Nutritional status and extended metabolic screening in Egyptian children with uncomplicated type 1 diabetes.

Nutritional status assessment, including amino acids, carnitine, and acylcarnitine profile, is an important component of diabetes care management, influencing growth and metabolic regulation. A designed case-control research included 100 Egyptian participants (50 T1DM and 50 healthy controls) aged 6 to 18 years old. The participants' nutritional status was assessed using the Body Mass Index (BMI) Z-score. Extended metabolic screening (EMS) was performed using a high-performance liquid chromatography-electrospray ionization-mass spectroscopy system to evaluate the levels of 14 amino acids, free carnitine, and 27 carnitine esters. T1DM children had considerably lower anthropometric Z-scores than the control group, with 16% undernutrition and 32% short stature. Total aromatic amino acids, phenylalanine, phenylalanine/tyrosine ratio, proline, arginine, leucine, isoleucine, free carnitine, and carnitine esters levels were considerably lower in the diabetic group, suggesting an altered amino acid and carnitine metabolism in type 1 diabetes. BMI Z-score showed a significant positive correlation with Leucine, Isoleucine, Phenylalanine, Citrulline, Tyrosine, Arginine, Proline, free carnitine, and some carnitine esters (Acetylcarnitine, Hydroxy-Isovalerylcarnitine, Hexanoylcarnitine, Methylglutarylcarnitine, Dodecanoylcarnitine, Tetradecanoylcarnitine, and Hexadecanoylcarnitine). HbA1c% had a significant negative correlation with Total aromatic amino acids, Branched-chain amino acid/Total aromatic amino acids ratio, Glutamic Acid, Citrulline, Tyrosine, Arginine, Proline, and certain carnitine esters (Propionylcarnitine, Methylglutarylcarnitine, Decanoylcarnitine, Octadecanoylcarnitine and Octadecenoylcarnitine), suggest that dysregulated amino acid and carnitine metabolism may be negatively affect the glycaemic control in children with TIDM. In conclusion, regular nutritional assessments including EMS of T1DM patients are critical in terms of diet quality and protein content for improved growth and glycemic management.

Metabolic profiling of psoriasis vulgaris and palmoplantar pustulosis.

Psoriasis is a chronic inflammatory skin disorder with various subtypes, including psoriasis vulgaris (PV) and palmoplantar pustulosis (PPP). Metabolomics studies have provided insights into psoriasis pathogenesis. However, research on metabolomic alterations in PV and PPP patients is limited. We aimed to explore and compare the metabolic profiles of patients with PV and PPP to those of healthy volunteers (HVs). A single-centre retrospective cohort was constructed, comprising Korean patients with psoriasis and HVs matched by age and sex. Clinical information including demographics, disease severity, and comorbidities were collected. Plasma samples were subjected to targeted metabolic analysis using an Absolute IDQ®p180 kit, which quantified 188 metabolites, including amino acids and carnitines. Statistical significance was assessed using an independent t-test and chi-square test, with p-values adjusted by the Benjamini-Hochberg procedure. Pathway analyses were employed to gain a comprehensive understanding of the metabolite profile. This study included 93 patients (73 PV and 20 PPP) and an equal number of HVs. PV patients showed increased levels of sarcosine, serotonin, propionylcarnitine, proline, aspartic acid, tyrosine, taurine, spermine and ornithine, but exhibited a decreased level of acetylcarnitine than matched HVs. Notably, sarcosine levels were significantly elevated in PPP patients. Furthermore, the sarcosine/glycine ratio was significantly higher in both PV and PPP patients than in HVs. Pathway analysis showed significant increases in metabolites involved in amino acid metabolism and the urea cycle in PV patients. In conclusion, this study demonstrated distinct metabolic profiles in PV and PPP patients compared to HVs, suggesting sarcosine as a potential biomarker for psoriasis.

Lipidome and metabolome profiling of longissimus lumborum beef with different ultimate pH and postmortem aging.

