Search

Variation in the size and relative proportion of type 1 and type 2 muscle fibers can occur in a number of conditions, including structural myopathies, neuropathies, and various syndromes. In most cases, the pathogenesis of such fiber type changes is unknown and the etiology is heterogeneous. Skeletal muscle mitochondrial respiratory chain analysis was performed in 10 children aged 3 weeks to 5 years with abnormalities in muscle fiber type, size, and proportion. Five children were classified as having definite, four as probable, and one as possible mitochondrial disease. Type 1 fiber predominance was the most common histological finding (six of 10). On light microscopy, four cases had subtle concomitants of a mitochondriopathy, including mildly increased glycogen, lipid, and/or succinate dehydrogenase staining, and one case had more prominent evidence of underlying mitochondrial disease with marked subsarcolemmal staining. Most cases (nine of 10) had abnormal mitochondrial morphology on electron microscopy. All were found to have mitochondrial electron transport chain (ETC) abnormalities and met diagnostic criteria for mitochondrial disease. We did not ascertain any patients who had isolated fiber type abnormalities and normal respiratory chain analysis during the period of study. We conclude that mitochondrial ETC disorders may represent an etiology of at least a subset of muscle fiber type abnormalities. To establish an etiologic diagnosis and to determine the frequency of such changes in mitochondrial disease, we suggest analysis of ETC function in individuals with fiber type changes in skeletal muscle, even in the absence of light histological features suggestive of mitochondrial disorders.

Early phase evaluation of anticancer drugs has traditionally used toxicity (usually hematological) rather than efficacy end points to establish appropriate dosing schedules. To establish a biochemical efficacy end point for overcoming alkylguanine DNA alkyltransferase (AGT)-mediated tumor cell resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea, we performed a novel dose escalation clinical trial for the AGT-depleting agent O6-benzylguanine (BG). The dose of BG required to deplete AGT to undetectable levels (BMD(T)) in sequential computed tomography-guided tumor tissue biopsies before BG and 18 h after BG was determined. Thirty patients received doses of BG ranging from 10 to 120 mg/m2. In tumor tissue, AGT depletion86% of baseline was demonstrated at all doses tested. Residual tumor AGT activity, present 18 h after BG doses of 10-80 mg/m2, was eliminated at the 120 mg/m2 dose and is thus the BMD(T) of BG. BG pharmacokinetics are characterized by the rapid, dose-independent clearance of BG from plasma Metabolism of BG to its biologically active metabolite, 8-oxo-benzylguanine (8-oxo-BG), was found. The t(1/2) of 8-oxo-BG is longer than BG. Plasma concentrations of 8-oxo-BG well above 200 ng/ml 18 h after the end of the BG infusion were observed at the highest dose levels tested and appeared to correlate with depletion of AGT activity to undetectable levels in tumor tissue. AGT activity in peripheral blood mononuclear cells at baseline did not correlate with tumor tissue AGT activity. Depletion of AGT activity to undetectable levels in peripheral blood mononuclear cells occurred at lower doses and was not a reliable predictor for tumor tissue depletion. No serious side effects were observed with administration of BG alone or in combination with 13 mg/m2 1,3-bis(2-chloroethyl)-1-nitrosourea. This is the first clinical study in which biochemical analyses from pre- and posttreatment tumor biopsies have been used as an efficacy end point for the clinical development of an anticancer agent. From our tumor tissue biopsy data, we have established that a BG dose of 120 mg/m2 infused over 1 h should be used in Phase II clinical trials.

3-Hydroxydicarboxylic acids are major urinary metabolites derived from fatty acid metabolism. These compounds are produced from the omega-oxidation of 3-hydroxy fatty acids. The production of the precursor 3-hydroxy fatty acids from incomplete beta-oxidation of fatty acids in rat liver mitochondria was investigated. Independent of the chain length or the concentration of fatty acid substrates, the accumulation of 3-hydroxyacyl intermediates was relatively constant at the concentration of 3-5 nmol/mg of mitochondrial protein. The extent of the incomplete oxidation was the same in Percoll gradient-purified mitochondria. Rotenone treatment increased the production of 3-hydroxy fatty acids. 3-Hydroxy fatty acids did not exist as pure L-enantiomer as expected from beta-oxidation. Instead, these metabolites were epimerized to a near racemic mixture of D- and L-isomers with a slightly dominant D-isomer (58 +/- 3%). By using deuterium-isotope labeling, the mechanism of epimerizartion was shown to be a rapid dehydration-rehydration through trans-2-enoyl-CoA. In addition, cis-3 and trans-3 fatty acids were produced; these metabolites were derived from the isomerization of trans-2-enoyl-CoA. Epimerase and isomerase were thought to be enzymes involved in the oxidation of unsaturated fatty acids. Current data have shown that the metabolism of these acids is actually through NADPH-dependent reduction pathways. The activities of epimerase and isomerase detected in rat liver mitochondria possibly function mainly in the metabolism of saturated fatty acids in a reverse role to the conventional concept.

