Medium-chain Fatty Acid Oxidation Research Articles

Disorders of mitochondrial β‐oxidation: Prenatal and early postnatal diagnosis and their relevance to Reye's syndrome and sudden infant death

There are still many problems with the diagnosis and classification of inherited disorders of mitochondrial beta-oxidation. At present only the acyl-CoA dehydrogenase step of the beta-oxidation spiral has been explored in any detail and a large number of patients have disorders that cannot be properly characterized. beta-Oxidation defects may present in a wide variety of ways, the most dramatic being acute encephalopathy with hepatic involvement (atypical Reye's syndrome) or 'sudden' death. Investigations may include urinary and plasma organic acids, metabolic stress tests and assays of overall metabolic pathways or of specific enzymes in cultured fibroblasts, lymphocytes, or other material. Early postnatal diagnosis presents particular difficulties but in medium-chain acyl-CoA dehydrogenase deficiency the diagnosis may be apparent from careful examination of urine. There is as yet little general experience in prenatal diagnosis of this group of disorders except for glutaric aciduria type II. Single prenatal diagnoses of medium-chain acyl-CoA dehydrogenase deficiency and of an incompletely characterized defect of medium-chain fatty acid oxidation have been performed.

Cis-4-decenoic acid in plasma: a characteristic metabolite in medium-chain acyl-CoA dehydrogenase deficiency.

The profile of organic acids in plasma of patients with a deficiency of medium-chain acyl-CoA dehydrogenase (EC 1.3.99.3) was determined by gas-liquid chromatography of trimethylsilylated derivatives of the acids isolated by ethyl acetate extraction. All 13 patients had increased concentrations of free octanoate, cis-4-decenoate, and decanoate in their plasma. Cis-4-decenoate, an intermediary metabolite of linoleic acid, is pathognomonic of medium-chain acyl-CoA dehydrogenase deficiency. This metabolite does not accumulate in plasma after oral loading with medium-chain triglycerides, in contrast to octanoate and decanoate. Two postmortem plasma samples from victims of infant sudden-death syndrome had detectable octanoate and decanoate, but cis-4-decenoate could not be detected. The identification of cis-4-decenoate in plasma may be an aid in the diagnosis of an inherited defect in oxidation of medium-chain fatty acids.

Effect of salicylic acid on mitochondrial-peroxisomal fatty acid catabolism.

To understand the possible role of salicylic acid in the pathogenesis of Reye's syndrome, we examined its effect on the oxidative metabolism of fatty acids in the rat liver mitochondrial-peroxisomal fraction. Fatty acids of different chain lengths are oxidized in different organelles. Octanoic acid is oxidized in mitochondria, lignoceric acid in peroxisomes, and palmitic acid in both mitochondria and peroxisomes. Salicylic acid (up to 1 mM concentration) had no effect on the oxidation of [1-14C]lignoceric acid. However, at the same concentration it inhibited the oxidation of [1-14C]palmitic acid by 26% and [1-14C] octanoic acid by 42%. The apparent Ki for the oxidation of [1-14C] octanoic acid, [1-14C]palmitic acid and [1-14C]lignoceric acid were 0.27, 6.0, and 14.8 mM, respectively. This selective inhibition of mitochondrial oxidation of medium-chain (octanoic acid) and long-chain (palmitic acid) fatty acids by salicylic acid may potentiate the accumulation of fatty acids in plasma in Reye's syndrome patients.

Sudden Child Death and `Healthy' Affected Family Members With Medium-Chain Acyl-Coenzyme A Dehydrogenase Deficiency

A family is described in which the father and three (and probably all four) of his children had a decreased capacity for the oxidation of medium-chain fatty acids. One of the children suddenly died at the age of 16 months following an episode of a rapidly deteriorating Reye syndrome-like illness with hypoketotic hypoglycemia and dicarboxylic aciduria, but without any previous alarming symptoms. The eldest sibling had died at the age of 19 months under similar conditions. The other family members had always been healthy. On fasting, all affected family members accumulated in their plasma the medium-chain fatty acids octanoic, decanoic, and cis-4-decenoic acids. Their urinary organic acid excretion profile could be characterized as "dicarboxylic aciduria." A deficiency of medium-chain acyl-coenzyme A dehydrogenase was demonstrated in a postmortem liver sample of the index patient. Cultured fibroblasts from the father and the two healthy children had a decreased rate of [14C]octanoate oxidation. It is suggested that a deficiency of medium-chain acyl-coenzyme A dehydrogenase may lead to a life-threatening illness when other complicating factors such as diarrhea and vomiting result in an abnormal depletion of the body's glycogen stores. Careful monitoring of at-risk patients during a minor illness is necessary.

