Riboflavin therapy. Biochemical heterogeneity in two adult lipid storage myopathies.

Two unrelated adult males, aged 36 (patient 1) and 25 (patient 2) years, presented with subacute carnitine-deficient lipid storage myopathy that was totally and partly responsive to riboflavin supplementation in the two patients, respectively. Plasma acyl-carnitine and urinary organic acid profiles indicated multiple acyl coenzyme A dehydrogenase deficiency, which was mild in patient 1 and severe in patient 2. The activities of short-chain and medium-chain acyl coenzyme A dehydrogenases in mitochondrial fractions were decreased, especially in patient 2. This was in agreement with Western blotting results. Flavin-dependent complexes I and II were studied by immunoblotting and densitometric quantification of two-dimensional electrophoresis with comparable results. Complex I was present in normal amounts in both patients, whereas complex II was decreased only in the pretherapy muscle of patient 2. Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) concentrations in muscle and isolated mitochondria, and the activity of mitochondrial FAD pyrophosphatase, showed that patient 1 had low levels of FAD (46%) and FMN (49%) in mitochondria, with a significant increase (P < 0.01) in mitochondrial FAD pyrophosphatase (273%) compared with controls. Patient 2 had similar low levels of FAD and FMN in both total muscle (FAD and FMN 22% of controls) and mitochondria (FAD 26%; FMN 16%) and normal activity of mitochondrial FAD pyrophosphatase. All of these biochemical parameters were either totally or partly corrected after riboflavin therapy.

Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels.

Blue native-polyacrylamide gel electrophoresis is a powerful technique that enables the separation of intact multi-subunit complexes. However, positive identification of particular enzymes generally requires further separation in a second dimension on a denaturing polyacrylamide gel. Histochemical staining is widely used to demonstrate enzyme activities in tissues, including oxidative phosphorylation enzymes. In this report, we demonstrate that the two techniques can be combined to quantify in situ mitochondrial enzymes, separated on nondenaturing polyacrylamide gels. The method gives quantitative results with human skeletal muscle as well as heart that contains higher mitochondrial numbers. Comparison of muscle from patients with oxidative phosphorylation enzyme deficiencies, such as those of two riboflavin-responsive patients, before and after vitamin treatment, gives results in agreement with those obtained by analyzing the activity of the mitochondrial enzymes in muscle homogenates.

Post-hypoxic recovery of acetylcholine release: different sensitivity of guinea pig neocortical and striatal slices.

The release of endogenous acetylcholine (ACh) was measured in superfused guinea-pig cortical and striatal slices, kept at rest or electrically stimulated at different frequencies, before and during severe hypoxic conditions as well as after reoxygenation. In the cortex the basal release was unchanged by 30-60 min of hypoxia while it was inhibited in the striatum. The release evoked by short-term (2 min) stimulation at 0.5 Hz was moderately reduced (to 76%) by 30 min hypoxia in the cortex and in the striatum, but fully recovered after reoxygenation. The release evoked by continuous stimulation (from 5 to 10 to 20 Hz) was strongly inhibited (to 12-30%) in both areas after 30 min of hypoxia. After 30 min of reoxygenation, the recovery was complete in the cortex (mainly provided with cholinergic axons), but it was incomplete in the striatum (rich in cholinergic interneurones). The extent of the recovery in the latter area (i) was inversely related to stimulation frequency, (ii) did not depend on the depletion of neurotransmitter stores, because ACh tissue levels were fully restored by reoxygenation, and (iii) was consistently facilitated by excitatory aminoacid antagonists, slightly improved by the adenosine agonist R-phenylisopropyladenosine and unaffected by reducing the concentrations of radical species with catalase and superoxide dismutase or N omega-nitro-L-arginine. These results emphasize (i) the different vulnerability of the cortical and striatal cholinergic structures, (ii) the high sensitivity of the striatal interneurones to the frequency of stimulation during the posthypoxic recovery, and (iii) the relevant role played by endogenous glutamate on activity-dependent neurosecretory failure.