The objective of this exploratory study was to assess the changes on lipidome and metabolome profiling of Longissimus lumborum bull muscle with different ultimate pH (pHu) and aging periods. The bull muscles classified as normal, intermediate, or high pHu were collected from a Brazilian commercial slaughterhouse, cut into steaks, individually vacuum-packaged, and aged for 3 days (3-d) or 21 days (21-d) at 2 °C. Muscle extracts were analyzed for the profiles of both lipids, by mass spectrometry (via direct flow-injection), and metabolites, by nuclear magnetic resonance, with downstream multivariate data analysis. As major results, pairwise comparisons identified C12:0 and C14:0 acylcarnitines as potential biomarkers of the intermediate pHu-muscle, which are related to lipid catabolism for alternative energy metabolism and indicate less protein breakage postmortem. Interestingly, the concentration of arginine at early postmortem aging (3-d) may influence the previously reported improved tenderness in normal and high pHu-muscles. Moreover, upregulation of fumarate, formate, and acetate with increased pHu muscle at 21-d aging indicate more intense tricarboxylic acid cycle, amino acid degradation, and pyruvate oxidation by reactive oxygen species, respectively. These three compounds (fumarate, formate, and acetate) discriminated statistically the muscle with high pHu at 21-d aging. The normal pHu-muscle showed higher concentrations of glycogenolysis and glycolysis metabolites, including glucose, mannose, and pyruvate. Hence, our results enhance knowledge of postmortem biochemical changes of beef within different pHu groups aged up to 21 days, which is essential to understand the mechanisms underpinning bull meat quality changes.

shRNA-mediated down-regulation of Acsl1 reverses skeletal muscle insulin resistance in obese C57BL6/J mice.

Prolonged consumption of diet rich in fats is regarded as the major factor leading to the insulin resistance (IR) and type 2 diabetes (T2D). Emerging evidence link excessive accumulation of bioactive lipids such as diacylglycerol (DAG) and ceramide (Cer), with impairment of insulin signaling in skeletal muscle. Until recently, little has been known about the involvement of long-chain acyl-CoAs synthetases in the above mechanism. To examine possible role of long-chain acyl-coenzyme A synthetase 1 (Acsl1) (a major muscular ACSL isoform) in mediating HFD-induced IR we locally silenced Acsl1 in gastrocnemius of high-fat diet (HFD)-fed C57BL/6J mice through electroporation-delivered shRNA and compared it to non-silenced tissue within the same animal. Acsl1 down-regulation decreased the content of muscular long-chain acyl-CoA (LCACoA) and both the Cer (C18:1-Cer and C24:1-Cer) and DAG (C16:0/18:0-DAG, C16:0/18:2-DAG, C18:0/18:0-DAG) and simultaneously improved insulin sensitivity and glucose uptake as compared with non-silenced tissue. Acsl1 down-regulation decreased expression of mitochondrial ß-oxidation enzymes, and the content of both the short-chain acylcarnitine (SCA-Car) and short-chain acyl-CoA (SCACoA) in muscle, pointing towards reduction of mitochondrial FA oxidation. The results indicate, that beneficial effects of Acsl1 partial ablation on muscular insulin sensitivity are connected with inhibition of Cer and DAG accumulation, and outweigh detrimental impact of decreased mitochondrial fatty acids metabolism in skeletal muscle of obese HFD-fed mice.

Biomarker patterns and mechanistic insights into hypothermia from a postmortem metabolomics investigation.

Postmortem metabolomics holds promise for identifying crucial biological markers relevant to death investigations and clinical scenarios. We aimed to assess its applicability in diagnosing hypothermia, a condition lacking definitive biomarkers. Our retrospective analysis involved 1095 postmortem femoral blood samples, including 150 hypothermia cases, 278 matched controls, and 667 randomly selected test cases, analyzed using UHPLC-QTOF mass spectrometry. The model demonstrated robustness with an R2 and Q2 value of 0.73 and 0.68, achieving 94% classification accuracy, 92% sensitivity, and 96% specificity. Discriminative metabolite patterns, including acylcarnitines, stress hormones, and NAD metabolites, along with identified pathways, suggest that metabolomics analysis can be helpful to diagnose fatal hypothermia. Exposure to cold seems to trigger a stress response in the body, increasing cortisol production to maintain core temperature, possibly explaining the observed upregulation of cortisol levels and alterations in metabolic markers related to renal function. In addition, thermogenesis seems to increase metabolism in brown adipose tissue, contributing to changes in nicotinamide metabolism and elevated levels of ketone bodies and acylcarnitines, these findings highlight the effectiveness of UHPLC-QTOF mass spectrometry, multivariate analysis, and pathway identification of postmortem samples in identifying metabolite markers with forensic and clinical significance. The discovered patterns may offer valuable clinical insights and diagnostic markers, emphasizing the broader potential of postmortem metabolomics in understanding critical states or diseases.

The potential role of lung microbiota and lauroylcarnitine in T-cell activation associated with checkpoint inhibitor pneumonitis.

