Short-chain Fatty Acid Oxidation Research Articles

Syntrophic microbes involved in the oxidation of short-chain fatty acids in continuous-flow anaerobic digesters treating waste activated sludge with hydrochar.

The rapid degradation of short-chain fatty acids (SCFAs) is an essential issue of anaerobic digestion (AD), in which SCFA oxidizers could generally metabolize in syntrophy with methanogens. The dynamic responses of active metagenome-assembled genomes to low concentrations of propionate and acetate were analyzed to identify specific syntrophic SCFA oxidizers and their metabolic characteristics in continuous-flow AD systems treating waste activated sludge with and without hydrochar. In this study, hydrochar increased methane production by 19%, possibly due to hydrochar enhancing acidification and methanogenesis processes. A putative syntrophic propionate oxidizer and two acetate oxidizers contributed substantially to the syntrophic degradation of SCFAs, and hydrochar positively regulated their functional gene expressions. A significant relationship was established between the replication rate of SCFA oxidizers and their stimulation-related transcriptional activity. Acetate was degraded in the hydrochar group, which might be mainly through the syntrophic acetate oxidizer from the genus Desulfallas and methanogens from the genus Methanosarcina.IMPORTANCEShort-chain fatty acid (SCFA) degradation is an important process in the methanogenic ecosystem. However, current knowledge of this microbial mechanism is mainly based on studies on a few model organisms incubated as mono- or co-cultures or in enrichments, which cannot provide appropriate evidence in complex environments. Here, this study revealed the microbial mechanism of a hydrochar-mediated anaerobic digestion (AD) system promoting SCFA degradation at the species level and identified key SCFA oxidizing bacteria. Our analysis provided new insights into the SCFA oxidizers involved in the AD of waste activated sludge facilitated by hydrochar.

Mitochondrial morphology controls fatty acid utilization by changing CPT1 sensitivity to malonyl-CoA.

Changes in mitochondrial morphology are associated with nutrient utilization, but the precise causalities and the underlying mechanisms remain unknown. Here, using cellular models representing a wide variety of mitochondrial shapes, we show a strong linear correlation between mitochondrial fragmentation and increased fatty acid oxidation (FAO) rates. Forced mitochondrial elongation following MFN2 over-expression or DRP1 depletion diminishes FAO, while forced fragmentation upon knockdown or knockout of MFN2 augments FAO as evident from respirometry and metabolic tracing. Remarkably, the genetic induction of fragmentation phenocopies distinct cell type-specific biologicalfunctions of enhanced FAO. These include stimulation of gluconeogenesis in hepatocytes, induction of insulin secretion in islet β-cells exposed to fatty acids,and survival of FAO-dependent lymphoma subtypes. We find that fragmentation increases long-chain but not short-chain FAO,identifying carnitine O-palmitoyltransferase 1 (CPT1) as thedownstream effector of mitochondrial morphology in regulation of FAO.Mechanistically, we determined that fragmentation reduces malonyl-CoA inhibition of CPT1, while elongation increasesCPT1sensitivity to malonyl-CoA inhibition. Overall, these findings underscore a physiologic role for fragmentation as a mechanism whereby cellular fuel preference and FAO capacity are determined.

Dietary Fiber Increases Oxidative Metabolism in Colonocytes but Not in Distal Small Intestinal Enterocytes Isolated from Rats

Colonocyte and distal small intestinal enterocyte metabolism was studied in rats fed either an elemental diet or an elemental diet supplemented with 30% mixed dietary fiber for 14 d. Cells were incubated in RPMI 1640 culture media containing 14C-labeled glutamine (1 mmol/L), glucose (11 mmol/L), and short-chain fatty acids: acetate, propionate and butyrate (5 mmol/L). Substrate oxidation and product formation were measured. Colonocytes from the fiber-fed group had 22–51% higher oxidation rates than the elementalfed group for all substrates tested. The group consuming the fiber diet had 28% less glutamate formation from glutamine by isolated colonocytes than the group consuming the elemental diet. Enterocyte acetate oxidation and lactate formation rates were lower (60 and 30%) in the fiber-fed animals vs. the elemental-fed group. Including short-chain fatty acids and ketone bodies in incubation media differentially affected acetate, glutamine and glucose metabolism in isolated intestinal cells, depending on segment and diet. Short-chain fatty acid and glucose oxidation rates were higher for colonocytes than enterocytes and glutamate formation was greater in enterocytes than colonocytes. Fiber consumption increased this segmental disparity. This study demonstrates that dietary fiber consumption increases substrate oxidation by isolated colonocytes but not distal small intestinal enterocytes.