Octanoylglucuronide excretion in patients with a defective oxidation of medium-chain fatty acids

Octanoyl-β- d-glucuronide was identified in the urine of five patients with hypoketotic hypoglycemia and dicarboxylic aciduria due to a defective β-oxidation of medium-chain fatty acids. Two subjects who ingested large amounts of medium-chain triglycerides also excreted large amounts of the glucuronide. The substance was extracted from the urine with ethyl acetate and analyzed by: (1) gas chromatography/mass spectrometry (GC-MS) of the trimethylsilyl derivative and (2) preparative one-dimensional thin-layer chromatography followed by enzymatic hydrolysis with β-glucuronidase and again GC-MS. A quantitative analysis was performed indirectly by measuring the urinary bound octanoate after the removal of octanoylcarnitine. Octanoylglucuronide represents an additional mechanism for the detoxification of octanoate; its formation may be of help for the maintenance of carnitine homeostasis in patients with medium-chain acyl-CoA dehydrogenase deficiency.

FAMILIAL SUDDEN CHILD DEATH IN A FAMILY WITH PSEUDO-DOMINANT DICARBOXYLIC ACIDURIA AND HYPOKETOTIC HYPOGLYCEMIA

A family is described in which the father and three (and probably all four) children had a decreased capacity for the oxidation of medium-chain fatty acids resulting in a defective ketogenesis. The 4th child died at the age of 16 months during an episode of hypoketotic hypoglycemia (plasma glucose 0.7 mmole/l; 3-hydroxybutyrate 1.7 mmoles/1). Previously the child had always been in good health. Medium-chain fatty acids accumulated in the patient's plasma; the urinary organic acid profile showed large amounts of dicarboxylic acids and fatty acid conjugates such as hexanoylglycine, octanoylcarnitine, and octanoylglucuronide. The activity of medium-chain acyl-CoA dehydrogenase in an autopsy liver sample was only 15% of the mean control. The first child had died at the age of 19 months under similar conditions. When subjected to a 17 h fast the father and the 2nd and 3rd child, but not the mother, accumulated octanoic, decanoic and cis-4-decenoic acids in their plasma.Their urinary organic acid profile resembled that of the deceased 4th child, but excretions were at a lower level. Measurement of [14-C]-octanoate degradation in cultured fibroblasts revealed low values for the father and the two ‘healthy’ children as compared to the controls. It is suggested that a -partial- deficiency of medium-chain acyl-CoA dehydrogenase may lead to life-threatening illness when gastrointestinal abnormalities eventually lead to a severe depletion of the glycogen stores. Careful monitoring of at-risk patients is indicated under these circumstances.

Octanoic acidemia and octanoylcarnitine excretion with dicarboxylic aciduria due to defective oxidation of medium-chain fatty acids

Five patients aged 7 to 21 months are described who developed attacks of coma after a short prodromal illness with diarrhea or vomiting or both. Four had concomitant hypoglycemia, and all had hypoketonemia, with excessive urinary excretion of medium-chain dicarboxylic acids, medium-chain (omega-1)-hydroxyacids, suberylglycine, hexanoylglycine, and octanoylcarnitine. All patients accumulated octanoic acid, decanoic acid, and cis-4-decenoic acid in plasma. Fibroblasts from three patients showed a decreased rate of octanoate oxidation (10%, 12%, and 29% of control values, respectively). These findings suggest a deficiency of medium-chain acyl-CoA dehydrogenase, most probably an autosomal recessive inherited metabolic disorder. Two of the patients died during an acute attack, and a third had severe neurologic sequelae; the two remaining patients recovered. Plasma free carnitine levels were low, but total carnitine was normal. The three surviving patients underwent a fasting test, which did not lead to hypoglycemia, although hypoketonemia, dicarboxylic aciduria, and excessive mobilization of fatty acids did occur. The surviving patients were maintained on frequent carbohydrate-enriched meals.

Medium-chain acyl-CoA dehydrogenase deficiency in two siblings with a Reye-like syndrome

An increasing number of reports indicate that patients with some inherited metabolic diseases may have symptoms resembling those of Reye syndrome. We describe two siblings who developed a Reye-like syndrome at ages 16 and 18 months, respectively, after a viral illness and salicylate therapy. Both had fasting hypoglycemia and hypoketonemia. At the time of the acute episode and after ingestion of a medium-chain triglyceride load, one of them excreted large amounts of abnormal metabolites derived from the omega- and (omega-1)-oxidation of medium-chain fatty acids. Medium-chain acyl-CoA dehydrogenase activity was lower than 20% of control values in fibroblasts from both patients. This enzyme defect should be considered in children with a Reye-like syndrome with these distinctive manifestations.