BACKGROUND: Checkpoint inhibitor pneumonitis (CIP) is a potentially fatal adverse event characterized by new pulmonary infiltrates in cancer patients receiving immune checkpoint inhibitor therapy. This study aims to explore the interplay between lung microbiota, dysregulated metabolites, and host immunity in CIP. METHODS: We recruited thirteen hospitalized CIP patients, eleven idiopathic pulmonary fibrosis (IPF) patients, and ten new-onset non-small cell lung cancer patients. Bronchoalveolar lavage fluid samples were collected for 16S rRNA gene sequencing. The percentages of immune cells were determined using manual counting and flow cytometry. Interactions among microbiota, metabolites, and lymphocytes were analyzed using cultured mouse splenocytes and human T cells. FINDINGS: Proteobacteria emerged as the dominant phylum, notably abundant in both the CIP and IPF groups. Vibrio, Halomonas, Mangrovibacter, and Salinivibrio were the predominant microbiota because of their discriminative abundance patterns. Vibrio (r = 0.72, P-adj = 0.007) and Halomonas (r = 0.65, P-adj = 0.023) demonstrated strong correlations with lymphocytes. Vibrio metschnikovii and Mangrovibacter plantisponsors were more abundant in the CIP group than in the IPF group. Lauroylcarnitine, a key intermediary metabolite co-occurring with the predominant microbiota, exhibited a potent effect on cytokine secretion by mouse and human T cells, notably enhancing IFN-γ and TNF-α production from CD4 and CD8 cells in vitro. INTERPRETATION: Lauroylcarnitine, co-occurring with the predominant lung microbiota in CIP, could activate T cells in vitro. These findings suggest potential involvement of lung microbiota and acylcarnitine metabolism dysregulation in the pathogenesis of CIP. FUNDING: This work was supported by Peking University People's Hospital Scientific Research Development Funds (RDJ2022-15) and Provincial Key Clinical Specialty Capacity Building Project 2020 (Department of the Respiratory Medicine).

Imatinib therapy of chronic myeloid leukemia significantly reduces carnitine cell intake, resulting in adverse events.

OBJECTIVE: A prominent, safe and efficient therapy for patients with chronic myeloid leukemia (CML) is inhibiting oncogenic protein BCR::ABL1 in a targeted manner with imatinib, a tyrosine kinase inhibitor. A substantial part of patients treated with imatinib report skeletomuscular adverse events affecting their quality of life. OCTN2 membrane transporter is involved in imatinib transportation into the cells. At the same time, the crucial physiological role of OCTN2 is cellular uptake of carnitine which is an essential co-factor for the mitochondrial ß-oxidation pathway. This work investigates the impact of imatinib treatment on carnitine intake and energy metabolism of muscle cells. METHODS: HTB-153 (human rhabdomyosarcoma) cell line and KCL-22 (CML cell line) were used to study the impact of imatinib treatment on intracellular levels of carnitine and vice versa. The energy metabolism changes in cells treated by imatinib were quantified and compared to changes in cells exposed to highly specific OCTN2 inhibitor vinorelbine. Mouse models were used to test whether in vitro observations are also achieved in vivo in thigh muscle tissue. The analytes of interest were quantified using a Prominence HPLC system coupled with a tandem mass spectrometer. RESULTS: This work showed that through the carnitine-specific transporter OCTN2, imatinib and carnitine intake competed unequally and intracellular carnitine concentrations were significantly reduced. In contrast, carnitine preincubation did not influence imatinib cell intake or interfere with leukemia cell targeting. Blocking the intracellular supply of carnitine with imatinib significantly reduced the production of most Krebs cycle metabolites and ATP. However, subsequent carnitine supplementation rescued mitochondrial energy production. Due to specific inhibition of OCTN2 activity, the influx of carnitine was blocked and mitochondrial energy metabolism was impaired in muscle cells in vitro and in thigh muscle tissue in a mouse model. CONCLUSIONS: This preclinical experimental study revealed detrimental effect of imatinib on carnitine-mediated energy metabolism of muscle cells providing a possible molecular background of the frequently occurred side effects during imatinib therapy such as fatigue, muscle pain and cramps.

Metabolomic Profiling of Tumor Tissues Unveils Metabolic Shifts in Non-Small Cell Lung Cancer Patients with Concurrent Diabetes Mellitus.

