Fatty Acid Oxidation Intermediates Research Articles

Decreased long-chain acylcarnitine content increases mitochondrial coupling efficiency and prevents ischemia-induced brain damage in rats

Long-chain acylcarnitines (LCACs) are intermediates of fatty acid oxidation and are known to exert detrimental effects on mitochondria. This study aimed to test whether lowering LCAC levels with the anti-ischemia compound 4-[ethyl(dimethyl)ammonio]butanoate (methyl-GBB) protects brain mitochondrial function and improves neurological outcomes after transient middle cerebral artery occlusion (MCAO). The effects of 14 days of pretreatment with methyl-GBB (5 mg/kg, p.o.) on brain acylcarnitine (short-, long- and medium-chain) concentrations and brain mitochondrial function were evaluated in Wistar rats. Additionally, the mitochondrial respiration and reactive oxygen species (ROS) production rates were determined using ex vivo high-resolution fluorespirometry under normal conditions, in models of ischemia-reperfusion injury (reverse electron transfer and anoxia-reoxygenation) and 24 h after MCAO. MCAO model rats underwent vibrissae-evoked forelimb-placing and limb-placing tests to assess neurological function. The infarct volume was measured on day 7 after MCAO using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Treatment with methyl-GBB significantly reduced the LCAC content in brain tissue, which decreased the ROS production rate without affecting the respiration rate, indicating an increase in mitochondrial coupling. Furthermore, methyl-GBB treatment protected brain mitochondria against anoxia–reoxygenation injury. In addition, treatment with methyl-GBB significantly reduced the infarct size and improved neurological outcomes after MCAO. Increased mitochondrial coupling efficiency may be the basis for the neuroprotective effects of methyl-GBB. This study provides evidence that maintaining brain energy metabolism by lowering the levels of LCACs protects against ischemia-induced brain damage in experimental stroke models.

Divergent Metabolic Effects of Metformin Merge to Enhance Eicosapentaenoic Acid Metabolism and Inhibit Ovarian Cancer In Vivo

Simple SummaryAlthough the anticancer effects of metformin are well studied, its effect on energy metabolism of cancer cells remains elusive. Metformin can alter the metabolism of cells either by activating AMPK or inhibiting the mitochondrial respiratory chain complex. The aim of the study was to explore the distinct metabolic profiles of ovarian cancer cell lines post-metformin treatment and to understand metformin’s metabolism-based anti-growth effect on ovarian cancer cell lines. In this study, using the untargeted metabolomics approach, we found that metformin treatment promoted omega-3 fatty acid metabolism in three different ovarian cancer cell lines, and treating ovarian xenografts with specific omega-3 fatty acids, especially EPA, reduced ovarian tumor growth. Thus, our study adds an additional potential therapeutic mechanism to the multi-potent anticancer effects of metformin.Metformin is being actively repurposed for the treatment of gynecologic malignancies including ovarian cancer. We investigated if metformin induces analogous metabolic changes across ovarian cancer cells. Functional metabolic analysis showed metformin caused an immediate and sustained decrease in oxygen consumption while increasing glycolysis across A2780, C200, and SKOV3ip cell lines. Untargeted metabolomics showed metformin to have differential effects on glycolysis and TCA cycle metabolites, while consistent increased fatty acid oxidation intermediates were observed across the three cell lines. Metabolite set enrichment analysis showed alpha-linolenic/linoleic acid metabolism as being most upregulated. Downstream mediators of the alpha-linolenic/linoleic acid metabolism, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), were abundant in all three cell lines. EPA was more effective in inhibiting SKOV3 and CaOV3 xenografts, which correlated with inhibition of inflammatory markers and indicated a role for EPA-derived specialized pro-resolving mediators such as Resolvin E1. Thus, modulation of the metabolism of omega-3 fatty acids and their anti-inflammatory signaling molecules appears to be one of the common mechanisms of metformin’s antitumor activity. The distinct metabolic signature of the tumors may indicate metformin response and aid the preclinical and clinical interpretation of metformin therapy in ovarian and other cancers.

