Long-chain Acylcarnitine Levels Research Articles

Abstract P259: Favorable Ventricular-Arterial Mechanics Among Women is Associated With Better Mitochondrial Oxidation in the Left Ventricle

Background: Traditional risk factors insufficiently characterize the biological phenomena underlying sex differences in ventriculoarterial coupling (VAC). We hypothesize that sex differences in VAC are due to differences in left ventricular global longitudinal strain (LVGLS), a fuel-dependent chamber closely linked to mitochondrial fuel oxidation pathways. Methods: Community older adults without cardiovascular disease were prospectively recruited for cardiac magnetic resonance imaging and concurrent serum metabolomics sampling. VAC was determined as aortic pulse wave velocity (PWV) divided by LVGLS. Multivariable models adjusted for clinical risk factors and metabolites. Results: Among 202 participants (46.0% female), fewer women were smokers compared to men (3.2% vs 25.7% p<0.001). Despite similarities in age, (women: 69.7 vs men: 70.7 p=0.432) women had better VAC (-0.39 vs -0.47 p = 0.003), LVGLS (-21.9 vs -20.1% p <0.001), and LV mass index (LVMI) (41.7 vs 49.5 g/m 2 p <0.001) compared to men. Both sexes had similar PWV (8.41 vs 9.13 m/s p = 0.087). LVGLS was inversely related to LVMI (β = -0.09 p <0.001). Among women, metabolomic determinants of better LVGLS, independent of LVMI, were lower levels of long chain acylcarnitines (LCACs) [C12:2-OH/C10:2-DC (OR 0.830 adj. p=0.020), C16:3-OH/C14:3-DC (OR 0.334 adj. p=0.048), C16-OH (OR 0.756 adj. p=0.029) and higher levels of C20:2 (OR 1.987 adj. p=0.033)] (Figure A). Among men, determinants of better LVGLS included lower levels of C12:2-OH/C10:2-DC (OR 0.864 adj. p=0.020), C12:1-OH (OR 0.969 adj. p=0.016), C16:1 (OR 0.923 adj. p=0.025), C16:3-OH/C14:3-DC (OR 0.566 adj. p=0.028)] and higher levels of C5:1 (OR 1.096 adj. p=0.026). Conclusion: Better LV longitudinal function contributed to better VAC among older women. Lower LCAC fatty acid metabolism intermediaries was linked to better LVGLS, implying improved mitochondrial β-oxidation. Circulating metabolites may represent novel precision medicine factors for sex-based stratification of vascular risks.

Circulating metabolic profile in idiopathic pulmonary fibrosis: data from the IPF-PRO Registry

BackgroundThe circulating metabolome, reflecting underlying cellular processes and disease biology, has not been fully characterized in patients with idiopathic pulmonary fibrosis (IPF). We evaluated whether circulating levels of metabolites correlate with the presence of IPF, with the severity of IPF, or with the risk of clinically relevant outcomes among patients with IPF.MethodsWe analyzed enrollment plasma samples from 300 patients with IPF in the IPF-PRO Registry and 100 individuals without known lung disease using a set of targeted metabolomics and clinical analyte modules. Linear regression was used to compare metabolite and clinical analyte levels between patients with IPF and controls and to determine associations between metabolite levels and measures of disease severity in patients with IPF. Unadjusted and adjusted univariable Cox regression models were used to evaluate associations between circulating metabolites and the risk of mortality or disease progression among patients with IPF.ResultsLevels of 64 metabolites and 5 clinical analytes were significantly different between patients with IPF and controls. Among analytes with greatest differences were non-esterified fatty acids, multiple long-chain acylcarnitines, and select ceramides, levels of which were higher among patients with IPF versus controls. Levels of the branched-chain amino acids valine and leucine/isoleucine were inversely correlated with measures of disease severity. After adjusting for clinical factors known to influence outcomes, higher levels of the acylcarnitine C:16-OH/C:14-DC were associated with all-cause mortality, lower levels of the acylcarnitine C16:1-OH/C14:1DC were associated with all-cause mortality, respiratory death, and respiratory death or lung transplant, and higher levels of the sphingomyelin d43:2 were associated with the risk of respiratory death or lung transplantation.ConclusionsIPF has a distinct circulating metabolic profile characterized by increased levels of non-esterified fatty acids, long-chain acylcarnitines, and ceramides, which may suggest a more catabolic environment that enhances lipid mobilization and metabolism. We identified select metabolites that were highly correlated with measures of disease severity or the risk of disease progression and that may be developed further as biomarkers.Trial registrationClinicalTrials.gov; No: NCT01915511; URL: www.clinicaltrials.gov.

