Determination of high molecular mass Al species in serum and spent CAPD fluids of dialysis patients combining SEC and anion-exchange FPLC with ETAAS detection

Abstract

Speciation of high molecular mass Al compounds (HMM-Al) in human serum is commonly performed by size-exclusion or ion-exchange chromatography. In general, size-exclusion chromatographic (SEC) columns are not able to completely separate transferrin from albumin. Fast protein liquid chromatography (FPLC), using an anion-exchange column, enables separation of transferrin from albumin. However, the possibility exists of coelution of LMM–Al species (e.g., citrate, phosphate) with HMM–Al species. In this case the concentration of HMM–Al species could be overestimated. It is therefore important to remove LMM–Al species before anion-exchange FPLC separation. For this purpose a novel analytical approach was developed for speciation of HMM–Al species in human serum by combination of SEC (Superdex 75 HR 10/30) and anion-exchange FPLC (Mono Q HR 5/5 anion-exchange FPLC) with UV detection at 278 nm. 1 cm³ of serum was injected onto the SEC column. Isocratic elution using 0.05 mol dm⁻³ TRIS-HCl + 0.03 mol dm⁻³ NaHCO₃ was applied. It was experimentally proved that proteins were eluted in a 5 cm³ peak that was collected into a polyethylene cup. A 0.5 cm³ aliquot of the sample was then injected onto the anion-exchange FPLC column. The separation of serum proteins was obtained by applying linear gradient elution from 100% buffer A (0.05 mol dm⁻³ TRIS-HCl + 0.03 mol dm⁻³ NaHCO₃) to 100% buffer B (A + 0.25 mol dm⁻³ NaCl). Well-resolved protein peaks were obtained. 0.25 cm³ fractions were collected during the chromatographic run and Al determined by ETAAS. It was experimentally proved that 90 ± 5% of Al in spiked serum from a dialysis patient was eluted under the transferrin peak which was identified, not only on the basis of the retention volume, but also by the SDS-PAGE electrophoresis. The same procedure may be applied for speciation of Al in spent CAPD fluids of dialysis patients if the concentration of Al is higher than10 ng cm⁻³. The proposed speciation procedure removes LMM–Al species and enables reliable determination of the concentration and composition of Al bound to proteins by anion-exchange FPLC-ETAAS.