Increased circulating butyrate and ursodeoxycholate during probiotic intervention in humans with type 2 diabetes

BackgroundAn increasing body of evidence implicates the resident gut microbiota as playing a critical role in type 2 diabetes (T2D) pathogenesis. We previously reported significant improvement in postprandial glucose control in human participants with T2D following 12-week administration of a 5-strain novel probiotic formulation (‘WBF-011’) in a double-blind, randomized, placebo controlled setting (NCT03893422). While the clinical endpoints were encouraging, additional exploratory measurements were needed in order to link the motivating mechanistic hypothesis - increased short-chain fatty acids - with markers of disease.ResultsHere we report targeted and untargeted metabolomic measurements on fasting plasma (n = 104) collected at baseline and end of intervention. Butyrate and ursodeoxycholate increased among participants randomized to WBF-011, along with compelling trends between butyrate and glycated haemoglobin (HbA1c). In vitro monoculture experiments demonstrated that the formulation’s C. butyricum strain efficiently synthesizes ursodeoxycholate from the primary bile acid chenodeoxycholate during butyrogenic growth. Untargeted metabolomics also revealed coordinated decreases in intermediates of fatty acid oxidation and bilirubin, potential secondary signatures for metabolic improvement. Finally, improvement in HbA1c was limited almost entirely to participants not using sulfonylurea drugs. We show that these drugs can inhibit growth of formulation strains in vitro.ConclusionTo our knowledge, this is the first description of an increase in circulating butyrate or ursodeoxycholate following a probiotic intervention in humans with T2D, adding support for the possibility of a targeted microbiome-based approach to assist in the management of T2D. The efficient synthesis of UDCA by C. butyricum is also likely of interest to investigators of its use as a probiotic in other disease settings. The potential for inhibitory interaction between sulfonylurea drugs and gut microbiota should be considered carefully in the design of future studies.

Direct enantioselective gradient reversed-phase ultra-high performance liquid chromatography tandem mass spectrometry method for 3-hydroxy alkanoic acids in lipopeptides on an immobilized 1.6μm amylose-based chiral stationary phase.

3-Hydroxy fatty acids are important chiral building blocks of lipopeptides and metabolic intermediates of fatty acid oxidation, respectively. The analysis of the stereochemistry of such biomolecules has significant practical impact to elucidate and assign the enzymatic specificity of the biosynthesis machinery. In this work, a new mass spectrometry compatible direct chiral ultra high performance liquid chromatography separation method for 3-hydroxy fatty acids without derivatization is presented. The application of amylose tris(3,5-dimethylphenyl carbamate) based polysaccharide chiral stationary phase immobilized on 1.6μm silica particles (CHIRALPAK IA-U) allows the enantioseparation of 3-hydroxy fatty acids under generic electrospray ionization mass spectrometry friendly reversed phase gradient elution conditions. Adequate separation factors were achieved with both acetonitrile and methanol as organic modifiers, covering hydrocarbon chain lengths between C6 and C14 . Elution orders were derived from rhamnolipid (R-95) of which enantiomerically pure or enriched (R)-3-hydroxy fatty acids were recovered after ester hydrolysis. The S-configured acids consistently eluted before the respective R-enantiomers. The method was successfully applied for the elucidation of the absolute configuration of 3-hydroxy fatty acids originating from a novel lipopeptide with unknown structure. The work furthermore demonstrates that gradient elution is a viable option also in enantioselective (ultra)high performance liquid chromatography, even for analytes with modest separation factors, although less commonly exploited.

SQSTM1/p62 Inhibits whereas Nrf2 Promotes Tumorigenesis by Inducing Cell Population Remodeling and Metabolic Reprograming in Mouse Livers with mTORC1 Activation and Defective Autophagy