Dissimilatory manganese reduction facilitates synergistic cooperation of hydrolysis, acidogenesis, acetogenesis and methanogenesis via promoting microbial interaction during anaerobic digestion of waste activated sludge

Anaerobic digestion (AD) of waste activated sludge (WAS) is commonly limited to poor synergistic cooperation of four stages including hydrolysis, acidogenesis, acetogenesis and methanogenesis. Dissimilatory metal reduction that induced by metal-based conductive materials is promising strategy to regulate anaerobic metabolism with the higher metabolic driving force. In this study, MnO2 as inducer of dissimilatory manganese reduction (DMnR) was added into WAS-feeding AD system for mediating complicated anaerobic metabolism. The results demonstrated that main operational performances including volatile solid (VS) degradation efficiency and cumulative CH4 production with MnO2 dosage of 60 mg/g·VS reached up to maximum 53.6 ± 3.4% and 248.2 ± 10.1 mL/g·VS while the lowest operational performances in control group (38.5 ± 2.8% and 183.5 ± 8.5 mL/g·VS) was originated from abnormal operation of four stages. Furthermore, high-throughput 16 S rRNA pyrosequencing revealed that enrichment of dissimilatory manganese-reducing contributors and methanogens such as Thermovirga, Christensenellaceae_R_7_group and Methanosaeta performed the crucial role in short-chain fatty acids (SCFAs) oxidation and final methanogenesis, which greatly optimized operational environment of hydrolysis, acidogenesis and acetogenesis. More importantly, analysis of functional genes expression proved that abundances of genes encoding enzymes participated in acetate oxidation, direct interspecies electron transfer (DIET) and CO2 reduction pathway were simultaneously up-regulated with the optimum MnO2 dosage, suggesting that DMnR with SCFAs oxidation as electron sink could benefit stable operation of four stages via triggering effective DIET-based microbial interaction mode.

Short-Chain Fatty Acids Outpace Ketone Oxidation in the Failing Heart

The failing heart is energy starved with impaired oxidation of long-chain fatty acids (LCFAs) at the level of reduced CPT1 (carnitine palmitoyltransferase 1) activity at the outer mitochondrial membrane. Recent work shows elevated ketone oxidation in failing hearts as an alternate carbon source for oxidative ATP generation. We hypothesized that another short-chain carbon source, short-chain fatty acids (SCFAs) that bypass carnitine palmitoyltransferase 1, could similarly support energy production in failing hearts. Cardiac hypertrophy and dysfunction were induced in rats by transverse-aortic constriction (TAC). Fourteen weeks after TAC or sham operation, isolated hearts were perfused with either the 4 carbon, 13C-labeled ketone (D3-hydroxybutyrate) or the 4 carbon, 13C-labeled SCFA butyrate in the presence of glucose and the LCFA palmitate. Oxidation of ketone and SCFA was compared by in vitro 13C nuclear magnetic resonance spectroscopy, as was the capacity for short-chain carbon sources to compensate for impaired LCFA oxidation in the hypertrophic heart. Adaptive changes in enzyme expression and content for the distinct pathways of ketone and SCFA oxidation were examined in both failing rat and human hearts. TAC produced pathological hypertrophy and increased the fractional contributions of ketone to acetyl coenzyme-A production in the tricarboxylic acid cycle (0.60±0.02 sham ketone versus 0.70±0.02 TAC ketone; P<0.05). However, butyrate oxidation in failing hearts was 15% greater (0.803±0.020 TAC SCFA) than ketone oxidation. SCFA was also more readily oxidized than ketone in sham hearts by 15% (0.693±0.020 sham SCFA). Despite greater SFCA oxidation, TAC did not change short-chain acyl coenzyme-A dehydrogenase content. However, failing hearts of humans and the rat model both contain significant increases in acyl coenzyme-A synthetase medium-chain 3 enzyme gene expression and protein content. The increased oxidation of SCFA and ketones occurred at the expense of LCFA oxidation, with LCFA contributing less to acetyl coenzyme-A production in failing hearts perfused with SCFA (0.190±0.012 TAC SCFA versus 0.3163±0.0360 TAC ketone). SCFAs are more readily oxidized than ketones in failing hearts, despite both bypassing reduced CPT1 activity and represent an unexplored carbon source for energy production in failing hearts.