A comprehensive understanding of the exact influence of type 2 diabetes mellitus (T2DM) on the metabolic status of non-small cell lung cancer (NSCLC) is still lacking. This study explores metabolic alterations in tumor tissues among patients with coexisting NSCLC and T2DM in comparison with NSCLC patients. A combined approach of clinical analysis and metabolomics was employed, including 20 NSCLC patients and 20 NSCLC+T2DM patients. Targeted metabolomics analysis was performed on tumor tissues using the liquid chromatography-mass spectrometry (LC-MS) approach. A clear segregation was observed between NSCLC+T2DM and matched NSCLC tissue samples in Orthogonal Partial Least Squares Discrimination Analysis (OPLS-DA). Furthermore, the levels of 7 metabolites are found to be significantly different between diabetes/nondiabetes tumor tissue samples. The related pathways included arginine biosynthesis, glutathione metabolism, arginine and proline metabolism, purine metabolism, biotin metabolism, and histidine metabolism. 3-Phenyllactic acid, carnitine-C5, carnitine-C12, and serotonin showed a positive linear correlation with fasting blood glucose levels in NSCLC patients. Uridine, pipecolic acid, cytosine, and fasting blood glucose levels were found to have a negative correlation. Our results suggest that NSCLC patients with concurrent T2DM exhibit distinct metabolic shifts in tumor tissues compared to those of solely NSCLC patients.

Characterising the urinary acylcarnitine and amino acid profiles of HIV/TB co-infection, using LC-MS metabolomics.

INTRODUCTION: The human immunodeficiency virus (HIV) and tuberculosis (TB) co-infection presents significant challenges due to the complex interplay between these diseases, leading to exacerbated metabolic disturbances. Understanding these metabolic profiles is crucial for improving diagnostic and therapeutic approaches. OBJECTIVE: This study aimed to characterise the urinary acylcarnitine and amino acid profiles, including 5-hydroxyindoleacetic acid (5-HIAA), in patients co-infected with HIV and TB using targeted liquid chromatography mass spectrometry (LC-MS) metabolomics. METHODS: Urine samples, categorised into HIV, TB, HIV/TB co-infected, and healthy controls, were analysed using HPLC-MS/MS. Statistical analyses included one-way ANOVA and a Kruskal-Wallis test to determine significant differences in the acylcarnitine and amino acid profiles between groups. RESULTS: The study revealed significant metabolic alterations, especially in TB and co-infected groups. Elevated levels of medium-chain acylcarnitines indicated increased fatty acid oxidation, commonly associated with cachexia in TB. Altered amino acid profiles suggested disruptions in protein and glucose metabolism, indicating a shift towards diabetes-like metabolic states. Notably, TB was identified as a primary driver of these changes, affecting protein turnover, and impacting energy metabolism in co-infected patients. CONCLUSION: The metabolic profiling of HIV/TB co-infection highlights the profound impact of TB on metabolic pathways, which may exacerbate the clinical complexities of co-infection. Understanding these metabolic disruptions can guide the development of targeted treatments and improve management strategies, ultimately enhancing the clinical outcomes for these patients. Further research is required to validate these findings and explore their implications in larger, diverse populations.

Exploring the interplay between kidney function and urinary metabolites in young adults: the African-PREDICT study.

The exposure to modifiable risk factors at young ages have been linked to premature fatal and non-fatal cardiovascular and kidney outcomes. The use of urinary metabolomics has shown strong predictability of kidney function and cardiovascular disease (CVD). We therefore determined the associations between estimated glomerular filtration rate (eGFR) and urinary metabolites in young adults with and without CVD risk factors. Apparently healthy Black and White sexes were included (aged 20-30 years) and categorised by the presence or absence of risk factors, i.e., obesity, physical inactivity, smoking, excessive alcohol intake, masked hypertension, hyperglycemia, dyslipidemia and low socio-economic status, forming the CVD risk group (N = 1036), CVD risk clusters (i.e. presenting with 1 CVD risk factor (N = 344), 2 CVD risk factors (N = 360) and 3 + CVD risk factors (N = 332)) and the control group (N = 166). eGFR was calculated with CKD-EPI equations. A targeted metabolomics approach using liquid chromatography-tandem mass spectrometry was used to measure amino acids and acylcarnitines. Lower cystatin C-based eGFR were indicated in the CVD risk group, 2 and 3 + CVD risk clusters compared to the control group (all P ≤ 0.033). In the CVD risk group, eGFR associated positively with histidine, lysine, asparagine, glycine, serine, glutamine, dimethylglycine, threonine, alanine, creatine, cystine, methionine, tyrosine, pyroglutamic acid, leucine/isoleucine, aspartic acid, tryptophan, glutamic acid, free carnitine, acetylcarnitine, propionylcarnitine, isovalerylcarnitine, octanoylcarnitine and decanoylcarnitine (all P ≤ 0.044), with similar results found in the CVD risk clusters, particularly the 2 CVD risk cluster. eGFR was positively associated with metabolites linked to aromatic amino acid and branched-chain amino acid metabolism, energy metabolism and oxidative stress. These findings may indicate altered reabsorption of these metabolites or altered metabolic regulation to preserve renal health in the setting of CVD risk factors at this young age without established CVD.

