title: Spectrophotometric enzymatic assay for S-3-hydroxyisobutyrate.
abstract: An enzymatic spectrophotometric end-point assay has been developed for determination of S-3-hydroxyisobutyrate in biological fluids. The assay measures NADH production at 340 nm after initiation of the reaction with rabbit liver 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31). The assay is not affected by R-3-hydroxyisobutyrate, lactate, malate, 3-hydroxybutyrate, 2-methyl-3-hydroxybutyrate, 3-hydroxyisovalerate, 3-hydroxy-n-valerate, 2-methyl-3-hydroxy-valerate, and 3-hydroxypropionate. The assay does measure 2-ethyl-3-hydroxypropionate, a minor metabolite produced by catabolism of alloisoleucine. Application of the method to measure S-3-hydroxyisobutyrate in plasma obtained from normal, 48-h starved, and mildly and severely diabetic rats gave levels of 28, 42, 112, and 155 microM, respectively.
authors.affiliation: Department of Biochemistry, Indiana University School of Medicine, Indianapolis 46202-5122.
authors.forename: P M
authors.initials: PM
authors.lastname: Rougraff
authors.forename: R
authors.initials: R
authors.lastname: Paxton
authors.forename: G W
authors.initials: GW
authors.lastname: Goodwin
authors.forename: R G
authors.initials: RG
authors.lastname: Gibson
authors.forename: R A
authors.initials: RA
authors.lastname: Harris
mesh_terms: D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D003921:Diabetes Mellitus, Experimental / Q000097:blood; D006885:Hydroxybutyrates / Q000097:blood*; D008297:Male; D009243:NAD / Q000032:analysis; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D013053:Spectrophotometry; D013379:Substrate Specificity
chemical_list: D006885:Hydroxybutyrates; D009243:NAD; D000429:Alcohol Oxidoreductases; C021398:3-hydroxyisobutyrate dehydrogenase; C020757:3-hydroxyisobutyric acid