Novel syntrophic bacteria in full-scale anaerobic digesters revealed by genome-centric metatranscriptomics

Short-chain fatty acid (SCFA) degradation is an important process in methanogenic ecosystems, and is usually catalyzed by SCFA-oxidizing bacteria in syntrophy with methanogens. Current knowledge of this functional guild is mainly based on isolates or enrichment cultures, but these may not reflect the true diversity and in situ activities of the syntrophs predominating in full-scale systems. Here we obtained 182 medium to high quality metagenome-assembled genomes (MAGs) from the microbiome of two full-scale anaerobic digesters. The transcriptomic response of individual MAG was studied after stimulation with low concentrations of acetate, propionate, or butyrate, separately. The most pronounced response to butyrate was observed for two MAGs of the recently described genus Candidatus Phosphitivorax (phylum Desulfobacterota), expressing a butyrate beta-oxidation pathway. For propionate, the largest response was observed for an MAG of a novel genus in the family Pelotomaculaceae, transcribing a methylmalonyl-CoA pathway. All three species were common in anaerobic digesters at Danish wastewater treatment plants as shown by amplicon analysis, and this is the first time their syntrophic features involved in SCFA oxidation were revealed with transcriptomic evidence. Further, they also possessed unique genomic features undescribed in well-characterized syntrophs, including the metabolic pathways for phosphite oxidation, nitrite and sulfate reduction.

Anaerobic digestion of sludge filtrate using anaerobic baffled reactor assisted by symbionts of short chain fatty acid-oxidation syntrophs and exoelectrogens: Pilot-scale verification

The growing amount of sewage sludge from wastewater treatment plant is an emerging challenge in China. The efficient anaerobic digestion of sludge filtrate generated from hydrothermally pretreated sewage sludge can promote the disposal of sewage sludge. Herein, a pilot-scale anaerobic baffled reactor (ABR) assisted by symbionts of short chain fatty acid-oxidation syntrophs (SFAS) and exoelectrogens was developed to improve its stability and efficiency for filtrate treatment. The results demonstrated that the symbionts of exoelectrogens and SFAS, which were enriched by introduction of electrodes in the ABR system, promoted the degradation of butyric, propionic and acetic acids. Therefore, the COD removal efficiency increased from 74.1% to 86.6% and the methane content increased from 81.5% to 92.2% with methane production rising from 241 to 282 mL/g CODremoved. Furthermore, the economic evaluation indicated that the energy consumption of electrodes was 0.600 kWh/m3 of sludge filtrate, the net energy profited from increased methane was 2.344 kWh/m3 of sludge filtrate. These results confirmed that the ABR system assisted by symbionts of SFAS and exoelectrogens was feasible for treatment of sludge filtrate in terms of both technical and economic level through pilot-scale verification.

Anaerobic digestion of sludge filtrate assisted by symbionts of short chain fatty acid-oxidation syntrophs and exoelectrogens: Process performance, methane yield and microbial community

