Short-chain Acylcarnitines Research Articles

Cerebrospinal fluid ctDNA and metabolites are informative biomarkers for the evaluation of CNS germ cell tumors

Serum and cerebrospinal fluid (CSF) levels of α-fetoprotein and β-subunit of human chorionic gonadotropin are used as biomarkers for the management of central nervous system (CNS) germ cell tumors (GCTs). However, additional discriminating biomarkers are required. Especially, biomarkers to differentiate non-germinomatous germ cell tumors (NGGCTs) from germinomas are critical, as these have a distinct prognosis. We investigated CSF samples from 12 patients with CNS-GCT patients (8 germinomas and 4 NGGCTs). We analyzed circulating tumor DNA (ctDNA) in CSF to detect mutated genes. We also used liquid chromatography-mass spectrometry to characterize metabolites in CSF. We detected KIT and/or NRAS mutation, known as frequently mutated genes in GCTs, in 3/12 (25%) patients. We also found significant differences in the abundance of 15 metabolites between control and GCT, with unsupervised hierarchical clustering analysis. Metabolites related to the TCA cycle were increased in GCTs. Urea, ornithine, and short-chain acylcarnitines were decreased in GCTs. Moreover, we also detected several metabolites (e.g., betaine, guanidine acetic acid, and 2-aminoheptanoic acid) that displayed significant differences in abundance in patients with germinomas and NGGCTs. Our results suggest that ctDNA and metabolites in CSF can serve as novel biomarkers for CNS-GCTs and can be useful to differentiate germinomas from NGGCTs.

Perinatal free carnitine and short chain acylcarnitine blood concentrations in 12,000 full-term breastfed newborns in relation to their birth weight

Free carnitine (C0) and short chain acylcarnitine (SCA) blood concentrations play a significant role in fatty acid oxidation process during the first days of life. The aim of this study was to demonstrate C0 and SCA concentrations in Dried Blood Spots (DBS) of full term breastfed infants in relation to their birth weight (BW) perinatally. Breastfed full term infants (n=12,000, 6000 males, 6000 females) with BW 2000-4000g were divided into 4 equal groups: Group A, 2000-2500g, B 2500-3000g, C 3000-3500g and D 3500-4000g. Blood samples in the form of DBS were collected on the 3rd day of life and analyzed via a liquid chromatography tandem mass spectrometry (LC-MS/MS) protocol. BW-related C0 and SCAs were found as follows: C0 was determined to be statistically significantly higher in group A (BW 2000-2500g) in both males and females. Lower acetylcarnitine (C2) and hydroxybutyrylcarnitine (C4OH) blood concentrations were detected in group A of both sexes, whereas butyrylcarnitine (C4) concentrations were found to be lower in the same group of males only. Furthermore, high concentrations of C2 and C4OH were shown in group D (BW 3500-4000g) in both sexes. SCA sum of means±SD values in males and females of group A were statistically significantly lower as compared to other study groups. Due to the number of the samples, data from this study could be applied as neonatal screening reference values for full term breastfed newborns in relation to their birth weight.

Intramuscular short-chain acylcarnitines in elderly people are decreased in (pre-)frail females, but not in males.

This study tested the hypothesis that in human aging, a decreased intramuscular acylcarnitine status is associated with (pre-)frailty, reduced physical performance, and altered mitochondrial function. We used a cross-sectional study design with well-matched fit and (pre-)frail old males and females, using young males and females as healthy controls. Frailty was assessed according to the Fried criteria and physical performance was determined by 400m walk test, short physical performance battery and handgrip strength. Muscle and plasma acylcarnitine status, and muscle mitochondrial gene expression was analyzed. Results showed that intramuscular total carnitine levels and short-chain acylcarnitine levels were lower in (pre-)frail old females compared to fit old females and young females, whereas no differences were observed in males. The low intramuscular short-chain acylcarnitine levels in females correlated with low physical performance, even after correction for muscle mass (%), and were accompanied with lowered expression of genes involved in mitochondrial energy production and functionality. It is, therefore, concluded that in (pre-)frail old females, intramuscular total carnitine levels and short-chain acylcarnitine levels are decreased, and this decrease is associated with reduced physical performance and low expression of a wide range of genes critical for mitochondrial function. The results stress the importance of taking sex differences into account in aging research.

Preliminary evidence of effects of potassium chloride on a metabolomic path to diabetes and cardiovascular disease.