Alterations of Plasma Metabolomics Profile in Thalassemia Patients with Low Bone Mineral Density

Background: Osteoporosis is commonly found in thalassemia patients; however, its pathogenesis is not thoroughly understood. Metabolomics is the study of metabolites in biofluids, which help to elucidate the underlying mechanism of several metabolic diseases. Nevertheless, plasma metabolomics profiling has not been carefully investigated in thalassemia patients with low bone mineral density (BMD). Objective: To investigate the alterations of plasma targeted metabolomics in adult thalassemia patients with low BMD. Methods: We conducted a cross-sectional study from February 2022 to January 2023 at Outpatient Hematology Clinic, Chiang Mai University. Thalassemia patients, confirmed by HPLC hemoglobin typing, who aged 19-50 years (male) or more than 19 years (premenopausal female) were eligible. Exclusion criteria were (1) current use or history of any bone modifying agent use within 1 year, (2) severe hepatic or renal impairment, (3) active malignancy or infection. Age- and sex-matched with normal body mass index were also recruited as controls. Two-point BMD, plasma targeted metabolomics by using liquid chromatography coupled with mass spectrometry (LC/MS), and serum bone turnover markers, including bone resorption using CTX, and bone formation using P1NP, were assessed. Interested metabolomes included 19 amino acids, 7 free fatty acids, 20 acylcarnitine intermediates of branched-chain amino acids and fatty acids, 37 phospholipid derivatives, and 4 Krebs' cycle metabolomes. Low BMD was defined as a Z-score of less than -2.0 at any site. Outcomes were the alterations of plasma levels of metabolomes among control, thalassemia with normal BMD, and thalassemia with low BMD. Results: There were a total of 80 thalassemia patients and 40 controls recruited. The mean age of patients was 34.8 years (SD 8.7). Twenty-six patients (32.5%) were male. Forty-seven patients (58.8%) were transfusion-dependent thalassemia. Low BMD was detected in 37 patients (47.5%). We found several plasma metabolomes that were significantly changed between controls and thalassemia and further altered when compared between thalassemia with normal BMD and low BMD (Figure 1). Considering plasma amino acids, glutamate level was significantly increased while glutamine level was decreased. Regarding fatty acid-related metabolomes, arachidonic acid level was increased in thalassemia with low BMD. In addition, several fatty acid-derived medium- and long-chain acylcarnitine levels were higher in plasma of thalassemia with low BMD, particularly C14:0 carnitine. Among plasma phospholipid derivatives, phosphatidylinositol (PI) and lysoPI levels were significantly increased in thalassemia with low BMD. Four plasma Krebs' cycle metabolites were not different among groups. Surprisingly, two serum bone turnover markers (CTX and P1NP) were not sensitive for detecting low bone mineral density in thalassemia (Control vs Thalassemia with normal BMD vs Thalassemia with low BMD: CTX 0.27±0.17 vs 0.36±0.20 vs 0.34±0.19 ng/mL, p=0.079 and P1NP 57.2±25.0 vs 57.2±34.0 vs 53.7±26.4 ng/mL, p=0.824). Conclusion: There were several changes in plasma metabolome levels among thalassemia with low BMD, including altered glutamate-glutamine metabolism, impaired fatty acid oxidation, and increased plasma phosphatidylinositol. Interestingly, these changes were found in patients with normal BMD and further increased in patients with low BMD. Our findings suggest the role of plasma metabolomics as non-invasive biomarkers for osteoporotic condition in thalassemia patients.

Biliverdin Reductase B Protects Against Primaquine Induced Hemolysis - Identification of a Novel Anti-Oxidant Pathway in Murine Erythrocytes

Background: Biliverdin Reductase B (BlvrB) is the fourth most abundant enzyme in red blood cells (RBCs) and is widely expressed in other tissues. In normal heme catabolism, porphyrin is converted to biliverdin-alpha (by hemoxygenase (HO)) and then reduced to bilirubin-alpha (by biliverdin reductase A (BlvrA)). Bilirubin-alpha is then glucuronidated and excreted. The large body of literature investigating heme metabolism, bilirubin-based pathologies, and the oxidative biology of bilirubin/biliverdin in adults focuses almost exclusively on BlvrA and the alpha isomers of biliverdin and bilirubin. However, porphyrin is a tetrapyrrole with 4 distinct meso-carbon bridges, each of which can be broken to generate a distinct biliverdin isomer (alpha, beta, gamma, or delta). Although HO is exquisitely specific for the alpha-bond (generating only biliverdin-alpha), chemical coupled oxidation of heme yields all four biliverdin isomers. BlvrB (and not BlvrA) can reduce each of non-alpha biliverdin isomers into their corresponding bilirubin isomers (bilirubin-beta is prevalent in neonatal jaundice and kernicterus). BlvrB also reduces flavins and additional quinone metabolites. Despite BlvrB's abundance and evolutionary conservation, its only known function is a subtle effect on hematopoietic lineage fate (megakaryocyte vs. erythroid). BlvrB has no known function in mature RBCs. We hypothesized that BlvrB plays an important antioxidant role that would only become obvious when other redundant anti-oxidant pathways were limited. Methods: A novel BlvrB knockout mouse was generated on a C57BL/6 background. A model of primaquine (PQ) induced oxidative hemolytic anemia in G6PD deficient mice was used as a clinically relevant scenario in which compromised antioxidant biology leads to treatment limiting pharmacotoxicity in RBCs. BlvrB-KO mice were crossed with two strains of mice that had murine G6PD replaced with either the A- deficient variant of human G6PD (hG6PD(A-)) or the non-deficient form of human G6PD (hG6PD(ND)). Four mouse strains (i.e., hG6PD(ND) and hG6PD(A-) mice with or without BlvrB deletion) received either a 4-day course of PQ or control PBS injections. Anemia was monitored by hematocrit (HCT). Reticulocyte count (RET) was monitored by flow cytometry as CD71+, thiazole orange+ erythrocytes. Peripheral blood was subjected to high resolution metabolomics to elucidate biochemical changes. Results: All mouse strains were healthy at baseline, with normal measured hematological parameters (i.e., Hct 40, RET 1.5%). No change in HCT or RET was observed in hG6PD(ND) or BlvrB-KOxhG6PD(ND) mice receiving PQ or in any strain receiving PBS. PQ induced hemolytic anemia in hG6PD(A-) mice, with an average HCT of 32.7 (18% drop) with RET of 3.1%. A significant exacerbation of PQ induced hemolysis was observed in BlvrB-KOxhG6PD(A-) mice that had an average HCT of 22.5 (44% drop) with RET of 5.3%. (n= 11 mice per group, p <0.001 between groups). Blood from PQ-treated hG6PD(A-)xBlvrBKO mice showed defective glycolysis at the level of glyceraldehyde 3-phosphate dehydrogenase, increased levels of multiple long-chain acylcarnitines, altered redox metabolism, and specific increases of hypoxanthine and sphingosine-1-phosphate (S1P). Three separate experiments were performed with similar results. Discussion: These data demonstrate that BlvrB deletion exacerbates oxidative injury and suggest that BlvrB provides a novel and hitherto undescribed antioxidant pathway in RBCs. Because approximately half a billion humans exhibit some form of G6PD deficiency, these data are also directly relevant to a large population of humans, which were modeled using hG6PD(A-) mice. These studies also define the metabolic lesion in peripheral blood. For example, GAPDH is well known to be highly sensitive to inactivation by oxidative damage, resulting in impaired glycolysis. Changes in hypoxanthine and S1P are known to be associated with decreased RBC function and survival. Finally, altered carnitine metabolism is associated with repair of lipid peroxidation in RBCs via the Lands Cycle. Although the mechanism by which BlvrB facilitates antioxidation is not yet clear, we hypothesize it involves redox cycling of non-alpha isomers of biliverdin and bilirubin. However, the mechanisms may be through reduction of flavins and/or other endogenous substrates. Ongoing studies are testing these hypotheses.

