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A convenient and rapid micro-anion exchange liquid chromatography (LC) tandem electrospray mass spectrometry (MS) procedure was developed for quantitative analysis in serum of O-isopropyl methylphosphonic acid (IMPA), the hydrolysis product of the nerve agent sarin. The mass spectrometric procedure involves negative or positive ion electrospray ionization and multiple reaction monitoring (MRM) detection. The method could be successfully applied to the analysis of serum samples from victims of the Tokyo subway attack and of an earlier incident at Matsumoto, Japan. IMPA levels ranging from 2 to 135 ng/ml were found. High levels of IMPA appear to correlate with low levels of residual butyrylcholinesterase activity in the samples and vice versa. Based on our analyses, the internal and exposure doses of the victims were estimated. In several cases, the doses appeared to be substantially higher than the assumed lethal doses in man.

The utility was explored of a new approach to detect retrospectively exposure to nerve agents by means of conversion of the inhibitor moiety bound to the active site of the enzyme BuChE in plasma with fluoride ions into a phosphofluoridate which is subsequently analyzed by means of gas chromatography (GC). This quantifies >or=0.01% inhibition of BuChE and identifies the structure of the inhibitor except for the original leaving group. A three-tiered approach was followed involving the five classical nerve agents GA, GB, GF, GD, and VX, as well as the active metabolite of parathion, i.e., paraoxon: in vivo experiments in rhesus monkeys after iv administration of a sign-free dose of agent and concomitant in vitro experiments in plasma of rhesus monkeys and humans should allow an assessment of in vivo retrospectivity in humans. A systematic investigation was performed in order to find a single set of reaction conditions which yields a maximum amount of phosphofluoridate for all nerve agents. Fluoride-induced reactivation at 25 degrees C at a final concentration of 250 mM KF during 15 min in a pH-range between 4 and 6 appears to be effective. The in vitro decrease with time in reactivatibility of inhibited BuChE in plasma from humans and rhesus monkeys was largely due to aging of the phosphyl moiety, except for VX where spontaneous reactivation was a major cause. The decrease followed first-order except for a biphasic course in the case of GF in human and rhesus monkey plasma as well as of GD in rhesus plasma. In vitro half-lifes in human plasma ranged between ca. 14 h for GB and ca. 63 h for GA. A comparison of the in vivo data from rhesus monkeys and the in vitro data is complicated by the observation that the in vivo decrease with time of fluoride-reactivated phosphofluoridate is biphasic for all nerve agents. The terminal in vivo phase pertains to a small fraction of the amount of initially regenerated phosphofluoridate but is responsible for a considerable degree of retrospectivity, ranging between 14 and 56 days for GF and GB, respectively. The new procedure can be used in a variety of practical applications, e.g., (i) biomonitoring in health surveillance at exposure levels that are several orders of magnitude lower than presently possible; (ii) diagnosis in case of alleged exposure to nerve agents in time of war or after terrorist attacks; (iii) in forensic cases against suspected terrorists that have handled organophosphate anticholinesterases; and (iv) in research applications such as investigations on lowest observable effect levels of exposure to nerve agents.

The relationship between hepatic glutathione content and hepatic glutathione conjugation rate in the rat in vivo was investigated. As substrate for glutathione conjugation, racemic (R,S)-2-bromoisovalerylurea (BIU) was used which gives rise to the biliary excretion of two diastereoisomeric glutathione conjugates and the urinary excretion of two diastereoisomeric mercapturates. The excretion rate of the glutathione conjugate in bile reflects hepatic conjugation exclusively. An intravenous infusion of BIU was given and the excretion rates of the metabolites in bile and urine were determined. The glutathione concentration in the liver was followed by taking biopsies every hour. Glutathione was depleted by the infused substrate; in rats that were pretreated with the inhibitor of glutathione biosynthesis, buthionine sulphoximine (BSO), the depletion of the glutathione content was more rapid. The rate of excretion of the glutathione conjugate in bile was plotted against hepatic glutathione content. These results indicate that the 'organ Km' for glutathione in the liver is approximately 0.5 mumol/g of liver, so that the hepatic glutathione conjugation rate is decreased only at severe glutathione depletion.

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