Disorders of fatty acid metabolism and imbalance in the ratio of monounsaturated fatty acids promote the development of pulmonary fibrosis.

OBJECTIVE: Although some studies suggested that metabolic abnormalities may contribute to the development of pulmonary fibrosis, there are no studies that have reported a clear causal relationship between them, and the aim of this study was to explore the causal relationship between plasma metabolites and pulmonary fibrosis using Mendelian randomization (MR) combined with metabolomics analysis. METHODS: Firstly, we explored the causal relationship between 1400 metabolites and pulmonary fibrosis using MR analysis, and detected plasma metabolites in mice with pulmonary fibrosis using metabolomics technology, thus validating the results of MR analysis. In addition, we again used MR to explore the causal relationship between the results of the differential metabolite KEGG in metabolomics and pulmonary fibrosis. RESULTS: A total of 52 metabolites were screened for association with pulmonary fibrosis in the MR analysis of 1400 plasma metabolites with pulmonary fibrosis, based on P < 0.05 for the IVW method, with consistent OR directions for all methods. Four of them were validated in the plasma of mice with pulmonary fibrosis, namely carnitine c18:2 levels (negative correlation), Glutamine degradant levels (positive correlation), Propionylcarnitine (c3) levels (negative correlation), carnitine to palmitoylcarnitine (c16) ratio (negative correlation). In addition, KEGG analysis of plasma differential metabolites revealed that the signaling pathway of biosynthetic of unsaturated fatty acids was most affected in mice with pulmonary fibrosis, and MR analysis showed that imbalance in the ratio of monounsaturated fatty acids was significantly associated with pulmonary fibrosis. CONCLUSIONS: Our study suggests that abnormal fatty acid levels due to reduced levels of carnitine-like metabolites, and an imbalance in the ratio of monounsaturated, promote the development of pulmonary fibrosis. This study reveals the marker metabolites and metabolic pathways affecting the development of pulmonary fibrosis to provide a basis for the development of new drugs for the treatment of pulmonary fibrosis.

Rats exposed to Alternaria toxins in vivo exhibit altered liver activity highlighted by disruptions in riboflavin and acylcarnitine metabolism.

Natural toxins produced by Alternaria fungi include the mycotoxins alternariol, tenuazonic acid and altertoxins I and II. Several of these toxins have shown high toxicity even at low levels including genotoxic, mutagenic, and estrogenic effects. However, the metabolic effects of toxin exposure from Alternaria are understudied, especially in the liver as a key target. To gain insight into the impact of Alternaria toxin exposure on the liver metabolome, rats (n = 21) were exposed to either (1) a complex culture extract with defined toxin profiles from Alternaria alternata (50 mg/kg body weight), (2) the isolated, highly genotoxic altertoxin-II (ATX-II) (0.7 mg/kg of body weight) or (3) a solvent control. The complex mixture contained a spectrum of Alternaria toxins including a controlled dose of ATX-II, matching the concentration of the isolated ATX-II. Liver samples were collected after 24 h and analyzed via liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Authentic reference standards (> 100) were used to identify endogenous metabolites and exogenous compounds from the administered exposures in tandem with SWATH-acquired MS/MS data which was used for non-targeted analysis/screening. Screening for metabolites produced by Alternaria revealed several compounds solely isolated in the liver of rats exposed to the complex culture, confirming results from a previously performed targeted biomonitoring study. This included the altersetin and altercrasin A that were tentatively identified. An untargeted metabolomics analysis found upregulation of acylcarnitines in rats receiving the complex Alternaria extract as well as downregulation of riboflavin in rats exposed to both ATX-II and the complex mixture. Taken together, this work provides a mechanistic view of Alternari toxin exposure and new suspect screening insights into hardly characterized Alternaria toxins.

Plasma metabolomics reveals the shared and distinct metabolic disturbances associated with cardiovascular events in coronary artery disease.