Sludge filtrate is a kind of special organic wastewater generated from hydrothermally pretreated sewage sludge. The efficient treatment of sludge filtrate can promote the development of sludge recycling technology. Herein, the anaerobic baffled reactor (ABR) assisted by symbionts of short chain fatty acid-oxidation syntrophs (SFAS) and exoelectrogens was applied to treat the sludge filtrate. The influence of fermentation temperature and promotion of methanogenesis via symbionts were focused. The results showed that the COD removal efficiency and methane yield of the ABR system assisted by symbionts at 35 °C (R3) were 11.7% and 11.0% higher than the one at 55 °C (R2), respectively. And the COD removal efficiency and methane yield of the R2 system were 9.1% and 12.9% higher than the traditional ABR system at 55 °C (R1), respectively. Large abundances of exoelectrogens such as Thermincola and Geobacter were found in the R2 and R3 systems, respectively. Moreover, ample Syntrophobacter, Syntrophomonas and Methanobacterium were detected in both R2 and R3 systems. The present research revealed the importance of SFAS, exoelectrogens and hydrogenotrophic methanogens for the improvement of methanogenesis. Besides, the mesophilic condition is conducive to enhancing the methanogenesis rate of sludge filtrate.

Cardiac metabolic modulation upon low-carbohydrate low-protein ketogenic diet in diabetic rats studied in vivo using hyperpolarized 13 C pyruvate, butyrate and acetoacetate probes.

To investigate the effects of long-term low-carbohydrate low-protein ketogenic diet (KD) on cardiac metabolism and diabetic cardiomyopathy status in lean diabetic Goto-Kakizaki (GK) rats. Diabetic GK rats were fed with KD for 62 weeks. Cardiac function and metabolism were assessed using magnetic resonance imaging and 13 C magnetic resonance spectroscopy (13 C-MRS), at rest and under dobutamine stress. 13 C-MRS was performed following injection of hyperpolarized [3-13 C]acetoacetate, [1-13 C]butyrate or [1-13 C]pyruvate to assess ketone body, short-chain fatty acid or glucose utilization, respectively. Protein expression and cardiomyocyte structure were determined via Western blotting and histology, respectively. KD lowered blood glucose, triglyceride and insulin levels while increasing blood ketone body levels. In KD-fed diabetic rats, myocardial ketone body and glucose oxidation were lower than in chow-fed diabetic rats, while myocardial glycolysis and short-chain fatty acid oxidation were unaltered. Dobutamine stress revealed an increased cardiac preload and reduced cardiac compliance in KD-fed diabetic rats. Dobutamine-induced stimulation of myocardial glycolysis was more enhanced in KD-fed diabetic rats than in chow-fed diabetic rats, which was potentially facilitated via an upregulation in basal expression of proteins involved in glucose transport and glycolysis in the hearts of KD-fed rats. The metabolic profile induced by KD was accompanied by cardiac hypertrophy, a trend for increased myocardial lipid and collagen content, and an increased marker of oxidative stress. KD seems to exacerbate diabetic cardiomyopathy in GK rats, which may be associated with maladaptive cardiac metabolic modulation and lipotoxicity.

Stimulation of carbon nanomaterials on syntrophic oxidation of butyrate in sediment enrichments and a defined coculture

It remains elusive if direct interspecies electron transfer (DIET) occurs in canonical syntrophy involving short-chain fatty acids oxidation. In the present study, we determined the effects of carbon nanomaterials on syntrophic oxidation of butyrate in two lake sediment enrichments and a defined coculture comprising Syntrophomonas wolfei and Methanococcus Maripaludis. After four continuous transfers of enrichment cultivation, Syntrophomonas dominated the bacterial populations in enrichments, and the dominated methanogens comprised Methanosarcina and Methanospirillum in one enrichment (from Weiming Lake) and Methanoregula and Methanospirillum in another (from Erhai Lake). Butyrate oxidation and CH4 production was significantly accelerated by carbon nanotubes (CNTs) in both enrichments. Replacement of CNTs by magnetite caused similar stimulating effect. For the defined coculture, two carbon nanomaterials, CNTs and reduced graphene oxide (rGO), were tested, both showed consistently stimulating effects on butyrate oxidation. Addition of kaolinite, an electric nonconductive clay mineral, however, revealed no effect. The test on M. maripaludis in pure culture showed no effect by rGO and a negative effect by CNTs (especially at a high concentration). Fluorescence in situ hybridization (FISH) and scanning electron microscopy (SEM) revealed that microbial cells were interwoven by CNTs forming cell-CNT mixture aggregates, and in case of rGO, cells were attached to surface or wrapped-up by rGO thin sheets. Collectively, our data suggest that the presence of conductive nanomaterials likely induces DIET in syntrophic butyrate oxidation.