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Introduction

Aluminium (Al) overload in the human body, especially in renal patients, is related to many clinical disorders such as dialysis encephalopathy¹ and osteomalacia.² Although Al overload in patients with chronic renal failure has been greatly reduced since the quality of water used for dialysis has significantly improved,³ the absorption and accumulation of Al, particularly via consumption of Al-based drugs,⁴ still remains the problem in nephrology. To understand the toxicity of Al in humans and its transport through the body it is important to identify and quantify the Al species in serum. Blood serum is a very complex matrix containing high molecular mass (HMM) and low molecular mass (LMM) compounds and a high concentration of salts. To determine the proportion of HMM–Al and LMM–Al species and to identify their composition various analytical techniques have been used. Ultrafiltration and microultrafiltration were first applied to determine the percentage of Al bound to HMM and LMM species.^5–9 The results in general suggested that about 90% of Al was bound to HMM proteins. Fractionation of Al was also investigated by size-exclusion chromatography (SEC).^10–13 Some authors reported problems with an excess of spiked Al eluting from the column,^10,13 most probably due to contamination from the column support. By use of SEC techniques it was demonstrated that Al was eluted under the elution profile of transferrin and albumin. The same observations resulted from the investigation on the fractionation of Al by SEC with UV and ETAAS detection in spent continuous ambulatory peritoneal dialysis (CAPD) fluids.¹⁴ More selective separation of proteins was obtained by an anion-exchange high-performance liquid chromatograph using a Protein-Pak DEAE-5PW column.¹⁵ In combination with SDS-PAGE electrophoresis and final element detection of Al by ETAAS, it has been demonstrated that the transferrin is the only binding Al protein in human serum. The main problem, however, was the contamination originating from the chromatographic system and eluents. The progress in Al speciation of positively¹⁶ and negatively charged Al complexes¹⁷ has been achieved by the use of the robust cation–Mono S and anion-exchange Mono Q fast protein liquid chromatographic columns (FPLC). These polyether resin based columns enabled quantitative separation of Al species, which is a great advantage in comparison to silica-based columns on which the adsorption of Al on the column support was evident.¹⁸ An anion-exchange Mono Q FPLC has been successfully applied in further investigations of the percentage and the composition of both LMM–^19–21 and HMM–Al species.^22–24 Various cleaning procedures of the column resin as well as eluents were employed,^19–24 all resulting in substantial lowering of the risk of contamination with extraneous Al. In investigations of LMM–Al binding ligands in serum, microultrafiltration (cut-off 30 000 Da) was first applied to separate ultrafiltrable Al from HMM–Al species. Ultrafiltrable Al was then injected on the FPLC column. On the basis of the elution time and identification of the Al binding ligands by ES-MS-MS it has been demonstrated that the main LMM–Al species were Al–citrate, Al–phosphate and ternary Al–citrate–phosphate complexes.^20,21 In these investigations it was also found experimentally that the amount of LMM species in spiked serum of healthy subjects and in serum of dialysis patients was individually variable and ranged in general between 14 and 50%. When HMM–Al species were investigated serum was injected either 5 times diluted or non-diluted on the FPLC column without prior separation of LMM–Al binding compounds.^22–24 Different eluents and various types of gradient elution were applied. The eluting peaks were identified by UV detection and Al determined by ETAAS²² or HR-ICP-MS.^23,24 These studies confirmed again that about 90% of Al was eluted under the elution volume of transferrin. It has also been demonstrated that the preferred binding site of Al in transferrin is the N-lobe site.²⁴ Recently, chemical speciation in human serum was critically reviewed.²⁵ Although analytical procedures have appreciably improved in the last decade, speciation of Al in serum and other biological samples is still in a state of development. On the basis of knowledge that we gained on the elution profiles of LMM–Al complexes in human serum on the Mono Q FPLC column^20,21 and data reported for HMM–Al species separated on the same column,^22–24 the possibility exists on the coelution of LMM–Al species (Al–citrate, Al–phosphate, ternary Al–citrate–phosphate complexes) with the Al–transferrin complex. This may result in overestimation of the concentration of Al bound to transferrin. In order to obtain reliable data there is a need for pre-separation of LMM–Al complexes before using an anion-exchange FPLC in the speciation of HMM–Al compounds. Therefore, the aim of our work was to develop an analytical procedure using SEC prior to an anion-exchange FPLC separation. Pre-separation and separation procedures were followed by the UV detection and Al determined “off-line” by ETAAS. Identification of the Al-binding protein eluting from the FPLC column was performed not only on the basis of the retention volume, but also by SDS-PAGE electrophoresis. The spiked serum of a CAPD patient and spent CAPD fluid were analysed under the proposed analytical protocol.

Experimental

Instrumentation

The chromatographic system consisted of a Varian (Mulgrave, Victoria, Australia), Model 9010, HPLC inert Star Gradient Solvent Delivery System, equipped with a Varian Polychrom Model 9065 UV diode array detector and a Rheodyne (Cotati, CA, USA) Model 7161 injector using 1 or 0.5 cm³ loop. SEC was performed on a Superdex 75 HR 10/30 column (Amersham, Uppsala, Sweden) (column dimensions 10 × 300 mm, 13 µm beaded composite of cross-linked agarose and dextran, pH stability 3–12, molecular permeation range from 3000 to 100 000). A strong anion-exchange FPLC column of Mono Q HR 5/5 (Pharmacia, Uppsala, Sweden) (column dimensions 5 × 50 mm, 10 µm beaded polyether resin, pH stability 2–12) was employed for the separation of Al species bound to proteins. Total Al in human serum as well as the concentration of Al in separated fractions was determined by ETAAS on a Hitachi (Hitachi, Tokyo, Japan) Z-8270 polarized Zeeman atomic absorption spectrometer equipped with an autosampler at 309.3 nm. The chromatographic runs were also followed by UV spectrometry at 278 nm, while the identification of proteins in separated fractions after the FPLC separation was performed by the SDS-PAGE electrophoresis on an Amersham Biosciences PhastSystem. A WTW (Weilheim, Germany) 330 pH meter was employed to determine the pH.

