Medium-chain Fatty Acid Oxidation Research Articles

Medium-chain fatty acid oxidation is independent of l-carnitine in liver and kidney but not in heart and skeletal muscle.

Medium-chain fatty acid (MCFA) consumption confers a wide range of health benefits that are highly distinct from long-chain fatty acids (LCFAs). A major difference between the metabolism of LCFAs compared with MCFAs is that mitochondrial LCFA oxidation depends on the carnitine shuttle, whereas MCFA mitochondrial oxidation is not. Although MCFAs are said to range from 6 to 14 carbons long based on physicochemical properties in vitro, the biological cut-off length of acyl chains that can bypass the carnitine shuttle in different mammalian tissues is unknown. To define the range of acyl chain length that can be oxidized in the mitochondria independent of carnitine, we determined the oxidative metabolism of free fatty acids (FFAs) from 6 to 18 carbons long in the liver, kidney, heart, and skeletal muscle. The liver oxidized FFAs 6 to 14 carbons long, whereas the kidney oxidized FFAs from 6 to 10 carbons in length. Heart and skeletal muscle were unable to oxidize FFAs of any chain length. These data show that while the liver and kidney can oxidize MCFAs in the free form, the heart and skeletal muscle require carnitine for the oxidative metabolism of MCFAs. Together these data demonstrate that MCFA oxidation independent of carnitine is tissue-specific.NEW & NOTEWORTHY This work demonstrates that the traditional concept of mitochondrial medium-chain fatty acid oxidation as unregulated and independent of carnitine applies only to liver metabolism, and to kidney to a lesser extent, but not the heart or skeletal muscle. Thus, the benefits of dietary medium-chain fatty acids are set by liver metabolic activity and peripheral tissues are unlikely to receive direct benefits from medium-chain fatty acid metabolism, but rather metabolic byproducts of liver's medium-chain oxidative metabolism.

The hepatic compensatory response to elevated systemic sulfide promotes diabetes.

SummaryImpaired hepatic glucose and lipid metabolism are hallmarks of type 2 diabetes. Increased sulfide production or sulfide donor compounds may beneficially regulate hepatic metabolism. Disposal of sulfide through the sulfide oxidation pathway (SOP) is critical for maintaining sulfide within a safe physiological range. We show that mice lacking the liver- enriched mitochondrial SOP enzyme thiosulfate sulfurtransferase (Tst−/− mice) exhibit high circulating sulfide, increased gluconeogenesis, hypertriglyceridemia, and fatty liver. Unexpectedly, hepatic sulfide levels are normal in Tst−/− mice because of exaggerated induction of sulfide disposal, with associated suppression of global protein persulfidation and nuclear respiratory factor 2 target protein levels. Hepatic proteomic and persulfidomic profiles converge on gluconeogenesis and lipid metabolism, revealing a selective deficit in medium-chain fatty acid oxidation in Tst−/− mice. We reveal a critical role of TST in hepatic metabolism that has implications for sulfide donor strategies in the context of metabolic disease.

Physiological Perspectives on the Use of Triheptanoin as Anaplerotic Therapy for Long Chain Fatty Acid Oxidation Disorders.