Butyrate decreases its own oxidation in colorectal cancer cells through inhibition of histone deacetylases.

Colorectal cancer is characterized by an increase in the utilization of glucose and a diminishment in the oxidation of butyrate, which is a short chain fatty acid. In colorectal cancer cells, butyrate inhibits histone deacetylases to increase the expression of genes that slow the cell cycle and induce apoptosis. Understanding the mechanisms that contribute to the metabolic shift away from butyrate oxidation in cancer cells is important in in understanding the beneficial effects of the molecule toward colorectal cancer. Here, we demonstrate that butyrate decreased its own oxidation in cancerous colonocytes. Butyrate lowered the expression of short chain acyl-CoA dehydrogenase, an enzyme that mediates the oxidation of short-chain fatty acids. Butyrate does not alter short chain acyl-CoA dehydrogenase levels in non-cancerous colonocytes. Trichostatin A, a structurally unrelated inhibitor of histone deacetylases, and propionate also decreased the level of short chain acyl-CoA dehydrogenase, which alluded to inhibition of histone deacetylases as a part of the mechanism. Knockdown of histone deacetylase isoform 1, but not isoform 2 or 3, inhibited the ability of butyrate to decrease short chain acyl-CoA dehydrogenase expression. This work identifies a mechanism by which butyrate selective targets colorectal cancer cells to reduce its own metabolism.

Nrf2 affects the efficiency of mitochondrial fatty acid oxidation.

Transcription factor Nrf2 (NF-E2 p45-related factor 2) regulates the cellular redox homoeostasis and cytoprotective responses, allowing adaptation and survival under conditions of stress. The significance of Nrf2in intermediary metabolism is also beginning to be recognized. Thus this transcription factor negatively affects fatty acid synthesis. However, the effect of Nrf2 on fatty acid oxidation is currently unknown. In the present paper, we report that the mitochondrial oxidation of long-chain (palmitic) and short-chain (hexanoic) fatty acids is depressed in the absence of Nrf2 and accelerated when Nrf2 is constitutively active. Addition of fatty acids stimulates respiration in heart and liver mitochondria isolated from wild-type mice. This effect is significantly weaker when Nrf2 is deleted, whereas it is stronger when Nrf2 activity is constitutively high. In the absence of glucose, addition of fatty acids differentially affects the production of ATP in mouse embryonic fibroblasts from wild-type, Nrf2-knockout and Keap1 (Kelch-like ECH-associated protein 1)-knockout mice. In acute tissue slices, the rate of regeneration of FADH2 is reduced when Nrf2 is absent. This metabolic role of Nrf2 on fatty acid oxidation has implications for chronic disease conditions including cancer, metabolic syndrome and neurodegeneration.

Measurement of tissue acyl-CoAs using flow-injection tandem mass spectrometry: acyl-CoA profiles in short-chain fatty acid oxidation defects

The primary accumulating metabolites in fatty acid oxidation defects are intramitochondrial acyl-CoAs. Typically, secondary metabolites such as acylcarnitines, acylglycines and dicarboxylic acids are measured to study these disorders. Methods have not been adapted for tissue acyl-CoA measurement in defects with primarily acyl-CoA accumulation. Our objective was to develop a method to measure fatty acyl-CoA species that are present in tissues of mice with fatty acid oxidation defects using flow-injection tandem mass spectrometry.Following the addition of internal standards of [13C2] acetyl-CoA, [13C8] octanoyl-CoA, and [C17] heptadecanoic CoA, acyl-CoA's are extracted from tissue samples and are injected directly into the mass spectrometer. Data is acquired using a 506.9 neutral loss scan and multiple reaction-monitoring (MRM).This method can identify all long, medium and short-chain acyl-CoA species in wild type mouse liver including predicted 3-hydroxyacyl-CoA species. We validated the method using liver of the short-chain-acyl-CoA dehydrogenase (SCAD) knock-out mice. As expected, there is a significant increase in [C4] butyryl-CoA species in the SCAD −/− mouse liver compared to wild type. We then tested the assay in liver from the short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) deficient mice to determine the profile of acyl-CoA accumulation in this less predictable model. There was more modest accumulation of medium chain species including 3-hydroxyacyl-CoA's consistent with the known chain-length specificity of the SCHAD enzyme.

