2-Methylacetoacetic acid is a metabolite that has an increased excretion in patients with acetoacetyl-CoA thiolase deficiency. Thiolases are ubiquitous and important enzymes. Several isoenzymes are known, which can occur in the cytosol, the mitochondria, or the peroxisomes. Thiolases are CoA-dependent enzymes which catalyze the formation of a carbon-carbon bond in a Claisen condensation step and its reverse reaction via a thiolytic degradation mechanism.

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of metabolism that affects the catabolism of isoleucine and ketone bodies. This disorder is characterized by intermittent ketoacidotic episodes.

Mitochondrial acetoacetyl-coenzyme A (CoA) thiolase (T2) is important in the pathways for the synthesis and degradation of ketone bodies as well as for the degradation of 2-methylacetoacetyl-CoA.

Moreover, 2-methylacetoacetic acid is found to be associated with beta-ketothiolase deficiency, which is also an inborn error of metabolism. 2-Methylacetoacetic acid is found in urine and can be used as a biomarker for the diagnosis of beta-ketothiolase deficiency.

Beta-ketothiolase deficiency is an inherited disorder in which the body cannot effectively process a protein building block (amino acid ) called isoleucine. This disorder also impairs the body's ability to process ketones, which are molecules produced during the breakdown of fats.