This R21 application request support for the development of a rapid method for: gamma-hydroxybutyrate (GHB) detection and measurement in biological fluids. An increasing number of reports indicate that the abuse of GHB is widespread. GHB is regularly encountered in cases of intoxicated drivers and fatal GHB overdoses are reported throughout the country. In Dade County, Florida, a number of fatal intoxications have been documented, some of them due to GHB alone. There are also reports suggesting that GHB is often used to facilitate sexual assault. Particularly in suspected poisoning cases, the need for a rapid method to detect and quantify GHB is obvious, since the symptomatology may be similar to, but the management and treatment different from, that of other kinds of intoxications. The analytical methods currently available are mainly based on gas chromatography with flame ionization (GC-FID) or mass spectrometric (GC-MS) techniques that involve laborious extraction steps for sample preparation. These methods are very time-consuming and inexpedient for use in emergency settings. We propose to develop an enzymatic assay based on the conversion of GHB to succinic semialdehyde as a rapid and specific method for the detection and measurement of GHB in biological fluids. This reaction is catalysed by a group of highly selective GHB dehydrogenase enzymes that utilize either NADP+ or NAD+ as cofactors. The NADPH or NADR produced in stoichiometric quantity by the reaction can be measured spectrophotometrically. Our approach is based upon the same principles as the classic alcohol dehydrogenase method for analysis of ethanol with substitution of highly selective GHB dehydrogenase. The GHB dehydrogenase of Syrian hamster liver, which has been purified and well characterized, will be used to evaluate the accuracy and reliability of the rapid spectrophotometric method of analysis. The rapid and sensitive analytical approach based on 4-hydroxybutyrate catabolism and spectrophotometric detection of the reaction product will be evaluated by comparing the results with a highly specific GC-MS method, currently in use at the toxicology unit of the Miami-Dade County Medical Examiner Office under the direction of Dr. W. Lee Ream. A novel approach will be to extend the methods developed in hamster liver for use with GHB dehydrogenase of Clostridium kluyveri. In this approach, a plasmid vector containing genes that express stable levels of 4-hydroxybutyrate dehydrogenase is grown and expressed in Escherichia coli. The enzyme will be isolated from the culture medium and employed in a reagent system adaptable to automated chemistry analyzers of the types used in medical and drug screening laboratories. In addition, we plan to extend the enzymatic method to develop a rapid spot test based on the same principle. Proof-of-concept and inter-assay validity will be evaluated by comparing the results of both these methods with GC-MS. Both of these methods will be tested on GHB-spiked controls and residual blood and urine specimens obtained from the Miami-Dade County Medical Examiner Department and the Emergency Department of Jackson Memorial Hospital.