Inborn errors of mitochondrial fatty acid oxidation (FAO) comprise the most common group of disorders identified through expanded newborn screening mandated in all 50 states in the United States, affecting 1:10,000 newborns. While some of the morbidity in FAO disorders (FAODs) can be reduced if identified through screening, a significant gap remains between the ability to diagnose these disorders and the ability to treat them. At least 25 enzymes and specific transport proteins are responsible for carrying out the steps of mitochondrial fatty acid metabolism, with at least 22 associated genetic disorders. Common symptoms in long chain FAODs (LC-FAODs) in the first week of life include cardiac arrhythmias, hypoglycemia, and sudden death. Symptoms later in infancy and early childhood may relate to the liver or cardiac or skeletal muscle dysfunction, and include fasting or stress-related hypoketotic hypoglycemia or Reye-like syndrome, conduction abnormalities, arrhythmias, dilated or hypertrophic cardiomyopathy, and muscle weakness or fasting- and exercise-induced rhabdomyolysis. In adolescent or adult-onset disease, muscular symptoms, including rhabdomyolysis, and cardiomyopathy predominate. Unfortunately, progress in developing better therapeutic strategies has been slow and incremental. Supplementation with medium chain triglyceride (MCT; most often a mixture of C8–12 fatty acids containing triglycerides) oil provides a fat source that can be utilized by patients with long chain defects, but does not eliminate symptoms. Three mitochondrial metabolic pathways are required for efficient energy production in eukaryotic cells: oxidative phosphorylation (OXPHOS), FAO, and the tricarboxylic (TCA) cycle, also called the Krebs cycle. Cell and mouse studies have identified a deficiency in TCA cycle intermediates in LC-FAODs, thought to be due to a depletion of odd chain carbon compounds in patients treated with a predominantly MCT fat source. Triheptanoin (triheptanoyl glycerol; UX007, Ultragenyx Pharmaceuticals) is chemically composed of three heptanoate (seven carbon fatty acid) molecules linked to glycerol through ester bonds that has the potential to replete TCA cycle intermediates through production of both acetyl-CoA and propionyl-CoA through medium chain FAO. Compassionate use, retrospective, and recently completed prospective studies demonstrate significant reduction of hypoglycemic events and improved cardiac function in LC-FAOD patients, but a less dramatic effect on muscle symptoms.

Dietary Fatty Acid Composition Influences Growth Performance and Hepatic Metabolism in Neonatal Pigs

Medium‐chain fatty acids (MCFA) are often included in neonatal milk formulas as a more readily oxidized source of energy compared with long‐chain fatty acids (LCFA). This rapid oxidation of MCFA is thought to enhance growth. Thus, the objectives of this study were to evaluate the effect of energy supplementation and fat source on growth and body composition, and determine the rate of Krebs Cycle substrate utilization in neonatal pigs. Eleven three‐day‐old pigs were allocated to one of three sow milk replacer formulas: a control (CONT) and diets rich in either LCFA or MCFA. The CONT formula provided 80%, whereas the MCFA and LCFA formulas provided 120%, of the metabolizable energy requirements of neonatal pigs. Pigs were fed for 20 days and body composition measured by DXA before initiation and on day 20 of feeding. At the end of the study, pigs were euthanized and muscles and organs collected and weighed. Livers were digested with collagenase and isolated hepatocytes were incubated in media containing all amino acids, glucose, and short‐chain fatty acids in the presence of [U13C] labeled alanine, glucose, propionate, and glutamate for 90 min. The enrichment of Krebs Cycle intermediates was determined using gas chromatography/mass spectrometry. Body weight of pigs in the LCFA group was greater than those in the MCFA and CONT groups on days 18 and 20. In addition, percent fat measured by DXA was greater for MCFA (8%) and LCFA (7%) than those in the CONT (4%) group, and this increase in fat deposition occurred at the expense of lean tissue (P < 0.05). Longissimus dorsi and heart weights as percent of body weight were similar for all groups; however, soleus weight was less for MCFA compared with those in the CONT and LCFA groups. In addition, liver and kidney weights as a percentage of body weight were greater for pigs in the MCFA group compared with those in the CONT group with LCFA being intermediate (P< 0.05). There was no effect of formula feeding on glucose and alanine contribution to lactate synthesis in isolated hepatocytes, but this contribution was greater for glucose (36%) than for alanine (15%) (P<0.05). Conversely, the contribution of alanine (35%) carbon to pyruvate was greater than the contribution of glucose (7%) (P<0.05). Furthermore, in hepatocytes of pigs fed CONT formula 39% of pyruvate carbon was derived from alanine and 31–34% for pigs in the LCFA and MCFA groups (P<0.05). Lastly, glutamate contributed 64% of α‐ketoglutarate carbon in hepatocytes of CONT and MCFA and only 40% for LCFA pigs. These data suggest that dietary MCFA increase body fat without increasing lean tissue accretion compared with CONT. In addition, anaplerosis from glutamate was greater for pigs fed MCFA and CONT than for those fed LCFA.

