Acetoacetate (AcAc), a Beta-ketoacid, is one of the three ketone bodies and is formed via condensation of two molecules of acetyl-CoA in liver mitochondria. AcAc can be enzymatically reduced to 3-Beta-hydroxybutyrate (Beta-HB), or decarboxylated producing acetone (CH3)2CO). Ketone bodies (BetaHB: 78%; AcAc: 20% & CH3)2CO: 2%) are mainly used as an alternative energy source when glucose cannot be delivered to the system. Excessive concentration of ketone bodies (ketoacidosis) is observed in patients with Type I diabetes, severe starvation or alcoholism. Traditionally, AcAc levels have been qualitatively detected using dipsticks that use sodium nitroferricyanide as a chromophore. Assay Genie's Acetoacetate Assay Kit has adapted that principle with a modification that provides a sensitive method to quantitate endogenous levels of AcAc in human blood, and urine. In this non-enzymatic assay, AcAc reacts with a substrate to generate a colored product that can be measured at 550 nm. The reaction is specific for AcAc and does not detect 3-Beta-hydroxybutyrate. The assay kit can detect samples containing acetoacetate as low as 25 µM.

Figure: a) Time course of color development at 4°C and 25°C (100 nmol Acetoacetate). b) Standard Curve for equimolar amounts of Acetoacetate, β-HB and Acetone (4°C, 100 min.). The other two ketone bodies β-hydroxybutyrate or Acetone are not detected in the assay due to much lower sensitivity (β-HB) and much lower concentration (Acetone). c) Measurement of Acetoacetate in urine. Samples were deproteinized by spin filtering. 100 µl of undiluted urine was spiked with known amounts of AcAc (30 nmol). Assays were performed following the kit protocol.