Respiratory complex II defect in siblings associated with a symptomatic secondary block in fatty acid oxidation.

The mitochondrial oxidative phosphorylation and fatty acid oxidation pathways have traditionally been considered independent major sources of cellular energy production; however, case reports of patients with specific enzymatic defects in either pathway have suggested the potential for a complex interference between the two. This study documents a new site of interference between the two pathways, a site in respiratory complex II capable of producing clinical signs of a block in fatty acid oxidation and reduced in vitro activity of acyl-CoA dehydrogenases. The initial patient, and later her newborn sibling, had mildly dysmorphic features, lactic acidosis and a defect in mitochondrial respiratory complex II associated with many biochemical features of a block in fatty acid oxidation. Results of in vitro probing of intact fibroblasts from both patients with methyl[2H3]palmitate and L-carnitine revealed greatly increased [2H3]butyrylcarnitine; however, the ratio of dehydrogenase activity with butyryl-CoA with anti-MCAD inactivating antibody (used to reveal SCAD-specific activity) to that with octanoyl-CoA was normal, excluding a selective SCAD or MCAD deficiency. Respiratory complex II was defective in both patients, with an absent thenoyltrifluoroacetone-sensitive succinate Q reductase activity that was partially restored by supplementation with duroquinone. Although secondary, the block in fatty acid oxidation was a major management problem since attempts to provide essential fatty acids precipitated acidotic decompensations. This study reinforces the need to pursue broadly the primary genetic defect within these two pathways, making full use of increasingly available functional and molecular diagnostic tools.

Nuclear DNA origin of mitochondrial complex I deficiency in fatal infantile lactic acidosis evidenced by transnuclear complementation of cultured fibroblasts.

We have studied complex I (NADH-ubiquinone reductase) defects of the mitochondrial respiratory chain in 2 infants who died in the neonatal period from 2 different neurological forms of severe neonatal lactic acidosis. Specific and marked decrease in complex I activity was documented in muscle, liver, and cultured skin fibroblasts. Biochemical characterization and study of the genetic origin of this defect were performed using cultured fibroblasts. Immunodetection of 6 nuclear DNA-encoded (20, 23, 24, 30, 49, and 51 kDa) and 1 mitochondrial DNA-encoded (ND1) complex I subunits in fibroblast mitochondria revealed 2 distinct patterns. In 1 patient, complex I contained reduced amounts of the 24- and 51-kDa subunits and normal amounts of all the other investigated subunits. In the second patient, amounts of all the investigated subunits were severely decreased. The data suggest partial or extensive impairment of complex I assembly in both patients. Cell fusion experiments between 143B206 rho degrees cells, fully depleted of mitochondrial DNA, and fibroblasts from both patients led to phenotypic complementation of the complex I defects in mitochondria of the resulting cybrid cells. These results indicate that the complex I defects in the 2 reported cases are due to nuclear gene mutations.

Detection of pyruvate metabolism disorders by culture of skin fibroblasts with dichloroacetate.

For use in screening for disorders of pyruvate metabolism, a sensitive assay method was developed for measuring the rate of decarboxylation of [1-14C]pyruvate during in vitro culture of skin fibroblasts with dichloroacetate (DCA). The rate of decarboxylation of [1-14C]pyruvate by skin fibroblasts from control subjects increased from 59.6 +/- 13.2 to 97.3 +/- 12.0 nmol/h/mg protein during in vitro culture in medium supplemented with 10 mM DCA for 3 days. In contrast, the rate hardly increased in cells from four of 20 patients with congenital lactic acidosis of unknown cause during in vitro culture with DCA. On day 3 of culture, the values for the four patients did not overlap those of control cells and so these four patients could be clearly distinguished from control subjects. Measurements of the original activity and the activity of the pyruvate dehydrogenase (PDH) complex after activation with a broad specificity protein phosphatase and DCA suggested that in three of the patients the aberration was a disorder in the mechanism for activation of PDH, including deficiency of PDH phosphatase or a mutation of PDH itself, whereas that in the fourth patient it might be a disorder of the mitochondrial transport system for pyruvate. Thus, measurement of the rate of decarboxylation of [1-14C]pyruvate by skin fibroblasts cultured in medium supplemented with 10 mM DCA for 3 days is a useful method for screening for disorders of pyruvate metabolism in cultured skin fibroblasts.