Detection of myocardial medium-chain fatty acid oxidation and tricarboxylic acid cycle activity with hyperpolarized [1-13 C]octanoate.

Under normal conditions, the heart mainly relies on fatty acid oxidation to meet its energy needs. Changes in myocardial fuel preference are noted in the diseased and failing heart. The magnetic resonance signal enhancement provided by spin hyperpolarization allows the metabolism of substrates labeled with carbon-13 to be followed in real time in vivo. Although the low water solubility of long-chain fatty acids abrogates their hyperpolarization by dissolution dynamic nuclear polarization, medium-chain fatty acids have sufficient solubility to be efficiently polarized and dissolved. In this study, we investigated the applicability of hyperpolarized [1-13 C]octanoate to measure myocardial medium-chain fatty acid metabolism in vivo. Scanning rats infused with a bolus of hyperpolarized [1-13 C]octanoate, the primary metabolite observed in the heart was identified as [1-13 C]acetylcarnitine. Additionally, [5-13 C]glutamate and [5-13 C]citrate could be respectively resolved in seven and five of 31 experiments, demonstrating the incorporation of oxidation products of octanoate into the tricarboxylic acid cycle. A variable drop in blood pressure was observed immediately following the bolus injection, and this drop correlated with a decrease in normalized acetylcarnitine signal (acetylcarnitine/octanoate). Increasing the delay before infusion moderated the decrease in blood pressure, which was attributed to the presence of residual gas bubbles in the octanoate solution. No significant difference in normalized acetylcarnitine signal was apparent between fed and 12-hour fasted rats. Compared with a solution in buffer, the longitudinal relaxation of [1-13 C]octanoate was accelerated ~3-fold in blood and by the addition of serum albumin. These results demonstrate the potential of hyperpolarized [1-13 C]octanoate to probe myocardial medium-chain fatty acid metabolism as well as some of the limitations that may accompany its use.

Structural insights into oxidation of medium-chain fatty acids and flavanone by myxobacterial cytochrome P450 CYP267B1.

Oxidative biocatalytic reactions performed by cytochrome P450 enzymes (P450s) are of high interest for the chemical and pharmaceutical industries. CYP267B1 is a P450 enzyme from myxobacterium Sorangium cellulosum So ce56 displaying a broad substrate scope. In this work, a search for new substrates was performed, combined with product characterization and a structural analysis of substrate-bound complexes using X-ray crystallography and computational docking. The results demonstrate the ability of CYP267B1 to perform in-chain hydroxylations of medium-chain saturated fatty acids (decanoic acid, dodecanoic acid and tetradecanoic acid) and a regioselective hydroxylation of flavanone. The fatty acids are mono-hydroxylated at different in-chain positions, with decanoic acid displaying the highest regioselectivity towards ω-3 hydroxylation. Flavanone is preferably oxidized to 3-hydroxyflavanone. High-resolution crystal structures of CYP267B1 revealed a very spacious active site pocket, similarly to other P450s capable of converting macrocyclic compounds. The pocket becomes more constricted near to the heme and is closed off from solvent by residues of the F and G helices and the B-C loop. The crystal structure of the tetradecanoic acid-bound complex displays the fatty acid bound near to the heme, but in a nonproductive conformation. Molecular docking allowed modeling of the productive binding modes for the four investigated fatty acids and flavanone, as well as of two substrates identified in a previous study (diclofenac and ibuprofen), explaining the observed product profiles. The obtained structures of CYP267B1 thus serve as a valuable prediction tool for substrate hydroxylations by this highly versatile enzyme and will encourage future selectivity changes by rational protein engineering.

