Acute valproate poisoning: pharmacokinetics, alteration in fatty acid metabolism, and changes during therapy.

The clinical features, complications, and pharmacokinetics of intentional acute valproic acid (VPA) overdoses are described. Alteration in fatty acid metabolism is evaluated and therapy-induced changes are discussed. Central nervous system features were the predominant clinical manifestations (6/6), followed by respiratory failure (5/6) and multiorgan failure (2/6). Mechanical ventilation was required in 5 of 6 patients because of respiratory depression or deep coma. Hemodialysis was applied in 4/6 of the cases due to hyperammonemia, worsening neurologic condition, or organ dysfunction. Cerebral edema and hemorrhagic pancreatitis ensued in 2/6 of the patients and ICU mortality was 2/6. VPA peak levels ranged from 520 to 1700 mg/L with a mean of 1127 mg/L. Ammonia was elevated in all cases with a mean of 550 microg/dL. All patients showed signs of impaired mitochondrial beta-oxidation with increase of medium- and long-chain acylcarnitines in serum. Severe VPA overdose is associated with a high mortality rate requiring early medical interventions. Beside supportive intensive care, hemodialysis can be considered as an adjunctive measure.

Wolfram syndrome: structural and functional analyses of mutant and wild-type wolframin, the WFS1 gene product.

Mutations of the WFS1 gene are responsible for Wolfram syndrome, a rare, recessive disorder characterized by early-onset, non-autoimmune diabetes mellitus, optic atrophy and further neurological and endocrinological abnormalities. The WFS1 gene encodes wolframin, a putative multispanning membrane glycoprotein of the endoplasmic reticulum. The function of wolframin is completely unknown. In order to characterize wolframin, we have generated polyclonal antibodies against both hydrophilic termini of the protein. Wolframin was found to be ubiquitously expressed with highest levels in brain, pancreas, heart and insulinoma beta-cell lines. Analysis of the structural features provides experimental evidence that wolframin contains nine transmembrane segments and is embedded in the membrane in an N(cyt)/C(lum) topology. Wolframin assembles into higher molecular weight complexes of approximately 400 kDa in the membrane. Pulse-chase experiments demonstrate that during maturation wolframin is N-glycosylated but lacks proteolytical processing. Moreover, N-glycosylation appears to be essential for the biogenesis and stability of wolframin. Here we investigate, for the first time, the molecular mechanisms that cause loss-of-function of wolframin in affected individuals. In patients harboring nonsense mutations complete absence of the mutated wolframin is caused by instability and rapid decay of WFS1 nonsense transcripts. In a patient carrying a compound heterozygous missense mutation, R629W, we found markedly reduced steady-state levels of wolframin. Pulse-chase experiments of mutant wolframin expressed in COS-7 cells indicated that the R629W mutation leads to instability and strongly reduced half-life of wolframin. Thus, the Wolfram syndrome in patients investigated here is caused by reduced protein dosage rather than dysfunction of the mutant wolframin.

Tandem mass spectrometric assay for the determination of carnitine palmitoyltransferase II activity in muscle tissue.

Carnitine palmitoyltransferase II (CPT-II) mediates the import of long-chain fatty acids into the mitochondrial matrix for subsequent beta-oxidation. Defects of CPT-II manifest as a severe neonatal hepatocardiomuscular form or as a mild muscular phenotype in early infancy or adolescence. CPT-II deficiency is diagnosed by the determination of enzyme activity in tissues involving the time-dependent conversion of radiolabeled CPT-II substrates (isotope-exchange assays) or the formation of chromogenic reaction products. We have established a mass spectrometric assay (MS/MS) for the determination of CPT-II activity based on the stoichiometric formation of acetylcarnitine in a coupled reaction system. In this single-tube reaction system palmitoylcarnitine is converted by CPT-II to free carnitine, which is subsequently esterified to acetylcarnitine by carnitine acetyltransferase. The formation of acetylcarnitine directly correlates with the CPT-II activity. Comparison of the MS/MS method (y) with our routine spectrophotometric assay (x) revealed a linear regression of y = 0.58x + 0.12 (r = 0.8369). Both assays allow one to unambiguously detect patients with the muscular form of CPT-II deficiency. However, the higher specificity and sensitivity as well as the avoidance of the drawbacks inherent in the use of radiolabeled substrates make this mass spectrometric method most suitable for the determination of CPT-II activity.

The C66W mutation in the deafness dystonia peptide 1 (DDP1) affects the formation of functional DDP1.TIM13 complexes in the mitochondrial intermembrane space.

Mohr-Tranebjaerg syndrome is a progressive, neurodegenerative disorder caused by loss-of-function mutations in the DDP1/TIMM8A gene. DDP1 belongs to a family of evolutionary conserved proteins that are organized in hetero-oligomeric complexes in the mitochondrial intermembrane space. They mediate the import and insertion of hydrophobic membrane proteins into the mitochondrial inner membrane. All of them share a conserved Cys(4) metal binding site proposed to be required for the formation of zinc fingers. So far, the only missense mutation known to cause a full-blown clinical phenotype is a C66W exchange directly affecting this Cys(4) motif. Here, we show that the mutant human protein is efficiently imported into mitochondria and sorted into the intermembrane space. In contrast to wild-type DDP1, it does not complement the function of its yeast homologue Tim8. The C66W mutation impairs binding of Zn(2+) ions via the Cys(4) motif. As a consequence, the mutated DDP1 is incorrectly folded and loses its ability to assemble into a hetero-hexameric 70-kDa complex with its cognate partner protein human Tim13. Thus, an assembly defect of DDP1 is the molecular basis of Mohr-Tranebjaerg syndrome in patients carrying the C66W mutation.