Low potassium intake can affect cardiovascular disease (CVD) risk and cardiometabolic risk factors. We hypothesize that potassium chloride (KCl) supplementation can improve cardiovascular risk metabolomic profile. In this secondary analysis of a pilot randomized clinical trial (RCT) of 26 participants with prediabetes randomized to KCl or placebo, we performed targeted mass-spectrometry-based metabolomic profiling on baseline and 12-week (end-of-study) plasma samples. Principal component analysis (PCA) was used to reduce the many correlated metabolites into fewer, independent factors that retain most of the information in the original data. Those taking KCl had significant reductions (corresponding to lower cardiovascular risk) in the branched-chain amino acids (BCAA) factor (P = 0.004) and in valine levels (P = 0.02); and non-significant reductions in short-chain acylcarnitines (SCA) factor (P = 0.11). KCl supplementation may improve circulating BCAA levels, which may reflect improvements in overall cardiometabolic risk profile. Clinicaltrials.gov identifier: NCT02236598; https://clinicaltrials.gov/ct2/show/NCT02236598.

Relationship between fermented dairy consumption, circulating short-chain acylcarnitines and angiographic severity of coronary artery disease

Background and aimsCurrent epidemiologic data suggest beneficial cardiovascular effects of fermented dairy products (FDP). However, the relationship between FDP consumption and angiographic coronary status has not been previously studied. Furthermore, the role of novel metabolomic biomarkers of cardiovascular risk in this context is unclear. We hypothesize that short-chain acylcarnitines (SCA) reflect the link between FDP intake and angiographic extent of stable coronary artery disease (CAD). Methods and resultsWe recruited 1185 patients admitted for suspected CAD [median age 62 years (interquartile range: 54–69); 714 men (60.3%)]. Prior to coronary angiography, each patient completed a validated Food Frequency Questionnaire. In addition, venous blood was collected from each patient for whole blood metabolomic analysis, using targeted mass-spectrometry. CAD was defined by the presence of ≥1 coronary stenosis ≥50%. Patients with CAD (n = 441) reported lower median FDP intake [86.8 g/day (IQR: 53.4–127.6)] than patients without CAD [n = 744; 103.9 g/day (IQR: 62.9–152.7); p < 0.001]. Upon adjustment for relevant confounders, increased circulating SCA, particularly level of acetylcarnitine (C2) associated with both higher CAD probability [SCA:β(SE) = 0.584 (0.235), p = 0.013; C2:β(SE) = 0.575 (0.242), p = 0.017] and decreased FDP consumption [SCA:β/100 g FDP-increment/day (SE) = −0.785 (0.242), p = 0.001; C2:β(SE) = −0.560 (0.230), p = 0.015]. By mediation analysis, neither SCA nor C2 showed relevant mediator effect linking FDP consumption to the risk of CAD. ConclusionIncreased consumption of fermented milk was associated with lower prevalence of CAD and correlated inversely with circulating SCA, in particular with acetylcarnitine. No substantial mediator effect of SCA linking fermented milk intake with risk of CAD was found. Clinical trial registryNCT00497887.

531-P: Metabolomics Identifies Novel Plasma Metabolomic Signatures Associated with Diabetic Neuropathy in a Cohort with Screen-Tested Type 2 Diabetes: ADDITION-Denmark

