Acylcarnitines Research Articles

Abstract 4137620: Serum Acylcarnitines Predict Longitudinal Deteriorations in Cardiac Function and Adverse Outcomes in Older Adults

Background: Circulating acylcarnitines (ACs), biomarkers of mitochondrial β-oxidative function, are linked to cardiovascular diseases (CVD) yet the extent to which ACs relate to prospective cardiac aging remains uncertain. We investigate the longitudinal associations between ACs, cardiac function, and clinical outcomes. Methods: A population-based sample of community older adults without CVD underwent baseline serum metabolomic profiling and interval echocardiography at five years. Incident hospitalizations were prospectively tracked from 2014 to 2021. Multiple linear regression adjusted for significant clinical variables. Results: Of 202 participants (baseline 65±11.9 years, 49.3% women), we observed interval increases in risk factors (hypertension 39% vs 52.2% p<0.001; dyslipidemia 39% vs 50% p=0.034; diabetes 11.7% vs 18.8% p=0.035; smoking 13.2% vs 24.7% p=0.003; waist circumference 81±10.1 vs 86±9.9cm p<0.001) during follow-up (4.9±0.4 years). These changes paralleled E/e’ increases (8.9±2.6 vs 11.1±3.3 p<0.001) and E/A decreases (1.09±0.4 vs 1.0±0.4 p<0.001). Baseline C8:1 (β=0.182 p=0.011), C22:3 (β=0.148 p=0.047), C22:5 (β=0.210 p=0.003), C5:1 (β=-0.142 p=0.048), and C16-OH (β=-0.156 p=0.028) levels were associated with E/e’ change over time while C6 (β=0.163 adj. p=0.019), C8:1 (β=0.137 adj. p=0.047), C10:2 (β=0.153 adj. p=0.035), C14:3 (β=0.147 adj. p=0.031), and C16:1-OH/C14:1-DC (β=0.158 adj. p=0.022) were associated with E/A change. At follow-up, baseline C8:1 (β=0.178 adj. p=0.006) and C10:3 (β=0.145 adj. p=0.024) were associated with E/e’ while C4-DC/C6-OH (β=-0.004 adj. p=0.034) was associated with E/A. Baseline C22:2 predicted incident hospitalizations (HR=1.20 95%CI 1.03-1.39, adj. p=0.018). Conclusion: Circulating ACs independently predicted declining cardiac function and adverse outcomes over five years among older adults without CVD. The prognostic effect of ACs should be translated to strategies targeting mitochondrial health in cardiac aging prevention.

Circulating medium- and long-chain acylcarnitines are associated with plasma P-tau181 in cognitively normal older adults.

Alzheimer's disease (AD) pathogenesis involves dysregulation in diverse biochemical processes. Nevertheless, plasma tau phosphorylated at threonine 181 (P-tau181), a recognised AD biomarker, has been described to reflect early-stage cortical amyloid-β (Aβ) deposition in cognitively normal (CN) adults. Therefore, identifying changes in plasma metabolites associated with plasma P-tau181 at the pre-clinical stage may provide insights into underlying biochemical mechanisms to better understand initial AD pathogenesis. In the current study, plasma P-tau181, quantified via single molecule array (Simoa) technology, and plasma metabolites, quantified via targeted-mass spectrometry, were investigated for associations in CN older adults and upon stratification by positron emission tomography (PET)-Aβ load. In addition, the P-tau181-linked metabolites were evaluated for cognitive performance and neuroimaging markers of AD and the potential to distinguish between CN Aβ- and CN Aβ+ individuals. Significant positive associations of medium- and long-chain acylcarnitines (ACs) were observed with P-tau181 in the entire cohort, CN Aβ- and CN Aβ+, suggesting a link between initial Aβ pathology and fatty acid oxidation-mediated energy metabolism pathways. However, in CN Aβ-, additional linear associations of P-tau181 were observed with muscle metabolism and nitric oxide homeostasis-associated metabolites. Upon investigating the P-tau181-linked metabolites for cognitive performance, significant inverse correlations of the verbal and visual episodic memory and the global composite score were noted in CN Aβ+ with medium- and long-chain ACs, suggesting prognostic value of ACs accompanying weaker cognitive performance. While investigating neuroimaging markers, ACs had positive associations with PET-Aβ load and inverse associations with hippocampal volume in CN Aβ+, indicating connections of ACs with initial AD pathogenesis. Furthermore, based on receiver operating characteristics analysis, the associated ACs potentially classified PET-Aβ status in older adults. Therefore, plasma P-tau181-linked circulating ACs may serve as potential prognostic markers for initial AD pathogenesis in CN older adults. However, further cross-sectional and longitudinal research in highly characterised AD cohorts is needed to validate current findings.

Dietary lipid is largely deposited in skin and rapidly affects insulating properties.

Skin has been shown to be a regulatory hub for energy expenditure and metabolism: mutations of skin lipid metabolism enzymes can change the rate of thermogenesis and susceptibility to diet-induced obesity. However, little is known about the physiological basis for this function. Here we show that the thermal properties of skin are highly reactive to diet: within three days, a high fat diet reduces heat transfer through skin. In contrast, a dietary manipulation that prevents obesity accelerates energy loss through skins. We found that skin was the largest target in a mouse body for dietary fat delivery, and that dietary triglyceride was assimilated both by epidermis and by dermal white adipose tissue. Skin from mice calorie-restricted for 3 weeks did not take up circulating lipids and showed a highly depleted stratum corneum. Dietary triglyceride acyl groups persist in skin for weeks after feeding. Using multi-modal lipid profiling, we have implicated both keratinocytes and sebocytes in the altered lipids which correlate with thermal function. In response to high fat feeding, wax diesters and ceramides accumulate, and triglycerides become more saturated. In contrast, in response to the dramatic loss of adipose tissue that accompanies restriction of the branched chain amino acid isoleucine, skin becomes more heat-permeable, resisting changes induced by Western diet feeding, with a signature of depleted signaling lipids. We propose that skin should be routinely included in physiological studies of lipid metabolism, given the size of the skin lipid reservoir and its adaptable functionality.

