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Acylcarnitine
Acylcarnitine
Acylcarnitines are a class of organic compounds that play a crucial role in the transport of fatty acids across the mitochondrial membrane for energy production.
They are formed by the esterification of carnitine with various acyl groups, enabling the entry of long-chain fatty acids into the mitochondria.
Acylcarnitines are important biomarkers for the diagnosis and monitoring of metabolic disorders, such as fatty acid oxidation defects and carnitine deficiency.
They also have potential applications in the study of energy metabolism, signaling pathways, and therapeutic interventions.
Researchers can optimie their Acylcarnitine studies by utilizing PubCompare.ai, an AI-driven platform that helps locate the most reliable and effective research protocols from literature, preprints, and patents.
This tool enhances reproducibility and accuracy, ensuring researchers find the most suitable methods for their Acylcarnitine investigations and take their studies to the next levl.
They are formed by the esterification of carnitine with various acyl groups, enabling the entry of long-chain fatty acids into the mitochondria.
Acylcarnitines are important biomarkers for the diagnosis and monitoring of metabolic disorders, such as fatty acid oxidation defects and carnitine deficiency.
They also have potential applications in the study of energy metabolism, signaling pathways, and therapeutic interventions.
Researchers can optimie their Acylcarnitine studies by utilizing PubCompare.ai, an AI-driven platform that helps locate the most reliable and effective research protocols from literature, preprints, and patents.
This tool enhances reproducibility and accuracy, ensuring researchers find the most suitable methods for their Acylcarnitine investigations and take their studies to the next levl.
Most cited protocols related to «Acylcarnitine»
3-Hydroxybutyrate
Acids
acylcarnitine
Acyl Coenzyme A
Amino Acids
BLOOD
Capillaries
Cholesterol
Diagnosis
Electrons
Fatty Acids, Esterified
Gas Chromatography-Mass Spectrometry
Glycerin
Isoleucine
Isotopes
Keto Acids
Ketogenic Diet
Ketones
Lactates
Leucine
Liver
Muscle, Gastrocnemius
Plasma
Tandem Mass Spectrometry
Technique, Dilution
Triglycerides
Valine
Acetylcarnitine
acylcarnitine
Acyl Coenzyme A
Fatty Acids, Esterified
Heart
Insulin
Isotopes
Plasma
Tandem Mass Spectrometry
Technique, Dilution
Tissues
Triglycerides
Acetonitrile (high-performance liquid chromatography-grade) and high-purity water were obtained from Thermo Fisher (Waltham, MA, USA). 1-Butanol and acetyl chloride were commercially acquired from Sigma-Aldrich (St Louis, MO, USA). Isotope-labeled internal standards of 12 amino acids (NSK-A) and eight acylcarnitine (NSK-B) from Cambridge Isotope Laboratories (Tewksbury, MA, USA) were used for absolute quantification purpose. All the standards were mixed and dissolved in 2 mL pure methanol and stored at 4°C. Working solution was prepared through 100-fold dilution for metabolite extraction. Amino acids and carnitines quality control (QC) standards were provided by Chromsystems (Grafelfing, Germany). The QC samples were treated as real samples and processed according to the provided instructions to ensure the analysis stability.
acetonitrile
acetyl chloride
acylcarnitine
Amino Acids
Butyl Alcohol
Carnitine
High-Performance Liquid Chromatographies
Isotopes
Methanol
Technique, Dilution
Frozen plasma samples were used to quantitatively measure targeted metabolites, including 37 acylcarnitine species, 15 amino acids, 9 free fatty acids and conventional analytes, ketones and C-reactive protein (CRP). Sample preparation and coefficients of variation have been reported (Haqq et al, 2005 (link)). The laboratory was blinded to family identifiers and case–control status. Assay ranges are 0.05–40 μM (acylcarnitines); 5–1000 μM (amino acids) and 1–1000 mmol/l (fatty acids). For simplicity, the clinical shorthand of metabolites is used (Supplementary information ). Intra-individual variability was assessed in samples from five individuals for which repeat profiling was performed on the same sample on five separate days. Coefficients of variation and correlation confirmed minimal inter-assay variability (Supplementary information ).
