Jurgen De Kimpe

Speciation of toxicologically important arsenic species in human serum by liquid chromatography–hydride generation atomic absorption spectrometry

An on-line method was developed for the speciation of trivalent and pentavalent inorganic arsenic (As), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) in serum. The method is based on HPLC separation of As species and continuous HGAAS for the measurement of As in the HPLC eluate. Three types of HPLC system were evaluated in order to find a method that was applicable to serum samples: reversed-phase ion-pair chromatography, polymer-based anion-exchange chromatography and silica-based anion-exchange chromatography. As the last-named appeared more suitable, the analytical conditions were optimized, and a procedure is proposed for the speciation of As species in serum. Phosphate buffer of pH 4.5 at a concentration of 30 mmol l–1 was used as the mobile phase. Serum samples were adjusted to the same pH as that of the mobile phase before injecting them onto the column. The detection limits were 0.49, 0.44, 0.92 and 0.40 µg l–1 for AsIII, AsV, MMA and DMA in serum, respectively. The HPLC separation, hydride generation and AAS measurement could be completed on-line within 10 min. The method was applied to the speciation of As in the serum of patients with chronic renal disease. The only As species that could be measured in the serum of the patients was DMA.

In Vitro Methylation of Arsenite by Rabbit Liver Cytosol: Effect of Metal Ions, Metal Chelating Agents, Methyltransferase Inhibitors and Uremic Toxins

The methylation of carrier-free 74As-arsenite by liver cytosol of Flemish Giant rabbits is highly susceptible to additions of trace elements. In vitro supplementation of essential trace elements like zinc (Zn2+), vanadium (V5+), iron (Fe2+), copper (Cu2+) and selenate was shown to increase the methylation efficiency. Trivalent metal ions (e.g. Al3+, Cr3+ and Fe3+), Hg2+, Tl+ and SeO3(2-) had a deleterious effect. The inhibitory effect of EDTA, oxime and many divalent cations (Ca2+, Mg2+, Sr2+, ...) suggest a co-factor role for a specific divalent metal ion, possibly Zn2+. Chelating agents used in clinical treatment of acute and chronic inorganic arsenic poisoning lower the methylation capacity of cytosol by rendering the trivalent arsenic unavailable for the methyltransferase enzymes. S-adenosylhomocysteine and periodate-oxidized adenosine, inhibitors of s-adenosylmethionine dependent methylation pathways, inhibit the methylation of arsenite. Pyrogallol, a catechol-O-methyltransferase inhibitor, blocks the action of arsenite- and monomethylarsonic methyltransferase enzymes, suggesting a close structural relationship between the active sites of the different enzymes. Some uraemic toxins, namely oxalate, p-cresol, hypoxanthine, homocysteine and myo-inositol, inhibit arsenic methylation.

Elemental speciation in biological fluids. Invited lecture

Elemental speciation in biological fluids implies investigation of the bond between the trace element and available ligands, mostly proteins or low relative molecular mass compounds, as a basis for kinetic and metabolic studies. Strategies of procedures are outlined and attention is drawn to the many difficulties that can be encountered. These include the complexity of the matrix, insufficient specificity of separation of the biocompounds, fortuitous contaminations with trace elements, breaking-up of the original metal–protein binding. Identification and quantitative determination of the biocompounds to which the trace element is linked and accurate measurement of the trace element in the fractions are quintessential. This is illustrated with As speciation studies in rat urine and red blood cell lysate, and with Cr speciation studies in serum using radiolabels.

Platinum speciation in clinical and environmental samples: Scrutiny of data obtained by using electrophoresis techniques (flatbed and capillary)

The commercially available and widely used flatbed electrophoresis apparatus PhastSystem and MultiPhor II (Amersham Pharmacia Biotech, Uppsala, Sweden) were checked for the possible release of significant amounts of platinum from the electrodes during isoelectric focusing (IEF) and native polyacrylamide gel electrophoresis (PAGE). Capillary electrophoresis (CE; Biofocus 3000; Bio‐Rad, Munich, Germany) in zone electrophoresis (CZE) mode was investigated for the same purpose. Platinum analysis was done by inductively coupled plasma mass spectrometry (quadrupole and magnetic sector field) either “off‐line” for all flatbed gels or “on‐line” for the CE measurements. The buffers and process chemicals did not significantly leach platinum from the electrodes. During flatbed electrophoresis, application of the electrical field, however, released high platinum amounts exceeding by far the amount of platinum originally present in the sample. For CE, no platinum was released from the electrodes. The results are strongly dependent on the system and conditions used. The results presented in this paper underline the necessity to replace the platinum electrodes with ultrapure gold electrodes whenever investigating platinum species. Previous literature data, in which electrophoresis was used for platinum speciation without mentioning the platinum recoveries, becomes questionable.

Capability of flatbed electrophoresis (IEF and native PAGE) combined with sector field ICP-MS and autoradiography for the speciation of Cr, Ga, In, Pt and V in incubated serum samples

The capability of flatbed electrophoresis combined with sector field ICP-MS (ICP-SFMS) and autoradiography is reported. Human serum and rabbit serum were separated by IEF and native PAGE using a PhastSystem unit (Amersham Pharmacia Biotech, Uppsala, Sweden). Rabbits (Flemish Giant) received intraperitoneal injections of ‘cold’ Ga(III) and In(III) in physiological buffer. Human serum was incubated in vitro with the radiotracers 51 Cr(III), 191 Pt(II) or carrier-free 48 V(V). Measurement of the protein-bound metals followed two different strategies: (A) ICP-SFMS, in which each rabbit serum sample was run twofold and, after separation, one gel was silver stained and the other replicate was cut into segments, which were digested and measured for Ga and In by ICP-SFMS; (B) autoradiography, in which, after separation, the gels were exposed to a phosphorus screen at –20 °C, an autoradiogram was obtained by laser densitometry and subsequently the proteins were detected by silver staining. Both strategies have advantages and drawbacks, but both are powerful and highly sensitive and allow the attribution of the respective metals to serum proteins. The suitability of these methods for trace element speciation depends strongly on the stability of the trace element-protein binding.

In vitro Methylation of Arsenite in Flemish giant rabbit Cytosol

The formation of 74As‐monomethyIarsonic acid and 74As‐dimethylarsinic acid from carrier‐free radiolabelled 74As‐arsenite was evaluated in an assay mixture containing 17.6% liver cytosol from the Flemish Giant rabbit. The optimal incubation temperature and pH were respectively, 39°C and 7.6. After a 2h incubation about 90% of 74As was protein bound. Protein bound 74As was released by hot 2 M HNO3 (1 min, 110°C). The treatment did not destroy methylated As‐species. Up to 70% of the carrier‐free 74As‐arsenite was methylated. Liver cytosol was stored, without loss of activity, in liquid nitrogen in the presence of 2 mM glutathione. The optimal s‐adenosyl‐methionine concentration was 1.7mM. Formation of 74As‐monomethylarsonic acid and 74As‐dimethylarsinic acid increased up to a glutathione concentration of respectively 10 and 20 mM. Methylation also went through in the presence of other reducing agents and proved to be ATP dependent.