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Speciation of aluminium and silicon in serum was studied by a reliable and sensitive high-performance liquid chromatographic-electrothermal atomic absorption spectrometric (HPLC-ETAAS) hybrid method, based on the use of a polymeric anion-exchange column (Protein-Pak DEAE-5PW). This polymer-based column minimizes the risk of aluminium losses and of silicon contamination from the column during separation. The results obtained were compared with the results of previous studies carried out using different, complementary techniques including ultramicrofiltration, gel filtration and silica-based column for HPLC. In order to ascertain which protein(s) of serum actually bind(s) aluminium, gel electrophoresis was employed for the further separation of the column fractions obtained by HPLC and aluminium was determined in separate aliquots of the same fractions. From all the experiments, it appears that transferrin (Tf) is the only serum protein that binds aluminium and it contains about 90% of total serum aluminium. It was also confirmed that in the presence of desferrioxamine (DFO). aluminium is partly displaced from its complex with transferrin to a low molecular mass AL-DFO complex. Aluminum citrate seems to be the main low molecular mass aluminium species in serum, amounting to about (12 +/- 5% of the total aluminium in an aluminium-loaded serum sample. The proposed speciation procedure permits the simultaneous identification and determination of three aluminium species in metal-spiked serum (Al-Tf, Al-DFO and AI-citrate). The result for silicon suggest that it seems to be unspecifically adsorbed to several serum proteins and its speciation is not affected by the presence of DFO.(ABSTRACT TRUNCATED AT 250 WORDS)

Fast protein liquid chromatography (FPLC) was used with electrothermal atomic absorption spectrometric (ETAAS) detection for quantitative studies of aluminium binding species in unspiked human uremic serum. A rapid and reproducible separation of human serum proteins and other aluminium binders (citrate and desferroxiamine) was achieved on a Mono Q (HR 5/5) anion-exchange column using a sodium chloride gradient (0-0.25 mol l-1) at the physiological human serum pH of 7.4 (0.05 mol l-1 buffer TRIS-HCl). The aluminium distribution in the column fractions was determined by ETAAS. Aluminium contamination was avoided by using an inert chromatographic system equipped with an on-line aluminium-chelating scavenger column (Kelex 100-impregnated silica C18). The sensitivity of the proposed method (detection limit for Al in serum = 5 micrograms l-1) allowed aluminium speciation studies at clinically relevant concentrations (unspiked serum from dialysis patients). The results obtained confirmed that transferrin is the only serum protein binding aluminium and it contains about 90% of total serum aluminium (post-elution aluminium recovery = 105 +/- 5%). It was also confirmed that in the presence of the chelating drug desferrioxamine (DFO) most of the serum aluminium (80%) is bound to DFO.

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The analytical potential of fast protein liquid chromatography (FPLC ) coupled with a double focusing inductively coupled plasma mass spectrometer (DF-ICP-MS ) was evaluated for the multielemental speciation of trace elements in human serum. The separation of the main serum proteins was performed in a MonoQ (HR5/5) anion exchange column using an ammonium acetate gradient (0-0.5 mol l−1) at the physiological pH of 7.4 (0.05 mol l−1 TRIS-HCl buffer) as the mobile phase. Proteins were first detected on-line spectrophotometrically at 295 nm and specific detection of metals was also carried out on-line by using a double focusing ICP-MS working at both low (m/Dm= 300) and medium resolution (m/Dm=3000) in order to avoid possible polyatomic interferences. The use of variable resolution to carry out multielement speciation studies allowed the detection of Ca, Sr, Fe, Cu, Zn, Se, Mn, Cr, Pb, Al and Sn in different serum samples and introduces a new dimension to this area of investigation. From the multielemental study it was observed that detectable levels of Cr, Al, Pb and Sn were present in uraemic sera while those elements could not be detected in healthy subjects.Using resolution settings (m/Dm) up to 3000, many well known

Serum transferrin (Tf) is an iron binding glycoprotein that plays a central role in the metabolism of this essential metal but it also binds other metal ions. Four main transferrin forms containing different iron binding states can be distinguished in human serum samples: monoferric (C-site or N-site), holotransferrin (with two Fe atoms) and apotransferrin (with no metal). Recently, it has been reported that Tf binds also Ti even more tightly than does Fe, in artificially Ti(iv) spiked solutions. However, very limited work has been done on the Ti binding to Tf at physiological concentrations in patients carrying intramedullary Ti nails. Here we report the chemical association of Ti to Tf "in vivo" under different chromatographic conditions by elemental mass spectrometry using double focusing inductively coupled plasma (DF-ICP-MS) as detector. For the separation of the Ti/Fe-Tf forms different gradient conditions have been explored. The observed results reveal that human serum Ti (from patients carrying intramedullary Ti nails) is uniquely associated to the N-lobe of Tf. The investigation of the influence of sialic acid in the carbohydrate chain of human serum Tf, studied by incubating the protein with neuraminidase (sialidase) to obtain the monosialilated species, revealed that the binding affinity of Ti was similar for monosialo-Tf and for native-Tf and occurs in the N-lobe. These results suggest that the species Fe(C)Ti(N)-TF might provide a route for Ti entry into cells via the transferrin receptors after the release of the metal from its implants.