Reagents

Merck (Darmstadt, Germany) Suprapur acids and water doubly distilled in quartz were used for the preparation of samples and standard solutions. All other reagents were of analytical reagent grade.

A stock standard solution of Al (1000 ± 0.002 mg dm⁻³ in 5% HNO₃) used for calibration in ETAAS determinations was obtained from Merck. A stock Al³⁺ solution (100 µg Al cm⁻³) used for spiking of serum sample was prepared in a 100 cm³ calibration flask by dissolving 0.1388 g of Al(NO₃)₃·9H₂O (Riedel-de Haën, Hannover, Germany) in water. Fresh working standard solutions were prepared daily by dilution of stock solutions with water.

Buffer A consisted of 0.05 mol dm⁻³ TRIS-HCl buffer (Merck) + 0.03 mol dm⁻³ NaHCO₃ (Merck). The pH of buffer A was adjusted to 7.4 with an appropriate amount of 1 mol dm⁻³ of hydrochloric acid. Buffer B contained 0.25 mol dm⁻³ of sodium chloride (Merck) and was prepared by dissolving 14.60 g NaCl in 1 dm³ of buffer A.

Standard proteins (Sigma–Aldrich, Steinheim, Germany) of known molecular masses [albumin (66 000 Da), transferrin (77 000 Da), Imunoglobulin G (IgG) (150 000 Da)] (0.5–5 mg cm⁻³) were used for calibration of the FPLC Mono Q column. Blue dextran with a molecular mass 200 000 Da (1 mg cm⁻³) was used to determine the void volume of the size exclusion chromatographic column.

Chelex 100 (Na⁺ form, 100–200 mesh) chelating ion-exchange resin (Sigma) and a silica based LiChrosorb RP-18 HPLC column (150 × 4.6 mm id) were used for purification of the eluents.¹⁹

Centricon 30 concentrators (Amicon, Beverly, MA, USA) with a nominal cut-off 30 000 Da were used to concentrate fractions containing proteins of spent CAPD fluid before applying SDS-PAGE electrophoresis.

Sample preparation

In order to study the separation of proteins at pH 7.4 standard proteins were dissolved in buffer A (albumin 25 mg cm⁻³, IgG 5 mg cm⁻³ and transferrin 2.5 mg cm⁻³) and first injected onto the SEC column. After that an aliquot of the protein peak was injected onto the FPLC MonoQ column.

Venous blood (venous puncture) from a CAPD patient without peritonitis was taken during clinical examination after informed consent was obtained. It was collected into Al-free Becton–Dickinson vacutainers without additives. Sample was centrifuged for 10 min at 855g. Serum aliquots were transferred into 5 cm³ polyethylene tubes with a polyethylene pipette and stored in a freezer at −20 °C. Spent CAPD fluid of the same dialysis patient was collected at the out-patient department after the night dwell into 5 cm³ polyethylene tubes and stored in a freezer at −20 °C. Before analysis samples were equilibrated to room temperature. Total Al was first determined. In order to study the speciation of HMM–Al compounds, 3 cm³ of serum was spiked with 0.05 cm³ of Al³⁺ solution (Al nitrate salt), so that the final concentration of Al in the spiked serum ranged from 250 to 300 ng cm⁻³. Spiked serum was left to equilibrate at room temperature for 5 h.^19,20 After that it was injected onto the SEC column (1 cm³ loop). Speciation of Al was then performed following the recommended analytical procedures. It was experimentally proven that freezing of samples did not influence the speciation of Al.

Recommended procedures

Sample preparation, chromatographic separations and determination of Al by ETAAS were carried out under clean-room conditions (class 10000). To avoid contamination by extraneous Al, polyethylene or Teflon ware was treated with 10% HNO₃ for 24 h, rinsed well with water and dried at room temperature. In order to lower the blank, the eluents were purified by the cleaning procedure reported previously.¹⁹ For the determination of the total Al concentration in serum and spent CAPD fluids by ETAAS, 5 mm³ of 32% nitric acid were added into the graphite furnace tube before each determination in order to reduce matrix effects of proteins.¹⁴