Fatty acids and inflammatory stimuli induce expression of long-chain acyl-CoA synthetase 1 to promote lipid remodeling in diabetic kidney disease

Fatty acid handling and complex lipid synthesis are altered in the kidney cortex of diabetic patients. We recently showed that inhibition of the renin-angiotensin system without changes in glycemia can reverse diabetic kidney disease (DKD) and restore the lipid metabolic network in the kidney cortex of diabetic (db/db) mice, raising the possibility that lipid remodeling may play a central role in DKD. However, the roles of specific enzymes involved in lipid remodeling in DKD have not been elucidated. In the present study, we used this diabetic mouse model and a proximal tubule epithelial cell line (HK2) to investigate the potential relationship between long-chain acyl-CoA synthetase 1 (ACSL1) and lipid metabolism in response to fatty acid exposure and inflammatory signals. We found ACSL1 expression was significantly increased in the kidney cortex of db/db mice, and exposure to palmitate (PA) or tumor necrosis factor (TNF-α) significantly increased Acsl1 mRNA expression in HK-2 cells. In addition, PA treatment significantly increased the levels of long-chain acylcarnitines and fatty acyl CoAs in HK2 cells, and these increases were abolished in HK2 cell lines with specific deletion of Acsl1(Acsl1KO cells), suggesting a key role for ACSL1 in fatty acid β-oxidation. In contrast, TNF-α treatment significantly increased the levels short-chain acylcarnitines and long-chain fatty acyl CoAs in HK2 cells but not in Acsl1KO cells, consistent with fatty acid channeling to complex lipids. Taken together, our data demonstrate a key role for ACSL1 in regulating lipid metabolism, fatty acid partitioning, and inflammation.

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.

A multi-omics study to investigate the progression of the Correa pathway in gastric mucosa in the context of cirrhosis

BackgroundPatients with liver cirrhosis (LC) are prone to gastric mucosa damage. We investigated the alterations of gastric mucosa in LC patients and their possible mechanisms through multi-omics.ResultsWe observed significant gastric mucosa microbial dysbiosis in LC subjects. Gastric mucosal microbiomes of LC patients contained a higher relative abundance of Streptococcus, Neisseria, Prevotella, Veillonella, and Porphyromonas, as well as a decreased abundance in Helicobacter and Achromobacter, than control subjects. The LC patients had higher levels of bile acids (BAs) and long-chain acylcarnitines (long-chain ACs) in serum. The gastric mucosal microbiomes were associated with serum levels of BAs and long-chain ACs. Transcriptome analyses of gastric mucosa revealed an upregulation of endothelial cell specific molecule 1, serpin family E member 1, mucin 2, caudal type homeobox 2, retinol binding protein 2, and defensin alpha 5 in LC group. Besides, the bile secretion signaling pathway was significantly upregulated in the LC group.ConclusionsThe alterations in the gastric mucosal microbiome and transcriptome of LC patients were identified. The impaired energy metabolism in gastric mucosal cells and bile acids might aggravate the inflammation of gastric mucosa and even exacerbate the Correa’s cascade process. The gastric mucosal cells might reduce bile acid toxicity by bile acid efflux and detoxification.Trial registration: ChiCTR2100051070.

