3-hydroxybutyrate 3-HB beta-hydroxybutyrate enzymatic assay R1 serum plasma 3-1000µmol/L standard 0.2-200µmol/L High-Sens Wako

3-hydroxybutyrate (3-HB) R1 serum manufactured by FUJIFILM Wako Pure Chemical Corporation

Ketones are a product of fat metabolism. Whilst glucose is the preferred energy source for cells when this is not available the body will switch to fat metabolism to produce energy. This leads to a build up of ketones in the blood, firstly producing ketosis and then progressing to ketoacidosis. Ketoacidosis is a form of metabolic acidosis most often seen is individuals with uncontrolled type I diabetes, although it can occur in type II diabetes, during pregnancy, illness and severe infections. There are three ketone bodies - acetoacetate (Acacia), acetone and beta-hydroxybutyrate (a.k.a. 3-HB). Beta-hydroxybutyrate (3-Hydroxybutyrate) is the main ketone present in severe diabetic ketoacidosis and measurement of 3-HB and Total Ketone bodies can be useful in the diagnosis and monitoring of diabetes. The AcAc/3-HB ratio in arterial blood reflects liver reserve capacity so may also be useful in the field of liver transplantation.

The Wako 3-HB assay is a kinetic method utilising cyclic enzymatic reactions. To begin the sample is mixed with Reagent 1 containing acetoacetate decarboxylase (AADC) which catalyses the breakdown of acetoacetate into acetone. On addition of Reagent 2, which contains 3-hydroxybutyrate dehydrogenase (3-HBDH) and Thio-NAD, the 3-HB present in the sample is oxidised and a cyclic reaction triggered. 3-HB is oxidised to give AcAc (reduction of Thio-NAD to Thio-NADH) and AcAc is reduced to give 3-HB (oxidation of NADH to NAD). As any AcAc originally present in the sample was removed by AADC only the 3-HB is assayed by measuring the rate of Thio-NADH production at 405nm. Wako's 3-HB assay can be used with serum or plasma (heparin, citrate oxalate, EDTA or sodium fluoride) samples and has a standard measuring range of 3-1000µmol/L (a high-sensitivity method using increased sample gives a range of 0.2-200µmol/L.

The 3-HB assay utilises two reagents: this reagent is 3-HB Reagent 1 (417-73501) and prepared by reconstituting the lyophilised Thio-NAD with phosphate buffer containing AADD enzyme, this solution is stable for up to 3 weeks when stored at 2-10C. This must be used with 3-HB Reagent 2 (417-73501) which is prepared by combining lyophilised 3-HBDH and NADH with Good's Buffer; the reconstituted reagent is stable for up to 3 weeks when stored at 2-10C. The 3-HB assay should be calibrator with the Ketone Body Calibrator (412-73791).

Total Ketone Bodies can be determined by using Total Ketone Bodies Reagent 1 (415-73301) with 3-HB Reagent 2 (417-73501).