J. De Kimpe

Trace elements in medicine: Speciation: the new frontier

Abstract Speciation of trace elements in body fluids and tissues of defining the many biocomponents to which the trace elements are bound and to explain their mobility, storage, retention and toxicity. Such new frontiers are now open for interdisciplinary research. It is necessary to maintain the integrity of the metal-ligand binding and to check the mass balance of the protein and the trace element throughout the isolation steps. A distinction is made between chemical speciation of the trace element and speciation as a part of cytology. Some methods are discussed for the speciation of As and Cr in serum, together with the limitations of the speciation procedure. It is assumed that the biomolecule and the trace element which are subsequently detected in the same fraction, are indeed associated with one another. This is not necessarily the case, so that confirmation with different means of identification of the biocomponent is imperative. A major problem in speciation work is due to the fact that immense hazards exist for contamination and losses of the trace element. In cytology, meaningful speciation cannot be achieved by ultracentrifugation of the different cell organelles. The best approach nowadays is to use microbeam techniques which are able to locate the trace element in situ, and even to attribute a semiquantitative value to its presence.

Arsenic speciation in serum of uraemic patients based on liquid chromatography with hydride generation atomic absorption spectrometry and on-line UV photo-oxidation digestion

Abstract An on-line method was developed for the speciation of arsenic species in human serum, including monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenobetaine (AsB) and arsenocholine (AsC). The method is based on cation-exchange liquid chromatography (LC) separation, UV-photo-oxidation for sample digestion and continuous hydride generation (HG) atomic absorption spectrometry (AAS) for the measurement of arsenic in the LC eluent. By developing the technique of argon segmented flow in the post-column eluent, an substantial improvement in Chromatographic resolution for the separation of these four arsenic species was obtained. The LC separation, photo-oxidation, hydride generation and AAS measurement could be completed on-line within 10 min. The detection limits were 1.0, 1.3, 1.5 and 1.4 μg/l of arsenic for MMA, DMA, AsB and AsC, in serum respectively. The concentration of the 4 species was determined in serum samples of patients with chronic renal insufficiency. Only AsB and DMA were significantly detected by the present method. The main part of arsenic in human serum is AsB. No MMA, AsC and inorganic arsenic were detected in these 6 samples.

More than Tenfold Increase of Arsenic in Serum and Packed Cells of Chronic Hemodialysis Patients

Arsenic concentrations were determined in serum and packed cells of 7 chronic hemodialysis patients, in fresh dialysate and in a heparin solution. The analytical technique was radiochemical neutron activation analysis. The accuracy of the method was tested by the analysis of As in certified reference materials. Patients showed elevated serum and packed cell arsenic concentrations compared with controls (serum, range: 2.3-79.8 ng As/ml, median: 11.5 ng As/ml; versus range: 0.132-4.783 ng As/ml, median: 0.38 ng As/ml; packed cells, range: 2.1-68.4 ng As/g, median: 9.5 ng As/g; versus range: 0.51-14.44 ng As/g, median: 3.17 ng As/g). Arsenic concentrations remained unaltered, before versus after a single hemodialysis treatment. The arsenic contents of serum and packed cells were significantly correlated (n = 7, r = 0.96, p < 0.05). No arsenic could be detected in the heparin solution or in the dialysate.

Trace elements in clinical samples revisited—speciation is knocking at the door. Sample preparation, separation of the species and measurements methods

The measurement of the different species of the trace elements in clinical samples requires a sample preparation and handling that is specially designed for that purpose. The present article is an attempt to give background information about the various procedures. Some of the difficulties that may affect the separation and the stability of the species will be highlighted with practical examples. The intent is not to present a review of the existing knowledge about all the different species of the trace elements, neither to give procedural details in various biological fluids and tissues, but rather to initiate the reader who wants to start research on this topic.

74As-arsenate metabolism in flemish giant rabbits with renal insufficiency

The metabolic management of carrier-free 74As-arsenate (As(V)) by uremic rabbits of the strain Flemish Giant was studied. Renal insufficiency was induced by nephrectomy of respectively 1 kidney (3/6 nephrectomy) and 1 kidney + 2/3 remaining kidney (5/6 nephrectomy). Marginal renal insufficiency developed in the 3/6 nephrectomized group, while animals of the 5/6 group became severely uremic. Renal excretion of 74As was reduced by 90% in 5/6 nephrectomized animals 4 h after intraperitoneal injection (i.p.) of the animals. The associated uremic syndrome caused a strong decrease in methylation capacity of inorganic arsenic (Asi). The second methylation step from monomethylarsonic acid (MMA) to dimethylarsinic acid (DMA) was more strongly affected than the first one, from arsenite (As(III)) to MMA. The increased availability of Asi led to more extensive binding to insoluble tissue constituents after 5/6 nephrectomy. The decreased renal reduction of As(V) led to a decrease in As(III) and an increase of As(V) and the associated As(V)-transferrin binding in plasma. Uptake of 74As-transferrin by the bone marrow might contribute to uremic anemia.

Dose Dependent Changes in 74As-Arsenate Metabolism of Flemish Giant Rabbits

The metabolic handling of 74As-arsenate (As(V)) was studied in rabbits injected intraperitoneally (i.p.) with increasing doses of As(V) (0.00 to 1.00 mg As(V)/kg/day) over a period of 10 days. Plasma, packed cells, urine from the bladder and several tissues were analyzed for their 74As content and presence of 74As-As(V) metabolites 4 h after administration of 74As-As(V). 74As showed strong increases with increasing As(V) dose in nails and bone whereas in fat, thyroid and kidneys it decreased. Also with increasing As(V) dose, arsenate was less efficiently methylated to dimethylarsenic acid (DMA) and became more bound to insoluble tissue constituents. As a result 74As-DMA levels in tissues were systematically lower in the groups of rabbits receiving the higher doses, be it with a wide variation from one type of tissue to the other. The behaviour of 74As-monomethylarsonic acid (MMA) was different. The levels did not decrease significantly, occasionally even increased compared to the control group, indicating that especially the second methylation step is sensitive towards increasing doses of As(V). 74As-arsenite (As(III)), formed by in vivo reduction of As(V), reached maximal levels in the 0.25 mg As(V)/kg/day group as a result of the inhibited methylation. At doses > 0.25 mg As(V)/kg/day the amount of 74As-As(V) increased especially in plasma, packed cells and the urine in the bladder, indicative for a less efficient reduction of As(V).