Metabolic profiles reflect weight loss maintenance and the composition of diet after very-low-energy diet

Diet and weight loss affect circulating metabolome. However, metabolite profiles induced by different weight loss maintenance diets and underlying longer term weight loss maintenance remain unknown. Herein, we investigated after-weight-loss metabolic signatures of two isocaloric 24-wk weight maintenance diets differing in satiety value due to dietary fibre, protein and fat contents and identified metabolite features that associated with successful weight loss maintenance. Non-targeted LC-MS metabolomics approach was used to analyse plasma metabolites of 79 women and men (mean age±SD 49.7±9.0 years; BMI 34.2±2.5kg/m2) participating in a weight management study. Participants underwent a 7-week very-low-energy diet (VLED) and were thereafter randomised into two groups for a 24-week weight maintenance phase. Higher satiety food (HSF) group consumed high-fibre, high-protein, and low-fat products, while lower satiety food (LSF) group consumed isocaloric low-fibre products with average protein and fat content as a part of their weight maintenance diets. Plasma metabolites were analysed before the VLED and before and after the weight maintenance phase. Metabolite features discriminating HSF and LSF groups were annotated. We also analysed metabolite features that discriminated participants who maintained ≥10% weight loss (HWM) and participants who maintained <10% weight loss (LWM) at the end of the study, irrespective of the diet. Finally, we assessed robust linear regression between metabolite features and anthropometric and food group variables. We annotated 126 metabolites that discriminated the HSF and LSF groups and HWM and LWM groups (p<0.05). Compared to LSF, the HSF group had lower levels of several amino acids, e.g. glutamine, arginine, and glycine, short-, medium- and long-chain acylcarnitines (CARs), odd- and even-chain lysoglycerophospholipids, and higher levels of fatty amides. Compared to LWM, the HWM group in general showed higher levels of glycerophospholipids with a saturated long-chain and a C20:4 fatty acid tail, and unsaturated free fatty acids (FFAs). Changes in several saturated odd- and even-chain LPCs and LPEs and fatty amides were associated with the intake of many food groups, particularly grain and dairy products. Increase in several (lyso)glycerophospholipids was associated with decrease in body weight and adiposity. Increased short- and medium-chain CARs were related to decreased body fat-free mass. Our results show that isocaloric weight maintenance diets differing in dietary fibre, protein, and fat content affected amino acid and lipid metabolism. Increased abundances of several phospholipid species and FFAs were related with greater weight loss maintenance. Our findings indicate common and distinct metabolites for weight and dietary related variables in the context of weight reduction and weight management. The study was registered in isrctn.org with identifier 67529475.

Abstract 295: Targeting spermine synthase triggers lipid metabolism reprogramming as a new therapeutic option to combat colorectal cancer

Abstract Dysregulation of polyamine metabolism has been linked to the development of colorectal cancer (CRC). Our recent work demonstrates that spermine synthase (SMS), a polyamine biosynthetic enzyme that converts spermidine to spermine, is overexpressed in CRC, which is required for balancing cellular spermidine levels to facilitate CRC tumorigenesis (Nat Commun 11:3243, 2020). Our findings reveal SMS as an attractive therapeutic target in CRC; yet, genetic depletion of SMS expression only shows a limited antitumor effect. Using unbiased metabolomics and transcriptomics analyses, we identified a lipid metabolism reprograming as among the most impacted metabolic change by SMS depletion in CRC cells. Specifically, SMS depletion significantly altered long-chain fatty acid, triacylglycerol and phospholipid metabolism. Furthermore, targeted inhibition of SMS significantly increased the number of lipid droplets and the levels of long-chain fatty acid acylcarnitines for oxidative phosphorylation in mitochondria, and upregulated expression of genes associated with increased mobilization of polyunsaturated fatty acids and the genes associated with lipid peroxidation for induction of ferroptosis, an iron-dependent form of nonapoptotic cell death. The glutathione peroxidase 4 (GPX4) is a key negative regulator of ferroptosis by neutralizing lipid peroxides. Notably, pharmacological inhibition of GPX4 or its upstream regulator system xc − in combination with genetic depletion or pharmacologic inhibition of SMS synergistically caused lipid peroxidation leading to ferroptosis induction and marked suppression of CRC cell growth. Collectively, these findings highlight lipid metabolism reprograming as an adaptive response to targeted inhibition of SMS to enable CRC cell survival, which represents an Achilles’ heel that can be exploited for potential effective therapy for CRC. Citation Format: Murong Ma, Yubin Guo, Qing Ye, Pan Deng, Daheng He, Chi Wang, Qing-Bai She. Targeting spermine synthase triggers lipid metabolism reprogramming as a new therapeutic option to combat colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 295.

Pasture-finishing of bison improves animal metabolic health and potential health-promoting compounds in meat