Tsc1 (Tuberous Sclerosis 1) and Tsc2 are tumor suppressor genes and negative regulators of mTORC1, and the mutation of Tsc1 or Tsc2 is associated with various cancers including hepatocellular carcinoma (HCC). mTORC1 activation promotes anabolic protein synthesis and inhibits catabolic autophagy process. Either hepatic persistent activation of mTORC1 due to the loss of Tsc1 or impaired hepatic autophagy due to the loss of Atg5 leads to spontaneous liver tumorigenesis in mice. However, how autophagy impacts mTORC1-mediated liver metabolic changes and tumorigenesis is less clear. Here we show that deletion of Atg5 in liver-specific Tsc1 knockout (L-Tsc1 KO) mice inhibited liver tumorigenesis but increased mortality of L-Tsc1 KO mice. Histological and unbiased RNA-seq as well as metabolomic analysis revealed that lack of hepatic autophagy dramatically induced the remodeling of liver cell population and hepatic metabolic reprogramming in L-Tsc1 KO mice. RNA-seq analysis revealed increased cholangiocyte, hepatic stellate cell and macrophage but decreased hepatocyte gene signatures in liver-specific Tsc1 and Atg5 double knockout (DKO) mice. Histologically, Atg5 and Tsc1 DKO mice had increased hepatomegaly, ductular reactions, increased number of macrophage/Kupffer cells and infiltrated neutrophils as well as massive fibrosis, and the DKO mice died at the age of approximately 8-months-old with no detectable liver tumors. Metabolomic analysis revealed marked increased hepatic glutathione and glutathione metabolites but decreased hepatic levels of glucose and lipid with accumulation of TCA cycle intermediate metabolites and fatty acid oxidation intermediates in Atg5 and Tsc1 DKO mice. Deletion of hepatic p62 partially but deletion of Nrf2 almost completely rescued the liver cell type remodeling and metabolic reprogramming and the mortality of Atg5 and Tsc1 DKO mice. Deletion of p62 promoted whereas deletion of Nrf2 suppressed the liver tumorigenesis of Atg5 and Tsc1 DKO mice. These results reveal a critical role of Nrf2 in regulating liver cell type remodeling and metabolic reprogramming in mice lack of hepatic autophagy with mTORC1 activation. Our data may provide a mechanistic basis for a precision medicine to target Nrf2 pathway for treating as subset of HCC, especially in HCV- or alcohol-associated liver cancers that may have defective autophagy and mTOR activation.

Isolated Plin5-deficient cardiomyocytes store less lipid droplets than normal, but without increased sensitivity to hypoxia

Plin5 is abundantly expressed in the heart where it binds to lipid droplets (LDs) and facilitates physical interaction between LDs and mitochondria. We isolated cardiomyocytes from adult Plin5+/+ and Plin5−/− mice to study the role of Plin5 for fatty acid uptake, LD accumulation, fatty acid oxidation, and tolerance to hypoxia. Cardiomyocytes isolated from Plin5−/− mice cultured with oleic acid stored less LDs than Plin5+/+, but comparable levels to Plin5+/+ cardiomyocytes when adipose triglyceride lipase activity was inhibited. The ability to oxidize fatty acids into CO2 was similar between Plin5+/+ and Plin5−/− cardiomyocytes, but Plin5−/− cardiomyocytes had a transient increase in intracellular fatty acid oxidation intermediates. After pre-incubation with oleic acids, Plin5−/− cardiomyocytes retained a higher content of glycogen and showed improved tolerance to hypoxia compared to Plin5+/+. In isolated, perfused hearts, deletion of Plin5 had no important effect on ventricular pressures or infarct size after ischemia. Old Plin5−/− mice had reduced levels of cardiac triacylglycerides, increased heart weight, and apart from modest elevated expression of mRNAs for beta myosin heavy chain Myh7 and the fatty acid transporter Cd36, other genes involved in fatty acid oxidation, glycogen metabolism and glucose utilization were essentially unchanged by removal of Plin5. Plin5 seems to facilitate cardiac LD storage primarily by repressing adipose triglyceride lipase activity without altering cardiac fatty acid oxidation capacity. Expression of Plin5 and cardiac LD content of isolated cardiomyocytes has little importance for tolerance to acute hypoxia and ischemia, which contrasts the protective role for Plin5 in mouse models during myocardial ischemia.

Electrophysiological Abnormalities in VLCAD Deficient hiPSC-Cardiomyocytes Can Be Improved by Lowering Accumulation of Fatty Acid Oxidation Intermediates.

Patients with very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) can present with life-threatening cardiac arrhythmias. The pathophysiological mechanism is unknown. We reprogrammed fibroblasts from one mildly and one severely affected VLCADD patient, into human induced pluripotent stem cells (hiPSCs) and differentiated these into cardiomyocytes (VLCADD-CMs). VLCADD-CMs displayed shorter action potentials (APs), more delayed afterdepolarizations (DADs) and higher systolic and diastolic intracellular Ca2+ concentration ([Ca2+]i) than control CMs. The mitochondrial booster resveratrol mitigated the biochemical, electrophysiological and [Ca2+]i changes in the mild but not in the severe VLCADD-CMs. Accumulation of potentially toxic intermediates of fatty acid oxidation was blocked by substrate reduction with etomoxir. Incubation with etomoxir led to marked prolongation of AP duration and reduced DADs and [Ca2+]i in both VLCADD-CMs. These results provide compelling evidence that reduced accumulation of fatty acid oxidation intermediates, either by enhanced fatty acid oxidation flux through increased mitochondria biogenesis (resveratrol) or by inhibition of fatty acid transport into the mitochondria (etomoxir), rescues pro-arrhythmia defects in VLCADD-CMs and open doors for new treatments.