Priority use of medium-chain fatty acids during high-intensity exercise in cross-country skiers

BackgroundOne of the topics discussed in sports science is the use of medium-chain saturated fat as an energy-saving nutrient additive when approaching high-intensity exercise. The purpose of this study was to compare the blood concentrations of medium-chain and long-chain fatty acids (FAs) across different intensity loads.MethodsFifteen male highly trained athletes from the Russian cross-country skiing team participated in the study. Blood samples were drawn at rest, at the peak of veloergometric test with a growing exercise load till exhaustion (97–100% VO2max), and after competitions. The plasma FA profile was determined using gas-liquid chromatography.ResultsWe observed a substantial increase in the concentrations of capric acid (С10:0) (+ 164.1%), lauric acid (С12:0) (+ 223.9%), and myristic acid (С14:0) (+ 130.2%) in skiers after a sprint distance (1.3 km). A less intense increase in the concentrations of these acids (p < 0.05) was observed after a middle length distance or cycle exercise «until exhaustion». No significant differences in long-chain saturated FA content relative to baselines during exercise tests or competitions were revealed.ConclusionsIn conclusion, the obtained results demonstrate activation of the lipolysis and the oxidation of medium-chain FA involved in the energy supply for highly trained athletes at maximum exercise loads.

Consumption of Whey in Combination with Dairy Medium‐Chain Fatty Acids (MCFAs) may Reduce Lipid Storage due to Urinary Loss of Tricarboxylic Acid Cycle Intermediates and Increased Rates of MCFAs Oxidation

The aim of the paper is to investigate whether changes in the metabolome could explain observed changes in body composition in overweight adults after consumption of butter with high level of medium-chain fatty acids (MCFAs) in combination with casein or whey. With GC-TOF and LC-Q/MS, metabolites in plasma and urine from a 12-week randomized double-blinded human intervention including 52-abdominally overweight adults were analyzed. The participants consumed 63 g per day of milk fat (high or low in MCFAs) and 60 g per day of protein (whey or casein). Urinary loss of the tricarboxylic acid cycle metabolites and a concomitantly increase of glycerol in blood were observed in the whey + high-MCFAs group, indicating potential lower anabolic processes, such as lipogenesis, by draining substrates. High intake of MCFAs resulted in elevated level of urinary adipic (independently of protein type) and plasma sebacic acid (with whey), indicating a potential increase in oxidation of MCFAs, which might lead to energy loss. The type of protein showed highest effect on the overall metabolic profiles, but ω-oxidation of MCFAs in the liver seemed to be the main reason for the observed reduction in body fat mass after consumption of high MCFAs, independent of type of protein.

Aspirin increases mitochondrial fatty acid oxidation

The metabolic effects of salicylates are poorly understood. This study investigated the effects of aspirin on fatty acid oxidation. Aspirin increased mitochondrial long-chain fatty acid oxidation, but inhibited peroxisomal fatty acid oxidation, in two different cell lines. Aspirin increased mitochondrial protein acetylation and was found to be a stronger acetylating agent in vitro than acetyl-CoA. However, aspirin-induced acetylation did not alter the activity of fatty acid oxidation proteins, and knocking out the mitochondrial deacetylase SIRT3 did not affect the induction of long-chain fatty acid oxidation by aspirin. Aspirin did not change oxidation of medium-chain fatty acids, which can freely traverse the mitochondrial membrane. Together, these data indicate that aspirin does not directly alter mitochondrial matrix fatty acid oxidation enzymes, but most likely exerts its effects at the level of long-chain fatty acid transport into mitochondria. The drive on mitochondrial fatty acid oxidation may be a compensatory response to altered mitochondrial morphology and inhibited electron transport chain function, both of which were observed after 24 h incubation of cells with aspirin. These studies provide insight into the pathophysiology of Reye Syndrome, which is known to be triggered by aspirin ingestion in patients with fatty acid oxidation disorders.

