A light addressable potentiometric sensor (LAPS) was used to detect organophosphate and carbamate anticholinesterases (anti-ChEs), using eel acetylcholinesterase (AChE) as the biological sensing element.Biotinylated AChE was preincubated with inhibitor or buffer alone and then captured on biotinylated nitrocellulose membrane via streptavidin cross-linking, or AChE was preimmobilized on the capture membrane and then a sample containing the anti-ChE was filtered through the capture membrane. Hydrolysis of acetylcholine (ACh) by the captured AChE resulted in a strong potentiometric signal, and the immobilized AChE retained its affinity for ACh and anti-ChEs. IC50 values for inhibition of captured AChE obtained by the LAPS agreed with those obtained by a spectrophotometric method or a fiber optic evanescent fluorosensor. Paraoxon and bendiocarb were detected at 10 nM, while higher concentrations were required for monocrotophos, dicrotophos, dichlorvos, phosdrin, diazinon, tetraethyl pyrophosphate, aldicarb, and methomyl. Important features of the LAPS for detection of anti-ChEs are speed (eight samples assayed simultaneously in minutes), precision, and reusability. INTRODUCTIONDetection of anticholinesterases (anti-ChEs) is of major concern to the agriculture chemical industry, regulating agencies (e.g., FDA, EPA), health care professionals, and the Department of Defense. In addition to the physical and chemical assay methods [e.g., gas chromatography, high-pressure liquid chromatography (Conaway, 1991;Sharma et al., 1990), and immunoassay (Kaufman and Clower, 1991)], several methods using acetylcholinesterase (AChE) have been developed. These enzymatic assays include methods dependent on a change in pH, measured electrometrically (Michel, 1949) or titermetrically (Jacobsen et al., 1957), or on a change in color [e.g., Ellman etal. (1961)]. Other methods used to assay cholinesterase (ChE) activity radiometrically (Potter, 1967;Lewis and Eldefrawi, 1974) {ire too slow and use hazardous radioactive materials, while the histochemical method (Koelle and Friedenwald, 1949) is not applicable for screening or detection purposes in body fluids or environmental samples.The advent of biosensor technology has renewed interest in developing better detection methods for anti-ChEs using AChE as the sensing element. AChE enzyme electrodes were developed by immobilizing the enzyme protein on glass electrodes and were used to detect anti-ChEs (Baum and Ward, 1971;Durand et al., 1984). More sensitive fiber optic biosensors, using ChEs as their sensing elements, have also been developed to detect anti-ChEs (Rogers et al., 1991a;Hobel and Polster, 1992;Skladal and Mascini, 1992;Marty et al., 1992).Recently, a light addressable potentiometric sensor (LAPS) (Molecular Devices Corp.,

—Three weeks after porto‐caval anastomosis, tryptophan and 5‐hydroxyindolylacetic acid concentrations were‐greatly increased in rat brain regions. 5‐Hydroxytryptamine showed smaller increases. Midbrain tyrosine and muscle tyrosine and tryptophan concentrations were also increased. Striatal dopa‐mine concentration was not significantly changed. Unlike previous results from acute liver failure, brain tryptophan changes in this chronic study did not simply reflect plasma‐free tryptophan changes. Midbrain tryptophan/plasma‐free tryptophan ratio and midbrain tyrosine/plasma tyrosine ratio both rose, suggesting increased effectiveness of uptake of these amino acids from plasma by brain. Corresponding muscle/plasma ratios were unaltered by the porto‐caval anastomosis. Uptake of tryptophan from buffer by cerebral cortex slices was unaffected. Results on control animals illustrate the importance of plasma‐free tryptophan in the normal physiological control of brain tryptophan.

The effect of ad libitum dietary exposure (as occurs in the field) to parathion for 14 d was investigated on the muscarinic acetylcholine receptor (mAChR) in brains and submaxillary glands of adults of a field species, the white-footed mouse Peromyscus leucopus. Immunoprecipitation using subtype selective antibodies revealed that the relative ratios of the m1-m5 mAChR subtypes in Peromyscus brain were similar to those in rat brain. There was little variability in acetylcholinesterase (AChE) activity in control mice brains but large variability in 39 exposed mice, resulting from differences in food ingestion and parathion metabolism. Accordingly, data on radioligand binding to mAChRs in each mouse brain were correlated with brain AChE activity in the same mouse, and AChE inhibition served as a biomarker of exposure reflecting in situ paraoxon concentrations. Exposure to parathion for 14 d reduced maximal binding (Bmax) of [3H]quinuclidinyl benzilate ([3H]QNB), [3H]-N-methylscopolamine ([3H]NMS), and [3H]-4-diphenylacetoxy-N-methylpiperidine methiodide ([3H]-4-DAMP) by up to approximately 58% without affecting receptor affinities for these ligands. Maximal reduction in Bmax of [3H]QNB and [3H]-4-DAMP binding occurred in mice with highest AChE inhibition, while equivalent maximal reduction in Bmax of [3H]NMS occurred in mice with only approximately 10% AChE inhibition, without further change at higher parathion doses. This is believed to be due to the hydrophilicity of [3H]NMS, which limits its accessibility to internalized desensitized receptors. In submaxillary glands (mAChRs are predominantly m3 subtype), there were significant dose-dependent reductions in [3H]QNB binding and m3 mRNA levels in exposed mice, revealed by Northern blot analyses. The reduction in m3 receptors is suggested to result mostly from reduced synthesis at the transcription level, rather than from translational or posttranslational events. The data suggest that down-regulation of mAChRs occurs after dietary exposure for 14 d to sublethal concentrations of parathion in a field rodent species, and that significant though incomplete recovery in AChE and mAChRs occurs in 7 d following termination of exposure.

I An investigation was made into the effects of drugs which alter insulin secretion on the concentrations Of to hanWandacids in plasma and brain and on 5-hydroxv- streptozotocin, propranolol, tolbutamide and phentolamine. 2 Tolbutamide and phentolamine increased the plasma insulin concentrations by 100% and 300% respectively but with little effect on the brain/plasma ratios for the aromatic amino acids. Previously propranolol was found to decrease plasma insulin by 50% without altering the above ratios. The ratios were decreased by streptozotocin but only when plasma insulin fell by more than 50%. 3 Phentolamine and propranolol did not alter the brain/plasma ratios for the aromatic amino acids in streptozotocin-treated rats. 4 The results suggest that only large changes of insulin secretion e.g. those associated with food intake or aminophylline injection are likely to alter appreciably the brain/plasma ratios for the aromatic amino acids. STolbutamide displaced tryptophan from its binding to plasma albumin and increased brain 5-HIAA probably by inhibiting 5-HIAA efflux from brain. The other drugs did not alter brain 5-HT or 5-HIAA concentrations.

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