6828 Exploring Metabolomic Clues in Diabetic Retinopathy

Abstract Disclosure: M.W. Simonson: None. Y. Li: None. J.J. McAnany: None. B. Prasad: None. E.C. Hanlon: None. S. Pannain: None. B.T. Layden: None. E. Van Cauter: None. J.C. Park: None. S.J. Crowley: None. F.Y. Chau: None. K.K. Danielson: None. H. Chen: None. G.E. Chlipala: None. C. Martinez: None. S. Reutrakul: Speaker; Self; Eli Lilly & Company. Background: Diabetic retinopathy (DR) is a common microvascular complication of diabetes. Metabolomics offers the possibility for novel biomarker discovery. The purpose of this study was to determine metabolomic differences that characterized patients with versus without DR. Methods: A total of 62 serum samples were collected from 36 type 2 diabetes (T2D) patients with DR and 26 T2D patients without DR, and analyzed via UPLC-MS. The metabolite data were normalized using median normalization. The differential expression of metabolites was compared among the groups using the limma package in R. The covariates (age, sex, BMI and sleep apnea severity) were accounted for by using a generalized linear model fitted to the data. Significantly different metabolites were filtered by an absolute log2 Fold Change (FC) of 1.5 or greater and Q values < 0.05. Results: Mean (SD) age for DR and no-DR group was 55 (±5.9) versus 54 (±6.7) years, while BMI was 33 (±5.8) versus 33 (±6.3) kg/m2 respectively. Both DR and no-DR groups were 58% female. A1C levels in DR group was marginally higher than in no-DR group (p=0.09). There were significant differences in metabolomic changes between DR versus no-DR, with 166 total differential metabolites after adjusting for covariates and filtering by FC. Notable metabolites included those involved in fatty acid metabolism, acyl carnitines, prostaglandins, and phospholipids. There was an overall upregulation of serum acyl carnitines in DR as compared to no-DR, including 2,6 dimethylheptanoyl carnitine (FC = 3.0) and cis-5-Tetradecenoylcarnitine (FC = 2.0). Additionally, there was a decrease in long chain fatty acids in DR as compared to no-DR, which included 6-hydroxypentadecanedioic acid (FC = -6.6), 10,16-dihydroxy-palmitic acid (FC = -6.0), and tetracosatetraenoyl CoA (FC = -6.1). The 11-beta prostaglandin F2 (11-PGF2) was increased in DR (FC = 1.9). Circulating phospholipids including lysophosphatidylcholine (22:2) (lysoPC) (FC = 1.8) and phosphatidylcholine (24:1/24:1) (PC) (FC = 5.9) were also significantly dysregulated. Conclusions: We identified a subset of metabolites that discriminate T2D patients with or without DR. The changes in acyl carnitines and long chain fatty acids may reflect mitochondrial dysfunction in DR. The presence of certain prostaglandins highlights the inflammatory environment which may facilitate DR progression. Increases in phospholipids such as lysoPC suggest an atherogenic state and higher LDL oxidation, while increases in PC may indicate altered lipogenesis in DR. These findings may serve as new potential serum biomarkers to promote efficient preventive care and provide mechanistic insight into understanding DR pathophysiology. Presentation: 6/2/2024

7286 Higher Order Associations Linking Tryptophan, Kynurenine, And Tyrosine As Serum Markers Of Glucocorticoid Downstream Action In Adipose Tissue

Abstract Disclosure: K. Bennett: None. T. Garner: None. D. Chantzichristos: Speaker; Self; Sanofi, Takeda. P. Jansson: None. A. Stevens: None. G. Johannsson: Consulting Fee; Self; Novo Nordisk, Shire, AstraZeneca. Speaker; Self; Novo Nordisk, Pfizer, Inc.. Introduction: Treatment of Adrenal Insufficiency (AI) by glucocorticoid (GC) replacement therapy is mainly guided by clinical judgement, as there is no biomarker of GC action to individualise its dosage. Hypergraphs are networks modelling higher order associations (HOAs), which are interactions between more than two ‘omic elements. The aim of this study was to use hypergraphs to identify serum markers that reflect the action of GCs in adipose tissue (AT). Methods: In a randomised, cross-over trial, 10 patients with primary AI received saline i.v. (GC withdrawal), or circadian infusion of hydrocortisone (GC exposure), with > 2 weeks apart. After 26 hours (8AM), AT biopsy and PBMCs were collected for transcriptomic analysis, and abdominal subcutaneous microdialysis and PBMCs were collected for metabolomic profiling using LC/MS. Hypergraph models were generated to integrate serum and AT transcriptomics and metabolomics, identifying clusters of putatively causally linked metabolites which could represent serum markers of downstream GC action in AT. Hypergraph structure was assessed in AT and serum using entropy to infer associations within pre-defined metabolic pathways. Differences in entropy between the interventions was measured using Bayes Factor. Univariate ROC curve analysis was used to calculate AUCs for the serum markers. Results: In AT dialysate, 22 metabolites were identified as significantly different between GC exposure and withdrawal states. These included metabolites found in lipolysis and proteolysis pathways, such as acyl carnitines, lysophosphatidylcholines, fatty acids, and peptide derivatives. Nine metabolites were significantly different between the interventions in the serum. DEGs in AT (n=2048) were found to be enriched in metabolic pathways. Hypergraphs were used to refine clusters in the serum based on their HOAs with AT differentially expressed genes and metabolites. Tryptophan, Kynurenine, and Tyrosine clustered together, demonstrating a relationship between their expression in serum, and the metabolomic and transcriptomic profile of AT. These HOAs were shared with cortisol in the exposure intervention, but not in GC withdrawal, suggesting GC dependent regulation. All 3 metabolites had AUC-ROCs > 0.8, with Kynurenine performing the best at 0.86. Tissue specific differences were seen in the entropy of metabolic pathways between GC exposure and withdrawal, with similar changes seen in lipid and proteolysis pathways, but no difference seen in serum in carbohydrate and amino acid biosynthesis. Conclusions: During near physiological exposure, we identified changes to hypergraph structure and metabolite concentrations in lipid, protein, and carbohydrate pathways in AT and serum, compared to GC withdrawal. Hypergraph integration of multi-omic data identified tryptophan, kynurenine, and tyrosine as potential serum markers reflecting GC action in AT. Presentation: 6/3/2024