Peroxisomes contribute to the acylcarnitine production when the carnitine shuttle is deficient

Fatty acid β-oxidation may occur in both mitochondria and peroxisomes. While peroxisomes oxidize specific carboxylic acids such as very long-chain fatty acids, branched-chain fatty acids, bile acids, and fatty dicarboxylic acids, mitochondria oxidize long-, medium-, and short-chain fatty acids. Oxidation of long-chain substrates requires the carnitine shuttle for mitochondrial access but medium-chain fatty acid oxidation is generally considered carnitine-independent. Using control and carnitine palmitoyltransferase 2 (CPT2)- and carnitine/acylcarnitine translocase (CACT)-deficient human fibroblasts, we investigated the oxidation of lauric acid (C12:0). Measurement of the acylcarnitine profile in the extracellular medium revealed significantly elevated levels of extracellular C10- and C12-carnitine in CPT2- and CACT-deficient fibroblasts. The accumulation of C12-carnitine indicates that lauric acid also uses the carnitine shuttle to access mitochondria. Moreover, the accumulation of extracellular C10-carnitine in CPT2- and CACT-deficient cells suggests an extramitochondrial pathway for the oxidation of lauric acid. Indeed, in the absence of peroxisomes C10-carnitine is not produced, proving that this intermediate is a product of peroxisomal β-oxidation. In conclusion, when the carnitine shuttle is impaired lauric acid is partly oxidized in peroxisomes. This peroxisomal oxidation could be a compensatory mechanism to metabolize straight medium- and long-chain fatty acids, especially in cases of mitochondrial fatty acid β-oxidation deficiency or overload.

Harmful effects of usnic acid on hepatic metabolism

Usnic acid is a naturally occurring dibenzofuran derivative found in several lichen species. The compound has been marketed as an ingredient of food supplements for weight reduction. There is evidence that the compound acts as an uncoupler of mitochondrial oxidative phosphorylation and it is also clear that consumption of the drug can lead to severe hepatotoxicity depending on the doses. Based on these and other ideas the objective of the present work was to investigate the possible effects of usnic acid on liver metabolism. Livers of male Wistar rats were perfused in a non-recirculating system. Usnic acid stimulated oxygen consumption at low concentrations, diminished the cellular ATP levels, increased the cytosolic but diminished the mitochondrial NADH/NAD+ ratio, strongly inhibited gluconeogenesis from three different substrates (IC50 between 1.33 and 3.61μM), stimulated glycolysis, fructolysis, glycogenolysis and ammoniagenesis and inhibited ureogenesis. The 14CO2 production from [1-14C]octanoate and [1-14C]oleate was increased by usnic acid, but ketogenesis from octanoate was diminished and that from oleate was not affected. It may be concluded that the effects of usnic acid up to 2.5μM reflect predominantly its activity as an uncoupler. At higher concentrations, however, several other effects may become significant, including inhibition of mitochondrial electron flow and inhibition of medium-chain fatty acid oxidation. In metabolic terms, toxicity of usnic acid can be predicted to be especially dangerous in the fasted state due to the combination of several deleterius events such as diminished hepatic glucose and ketone bodies output to the brain and increased ammonia production.

Decreased mitochondrial oxidative phosphorylation capacity in the human heart with left ventricular systolic dysfunction