Peripheral neuropathy (PN) is a common complication of diabetes that develops in up to 50% of people with diabetes. Obesity and the metabolic syndrome are risk factors for PN in patients with type 2 diabetes (T2D). However, the lipid metabolites associated with PN development are unknown. We compared the metabolomic profile of plasma from T2D patients with and without PN compared to lean control patients to identify metabolic changes that underlie PN associated with T2D. PN was evaluated in a cohort of T2D patients from the Danish portion of the Anglo-Danish-Dutch study of Intensive Treatment of Diabetes in Primary Care (ADDITION). The Toronto criteria was used to assess PN based on symptoms of neuropathy and abnormal nerve conduction studies. To identify metabolomic signatures that associate with PN, plasma samples from 9 lean control, 49 T2D, and 48 T2D patients with PN were submitted to Metabolon® for a global metabolomics analysis. We used least absolute shrinkage and selection operator (LASSO) and Welch’s two-sample t-test to identify metabolites that differed between PN and non-PN subjects. Metabolomics identified plasma lipid metabolites that were dysregulated in T2D including cholesterol, sphingolipids, and acylcarnitines. Cholesterol and three classes of sphingolipid metabolites (dihydrosphingomyelin, glucosylceramide, and sphingomyelin) were significantly lower in plasma from T2D patients irrespective of PN compared to lean controls. Interestingly, long-chain and very long-chain acylcarnitines were also reduced in T2D patients while short-chain acylcarnitines were unchanged. Only a small number of metabolites were uniquely regulated by PN; of these, two very long-chain acylcarnitine species (C26:1 and C24) were significantly diminished in T2D patients with PN compared to those without. These results suggest an association with lipid markers of impaired mitochondrial β-oxidation and PN in T2D. Disclosure A.E. Rumora: None. F. Alakwaa: None. S.T. Andersen: None. M.E. Jørgensen: Research Support; Self; Amgen, AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Sanofi-Aventis. Stock/Shareholder; Self; Novo Nordisk A/S. M. Charles: Other Relationship; Self; Novo Nordisk A/S. B.C. Callaghan: None. T.S. Jensen: None. E.L. Feldman: Consultant; Self; Novartis Pharmaceuticals Corporation. Funding American Diabetes Association (7-12-BS-045 to E.L.F.); Novo Nordisk Foundation (NNF14OC0011633); National Institutes of Health (1R24082841, 1DP3DK094292); National Institute of Diabetes and Digestive and Kidney Diseases (T32DK101357, F32DK112642, K99DK119366); NeuroNetwork for Emerging Therapies; A. Alfred Taubman Medical Research Institute

Metabolic profile in Mexican young women breast cancer (YWBC) to predict pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC).

e12652 Background: YWBC is associated with aggressive biology with a higher mortality rate and recurrence risk. Similar to older patients, pCR after NAC is a surrogate marker of disease-free survival and overall survival in YWBC. However, YWBC who did not achieve pCR did significantly worse. Metabolic profile (MP) has been used for biomarker discovery to monitor chemotherapy response. Few studies focus on using metabolomics to predict pCR in BC. The study aimed to identify MP in YWBC, before NAC to predict response to chemotherapy. Methods: A targeted metabolomics approach was employed to determine the concentration of 11 AAs, free carnitine, and 30 ACCs using electrospray tandem mass spectrometry in plasma of 30 YWBC patients, before NAC to select plasma biomarkers with the most predictive power to identify pCR in YWBC. Patients were classified in responders (R) and non-responders (NR), based on pCR or not-pCR reported by the pathologist department. The differences between R and NR groups were evaluated using an analysis of Random Forest decision trees according to the distribution of the variables. To evaluate the diagnostic power of each biomarker alone, we performed univariate receiver operating characteristics (ROC) analysis on each biomarker to obtain its ROC curve, ROC area under the curve (AUC), and standard error (SE) of the AUC. Results: We observed differences in MP between R and NR YWBC. Ten metabolites contributed most to differentiating between both groups. We developed a model that included two short-chain acyl-carnitines (AC4 and AC5:1) that allowed an AUC of 0.851 (0.767-0.921) and AUC of 0.737 (0.806-0.844) respectively, and ornithine with AUC of 0.723 (0.6-0.626). Conclusions: There are different MP among Mexican YWBC patients classified as R and NR to NAC. To our understanding, this is one of the first studies that evaluate MP in YWBC in Mexico. Further studies are needed to validate a metabolic signature to predict pCR in this patient group.

Circulating plasma metabolites and risk of rheumatoid arthritis in the Nurses' Health Study.

RA develops slowly over years. We tested for metabolic changes prior to RA onset using a large non-targeted metabolomics platform to identify novel pathways and advance understanding of RA development. Two hundred and fifty-four incident RA cases with plasma samples drawn pre-RA onset in the Nurses' Health Study (NHS) cohorts were matched 1:2 to 501 controls on age, race, menopause/post-menopausal hormone use and blood collection features. Relative abundances of 360 unique, known metabolites were measured. Conditional logistic regression analyses assessed associations between metabolites and incidence of RA, adjusted for age, smoking and BMI, accounting for multiple comparisons. Subgroup analyses investigated seropositive (sero+) RA and RA within 5 years of sample collection. Significant metabolites were then tested in a female military pre-RA case-control study (n = 290). In the NHS, metabolites associated with RA and sero+RA in multivariable models included 4-acetamidobutanoate (odds ratio (OR) = 0.80/S.d., 95% CI: 0.66, 0.95), N-acetylputrescine (OR = 0.82, 95% CI: 0.69, 0.96), C5 carnitine (OR = 0.84, 95% CI: 0.71, 0.99) and C5:1 carnitine (OR = 0.81, 95% CI: 0.68, 0.95). These were involved primarily in polyamine and leucine, isoleucine and valine metabolism. Several metabolites associated with sero+RA within 5 years of diagnosis were replicated in the independent military cohort: C5 carnitine (OR = 0.55, 95% CI: 0.33, 0.92), C5:1 carnitine (OR = 0.62, 95% CI: 0.39, 0.99) and C3 carnitine (OR = 0.57, 95% CI: 0.36, 0.91). Several metabolites were inversely associated with incidence of RA among women. Three short-chain acylcarnitines replicated in a smaller dataset and may reflect inflammation in the 5-year period prior to sero+RA diagnosis.

