ABSTRACT
Hepatocyte cytosol contains a multitude of proteins, but also comparatively high concentrations of l-glutathione (GSH, ~5.0 mM) and L-cysteine (Cys, ~0.5 mM). Since Hg(2+), CH(3)Hg(+) and Cd(2+) have a high affinity for thiols, their coordination to these thiols is likely involved in their intracellular transport. The comparative coordination behavior of these metal species towards mixtures of Cys and GSH, however, has not been studied under near physiological conditions. To probe these toxicologically relevant interactions, each metal species was separately injected onto a C(18)-HPLC column (37°C) that had been equilibrated with phosphate buffered saline (PBS) that contained 5.0 mM GSH (mobile phase) and detected with an inductively coupled plasma atomic emission spectrometer. The incremental increase of the Cys concentration in the mobile phase (in 0.5 or 1.0 mM steps) up to 10mM followed by the chromatography of each metal species decreased the retention of Hg(2+) and CH(3)Hg(+) albeit in a different manner. This behavior was rationalized in terms of the replacement of hydrophobic GS-moieties coordinated to each mercurial by less hydrophobic Cys-moieties. In contrast, a Cd-peak eluted close to the void volume with all investigated mobile phases. Using X-ray absorption spectroscopy, the Cd-compound that eluted with a PBS-buffer that contained 5.0 mM GSH was structurally characterized as tetrahedral (GS)(4)Cd. Thus, the in vivo formation of (GS)(4)Cd must be considered and HPLC-ICP-AES is identified as a useful tool to probe dynamic bioinorganic processes which involve the interaction of a metal ion with multiple ligands under physiologically relevant conditions.
Subject(s)
Cadmium/chemistry , Chromatography, High Pressure Liquid/methods , Mercury/chemistry , Methylmercury Compounds/chemistry , Spectrophotometry, Atomic/methods , Sulfhydryl Compounds/analysis , X-Ray Absorption Spectroscopy/methods , Cations, Divalent/chemistry , Cysteine/analysis , Cysteine/chemistry , Glutathione/analysis , Glutathione/chemistry , Glutathione Disulfide/analysis , Hydrophobic and Hydrophilic Interactions , Sulfhydryl Compounds/chemistryABSTRACT
Arsenobetaine, which is frequently ingested by humans via the consumption of seafood, is rapidly excreted unchanged in urine, but not much is known about its transport in the mammalian bloodstream. To assess whether this transport involves binding to plasma proteins, rabbit and human plasma were spiked with arsenobetaine and the mixture was analyzed (after 5 min and again after 6 h) by size-exclusion chromatography (SEC) coupled on-line to an inductively coupled plasma atomic emission spectrometer (ICP-AES). Simultaneous monitoring of the emission lines of As, Cu, Fe and Zn in the column effluent allowed us to determine the elution of arsenobetaine relative to that of the major Cu, Fe and Zn-containing metalloproteins. Over the investigated time period, a single As peak eluted near the inclusion volume on two different SEC columns with fractionation ranges of 600-10 KDa and 7000-100 Da. These results indicate that arsenobetaine did not bind to plasma proteins and that SEC-ICP-AES is a useful tool to rapidly probe toxicologically and pharmacologically-relevant interactions between organometalloid compounds and mammalian blood plasma constituents in vitro.