Heart failure (HF) with left ventricular systolic dysfunction (LVSD) is associated with a shift in substrate utilization and a compromised energetic state. Whether these changes are connected with mitochondrial dysfunction is not known. We hypothesized that the cardiac phenotype in LVSD could be caused by reduced mitochondrial oxidative phosphorylation (OXPHOS) capacity and reduced mitochondrial creatine kinase (miCK) capacity. The study aim was to test mitochondrial OXPHOS capacity in LVSD myocardium compared with OXPHOS capacity in a comparable patient group without LVSD. Myocardial biopsies were obtained from the left ventricle during cardiac valve or left ventricular assist device (LVAD) surgery. Patients were stratified according to left ventricular ejection fraction (LVEF) into LVSD (LVEF <45%, n = 14) or CONTROL (LVEF >45%, n = 15). Mitochondrial respiration was measured in muscle fibres with addition of non-fatty acid substrates or octanoyl-l-carnitine, a medium chain fatty acid (MCFA). The in situ enzyme capacity of miCK was determined from APD titrations in the presence or absence of creatine. Maximal OXPHOS capacity with non-fatty acid substrates was lower in the LVSD group compared with the CONTROL group (P ≤ 0.05). ADP sensitivity always increased significantly (P ≤ 0.05) with the addition of creatine, after which the sensitivity was highest (P ≤ 0.05) in LVSD compared with CONTROL. The stimulation of OXPHOS from octanoyl-l-carnitine titrations elicited ∼40% lower respiration in LVSD compared with CONTROL (P ≤ 0.05). Human LVSD is associated with markedly diminished OXPHOS capacity, particularly in MCFA oxidation. This offers a candidate mechanism for a compromised energetic state and decreased reliance on fatty acid utilization in HF.

Effects of labor on placental fatty acid β oxidation

Objective: To measure the effect labor exerts on fatty acid (FA) oxidation in term human placentas, and to compare enzymes expression and activity between placenta and liver. Methods: Placental samples were collected: (a) scheduled non-labored cesarean section and (b) normal vaginal delivery at or beyond 37 weeks. Long and medium-chain FA oxidation were measured using 3H-labeled FA, ATP concentration was measured via commercial kit. Activity and expression levels of 11 FA enzymes were measured and results compared to both human and mouse liver. Results: Placentas undergoing labor had significantly decreased palmitate oxidation and ATP levels. Octanoic acid oxidation was 10-fold higher than palmitic acid oxidation. No difference in expression or activity level was detected between the groups. Conclusion: Term human placentas express all the enzymes required to oxidize FA, at a rate 20-fold lower than liver. FA Oxidation is not likely an important placental energy source during labor. Further work is needed to determine the functionality of this pathway in placenta.

Impaired myogenesis in estrogen‐related receptor γ (ERRγ)‐deficient skeletal myocytes due to oxidative stress

Specialized contractile function and increased mitochondrial number and oxidative capacity are hallmark features of myocyte differentiation. The estrogen-related receptors (ERRs) can regulate mitochondrial biogenesis or mitochondrial enzyme expression in skeletal muscle, suggesting that ERRs may have a role in promoting myogenesis. Therefore, we characterized myogenic programs in primary myocytes isolated from wild-type (M-ERRγWT) and muscle-specific ERRγ(-/-) (M-ERRγ(-/-)) mice. Myotube maturation and number were decreased throughout differentiation in M-ERRγ(-/-) primary myocytes, resulting in myotubes with reduced mitochondrial content and sarcomere assembly. Compared with M-ERRγWT myocytes at the same differentiation stage, the glucose oxidation rate was reduced by 30% in M-ERRγ(-/-) myotubes, while medium-chain fatty acid oxidation was increased by 34% in M-ERRγ(-/-) myoblasts and 36% in M-ERRγ(-/-) myotubes. Concomitant with increased reliance on mitochondrial β-oxidation, H(2)O(2) production was significantly increased by 40% in M-ERRγ(-/-) myoblasts and 70% in M-ERRγ(-/-) myotubes compared to M-ERRγWT myocytes. ROS activation of FoxO and NF-κB and their downstream targets, atrogin-1 and MuRF1, was observed in M-ERRγ(-/-) myocytes. The antioxidant N-acetyl cysteine rescued myotube formation and atrophy gene induction in M-ERRγ(-/-) myocytes. These results suggest that loss of ERRγ causes metabolic defects and oxidative stress that impair myotube formation through activation of skeletal muscle atrophy pathways.

