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This is the first report of the effect of prostaglandins on the biochemical pathways for fatty acid synthesis. PGE2 and PGF2α inhibited fatty acid elongation in a lung microsomal fraction. Neither prostaglandin affected the de novo, or soluble, system for fatty acid synthesis (i.e. acetyl CoA carboxylase or fatty acid synthetase). The results also suggest that the initial inhibition of fatty acid synthesis leads to a decrease in free fatty acids available for esterification into phospholipids. The site and possible mechanisms of inhibition are discussed.

Rat atrial metabolism was monitored by measuring the production of 14 CO 2 from 14 C-labeled substrates. D -Glucose and D -mannose metabolism were depressed by low concentrations of halothane (1 mM) which did not significantly affect the metabolism of pyruvate, D -fructose, DL -beta-hydroxybutyrate or octanoate. Halothane (1 mM) did not alter the uptake of 3- O -methyl glucose by rat atria. It is concluded that halothane blocks an early step(s) in glycolysis. The most likely sites are the phosphoglucose isomerase (PGI) and phosphomannose isomerase (PMI) steps. The incorporation of D -glucose, D -mannose and D -fructose into glycogen were significantly inhibited by 1 mM halothane, although the total glycogen content was not affected. We conclude that halothane inhibits glycogen turnover. Higher concentrations of halothane (8 and 16 mM) required to inhibit the metabolism of pyruvate and D -fructose. This action of halothane is attributed to the known inhibition by halothane of electron transport processes. Neither DL -beta-hydroxybutyrate nor octanoate metabolism to CO 2 was affected by 1 mM halothane, although higher concentrations of halothane produced an inhibition. It is concluded that some of the steps in fatty acid oxidation are unaffected by low concentrations of halothane.

1. 4-Pentenoic acid, infused into the left renal artery of the dog (6.6 μM/min) caused a marked increase of sodium, potassium, phosphate and glucose excretion in all experiments. Amino acid excretion was only slightly augmented in 2 of 4 experiments. 2. The enhanced excretion of these substances took place even when GFR was diminished by about 50%. 3. Pentanoic acid and cyclopropanecarboxylic acid which do not inhibit long chain fatty acid oxidation in comparable doses, had no influence on renal function in doses up to 528 μM/min. Higher doses caused hemoglobinuria and anuria on the infused side. 4. These findings are in good agreement with the view that 4-pentenoic acid diminishes reabsorption of the substances tested in this study by blocking long chain fatty acid oxidation, a source of energy for transport.

In confirmation of previous findings it was shown that perfused livers from fasted rats converted oleic acid into ketone bodies far more efficiently than did livers from fed animals, whereas differences in rates of ketogenesis from octanoate were much less pronounced. However, relative rates of ketone body production from (-)-octanoylcarnitine resembled those seen with oleic acid rather than those obtained with free octanoic acid as substrate. In addition, (+)-octanoylcarnitine, an inhibitor of carnitine acyltransferase, was without effect on the oxidation of octanoic acid, but caused a profound and quantitatively similar depression in the oxidation of both oleic acid and (-)-octanoylcarnitine. The data support the concept that the carnitine acyltransferase system of liver is under strict dietary, or hormonal control, or both, and that it may constitute a primary site for the regulation of hepatic fatty acid oxidation and ketogenesis.

Feeding trials indicated that supplementing a diet adequate in vitamins with a four-fold excess of the National Research Council recommendations for vitamins afforded no protection against the growth inhibitory effect of aflatoxin in chickens. On the other hand, when the combined effects of dietary aflatoxin and vitamin deficiencies were studied the vitamin deficiencies investigated could be divided into three classes. Diets deficient in riboflavin or vitamin D3 made chickens sensitive to levels of aflatoxin normally too small to influence their growth rate. In a diet adequate in vitamin D3, aflatoxin reduced the serum calcium level by 20 percent. The second class of vitamin deficiencies with regard to aflatoxin contained vitamin K3 and vitamin E whose dietary status had no influence on aflatoxicosis as measured by growth rate. The third class was represented by thiamine. A thiamine deficiency had a protective effect against the growth inhibitory effect of dietary aflatoxin. This unexpected protective effect can be rationalized on the basis that aflatoxin inhibits transport of fat from the liver while a thiamine deficiency stimulates fatty acid oxidation.

1. 1. Acetoacetate reduction by rat liver mitochondria in the presence of different succinate concentrations has been studied. 2. 2. At low succinate concentrations (0.5-1 mM) the oxidation of mitochondrial free fatty acids, sparked by succinate, plays a prominent role in the acetoacetate reduction. 3. 3. At higher succinate concentrations the reducing equivalents for acetoacetate reduction derive from succinate via the ‘ reversal’ pathway. 4. 4. Furthermore in the presence of high succinate concentrations (lomM) fatty acid oxidation is almost completely inhibited.