Alterations of meat quality, lipid composition and flavor in breast meat of laying hens with fatty liver hemorrhagic syndrome

Fatty liver hemorrhagic syndrome (FLHS) has a high incidence rate in laying hens, and lots of FLHS-affected meat products enter the market every year. At the same time, the meat of laying hens is an important component of the human diet. However, the impact of FLHS on meat of laying hens is unknown, which could have a negative impact on consumers. To explore the effects of FLHS on chicken breast meat, a total of 36 twenty-five-wk-old Jingfen laying hens were used in the experiment. The hens were randomly divided into Control group and Model group, with 6 replicates per group and 3 hens per replicate. All chickens were raised in double-story step cages with individual pens. After a 3-wk acclimation period, the formal experiment began at 28 wk of age and lasted for 8 wk. Blood, liver, and breast meat samples were collected for the study of FLHS effects on breast meat. The impact of FLHS on meat quality was assessed by measuring indicators such as water-holding capacity and tenderness of the breast meat. Absolute quantitative lipidomics was employed to reveal the impact of FLHS on the lipid composition of chicken breast meat, and then validated by using RT-qPCR. Moreover, the volatilomics was utilized to detect changes in volatile organic compounds (VOCs) in chicken breast meat and to elucidate the resulting flavor changes. This research results showed that the meat quality of chicken breast meat decreased under FLHS, mainly manifested as reduced water holding capacity and decreased tenderness. The lipid content in the breast meat of FLHS-affected hens was significantly increased (P < 0.05). Among the affected lipids, 38 triglycerides exhibited notable elevation, possibly linked to heightened gene expression, such as lysophosphatidylcholine acyltransferase 3. The breast meat of laying hens with FLHS demonstrated an increased presence of VOCs, with 20 differential VOCs identified. Notably, 14 VOCs, particularly in 2-Undecenal, trans-Geranylacetone and ethyl nonanoate, exhibited substantial increases. These 3 VOCs had been identified as playing an important role in the formation of flavor in the breast meat of FLHS-affected laying hens. Correlation analysis suggested that the increase in these 3 VOCs might be related to the increase in lipid molecules such as phosphatidylethanolamine (38;3e) and acyl carnitine (10:3). In summary, FLHS reduced the breast meat quality of laying hens, altered its lipid profiles, and enhanced its flavor. These findings underscore the profound impact of FLHS on lipid and VOC profiles in chicken breast meat, offering valuable insights for chicken meat quality affected by FLHS.

Association of Whole Blood Amino Acid and Acylcarnitine Metabolome with Anthropometry and IGF-I Serum Levels in Healthy Children and Adolescents in Germany.

Physiological changes of blood amino acids and acylcarnitines during healthy child development are poorly studied. The LIFE (Leipziger Forschungszentrum für Zivilisationserkrankungen) Child study offers a platform with a large cohort of healthy children to investigate these dynamics. We aimed to assess the intra-person variability of 28 blood metabolites and their associations with anthropometric parameters related to growth and excess body fat. Concentrations of 22 amino acids (AA), 5 acylcarnitines (AC) and free carnitine of 2213 children aged between 3 months and 19 years were analyzed using liquid chromatography/tandem mass spectrometry. Values were transformed into standard deviation scores (SDS) to account for sex- and age-related variations. The stability of metabolites was assessed through the coefficient of determination. Associations with parameters for body composition and insulin-like growth factor-I (IGF-I) SDS were determined by the Pearson correlation and linear regression. Our research revealed substantial within-person variation in metabolite concentrations during childhood and adolescence. Most metabolites showed a positive correlation with body composition parameters, with a notable influence of sex, pubertal status and weight group. Glycine exhibited negative associations with parameters of body fat distribution, especially in normal weight girls, overweight/obese boys and during puberty. Blood AA and AC measurements may contribute to elucidating pathogenesis pathways of adiposity-related comorbidities, but the specific timings and conditions of development during childhood and adolescence need to be taken into consideration.

Fat burning capacity in a mixed macronutrient meal protocol does not reflect metabolic flexibility in women who are overweight or obese.

