Investigation of low molecular weight Al complexes in human serum by fast protein liquid chromatography (FPLC)-ETAAS and electrospray (ES)-MS-MS techniques

Abstract

Speciation of low molecular weight (LMW) Al complexes was performed in human serum from eight healthy volunteers in order to investigate the individual variability in the percentage and composition of LMW-Al species. Spiked samples (100±120 ng cm 23 Al 3z ) were microultra®ltered through a membrane ®lter (cut-off 30 000 Da) to separate Al bound to transferrin from LMW-Al complexes. A 0.5 cm 3 volume of the ®ltrate was injected onto an anion-exchange fast protein liquid chromatography (FPLC) column and aqueous 4 mol dm 23 NH 4 NO 3 linear gradient elution was applied for 10 min to separate LMW-Al complexes. Fractions of 0.2 cm 3 were collected throughout the chromatographic run and Al was determined `off-line' by electrothermal atomic absorption spectrometry (ETAAS). The characterisation of LMW-Al species in spiked serum was performed not only on the basis of the retention time (ETAAS detection), but also by electrospray (ES)-MS-MS analysis. A tandem quadrupole mass spectrometer equipped with a Z spray ion source as LC-MS interface was used for the identi®cation of LMW ligands eluted under the chromatographic peaks. It was found experimentally that the amount of LMW-Al species in spiked serum ranged from 14 to 55%. On the basis of FPLC-ETAAS and ES-MS-MS analysis, it was found that the main LMW-Al species present in serum were Al-citrate, Alphosphate and ternary Al-citrate-phosphate complexes. The distribution of these species varied among particular individuals. In some of them Al-citrate and Al-phosphate were the main LMW-Al species in serum, while in others the ternary Al-citrate-phosphate complex was also present. The serum of some other individuals did not contain Al-phosphate and the main LMW-Al species were either Al-citrate and Al-citrate-phosphate complexes or Al-citrate species alone. The limit of detection for the separated Al species on the FPLC column was 5.0 ng cm 23 , while the RSD was found to be 8%.

Demonstration of the Importance of Metal Ion Speciation in Bioactive Systems

“…42 Harris et al, 36 using difference UV spectroscopy as the basic method and supported by pH-metric and ESI-MS measurements, have reported a model for the biospeciation of Al III in serum at %10 mM Al III concentration, which is much closer to the biologically relevant value, of %0:1{0:3 mM. 43,44 Their speciation model, given in Table 3, suggests that 93% of the total Al III is bound to transferrin. Of the pool of l.m.m.…”

“…A comparison of the NMR spectrum with the speciation curves calculated data acquired under the experimental conditions of the NMR measurements indicates practically complete agreement between the speciation results obtained by two independent methods. 44 have studied the speciation of l.m.m. Al III complexes in human serum, which were taken from healthy volunteers, by means of fast liquid chromatography and electrospray techniques.…”

Morin applied in speciation of aluminium in natural waters and biological samples by reversed-phase high-performance liquid chromatography with fluorescence detection

“…The most toxic Al in tea infusion was not detected, because all of Al in tea binds to high molecular weight (HMW) hydrolyzable polyphenols irrespective of the origin of the tea, and morin could not compete with the powerful natural organic ligands for free Al [33,34]. The main carriers of Al in serum were demonstrated to be transferrin (80-90%), citrate and phosphate (10-20%) and a very little Al exists freely [35,36]. The composition of cerebrospinal fluid is relative simple, in which there are rich adrenergic neurotransmitters [37,38], so important Al forms are complexes of these neurotransmitters with Al.…”

Identification, characterization and determination of metal-binding proteins by liquid chromatography. A review

“…Ion exchange chromatography is a widely used technique for both analytical [26,51] and preparative [52,53] separations of proteins.…”

“…He presents a summary of the evolution of the techniques applied to speciate aluminium in uremic samples, highlighting the use of IEC coupled to ICP-MS. Aluminium seems to be distributed among transferrin and some other smaller complexes such as Al-citrate. The structure of the different isoforms of Al-transferrin has been studied by Hamano and Maitani [67], using also HPLC coupled to high resolution ICP-MS. Studies have also been done on the smaller complexes of aluminium [51].…”