Inter-generational link of obesity in term and preterm births: role of maternal plasma acylcarnitines.

BACKGROUND/OBJECTIVESAcylcarnitines, intermediates of fatty acid oxidation, are known to be involved in obesity and insulin resistance. Since maternal prepregnancy overweight or obesity (OWO) is a recognized major risk factor for offspring OWO, we hypothesized that maternal plasma acylcarnitines may play a role in inter-generational OWO.SUBJECTS/METHODSThis study included 1402 mother-child pairs (1043 term, 359 preterm) recruited at birth from 1998–2013 and followed prospectively up to age 18 years at the Boston Medical Center. The primary outcomes were child OWO defined as BMI≥85th percentile for age and sex. The primary exposures were maternal prepregnancy OWO defined as BMI≥25 and maternal acylcarnitine levels measured in plasma samples collected soon after delivery using liquid chromatography–tandem mass spectrometry (LC-MS) in a targeted manner.RESULTSApproximately 40% of the children in this study were OWO by age 5. Maternal OWO had a significant association with childhood OWO, both in term and preterm births. β-hydroxybutyryl-carnitine (C4-OH) levels were significantly and positively associated with child OWO among term births after adjustment for potential confounders and multiple-comparisons. Children born to OWO mothers in the top tertile C4-OH levels were at highest risk of OWO: OR=3.78 (95%: 2.47, 5.79) as compared with those born to non-OWO mothers in the lowest tertile (P for interaction of maternal OWO and C4-OH= 0.035). In a four-way decomposition of mediation/interaction analysis, we estimated that C4-OH levels explained about 27% (se=0.08) of inter-generational OWO risk (P=0.001). In contrast, these associations were not observed in preterm births.CONCLUSIONSIn this U.S. urban low-income birth cohort, we provide further evidence of the inter-generational link of OWO and reveal the differential role of C4-OH in explaining the inter-generational obesity between term and preterm births. Further investigations are warranted to better understand and prevent the inter-generational transmission of OWO.

CDK9 Inhibition Induces a Metabolic Switch that Renders Prostate Cancer Cells Dependent on Fatty Acid Oxidation

Cyclin-dependent kinase 9 (CDK9), a key regulator of RNA-polymerase II, is a candidate drug target for cancers driven by transcriptional deregulation. Here we report a multi-omics-profiling of prostate cancer cell responses to CDK9 inhibition to identify synthetic lethal interactions. These interactions were validated using live-cell imaging, mitochondrial flux-, viability- and cell death activation assays. We show that CDK9 inhibition induces acute metabolic stress in prostate cancer cells. This is manifested by a drastic down-regulation of mitochondrial oxidative phosphorylation, ATP depletion and induction of a rapid and sustained phosphorylation of AMP-activated protein kinase (AMPK), the key sensor of cellular energy homeostasis. We used metabolomics to demonstrate that inhibition of CDK9 leads to accumulation of acyl-carnitines, metabolic intermediates in fatty acid oxidation (FAO). Acyl-carnitines are produced by carnitine palmitoyltransferase enzymes 1 and 2 (CPT), and we used both genetic and pharmacological tools to show that inhibition of CPT-activity is synthetically lethal with CDK9 inhibition. To our knowledge this is the first report to show that CDK9 inhibition dramatically alters cancer cell metabolism.

Abstract 11: Metformin exerts differential metabolic effects in ovarian cancer cell lines

