Biomedical subjects
Publications and source records attributed to C L Hoppel.
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The purpose of the present study was to measure the activity of carnitine palmitoyltransferase (CPT) in the lateral and fourth ventricular choroid plexus (LVCP, FVCP) as an example of an enzyme committed to mitochondrial long-chain fatty acid oxidation. The CPT activity measured with both assays determines total CPT activity, that is, the activity of both CPT-A (outer form) and CPT-B (inner form). CPT-assay 1 (forward reaction) activity was 893.3 +/- 44.5 mU/g and 803.3 +/- 71.3 mU/g (mean +/- S.E.M.) in LVCP and FVCP, respectively. CPT-assay 2 (backward reaction) activity was 3673.7 +/- 92.4 and 3417.0 +/- 277.1 mU/g in LVCP and FVCP, respectively. These data demonstrate that CPT activity is present in the rabbit choroid plexus, and that the activity is somewhat higher per gram wet weight of tissue than the activity observed in the skeletal muscle (CPT-assay 1 = 514.4 +/- 159, CPT-assay 2 = 2492 +/- 576 mU/g). CPT activity has been exclusively localized to the mitochondria of the liver, heart, kidney and the skeletal muscles. Our present study further demonstrates that the choroid plexus is another organ in which high activity of CPT exists.
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.
A method for the isolation and chromatography of butyrobetaine from plasma, urine, and liver is described. The recovery of [3H-methyl]butyrobetaine from spiked biological samples was from 76-80%. Spiked samples then were derivatized with 4'-bromophenacyl trifluoromethanesulfonate and the butyrobetaine 4'-bromophenacyl ester was isolated by high-performance liquid chromatography (HPLC). Radioactivity eluted in a single peak which co-chromatographed with authentic butyrobetaine 4'-bromophenacyl ester. Two identical liver specimens were treated according to this isolation procedure. Prior to derivatization, one specimen was treated with butyrobetaine hydroxylase. After derivatization, there was no butyrobetaine 4'-bromophenacyl ester peak in the specimen treated with butyrobetaine hydroxylase. The HPLC detection sensitivity to butyrobetaine 4'-bromophenacyl ester was 1 pmol injected with a signal-to-noise greater than 2:1.
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.
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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.
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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.
Changes in hepatic mitochondrial oxidative metabolism were examined during the development of severe riboflavin deficiency in mice, and during recovery from this deficiency. There was a marked reduction in oxidative rates for all substrates tested, with the decline being most pronounced with palmitoyl-1-carnitine. These effects were not enhanced by addition of galactoflavin to the riboflavin-deficient diet. Treatment of the deficient mice with riboflavin restored hepatic mitochondrial oxidation to normal within 24 hours in those mice fed a simple riboflavin-deficient diet, but required 72 hours in galactoflavin-supplemented mice. These metabolic changes in hepatic mitochondria appear to be temporally independent of the striking morphological changes occurring in these organelles during ariboflavinosis and recovery.
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Weaning mice were fed a powdered complete diet containing either 0.5% cuprizone, 0.5% cuprizone + 0.01% copper sulfate, or 0.01% copper sulfate. With cuprizone as the sole additive, hepatic mitochondria became greatly enlarged. When the diet contained cuprizone + copper sulfate, giant mitochondria were still present, and, in addition, numerous lysosome-like structures became evident. When only copper sulfate was added to the normal diet, the mitochondria were of normal size, but the hepatocytes contained abundant lysosomes. Dietary supplementation with cuprizone or with cuprizone + copper sulfate resulted in considerably depressed (20-50%) rates of mitochondrial oxidation. Supplementation solely with copper sulfate produced virtually no changes in oxidative activity. It may be concluded that the subcellular effects of cuprizone are not based on its ability to produce copper deficiency by chelation of copper, but on other properties of this drug.
Weanling mice were fed a riboflavin-deficient diet or the same diet with added galactoflavin. Both diets produced changes in hepatic mitochondrial morphology, the most striking of which was the development of giant mitochondria. The livers from these animals were fractionated, and the nuclear and mitochondrial fractions were examined by electron microscopy. The nuclear fraction contained giant mitochondria; the mitochondrial fraction contained the remaining normal to moderately enlarged mitochondria. Oxidative studies were carried out on the mitochondrial fractions. It was found that both experimental diets resulted in a marked reduction in fatty acid oxidation by the mitochondria. In addition, the mitochondria of mice with advanced riboflavin deficiency (induced simply by a riboflavin-free diet) showed a severely decreased state 3 (ADP-stimulated) respiration and depressed respiratory control ratios, but normal ADP/O ratios. In contrast, mitochondrial performance (aside from fatty acid oxidation) in galactoflavin-supplemented, riboflavin-deficient mice was related to the gross appearance, i.e., color, of the liver from which these organelles were derived. In mice fed this diet, the livers were either red or yellow. Mitochondria from yellow livers showed normal oxidative phosphorylation. Mitochondria from red livers showed a serious reduction in state 3 oxidation. This study demonstrates that in the mouse, riboflavin deficiency, however produced, not only results in altered mitochondrial morphology but also results in significantly impaired mitochondrial function.
The surgical operating room schedule has been produced automatically for more than 2 years in our large teaching hospital. In order to apply computer technology to the complex surgical scheduling problem a special programming approach was devised. We discuss this approach under the headings of Expand, Sort, Order and Assign. Consistent, reliable schedules, unaffected by weekends and holidays, are produced by a clerk trained to use a computer terminal. Our program is adapttable to other institutions once the scheduling parameters and operating priorities are delineated.