BackgroundWith rising concerns regarding the effects of red meat on human and environmental health, a growing number of livestock producers are exploring ways to improve production systems. A promising avenue includes agro-ecological practices such as rotational grazing of locally adapted ruminants. Additionally, growing consumer interest in pasture-finished meat (i.e., grass-fed) has raised questions about its nutritional composition. Thus, the goal of this study was to determine the impact of two common finishing systems in North American bison—pasture-finished or pen-finished on concentrates for 146 d—on metabolomic, lipidomic, and fatty acid profiles of striploins (M. longissimus lumborum).ResultsSix hundred and seventy-one (671) out of 1570 profiled compounds (43%) differed between pasture- and pen-finished conditions (n = 20 animals per group) (all, P < 0.05). Relative to pasture-finished animals, the muscle of pen-finished animals displayed elevated glucose metabolites (~ 1.6-fold), triglycerides (~ 2-fold), markers of oxidative stress (~ 1.5-fold), and proteolysis (~ 1.2-fold). In contrast, pasture-finished animals displayed improved mitochondrial (~ 1.3-fold higher levels of various Krebs cycle metabolites) and carnitine metabolism (~ 3-fold higher levels of long-chain acyl carnitines) (all P < 0.05). Pasture-finishing also concentrated higher levels of phenolics (~ 2.3-fold), alpha-tocopherol (~ 5.8-fold), carotene (~ 2.0-fold), and very long-chain fatty acids (~ 1.3-fold) in their meat, while having lower levels of a common advanced lipoxidation (4-hydroxy-nonenal-glutathione; ~ 2-fold) and glycation end-product (N6-carboxymethyllysine; ~ 1.7-fold) (all P < 0.05). In contrast, vitamins B5, B6, and C, gamma/beta-tocopherol, and three phenolics commonly found in alfalfa were ~ 2.5-fold higher in pen-finished animals (all P < 0.05); suggesting some concentrate feeding, or grazing plants rich in those compounds, may be beneficial.ConclusionsPasture-finishing (i.e., grass-fed) broadly improves bison metabolic health and accumulates additional potential health-promoting compounds in their meat compared to concentrate finishing in confinement (i.e., pen-finished). Our data, however, does not indicate that meat from pen-finished bison is therefore unhealthy. The studied bison meat—irrespective of finishing practice—contained favorable omega 6:3 ratios (< 3.2), and amino acid and vitamin profiles. Our study represents one of the deepest meat profiling studies to date (> 1500 unique compounds), having revealed previously unrecognized differences in animal metabolic health and nutritional composition because of finishing mode. Whether observed nutritional differences have an appreciable effect on human health remains to be determined.Graphical abstract

The association between acylcarnitine and amino acids profile and metabolic syndrome and its components in Iranian adults: Data from STEPs 2016.

Evidence, albeit with conflicting results, has suggested that cardiometabolic risk factors, including obesity, type 2 diabetes (T2D), dyslipidemia, and hypertension, are highly associated with changes in metabolic signature, especially plasma amino acids and acylcarnitines levels. Here, we aimed to evaluate the association of circulating levels of amino acids and acylcarnitines with metabolic syndrome (MetS) and its components in Iranian adults. This cross-sectional study was performed on 1192 participants from the large-scale cross-sectional study of Surveillance of Risk Factors of non-communicable diseases (NCDs) in Iran (STEP 2016). The circulating levels of amino acids and acylcarnitines were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in individuals with MetS (n=529) and without MetS (n=663). The higher plasma levels of branched-chain amino acids (Val, Leu), aromatic amino acids (Phe, Tyr), Pro, Ala, Glu, and the ratio of Asp to Asn were significantly associated with MetS, whereas lower circulating levels of Gly, Ser, His, Asn, and citrulline were significantly associated with MetS. As for plasma levels of free carnitine and acylcarnitines, higher levels of short-chain acylcarnitines (C2, C3, C4DC), free carnitine (C0), and long-chain acylcarnitines (C16, C18OH) were significantly associated with MetS. Principal component analysis (PCA) showed that factor 3 (Tyr, Leu, Val, Met, Trp, Phe, Thr) [OR:1.165, 95% CI: 1.121-1.210, P<0.001], factor 7 (C0, C3, C4) [OR:1.257, 95% CI: 1.150-1.374, P<0.001], factor 8 (Gly, Ser) [OR:0.718, 95% CI: 0.651-0.793, P< 0.001], factor 9 (Ala, Pro, C4DC) [OR:1.883, 95% CI: 1.669-2.124, P<0.001], factor 10 (Glu, Asp, C18:2OH) [OR:1.132, 95% CI: 1.032-1.242, P= 0.009], factor 11 (citrulline, ornithine) [OR:0.862, 95% CI: 0.778-0.955, P= 0.004] and 13 (C18OH, C18:1 OH) [OR: 1.242, 95% CI: 1.042-1.480, P= 0.016] were independently correlated with metabolic syndrome. Change in amino acid, and acylcarnitines profiles were seen in patients with MetS. Moreover, the alteration in the circulating levels of amino acids and acylcarnitines is along with an increase in MetS component number. It also seems that amino acid and acylcarnitines profiles can provide valuable information on evaluating and monitoring MetS risk. However, further studies are needed to establish this concept.

Metabolomic exploration of the effects of habituation to livestock trailer and extended transportation in goats.

Goats raised for meat production are often transported long distances. Twelve-month-old male Spanish goats were used to determine the effects of habituation to trailers on plasma metabolomic profiles when transported for extended periods. In a split-plot design, 168 goats were separated into two treatment (TRT; whole plot) groups and maintained on two different paddocks. Concentrate supplement was fed to one group inside two livestock trailers (habituated group, H), while the other group received the same quantity of concentrate, but not inside the trailers (non-habituated, NH). Goats were subjected to a 10-h transportation stress in 4 replicates (n = 21 goats/replicate/TRT) after 4 weeks of habituation period. Blood samples were collected prior to loading, 20min after loading (0h), and at 2, 4, 6, 8, and 10h of transportation (Time; subplot). A targeted quantitative metabolomics approach was employed to analyze the samples. The data were analyzed using R software and MIXED procedures in SAS. Several amino acids (alanine, serine, glycine, histidine, glutamate, trans-hydroxyproline, asparagine, threonine, methylhistidine, ornithine, proline, leucine, tryptophan) were higher (p < 0.05) in the H group compared to the NH group. Six long-chain acylcarnitines were higher (p < 0.05), while free (C0) and short-chain (C3, C5) carnitines were lower (p < 0.05) in the NH goats compared to the H goats. In general, amino acid concentrations decreased and long-chain acylcarnitine (>C10) levels increased with transportation time (p < 0.05). Butyric acid, α-ketoglutaric acid, and α-aminoadipic acid concentrations were lower (p < 0.05) and β-hydroxybutyric acid concentrations were higher in the NH goats compared to the H goats. Plasma glucose, non-esterified fatty acid (NEFA) and urea nitrogen concentrations were significantly influenced by Time (p < 0.01). Plasma NEFA concentrations were significantly lower (p < 0.01) in the H group than the NH group. Habituation to trailers can be beneficial in enhancing stress coping abilities in goats due to higher concentrations of metabolites such as butyrate and certain amino acids that support antioxidant activities and immune function. Plasma long-chain acylcarnitines may be good indicators of stress during long-distance transportation in goats.