Brain Transcriptional Responses to High-Fat Diet in Acads-Deficient Mice Reveal Energy Sensing Pathways

BackgroundHow signals from fatty acid metabolism are translated into changes in food intake remains unclear. Previously we reported that mice with a genetic inactivation of Acads (acyl-coenzyme A dehydrogenase, short-chain), the enzyme responsible for mitochondrial beta-oxidation of C4–C6 short-chain fatty acids (SCFAs), shift consumption away from fat and toward carbohydrate when offered a choice between diets. In the current study, we sought to indentify candidate genes and pathways underlying the effects of SCFA oxidation deficiency on food intake in Acads−/− mice.Methodology/Principal FindingsWe performed a transcriptional analysis of gene expression in brain tissue of Acads−/− and Acads+/+ mice fed either a high-fat (HF) or low-fat (LF) diet for 2 d. Ingenuity Pathway Analysis revealed three top-scoring pathways significantly modified by genotype or diet: oxidative phosphorylation, mitochondrial dysfunction, and CREB signaling in neurons. A comparison of statistically significant responses in HF Acads−/− vs. HF Acads+/+ (3917) and Acads+/+ HF vs. LF Acads+/+ (3879) revealed 2551 genes or approximately 65% in common between the two experimental comparisons. All but one of these genes were expressed in opposite direction with similar magnitude, demonstrating that HF-fed Acads-deficient mice display transcriptional responses that strongly resemble those of Acads+/+ mice fed LF diet. Intriguingly, genes involved in both AMP-kinase regulation and the neural control of food intake followed this pattern. Quantitative RT-PCR in hypothalamus confirmed the dysregulation of genes in these pathways. Western blotting showed an increase in hypothalamic AMP-kinase in Acads−/− mice and HF diet increased, a key protein in an energy-sensing cascade that responds to depletion of ATP.ConclusionsOur results suggest that the decreased beta-oxidation of short-chain fatty acids in Acads-deficient mice fed HF diet produces a state of energy deficiency in the brain and that AMP-kinase may be the cellular energy-sensing mechanism linking fatty acid oxidation to feeding behavior in this model.

Loss of ETHE1, a mitochondrial dioxygenase, causes fatal sulfide toxicity in ethylmalonic encephalopathy

Ethylmalonic encephalopathy is an autosomal recessive, invariably fatal disorder characterized by early-onset encephalopathy, microangiopathy, chronic diarrhea, defective cytochrome c oxidase (COX) in muscle and brain, high concentrations of C4 and C5 acylcarnitines in blood and high excretion of ethylmalonic acid in urine. ETHE1, a gene encoding a beta-lactamase-like, iron-coordinating metalloprotein, is mutated in ethylmalonic encephalopathy. In bacteria, ETHE1-like sequences are in the same operon of, or fused with, orthologs of TST, the gene encoding rhodanese, a sulfurtransferase. In eukaryotes, both ETHE1 and rhodanese are located within the mitochondrial matrix. We created a Ethe1(-/-) mouse that showed the cardinal features of ethylmalonic encephalopathy. We found that thiosulfate was excreted in massive amounts in urine of both Ethe1(-/-) mice and humans with ethylmalonic encephalopathy. High thiosulfate and sulfide concentrations were present in Ethe1(-/-) mouse tissues. Sulfide is a powerful inhibitor of COX and short-chain fatty acid oxidation, with vasoactive and vasotoxic effects that explain the microangiopathy in ethylmalonic encephalopathy patients. Sulfide is detoxified by a mitochondrial pathway that includes a sulfur dioxygenase. Sulfur dioxygenase activity was absent in Ethe1(-/-) mice, whereas it was markedly increased by ETHE1 overexpression in HeLa cells and Escherichia coli. Therefore, ETHE1 is a mitochondrial sulfur dioxygenase involved in catabolism of sulfide that accumulates to toxic levels in ethylmalonic encephalopathy.