acylcarnitine
Amino Acids
Biological Assay
C Reactive Protein
Fatty Acids
Freezing
Ketones
Nonesterified Fatty Acids
Plasma
A thorough literature search was performed to obtain an in-depth knowledge of all the major metabolic pathways known to occur in an epithelial cell of the small intestine. We then retrieved the corresponding reactions and genes from the human metabolic reconstruction (13 (link)), which is accessible through the BiGG database (70 (link)), to compile a draft reconstruction. Missing transport and metabolic reactions for peptides and for dietary fibers were added to the initial draft reconstruction upon detailed manual gap analysis and further review of the corresponding literature. Genome annotations from the EntrezGene database (71 (link)) as well as protein information from the UniProt (72 (link)) and BRENDA database (73 (link)) were used in addition to the information retrieved from the scientific literature to assign GPR associations to the reactions not present in Recon 1. For the reactions that were extracted from the Recon 1, GPR associations were kept as reported in Recon 1, since no comprehensive transcriptomic data are available for sIECs. The sIEC metabolic reconstruction was assembled and converted to a mathematical model using rBioNet as a reconstruction environment (74 (link)) and an established protocol (75 ).
We used the global human metabolic network, Recon 1 (13 (link)), as reaction database, but adjusted sub-cellular and extracellular location, reaction stoichiometry and directionality according to literature evidence. Only those reactions and pathways with literature evidence for their occurrence in human small intestinal enterocytes were incorporated into hs_sIEC611 from the global human metabolic reconstruction Recon 1, which captures metabolic capabilities known to occur in any human cell. Moreover, we added 262 transport and 50 metabolic reactions, which were not present in Recon 1, but for which supporting information for their presence in sIECs could be found in the scientific literature (Fig.1 E, Supplementary Material, Table S2 ). These reactions included many transport systems specific for enterocytes and metabolic pathways for sulfo-cysteine metabolism, dietary fiber metabolism, di- and tri-peptide degradation and cholesterol-ester synthesis (Fig. 1 E, Supplementary Material, Table S2 ). In addition to these reactions, 73 reactions were added from our recently published acylcarnitine/fatty acid oxidation module for the human metabolic reconstruction (20 (link)). We added further 95 reactions, which were present in Recon 1, but for which the compartment was adjusted by placing them into the lumen compartment. The stoichiometry of the reactions catalyzed by the glucose 6-phosphate dehydrogenase (E.C. 1.1.1.49), the 6-phosphogluconolactonase (E.C. 3.1.1.31) and the phosphogluconate dehydrogenase (E.C. 1.1.1.44) was changed to three, since they were required to generate three molecules each of 6-phospho-d -glucono-1,5-lactone, 6-Phospho-d -gluconate and ribulose-5-phosphate simultaneously (76 ). The directionality of ATP requiring fatty acid activation reactions catalyzed by the fatty acyl-CoA ligase (E.C. 6.2.1.3) was changed in agreement with a recent report (76 ). Also, the cofactor requirement and sub-cellular localization of reactions included in the cholesterol synthesis pathway, which are catalyzed by the desmosterol reductase (E.C. 1.3.1.72) and HMG-CoA reductase (E.C. 2.3.3.10) reactions, were updated in accordance with the current literature evidence (76 ).
We used the global human metabolic network, Recon 1 (13 (link)), as reaction database, but adjusted sub-cellular and extracellular location, reaction stoichiometry and directionality according to literature evidence. Only those reactions and pathways with literature evidence for their occurrence in human small intestinal enterocytes were incorporated into hs_sIEC611 from the global human metabolic reconstruction Recon 1, which captures metabolic capabilities known to occur in any human cell. Moreover, we added 262 transport and 50 metabolic reactions, which were not present in Recon 1, but for which supporting information for their presence in sIECs could be found in the scientific literature (Fig.