Risk prediction for subsequent cardiovascular events remains an unmet clinical issue in patients with coronary artery disease. We aimed to investigate prognostic metabolic biomarkers by considering both shared and distinct metabolic disturbance associated with the composite and individual cardiovascular events. Here, we conducted an untargeted metabolomics analysis for 333 incident cardiovascular events and 333 matched controls. The cardiovascular events were designated as cardiovascular death, myocardial infarction/stroke and heart failure. A total of 23 shared differential metabolites were associated with the composite of cardiovascular events. The majority were middle and long chain acylcarnitines. Distinct metabolic patterns for individual events were revealed, and glycerophospholipids alteration was specific to heart failure. Notably, the addition of metabolites to clinical markers significantly improved heart failure risk prediction. This study highlights the potential significance of plasma metabolites on tailed risk assessment of cardiovascular events, and strengthens the understanding of the heterogenic mechanisms across different events.

Acylcarnitines are associated with lower depressive symptomatology in a mainland puerto rican cohort.

INTRODUCTION: Recent studies have implicated acetyl-L-carnitine as well as other acylcarnitines in depression. To our knowledge, no untargeted metabolomics studies have been conducted among US mainland Puerto Ricans. OBJECTIVES: We conducted untargeted metabolomic profiling on plasma from 736 participants of the Boston Puerto Rican Health Study. METHODS: Using Weighted Gene Co-expression Network Analysis, we identified metabolite modules associated with depressive symptomatology, assessed via the Center for Epidemiologic Studies Depression scale. We identified metabolites contributing to these modules and assessed the relationship between these metabolites and depressive symptomatology. RESULTS: 621 annotated metabolites clustered into eight metabolite modules, of which one, the acylcarnitine module, was significantly inversely associated with depressive symptomatology (ß = - 27.7 (95% CI (- 54.5-0.8); p = 0.043). Several metabolite hub features in the acylcarnitine module were significantly associated with depressive symptomatology, after correction for multiple comparisons. CONCLUSIONS: In this untargeted plasma metabolomics study among mainland Puerto Rican older adults, acylcarnitines, as a metabolite module were inversely associated with depressive symptomatology.

Simultaneous determination of choline, L-carnitine, betaine, trimethylamine, trimethylamine N-oxide, and creatinine in plasma, liver, and feces of hyperlipidemic rats by UHPLC-MS/MS.

BACKGROUND: Due to the close correlation between choline, L-carnitine, betaine and their intestinal microbial metabolites, including trimethylamine (TMA) and trimethylamine N-oxide (TMAO), and creatinine, there has been an increasing interest in the study of these compounds in vivo. METHODS: In this study, a rapid stable isotope dilution (SID)-UHPLC-MS/MS method was developed for the simultaneous determination of choline, L-carnitine, betaine, TMA, TMAO and creatinine in plasma, liver and feces of rats. The method was validated using quality control (QC) samples spiked at low, medium and high levels. Second, we applied the method to quantify the effects of Rosa Roxburghii Tratt juice (RRTJ) on plasma, liver, and fecal levels of choline, L-carnitine, betaine, TMA, TMAO, and creatinine in high-fat diet-induced hyperlipidemic rats, demonstrating the utility of the method. RESULTS: The limits of detection (LOD) were 0.04-0.027 µM and the limits of quantification (LOQ) were 0.009-0.094 µM. The linear ranges for each metabolite in plasma were choline1.50-96 µM; L-carnitine: 2-128 µM; betaine: 3-192 µM; TMA: 0.01-40.96 µM; TMAO: 0.06-61.44 µM and creatinine: 1-64 µM (R2 ≥ 0.9954). The linear ranges for each metabolite in liver were Choline: 12-768 µM; L-carnitine: 1.5-96 µM; betaine: 10-640 µM; TMA: 0.5-32 µM; TMAO: 0.02-81.92 µM and creatinine: 0.2-204.8 µM (R2 ≥ 0.9938). The linear ranges for each metabolite in feces were choline: 1.5-96 µM; L-carnitine: 0.01-40.96 µM; Betaine: 1.5-96 µM; TMA: 1-64 µM; TMAO: 0.02-81.92 µM and Creatinine: 0.02-81.92 µM (R2 ≥ 0.998). The intra-day and inter-day coefficients of variation were < 8 % for all analytes. The samples were stabilized after multiple freeze-thaw cycles (3 freeze-thaw cycles), 24 h at room temperature, 24 h at 4 °C and 20 days at -80 °C. The samples were stable. The average recovery was 89 %-99 %. This method was used to quantify TMAO and its related metabolites and creatinine levels in hyperlipidemic rats. The results showed that high-fat diet led to the disorder of TMAO and its related metabolites and creatinine in rats, which was effectively improved after the intervention of Rosa Roxburghii Tratt juice（RRTJ）. CONCLUSIONS: A method for the determination of choline, L-carnitine, betaine, TMA, TMAO and creatinine in plasma, liver and feces samples was established, which is simple, time-saving, high precision, accuracy and recovery.