Rats treated with hydroxycobalamin[c-lactam] (HCCL), a cobalamin analogue that induces methylmalonic aciduria, have increased hepatic mitochondrial content and increased oxidative metabolism of pyruvate and palmitate per hepatocyte. The present studies were undertaken to characterize oxidative metabolism in isolated liver mitochondria from rats treated with HCCL. After 5-6 weeks, state 3 oxidation rates for diverse substrates are reduced in mitochondria from HCCL-treated rats. Similar reductions of mitochondrial oxidation rates are obtained with dinitrophenol-uncoupled mitochondria excluding defective phosphorylation as a cause for the observed decrease in mitochondrial oxidation. The activities of mitochondrial oxidases are reduced in HCCL-treated rats and demonstrate a defect in complex IV. Investigation of the complexes of the respiratory chain reveals a 32% decrease of ubiquinol:ferricytochrome c oxidoreductase (complex III) activity and a 72% decrease of ferrocytochrome c:oxygen oxidoreductase (complex IV) activity in mitochondria from 5-6-week HCCL-treated rats as compared with controls. Liver mitochondria from HCCL-treated rats also demonstrate decreased cytochrome content per mg of mitochondrial protein (25% decrease of cytochrome b and 52% decrease of cytochrome a + a3 as compared with control rats). The HCCL-treated rat represents an animal model for the study of the consequences of respiratory chain defects in liver mitochondria.

Purpose To identify molecular defects in a girl with clinical features of MELAS (mitochondrial encephalomyopathy and lactic acidosis) and MERRF (ragged-red fibres) syndromes. Methods The enzyme complex activities of the mitochondrial respiratory chain were assayed. Temporal temperature gradient gel electrophoresis was used to scan the entire mitochondrial genome for unknown mitochondrial DNA (mtDNA) alterations, which were then identified by direct DNA sequencing. Results A novel heteroplasmic mtDNA mutation, G12207A, in the tRNA(Ser(AGY)) gene was identified in the patient who had a history of developmental delay, feeding difficulty, lesions within her basal ganglia, cerebral atrophy, proximal muscle weakness, increased blood lactate, liver dysfunction, and fatty infiltration of her muscle. Muscle biopsy revealed ragged red fibres and pleomorphic mitochondria. Study of skeletal muscle mitochondria revealed complex I deficiency associated with mitochondrial proliferation. Real time quantitative PCR analysis showed elevated mtDNA content, 2.5 times higher than normal. The tRNA(Ser(AGY)) mutation was found in heteroplasmic state (92%) in the patient's skeletal muscle. It was not present in her unaffected mother's blood or in 200 healthy controls. This mutation occurs at the first nucleotide of the 5' end of tRNA, which is involved in the formation of the stem region of the amino acid acceptor arm. Mutation at this position may affect processing of the precursor RNA, the stability and amino acid charging efficiency of the tRNA, and overall efficiency of protein translation. Conclusion This case underscores the importance of comprehensive mutational analysis of the entire mitochondrial genome when a mtDNA defect is strongly suggested.

Intact mitochondria and inverted submitochondrial vesicles were prepared from the liver of fed, starved (48 h) and streptozotocin-diabetic rats in order to characterize carnitine palmitoyltransferase kinetics and malonyl-CoA sensitivity in situ. In intact mitochondria, both starved and diabetic rats exhibited increased Vmax., increased Km for palmitoyl-CoA, and decreased sensitivity to malonyl-CoA inhibition. Inverted submitochondrial vesicles also showed increased Vmax. with starvation and diabetes, with no change in Km for either palmitoyl-CoA or carnitine. Inverted vesicles were uniformly less sensitive to malonyl-CoA regardless of treatment, and diabetes resulted in a further decrease in sensitivity. In part, differences in the response of carnitine palmitoyltransferase to starvation and diabetes may reside in differences in the membrane environment, as observed with Arrhenius plots, and the relation of enzyme activity and membrane fluidity. In all cases, whether rats were fed, starved or diabetic, and whether intact or inverted vesicles were examined, increasing membrane fluidity was associated with increasing activity. Malonyl-CoA was found to produce a decrease in intact mitochondrial membrane fluidity in the fed state, particularly at pH 7.0 or less. No effect was observed in intact mitochondria from starved or diabetic rats, or in inverted vesicles from any of the treatment groups. Through its effect on membrane fluidity, malonyl-CoA could regulate carnitine palmitoyltransferase activity on both surfaces of the inner membrane through an interaction with only the outer surface.