Abstract Metformin is being actively repurposed for treatment of gynecologic malignancies including ovarian cancer, with various clinical trials underway. Metformin is known to alter the cancer cell metabolism, primarily by inhibiting oxidative phosphorylation and inducing glycolysis. Our aim was to investigate if metformin induces similar metabolic changes across ovarian cancer cells. Untargeted global metabolite assay, by ultra-high performance Liquid Chromatography and Gas Chromatography Mass Spectroscopy, was performed on A2780, C200, and SKOV3ip cell lines with and without metformin treatment (10mM for 48 hours). Per-metabolite comparisons were made across conditions. Interpretive analysis was performed using the KEGG molecular pathways (Kyoto Encyclopedia of Genes and Genomes) and the Ingenuity molecule library with a focus on energy pathways of glycolysis, oxidative phosphorylation and also other metabolic pathways. Additionally, glycolytic and mitochondrial respiration were measured using the Seahorse XFe Analyzer. Specific analysis of the glycolysis metabolites revealed that while glycolysis was increased by metformin in all the cells, the intracellular levels of glucose, lactose and pyruvate varied significantly across the cell lines and were differentially affected by metformin treatment. Metformin had little impact on the TCA cycle intermediates in the A2780 cells, which were significantly decreased in C200 and in contrast increased in SKOV3ip cells. Functional analysis showed the oxygen consumption rate to be significantly inhibited by metformin in all the three cell lines, while the increased fatty acid oxidation intermediates were observed across all the three cell lines albeit to a varying extent. Exploration of the global metabolite changes by metformin across the three cell lines revealed 57 common altered metabolites, of which 30 had consistent direction change, with 16 metabolite being up and 14 being downregulated by metformin treatment. Metabolite Set Enrichment analysis showed linolenic acid and methionine metabolism as most enriched in metabolites being increased by metformin, and RNA transcription and pyrimidine metabolism as most enriched in the metabolites downregulated by metformin treatment. Ingenuity analysis indicated cellular proliferation and signaling as the top common network pathway modulated by metformin. In conclusion metformin treatment had a significant and wide-spread effect on metabolism of ovarian cancer cell lines. While metformin resulted in certain consistent metabolic changes, it had cell line specific modulation on glycolysis and oxidative phosphorylation metabolites. These differential metabolic changes could indicate the degree of metformin response and suggest it to be context-dependent. Thus information about the cancer metabolism will aid in preclinical and clinical interpretation of metformin therapy in ovarian and other cancers. Citation Format: Adnan Munkarah, Laila Poisson, Seongho Kim, Jasdeep Chhina, Shailendra Giri, Ramandeep Rattan. Metformin exerts differential metabolic effects in ovarian cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 11.

Accumulation of Palmitoylcarnitine and Its Effect on Pro‐Inflammatory Pathways and Calcium Influx in Prostate Cancer

BACKGROUNDAcylcarnitines are intermediates of fatty acid oxidation and accumulate as a consequence of the metabolic dysfunction resulting from the insufficient integration between β‐oxidation and the tricarboxylic acid (TCA) cycle. The aim of this study was to investigate whether acylcarnitines accumulate in prostate cancer tissue, and whether their biological actions could be similar to those of dihydrotestosterone (DHT), a structurally related compound associated with cancer development.METHODSLevels of palmitoylcarnitine (palcar), a C16:00 acylcarnitine, were measured in prostate tissue using LC‐MS/MS. The effect of palcar on inflammatory cytokines and calcium (Ca2+) influx was investigated in in vitro models of prostate cancer.RESULTSWe observed a significantly higher level of palcar in prostate cancerous tissue compared to benign tissue. High levels of palcar have been associated with increased gene expression and secretion of the pro‐inflammatory cytokine IL‐6 in cancerous PC3 cells, compared to normal PNT1A cells. Furthermore, we found that high levels of palcar induced a rapid Ca2+ influx in PC3 cells, but not in DU145, BPH‐1, or PNT1A cells. This pattern of Ca2+ influx was also observed in response to DHT. Through the use of whole genome arrays we demonstrated that PNT1A cells exposed to palcar or DHT have a similar biological response.CONCLUSIONSThis study suggests that palcar might act as a potential mediator for prostate cancer progression through its effect on (i) pro‐inflammatory pathways, (ii) Ca2+ influx, and (iii) DHT‐like effects. Further studies need to be undertaken to explore whether this class of compounds has different biological functions at physiological and pathological levels. Prostate 76:1326–1337, 2016. © 2016 The Authors. The Prostate published by Wiley Periodicals, Inc.

Abstract PD3-05: Inhibition of fatty-acid oxidation as a therapy for triple-negative breast cancer