The effects of octanoate and oleate were studied in the isolated fasted rat liver perfused without substrate and were compared with the effects of lactate. Measurements were made of hepatic adenine nucleotide content, formation of ketone bodies, urea and glucose and uptake of fatty acids, lactate and oxygen. Calculations of the Krebs cycle activity and the flux through the oxidation at C-3 were carried out. Comparing the extra oxygen consumption with extra ATP needs after addition of the fatty acids or lactate, ADP: O ratios were estimated for fatty acid oxidation and lactate gluconeogenesis. 1. Octanoate as well as oleate induced a net decrease in ATP and an increase in AMP content of the liver. Total adenine nucleotides were unaltered. ATP:ADP ratios were lowered while the adenylate kinase mass-action ratio increased. 2. The Krebs cycle activity was suppressed by both fatty acids and enhanced by lactate. Oxidation at C-3 was strongly stimulated by the fatty acids and was unaffected by lactate. 3. An apparent ADP:O ratio of 2.7 was obtained after lactate addition, indicating a tightly coupled oxidative phosphorylation for the liver preparations used. Octanoate and oleate gave extremely low ratios of near one and near zero respectively. 4. Perfusion with oligomycin caused a severe drop in oxygen uptake by the liver, which was unaltered after addition of fatty acids. Oligomycin added after the fatty acids caused an immediate fall in oxygen uptake to the level observed with oligomycin alone. 2,4-Dinitrophenol was able to stimulate the oligomycin-depressed respiration, both in the absence and in the presence of fatty acids. These results indicate that under our experimental conditions the fatty acids had no uncoupling effect and that microsomal fatty acid oxidation had to be minimal. 5. Incorporation of [3H]leucine into proteins in liver and medium remained unchanged after addition of fatty acids, indicating that the low apparent ADP:O ratios are not the result of an enhanced synthesis of proteins. 6. The possible relationship between fatty acid transport, energy-wasting futile cycles and the low apparent ADP:O ratios observed during fatty acid oxidation is discussed.

Crystals of l -3-hydroxyacyl coenzyme A dehydrogenase from pig heart that are suitable for X-ray analysis have been prepared and characterized. The enzyme is a mitochondrial protein important in the beta oxidation of fatty acids. It is of special interest because it requires coenzyme A-related molecules for its catalytic activity. For one of the crystalline forms of the enzyme, the X-ray data tend to confirm chemical observations that the molecule is a dimer of identical subunits.

1. 1. Carnitine added to mitochondria isolated from human newborn white adipose tissue by a new method enhances oxygen consumption, while deoxycarnitine inhibits it. 2. 2. These effects are not seen in adult tissue. 3. 3. Isolated adipose tissue cells from the newborn can be separated into large and small cells. Mitochondria isolated from the former are insensitive to carnitine, while mitochondria isolated from the small cells show increased oxygen consumption on addition of carnitine. 4. 5. It is suggested that these small cells may produce heat by fatty acid oxidation.

1. During fatty acid oxidation by rat liver mitochondria, the rate of β-oxidation is dependent on the relative amounts of substrate and mitochondrial protein, on the energy state of the mitochondria, on the chain length and the number of double bonds of the fatty acid and on the concentration of various compounds in the reaction medium ( l -carnitine, CoASH, hexokinase, albumin). 2. The rate of β-oxidation of long-chain fatty acids decreases when the ratio of albumin over fatty acid is increased. This effect is most marked in the absence of added carnitine. 3. Addition of excess hexokinase decreases the rate of β-oxidation in the presence of added carnitine. 4. Maximal rates of β-oxidation are observed with octanoate and decanoate (40–60 nmoles acetyl-CoA/min per mg mitochondrial protein at 25 °C). 5. Odd-numbered fatty acids are oxidized at a much lower rate than the even-numbered homologues. In a low-energy state propionyl-CoA accumulates; in a high-energy state in the presence of bicarbonate, Krebs-cycle intermediates accumulate. 6. l -Carnitine enhances the rate of β-oxidation of all fatty acids except butyrate. The stimulatory effect is most pronounced with odd-numbered and with long-chain fatty acids. 7. In the absence of added carnitine the rate of β-oxidation of long-chain fatty acids decreases with the chain length and increases with the number of double bonds. It is suggested that the solubility of the long-chain fatty acids in the aqueous medium is the rate-limiting factor under these conditions. 8. In the presence of carnitine and albumin, palmitate, oleate, linoleate and linolenate are all oxidized at about the same rate (25–30 nmoles/min per mg protein at 25 °C). 9. Propionyl-CoA is not formed as an intermediate during oxidation of unsaturated fatty acids.

A study was conducted on the metabolism of ketone bodies in tissue preparations from normal and dystrophic chicks. The data indicated that the production of ketone bodies in liver homogenates, as a result of fatty acid oxidation, was not markedly altered by development of the dystrophic condition. Whereas acetoacetate was oxidized by normal and degenerative pectoral muscle to approximately the same extent, utilization of β-hydroxybutyrate in dystrophic muscle was markedly poorer. In view of present concepts of the reactions involved in the metabolism of ketone bodies the results suggest that in the chick myopathy the conversion of β-hydroxybutyrate to acetoacetate may be impaired.

The metabolic fate of glucose in isolated muscle after addition of insulin was shown to be dependent upon the functional state of the tissue. While nonstimulated muscle responded primarily with an increased incorporation of glucose into glycogen, stimulated muscle showed predominantly an increased conversion of labeled glucose to CO2 following insulin addition. The oxidation of palmitic-1-C14 acid by muscle was not influenced by the presence of insulin. Ryanodine, used as a chemical agent for inducing contraction of diaphragm, resulted in stimulation of oxygen consumption, fatty acid oxidation and glucose oxidation to an extent comparable to that previously achieved with electrical stimulation of muscle. The conclusion was reached that insulin increased the oxidation of glucose but not of palmitate, and that the specific metabolic fate of intracellular glucose is not influenced by insulin. The data are discussed in relation to the prevailing theory that insulin acts by increasing permeability of muscle cell membranes to certain substrates.