Metabolic flexibility, the ability to switch from glucose to fat as a fuel source, is considered a marker of metabolic health. Higher fat oxidation is often associated with greater flexibility and insulin sensitivity, while lower fat oxidation is linked to metabolic inflexibility and insulin resistance. However, our study challenges the universal validity of this relationship, uncovering a more nuanced understanding of the complex interplay between fuel source switching and fat oxidation, especially in the presence of insulin resistance. In an 8-week controlled feeding intervention, overweight to obese women with insulin resistance (as defined by McAuley's index) were randomized to consume either a diet based on the Dietary Guidelines for Americans 2010 (DGA) or a 'Typical' American Diet (TAD), n = 22 each. Participants were given a high-fat mixed macronutrient challenge test (MMCT) (60% fat, 28% carbohydrates, and 12% protein) at weeks 0, 2, and 8. Plasma lipids, metabolome, and lipidome were measured at 0, 0.5, 3, and 6h postprandial (PP); substrate oxidation measures were also recorded at 0,1 3, and 6h PP. Metabolic flexibility was evaluated as the change in fat oxidation from fasting to PP. Mixed model and multivariate analyses were used to evaluate the effect of diet on these outcomes, and to identify variables of interest to metabolic flexibility. Intervention diets (DGA and TAD) did not differentially affect substrate oxidation or metabolic flexibility, and equivalence tests indicated that groups could be combined for subsequent analyses. Participants were classified into three groups based on the % of consumed MMCT fat was oxidized in the 6h post meal period at weeks 0, 2 and 8. Low fat burners (LB, n = 6, burned <30% of fat in MMCT) and high fat burners (HB, n = 7, burned > 40% of fat in MMCT) at all weeks. Compared to LB, HB group had higher fat mass, total mass, lean mass, BMI, lower HDLc and lower RER (p < 0.05), but not different % body fat or % lean mass. During week 0, at 1h PP, LB had an increase in % fat oxidation change from 0h compared to HB (p<0.05), suggesting higher metabolic flexibility. This difference disappeared later in the PP phase, and we did not detect this beyond week 0. Partial least squares discriminant analysis (PLSDA (regular and repeated measures (sPLSDA)) models identified that LB group, in the late PP phase, was associated with higher rates of disappearance of acylcarnitines (AC) and lysophosphatidylcholines (LPC) from plasma (Q2: 0.20, R2X: 0.177, R2Y: 0.716). In women with insulin resistance, a high fat burning capacity does not imply high metabolic flexibility, and not all women with insulin resistance are metabolically inflexible. LPCs and ACs are promising biomarkers of metabolic flexibility.

Acylcarnitines metabolism in depression: association with diagnostic status, depression severity and symptom profile in the NESDA cohort.

Acylcarnitines (ACs) are involved in bioenergetics processes that may play a role in the pathophysiology of depression. Previous genomic evidence identified four ACs potentially linked to depression risk. We carried forward these ACs and tested the association of their circulating levels with Major Depressive Disorder (MDD) diagnosis, overall depression severity and specific symptom profiles. The sample from the Netherlands Study of Depression and Anxiety included participants with current (n = 1035) or remitted (n = 739) MDD and healthy controls (n = 800). Plasma levels of four ACs (short-chain: acetylcarnitine C2 and propionylcarnitine C3; medium-chain: octanoylcarnitine C8 and decanoylcarnitine C10) were measured. Overall depression severity as well as atypical/energy-related (AES), anhedonic and melancholic symptom profiles were derived from the Inventory of Depressive Symptomatology. As compared to healthy controls, subjects with current or remitted MDD presented similarly lower mean C2 levels (Cohen's d = 0.2, p ≤ 1e-4). Higher overall depression severity was significantly associated with higher C3 levels (ß=0.06, SE = 0.02, p = 1.21e-3). No associations were found for C8 and C10. Focusing on symptom profiles, only higher AES scores were linked to lower C2 (ß=-0.05, SE = 0.02, p = 1.85e-2) and higher C3 (ß=0.08, SE = 0.02, p = 3.41e-5) levels. Results were confirmed in analyses pooling data with an additional internal replication sample from the same subjects measured at 6-year follow-up (totaling 4141 observations). Small alterations in levels of short-chain acylcarnitine levels were related to the presence and severity of depression, especially for symptoms reflecting altered energy homeostasis. Cellular metabolic dysfunctions may represent a key pathway in depression pathophysiology potentially accessible through AC metabolism.

Abstract Tu114: Prostaglandin E2 Alters Mitochondrial Energy Metabolism in the Murine Heart

Prostaglandin E 2 (PGE2) is an autacoid that acts via 4 receptors (EP1-EP4), with EP3 and EP4 being the most prominent in the heart. We previously reported that cardiomyocyte specific knockout of EP4 (EP4 KO) results in dilated cardiomyopathy. Additionally, transgenic mice that overexpress EP3 in the cardiomyocyte (EP3 TG) develop heart failure. Recently published data from our laboratory suggests EP4 KO results in mitochondrial dysfunction, likely via EP3 receptor signaling. We therefore hypothesized that PGE2 alters energy metabolism in the heart via EP3. To test our hypothesis, we performed gene array analysis on left ventricles from adult EP4 KO and wild type (WT) mice. Significant reductions (2.1-fold) in carnitine palmitoyltransferase 2 ( Cpt2 ) were observed in EP4 KO vs. EP4 WT. CPT2 is a key enzyme in fatty acid oxidation and deficiencies in humans have been shown to cause cardiomyopathy. Substantial reductions in CPT2 were also observed in C57 mice after 2 weeks of myocardial infarction (0.22 ± 0.04 a.u. in sham controls vs. 0.039 ± 0.06 a.u. after MI. p&lt;0.01) by western blot analysis. Moreover, treatment with the EP3 receptor agonist, sulprostone, reduced CPT2 protein expression significantly in cardiomyocytes isolated from C57 mice (1.0 ± 0.0 a.u. in vehicle control vs. 0.67 ± 0.07 a.u. in sulprostone treated. p&lt;0.005). These data suggest that PGE2 via its EP3 receptor reduces CPT2 expression. In fact, RNAseq analysis on left ventricles from EP3 TG and EP3 WT mice showed significant reductions in Cpt 2 expression as early as 5 wks of age (log 2 FC = -1.17. p&lt;0.01). RNAseq also showed reductions in other key fatty acid oxidation/transport genes in EP3 TG mice ( Acsl1 ; log 2 FC= -0.73, Acads , log 2 FC= -0.59, Cd36 , log 2 FC= -0.64. p&lt;0.005). Lastly, to further investigate the role of the EP receptors on energy metabolism in the heart, we isolated cardiomyocytes from EP4 KO and EP4 WT mice and performed metabolomics. As anticipated, we observed significant reductions in L-carnitine (7.6 ± 1.13 fmol/cell in EP4 WT vs. 2.1 ± 0.32 fmol/cell in EP4 KO. p&lt;0.005) and several acyl carnitine products. Altogether our data supports our hypothesis that PGE2 signaling via its EP3 receptor impairs mitochondrial energy metabolism in the heart by reducing fatty acid oxidation.