Evidence that medium-chain fatty acid oxidation can support an active gluconeogenesis in the suckling newborn rat.

Long-chain fatty acid oxidation was inhibited in 1-day-old suckling rats by 2-tetradecylglycidic acid. Within 6 h it induced profound hypoketonemia and hypoglycemia together with a decreased rate of gluconeogenesis estimated from [U-14-C]-lactate incorporation into glucose. Medium-chain triglyceride feeding restored, within 3 h, normal blood concentrations of ketones and glucose as well as a high gluconeogenic rate from [U-14-C]-lactate. It is concluded that in the suckling newborn rat 2-tetradecylglycidic acid is effective in inhibiting selectively long-chain fatty acid oxidation and that medium-chain fatty acid oxidation can support an active gluconeogenesis.

Ketogenesis in mitochondria isolated from liver biopsies of normal and starved dogs: Comparison with rat-liver mitochondria

1. 1. Fatty acid oxidation and ketogenesis were studied in isolated dog-liver mitochondria in order to investigate whether the absence of hyperketonemia in fasting dogs results from a low capacity of hepatic ketogenesis. Isolated rat-liver mitochondria were used as reference. 2. 2. The results indicate that: 2.1. (a) Dog-liver mitochondria oxidize long-chain fatty acids and produce ketone bodies at about equal rates as rat-liver mitochondria. No differences were detected in the regulation of ketogenesis. 2.2. (b) Rates of oxidation of medium-chain fatty acids are significantly lower in dog-liver mitochondria than in rat-liver mitochondria. 2.3. (c) Fasting does not influence the capacity of liver mitochondria for fatty acid oxidation but their ketogenic capacity is slightly enhanced in both species. 2.4. 3. The regulation of the energy metabolism in the fasting dog is discussed and compared with that in other mammalian species.

Inhibition of medium and short-chain fatty acid oxidation in rat heart mitochondria by dichloroacetate

The effect of dichloroacetate (DCA) on the oxidation of medium-chain and short-chain fatty acids was studied in rat heart mitochondria. Dichloroacetate (1–10 mM) markedly decreased medium-chain and short-chain fatty acid oxidation (oxygen uptake and release of 14CO 2 from labelled fatty acids) by heart mitochondria; the oxidation of long-chain fatty acid, however, was unaffected by the compound. The oxidation of acylcarnitines was not influenced by DCA. Dichloroacetate inhibited medium-chain and short-chain fatty acid oxidation by inhibiting the activity of mitochondrial acyl-CoA synthases. Inhibition of the enzyme activity by DCA was rapid and not due to its effect on pyruvate dehydrogenase.

A comparison of the utilization of medium and long-chain fatty acids for oxidation and ketogenesis in the suckling rat: In vivo and in vitro studies

Fatty acid metabolism was studied both in vivo and in perfused liver of suckling rats. In vivo studies showed that [ 14C]decanoate was oxidized to 14CO 2 at a faster rate than was [ 14C]palmitate. This difference was due in part to the different modes of absorption of medium- and long-chain fatty acids. [1- 14C]- and [16- 14C]palmitate were oxidized at similar rates indicating complete oxidation of the fatty acid. Analysis of plasma acetoacetate and β-hydroxybutyrate showed that ketone body production was three times as great from labeled decanoate as from palmitate. Perfusion of livers from suckling rats with physiological concentration of fatty acid showed that ketone bodies were generated from decanoate at three times the rate observed with palmitate. The apparent K m for ketogenesis from decanoate was 0.4 m m while that from palmitate was 2.9 m m. The V for palmitate was 2.9 μmol/min/g liver while that for decanoate was 1.5 μmol/min/g liver. Compared to decanoate, palmitate was preferentially converted to triglycerides and phospholipids both in vivo and in vitro. These data suggest that medium-chain fatty acid oxidation in the neonatal rat spares long-chain fatty acids for utilization in complex lipid synthesis. Although medium-chain fatty acid catabolism appears similar to that in the adult rat, it is physiologically relevant during the neonatal period since rat milk, the major source of food, is rich in triglycerides which contain about 35% medium-chain fatty acids.

