A chemiluminescence-flow injection analysis of serum 3-hydroxybutyrate using a bioreactor consisting of 3-hydroxybutyrate dehydrogenase and NADH oxidase.
We describe a simple method for the highly sensitive chemiluminescence--flow injection analysis of 3-hydroxybutyrate in serum using a bioreactor column consisting of the two immobilized enzymes, 3-hydroxybutyrate dehydrogenase and NADH oxidase. The method was based on measuring the level of chemiluminescence formed by the reaction of a luminol-hexacyanoferrate mixture with hydrogen peroxide. The hydrogen peroxide was produced by the NADH oxidase reaction from NADH which was formed in the conversion of 3-hydroxybutyrate to acetoacetate by the 3-hydroxybutyrate dehydrogenase reaction. Among three immobilized enzyme columns, a coimmobilized, small 3-hydroxybutyrate dehydrogenase/NADH oxidase bioreactor alone (2 x 20 mm i.d.) readily hydrolyzed all of the injected 3-hydroxybutyrate into acetoacetate, although 3-hydroxybutyrate dehydrogenase catalyzed the reversible reaction. The present method generated linearity of the data up to 1.5 mM 3-hydroxybutyrate with satisfactory precision, reproducibility, and accurate reaction recoveries. The results from 3-hydroxybutyrate correlated satisfactorily with those obtained by other well-established methods. The coimmobilized 3-hydroxybutyrate dehydrogenase/NADH oxidase reactor unit showed good operational stability over a 5-week period, during which it was repeatedly used for 1500 analyses.
Tabata
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College of Medical Technology, Kyoto University, Japan.
Totani
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D020155:3-Hydroxybutyric Acid; D015374:Biosensing Techniques; D001709:Biotechnology; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D005069:Evaluation Studies as Topic; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006884:Hydroxybutyrate Dehydrogenase; D006885:Hydroxybutyrates / Q000097:blood*; D008163:Luminescent Measurements; D009097:Multienzyme Complexes; D009247:NADH, NADPH Oxidoreductases; D012680:Sensitivity and Specificity
D004800:Enzymes, Immobilized; D006885:Hydroxybutyrates; D009097:Multienzyme Complexes; D006884:Hydroxybutyrate Dehydrogenase; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D020155:3-Hydroxybutyric Acid