Abstract Expression of the oncogenic transcription factor MYC is disproportionately elevated in triple-negative breast cancer (TNBC) compared to estrogen, progesterone and/or human epidermal growth factor 2 receptor-positive (RP) breast tumors. We and others have shown that MYC alters metabolism during tumorigenesis. However, the role of MYC in TNBC metabolism remains largely unexplored. We hypothesized that pharmacologic inhibition of MYC-driven metabolic pathways may serve as a therapeutic strategy for this clinically challenging subtype of breast cancer. Using an unbiased metabolomics approach, we identified fatty acid oxidation intermediates as dramatically up-regulated in MYC-driven models of TNBC. A lipid metabolism gene signature was identified in patients with TNBC in the TCGA and multiple other clinical datasets, implicating fatty acid oxidation as a deregulated pathway critical for TNBC metabolism. We find that MYC-overexpressing TNBC, including transgenic models and patient-derived xenografts (PDX), display increased bioenergetic reliance upon fatty-acid oxidation (FAO). Pharmacologic inhibition of FAO catastrophically decreases energy metabolism of MYC over-expressing breast cancer, blocks growth of a MYC-driven transgenic TNBC model and MYC-overexpressing patient-derived xenografts. In vivo inhibition of FAO induced proliferation arrest and increased cell death in PDX models of TNBC. Our results demonstrate that inhibition of FAO is a novel therapeutic strategy against TNBCs that over-express MYC. Citation Format: Goga A, Camarda R, Zhou AY, Kohnz RA, Balakrishnan S, Anderton B, Mahieu C, Eyob H, Krings G, Nomura DK. Inhibition of fatty-acid oxidation as a therapy for triple-negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD3-05.

Metabolic Therapies Improve Mitochondrial Morphology and Function

In diseases where glucose utilization is impaired ketone bodies and TCA cycle intermediates serve as alternative fuel. Since these diseases are linked pathophysiologically to impaired mitochondrial function, we hypothesized that nutritional ketosis induced by ketogenic precursors or TCA cycle intermediate supplements promote mitochondrial biogenesis and preservation of existing mitochondria.Exogenous ketone supplementation was used in Sprague Dawley (SD) rats and in SOD1 G93A mice. In SD rats, ketone ester and ketone salt‐MCT oil mixture was gavaged daily, whereas the Deanna protocol (DP) supplementation containing tricarboxylic acid (TCA) cycle intermediates was added to standard rodent chow fed to SOD1 G93A mice. Plasma was collected at endpoint and global metabolomic profiles were compared by Metabolon. Morphology, density and distribution of skeletal muscle mitochondria were studied by TEM and SEM.Exogenous ketone supplementation and the DP supplementation dose‐dependently increased compounds associated with glycolysis and the TCA cycle. Elevations of intermediates of fatty acid oxidation (FAO) and FAO substrates indicated increased beta‐oxidation in response to the DP supplementation. Higher density of mitochondria and mitochondrial cristae could be observed in skeletal muscle in response to supplementation. These processes together likely result in improved ATP production, enhanced motor function and slower progression of diseases associated with metabolic dysfunction.

Increased TCA cycle intermediates in response to diet with Deanna protocol in ALS mouse model (578.3)

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting motor neurons and is characterized by progressive muscle weakness, paralysis, and, eventually, death. There is no cure for ALS. This metabolic profiling study was conducted to determine the metabolic impacts of a multi‐component experimental nutritional regimen known as the Deanna Protocol (DP) to gain insights into possible mechanisms by which this diet is reported to help slow the progression of ALS.In this 30 sample study SOD1 G93A mice were fed for 6 weeks a standard diet (CTRL) or diets representing a low (12%) or high (22%) dose of DP. Comparison of the plasma global biochemical profiles were performed at endpoint by Metabolon that revealed key metabolic differences.The DP diet dose‐dependently increased compounds associated with glycolysis and the tricarboxylic acid (TCA) cycle. Alpha‐ketoglutarate, a key ingredient of DP, pyruvate, lactate succinate, fumarate, malate and phosphatidylcholine (PC) metabolites were elevated in the high DP group relative to the CTRL. Elevations of intermediates of fatty acid oxidation (FAO) and ready FAO substrates indicated a likely increased beta‐oxidation in response to the DP diet.Substantial build‐up of TCA cycle components combined with acetyl‐CoA provided by increased beta‐oxidation, likely led to greater mitochondrial ATP production, which might be the reason of improved motor function and slower progression of disease.Grant Funding Source: Winning the Fight Foundation

13C-labelled palmitate and metabolomics/lipidomics analyses reveal the fate of free fatty acids in fasting mice

Aims: During fasting, free fatty acids (FFA) released from adipose triglyceride stores become the major source of energy. Because lipolysis is in excess over utilization, plasma FFA are considerably elevated despite an increased utilization by skeletal muscle and other peripheral tissues. The plasma concentration of acylcarnitines, intermediates of fatty acid oxidation, also increases during fasting and exercise, but it is unclear if they originate from skeletal muscle or from the liver, the key metabolic regulator and short-term buffer for excess circulating FFA. Using a stable 13C-isotope labelled fatty acid tracer in mice, we tested the hypothesis that excess palmitate is incorporated into acylcarnitines in the muscle and into triglycerides and other lipids in the liver in the fasted state.