SEC procedure. 1 cm³ of spiked serum was injected onto the column. The chromatographic run was carried out at a flow rate of 1 cm³ min⁻¹. Isocratic elution with buffer A was applied for 15 min. From 15 to 16 min linear gradient elution from 100% buffer A to 100% buffer B followed. Elution with 100% buffer B was kept up to 29 min. From 29 to 30 min linear gradient elution from 100% buffer B to 100% buffer A continued and from 30 to 40 min the column was rinsed with 100% buffer A. The protein peak was collected from 7 to 12 min into a polyethylene sample cup. The concentration of Al in fractions after the chromatographic separation was determined “off line” by ETAAS using eluent-matched standards for calibration. 32% nitric acid was introduced into the graphite tube before each determination to eliminate the matrix effect of proteins.¹⁴

FPLC Mono Q procedure. The protein fraction from the SEC column was subjected to an anion-exchange FPLC. 0.5 cm³ of the sample aliquot was injected onto the column. The chromatographic run was carried out at a flow rate of 1 cm³ min⁻¹. Linear gradient elution from 100% buffer A to 100% buffer B was applied for 15 min. The column was rinsed with 100% buffer B for 1 min and from 16 to 25 min with buffer A. 0.25 cm³ fractions were collected during the chromatographic run into the graphite furnace sample cups. The concentration of Al in fractions after the chromatographic separation was determined “off line” by ETAAS using eluent-matched standards for calibration. 32% nitric acid was introduced into the graphite tube before each determination to eliminate the matrix effect of proteins.¹⁴

Cleaning procedures of the SEC and FPLC Mono Q columns. The cleaning of columns was performed at a flow rate of 1 cm³ min⁻¹ in the opposite direction from the chromatographic separation. Columns were first rinsed with 15 cm³ of water. Then, 1 mol dm⁻³ NaOH was injected (1 cm³ onto the SEC and 0.5 cm³ onto the FPLC Mono Q, respectively). Rinsing of the columns with 30 cm³ of water followed. The procedure was repeated twice. After that 10 cm³ of 2 mol dm⁻³ citric acid was applied and columns rinsed with 60 cm³ of water. Finally, the columns were turned back to the original flow direction and rinsed with 15 cm³ of buffer A before the next chromatographic separation.

Results and discussion

Optimisation of the cleaning procedures

Efficient cleaning of eluents and chromatographic columns are of crucial importance in speciation of Al in serum and spent CAPD fluids. By combination of chelating ion-exchange chromatography (Chelex 100, Na⁺ form, batch procedure) and silica based reversed-phase HPLC,¹⁹ effective cleaning of the eluents was obtained, lowering the blanks down to 1 ng cm⁻³ of Al. A more difficult task is to remove traces of Al from the support of the chromatographic columns. In our previous investigations it was experimentally proved that the application of 2 mol dm⁻³ citric acid efficiently removed traces of Al from column supports when speciation of LMM–Al complexes in serum¹⁹ and fractionation of Al in spent CAPD fluids¹⁴ were investigated. However, in the speciation of HMM–Al compounds a more rigorous cleaning procedure is required, due to the very high affinity of serum proteins for mobilizing Al from the column support. This results in high blank values. In addition, it was also found experimentally that after application of more than three successive serum samples on the SEC or FPLC Mono Q columns, the pressure on the columns has risen sharply. To overcome the pressure problems and to efficiently remove the traces of Al from the column support, the cleaning of the column in the reverse direction of the chromatographic run, as described under Recommended procedures, was applied. Using the proposed cleaning protocol the blanks from the column support were lowered down to 1 ng cm⁻³ Al.

Parameters influencing speciation of Al bound to serum proteins by combining SEC and anion-exchange FPLC

