhibition. Whether the reduction of complement components may have a beneficial effect on prevention and treatment of atherosclerosis remains to be determined. On the other hand, complement removal may explain the susceptibility to infections in LDL-A patients [36].

Most proteins captured by the HELP® precipitate column had, not unexpectedly, a pH value >5. However, some proteins were detected in the pH range 5–10, indicating a clearance independent of the precipitation by the sodium acetate buffer at pH 4.85. Although highly speculative, this may be dedicated to additional physicochemical interactions between plasma and columns than the capture of precipitated proteins alone. Among these, we found atherosclerosis relevant proteins, e.g. complement components or matrix proteins. The eluate of the EC50 column (DFPP) consisted, in the majority of proteins, with an MW between 37 and 100 kDa. However, there were two protein clusters with an MW of 25 and below 15 kDa, which were also captured by DFPP. Among these atherosclerosis relevant proteins, e.g. complement components, were identified. DFPP is known to capture proteins depending on their size. Plasma proteins with a tertiary structure >15 nm are eliminated by the lipid filter [37]. Although the correlation between protein MW and diameter is not significant, it is tempting to speculate an additional clearance in DFPP independent of the protein size alone. DALI eluates showed additionally large amounts of proteins at low molecular weight with the identification of carbonic anhydrase as well as actin indicating, not unexpectedly, the presence of red blood cells within this fraction.

All three LDL-A methods under investigation were found to capture a broad series of proteins suggesting a certain degree of ‘unspecifity’. If this is beneficial or not is fully open. On the one hand, this ‘unspecifity’ with reduction components of blood viscosity may explain some beneficial effects like the successful use of HELP and DFPP system in treatment of non-LDL-C-associated microvascular diseases, e.g. sudden hearing loss or non-arteritic acute anterior ischaemic optic neuropathy [26–28]. On the other hand, side effects like the development of iron deficiency anaemia by LDL-A (Koziolek et al., manuscript in preparation) may also be explainable. However, although the mapping of protein adsorption by LDL-A offers qualitative differences between different methods, it gives no evidence about the superiority of one particular LDL-A method in reducing cardiovascular risk.



   Acknowledgments
 
Parts of the present work have been presented in abstract form at the 36th congress of the German Society of Nephrology, Saarbrücken, Germany, September 2005. This work was supported by a research grant of Diamed (Cologne, Germany) to M. Koziolek and F. Strutz.

Conflict of interest statement. The authors declare lecture fees frin Diamed (Cologne, Germany) to R. Klingel and G.A. Müller, from B. Braun (Melsungen, Germany) to M. Koziolek and frin Fresenius Medical Care (Bad Homburg, Germany) to G.A. Müller and travel fundings from Diamed (Cologne, Germany) to M. Koziolek, R. Klingel and G.A. Müller.



   Notes
 
* Contributed equally to this paper. Back



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 12.11.07
Accepted in revised form: 15. 2.08


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