Adiposity in mares induces insulin dysregulation and mitochondrial dysfunction which can be mitigated by nutritional intervention.

Obesity is a complex disease associated with augmented risk of metabolic disorder development and cellular dysfunction in various species. The goal of the present study was to investigate the impacts of obesity on the metabolic health of old mares as well as test the ability of diet supplementation with either a complex blend of nutrients designed to improve equine metabolism and gastrointestinal health or L-carnitine alone to mitigate negative effects of obesity. Mares (n = 19, 17.9 ± 3.7 years) were placed into one of three group: normal-weight (NW, n = 6), obese (OB, n = 7) or obese fed a complex diet supplement for 12 weeks (OBD, n = 6). After 12 weeks and completion of sample collections, OB mares received L-carnitine alone for an additional 6 weeks. Obesity in mares was significantly associated with insulin dysregulation, reduced muscle mitochondrial function, and decreased skeletal muscle oxidative capacity with greater ROS production when compared to NW. Obese mares fed the complex diet supplement had better insulin sensivity, greater cell lipid metabolism, and higher muscle oxidative capacity with reduced ROS production than OB. L-carnitine supplementation alone did not significantly alter insulin signaling, but improved lipid metabolism and muscle oxidative capacity with reduced ROS. In conclusion, obesity is associated with insulin dysregulation and altered skeletal muscle metabolism in older mares. However, dietary interventions are an effective strategy to improve metabolic status and skeletal muscle mitochondrial function in older mares.

The Multifaceted Cause of Lipid Storage Myopathies, Genetics, and Treatment.

Several inherited metabolic fatty acid disorders present with myopathies. Skeletal muscle accounts for 40% of the body and is important for metabolism, exercise, and movement. Muscle energy failure is manifested by metabolic crises with muscle weakness, sometimes associated with muscle fatigue and failure resulting in acute necrosis or rhabdomyolysis/myoglobinuria episodes. Lack of energy leads to muscle necrosis. Other presentations are weakness and myalgias with lipid storage myopathies in the biopsy. The biomarkers of such disorders are acyl-carnitine with various profiles and need to be carefully evaluated to plan supplementary therapy and specific diets. If red flags are not distinctly followed and diagnosed in time they might lead to a metabolic or cardiac failure.

Creatine and L-carnitine attenuate muscular laminopathy in the LMNA mutation transgenic zebrafish.

Lamin A/C gene (LMNA) mutations contribute to severe striated muscle laminopathies, affecting cardiac and skeletal muscles, with limited treatment options. In this study, we delve into the investigations of five distinct LMNA mutations, including three novel variants and two pathogenic variants identified in patients with muscular laminopathy. Our approach employs zebrafish models to comprehensively study these variants. Transgenic zebrafish expressing wild-type LMNA and each mutation undergo extensive morphological profiling, swimming behavior assessments, muscle endurance evaluations, heartbeat measurement, and histopathological analysis of skeletal muscles. Additionally, these models serve as platform for focused drug screening. We explore the transcriptomic landscape through qPCR and RNAseq to unveil altered gene expression profiles in muscle tissues. Larvae of LMNA(L35P), LMNA(E358K), and LMNA(R453W) transgenic fish exhibit reduced swim speed compared to LMNA(WT) measured by DanioVision. All LMNA transgenic adult fish exhibit reduced swim speed compared to LMNA(WT) in T-maze. Moreover, all LMNA transgenic adult fish, except LMNA(E358K), display weaker muscle endurance than LMNA(WT) measured by swimming tunnel. Histochemical staining reveals decreased fiber size in all LMNA mutations transgenic fish, excluding LMNA(WT) fish. Interestingly, LMNA(A539V) and LMNA(E358K) exhibited elevated heartbeats. We recognize potential limitations with transgene overexpression and conducted association calculations to explore its effects on zebrafish phenotypes. Our results suggest lamin A/C overexpression may not directly impact mutant phenotypes, such as impaired swim speed, increased heart rates, or decreased muscle fiber diameter. Utilizing LMNA zebrafish models for drug screening, we identify L-carnitine treatment rescuing muscle endurance in LMNA(L35P) and creatine treatment reversing muscle endurance in LMNA(R453W) zebrafish models. Creatine activates AMPK and mTOR pathways, improving muscle endurance and swim speed in LMNA(R453W) fish. Transcriptomic profiling reveals upstream regulators and affected genes contributing to motor dysfunction, cardiac anomalies, and ion flux dysregulation in LMNA mutant transgenic fish. These findings faithfully mimic clinical manifestations of muscular laminopathies, including dysmorphism, early mortality, decreased fiber size, and muscle dysfunction in zebrafish. Furthermore, our drug screening results suggest L-carnitine and creatine treatments as potential rescuers of muscle endurance in LMNA(L35P) and LMNA(R453W) zebrafish models. Our study offers valuable insights into the future development of potential treatments for LMNA-related muscular laminopathy.