Bilirubin metabolism in early life and respiratory health during preschool age: A combined analysis of two independent birth cohorts.

Bilirubin has antioxidant properties, and elevated levels within the normal range have been associated with improved lung function and decreased risk of asthma in adults, but studies of young children are scarce. Here, we investigate associations between bilirubin in early life and respiratory health endpoints during preschool age in two independent birth cohorts. Bilirubin metabolites were assessed at ages 0.5, 1.5, and 6 years in COPSAC2010 (Copenhagen Prospective Studies on Asthma in Childhood 2010) and ages 1, 3, and 6 years in the VDAART (The Vitamin D Antenatal Asthma Reduction Trial) cohort. Meta-analyses were done to summarize the relationship between levels of bilirubin metabolites and asthma, infections, lung function, and allergic sensitization until age 6 across the cohorts. Interaction with the glucuronosyltransferase family 1 member A1 (UGT1A) genotype encoding for an enzyme in the bilirubin metabolism was explored, and metabolomics data were integrated to study underlying mechanisms. Increasing bilirubin (Z,Z) at ages 1.5-3 years was associated with an increased risk of allergic sensitization (adjusted relative risk [aRR]= 1.85 [1.20-2.85], p= 0.005), and age 6 bilirubin (Z,Z) also showed a trend of association with allergic sensitization at age 6 (aRR= 1.31 [0.97-1.77], p= 0.08), which showed significant interaction for the age 6 bilirubin (Z,Z)xUGT1A genotype. Further, increasing bilirubin (E,E), bilirubin (Z,Z), and biliverdin at ages 1.5-3 years was associated with a lower forced expiratory volume at age 6 (aRRrange= 0.81-0.91, p<0.049) but without a significant interaction with the UGT1A genotype (pinteractions>0.05). Network analysis showed a significant correlation between bilirubin metabolism and acyl carnitines. There were no associations between bilirubin metabolites and the risk of asthma and infections. Bilirubin metabolism in early life may play a role in childhood respiratory health, particularly in children with specific UGT1A genotypes. The Lundbeck Foundation (Grant no R16-A1694), The Ministry of Health (Grant no 903516), Danish Council for Strategic Research (Grant no 0603-00280B), and The Capital Region Research Foundation have provided core support to the COPSAC research center. This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No. 946228). The Vitamin D Antenatal Asthma Reduction Trial (VDDART, ClinicalTrials.gov identifier: NCT00920621) was supported by grant U01HL091528 from NHLBI, U54TR001012 from the National Centers for Advancing Translational Sciences (NCATS). Metabolomics work by VDAART was supported by the National Heart, Lung, and Blood Institute (NHLBI) grant R01HL123915 and R01HL141826. S.T.W. was supported by R01HL091528 from the NHLBI, UG3OD023268 from Office of The Director, National Institute of Health, and P01HL132825 from the NHLBI.

Disturbed carnitine metabolism is independently correlated with sarcopenia and prognosis in patients on hemodialysis

Background & AimsSarcopenia is frequent in hemodialysis patients and associated with an increased likelihood of adverse outcomes. Early identification of the risk of sarcopenia and effective intervention are of great importance for dialysis patients. However, little research has been carried out on potential biomarkers of sarcopenia in hemodialysis patients. The aim of this study was to investigate whether serum carnitine or acylcarnitine levels are biomarkers of sarcopenia in hemodialysis patients, and whether these are prognostic factors for occurrence of complications. MethodsThis prospective clinical pilot study enrolled patients (n=259) who were treated in the Blood Purification Center from May 2021 to July 2022, all participants were followed-up for 1- year. Serum carnintine and acylcarnitine (AC) were measured using our previously reported targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The correlations between carnitine or acylcarnitine levels with sarcopenia and prognosis in patients were analysed. ResultsThe C0 (Free carnitine, FC) and total carnitine (TC) levels were significantly lower in the sarcopenia group than in the nonsarcopenia group (nonsarcopenia vs. sarcopenia: 20.97 (16.96, 25.83) vs. 17.77 (14.30, 22.78); p =0.002) and (nonsarcopenia vs. sarcopenia: 30.12 (24.76, 36.62) vs. 26.03 (21.30, 32.01); p = 0.003). Besides, significant difference between the groups were noted in low free carnitine (C0 < 20 μmol/L) patients (nonsarcopenia vs. sarcopenia: 72 (42.4%) vs. 56 (62.9%); p = 0.002) and high C2/ C0 ratio (> 0.4) patients (nonsarcopenia vs. sarcopenia: 36 (21.2%) vs. 30 (33.7%); p = 0.028). By multivariable analysis, the disturbed CM defined as C0 deficient and/or C2/C0 carnitine ratio abnormal rise was independently and significantly correlated with the prevalence of sarcopenia after adjusting for some confounding factors, such as age, gender and dialysis duration (P values for trend <0.05).Hemodialysis patients with sarcopenia [OR: 3.214 (1.307,7.904)] and disturbed CM [OR: 3.217 (1.112,9.305)] both had a 3-fold increased risk of falling and fracture after one year follow up. In addition, age and sarcopenia [OR: 2.883 (1.321, 6.289)] were independently and positively associated with incidence of Cardio- and cerebro-vascular events. ConclusionDisturbed carnitine metabolism is independently correlated with sarcopenia and prognosis in patients with hemodialysis. Serum carnitine level and C0/C2 ratio has the potential to be a simple, objective, and quick test for sarcopenia assessment whether such an intervention should be carried out for dialysis patients.