Cross-generational effects of maternal exposure to imazalil on anaerobic components and carnitine absorption associated with OCTN2 expression in mice

Imazalil (IMZ) is a fungicide recommended by the Chinese ministry of agriculture. However, recent study was observed high level of IMZ by dietary exposure in pregnant women. To determine the cross-generational effects, C57BL/6 mice were exposed to IMZ at dietary levels of 0, 0.025‰, and 0.25‰ during the gestation and lactation periods. Then, we assessed the changes in growth phenotypes, carnitine levels, and gut microbiota in F0, F1 or F2 generations. The growth phenotypes of dams didn't observe significant difference, but there were significant changes in the offspring. Plasma samples revealed low levels of free carnitine (C0), long-chain acyl-carnitines and total carnitine. In particular, C0 may be regarded as relatively potential, specific markers by maternal IMZ exposure. Caco2 cell culture and animal experiment confirmed IMZ affected carnitine absorption through the organic cation transporter type-2 (OCTN2) protein encoded by solute carrier family 22A member 5 (SLC22A5) gene in colon. Maternal IMZ exposure also had a greater effect on gut microbiota in offspring, especially anaerobic bacteria, which positively correlated with C0 and acyl-carnitines. These results suggested that maternal IMZ exposure affected carnitine absorption through OCTN2 protein, which led to the decline of anaerobic bacteria and unbalanced intestinal homeostasis.

Dietary fat composition shapes bile acid metabolism and severity of liver injury in a pig model of pediatric NAFLD.

The objective of this study was to investigate the effect of dietary fatty acid (FA) composition on bile acid (BA) metabolism in a pig model of NAFLD, by using a multiomics approach combined with histology and serum biochemistry. Thirty 20-day-old Iberian pigs pair-housed in pens were randomly assigned to receive 1 of 3 hypercaloric diets for 10 wk: 1) lard-enriched (LAR; n = 5 pens), 2) olive oil-enriched (OLI; n = 5), and 3) coconut oil-enriched (COC; n = 5). Animals were euthanized on week 10 after blood sampling, and liver, colon, and distal ileum (DI) were collected for histology, metabolomics, and transcriptomics. Data were analyzed by multivariate and univariate statistics. Compared with OLI and LAR, COC increased primary and secondary BAs in liver, plasma, and colon. In addition, both COC and OLI reduced circulating fibroblast growth factor 19, increased hepatic necrosis, composite lesion score, and liver enzymes in serum, and upregulated genes involved in hepatocyte proliferation and DNA repair. The severity of liver disease in COC and OLI pigs was associated with increased levels of phosphatidylcholines, medium-chain triacylglycerides, trimethylamine-N-oxide, and long-chain acylcarnitines in the liver, and the expression of profibrotic markers in DI, but not with changes in the composition or size of BA pool. In conclusion, our results indicate a role of dietary FAs in the regulation of BA metabolism and progression of NAFLD. Interventions that aim to modify the composition of dietary FAs, rather than to regulate BA metabolism or signaling, may be more effective in the treatment of NAFLD.NEW & NOTEWORTHY Bile acid homeostasis and signaling is disrupted in NAFLD and may play a central role in the development of the disease. However, there are no studies addressing the impact of diet on bile acid metabolism in patients with NAFLD. In juvenile Iberian pigs, we show that fatty acid composition in high-fat high-fructose diets affects BA levels in liver, plasma, and colon but these changes were not associated with the severity of the disease.

Longitudinal associations of pre-pregnancy BMI and gestational weight gain with maternal urinary metabolites: an NYU CHES study.

