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Biomedical subjects

J Bremer

Publications and source records attributed to J Bremer.

At least 127 records · Page 7Linked to original sources
PubMed1977

A spectrophotometric procedure for rapid and sensitive measurements of beta-oxidation. Demonstration of factors that can be rate-limiting for beta-oxidation.

1. A spectrophotometric direct-reading assay for measurements of beta-oxidation by intact mitochondria is described. The procedure relies on the ability of ferricyanide to trap reducing equivalents generated by the acyl-CoA dehydrogenases (EC 1.3.99.3). The reduction of ferricyanide was recorded by using a dual-wavelength spectrophotometer. 2. Oxaloacetate or acetoacetate was used to stimulate the rate of beta-oxidation by rotenone-blocked mitochondria. Although both were effective with rat liver mitochondria, oxaloacetate gave about 75% more stimulation. With heart or kidney mitochondria, only oxaloacetate gave marked stimulation. Acetoacetate had no stimulatory effect with heart mitochondria, but a small stimulatory effect on the rate of beta-oxidation by kidney mitochondria. 3. The stoicheiometry of beta-oxidation-dependent reduction of ferricyanide was examined, and good correlations were found between experimental and theoretical amounts of ferricyanide reduced. 4. Ferricyanide appears as efficient a final electron acceptor as O2. With ferricyanide the rate of beta-oxidation by heart mitochondria can be measured without interference from the oxidation of tricarboxylic acid-cycle intermediates.

Acetoacetates↗
PubMed1977

Monoethlenic C20 and C22 fatty acids in marine oil and rapeseed oil. Studies on their oxidation and on their relative ability to inhibit palmitate oxidation in heart and liver mitochondria.

1. Carnitine esters of erucic acid (22:1 n-9 cis), cetoleic acid (22:1 n-11 cis), brassidic acid (22:1 n-9 trans), gadoleic acid (20:1 n-9 cis) and oleic acid (18:1 n-9 cis) have been compared as mitochondrial substrates and as inhibitors of palmitoylcarnitine oxidation in heart and liver mitochondria. 2. Both the rate of intramitochondrial-CoA acylation and the rate of beta-oxidation decreases as the chain length increases from C18 to C22. There are no significant differences among the three C22 isomers as oxidizable substrates. 3. All the tested acylcarnitines inhibit palmitoylcarnitine oxidation. The C18 and C20 acylcarnitines inhibit by virtue of being competing substrates; i.e. the respiration is not inhibited. The C22-isomers inhibit also respiration; this shows that the inhibition of palmitolycarnitine oxidation is not compensated for by oxidation of C22-acylcarnitines. Brassidoylcarnitine inhibits the oxidation of palmitoylcarnitine and respiration less than erucoyl-and cetoleoylcarnitine. The different behaviour of the C22-isomers is probably due to the difference in their competitive properties with respect to long-chain acyl-CoA dehydrogenase. 4. All C22 acylcarnitines seem to be relatively better oxidized in the liver than in the heart mitochondria while their inhibitory effect on the usage of the radioactive palmitoylcarnitine is very similar. 5. Palmitoylcarnitine inhibits almost completely the "endogenous" formation of acetyl-CoA presumably from malate via pyruvate in the liver mitochondria while the C22-acylcarnitines cause only a partial inhibiton of this acetyl-CaO formation.

Animals↗
PubMed1976

Active transport of butyrobetaine and carnitine into isolated liver cells.

1. The liver cells lose the major part of their carnitine during the commonly used isolation procedure by the collagenase-perfusion method. 2. The cells take up carnitine and the carnitine precursor butyrobetaine when these substances are added to the medium. The carnitine content of isolated liver cells can increase to about 15 mM with no apparent harm to the cells. 3. The data indicate the existence of a common carrier in the plasma membrane which mediates the uphill transport of both carnitine and butyrobetaine. The carrier has a high affinity for butyrobetaine (Km=0.5 mM) and a lower one for carnitine (Km=5.6 mM). 4. The intracellular butyrobetaine is hydroxylated to carnitine with a rate of approximately 0.33 mumol-g wet weight-1-h-1 which is sufficient to cover the turn over of carnitine in the whole rat. Carnitine is effectively esterified in the liver cells to acetylcarnitine and long-chain acylcarnitines. 5. Both carnitine and acetylcarnitine are released from the cells. The release of both compounds is probably physiological since it was found that acetylcarnitine constitutes a similar fraction of the total acid soluble carnitine both in the blood and liver of the intact rat.

Animals↗
PubMed1976

The glycerophosphateacyltransferases and their function in the metabolism of fatty acids.