Comparative Determination of Mitochondrial Biomarkers and Their Relationship With Insulin Resistance in Type 2 Diabetic Patients: An Observational Cross-Sectional Study.

Data suggest malfunctioning mitochondria reduce oxidation and adenosine triphosphate (ATP) production, disrupting insulin signalling. Cytochrome c(CC), acylcarnitine (AC) and citrate synthase (CS) are essential components of the mitochondria machinery and can be used as reliable biomarkers of mitochondrial dysfunction. This study aimed to determine whether mitochondrial biomarkers (AC, CS and CC) are altered in individuals with type 2 diabetes mellitus (T2DM) and to examine the association between these biomarkers and insulin resistance. A cross-sectional observational study that recruited 170 participants (88 with T2DM and 82 without DM) was conducted. Blood samples were collected from the recruits and analysed for levels of fasting glucose (FBG), AC, CS, CC, insulin, total cholesterol, triglycerides (TG), glycated haemoglobin (HbA1c) and magnesium. Blood pressure (BP) and anthropometric characteristics of participants were also taken. Appropriate formulas were used to determine %body fat, body mass index (BMI), waist-to-hip ratio (WHR), the homeostatic model assessment for insulin resistance (HOMA-IR) and insulin sensitivity (HOMA-β). Patients with T2DM had higher levels of CC, %body fat, FBG, TG, HbA1c, BMI and HOMA-IR than controls (p < 0.05, respectively). Results showed a significant relationship between circulating CC levels versus HOMA-β (r = -0.40, p = 0.001), CS (r = -0.70, p = 0.001) and AC (r = -0.72, p = 0.001) levels in patients with T2DM. The adjusted odds increased in the T2DM patients for VLDL (OR = 6.66, p = 0.002), HbA1c (OR = 6.50, p = 0.001), FPG (OR = 3.17, p = 0.001), TG (OR = 2.36, p = 0.010), being female (OR = 2.09, p = 0.020) and CC (OR = 1.14, p = 0.016). Overall, alterations in mitochondrial biomarkers, measured by AC, CC and CS, were observed in people with T2DM and showed a direct relationship with insulin resistance. These findings are potentially significant in Africa, although additional confirmation from a larger cohort is necessary.

Metabolic blood profile and response to treatment with the pyruvate kinase activator mitapivat in patients with sickle cell disease.

Mitapivat is an investigational, oral, small-molecule allosteric activator of pyruvate kinase (PK). PK is a regulatory glycolytic enzyme that is key in providing the red blood cell (RBC) with sufficient amounts of adenosine triphosphate (ATP). In sickle cell disease (SCD), decreased 2,3-DPG levels increase the oxygen affinity of hemoglobin, thereby preventing deoxygenation and polymerization of sickle hemoglobin. The PK activator mitapivat has been shown to decrease levels of 2,3-DPG and increase levels of ATP in RBCs in patients with SCD. In this phase 2, investigator-initiated, open-label study (https://www.clinicaltrialsregister.eu/ NL8517; EudraCT 2019-003438-18), untargeted metabolomics was used to explore the overall metabolic effects of 8-week treatment with mitapivat in the dose-finding period. In total, 1773 unique metabolites were identified in dried blood spots of whole blood from ten patients with SCD and 42 healthy controls (HCs). The metabolic phenotype of patients with SCD revealed alterations in 139/1773 (7.8%) metabolites at baseline when compared to HCs (false discovery rate-adjusted p < 0.05), including increases of (derivatives of) polyamines, purines, and acyl carnitines. Eight-week treatment with mitapivat in nine patients with SCD altered 85/1773 (4.8%) of the total metabolites and 18/139 (12.9%) of the previously identified altered metabolites in SCD (unadjusted p < 0.05). Effects were observed on a broad spectrum of metabolites and were not limited to glycolytic intermediates. Our results show the relevance of metabolic profiling in SCD, not only to unravel potential pathophysiological pathways and biomarkers in multisystem diseases but also to determine the effect of treatment.