Background/Objectives:Excessive gestational weight gain (GWG) and pre-pregnancy obesity affect a significant portion of the US pregnant population and are linked with negative maternal and child health outcomes. The objective of this study was to explore associations of pre-pregnancy body mass index (pBMI) and GWG with longitudinally measured maternal urinary metabolites throughout pregnancy.Subjects/Methods:Among 652 participants in the New York University Children’s Health and Environment Study, a longitudinal pregnancy cohort, targeted metabolomics were measured in serially collected urine samples throughout pregnancy. Metabolites were measured at median 10 (T1), 21 (T2), and 29 (T3) weeks gestation using the Biocrates AbsoluteIDQ® p180 Urine Extension kit. Acylcarnitine, amino acid, biogenic amine, phosphatidylcholine, lysophosphatidylcholine, sphingolipid, and sugar levels were quantified. Pregnant people 18 years or older, without type 1 or 2 diabetes and with singleton live births and valid pBMI and metabolomics data were included. GWG and pBMI were calculated using weight and height data obtained from electronic health records. Linear mixed effects models with interactions with time were fit to determine the gestational age-specific associations of categorical pBMI and continuous interval-specific GWG with urinary metabolites. All analyses were corrected for false discovery rate.Results:Participants with obesity had lower long-chain acylcarnitine levels throughout pregnancy and lower phosphatidylcholine and glucogenic amino acids and higher phenylethylamine concentrations in T2 and T3 compared with participants with normal/underweight pBMI. GWG was associated with taurine in T2 and T3 and C5 acylcarnitine species, C5:1, C5-DC, and C5-M-DC, in T2.Conclusions:pBMI and GWG were associated with the metabolic environment of pregnant individuals, particularly in relation to mid-pregnancy. These results highlight the importance of both preconception and prenatal maternal health.

A Preliminary Metabolomic Study of Yorkshire Terrier Enteropathy

Perturbations of metabolite profiles in human and canine enteropathies have been reported before. However, data in dogs are scarce and inconsistent. Currently, the metabolite profile in Yorkshire Terrier enteropathy (YTE) and the impact of treatment is unknown. The objective of this study was to investigate the plasma metabolome of 13 Yorkshire Terriers with YTE and compare it to 20 healthy Yorkshire Terriers. Furthermore, we studied the impact of treatment on the metabolome. In this prospective observational study, plasma metabolite profiles were analyzed by flow injection analysis-tandem mass spectrometry (FIA-MS/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a targeted metabolomics kit. Metabolite analysis revealed that YTE is accompanied by changes in lipid and bile acid metabolism. YTE was associated with a significant decrease of long-chain fatty acids (octadecenoic acid, eicosadienoic acid, eicosatrienoic acid) and lower levels of long-chain acylcarnitines (tetradecanoylcarnitine, hexadecanoylcarnitine, hexadecenoylcarnitine, octadecenoylcarnitine) compared with healthy controls. Furthermore, taurodeoxycholic acid, a secondary bile acid, was decreased in plasma from YTE patients. These changes might be breed-specific and might be involved in the pathogenesis of YTE. Interestingly, changes in metabolite levels were not recovered after treatment and differed considerably from healthy controls.

POS-331 L-carnitine ameliorates diabetic kidney disease by alleviating mitochondrial abnormality and fatty acid accumulation in SDT-fatty rats

Abnormal fatty acid metabolism is associated with the progression of diabetic kidney disease (DKD). Carnitine plays a central role in fatty acid β-oxidation and energy production by transporting long-chain fatty acids from the cytoplasm to the mitochondria. Recently, we found that significant ectopic fat was accumulated in the kidney tissue in DKD patients compared with those in minimal change kidney disease patients independent of proteinuria levels, suggesting the abnormal carnitine dynamics in DKD. Juvenile visceral steatosis (jvs) mice, described as an animal model for systemic carnitine deficiency caused by a mutation in slc22a5 gene, introduce a marked ectopic fat accumulation in the kidney. These findings suggest that abnormal carnitine dynamics in DKD cause ectopic fat accumulation due to abnormality of the fatty acid metabolism. However, the association between abnormalities of carnitine metabolism and the progression of DKD is unknown. Therefore, we investigated the kinetics of carnitine and the effect of L-carnitine therapy in type 2 DKD model rats. Spontaneously Diabetic Torii (SDT) fatty rat, an obese type 2 diabetic model was used. Unilateral nephrectomy was performed and 0.3% salt water was administered at 6 weeks of age, and created a renal dysfunction model in SDT fatty rat (DKD model). Age-matched male Sprague-Dawley (SD) rat was used as a control. Experiment 1: We compared 3 groups; ①SD rats (n=8) ②SDT fatty rats (n=8) ③DKD rats (n=8). Experiment 2: We compared 3 groups; ①SD rats with sham operation (n=8) ②DKD rats (n=7) ③DKD rats treated with L-carnitine administration (0.75% L-carnitine diet) for 10 weeks (n=6). All rats were sacrificed at 17 weeks old. Carnitine was measured by the enzyme cycling method. Acylcarnitine was analyzed by the tandem-mass spectrometry. Plasma and renal free carnitine levels were significantly decreased and long-chain acylcarnitine levels were significantly increased in DKD rats compared with those in SD and SDT fatty rats. The renal expression levels of organic cation / carnitine transporter 2 (OCTN2), carnitine palmitoyl transferase 1a (CPT1a), CPT2 and carnitine acetyltransferase (CrAT) were significantly decreased in DKD rats compared with those in SD and SDT fatty rats. Similarly, acetyl-CoA carboxylase (ACC) activity was significantly increased and peroxisome proliferator-activated receptor γ co-activator 1α (PGC1α) and peroxisome proliferator-activated receptor α (PPARα) activity were decreased in DKD rats. Further, marked ectopic fat accumulation in the renal tubules and glomeruli in DKD model rats was observed. L-carnitine administration significantly improved renal function, urinary albumin excretion, interstitial fibrosis and renal ectopic fat accumulation in DKD model rats. L-carnitine administration ameliorated the decrease of renal OCTN2, CPT1a, CPT2 and CrAT protein expression, and significantly improved ACC, PGC1α, and PPARα activity in DKD model rats. Furthermore, L-carnitine administration mitigated lipid peroxidation and mitochondrial dysfunction in DKD rats Abnormal carnitine kinetics and dynamics might play an important role in the progression of DKD. L-carnitine administration might be a promising therapeutic strategy for improving renal function by alleviating mitochondrial dysfunction in DKD rat model.