The time course of changes in a variety of physiological parameters concerned with carbohydrate and lipid metabolism has been studied both in vivo and in the isolated perfused liver during induction and reversal of starvation ketosis in the rat. The data obtained demonstrate that surprisingly brief periods of starvation and refeeding exert dramatic effects on glucose and fatty acid metabolism in the intact animal and that generally synchronous changes occur in the ketogenic and gluconeogenic capacities of the perfused liver. In agreement with previous findings it was shown that the enhanced conversion of labeled oleate into ketone bodies by livers from fasted rats was associated with a concomitant depression in its incorporation into triglycerides, and that the antiketogenic effect of lactate was accompanied by a diversion of the fatty acid from the β oxidation sequence into the esterification pathway. The key observation, however, was that blockade of long chain fatty acid oxidation by (+)-decanoylcarnitine, an inhibitor of long chain acylcarnitinetransferase, stopped ketone body formation and acutely changed the pattern of metabolism of oleic acid in livers from fasted rats to that exhibited by livers from normal animals, i.e. the fatty acid was now virtually completely esterified. The data are consistent with the view that hepatic fatty acid oxidation and ketogenesis are under strict dietary and hormonal control exerted primarily by regulation of an early step in the oxidative sequence, probably the acylcarnitinetransferase reaction. The possibility is also raised that the effects of lactate and other antiketogenic agents are related to interactions at this site.

The mechanism of alloisocitric acid fermentation by the fungus, Penicillium purpurogenum var. rubrisclerotium No. 1148, was studied using C 14 O 2 and citric acid-1,5-C 14 . By the aerobic incubation with glucose in the presence of C 14 O 2 , C 14 O 2 was incorporated into carbons 1 and 6 of alloisocitric acid in equal amounts. The pathway was assumed including the carboxylation of the C 3 compound to the C 4 -dicarboxylic acid, and the condensation of C 2 with the C 4 -dicarboxylic acid, which was randomized completely through the dicarboxylic acid shuttle. Isocitritase type condensation of C 4 with C 2 , Thunberg reaction, and the glyoxylic acid bypath seemed unlikely to be operative. The incorporation experiments of citric acid-1,5-C 14 showed that the labeling patterns of the residual citric acid and alloisocitric acid formed were completely the same. It suggested the presence of some direct conversion pathway from citric acid to alloisocitric acid without splitting of the carbon skeleton. From these with some other data, it was presumed that alloisocitric acid might be formed immediately from citric acid which was recycling through the tricarboxylic acid cycle. The presence of citric condensing enzyme and a few other enzymes of the tricarboxylic acid cycle was shown.

The effects of growth hormone administration on in vitro adipose tissue metabolism have been investigated using normal rats and hypophysectomized rats subjected either to fasting or to streptozotocin-induced diabetes.Growth hormone administration in normals increased animal weight gain and in vitro incorporation of fatty acid into tissue lipids but did not alter fat pad composition or fatty acid oxidation. In diabetic hypophysectomized rats given growth hormone weight gain, fat pad protein content and fatty acid utilization were increased, whereas fat pad lipid content and glucose utilization were decreased. In fasted-hypophysectomized rats growth hormone decreased weight loss and increased adipose tissue protein concentration. Fatty acid oxidation was not altered, whereas incorporation into adipose tissue lipids was increased.

Long-chain fatty acid synthesis by rat-liver microsomes increases strikingly following birth. After a peak of activity at 11 days of age, microsomal synthesis declines until, coincident with the time of weaning, there is a rapid increase in activity to adult levels. Product identification indicates that microsomal fatty acid synthesis occurs primarily by chain elongation.

SUMMARY: Low concentrations of fatty acids with less than 14 carbon atoms were oxidized without a lag phase over a wide range of pH values by mycelium of Penicillium roqueforti. The effect of the fatty acids upon oxygen uptake by a given weight of mycelium, and the nature of the products of oxidation, were dependent upon the concentration and chain length of the fatty acid and the pH value of the system. The C9-C12 fatty acids which showed the greatest inhibitory effect were not oxidized to the corresponding methyl ketone with one less carbon atom in such high yields as the less toxic C6-C8 acids. The C6-C8 fatty acids markedly inhibited endogenous respiration at low pH values but this inhibition was reversed by increasing the pH value. The toxic effect associated with some fatty acids was less pronounced against mycelium which had been previously shaken over an extended period in phosphate buffer. It is suggested that the cellular regulation of fatty acid oxidation and methyl ketone formation involves deacylation of β-oxo acyl thiolester which provides an alternative means of recycling coenzyme A when oxidation of acetyl CoA is impaired.

Escherichia coli grows on long-chain fatty acids after a distinct lag phase. Cells, preadapted to palmitate, grow immediately on fatty acids, indicating that fatty acid oxidation in this bacterium is an inducible system. This hypothesis is supported by the fact that cells grown on palmitate oxidize fatty acids at rates 7 times faster than cells grown on amino acids and 60 times faster than cells grown on a combined medium of glucose and amino acids. The inhibitory effect of glucose may be explained in terms of catabolite repression. The activities of the five key enzymes of β-oxidation [palmityl-coenzyme A (CoA) synthetase, acyl-CoA dehydrogenase, enoyl-CoA hydrase, β-hydroxyacyl-CoA dehydrogenase, and thiolase] all vary coordinately over a wide range of activity, indicating that they are all under unit control. The ability of a fatty acid to induce the enzymes of β-oxidation and support-growth is a function of its chain length. Fatty acids of carbon chain lengths of C 14 and longer induce the enzymes of fatty acid oxidation and readily support growth, whereas decanoate and laurate do not induce the enzymes of fatty acid oxidation and only support limited growth of palmitate-induced cells. Two mutants, D-1 and D-3, which grow on decanoate and laurate were isolated and were found to contain constitutive levels of the β-oxidation enzymes. Short-chain fatty acids (8 ) do not support growth of either the parent strain or the mutants D-1 and D-3. Evidence is also presented to show that decanoate is actively transported by the parent strain and by the mutants.

