ISSN: 0022-1198
CODEN: JFSCA
Published Online: 4 February 2004
Page Count: 9

Reliable, Sensitive, Rapid and Quantitative Enzyme-Based Assay for Gamma-Hydroxybutyric Acid (GHB)
Harris, DO
University of California, California

Bravo, DT
University of California, California

Parsons, SM
University of California, California

(Received 10 May 2003; accepted 18 October 2003)

Abstract

Several assays for gamma-hydroxybutyrate (4-hydroxybutyrate, GHB) have been developed based on the enzyme gamma-hydroxybutyrate dehydrogenase (GHB-DH). Enzymatic oxidation of GHB by NAD+ is coupled to diaphorase-mediated reduction of pro-dye to yield colored product. GHB-DH from Ralstonia eutropha was cloned and expressed as a stable fusion protein easily purified by affinity chromatography. Quantitative initial velocity and endpoint versions of the assay in solution are described. Michaelis-Menten parameters for oxidation of GHB and ethanol were estimated. A semi-quantitative “dipstick” version of the assay on paper also is described. Both solution endpoint and “dipstick” assays are sensitive to about 0.05 mg GHB/mL using 10 μL of sample. Ethanol at concentrations possible in urine and agents used to stabilize physiological fluids for forensics analysis do not interfere significantly. The “dipstick” assay also allows detection of GHB in alcoholic beverages after evaporation of about one-fourth drop of beverage before testing. The enzymatic assay for GHB is reliable, sensitive, inexpensive and rapid.

Keywords:
forensic science, GHB, gamma-hydroxybutyrate dehydrogenase, 4-hydroxybutyrate, tetrazolium pro-dye, enzymatic detection

Paper ID: JFS2003165
DOI: 10.1520/JFS2003165
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