To separate HMM–Al compounds from LMM–Al species SEC was first applied. Isocratic elution using 0.25 mol dm⁻³ NaCl in 0.05 mol dm⁻³ TRIS-HCl + 0.03 mol dm⁻³ NaHCO₃ (buffer B, pH 7.4) efficiently separated the proteins from LMM compounds. However, it was found experimentally that the high ionic strength of 0.25 mol dm⁻³ NaCl affected the stability of the Al–protein complexes. Al from the protein peak was eluted on the anion-exchange FPLC column with a solvent front as ionic species. In order to ensure the separation of proteins on the SEC column without influencing the chemical speciation of Al, isocratic elution with 0.05 mol dm⁻³ TRIS-HCl + 0.03 mol dm⁻³ NaHCO₃ (buffer A, pH 7.4) was then applied. The same shape of the protein peak was obtained as when using buffer B. After separation of proteins LMM–Al species were eluted from the SEC column with buffer B, as described in Recommended procedures. The UV chromatogram of the synthetic solution of standard proteins (IgG, transferrin, albumin) separated on the SEC column is presented in Fig. 1A. It is evident that proteins were eluted from 7 to 12 min. The 5 cm³ protein peak was collected into a polyethylene cup. Since 1 cm³ of sample was injected onto the SEC column this means that the proteins after the separation were diluted 5 times. A 0.5 cm³ aliquot of the protein peak was then injected onto the anion-exchange FPLC column and separation performed as described under Recommended procedures. The UV chromatogram is presented in Fig. 1B. Well resolved protein peaks were obtained. IgG was separated from 0.5 to 3.0 min, transferrin from 5 to 7 min and albumin from 10 to 16 min.

Fig. 1 Separation of a standard solution of proteins at a flow rate of 1 cm³ min⁻¹ followed by UV detection at 278 nm. 1 cm³ of sample containing 25 g dm⁻³ of albumin, 5 g dm⁻³ of IgG and 2.5 g dm⁻³ of transferrin was first injected onto the SEC column (A). A protein peak was collected from 7 to 12 min (5 cm³). 0.5 cm³ aliquot of a protein peak was then injected onto the anion-exchange FPLC column (B).

Speciation of Al in a spiked serum of a dialysis patient

A serum sample from a dialysis patient obtained during the venous puncture was first analysed for the total Al content under the optimum measurement conditions.¹⁴ The accuracy of determination of total Al was checked by the analysis of Seronom^™ Trace Elements Serum standard reference material obtained from Nycomed Pharma AS (Oslo, Norway). Good agreement between determined Al (62 ± 2 ng cm⁻³) and the reported certified value (63 ± 4 ng cm⁻³) was obtained. The concentration of total Al in serum (mean of three parallel analysis) was found to be 4.5 ± 0.5 ng cm⁻³. This concentration was too low to perform speciation analysis. Therefore, the sample was spiked with Al³⁺ solution (five individual spikes), so that the final concentration of Al in serum ranged between 250 and 300 ng cm⁻³ Al. 1 cm³ of spiked serum was injected onto the SEC column and speciation analysis performed as described under Recommended procedures. UV chromatograms of SEC and anion-exchange FPLC separations, as well as the percentage of Al eluted under the chromatographic peaks, are presented in Fig. 2A and 2B, respectively. From the data in Fig. 2A it is evident that 90% of total Al in spiked serum was eluted under the protein peak on the SEC column. The remaining 10% of Al was eluted as LMM species. These findings are in agreement with most of the reported literature data^{5–9,15,22,23} and are still in agreement with our findings on the individual variability of the percentage of the serum Al bound to proteins (50–86%).^19–21 It can be further seen from data in Fig. 2B that Al bound to proteins is quantitatively eluted on the anion-exchange FPLC column at the elution volume of the transferrin peak. Al appeared in fractions from 5.25 to 6.0 min with a maximum peak eluted from 5.5 to 5.75 min. The distribution of Al species in five individual spikes of a serum of a dialysis patient, successively analysed by combining SEC and anion-exchange FPLC separation with ETAAS detection, is presented in Table 1. The UV chromatograms for individual spiked samples gave the same shape of the chromatographic peaks as presented in Figs. 2A and 2B. Data in Table 1 indicate that, on the SEC column, in all spiked samples investigated 90 ± 5% of Al is eluted as HMM–Al species (Al bound to proteins) and that the remaining Al corresponded to LMM–Al species. Furthermore, speciation analysis of the HMM–Al fraction on the anion-exchange FPLC column confirmed that 97 ± 3% of Al in the protein fraction is eluted exclusively under the elution volume of the transferrin peak. This finding is again in agreement with the reported literature data.^22–24 However, it should be stressed that in the present work the spiked serum investigated contained a high proportion of the HMM–Al species (90 ± 5%). For that reason, there was no evident difference between the reported literature data when anion-exchange FPLC separation without the pre-separation of LMM-Al species from the protein fraction was applied^22–24 and our investigation where the pre-separation of LMM-Al species has been carried out first. However, to eliminate the possibility of overlapping of HMM–Al and LMM–Al species, the pre-separation of LMM–Al species is strongly recommended before the anion-exchange FPLC separation of proteins is performed.