Bile acylcarnitine profiles in pediatric liver disease do not interfere with the diagnosis of long-chain fatty acid oxidation defects

Background Plasma acylcarnitine measurement is an important diagnostic tool for inherited disorders of fatty acid and organic acid metabolism. Biliary excretion has been shown to be the primary route of excretion for acylcarnitines and analysis of bile acylcarnitine profiles may provide greater sensitivity for detecting metabolic disorders. Disorders of fatty acid oxidation frequently present with deranged liver function and the effect of hepatic disease on biliary acylcarnitine excretion are unknown. Methods We measured biliary acylcarnitine levels in pediatric patients aged 6 months to 1 year undergoing open liver biopsy with prospectively determined non-metabolic liver disease in order to determine the effect of the liver disease on acylcarnitine excretion. Bile was collected in syringes and was transported immediately and stored at − 70 °C until the time of testing. The disease patient population consisted of 2 patients with known defects in long- and short-chain fatty acid oxidation (long-chain L-3-hydroxy acyl-CoA dehydrogenase: LCHAD and short-chain L-3-hydroxy acyl-CoA dehydrogenase: SCHAD). The sample from the LCHAD patient was collected at autopsy and the patient with SCHAD deficiency was subsequently diagnosed as part of the prospective study and removed from the unknown etiology group. Acylcarnitine profiles were obtained for each specimen as butylated derivatives using tandem mass spectrometry. Results The non-metabolic liver disease had no effect on the diagnostic value of bile acylcarnitine levels for detecting LCHAD deficiency. The concentrations of bile long-chain acylcarnitine species analyzed from patients with non-metabolic liver disease were far lower than the levels seen in LCHAD deficiency which also demonstrated a characteristic pattern of 3-hydroxyacylcarnitine excretion. In SCHAD deficiency, for which pathognomonic markers have not yet been established, bile analysis did not improve the diagnostic ability. Conclusion The analysis of bile acylcarnitines for the diagnosis of long-chain fatty acid oxidation defects will provide unbiased information even in the presence of severe non-metabolic liver disease.

Establishing a Reference Interval For Measurement of Flux through the Mitochondrial Fatty Acid Oxidation Pathway in Cultured Skin Fibroblasts

Mitochondrial fatty acid oxidation (FAO) represents a normal metabolic response to increased energy demands during periods of fasting, febrile illness, or muscular exertion. FAO is a complex pathway involving activation of free fatty acids to acyl-CoA species, transport into mitochondria, and cyclic oxidation to break the long-chain fatty acids into acetyl-CoA and a two-carbon chain-shortened acyl-CoA, which is then recycled. Acetyl-CoA is used in the Krebs cycle and in liver as a substrate for ketogenesis (1). Measurement of FAO flux in cultured cells provides valuable information about the integrity of the enzymes and transporters involved in FAO at all stages. Clinical indications for the measurement of FAO flux in skin fibroblasts include fatty acid oxidation enzyme defects, unexplained myopathies, cardiomyopathies, hypoglycemia, liver disease, sudden unexplained infant death, and hypoketotic hypoglycemia. FAO flux is typically measured by monitoring the conversion of radiolabeled fatty acids to carbon dioxide and water. Labeling can be with 14C with final analysis of the rate of production of 14CO2, or with 3H and analysis of production of 3H2O (2)(3). Palmitic acid (C16) is used to detect long-chain defects, including deficiencies in very long chain acyl-CoA dehydrogenase, long-chain l-3-hydroxy acyl-CoA dehydrogenase, long-chain 3-keto acyl-CoA thiolase, carnitine/acylcarnitine translocase, carnitine pamitoyltransferase 1, and carnitine pamitoyltransferase 2. Myristic acid (C14) is used to detect defects in medium-chain FAO, such as medium-chain acyl-CoA dehydrogenase deficiency. We use tritiated fatty acids labeled in the (9,10) position. The assay will not detect defects in carnitine palmitoyl transferase 1B; short-chain FAO defects, including short-chain acyl-CoA dehydrogenase and short-chain l-3-hydroxy acyl-CoA dehydrogenase; and short-chain-3-keto acyl-CoA thiolase deficiencies. Historically, patient samples have been compared with batch controls, and a reference interval was not established for this assay. In the present study we have assigned …

Genetic organization of the catabolic plasmid pJP4 from Ralstonia eutropha JMP134 (pJP4) reveals mechanisms of adaptation to chloroaromatic pollutants and evolution of specialized chloroaromatic degradation pathways.