1. From different studies on the cellular localization, postional specificity, and regulatory properties of acyl-CoA: glycerophosphate acyltransferase (EC 2,3,1.15) AND ACYL-CoA: 1-ACYLGLYCEROPHOSPHATE ACYLTRANSFERASE (EC 2,3,1....) the following conclusions can be drawn: The glycerophosphate acyltransferase is localized in the endoplasmatic reticulum (microsomes) and in the outer membrane of the mitochondria of the animal cell. Its reaction product is 1-acylglycerophosphate (1-lysophosphatidic acid). The mitochondrial enzyme shows a high preference for saturated fatty acids while the microsomal enzyme is less specific (alternatively the microsomes contain more than one glycerophsophate acyltransferase). 2. The 1-acylglycerphosphate acyltransferase is localized in the endoplasmatic reticulum (microsomes) in the animal cell. Possibly a minor fraction of this enzyme is localized to the outer membrane of the mitochondria. This enzyme shows a strong preference for unsaturated fatty acids. 3. Both the microsomal and the mitochondrial dihydroxyacetonephosphate acyltransferase show similar fatty acid specificity as the corresponding glycerophosphate acyltransferases. It cannot be excluded that dihydroxy-acetonephosphate and glycerophosphate are acylated by the same enzymes. 4. The activity of the glycerophosphate acyltransferase(s) in the liver decreases in fasting or fat feeding and increases upon feeding of carbohydrate. The activity of carnitine palmityltransferase varies exacty opposit. These enzymes do not show dietary variations in heart and adipose tissue. 5. Under the otherwise identical conditions the rate of carnitine acylation in isolated mitochondria decreases more than the rate of glycerophosphate acylation when the concentration of palmityl-CoA is reduced. 6. In isolated liver cells (which has lost most of their carnitine) addition of carnitine increases the rate of fatty acid oxidation and decreases the rate of triglyceride formation. 7. Glycerol and fructose lower the rate of fatty acid oxidation, probably by lowering the levels of acyl-CoA and acyl-carnitine in the cells. 8. It is concluded that the relative activities of glycerophosphate acyltranse and carnitine palmityltransferase probably influence the fate of fatty acids in the cell.

Acyltransferases↗
PubMed1976

Phosphatidic acid biosynthesis in rat liver mitochondria and microsomal fractions. Regulation of fatty acid positional specificity.

The positional and fatty acid specificity of phosphatidic acid biosynthesis in rat liver mitochondria and microsomal fractions was studied by using acylcarnitines, CoA and an excess of carnitine palmitoyltransferase (EC 2.3.1.21) as the source of acyl-CoA. In the mitochondria, the preference for palmitic acid at the 1-position is increased at high acyl-CoA concentrations, whereas it is decreased in the microsomal fraction. There was no change in the fatty acid specificity at the 2-position with different acyl-CoA concentrations in any of the factions. The preference in mitochondria for linoleic acid at the 2-position is strongly increased at high concentrations of lysophosphatidic acid.

Acylation↗
PubMed1976

Suppression of the mitochondrial oxidation of (-)-palmitylcarnitine by the malate-aspartate and alpha-glycerophosphate shuttles.

Palmitylcarnitine oxidation by isolated liver mitochondria has been used to investigate the interaction of fatty acid oxidation with malate, glutamate, succinate, and the malate-aspartate shuttle. Mitochondria preincubated with fluorocitrate were added to a medium containing 2mM ATP and ATPase. This system, characterized by a high energy change, allowed titration of respiration to any desired rate between States 4 and 3 (Chance, B., and Williams, G. R. (1956) Adv. Enzymol. Relat. Areas Mol. Biol. 17, 65-134). When respiration (reference, with palmitylcarnitine and malate as substrates) was set at 75% of State 3, the oxidation of palmitylcarnitine was limited by acetoacetate formation. The addition of malate or glutamate approximately doubled the rate of beta oxidation. Malate circumvented this limitation by citrate formation, but the effect of glutamate apparently was due to enhancement of the capacity for ketogenesis. The rate of beta oxidation was curtailed when malate and glutamate were both present. This curtailment was more pronounced when the malate-aspartate shuttle was fully reconstituted. Among the oxidizable substrates examined, succinate was most effective in inhibiting palmitylcarnitine oxidation. Mitochondrial NADH/NAD+ ratios were correlated positively with suppression of beta oxidation. The degree of suppression of beta oxidation by the malate-aspartate shuttle (NADH oxidation) or by succinate oxidation was dependent on the respiratory state. Both substrates extensively reduced mitochondrial NAD+ and markedly suppressed beta oxidation as respiration approached State 4. Calculations of the rates of flux of hydrogen equivalents through beta oxidation show that the suppression of beta oxidation by glutamate or by the malate-aspartate shuttle is accounted for by increased flux of reducing equivalents through mitochondrial malic dehydrogenase. This increased Flux is accompanied by an increase in the steady state NADH/NAD+ ratio and a marked decrease in the synthesis of citrate. The alpha-glycerophosphate shuttle was reconstituted with mitochondria isolated from rats treated with L-thyroxine. This shuttle was about equal to the reconstructed malate-aspartate shuttle in supression of palmitylcarnitine oxidation. This interaction could not be demonstrated in euthyroid animals owing to the low activity of the mitochondrial alpha-glycerol phosphate dehydrogenase. It is concluded that beta oxidation can be regulated by the NADH/NAD+ ratio. The observed stimulation of flux through malate dehydrogenase both by glutamate and by the malate-aspartate shuttle results in an increased steady state NADH/NAD+ ratio, and is linked to a stoichiometric outward transport of aspartate. We suggest, therefore, that some of the reducing pressure exerted by the malate-aspartate shuttle and by glutamate plus malate is provided through the energy-linked, electrogenic transport of aspartate out of the mitochondria. These results are discussed with respect to the mechanism of the genesis of ethanol-induced fatty liver.

