Short-chain hydroxyacyl CoA dehydrogenase deficiency is an ill-defined, severe pediatric disorder of mitochondrial fatty acid P-oxidation of short-chain hydroxyacyl CoAs. To understand the relative contributions of the two known short-chain hydroxyacyl CoA dehydrogenases (HADH) tissue biopsies of six distinct family individuals were analyzed and kinetic parameters were compared. Steady-state kinetic constants for HADH I and HADH 2 suggest that type I is the major enzyme involved in mitochondrial P-oxidation of short-chain hydroxyacyl-CoAs. Two patients are heterozygous carriers of a HADH I polymorphism, whereas no mutation is detected in the HADH 2 gene of all patients. The data suggest that protein interactions rather than HADH mutations are responsible for the disease phenotype. Pull-down experiments of recombinant HADH I and 2 with human mitochondrial extracts reveal two proteins interacting with HADH 1, one of which was identified as glutamate dehydrogenase. This association provides a possible link between fatty acid metabolism and the hyperinsulinism/hyperammonia syndrome. (C) 2007 Elsevier Inc. All rights reserved.