Summary 1. The half-century of investigations directed towards an understanding of the mechanism of β-oxidation of fatty acids may be divided into three periods: (a) from 1904 to 1939 when the oxidation could be studied at the level of the intact animal or isolated organ or tissue slice; (b) from 1939 to 1952 when it could be studied at the mitochondrial level; and (c) from 1952 onwards when it could be reconstructed in non-mitochondrial and soluble enzyme systems. 2. The Knoop-Dakin theory of β-oxidation could not be directly confirmed owing to the non-accumulation of any intermediates. The theory was based on deductions from the nature of the end-products of the metabolism of phenyl fatty acids. 3. The study of fatty acid oxidation at the mitochondrial level led to the recognition that the fatty acids are not oxidized as such but only in the form of some derivative whose formation is tied up with oxidative phosphorylation and the production of adenosine triphosphate (ATP). 4. The transition from the mitochondrial system to soluble enzymes was facilitated first by the discovery that coenzyme A (CoA) was concerned in acyl transfer reactions and later by the recognition that the active fatty acids are indeed the fatty acyl derivatives of CoA. 5. There are four known enzymatic processes by which fatty acyl CoA's are formed: (a) oxidation of pyruvate to acetyl CoA; (b) conversion of fatty acids to fatty acyl CoA's by ATP; (c) replacement of the succinyl group of succinyl CoA by short-chain fatty acids; and (d) cleavage of β-ketoacyl CoA's by CoA with formation of a fatty acyl CoA and acetyl CoA. 6. Two separate enzymes are known to catalyse the oxidation of fatty acyl CoA's to their corresponding transα, β-unsaturated derivatives. The first is a green dehydrogenase containing copper and flavin as prosthetic groups which is active upon acyl CoA's from C3 to C8. The metal is essential for the interaction of this dehydrogenase with cytochrome c. The second is a yellow flavoprotein which is active upon acyl CoA's from C4 to C18. 7. Unsaturated fatty acyl CoA hydrase catalyses the hydration of transα, β- or β, γ-unsaturated CoA's to their corresponding l(+)-β-hydroxyacyl CoA derivatives. The enzyme acts upon all unsaturated derivatives from C4 to at least C12. At equilibrium (pH 9, 250) the ratio β-hydroxyacyl CoA:total unsaturated acyl CoA is 1. 4:1. 8. The β-hydroxyacyl CoA dehydrogenase catalyses the oxidation of l(+)-β-hydroxyacyl CoA by DPN+. The product of oxidation is the corresponding β-ketoacyl CoA. The enzyme is active over the entire range of fatty acid chain length. The E0of the reaction couple at pH 7.0 and 220 is – 0.224 V. The equilibrium point of the oxidation is strongly pH dependent. 9. The β-ketoacyl CoA cleavage enzyme catalyses the reversible cleavage of β-ketoacyl CoA's by another molecule of CoA to form acetyl CoA and a new acyl CoA with two carbon atoms less than the parent β-ketoacyl CoA. 10. The new fatty acyl CoA generated in the cleavage reaction undergoes a repeat cycle of β-oxidation while the C2 unit (acetyl CoA) undergoes condensation with oxalacetate to form citrate. 11. Each of the component reactions in the β-oxidation cycle has been shown to be reversible. 12. The asymmetric labelling of acetoacetate formed during oxidation of labelled fatty acids by liver homogenates or mitochondrial suspensions is a phenomenon which can readily be explained in terms of the mechanism of the β-ketoacyl CoA cleavage enzyme. 13. The factors which militate against the accumulation of intermediates during fatty acid oxidation are discussed. 14. The accumulation of acetoacetate in any tissue requires a combination of two essential conditions: (a) presence of acyl CoA deacylase; and (b) absence of a β-ketoacid activation enzyme. 15. Assuming that in the diabetic the operation of the citric acid cycle is subnormal by virtue of reduced conversion of glucose to pyruvate, it is possible to explain the accumulation of ketone bodies and its abolition by insulin in terms of the known enzyme reactions of the β-oxidation cycle. 16. The predominance of C16 and C18 fatty acids in lipids may be due to the fact that only the acyl CoA's of these particular acids dissociate to a sufficient degree from combination with the enzymes of the fatty acid oxidizing system as to become available for ester synthesis. It is a great pleasure to acknowledge my indebtedness to Dr Helmut Beinert for his advice and assistance in the preparation of the manuscript and to Drs H. R. Mahler, D. R. Sanadi and S. J. Wakil for their suggestions.

1. Measurements have been made of the activities of acyl-CoA dehydrogenase, enoyl-CoA hydratase, β-hydroxyacyl-CoA dehydrogenase and ketothiolase in the livers of rats treated for either 12hr. or 3 days with pituitary growth hormone. 2. There was a significant increase in the activity of acyl-CoA dehydrogenase in rats treated with the hormone for 3 days. 3. Measurements were also made of the lipogenic enzymes acetyl-CoA carboxylase and palmitate synthase in the livers of similarly treated animals. 4. There was a depression of the activity of both enzymes after 12hr. treatment and a further decline after 3 days. 5. The results are discussed in relation to the known increase in the rate of fatty acid oxidation and inhibition of fatty acid synthesis in rats treated with growth hormone.