Fig. 2 Separation of a spiked human serum (spike No. 1) of a dialysis patient (300 ng cm⁻³ Al) at a flow rate of 1 cm³ min⁻¹ followed by UV detection at 278 nm. 1 cm³ of spiked sample was first injected onto the SEC column (A). A protein peak was collected from 7 to 12 min (5 cm³). 0.5 cm³ aliquot of a protein peak was then injected onto the anion-exchange FPLC column (B).

Table 1 Distribution of Al species in five individual spikes of a serum of a dialysis patient, successively analysed by combining SEC and anion-exchange FPLC separation with UV and ETAAS detection

Sample		SEC column			Anion-exchange FPLC column
Spike no.	Concentration of Al in spiked serum/ng cm^−3	Concentration of HMM–Al species/ng cm^−3	Concentration of LMM–Al species/ng cm^−3	HMM–Al species (%)	Concentration of Al bound to transferrin/ng cm^−3	HMM-Al species bound to transferrin (%)
1	300 ± 8	272 ± 8	30 ± 2	91 ± 3	267 ± 8	98 ± 3
2	251 ± 6	213 ± 6	40 ± 2	85 ± 3	203 ± 6	95 ± 3
3	254 ± 6	236 ± 6	17 ± 2	93 ± 3	229 ± 6	97 ± 3
4	253 ± 6	223 ± 6	28 ± 2	88 ± 3	215 ± 6	96 ± 3
5	293 ± 9	278 ± 9	16 ± 2	95 ± 3	271 ± 8	97 ± 3

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To check the blanks of the analytical procedure speciation of non-spiked serum sample (4.5 ± 0.5 ng cm⁻³ of total Al) was also carried out. By using the recommended analytical procedures the concentrations of Al in the separated fractions were found to be below 1 ng cm⁻³ Al, indicating the efficient cleaning of eluents and both SEC as well as anion-exchange FPLC columns.

Speciation of Al in spent CAPD fluid

The concentration of total Al in a sample of spent CAPD fluid was found to be 2.5 ± 0.1 ng cm⁻³ and was too low to perform speciation analysis. Owing to its high salt content it was not possible to concentrate the CAPD fluid. Spiking of the sample with Al would also not reflect the original distribution between LMM–Al and HMM–Al species, because of its high salt content and the very low concentration of total serum proteins ranging between 0.5 and 1.5 mg cm⁻³.¹⁴ During the CAPD, excretion of Al from the serum, if it is present in elevated concentrations, takes place through the peritoneal membrane into the spent CAPD fluid. In our previous investigation it was proved that Al in spent CAPD fluid is mostly bound to serum proteins.¹⁴ It was presumed that the Al-binding protein was transferrin, but with the analytical procedure applied (SEC chromatography) it was not possible to confirm our predictions.¹⁴ In spite of low Al concentration in spent CAPD fluid, the speciation procedure combining SEC and anion-exchange FPLC with UV detection was nevertheless performed to estimate the ability to separate serum proteins. The corresponding UV chromatograms are presented in Fig. 3A and 3B. As in the case of separation of serum proteins (Fig. 2A and 2B), the protein peak is separated from LMM compounds on the SEC column (Fig. 3A). It is further evident that IgG, transferrin and albumin from the protein peak were completely separated on the anion-exchange FPLC column (Fig. 3B). These data indicate that speciation analysis of spent CAPD fluids by combining SEC and anion-exchange FPLC with UV and ETAAS detection may be performed when the total concentration of Al in spent CAPD fluid is higher than 10 ng cm⁻³ Al.

Fig. 3 Separation of a spent CAPD fluid at a flow rate of 1 cm³ min⁻¹ followed by UV detection at 278 nm. 1 cm³ of sample was first injected onto the SEC column (A). A protein peak was collected from 8.25 to 10.75 min (2.5 cm³). 0.5 cm³ aliquot of a protein peak was then injected onto the anion-exchange FPLC column (B).