Circulating long chain acylcarnitines and outcomes in diabetic heart failure: an HF-ACTION clinical trial substudy

BackgroundWhether differences in circulating long chain acylcarnitines (LCAC) are seen in heart failure (HF) patients with and without diabetes mellitus (DM), and whether these biomarkers report on exercise capacity and clinical outcomes, remains unknown. The objective of the current study was to use metabolomic profiling to identify biomarkers that report on exercise capacity, clinical outcomes, and differential response to exercise in HF patients with and without DM.MethodsTargeted mass spectrometry was used to quantify metabolites in plasma from participants in the heart failure: a controlled trial investigating outcomes of exercise training (HF-ACTION) trial. Principal components analysis was used to identify 12 uncorrelated factors. The association between metabolite factors, diabetes status, exercise capacity, and time to the primary clinical outcome of all-cause mortality or all-cause hospitalization was assessed.ResultsA total of 664 participants were included: 359 (54%) with DM. LCAC factor levels were associated with baseline exercise capacity as measured by peak oxygen consumption (beta 0.86, p = 2 × 10−7, and were differentially associated in participants with and without DM (beta 1.58, p = 8 × 10−8 vs. 0.67, p = 9 × 10−4, respectively; p value for interaction = 0.012). LCAC levels changed to a lesser extent in participants with DM after exercise (mean ∆ 0.09, p = 0.24) than in those without DM (mean ∆ 0.16, p = 0.08). In univariate and multivariate modeling, LCAC factor levels were associated with time to the primary outcome (multivariate HR 0.80, p = 2.74 × 10−8), and were more strongly linked to outcomes in diabetic participants (HR 0.64, p = 3.21 × 10−9 v. HR 0.90, p = 0.104, p value for interaction = 0.001). When analysis was performed at the level of individual metabolites, C16, C16:1, C18, and C18:1 had the greatest associations with both exercise capacity and outcomes, with higher levels associated with worse outcomes. Similar associations with time to the primary clinical outcome were not found in a control group of patients without HF from the CATHeterization GENetics (CATHGEN) study.ConclusionsLCAC biomarkers are associated with exercise status and clinical outcomes differentially in HF patients with and without DM. Impaired fatty acid substrate utilization and mitochondrial dysfunction both at the level of the skeletal muscle and the myocardium may explain the decreased exercise capacity, attenuated response to exercise training, and poor clinical outcomes seen in patients with HF and DM.Trial Registration clinicaltrials.gov Identifier: NCT00047437.

Inhibition of Fatty Acid Metabolism Increases EPA and DHA Levels and Protects against Myocardial Ischaemia-Reperfusion Injury in Zucker Rats.

Long-chain ω-3 polyunsaturated fatty acids (PUFAs) are known to induce cardiometabolic benefits, but the metabolic pathways of their biosynthesis ensuring sufficient bioavailability require further investigation. Here, we show that a pharmacological decrease in overall fatty acid utilization promotes an increase in the levels of PUFAs and attenuates cardiometabolic disturbances in a Zucker rat metabolic syndrome model. Metabolome analysis showed that inhibition of fatty acid utilization by methyl-GBB increased the concentration of PUFAs but not the total fatty acid levels in plasma. Insulin sensitivity was improved, and the plasma insulin concentration was decreased. Overall, pharmacological modulation of fatty acid handling preserved cardiac glucose and pyruvate oxidation, protected mitochondrial functionality by decreasing long-chain acylcarnitine levels, and decreased myocardial infarct size twofold. Our work shows that partial pharmacological inhibition of fatty acid oxidation is a novel approach to selectively increase the levels of PUFAs and modulate lipid handling to prevent cardiometabolic disturbances.

Long-Chain Acylcarnitines Decrease the Phosphorylation of the Insulin Receptor at Tyr1151 Through a PTP1B-Dependent Mechanism.

The accumulation of lipid intermediates may interfere with energy metabolism pathways and regulate cellular energy supplies. As increased levels of long-chain acylcarnitines have been linked to insulin resistance, we investigated the effects of long-chain acylcarnitines on key components of the insulin signalling pathway. We discovered that palmitoylcarnitine induces dephosphorylation of the insulin receptor (InsR) through increased activity of protein tyrosine phosphatase 1B (PTP1B). Palmitoylcarnitine suppresses protein kinase B (Akt) phosphorylation at Ser473, and this effect is not alleviated by the inhibition of PTP1B by the insulin sensitizer bis-(maltolato)-oxovanadium (IV). This result indicates that palmitoylcarnitine affects Akt activity independently of the InsR phosphorylation level. Inhibition of protein kinase C and protein phosphatase 2A does not affect the palmitoylcarnitine-mediated inhibition of Akt Ser473 phosphorylation. Additionally, palmitoylcarnitine markedly stimulates insulin release by suppressing Akt Ser473 phosphorylation in insulin-secreting RIN5F cells. In conclusion, long-chain acylcarnitines activate PTP1B and decrease InsR Tyr1151 phosphorylation and Akt Ser473 phosphorylation, thus limiting the cellular response to insulin stimulation.