Rat kidney homogenates metabolize N6-trimethyl-lysine to N-trimethylammoniobutyrate, but not to carnitine. The first step in this conversion is the hydroxylation of trimethyl-lysine to form 3-hydroxy-N6-trimethyl-lysine. An assay system was developed in which hydroxylation of trimethyl-lysine is linear with respect to both time and homogenate protein concentration. The rate is 5 nmol of 3-hydroxy-N6-trimethyl-lysine formed/min per mg of homogenate protein. The cofactors required are ascorbate, alpha-oxoglutarate, FeSO4, and O2. Catalase and dithiothreitol give a 20% stimulation. Ca2+ produces a 2-fold increase in specific activity and cannot be replaced by Mg2+, Mn2+ or Zn2+. These last three bivalent cations lead to a decreased activity. Subcellular distribution studies demonstrate that trimethyl-lysine hydroxylase activity parallels the distribution profile of succinate dehydrogenase and citrate synthase. Thus trimethyl-lysine hydroxylase has a mitochondrial localization. Distribution of trimethyl-lysine hydroxylase activity between cortex and medulla of kidney if 67 and 33% respectively, similar to mitochondrial distribution.

1-Carnitine was administered to fed rats and the changes in plasma beta-hydroxybutrate concentration and liver acid-insoluble acylcarnitine content were assessed. One hour following injection of carnitine in doses greater than 1 mumol/100 g of body weight there was a dose-dependent increase in liver acid-insoluble acylcarnitine content to levels comparable to those seen in fasting. These increased levels were maintained for a least 2 h following injection. During the period following carnitine administration there was no increase in ketogenesis as evidenced by plasma beta-hydroxybutyrate concentrations. Since acid-insoluble acylcarnitines represent the product of carnitine palmitoyltransferase A, the results are interpreted as contradictory to the theory that this enzyme is rate-limiting and regulatory for ketogenesis.

Two populations of mitochondria were observed upon ultrastructural examination of cardiac muscle tissue, one located directly beneath the sarcolemma (subsarcolemmal mitochondria) and another between the myofibrils (interfibrillar mitochondria). Subsarcolemmal mitochondria were released by treatment of heart muscle with a Polytron tissue processor, while interfibrillar mitochondria were released by nagarse digestion of the remaining tissue. These results were supported by electron microscopy of Polytron-treated heart tissue showing rupture and loss of sarcolemma with release of the underlying mitochondria but with retention of intact mitochondria between the myofibrils. Electron microscopy of the isolated mitochondria indicated that both mitochondrial types maintained their structural integrity throughout the isolation procedure. Specific activities of succinate dehydrogenase and citrate synthase were higher in the interfibrillar mitochondria as compared to the subsarcolemmal mitochondria, while those of carnitine palmitoyltransferase and alpha-glycerophosphate dehydrogenase were nearly the same in both. Interfibrillar mitochondria oxidized all substrates tested approximately 1.5 times faster than did the subsarcolemmal mitochondria. Thus the two mitochondrial types differed not only in their respective locations in the cell, but also in certain biochemical properties.

Hepatic mitochondrial carnitine palmitoyltransferase (CPT) properties, beta-oxidation of palmitoyl-CoA and membrane polarization were measured in lean and obese Zucker rats. The Vmax. of the ‘outer’ carnitine palmitoyltransferase (‘CPT-A‘) increased with starvation, with no change in the Km for either carnitine or palmitoyl-CoA. The Ki for malonyl-CoA increased with starvation in lean rats, but not in obese rats. The Vmax. of the ‘inner’ enzyme (‘CPT-B‘), as measured by using inverted submitochondrial vesicles, increased with starvation in obese rats only, with no change in the Km for either carnitine or palmitoyl-CoA. The Ki for malonyl-CoA was 2-5-fold higher in inverted vesicles than in intact mitochondria, and showed no alteration with starvation. The activities of both enzymes correlated positively with each other and with beta-oxidation, and inversely with membrane polarization. Malonyl-CoA had little effect on gross membrane fluidity in the Zucker rat, as reflected by diphenylhexatriene fluorescence polarization. The results indicate that both enzymes are related and respond similarly to alterations in membrane fluidity. Membrane fluidity may provide a mechanism for co-ordinated control of CPT activity on both sides of the mitochondrial inner membrane.