An untargeted analytical workflow based on Kendrick mass defect filtering reveals dysregulations in acylcarnitines in prostate cancer tissue.

BACKGROUND: Metabolomics is nowadays considered one the most powerful analytical for the discovery of metabolic dysregulations associated with the insurgence of cancer, given the reprogramming of the cell metabolism to meet the bioenergetic and biosynthetic demands of the malignant cell. Notwithstanding, several challenges still exist regarding quality control, method standardization, data processing, and compound identification. Therefore, there is a need for effective and straightforward approaches for the untargeted analysis of structurally related classes of compounds, such as acylcarnitines, that have been widely investigated in prostate cancer research for their role in energy metabolism and transport and ß-oxidation of fatty acids. RESULTS: In the present study, an innovative analytical platform was developed for the straightforward albeit comprehensive characterization of acylcarnitines based on high-resolution mass spectrometry, Kendrick mass defect filtering, and confirmation by prediction of their retention time in reversed-phase chromatography. In particular, a customized data processing workflow was set up on Compound Discoverer software to enable the Kendrick mass defect filtering, which allowed filtering out more than 90 % of the initial features resulting from the processing of 25 tumoral and adjacent non-malignant prostate tissues collected from patients undergoing radical prostatectomy. Later, a partial least square-discriminant analysis model validated by repeated double cross-validation was built on the dataset of 74 annotated acylcarnitines, with classification rates higher than 93 % for both groups, and univariate statistical analysis helped elucidate the individual role of the annotated metabolites. SIGNIFICANCE: Hydroxylation of short- and medium-chain minor acylcarnitines appeared to be a significant variable in describing tissue differences, suggesting the hypothesis that the neoplastic growth is linked to oxidation phenomena on selected metabolites and reinforcing the need for effective methods for the annotation of minor metabolites.

Liver sinusoidal endothelial cells rely on oxidative phosphorylation but avoid processing long-chain fatty acids in their mitochondria.

BACKGROUND: It is generally accepted that endothelial cells (ECs), primarily rely on glycolysis for ATP production, despite having functional mitochondria. However, it is also known that ECs are heterogeneous, and their phenotypic features depend on the vascular bed. Emerging evidence suggests that liver sinusoidal ECs (LSECs), located in the metabolically rich environment of the liver, show high metabolic plasticity. However, the substrate preference for energy metabolism in LSECs remains unclear. METHODS: Investigations were conducted in primary murine LSECs in vitro using the Seahorse XF technique for functional bioenergetic assays, untargeted mass spectrometry-based proteomics to analyse the LSEC proteome involved in energy metabolism pathways, liquid chromatography-tandem mass spectrometry-based analysis of acyl-carnitine species and Raman spectroscopy imaging to track intracellular palmitic acid. RESULTS: This study comprehensively characterized the energy metabolism of LSECs, which were found to depend on oxidative phosphorylation, efficiently fuelled by glucose-derived pyruvate, short- and medium-chain fatty acids and glutamine. Furthermore, despite its high availability, palmitic acid was not directly oxidized in LSEC mitochondria, as evidenced by the acylcarnitine profile and etomoxir's lack of effect on oxygen consumption. However, together with L-carnitine, palmitic acid supported mitochondrial respiration, which is compatible with the chain-shortening role of peroxisomal ß-oxidation of long-chain fatty acids before further degradation and energy generation in mitochondria. CONCLUSIONS: LSECs show a unique bioenergetic profile of highly metabolically plastic ECs adapted to the liver environment. The functional reliance of LSECs on oxidative phosphorylation, which is not a typical feature of ECs, remains to be determined.