Treatment With Lipopolysaccharide Induces Distinct Changes in Metabolite Profile and Body Weight in 129Sv and Bl6 Mouse Strains.

Mouse strains differ significantly in their behaviors and responses to pathogenic and pharmacological agents. This study seeks to characterize behavioral and metabolomic profiles of two widely used mouse lines, 129S6/SvEvTac (129Sv) and C57BL/6NTac (Bl6), to acute administration of lipopolysaccharide (LPS). LPS caused a significant suppression of locomotor activity and a decline in body weight (BW) in both strains within 24 h. However, the BW loss was more pronounced in Bl6 than in 129Sv. Comparison of strains revealed clear differences between their metabolomic profiles. According to the general linear model analysis (GLM), the 1.5 h LPS challenge in Bl6 caused a decrease of propionylcarnitine (C3), glucogenic amino acids, and acetylornithine (Ac-Orn), whereas the response of 129Sv included decreased concentrations of short-chain acylcarnitines (SCACs), citrulline, and elevation of glycerophospholipid (PCaa C42:0) and sphingolipid [SM(OH)C16:1]. 24 h after LPS administration, robust alterations in lipid profile were observed in both strains. LPS treatment caused elevation of sphingolipids, phosphatidylcholine diacyls (PCaa) as well as a decrease in lysophosphatidylcholines (LysoPC). However, the number of elevated PCaa and sphingolipids was considerably higher in 129Sv. In addition to lipids, 24 h LPS challenge in Bl6 mice induced increased levels of kynurenine (KYN), putrescine and decreased levels of citrulline, hexoses, Ac-Orn, and PC acyl-alkyl (PCae 38:2) as well as severe BW loss. In contrast, the 24 h LPS challenge in 129Sv mice induced increased levels of KYN, long-chain acylcarnitines (LCACs) and decreased levels of citrulline as well as moderate BW loss. Altogether, our study revealed both similarities and differences in response to LPS in Bl6 and 129Sv strains. For major differences, Bl6 mice showed stronger reduction of BW 24 h after LPS treatment, accompanied by significantly reduced levels of hexoses, the ratio between LysoPC16:1/LysoPC16:0, and elevated levels of neuroprotective putrescine. In 129Sv mice, the BW loss was milder, accompanied by increased levels of hydroxylated LCACs, probably reflecting shifts in oxidative metabolism of fatty acids. One may suggest that LPS caused stronger hypometabolic state in the Bl6 mice than in the 129Sv strain. Altogether, this study confirms that Bl6 and 129Sv mice display vastly distinct adaptation capacities independent from the nature of stressful challenge.

Functional changes of the liver in the absence of growth hormone (GH) action – Proteomic and metabolomic insights from a GH receptor deficient pig model