Ralstonia eutropha JMP134 (pJP4) is a useful model for the study of bacterial degradation of substituted aromatic pollutants. Several key degrading capabilities, encoded by tfd genes, are located in the 88 kb, self-transmissible, IncP-1 beta plasmid pJP4. The complete sequence of the 87,688 nucleotides of pJP4, encoding 83 open reading frames (ORFs), is reported. Most of the coding sequence corresponds to a well-conserved IncP-1 beta backbone and the previously reported tfd genes. In addition, we found hypothetical proteins putatively involved in the transport of aromatic compounds and short-chain fatty acid oxidation. ORFs related to mobile elements, including the Tn501-encoded mercury resistance determinants, an IS1071-based composite transposon and a cryptic class II transposon, are also present in pJP4. These mobile elements are inefficient in transposition and are located in two regions of pJP4 that are rich in remnants of lateral gene transfer events. pJP4 plasmid was able to capture chromosomal genes and form hybrid plasmids with the IncP-1 alpha plasmid RP4. These observations are integrated into a model for the evolution of pJP4, which reveals mechanisms of bacterial adaptation to degrade pollutants.

Glucose alleviates ammonia-induced inhibition of short-chain fatty acid metabolism in rat colonic epithelial cells.

Ammonia decreased metabolism by rat colonic epithelial cells of butyrate and acetate to CO2 and ketones but increased oxidation of glucose and glutamine. Ammonia decreased cellular concentrations of oxaloacetate for all substrates evaluated. The extent to which butyrate carbon was oxidized to CO2 after entering the tricarboxylic acid (TCA) cycle was not significantly influenced by ammonia, suggesting there was no major shift toward efflux of carbon from the TCA cycle. Ammonia reduced entry of butyrate carbon into the TCA cycle, and the proportion of CoA esterified with acetate and butyrate correlated positively with the production of CO2 and ketone bodies. Also, ammonia reduced oxidation of propionate but had no effect on oxidation of 3-hydroxybutyrate. Inclusion of glucose, lactate, or glutamine with butyrate and acetate counteracted the ability of ammonia to decrease their oxidation. In rat colonocytes, it appears that ammonia suppresses short-chain fatty acid (SCFA) oxidation by inhibiting a step before or during their activation. This inhibition is alleviated by glucose and other energy-generating compounds. These results suggest that ammonia may only affect SCFA metabolism in vivo when glucose availability is compromised.

Hypoglycaemia and elevated urine ethylmalonic acid in a child homozygous for the short‐chain acyl‐CoA dehydrogenase 625G &gt; A gene variation

The aim of this case report is to call attention to short‐chain acyl‐CoA dehydrogenase (SCAD) deficiency as a possible contributory factor to hypoglycaemia in childhood. We report on a previously healthy 14 mo‐old Danish boy who presented with hypoglycaemia and metabolic acidosis after a few days of upper airway infection. After two days on a normal diet, he recovered clinically and biochemically. A thorough biochemical examination did not reveal the cause of the hypoglycaemia. However, the excretion of ethylmalonic acid in two morning urine samples was moderately increased, and hence the SCAD gene was screened for mutations. We found the child homozygous for the G &gt; A SCAD gene variation at position 625. Conclusion: In this patient, reduced function of the SCAD protein is reflected in the excretion of ethylmalonic acid, a marker of intracellular accumulation of butyryl‐CoA and the cytotoxic butyric acid. Furthermore, gluconeogenesis might be compromised owing to lack of reducing equivalents from the oxidation of short‐chain fatty acids in the fasting or stressed state, thus contributing to the predisposition for fasting hypoglycaemia.