Octanoate and Nonaoate Oxidation Increases 50–80% Over the First Two Days of Life in Piglet Triceps Brachii and Gracilis Muscle Strips ,

An in vitro muscle strip incubation system was developed to measure the rate of catabolism of 1 mmol/L [1-14C]octanoate, 1 mmol/L [1-14C]nonanoate, 1 mmol/L [9-14C]nonanoate, and 10 mmol/L [U-14C]glucose by measuring the recovery of14CO2. Muscle strips (13 mm × 1.5 mm, ~50 mg) were isolated from triceps brachii and gracilis muscles of newborn and 2-d-old, small (<950 g) and large (>1450 g) piglets. The position of the14C label in the substrate affected the rate and amount of recovery in14CO2. Therefore, comparisons were made between age groups (0 vs. 2 d old) within substrates but limited across substrates to comparisons of [1-14C]–labeled fatty acids. The medium-chain fatty acid (MCFA) oxidation rates [pmol/(h · mg)] in muscle strips isolated from piglets from the 2 weight groups (<950 and >1450 g) did not differ (P> 0.99), there was a trend towards a difference between triceps brachii and gracilis muscle (P= 0.09; data not shown), and there were no significant interactions involving pig weight or muscle type; therefore, results were pooled across these factors. During the first 2 d of life, MCFA oxidation [pmol/(h · · mg muscle strip)] increased (P< 0.05) 50–80%, but the glucose oxidation rate did not change (P> 0.82). By d 2, the oxidation rate of nonanoate as represented by the one carbon was 25% greater than for octanoate (P< 0.05). The conversion of [9-14C]nonanoate to14CO2indicated that muscle had the capacity to oxidize the propionyl-CoA produced by β-oxidation of nonanoate and that odd-chain C-9 MCFA provided anabolic carbon to the citric acid cycle.

Effect of Randomly Interesterified Triacylglycerol Containing Medium- and Long-Chain Fatty Acids on Hepatic Fatty Acid Oxidation after a Single Administration to Rats

MLCTs, which are randomly interesterified triacylglycerol containing medium- and long-chain fatty acids in the same glycerol molecule, showed significantly higher acyl-CoA dehydrogenase activity when measured by using butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA as substrates than long-chain triacylglycerol one hour after a single administration to rats. These results suggest that not only medium-chain fatty acid oxidation, but also long-chain fatty acid oxidation were increased in the liver of rats administered with MLCT.

Women Have Higher Protein Content of β-Oxidation Enzymes in Skeletal Muscle than Men

It is well recognized that compared with men, women have better ultra-endurance capacity, oxidize more fat during endurance exercise, and are more resistant to fat oxidation defects i.e. diet-induced insulin resistance. Several groups have shown that the mRNA and protein transcribed and translated from genes related to transport of fatty acids into the muscle are greater in women than men; however, the mechanism(s) for the observed sex differences in fat oxidation remains to be determined. Muscle biopsies from the vastus lateralis were obtained from moderately active men (N = 12) and women (N = 11) at rest to examine mRNA and protein content of genes involved in lipid oxidation. Our results show that women have significantly higher protein content for tri-functional protein alpha (TFPα), very long chain acyl-CoA dehydrogenase (VLCAD), and medium chain acyl-CoA dehydrogenase (MCAD) (P<0.05). There was no significant sex difference in the expression of short-chain hydroxyacyl-CoA dehydrogenase (SCHAD), or peroxisome proliferator activated receptor alpha (PPARα), or PPARγ, genes potentially involved in the transcriptional regulation of lipid metabolism. In conclusion, women have more protein content of the major enzymes involved in long and medium chain fatty acid oxidation which could account for the observed differences in fat oxidation during exercise.