Effect of dichloroacetate on the oxidation of short-chain and medium-chain fatty acids by rat heart mitochondria

Effect of dichloroacetate on the oxidation of short-chain and medium-chain fatty acids by rat heart mitochondria

Relation of Ketosis to Metabolic Changes Induced by Acute Medium-Chain Triglyceride Feeding in Rats

Medium-chain triglycerides (MCT) induce ketosis in several mammalian species including man. To clarify the regulation of this metabolic alteration, we fed rats either MCT or long-chain triglyceride (corn oil) and then attempted to correlate ketosis with changes in (i) concentrations of selected metabolites in plasma and (ii) the synthetic and oxidative capacities of the liver. By 1 hour after MCT feeding, plasma levels of total ketone bodies had increased 18-fold, with a maximum value reached 1 hour later. By contrast, total plasma ketones in rats fed corn oil were increased only about 2-fold at 2 hours after feeding and did not exceed this value at later intervals. Hepatic concentrations of ketone bodies also increased after MCT or corn oil feeding. Although plasma concentrations of glucose decreased and insulin increased in rats fed MCT, they were not affected by corn oil feeding. MCT-induced ketosis was depressed by glucose administration. Neither MCT nor corn oil feeding impaired utilization of glucose by the liver. Hepatic lipogenesis was suppressed 50% and 90% by MCT and corn oil feeding, respectively. A marked increase of long-chain fatty acids in plasma was observed in rats fed corn oil but not in rats fed MCT. The pronounced increase of ketones in MCT-fed rats was closely related to an elevation of octanoate. In liver slices of MCT-fed rats, ketogenesis from octanoate was 10-fold higher than from palmitate, and octanoate was oxidized 4 times more rapidly than palmitate. The ketosis of MCT-fed rats was depressed by administration of 4-pentenoic acid, a potent inhibitor of fatty acid oxidation. These results support the concept that ketosis induced by MCT stems from rapid oxidation of medium-chain fatty acids. Hyperinsulinemia, hypoglycemia and depressed lipogenesis resulting from MCT feeding appear to potentiate but not initiate ketosis.

Experimental hyperosmolar diabetic syndrome. Ketogenic response to medium-chain triglycerides.

The clinical features of the experimental hyperosmolar diabetic (EHD) rat model resemble those seen in the human syndrome--extreme hyperglycemia without ketoacidosis is common to both. The absence of ketoacidosis in the syndrome has been ascribed to both substrate (free fatty acid) deficiency and to interference with hepatic ketone body synthesis. The potential for hepatic ketone body synthesis in the experimental model has been directly assessed by challenging the EHD animals with medium-chain triglycerides (MCT) administered intragastrically. This neutral lipid, largely consisting of C8 and C10 fatty acids, leads to a dose- and thime-related increase in the plasma concentration of acetoacetate and beta-hydroxybutyrate. The EHD rats respond to MCT with an increase in plasma ketone bodies that rises to levels that are twice as high as those observed in normal rats receiving MCT and are equivalent to the levels seen in untreated ketoacidotic animals. These data indicate that hepatic medium-chain fatty acid oxidation and ketogenesis are unimparied in the EHD animal. An analysis of the factors responsible for the greater ketogenic response in the EHD rat reveals that moderate diabetes and dehydration enhance MCT-induced ketone body accumulation, while cortisol is without effect. The plasma free fatty acid concentration in EHD animals does not differ from normal rats, but is significantly lower than that seen in diabetic ketoacidosis. These data support the concept that a principal reason for the absence of ketoacidosis in the EHD syndrome is the limitation in availiability of substrate, free fatty acids, for ketone body synthesis.

Enhancement of Fatty Acid Oxidation and Medium-Chain Fatty Acyl Coenzyme A Synthetase by Adenine Nucleotides in Rat Heart Homogenates

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.

In vitro enzymic omega oxidation of medium-chain fatty acids in mammalian tissue

Euzymic omega oxidation of medium-chain fatty acids, having 8–12 carbon atoms, to their corresponding dicarboxylic acids can be carried out by mammalian tissue homogenates, in vitro. The overall conversion of decanoic acid to decanedioic acid by homogenates of mammalian tissues requires ATP, Mg 2+, DPN +, TPN +, and oxygen. A mixture of liver microsomes (5000 – 25,000 g fraction) and a liver-soluble supernatant fraction (25,000 g supernatant fraction) can replace the whole homogenate in the overall conversion. The omega oxidation of medium-chain fatty acids to their corresponding dicarboxylic acids proceeds through a series of reactions. The first reaction is the direct hydroxylation of the omega methyl group by liver microsomes requiring TPNH and oxygen. The microsomal enzyme system can be called an omega fatty acid hydroxylase. In the second reaction, the omega hydroxy fatty acid is oxidized to the omega oxo fatly acid by a liver-soluble supernatant, fraction requiring DPN +. In the final reaction, the omega oxo fatly acid is oxidized to the dicarboxylic acid by a liver-soluble supernatant fraction, also requiring DPN +. The soluble supernatant enzymes involved in the final two steps of the reaction can be called omega fatty acid dehydrogenases.