Neonatal Screening by Tandem Mass Spectrometry

After completing this article, readers should be able to: 1. List the major categories of diseases detectable by expanded newborn screening. 2. Give examples of the benefits and limitations of screening by tandem mass spectrometry. 3. Describe the appropriate follow-up for an abnormal screening result. The screening of newborns for inherited metabolic disorders is a well-established public health activity, first implemented in the early 1960s for the presymptomatic identification of patients who have phenylketonuria (PKU). Since then, significant technological advances, most importantly the development of tandem mass spectrometry (MS/MS), have enabled the detection of an increasing number of metabolic disorders in newborn blood. This article reviews the basic technology of MS/MS and its application to newborn screening, with the aim of highlighting the benefits and pointing out some limitations of this powerful technology. Early screening for PKU used the bacterial inhibition assay of Guthrie, in which inhibition of bacterial growth on an agar medium, introduced by the addition of a phenylalanine analog, was overcome by the presence of dried blood discs containing high levels of phenylalanine (such as from a PKU patient). This approach later was adapted for the detection of leucine in patients who have maple syrup urine disease (MSUD), methionine in patients who have classic homocystinuria, and tyrosine in patients who have tyrosinemia. Over the years, other techniques have been implemented to screen for additional disorders, including congenital hypothyroidism, biotinidase deficiency, galactosemia, hemoglobinopathies, and congenital adrenal hyperplasia. The classic criteria used to determine whether a disorder is suitable for screening include: 1) the disorder is clinically and biochemically well defined, 2) it is associated with significant morbidity or mortality, 3) an effective treatment is available, and 4) there is a simple and safe screening test. (1) By the early 1990s, all states offered screening for at least PKU and congenital hypothyroidism, …

Accumulation of 3-hydroxy-fatty acids in the culture medium of long-chain L-3-hydroxyacyl CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein-deficient skin fibroblasts: implications for medium chain triglyceride dietary treatment of LCHAD deficiency.

Dietary management of long-chain l-3-hydroxyacyl CoA dehydrogenase (LCHAD) deficiency involves a regimen that contains adequate carbohydrate, protein, and essential lipids, and supplementation with medium-chain fatty acids in the form of medium-chain triglycerides, providing energy from fats that bypasses the long-chain metabolic block. This study analyzes the effects of dietary treatment of LCHAD deficiency in an in vitro model. Cultured skin fibroblasts from LCHAD-deficient and normal individuals were grown in media supplemented with physiologic combinations of medium-chain fatty acids octanoate and decanoate, and the long-chain palmitate. Medium was removed from the cells after various incubation times, and assayed for 3-hydroxy-intermediates of fatty acid oxidation. The 3-hydroxy-fatty acids were measured by stable-isotope dilution gas chromatography/mass spectrometry. We found that the addition of medium-chain fatty acids caused a decrease in the accumulation of long-chain fatty acid oxidation intermediates in LCHAD-deficient cells when the cells were incubated in untreated medium, and also when they were incubated in this medium with palmitate added. Medium with decanoate alone was better at achieving this effect than medium with only octanoate added. A 1:3 ratio of octanoate to decanoate worked best over an extended time period in LCHAD-deficient cells in untreated medium, whereas a 1:1 ratio of octanoate to decanoate worked best in the same cells incubated in medium containing palmitate. In all dietary medium-chain triglyceride preparations, the ratio of octanoate was greater than that of decanoate. Our results suggest that a medium-chain triglyceride preparation that is higher in decanoate may be more effective in reducing the accumulation of potentially toxic long-chain 3-hydroxy-fatty acids in LCHAD deficiency.

Analysis of mitochondrial fatty acid oxidation intermediates by tandem mass spectrometry from intact mitochondria prepared from homogenates of cultured fibroblasts, skeletal muscle cells, and fresh muscle.