The substrate specificity of bovine liver crotonase has been examined with seven Δ2, 3-trans-enoyl-CoA substrates, containing an even number of carbon atoms. The Vmax for this series decreases progressively from a value of about 340,000 moles per min per mole of enzyme for crotonyl-CoA, the C4 derivative, to 2,300 for the C16 derivative. The action of several CoA derivatives on crotonase has been tested. None were found to stimulate the enzyme and only one derivative, acetoacetyl-CoA, was found to be markedly inhibitory. Evidence was obtained that the enolate form of acetoacetyl CoA was the inhibitory species and acted as a competitive inhibitor with a Ki of 1.6 x 10-6 m, a value about ten times lower than the Km for the best substrate, crotonyl-CoA. The interaction of acetoacetyl-CoA with crotonase was studied by ultraviolet difference spectroscopy and it was found that 6 molecules of inhibitor were bound per molecule of enzyme, or an average of one per subunit. This suggests that there are six active sites per molecule of native enzyme. The binding constant for the inhibitor was about equal to the kinetically determined value for Ki. The catalytic properties of crotonase have been compared with the turnover numbers and substrate specificities of the other enzymes acting in β oxidation of fatty acyl-CoA derivatives. This comparison suggests that crotonase, by virtue of its substrate specificity and its sensitivity to feedback inhibition by acetoacetyl-CoA, may play a regulatory role in fatty acid oxidation. The effects of acetoacetyl-CoA on the rate of oxidation of butyric, octanoic, and palmitic acids by heart muscle or liver mitochondria were those expected if crotonase is acting, at least in part, to regulate fatty acid oxidation.

The oxidation of several sterols by mouse liver mitochondria has been found to be inhibited by mepyrapone; 26-hydroxycholesterol, cholesterol oxidation and to a lesser extent, 7α-hydroxycholesterol oxidation were inhibited by mepyrapone, whereas 3β-hydroxycholest-5-en-26-oic acid oxidation was hardly affected. The mitochondrial oxidation of several fatty acids was unaffected by added mepyrapone even at relatively high concentrations of the drug. Three purified NAD- linked dehydrogenases, including 26-hydroxysterol dehydrogenase, were found to be selectively inhibited by mepyrapone when compared with an analogue, 3-acetylpyridine. Several other drugs in the group which inhibit steroid hydroxylations in the adrenal cortex (SU series) also inhibited cholesterol oxidation but not fatty acid oxidation in the liver. Three drugs which inhibit cholesterolgenesis in the liver (SKF series) were found to inhibit sterol oxidation by liver mitochondria. These drugs differed from the SU series in their effects on fatty acid oxidation by both heart and liver mitochondria. The stimulation of fatty acid oxidation by SKF 525A appeared to mimic the effects of added dl -carnitine especially in heart mitochondria. Whereas carnitine stimulated the mitochondria oxidation of 3β-hydroxycholest-5-en-26-oic acid, it was found that SKF 525A inhibited the latter's oxidation. From detailed studies comparing the effects of SKF 525A and carnitine, it was concluded that SKF 525A may antagonize carnitine at low concentrations of the latter. These effects were observed mainly with palmitate oxidation in heart mitochondria.

α-Bromopalmitate inhibits the activation of long-chain fatty acids by rat liver preparations. This inhibitory effect is not related to the amount of protein, is reversible and appears to be of a competitive type. An α-bromo or an α-hydroxy substitution slowed the rate of activation to about 1 20th of that of the corresponding unsubstituted fatty acid. Oxidation of palmitate as well as palmitate incorporation into glycerides by homogenates of heart was also inhibited by α-bromopalmitate. Evidence obtained shows that these inhibitory effects are not related to the possible surfactant property of this long-chain free fatty acid. Thus the reported selective inhibitory effect of α-bromopalmitate on fatty acid oxidation in intact cells may involve inhibition of activation in addition to the known inhibition of carnitine palmitoyltransferase by CoA esters of α-bromo long-chain acids. Activation of palmitate and palmitate oxidation were also inhibited by phytanate. Hence it appears possible that the accumulation of phytanic acid as in Refsum's disease may interfere with the normal oxidation of fatty acids.

Rat liver cell suspensions oxidized [1-14C]palmitic acid and [1-14C]palmitoyl-l-carnitine efficiently to CO2. Oxidation of tracer amounts of palmitate was inhibited by α-bromo-palmitate (2 x 10-4 m). The oxidation of tracer amounts of palmitoyl-l-carnitine was not affected. α-Bromo-palmitoyl-dl-carnitine did not have any effect on the oxidation of either substrate. The inhibition of oxidation of tracer amounts of [1-14C]palmitate by the bromo-acid could be overcome by increasing the concentration of palmitic acid in the incubation medium. The oxidation of [1-14C]palmitic acid by liver mitochondria was also inhibited by α-bromo-palmitate but the inhibition could not be overcome by additional palmitate. α-Bromo-palmitoyl-dl-carnitine inhibited the oxidation of both palmitic acid and palmitoyl-l-carnitine by isolated mitochondria. The long chain acyl-CoA ligase activity of ultrasonically disrupted liver mitochondria was irreversibly inhibited by the α-bromo-palmitate but the long chain acyl-CoA-carnitine transferase was unaffected. Mitochondria isolated from liver cell which had been treated with α-bromo-palmitate were able to oxidize palmitic acid and had unimpaired long chain acyl-CoA ligase and acyl-CoA-carnitine transferase activities. The inhibition of fatty acid oxidation by cell suspensions in the presence of α-bromo-palmitic acid could be shown with palmitic, oleic, myristic, lauric, and decanoic acids but not with octanoic acid as substrates. From the results it is concluded that α-bromo-palmitic acid reversibly competes with a carrier mechanism of long chain fatty acid transport. It is suggested that long chain fatty acids do not cross the liver cell membrane by diffusion but probably by a mechanism involving fatty acid receptors on and across the membrane.