ObjectiveThe liver is a central target organ of growth hormone (GH), which stimulates the synthesis of insulin-like growth factor 1 (IGF1) and affects multiple biochemical pathways. A systematic multi-omics analysis of GH effects in the liver has not been performed. GH receptor (GHR) deficiency is a unique model for studying the consequences of lacking GH action. In this study, we used molecular profiling techniques to capture a broad spectrum of these effects in the liver of a clinically relevant large animal model for Laron syndrome. MethodsWe performed holistic proteome and targeted metabolome analyses of liver samples from 6-month-old GHR-deficient (GHR-KO) pigs and GHR-expressing controls (four males, four females per group). ResultsGHR deficiency resulted in an increased abundance of enzymes involved in amino acid degradation, in the urea cycle, and in the tricarboxylic acid cycle. A decreased ratio of long-chain acylcarnitines to free carnitine suggested reduced activity of carnitine palmitoyltransferase 1A and thus reduced mitochondrial import of fatty acids for beta-oxidation. Increased levels of short-chain acylcarnitines in the liver and in the circulation of GHR-KO pigs may result from impaired beta-oxidation of short-chain fatty acids or from increased degradation of specific amino acids. The concentration of mono-unsaturated glycerophosphocholines was significantly increased in the liver of GHR-KO pigs without morphological signs of steatosis, although the abundances of several proteins functionally linked to non-alcoholic fatty liver disease (fetuin B, retinol binding protein 4, several mitochondrial proteins) were increased. Moreover, GHR-deficient liver samples revealed distinct changes in the methionine and glutathione metabolic pathways, in particular, a significantly increased level of glycine N-methyltransferase and increased levels of total and free glutathione. Several proteins revealed a sex-related abundance difference in the control group but not in the GHR-KO group. ConclusionsOur integrated proteomics/targeted metabolomics study of GHR-deficient and control liver samples from a clinically relevant large animal model identified a spectrum of biological pathways that are significantly altered in the absence of GH action. Moreover, new insights into the role of GH in the sex-related specification of liver functions were provided.

Longitudinal metabolomics in dried bloodspots yields profiles informing newborn screening for succinic semialdehyde dehydrogenase deficiency.

Analyses of 19 amino acids, 38 acylcarnitines, and 3 creatine analogues (https://clir.mayo.edu) were implemented to test the hypothesis that succinic semialdehyde dehydrogenase deficiency (SSADHD) could be identified in dried bloodspots (DBS) using currently available newborn screening methodology. The study population included 17 post‐newborn SSADHD DBS (age range 0.8‐38 years; median, 8.2 years; 10 M; controls, 129‐353 age‐matched individuals, mixed gender) and 10 newborn SSADHD DBS (including first and second screens from 3 of 7 patients). Low (informative) markers in post‐newborn DBS included C2‐ and C4‐OH carnitines, ornithine, histidine and creatine, with no gender differences. For newborn DBS, informative markers included C2‐, C3‐, C4‐ and C4‐OH carnitines, creatine and ornithine. Of these, only creatine demonstrated a significant change with age, revealing an approximate 4‐fold decrease. We conclude that quantitation of short‐chain acylcarnitines, creatine, and ornithine provides a newborn DBS profile with potential as a first tier screening tool for early detection of SSADHD. This first tier evaluation can be readily verified using a previously described second tier liquid chromatography‐tandem mass spectrometry method for γ‐hydroxybutyric acid in the same DBS. More extensive evaluation of this first/second tier screening approach is needed in a larger population.

Mass spectrometry imaging-based metabolomics to visualize the spatially resolved reprogramming of carnitine metabolism in breast cancer.

New insights into tumor-associated metabolic reprogramming have provided novel vulnerabilities that can be targeted for cancer therapy. Here, we propose a mass spectrometry imaging (MSI)-based metabolomic strategy to visualize the spatially resolved reprogramming of carnitine metabolism in heterogeneous breast cancer.Methods: A wide carnitine coverage MSI method was developed to investigate the spatial alternations of carnitines in cancer tissues of xenograft mouse models and human samples. Spatial expression of key metabolic enzymes that are closely associated with the altered carnitines was examined in adjacent cancer tissue sections.Results: A total of 17 carnitines, including L-carnitine, 6 short-chain acylcarnitines, 3 middle-chain acylcarnitines, and 7 long-chain acylcarnitines were imaged. L-carnitine and short-chain acylcarnitines are significantly reprogrammed in breast cancer. A classification model based on the carnitine profiles of 170 cancer samples and 128 normal samples enables an accurate identification of breast cancer. CPT 1A, CPT 2, and CRAT, which are extensively involved in carnitine system-mediated fatty acid β-oxidation pathway were also found to be abnormally expressed in breast cancer. Remarkably, the expressions of CPT 2 and CRAT were found for the first time to be altered in breast cancer.Conclusion: These data not only expand our understanding of the complex tumor metabolic reprogramming, but also provide the first evidence that carnitine metabolism is reprogrammed at both the metabolite and enzyme levels in breast cancer.