The metabolome as a diagnostic for maximal aerobic capacity during exercise in type 1 diabetes

Aims/hypothesisOur aim was to characterise the in-depth metabolic response to aerobic exercise and the impact of residual pancreatic beta cell function in type 1 diabetes. We also aimed to use the metabolome to distinguish individuals with type 1 diabetes with reduced maximal aerobic capacity in exercise defined by V˙O2peak\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\dot{V}{\ ext{O}}_{\ ext{2peak}}$$\\end{document}.MethodsThirty participants with type 1 diabetes (≥3 years duration) and 30 control participants were recruited. Groups did not differ in age or sex. After quantification of peak stimulated C-peptide, participants were categorised into those with undetectable (<3 pmol/l), low (3–200 pmol/l) or high (>200 pmol/l) residual beta cell function. Maximal aerobic capacity was assessed by V˙O2peak\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\dot{V}{\ ext{O}}_{\ ext{2peak}}$$\\end{document} test and did not differ between control and type 1 diabetes groups. All participants completed 45 min of incline treadmill walking (60% V˙O2peak\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\dot{V}{\ ext{O}}_{\ ext{2peak}}$$\\end{document}) with venous blood taken prior to exercise, immediately post exercise and after 60 min recovery. Serum was analysed using targeted metabolomics. Metabolomic data were analysed by multivariate statistics to define the metabolic phenotype of exercise in type 1 diabetes. Receiver operating characteristic (ROC) curves were used to identify circulating metabolomic markers of maximal aerobic capacity (V˙O2peak\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\dot{V}{\ ext{O}}_{\ ext{2peak}}$$\\end{document}) during exercise in health and type 1 diabetes.ResultsMaximal aerobic capacity (V˙O2peak\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\dot{V}{\ ext{O}}_{\ ext{2peak}}$$\\end{document}) inversely correlated with HbA1c in the type 1 diabetes group (r2=0.17, p=0.024). Higher resting serum tricarboxylic acid cycle metabolites malic acid (fold change 1.4, p=0.001) and lactate (fold change 1.22, p=1.23×10−5) differentiated people with type 1 diabetes. Higher serum acylcarnitines (AC) (AC C14:1, F value=12.25, p=0.001345; AC C12, F value=11.055, p=0.0018) were unique to the metabolic response to exercise in people with type 1 diabetes. C-peptide status differentially affected metabolic responses in serum ACs during exercise (AC C18:1, leverage 0.066; squared prediction error 3.07). The malic acid/pyruvate ratio in rested serum was diagnostic for maximal aerobic capacity (V˙O2peak\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\dot{V}{\ ext{O}}_{\ ext{2peak}}$$\\end{document}) in people with type 1 diabetes (ROC curve AUC 0.867 [95% CI 0.716, 0.956]).Conclusions/interpretationThe serum metabolome distinguishes high and low maximal aerobic capacity and has diagnostic potential for facilitating personalised medicine approaches to manage aerobic exercise and fitness in type 1 diabetes.Graphical

Plasma Sphingomyelins and Carnitine Esters of Infants Consuming Whole Goat or Cow Milk-Based Infant Formulas or Human Milk

BackgroundInfant formulas are typically manufactured using skimmed milk, whey proteins, and vegetable oils, which excludes milk fat globule membranes (MFGM). MFGM contains polar lipids, including sphingomyelin (SM). ObjectiveThe objective of this study was comparison of infant plasma SM and acylcarnitine species between infants who are breastfed or receiving infant formulas with different fat sources. MethodsIn this explorative study, we focused on SM and acylcarnitine species concentrations measured in plasma samples from the TIGGA study (ACTRN12608000047392), where infants were randomly assigned to receive either a cow milk-based infant formula (CIF) with vegetable oils only or a goat milk-based infant formula (GIF) with a goat milk fat (including MFGM) and vegetable oil mixture to the age ≥4 mo. Breastfed infants were followed as a reference group. Using tandem mass spectrometry, SM species in the study formulas and SM and acylcarnitine species in plasma samples collected at the age of 4 mo were analyzed. ResultsTotal SM concentrations (∼42 μmol/L) and patterns of SM species were similar in both formulas. The total plasma SM concentrations were not different between the formula groups but were 15 % (CIF) and 21% (GIF) lower in the formula groups than in the breastfed group. Between the formula groups, differences in SM species were statistically significant but small. Total carnitine and major (acyl) carnitine species were not different between the groups. ConclusionsThe higher total SM concentration in breastfed than in formula-fed infants might be related to a higher SM content in human milk, differences in cholesterol metabolism, dietary fatty acid intake, or other factors not yet identified. SM and acylcarnitine species composition in plasma is not closely related to the formula fatty acid composition.This trial was registered at Australian New Zealand Clinical Trials Registry as ACTRN12608000047392

Acylcarnitines promote gallbladder cancer metastasis through lncBCL2L11-THOC5-JNK axis

BackgroundThe progression of gallbladder cancer (GBC) is accompanied by abnormal fatty acid β-oxidation (FAO) metabolism. Different types of lipids perform various biological functions. This study aimed to determine the role of acyl carnitines in the molecular mechanisms of GBC progression.MethodsDistribution of lipids in GBC was described by LC–MS-based lipidomics. Cellular localization, expression level and full-length of lncBCL2L11 were detected using fluorescence in situ hybridization (FISH) assays, subcellular fractionation assay and 5′ and 3′ rapid amplification of the cDNA ends (RACE), respectively. In vitro and in vivo experiments were used to verify the biological function of lncBCL2L11 in GBC cells. Methylated RNA Immunoprecipitation (MeRIP) was performed to detect the methylation levels of lncBCL2L11. RNA pull-down assay and RNA immunoprecipitation (RIP) assay were used to identify lncBCL2L11 interacting proteins. Co-Immunoprecipitation (Co-IP) and Western blot assay were performed to validate the regulatory mechanism of lncBCL2L11 and THO complex.ResultsAcylcarnitines were significantly up-regulated in GBC tissues. High serum triglycerides correlated to decreased survival in GBC patients and promoted tumor migration. LncBCL2L11 was identified in the joint analysis of highly metastatic cells and RNA sequencing data. LncBCl2L11 prevented the binding of THOC6 and THOC5 and causes the degradation of THOC5, thus promoting the accumulation of acylcarnitines in GBC cells, leading to the malignant progression of cancer cells. In addition, highly expressed acylcarnitines stabilized the expression of lncBCL2L11 through N6-methyladenosine methylation (m6A), forming a positive feedback regulation in tumor dissemination.ConclusionsLncBCL2L11 is involved in gallbladder cancer metastasis through FAO metabolism. High lipid intake is associated with poor prognosis of GBC. Therefore, targeting lncBCL2L11 and its pathway-related proteins or reducing lipid intake may be significant for the treatment of GBC patients.

