β-Hydroxybutyrate is a "ketone body" produced in the liver and exported to peripheral tissues for use as an energy source. "ketone body" includes three molecules: acetoacetate, β-Hydroxybutyrate, and acetone. β-Hydroxybutyrate and acetoacetate transport energy from the liver to other tissues, while acetone is generated by spontaneous decarboxylation of acetoacetate. High levels of ketone bodies can lead to ketosis, or as in extreme type 1 diabetes, ketoacidosis. Prolonged ketosis may lead to a life threatening metabolic acidosis. Pathological ketosis may indicate various conditions, including organ failure, hypoglycemia in children, diabetes, alcohol intoxication, corticosteroid or growth hormone insufficiency. The degree of ketosis can be determined by monitoring the blood levels of β-Hydroxybutyrate.

β-Hydroxybutyrate (Ketone Body) Assay Kit provides a simple, reproducible, and sensitive tool for measuring β-Hydroxybutyrate concentration in plasma, serum, cell lysates, urine, and other biological liquid samples. This assay employs an enzymatic cycling reaction. Initially, β-Hydroxybutyrate is oxidized to acetoacetate by the β-Hydroxybutyrate Enzyme. Concomitant with this oxidation, the cofactor NAD+ is reduced to NADH. Then NADH reacts with the colorimetric probe producing a colored product which can be measured at 450 nm. The optical density measured is directly proportional to the concentration of β-Hydroxybutyrate present in the sample.

• Microplate reader capable of measuring absorbance at 450 nm
• Precision pipettes to deliver 2 μL to 1 mL volumes
• Tubes to prepare sample dilutions
• 50 mL conical tubes

1. Add 50 µL of the diluted β-Hydroxybutyrate Standards or samples to the 96-well microtiter plate.
2. Initiate the reaction by adding 50 µL Working Solution to each well. Carefully shake the plate for a few seconds to mix.
3. Incubate the plate for 30 minutes at room temperature with mixing in the dark.
4. Measure the absorbance at 450nm using a plate reader.