The present study evaluated the hypothesis that a depression in the hepatic utilization of the fatty acids of liver triglycerides is a major factor in the genesis of the acute ethanol-induced fatty liver. Rats were killed from 2–24 hours after oral intubation of either ethanol, isocaloric glucose, or isotonic saline. The concentration of plasma FFA levels was employed as an index of the oxidation of hepatic fatty acids. Prior to the onset of fatty liver development, plasma FFA and blood ketone levels of ethanol-treated rats were similar to those levels observed in the fasting, saline-control group. During the onset of fatty liver development blood ketone levels of ethanol-treated rats were markedly increased and were associated with an increased fatty acid load presented to the liver. At the peak of fatty liver development blood ketones were markedly depressed in ethanol-gavaged rats. However, this effect was transient and as liver triglyceride levels of the ethanol group returned toward control levels, blood ketone concentrations increased and were comparable to those of the control groups. The data indicate that hepatic fatty acid utilization is unaffected by ethanol administration except for a transient inhibition observed during the peak of the acute ethanol-induced fatty liver.

Chick embryo heart cells in tissue culture actively oxidize [1-(14)C]palmitate to (14)CO(2). Fatty acid oxidation by cell monolayers was linear with time and increasing protein concentration. The addition of carnitine to the assay medium resulted in a 30-70% increase in the rate of fatty acid oxidation. The specific activity of palmitic acid oxidation did not change significantly with time in culture and was also the same in rapidly proliferating and density-inhibited cell cultures. Addition of unlabeled glucose to the assay medium resulted in a 50% decrease in (14)CO(2) production from [1-(14)C]palmitate. Conversely, palmitate had a similar sparing effect on [(14)C]glucose oxidation to (14)CO(2). Lactate production accounted for most of the glucose depleted from the medium and was not inhibited by the presence of palmitate in the assay. Thus, the sparing action of the fatty acids on glucose oxidation appears to be at the mitochondrial level. The results indicate that although chick heart cells in culture are primarily anaerobic, they can oxidize fatty acid actively.

The ability of the muscular tissue of fish to oxidize fatty acids has been studied on rainbow trout (Salmo gairdnerii). The rate of oxidation of Na hexanoate-1-C14, K octanoate-1-C14, and K myristate-1-C14by tissue slices from the lateral dark muscle and from the dorsal white muscle was determined at 25 °C by measuring the formation of C14O2. This transformation can be demonstrated in both the white and dark muscle; however, quantitatively a very pronounced difference exists between the two tissues, the dark muscle being more active.

We have investigated developmental profiles of ATP-dependent palmityl-CoA synthetase, acetyl-CoA synthetase, palmitylcarnitine transferase, and fatty acid oxidation in heart and liver of developing chicks and rats. Palmityl-CoA synthetase activity of rat liver and heart homogenates increased 6- to 10-fold during the first postnatal week. Chick embryo heart activity peaked between 13 and 16 days of development. The activity of embryonic chick livers was bimodal with highest activity seen at 7 and 16 days of development. Posthatching values were approximately 50–75% of the peak embryonic levels. Acetyl-CoA synthetase activity of rat liver and heart homogenates was low but also showed developmental increases following birth. Acetyl-CoA synthetase activity of chick embryonic hearts was greatest at 16 days of development. Palmitylcarnitine transferase activity of rat liver and heart homogenates showed a striking increase during the first week of life. Chick heart activity was similar to that observed for palmityl-CoA synthetase with a peak between 13 and 16 days of embryonic development. Coincident with the postnatal rise in fatty acid activation and palmitylcarnitine transferase activity in developing rats, the oxidation of palmityl-CoA plus carnitine and of palmitylcarnitine increased from barely measurable levels at birth to adult levels by 30 days of age. The increases that we observe probably relate to changes in the specific activity of the enzymes as well as to an increase in the absolute number of mitochondria during development.

Cyclic 3′,5′-adenosine monophosphate, 5′-adenosine monophosphate, or 2′-adenosine monophosphate markedly enhanced the rate of oxidation of medium-chain fatty acids by rat heart homogenates, as measured by oxygen utilization and carbon dioxide formation from 14C-labeled substrate. These nucleotides did not alter the rate of oxidation of medium-chain acylcoenzyme A derivatives. The activity of a medium-chain fatty acyl coenzyme A synthetase from rat heart homogenates was increased by these nucleotides, and it was suggested that the ability of the nucleotides to enhance fatty acid oxidation by heart homogenates was due to activation of acyl coenzyme A synthetase.

