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 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.
Bravo
Dawn T
DT
Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106, USA.
Harris
David O
DO
Parsons
Stanley M
SM
D002492:Central Nervous System Depressants / Q000032:analysis; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D020118:Cupriavidus necator; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D000431:Ethanol / Q000032:analysis; D005554:Forensic Medicine / Q000379:methods*; D005798:Genes, Bacterial; D006801:Humans; D006884:Hydroxybutyrate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D006885:Hydroxybutyrates / Q000737:chemistry / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D005680:gamma-Aminobutyric Acid / Q000031:analogs & derivatives* / Q000378:metabolism
D002492:Central Nervous System Depressants; D006885:Hydroxybutyrates; D009243:NAD; D000431:Ethanol; D005680:gamma-Aminobutyric Acid; D006884:Hydroxybutyrate Dehydrogenase; C009338:succinic semialdehyde