Acylcarnitine metabolomic profiles inform clinically-defined major depressive phenotypes

BackgroundAcylcarnitines have important functions in mitochondrial energetics and β-oxidation, and have been implicated to play a significant role in metabolic functions of the brain. This retrospective study examined whether plasma acylcarnitine profiles can help biochemically distinguish the three phenotypic subtypes of major depressive disorder (MDD): core depression (CD+), anxious depression (ANX+), and neurovegetative symptoms of melancholia (NVSM+). MethodsDepressed outpatients (n = 240) from the Mayo Clinic Pharmacogenomics Research Network were treated with citalopram or escitalopram for eight weeks. Plasma samples collected at baseline and after eight weeks of treatment with citalopram or escitalopram were profiled for short-, medium- and long-chain acylcarnitine levels using AbsoluteIDQ®p180-Kit and LC-MS. Linear mixed effects models were used to examine whether acylcarnitine levels discriminated the clinical phenotypes at baseline or eight weeks post-treatment, and whether temporal changes in acylcarnitine profiles differed between groups. ResultsCompared to ANX+, CD+ and NVSM+ had significantly lower concentrations of short- and long-chain acylcarnitines at both baseline and week 8. In NVSM+, the medium- and long-chain acylcarnitines were also significantly lower in NVSM+ compared to ANX+. Short-chain acylcarnitine levels increased significantly from baseline to week 8 in CD+ and ANX+, whereas medium- and long-chain acylcarnitines significantly decreased in CD+ and NVSM+. ConclusionsIn depressed patients treated with SSRIs, β-oxidation and mitochondrial energetics as evaluated by levels and changes in acylcarnitines may provide the biochemical basis of the clinical heterogeneity of MDD, especially when combined with clinical characteristics.

NOVEL METABOLOMICS MARKERS PREDICT 18-MONTH DECLINE IN HAND GRIP STRENGTH IN COMMUNITY-DWELLING OLDER ADULTS

Sarcopenia that accompanies aging necessitates early detection tools, ideally before the presentation of clinically evident symptoms. The acylcarnitines (ACs) are a class of metabolites generated by cellular fuel metabolism and their predictive utility in declining muscle strength in community-dwelling older adults is unknown. We aim to examine whether baseline acylcarnitines levels can predict changes in hand grip strength over 18 months in 121 community-dwelling older adults. We measured ACs by targeted plasma metabolomics profiling. We then performed a biologically-relevant classification of these markers. Hand grip strength was measured using a Smedley spring-type dynamometer. Multivariate linear regressions were performed to examine if: 1) there was an association between ACs and hand grip strength at baseline and 2) baseline ACs could significantly predict changes in hand grip strength over an 18-month period. At baseline, AC levels were not significantly associated with hand grip strength. We found an inverse association between baseline short-chain carboxyl and dihydroxl acylcarnitines (AC-DC/-OH) levels and 18-month changes in hand grip strength (p=0.047, β=-0.548, 95% CI=-1.088 to -0.008). Notably, a specific AC-DC/-OH species, C4-DC/C6-OH, accounts for the majority of the variance. The mean difference between Malay and Chinese ethnicity is 2.28kg (p=0.042, β=2.275, 95% CI=0.084 to 4.466). These findings suggest an association between metabolic markers and deterioration in hand grip strength. These results suggest that perturbations in fuel metabolism are detectable way before the emergence of clinically evident sarcopenia and frailty. The use of AC-DC/-OH panel as antecedent biomarkers may enable clinicians to risk stratify patients in the future.

Untargeted UPLC-MS metabolomics reveals multiple changes of urine composition in healthy adult volunteers after consumption of curcuma longa L. extract.

Curcuma longa L. is used as food supplement to prevent diseases, although limited studies have been performed on healthy subjects up to now. In the present work, an untargeted UPLC-MS metabolomics approach was applied to study the changes of 24-hours urinary composition on healthy volunteers due to a 28-days daily consumption of a dried C. longa extract containing a standardized amount of curcuminoids. Changes in the excretion of different metabolites were observed after supplementation. Curcumin and two metabolic derivatives (hexahydrocurcumin and dihydrocurcumin) were detected in urine, indicating the absorption of the main curcuminoid from the extract and its further metabolism by liver and gut microbiota. For the first time ar-turmerone, the main apolar constituent of curcuma, was detected in urine in intact form, and its presence was confirmed by a targeted GC-MS analysis. The increase of tetranor-PGJM and tetranor-PGDM, two prostaglandin-D2 metabolites, was observed, being related to the anti-inflammatory effect exerted by curcuma. The variation of the amounts of HPAG, PAG, proline-betaine and hydroxyphenyllactic acid indicate that the supplementation induced changes to the activity of gut microbiota. Finally, the reduced excretion of niacin metabolites (nicotinuric acid, trigonelline and 2PY) and medium- and short-chain acylcarnitines suggests that curcuma could induce the mitochondrial β-oxidation of fatty acids for energy production in healthy subjects. Overall, the results indicate that a prolonged daily consumption of a dried curcuma extract exerts multiple effects on healthy subjects, furthermore they show the opportunity offered by untargeted metabolomics for the study of the bioactivity of natural extracts in healthy human volunteers.