Diisopropylammonium dichloroacetate (DIPA) is known to produce a significant and prolonged hypoglycemic effect in alloxan-diabetic but not in normal rats. The active component of this compound is its acid moiety, dichloroacetate (DCA). Both DIPA and DCA stimulate glucose oxidation in diabetic muscle in vitro. DCA inhibits fatty acid oxidation in isolated diaphragms from diabetic rats. The present study shows that tissue concentrations of citrate are increased eight-fold in hearts and 4.6-fold in diaphragms of alloxan-diabetic rats. Alpha glycerol phosphate (α-GP) concentrations are reduced in hearts from diabetic rats. DIPA administered intraperitoneally markedly reduces tissue concentrations of citrate in hearts and diaphragms of alloxan-diabetic rats but has no effect on citrate in hearts and diaphragms of normal rats. The reduction of citrate in hearts of diabetic rats is accompanied by an increase in the intracellular concentration of α-GP. In hearts from diabetic animals treated with DIPA, both citrate and α-GP levels are similar to those found in hearts from untreated normal rats. DIPA administration has little effect on the concentrations of pyruvate or lactate in diabetic muscle. In diabetes excess fatty acid oxidation raises intracellular concentrations of citrate, a known inhibitor of phosphofructokinase (PFK). The results of the present investigation are consistent with the hypothesis that the selective hypoglycemic effect of DIPA is due, at least in part, to a suppression of fatty acid oxidation in diabetic muscle that results in a lowering of muscle citrate. The reduction in citrate levels reactivates PFK and promotes accelerated utilization of glucose.

1. Aqueous extracts of acetone-dried liver and kidney mitochondria, supplemented with NAD+, CoA and phenazine methosulphate, efficiently convert fatty-acyl-CoA compounds into acetyl-CoA; the process was followed with an O2 electrode. 2. Label from [1-14C]octanoyl-CoA appears in acetyl-CoA more rapidly than that from [8-14C]octanoyl-CoA. 3. Oxidation of [8-14C]octanoyl-CoA was terminated by addition of neutral ethanolic hydroxylamine and the resulting hydroxamates were separated chromatographically. Hydroxamate derivatives of 3-hydroxyoctanoyl-, hexanoyl-, butyryl- and acetyl-CoA were obtained. 4. These and other observations suggest that oxidation of octanoyl-CoA by extracts involves participation of free intermediates rather than uninterrupted complete degradation of individual molecules to acetyl-CoA by a multienzyme complex. 5. Intact liver mitochondria studied by the hydroxamate technique were also shown to form intermediates during oxidation of labelled octanoates. In addition to octanoylhydroxamate, [8-14C]octanoate gave rise to small amounts of hexanoyl-, butyryl- and 3-hydroxyoctanoyl-hydroxamate. In contrast with extracts, however, where the quantity of intermediates found was a significant fraction of the precursors, mitochondria oxidizing octanoate contained much larger quantities of octanoyl-CoA than of any other intermediate.

1. Butyryl-CoA dehydrogenase from Peptostreptococcus elsdenii forms very tightly bound complexes with various acyl-CoA compounds. Spectra in some cases merely show resolution of the 450nm band, but those with acetoacetyl-, pent-2-enoyl- and 4-methylpent-2-enoyl-CoA show long-wavelength bands similar to the 710nm band of native enzyme. These complexes are formed instantaneously by the yellow form of the enzyme and much more slowly by the green form. 2. An acid extract of the green enzyme reconverts the yellow into the green form. 3. Hydroxylamine makes irreversible the otherwise reversible conversion of the green enzyme into the yellow form by phenylmercuric acetate. 4. Amino acid analysis for taurine and β-alanine shows approx. 1mol of CoA/mol of flavin in green enzyme. Anaerobic dialysis of reduced enzyme removes the CoA. On acid precipitation of green enzyme the CoA is found only in the supernatant. 5. It is concluded that native green enzyme is probably complexed with unsaturated acyl-CoA. This is shown to be consistent with findings of other workers. Catalytic activity requires displacement of the acyl-CoA, which is therefore likely to be a potent inhibitor. 6. An explanation is offered for the irreversible conversion of green into yellow enzyme by sodium dithionite. 7. The enzyme displays a feeble, previously undetected, activity towards β-hydroxybutyryl-CoA. 8. The product of oxidation of pent-4-enoyl-CoA forms a complex with reduced enzyme and strongly inhibits reoxidation of the FAD. This may contribute to inhibition of fatty acid oxidation by pent-4-enoic acid in mammals.

1. I. Carboxyl-labeled fatty acids of the even series (C 8 C 16 ) and the triglycerides of carboxyl-labeled palmitic and capric acids were injected into fasted and carbohydrate-fed rats. The expired CO 2 was collected at various intervals for 24 hr., and the cumulative C 14 O 2 and the specific activity-time curves were derived for each rat. 1.1. 1. The conversion of the C 14 of the injected fatty acids to CO 2 was lower in the fed than in the fasted rat. This sparing action of carbohydrate was more pronounced in the case of the longer-chain fatty acids. Administered carbohydrate failed, under the conditions employed, to spare the oxidation of acetate to CO 2 . 1.2. 2. The sparing action of carbohydrate lasted several hours, and was followed by a secondary rise in the specific activity of the expired CO 2 . This secondary rise, suggestive of an increased breakdown of labeled fatty acid, was more pronounced in the case of the longer-chain fatty acids. 2. II. To study the mechanism of the action of carbohydrate in sparing the oxidation of palmitic acid to CO 2 , the triglyceride and the nonlipide fractions were isolated from carbohydrate-fed and fasted rats 5 hr. after the injection of palmitic acid-1-C 14 . The C 14 contents of the carboxyl carbon and decarboxylated moieties of the triglyceride 16-` and 18-carbon fatty acids and the C 14 contents of the nonlipides were determined. 2.1. 1. The results obtained indicate that the extent of utilization of acetyl-S-CoA, derived from palmitic acid breakdown, for fatty acid synthesis is far greater in the carbohydrate-fed than in the fasted rat. But diversion of the acetyl-S-CoA from an oxidative to a synthetic fate can account for only a small part of the difference in the C 14 O 2 recoveries observed in the fed and fasted rats. 2.2. 2. The results obtained for the C 14 nonlipides in the carbohydrate-fed and fasted rats indicate that carbohydrate feeding did not bring about a diversion of C 14 into this fraction. 2.3. 3. The conclusion was drawn that the principal action of carbohydrate in sparing the oxidation of palmitic acid is in restricting its breakdown. 2.4. 4. In both the carbohydrate-fed and fasted rats, the injected palmitic acid was recovered after 5 hr. chiefly as 16-carbon fatty acids. It was found, however, that a much greater proportion of the unoxidized palmitic acid was converted to 18-carbon fatty acids in the carbohydrate-fed than in the fasted rats. 2.5. 5. It is suggested that the degree of sparing, by carbohydrate, of the oxidation of fatty acids of chain lengths less than 16 carbons is related to the capacity of the organism to convert a particular fatty acid directly to 16- and 18-carbon fatty acids.