Identification of the transferrin by the SDS-PAGE electrophoresis after separation of proteins on the anion-exchange FPLC column

In order to identify the Al-binding protein in spiked serum of a dialysis patient after final separation of proteins on the anion-exchange FPLC column, Al-containing fractions eluting from 5.25 to 6.0 min (Fig. 2B) were combined together and the sample subjected to the SDS-PAGE electrophoresis. A similar experiment was carried out in a spent CAPD fluid sample. After final separation of proteins on the anion-exchange FPLC column, fractions eluted from 5.25 to 6.0 min (Fig. 3B) were also subjected to the SDS-PAGE electrophoresis. Since protein concentrations in spent CAPD fluids are 50–100 times lower than those in serum samples,¹⁴ the fractions of two successive chromatographic separations, eluted on the anion-exchange FPLC column between 5.25 and 6.0 min, were collected (1.5 cm³) and concentrated before the SDS-PAGE electrophoresis was applied. To concentrate the sample microultrafiltration (30 000 Da) was used. Concentrated sample was rinsed with 1 cm³ of water to remove most of the remaining salts from the protein fraction and microultrafiltered again until the volume of the protein fraction was reduced to 0.15 cm³. After that the volume was reduced 10 times more by the use of vacuum assisted concentration. So, the sample was concentrated 100 times before the SDS-PAGE electrophoresis was applied. Data from these investigations are presented in Fig. 4. From left to right standard serum proteins IgG, transferrin and albumin are followed by the protein fractions of spiked serum of a dialysis patient and spent CAPD fluid, eluted from 5.25 to 6.0 min after the anion-exchange FPLC separation. Next to the samples examined, the standard serum proteins transferrin, albumin and IgG again followed. It is clear that in the fractions of serum and spent CAPD fluid investigated the only present protein is exclusively the transferrin. These data confirmed efficient separation of transferrin from other serum proteins on the anion-exchange FPLC column.

Fig. 4 SDS-PAGE electrophoresis of standard serum proteins (IgG, transferrin, albumin) and selected fractions of a spiked human serum of a dialysis patient and spent CAPD fluid. Serum sample and spent CAPD fluid represent fractions 5.25–6.0 min collected from an anion-exchange FPLC column after pre-separation of proteins on SEC column (Fig. 2B and Fig. 3B, respectively). Spent CAPD fluid fraction was concentrated 100 times before being subjected to SDS-PAGE electrophoresis.

Conclusions

The developed analytical procedure combining SEC and anion-exchange FPLC with UV and ETAAS detection enables reliable determination of the concentration and the composition of the HMM–Al species in spiked human serum samples. If only anion-exchange FPLC is used in speciation of Al bound to serum proteins, the possibility exists of the co-elution of the LMM–Al species and HMM–Al compounds, which may result in overestimation of the proportion of the Al bound to proteins. Therefore, the important novelty of the proposed analytical procedure is the pre-separation of LMM–Al from the HMM–Al species on the SEC column before applying the anion-exchange FPLC separation of proteins. It is also important that by the use of the proposed cleaning protocol, the blanks arising from eluents and supports of the chromatographic columns, were lowered to concentrations below 1 ng cm⁻³ Al. The developed analytical procedure was applied to the speciation analysis of spiked serum of a dialysis patient. It was found experimentally that 90 ± 5% of Al in spiked serum investigated corresponded to Al–transferrin. The presence of transferrin in the separated Al-containing fractions, after the anion-exchange FPLC separation, was also identified by the SDS-PAGE electrophoresis. It was also demonstrated that the analytical procedure developed may be applied to the speciation of Al in spent CPAD fluids when the Al concentration in samples analysed exceeded 10 ng cm⁻³.

In a continuation of our work, the proposed analytical procedure will be applied in speciation of Al in serum and spent CAPD fluids of dialysis patients consuming Al-based drugs.

Acknowledgements

This work was supported by Grant PO-0530-0106 from the Ministry of Education, Science and Sport of the Republic of Slovenia. The authors thank Barbara Savicki-Ponikvar of the University Medical Centre, Ljubljana, for collecting serum and spent CAPD samples.

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