The Association between Alcohol Consumption and Serum Metabolites and the Modifying Effect of Smoking.

Alcohol consumption is an important lifestyle factor that is associated with several health conditions and a behavioral link with smoking is well established. Metabolic alterations after alcohol consumption have yet to be comprehensively investigated. We studied the association of alcohol consumption with metabolite patterns (MPs) among 2433 individuals from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam Study, and a potential modification by smoking. Alcohol consumption was self-reported through dietary questionnaires and serum metabolites were measured by a targeted approach. The metabolites were summarized as MPs using the treelet transform analysis (TT). We fitted linear models with alcohol consumption continuously and in five categories. We stratified the continuously modelled alcohol consumption by smoking status. All models were adjusted for potential confounders. Among men, alcohol consumption was positively associated with six MPs and negatively associated with one MP. In women, alcohol consumption was inversely associated with one MP. Heavy consumers differed from other consumers with respect to the “Long and short chain acylcarnitines” MP. Our findings suggest that long and short chain acylcarnitines might play an important role in the adverse effects of heavy alcohol consumption on chronic diseases. The relations seem to depend on gender and smoking status.

Higher dietary protein intake preserves lean body mass, lowers liver lipid deposition, and maintains metabolic control in participants with long-chain fatty acid oxidation disorders.

Medical nutrition therapy for long-chain fatty acid oxidation disorders (LC-FAODs) currently emphasizes fasting avoidance, restricted dietary long-chain fatty acid intake, supplementation with medium chain triglycerides, and increased carbohydrate intake. We hypothesize that increasing dietary protein intake relative to carbohydrate intake would preserve metabolic control yet induce physical benefits including reduced hepatic lipogenesis. Therefore, we compared two dietary approaches with similar fat intake but different carbohydrate to protein ratios in participants diagnosed with LC-FAODs. Thirteen participants were enrolled and randomized into either a high-protein (PRO) or a high-carbohydrate (CHO) diet for 4 months. Baseline and 4-month assessments included body composition, ectopic lipid deposition, and resting energy expenditure. End of study assessments also included total energy expenditure, metabolic responses to oral feedings, and whole-body fatty acid oxidation capacity. At the end of the dietary intervention, both groups had similar energy expenditure, fat and glucose oxidation rates, and glucolipid responses to mixed meal and oral glucose loads. Neither dietary group experienced worsening symptoms related to their LC-FAOD. Compared to the CHO group, the PRO group exhibited increased blood levels of short-chain acylcarnitines, reduced intrahepatic lipid content, and maintained lean body mass while the CHO group lost lean mass. In patients with LC-FAODs, increasing protein intake maintained metabolic control, reduced liver fat without risk of metabolic decompensation, and helped preserve lean body mass. We propose that a modest increase in dietary protein along with fasting avoidance and fat restriction may improve body composition and energy expenditure in patients with LC-FAODs.

2422-PUB: Short-Chain Acylcarnitines as Early Predictors of Gestational Diabetes in Mexican Population

Gestational diabetes (GD) represents the main metabolic alteration during pregnancy affecting 17% of pregnancies in Mexico. The clinical relevance of this pathology is the short and long-term complications such as obesity, diabetes, and hypertension; that affect mother and child. GD is often diagnosed between weeks 24 to 28 of gestation through an oral glucose tolerance test. The aim of the present study was to identify early pregnancy metabolites (under 18 weeks of gestation) present on women who were diagnosed with GD; in order, to generate an early prediction model to identify those women who will later be diagnosed with GD. We used targeted metabolomics (11 amino acids and 39 acylcarnitines) to select early serum biomarkers with the most predictive power to identify women who will later be diagnosed with GD on a Mexican cohort of pregnant women. We assessed 79 pregnant women, which 66 underwent a normal pregnancy and 13 were diagnosed with GD under the IADPSG criteria. Random forest analyses were performed to assess the contribution and association of the metabolites identified to the development of GD. We developed a model that included two short-chain acylcarnitines (namely AC4 and AC5:1) that allowed an AUC of 0.904 (0.843-0.965, 95 CI) for classification of gestational diabetes. This model allows the identification of women on early stages of pregnancy that will be diagnosed with gestational diabetes between weeks 24 to 28 of gestation. However, additional studies in a large sample of pregnant women are required. Disclosure M. Razo-Azamar: None. R. Nambo-Venegas: None. J. Delgadillo-Velazquez: None. B. Palacios-Gonzalez: None.

