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Dichloroacetate (DCA) is a structural analog of pyruvate that has been recommended for the treatment of primary lactic acidemia, particularly in patients with pyruvate dehydrogenase (PDHC) deficiency. Recent reports have demonstrated that the response to DCA may depend on the type of molecular abnormality. In this study, we investigated the response to DCA in various PDHC-deficient cell lines and tried to determine the mechanism involved. The effect of chronic 3-d DCA treatment on PDHC activity was assessed in two PDHC-deficient cell lines, each with a different point mutation in the E1␣ subunit gene (R378C and R88C), and one cell line in which an 8-bp tandem repeat was deleted (W383 del). Only two (R378C and R88C) of the three PDHC-deficient cell lines with very low levels of PDHC activity and unstable polypeptides were sensitive to chronic DCA treatment. In these cell lines, DCA treatment resulted in an increase in PDHC activity by 125 and 70%, respectively, with concomitant increases of 121 and 130% in steady-state levels of immunoreactive E1␣. DCA treatment reduced the turnover of the E1␣ subunit in R378C and R88C mutant cells with no significant effect on the E1␤ subunit. Chronic DCA treatment significantly improved the metabolic function of PDHC in digitonin-permeabilized R378C and R88C fibroblasts. The occurrence of DCA-sensitive mutations suggests that DCA treatment is potentially useful as an adjuvant to ketogenic and vitamin treatment in PDHC-deficient patients. PDHC deficiency is a nuclear-encoded mitochondrial disorder and a major recognized cause of neonatal encephalopathies associated with primary lactic acidosis (1). This multienzyme complex plays an important role in the irreversible oxidative decarboxylation of pyruvate to acetyl-CoA. E1, one of the subunits of the complex, is a thiamine pyrophosphatedependent pyruvate decarboxylase. It is a tetramer composed of two ␣ and two ␤ subunits, the ␣ subunit containing the thiamine pyrophosphate binding sites. E2 is a dihydrolipoamide acetyl transferase. E3 is a dihydrolipoamide dehydrogenase, and E3BP or protein X mediates the interaction between E2 and E3. Two elements regulate the complex: an E1 kinase and a phospho-E1 phosphatase, which phosphorylate and dephosphorylate, respectively, three serine residues in the E1 ␣ subunit, resulting in the deactivation and activation, respectively, of the complex (2).Defects in the PDHC are frequently attributed to deficiencies in the E1 ␣ component (3), encoded by chromosome X (Xp22.1-22.2) (4, 5). Considerable phenotypic and allelic heterogeneity has been observed for this defect. Previous studies have reported that a decrease in the stability of the E1 ␣ immunoreactive subunit may contribute to the expression of many E1 ␣ mutations (6, 7); very few studies to distinguish mutations that impair polypeptide stability from those impairing catalytic efficiency have been carried out to date (8).DCA is a structural analog of pyruvate that has been recommended for the treatment of primary lactic acidemia, particular...

2Investigation clinique, Hdpital R. Debrd, 75019, Paris; 3Inserm U75 and 4Clinique de gdn~tique mkdicale, Hdpital Necker, 75015 Paris, France Carnitine acylcarnitine translocase is one of the components necessary for the entry of long-chain fatty acids (LCFA) into the mitochondrial matrix, transferring acylcarnitines across the inner mitochondrial membrane in exchange for free carnitine. We have identified a new case ofcarnitine acylcarnitine translocase deficiency in a patient with impaired LCFA oxidation by complementation analysis. Restoration of release of tritiated water from [9,10(n)-3H]palmitate was used as the criterion for complementation in cultured fibroblasts.

Carnitine palmitoyltransferase (CPT) consists of two activities located in the outer (CPT I) and the inner (CPT II) mitochondrial membranes. CPT II deficiency in the adult as well as in the infantile form of the disease has been shown to result from mutations in the CPT II cDNA. Nothing is known regarding the genetic defect in CPT I deficiency. We carried out complementation experiments between CPT I- and infantile CPT II-deficient cell lines. Restoration of 3H2O release from [9,10(n)-3H]-palmitate was chosen as criterion of complementation. As expected, no complementation was observed in heteropolykaryons resulting from fusions between CPT II-deficient cells. Similar results were obtained in fusions between CPT I-deficient cells, suggesting that the enzymatic defect in these cell lines results from mutations in the same gene. Conversely, complementation was observed in fusions between CPT I- and CPT II-deficient cells. These data support that CPT I and CPT II defects result from mutations in distinct genes. Palmitate oxidation by control or CPT I-deficient cell lines was decreased when cocultured with infantile CPT II-deficient cell lines. This effect, not observed in coculture including an adult CPT II-deficient cell line, was carnitine-dependent. The possible mechanism of this effect, suppressed by a high carnitine concentration, is discussed.