Spectrophotometric enzymatic assay for S-3-hydroxyisobutyrate.
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.
Rougraff
P M
PM
Department of Biochemistry, Indiana University School of Medicine, Indianapolis 46202-5122.
Paxton
R
R
Goodwin
G W
GW
Gibson
R G
RG
Harris
R A
RA
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
D006885:Hydroxybutyrates; D009243:NAD; D000429:Alcohol Oxidoreductases; C021398:3-hydroxyisobutyrate dehydrogenase; C020757:3-hydroxyisobutyric acid