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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

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 and lactate dehydrogenase (EC 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.[1]


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