Plasma acylcarnitines and progression of carotid artery atherosclerosis in HIV infection.

To evaluate plasma acylcarnitine profiles and their relationships with progression of carotid artery atherosclerosis among individuals with and without HIV infection. Prospective cohort studies of 499 HIV-positive and 206 HIV-negative individuals from the Women's Interagency HIV Study and the Multicenter AIDS Cohort Study. Twenty-four acylcarnitine species were measured in plasma samples of participants at baseline. Carotid artery plaque was assessed using repeated B-mode carotid artery ultrasound imaging in 2004-2013. We examined the associations of individual and aggregate short-chain (C2-C7), medium-chain (C8-C14) and long-chain acylcarnitines (C16-C26) with incident carotid artery plaque over 7 years. Among 24 acylcarnitine species, C8-carnitines and C20 : 4-carnitines showed significantly lower levels comparing HIV-positive to HIV-negative individuals (false discovery rate adjusted P < 0.05); and C20-carnitines and C26-carnitines showed significantly higher levels in HIV positive using antiretroviral therapy than those without antiretroviral therapy (false discovery rate adjusted P < 0.05). In the univariate analyses, higher aggregated short-chain and long-chain acylcarnitine scores were associated with increased risk of carotid artery plaque [risk ratios (RRs) = 1.22 (95% confidence interval 1.02-1.45) and 1.20 (1.02-1.41) per SD increment, respectively]. The association for the short-chain acylcarnitine score remained significant [RR = 1.23 (1.05-1.44)] after multivariate adjustment (including traditional cardiovascular disease risk factors). This association was more evident in HIV-positive individuals without persistent viral suppression [RR = 1.37 (1.11-1.69)] compared with those with persistent viral suppression during follow-up [RR = 1.03 (0.76-1.40)] or HIV-negative individuals [RR = 1.02 (0.69-1.52)]. In two HIV cohorts, plasma levels of most acylcarnitines were not significantly different between HIV-positive and HIV-negative individuals. However, higher levels of aggregated short-chain acylcarnitines were associated with progression of carotid artery atherosclerosis.

Berberine alleviates nonalcoholic fatty liver induced by a high-fat diet in mice by activating SIRT3.

Berberine (BBR) shows promising effects in the treatment of nonalcoholic fatty liver disease (NAFLD) by influencing various metabolic aspects. Inhibition of mitochondrial β-oxidation (β-OX) participates in the pathogenesis of NAFLD. Silent mating-type information regulation 2 homolog 3 (SIRT3) has been reported to regulate mitochondrial β-OX by deacetylating its substrate, long-chain acyl-coenzyme A dehydrogenase (LCAD). This study aimed to explore whether BBR can promote mitochondrial β-OX and the role of SIRT3 as well as the mechanisms underlying the effects of BBR on hepatic lipid metabolism in mice fed a high-fat diet (HFD). BBR can significantly improve systematic and hepatic lipid metabolism in HFD-fed mice. Metabolomics analysis revealed that β-OX was inhibited in HFD-induced mice, as indicated by the reduced production of short and medium carbon chain acyl-carnitines, the activated form of free fatty acids, via β-OX, which was reversed by BBR intervention. Exploration of the mechanism found that BBR intervention reversed the down-regulation of SIRT3 and decreased the LCAD hyperacetylation level in HFD-fed mice. SIRT3 knockout (KO) mice were used to identify the role of SIRT3 in the BBR's influence of β-OX. The beneficial effects of BBR on systemic and hepatic metabolism were profoundly attenuated in KO mice. Moreover, the promotive effect of BBR on β-OX in HFD-induced mice was partially abolished in KO mice. These results suggested that BBR alleviates HFD-induced inhibition of fatty acid β-OX partly through SIRT3-mediated LCAD deacetylation, which may provide a novel mechanism and support BBR as a promising therapeutic for NAFLD.-Xu, X., Zhu, X.-P., Bai, J.-Y., Xia, P., Li, Y., Lu, Y., Li, X.-Y., Gao, X. Berberine alleviates nonalcoholic fatty liver induced by a high-fat diet in mice by activating SIRT3.