Low serum carnitine level is associated with increased urinary carnitine excretion in late pregnancy.

Carnitine is essential for fatty acid metabolism. Free carnitine (FCA) is excreted in the urine in the glomerulus, but is partly reabsorbed by a carnitine transporter. The mechanism underlying the decrease in serum carnitine level during pregnancy is unclear. To investigate whether low carnitine level is associated with increased renal excretion in pregnant women. We recruited 43 healthy pregnant and 25 non-pregnant women. Total carnitine (TCA) and FCA levels were measured using the enzymatic cycling method, and the acylcarnitine (ACA) level was calculated. Fractional excretion (FE) was calculated as carnitine clearance divided by creatinine clearance. The mean TCA, FCA, and ACA levels were lower at 12weeks of gestation in pregnant than non-pregnant women (P < .001); the levels decreased further at 36weeks, reaching 39%, 36%, and 52% of those in non-pregnant women, respectively (P < .001). The FEs were 3-4-fold higher in pregnant women than non-pregnant women. Pregnant women had a lower serum FCA/TCA ratio than non-pregnant women (0.788±0.098 vs 0.830±0.074, respectively; P < .05), whereas the urine FCA/TCA ratio was similar between the groups. Low carnitine level is associated with increased renal excretion during late pregnancy.

Pathophysiological roles of the serum acylcarnitine level and acylcarnitine/free carnitine ratio in patients with cardiovascular diseases

IntroductionL-carnitine exerts protective effects, such as maintaining mitochondrial functions and decreasing reactive oxygen species, while acylcarnitine (AC) is linked to the development of heart failure and atherosclerosis. HypothesisSerum carnitines play important pathophysiological roles in cardiovascular diseases. MethodsPre-operative biochemical data were obtained from 117 patients (71 men, average age 69.9 years) who underwent surgery for cardiovascular diseases. Measurements included pre-operative biochemical data including estimated glomerular filtration rate (eGFR), physical functions, skeletal muscle mass index (SMI) measured by bioelectrical impedance analysis, anterior thigh muscle thickness (MTh) measured by ultrasound, and routine echocardiography. Carnitine components were measured with the enzyme cycling method. Muscle wasting was diagnosed based on the Asian Working Group for Sarcopenia criteria. ResultsPlasma brain natriuretic peptide (BNP) level was correlated with serum free carnitine (FC) and AC level, and the acylcarnitine/free carnitine ratio (AC/FC). AC/FC was elevated with stage of chronic kidney disease. In multivariate analysis, log (eGFR) and log (BNP) were extracted as independent factors to define log (serum AC) (eGFR: β = 0.258, p = 0.008; BNP: β = 0.273, p = 0.011), even if corrected for age, sex and body mass index. AC/FC was negatively correlated with hand-grip strength (r = -0.387, p = 0.006), SMI (r = -0.314, p = 0.012), and anterior thigh MTh (r = -0.340, p = 0.014) in men. ConclusionsA significant association between serum AC level and AC/FC, and chronic kidney disease and heart failure exists in patients with cardiovascular diseases who have undergone cardiovascular surgery. Skeletal muscle loss and muscle wasting are also linked to the elevation of serum AC level and AC/FC.

Metabolomics as a Candidate for Endometriosis Biomarker: A Systematic Review

Background: To date, diagnosis is still a challenge in managing endometriosis. There is a need to develop a less invasive yet accurate approach, such as a biomarker, to help diagnose endometriosis earlier and more precisely. Metabolomics, the study of metabolites, are thought to provide a more accurate relation to phenotype compared to other omics technology and may help develop endometriosis biomarkers. This review aims to systematically summarize evidence regarding the use of metabolomics studies in the diagnosis of endometriosis. Methods: This review was conducted according to PRISMA 2020 guidelines. Including studies on endometriosis and metabolomics in Medline, Embase, and Cochrane Library to May 2022. Inclusion: Women of reproductive age, laparoscopically diagnosed — exclusion criteria: no full-text, languages other than English, and animal studies. Papers were screened and extracted using Covidence, appraised using Newcastle-Ottawa Scale (NOS), by two independent reviewers. Results: A total of 33 studies are included in this review, and 24 showed positive results regarding the metabolites identified and their association with endometriosis. Twenty-four studies reported good area under curve (AUC), sensitivity, and specificity scores in diagnosing endometriosis using a prediction model from the attained metabolites. However, the summary of these scores is not feasible due to the lack of standardization in reporting metabolomics studies. Both serum and endometriotic lesion microenvironments, such as peritoneal fluid, endometrioma, follicular fluid, and tissue lesions, give essential information on which metabolites are altered in endometriosis. Phosphatidylcholine (PC), acylcarnitines (AC), and sphingomyelins (SM) are these studies’ most frequent significant metabolites, but their levels vary. Although all studies had an overall good appraisal score, regarding confounders included, the variation in their methods of analysis and prediction model approach should be interpreted with caution. These predictions are also mostly done without using two different data groups for test and validation. Conclusions: Metabolomics studies may become an alternative for a less invasive approach to diagnosing endometriosis. This review revealed how encouraging results from metabolomics studies are. However, the need for more standardization in the study report and preliminary design for making a prediction model is still in the way of fully trusting metabolomics studies to account for endometriosis diagnostic biomarkers. Registration: PROSPERO CRD42022334916