Defects of mitochondrial fatty acid beta-oxidation are an important group of inherited metabolic disorders in children. Despite improved screening opportunities, diagnosis of these disorders is not often straightforward and requires enzyme analyses. Because therapy is effective in many of these disorders, rapid diagnosis is essential. We report a technique that allows analysis of fatty acid oxidation not only in cultured cells (fibroblasts, myoblasts, and myotubes) but also in fresh muscle homogenate. Fatty acid oxidation analysis was performed by incubating fresh muscle homogenate or harvested cultured cells with stable isotopically labeled palmitate. The intermediates generated were analyzed by tandem mass spectrometry. Results of patients with seven different beta-oxidation disorders were compared with controls. Acylcarnitine intermediates in patient samples could be easily differentiated from the control samples. The acylcarnitine profile of each beta-oxidation defect was compatible with localization of the enzyme defect. Both in patient and control samples, the same pattern of intermediates could be detected in fibroblasts, muscle cells, and fresh muscle homogenate. The procedure described allowed correct diagnosis of all the beta-oxidation defects studied. Utilization of fresh muscle samples reduces the delay in diagnosis related to tissue culture and is useful in diagnostic of patients with neuromuscular phenotype. Measurement of fatty acid oxidation intermediates from myoblasts or myotubes is an additional tool in investigating pathogenetic mechanisms of myopathy in beta-oxidation defects.

Addition of Quantitative 3-Hydroxy-Octadecanoic Acid to the Stable Isotope Gas Chromatography-Mass Spectrometry Method for Measuring 3-Hydroxy Fatty Acids

Mitochondrial fatty acid oxidation (FAO) is a catabolic pathway that supplies energy for the normal physiologic functioning of many tissues when glucose is unavailable, and it also supplies energy for some tissues even when glucose is available (1)(2). The FAO pathway is complex and not fully understood. Quantitative measurement of the concentrations of 3-hydroxy-fatty acids (3-OHFAs) in plasma or serum samples from individuals who are suspected of having a deficiency in FAO, especially in the enzyme step involving the l-3-hydroxyacyl-CoA-dehydrogenases, is a useful tool to aid in diagnosis (3)(4). This study adds the quantitative measurement of 3-hydroxy-octadecanoic acid (3-OH-C18) to the previously reported assay (4) that measures the six shorter chain-length FAO intermediates, 3-hydroxy-hexanoic acid (3-OH-C6), 3-hydroxy-octanoic acid (3-OH-C8), 3-hydroxy-decanoic acid (3-OH-C10), 3-hydroxy-dodecanoic (3-OH-C12), 3-hydroxy-tetradecanoic acid (3-OH-C14), and 3-hydroxy-hexadecanoic acid (3-OH-C16). 3-OH-C18 was synthesized by the method of Jones et al. (4), with the following changes. The precursor for 3-OH-C18 was not2commercially available; thus the 3-OH-C18 precursor, hexadecanal, was synthesized first by the method of Landini et al. (5). A saturated solution of potassium chromate (0.55 mol/L) in 300 mL/L aqueous sulfuric acid was reacted with 0.01 mol of 1-hexadecanal dissolved in 60 mL of methylene chloride in the presence of 0.001 mol of tetrabutylammonium hydrogen sulfate as a catalyst (ratio of 1-hexadecanol to catalyst, 10:1). The unlabeled and [1,2]-13C2-labeled 3-OH-C18 were then synthesized from the hexadecanal as described previously (4). The methylene chloride, 1-hexadecanol, and tetrabutylammonium hydrogen sulfate were obtained from Aldrich Chemical Co. Analysis of the naturally occurring and stable-isotope 3-OH-C18s after synthesis was also performed as described previously (4). This analysis demonstrated that the naturally occurring 3-OH-C18 was 89% pure with the expected composition, and the stable-isotope 3-OH-C18 was 95% pure with the expected composition. Mass spectra revealed …

Sudden Death of Chicken Embryos with Hereditary Riboflavin Deficiency

Riboflavin-binding protein (RfBP) mediates the deposition of riboflavin during the formation of eggs in birds. Hens of a strain of Single-Comb White Leghom chickens, which are genetically unable to produce RfBP, lay eggs containing insufficient riboflavin to sustain embryogenesis beyond 13 or 14 d of incubation. Embryos in these eggs grow normally until the day of death, and their heart rate is normal to within an hour of death. The effects of riboflavin-deficiency first appear after d 10 of incubation when embryos become severely hypoglycemic and begin to accumulate intermediates of fatty acid oxidation. Although the activities of flavin-dependent enzymes are reduced generally, the 80% reduction in the activity of mediumchain acyl-CoA dehydrogenase further suggests that the major metabolic consequence of riboflavin deficiency is a severe impairment of fatty acid oxidation. The riboflavin-deficient strain provides numerous insights into the metabolism of normal hens and chicken embryos and may be a useful model for sudden death syndromes in humans.