Diabetes and alcohol consumption are the common causes of ketoacidosis, and research has shown that acetoacetate/3-hydroxybutyrate (3-HB) ratio in arterial blood reflects stages of liver failure.
     When a sample is mixed with 3-HB R1, AcAc in the sample is broken down to acetone by acetoacetate decarboxylase. Upon addition of 3-HB R2, 3-HB in the sample is oxidized in the presence of 3-HBDH and Thio-NAD. This oxidation triggers a cyclic reaction. Since the original AcAc in the sample has been removed, only 3-HB is assayed by measuring the rate of Thio-NADH production spectrophotometrically.

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