Summary: A study of the mechanism of breakdown of w-phenyl-substituted fatty acids by Nocardia opaca has been made. Acids with an odd number of carbon atoms in the side chain (phenylpropionic, phenylvaleric and phenylheptylic acids) were converted to benzoic acid, and cinnamic acid was an intermediate. o-Hydroxy-phenylacetic acid was identified as a common product when acids with an even number of carbon atoms (phenylacetic, phenylbutyric, phenylcaproic and phenylcaprylic) were used. This evidence supports β-oxidation as a mechanism of breakdown of short chain fatty acids by N. opaca.

The effects of potassium decanoate on the phosphorylation associated with the oxidation of pyruvate by rat-kidney and rat-brain mitochondria have been investigated. The suggestion that these two processes may be uncoupled from each other in the presence of decanoate has been confirmed. Further, it has been shown that the decanoate-insensitive oxidation of pyruvate by rat-brain mitochondria, occurring in the absence of such stimulating agents as fumarate, is not associated with ATP synthesis. The fumarate-stimulated oxidation of pyruvate by rat-brain mitochondria, which is inhibited by decanoate, is associated with a phosphorylation process which is uncoupled by decanoate. When pyruvate oxidation by rat-kidney or by rat-brain mitochondria is uncoupled from phosphorylation, the extent of uncoupling is proportional to the amount of decanoate added.

Fatty acids inhibit the oxidation of pyruvate by rat-kidney mitochondria but the extent of inhibition depends upon the nature and amount of agent added to stimulate the oxidation. The longer chain fatty acids are more effective inhibitors and, in general, the even-numbered fatty acids show greater inhibitory effects than the adjacent odd-numbered fatty acids. Under conditions where 2, 4-dinitrophenol (DNOP) and the fatty acids separately have little effect on the respiratory activity of rat-kidney mitochondria with pyruvate as substrate, the addition of both fatty acid and DNOP results in an extensive inhibition. At low concentrations the fatty acids are oxidized by rat-kidney mitochondria but at concentrations of 10−3 M and higher they inhibit their own oxidation, the oxidation of pyruvate, and those of the acids of the tricarboxylic acid cycle. The oxidation of pyruvate by rat-brain mitochondria is insensitive to decanoate but both the fumarate- and DNOP-stimulated oxidations of pyruvate are sensitive to the presence of decanoate. In contrast, Nembutal inhibits both the oxidation of pyruvate alone and the fumarate-stimulated oxidation of pyruvate. Possible mechanisms for the observed inhibitory effects of fatty acids are discussed.

In the present study, ethanol oxidation by the perfused rat liver has been used to investigate the interrelationships between the pathways of glucose metabolism, fatty acid oxidation, and the citric acid cycle. In the absence of exogenous fatty acids, the production of glucose from alanine was stimulated 2-fold by 10 mm ethanol, whereas, in the presence of 1 mm oleate, ethanol caused an inhibition of net glucose production. Measurements of the rates of ethanol utilization and acetate formation showed that over 80% of the ethanol metabolized was converted to acetate. The increased rate of generation of reducing equivalents in the cytosol during ethanol oxidation increased the oxidation-reduction state of pyridine nucleotides in both the intra- and extramitochondrial compartments. This fact was established by analyses of the tissue content of pyridine nucleotides and substrate couple ratios, and directly by surface fluorometry. Changes of flavin and pyridine nucleotide fluorescence intensity from the surface of the liver showed that the transfer of reducing equivalents from cytosol to mitochondria during ethanol oxidation was very rapid. Analyses of intermediates in the gluconeogenic pathway of livers perfused in the absence of fatty acids indicated an activational site at the glyceraldehyde-3-P dehydrogenase step upon ethanol addition. The stimulatory effect of ethanol on gluconeogenesis from alanine, therefore, results from the increased availability of the NADH in the cytosol. On the other hand, when ethanol was added to livers perfused in the presence of oleate, an inhibitory site was observed between fructose-1, 6-di-P and fructose-6-P. Measurement of the tissue levels of the known modifiers of phosphofructokinase and fructose diphosphate phosphatase indicated that this effect was caused primarily by deinhibition of phosphofructokinase resulting from a fall of the citrate content. Oxidation of NADH produced during ethanol metabolism inhibited the activity of the citric acid cycle. Sites of inhibition were identified at the citrate synthase and iso-citrate dehydrogenase steps. The relative strengths of the inhibitory interactions at these sites were dependent on the rate of β oxidation. It is proposed that a coordinated inhibition of citrate synthase and isocitrate dehydrogenase is mediated primarily by the increased state of reduction of intramitochondrial pyridine nucleotides.