Animals↗
PubMed1976

Replenishment and depletion of citric acid cycle intermediates in skeletal muscle. Indication of pyruvate carboxylation.

The effects of various substrates on the concentrations of free amino acids, citric acid cycle intermediates and acylcarnitines were studies in perfused hindquarter of rat in presence of glucose and insulin in order to assess regulatory mechanisms of the level of citric acid cycle intermediates in skeletal muscle. 1. Acetate and acetoacetate effected a significant increase in the level of citrate cycle intermediates and accumulation of acetylcarnitine. These changes were accompanied by a reduction in the level of alanine. The concentration of AMP was significantly elevated. 2. Muscle mitochondria fixed 14CO2 in the presence of pyruvate. The products were identified as malate or citrate when whole and disintegrated mitochondria were used respectively. The fixation was greatly stimulated by acetylcarnitine. 3. Acetylcarnitine inhibited the production of pyruvate from malate by muscle mitochondria. 4. Perfusion with 2-oxoisocaproate and 2-oxoisovalerate promoted increases in the level of citric cycle intermediates, a drop in both alanine and glutamate, and accumulation of branched-chain acylcarnitines. 2-Oxoisocaproate also caused a reduction of alanine released from the muscle. 5. Perfusion with leucine and valine did not change the concentration of citric acid cycle intermediates, but elevated glutamate and still more the concentration of alanine. 6. It is concluded that citric cycle intermediate level in the perfused resting muscle is modified by a) conditions which change the concentration of acetyl-CoA and thereby modify the rate of pyruvate carboxylation and decarboxylation of malate via malic enzyme b) conditions which change the concentration of pyruvate cause changes in alanine and cycle intermediates in the same direction via transamination reactions c) conditions which change the concentrations of 2-oxoacids which are converted to cycle intermediates via oxidation.

Acetyl Coenzyme A↗
PubMed1976

The role of acyltransferases in fatty acid utilization.

Glycerophosphate and carnitine as competing acyl acceptors and the oxidation of different long chain acylcarnitines in isolated mitochondria have been investigated. In the presence of low concentrations of palmitate, glycerophosphate is the preferred acyl acceptor in isolated liver mitochondria, indicating that the glycerophosphate acyltransferase has a lower Michaelis constant (Km) for palmityl-CoA than has the carnitine palmityltransferase. Liver mitochondria from starved rats make more palmitylcarnitine and less palmitylglycerophosphate than do mitochondria from starved/refed rats. In heart mitochondria glycerophosphate has less effect on palmitylcarnitine formation because of glycerophosphate acyltransferase activity is lower and the carnitine palmityltransferase activity is higher in liver mitochondria. Carnitine esters of C22 fatty acids, especially erucic acid (22:1omega9,cis) are more slowly oxidized compared to palmitylcarnitine in heart than in liver mitochondria. They also inhibit palmitylcarnitine oxidation. The inhibition is relatively stronger in heart than in liver mitochondria. These observations are discussed in relation to organ differences in the utilization of fatty acids.

Acylation↗
PubMed1975

Studies on the active transfer of reducing equivalents into mitochondria via the malate-aspartate shuttle.

1. The effects of mitochondrial energy states onthe extramitochondrial NADH/NAD ratio via a reconstituted malate-aspartate shuttle have been investigated. 2. The transfer of reducing equivalents into isolated mitochondria is stimulated by ATP and by electron transport. The effect of ATP is inhibited by oligomycin. The effect of electron transport is inhibited by uncouplers. 3. Uncoupling of the mitochondria is required for rapid transfer of reducing equivalents out of the mitochondria. 4. A glutamate-stimulated entry of aspartate into energized mitochondria suggests that the malate-aspartate shuttle is to some extent reversible even in a high energy state of the mitochondria. 5. It is concluded that the malate-aspartate shuttle contributes to the formation of the skewed redox situation across the inner mitochondrial membrane, which has a more reduced inside.

Adenosine Triphosphate↗