Reliable, sensitive, rapid and quantitative enzyme-based assay for gamma-hydroxybutyric acid (GHB)

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 w...

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Bibliographic Details
Published in:Journal of forensic sciences 2004-03, Vol.49 (2), p.379-9
Main Authors: Bravo, Dawn T, Harris, David O, Parsons, Stanley M
Format: Article
Language:English
Subjects:
Central Nervous System Depressants - analysis
Chromatography, Affinity
Cloning, Molecular
Cupriavidus necator
Dyes
Escherichia coli - genetics
Escherichia coli - metabolism
Ethanol - analysis
Forensic Medicine - methods
Forensic radiography
Forensic sciences
gamma-Aminobutyric Acid - analogs & derivatives
gamma-Aminobutyric Acid - metabolism
Genes, Bacterial
Humans
Hydroxybutyrate Dehydrogenase - genetics
Hydroxybutyrate Dehydrogenase - metabolism
Hydroxybutyrates - chemistry
Hydroxybutyrates - metabolism
NAD - metabolism
Oxidation-Reduction
Reproducibility of Results
Sensitivity and Specificity
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Summary: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 microL 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.
ISSN:0022-1198
1556-4029
DOI:10.1520/jfs2003165