Multipoint kinetic method for simultaneously measuring the combined concentrations of acetoacetate-beta-hydroxybutyrate and lactate-pyruvate.
This is a multipoint kinetic method for simultaneously determining acetoacetate (AcAc) plus beta-hydroxybutyrate and lactate plus pyruvate in a single cuvette of the Multistat III centrifugal analyzer. In the first step, AcAc and pyruvate are completely reduced, using 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) and lactate dehydrogenase (EC 1.1.1.27) in the presence of excess NADH at pH 7.5, to beta-hydroxybutyrate and lactate, respectively. After dilution, the endogenous beta-hydroxybutyrate and lactate and that resulting from reduction are simultaneously oxidized by their respective dehydrogenases in the presence of excess NAD+ at pH 9.0. Adjustment of the relative enzyme concentrations allows simultaneous estimation of AcAc plus beta-hydroxybutyrate and lactate plus pyruvate by analyzing multipoint absorbance data, collected during the oxidation reaction, with use of a two-component linear-regression model. Total run-to-run CVs were 6.4% and 6.1% at 5 mmol/L beta-hydroxybutyrate and 5 mmol/L lactate, respectively. The method was designed to be useful for identifying the cause of an increased anion gap in serum.
Nuwayhid
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NF
Department of Pathology, College of Medicine, University of Iowa, Iowa City 52242.
Johnson
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Feld
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RD
D020155:3-Hydroxybutyric Acid; D000090:Acetoacetates / Q000097:blood*; D000136:Acid-Base Equilibrium; D000138:Acidosis / Q000097:blood; D002498:Centrifugation; D006801:Humans; D006885:Hydroxybutyrates / Q000097:blood*; D007700:Kinetics; D007773:Lactates / Q000097:blood*; D010084:Oxidation-Reduction; D011773:Pyruvates / Q000097:blood*
D000090:Acetoacetates; D006885:Hydroxybutyrates; D007773:Lactates; D011773:Pyruvates; C016635:acetoacetic acid; D020155:3-Hydroxybutyric Acid