Post‐mortem analysis for two prevalent β‐oxidation mutations in sudden infant death

Fatty acid oxidation disorders may cause sudden and unexpected infant death and are associated with the histological hallmark of hepatic steatosis. The goal of the present study was to assess the value of post-mortem molecular analysis for medium-chain acyl-coenzyme A dehydrogenase (MCAD) and mitochondrial trifunctional protein (MTP) defects in unexplained sudden infant death (SID) associated with fatty infiltration of the liver. MCAD catalyzes the first step of medium-chain fatty acid oxidation while MTP catalyzes the last three steps of long-chain fatty acid oxidation. In a retrospective study, 220 consecutive cases of sudden and unexplained infant death certified by medical examiners at Wake Forest University Medical Center were assessed for hepatic steatosis. Subjects with evidence of hepatic steatosis were screened for mutations in MCAD and MTPalpha-subunit using DNA isolated from paraffin-embedded liver tissue, single-strand conformation variance, and nucleotide sequence analyses. Sixteen cases (7.3%) were associated with diffuse micro-vesicular or mixed micro- and macro-vesicular hepatic steatosis. Two of these 16 cases (12.5%) had disease-causing mutations. One was homozygous for the prevalent MCAD A985G mutation. The second was a compound heterozygous for the prevalent MTP G1528C mutation and a novel 1 bp deletion in exon 18 of the MTPalpha-subunit gene. A significant proportion (7.3%) of SID is associated with hepatic steatosis. The present data support post-mortem molecular analysis for the MCAD A985G and MTP G1528C prevalent mutations in cases of sudden and unexplained infant death associated with hepatic steatosis.

Medium-chain fatty acids undergo elongation before β-oxidation in fibroblasts

Although mitochondrial fatty acid β-oxidation (FAO) is considered to be well understood, further elucidation of the pathway continues through evaluation of patients with FAO defects. The FAO pathway can be examined by measuring the 3-hydroxy-fatty acid (3-OHFA) intermediates. We present a unique finding in the study of this pathway: the addition of medium-chain fatty acids to the culture media of fibroblasts results in generation of 3-OHFAs which are two carbons longer than the precursor substrate. Cultured skin fibroblasts from normal and LCHAD-deficient individuals were grown in media supplemented with various chain-length fatty acids. The cell-free medium was analyzed for 3-OHFAs by stable-isotope dilution gas-chromatography/mass-spectrometry. Our finding suggests that a novel carbon chain-length elongation process precedes the oxidation of medium-chain fatty acids. This previously undescribed metabolic step may have important implications for the metabolism of medium-chain triglycerides, components in the dietary treatment of a number of disorders.

The anaplerotic potential of pentanoate and B‐ketopentanoate in pig heart in vivo

We previously showed (Roe et al., J Clin Invest 110: 259, 2002) that the cardiovascular and muscle strength status of patients with long-chain fatty acid oxidation disorders was greatly improved by a diet therapy with triheptanoin. The benefit of the treatment was ascribed to the production of anaplerotic propionyl-CoA from heptanoate. We hypothesized that pentanoate (as tripentanoin) and β-ketopentanoate (as a glycerol ester) might be used for the acute treatment of patients with long-chain or medium-chain fatty acid oxidation defects. We investigated the metabolism and anaplerotic potential of these compounds in live pig hearts. [3,4,5-13C3]Pentanoate, [1-13C]pentanoate or β-keto-[3,4,5-13C3]pentanoate were infused at various concentrations (0 to 1.5 mM) in the coronary artery of anesthetized pigs. The uptake of pentanoate and β-ketopentanoate increased linearly with substrate concentration, and there was no impairment of cardiac function at all concentrations infused. From the mass isotopomer distribution of intermediates of the propionyl-CoA pathway and the citric acid cycle, we calculated the rate of anaplerosis from pentanoate and β-ketopentanoate (6–10% of the citric acid cycle). The infusion of pentanoate or β-ketopentanoate decreased the production of endogenous propionyl-CoA, and thus the oxidation of proteins in the heart. Supported by NIH grants DK069752 and P01HL074237.