Most recents protocols related to «Acylcarnitine»
The pretreatment of samples for acylcarnitine analysis was conducted as previously published, with some modifications [28 (link)]. Briefly, 50 mg frozen tissue of mice were separately placed in the centrifuge tubes for extraction. Individual samples were added to 500 μL cold methanol and homogenized at 4 °C, and then the mixtures were sonicated for 15 min in ice water bath. Metabolite extracts were isolated by centrifugation at 12,000× g for 15 min at 4 °C, and the supernatants were separated and completely dried using lyophilization. The dried samples were re-dissolved into 20% methanol for targeted metabolomics analysis. Liquid chromatography–mass spectrometry-based targeted metabolite analysis was performed on a LC-QTRAP 5500+-MS/MS (Sciex, Concord, Vaughan, ON, USA). LC separations were carried out on a Kinetex C18 column (100 × 2.1 mm, particle size 2.6 μm, Phenomenex, Torrance, CA, USA) on reverse phase mode for 24 min. Column temperature and flow rate were set to 40 °C and 0.30 mL/min, respectively. The binary gradient system consisted of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). The linear gradient used for elution and equilibration of the initial gradient for subsequent runs was 5% B from 0 to 2 min, 5–45% B from 2 to 5 min, 45–100% B from 5 to 15 min, 100% B from 15 to 19 min, 100–5% B from 19 to 20 min, and 5% B from 20 to 24 min. The unlabeled and labeled Carnitine Standards Set B (Cambridge Isotope Laboratories, Tewksbury, MA, USA) were used for the standard.
We obtained a list of 32 acylcarnitine-associated SNPs (Supplementary Table 5 ) from a large GWAS study performed on a total of 7,824 individuals from the KORA and TwinsUK studies16 (link). Generalizability of effects of these SNPs to ADNI was tested using a targeted genetic association screening, where we tested for influences of all 32 SNPs against all 23 acylcarnitine species assuming an additive genetic model. Only age and sex were included as covariates, following the protocol of the reference study. Associations were considered to be significant if they had an FDR-adjusted p-value ≤ 0.05.
The reference GWAS further lists multi-SNP combinations of one lead SNP per associated locus that in concert explained the largest fraction of the heritable population variance of single acylcarnitine concentrations in blood. We used these multi-SNP combinations in epistatic modeling.
The reference GWAS further lists multi-SNP combinations of one lead SNP per associated locus that in concert explained the largest fraction of the heritable population variance of single acylcarnitine concentrations in blood. We used these multi-SNP combinations in epistatic modeling.
Blood samples were aseptically collected from both patients and control subjects following an overnight fast. The collected samples were subjected to a battery of tests, including CBC, INR, prothrombin time, blood chemistry, and acylcarnitine determination. In immediate succession to collection, 300 µL of each blood sample was dispensed onto three filter cards. These filter cards were then left to air-dry for 2-3 h in a dark room at room temperature. Each set of cards from individual samples was securely enclosed in an envelope, labeled with unique identifying codes, and preserved at -20 °C until required for the assay. Moreover, comprehensive assessments, encompassing liver function, renal function, serum cholesterol, and triglycerides, were performed using an automated biochemical analyzer (Bachman Ltd., London, UK).
The liquid chromatographic tandem mass spectrometric (LC-MS/MS) analyses were conducted on an Agilent Series system from Agilent Technologies (CA, USA), which was coupled with a 4000 Q-TRAP instrument from AB SCIEX (Foster City, CA, USA). The chromatographic separation was executed using a reverse-phase approach with mobile phases comprised of 0.1% (v/v) formic acid in bi-distilled water and 0.1% (v/v) formic acid in acetonitrile. The elution was carried out in a gradient mode following three different elution schemes as described in [25] . Acylcarnitines, including acetylcarnitine (AC2), propionylcarnitine (AC3), and others, were analyzed using standardized protocols for non-derivatized methods employing the NeoBase kit from Perki-nElmer (MA, USA). In brief, a single 5-mm DBS punch was placed in each well of a 96-well assay plate, and 100 μL of an extraction solution containing internal standards for acylcarnitines was added to each well. The plate was incubated at 45 °C for 45 min with a shaker set at 700 rpm. Subsequently, the supernatant was transferred to a new plate, followed by centrifugation at 1000 rpm for 5 min. Each well received 1 μL for injection into a C18 column (1.7 µm, 100 mm × 2.1 mm internal dimensions) of an ultra-performance liquid chromatography system with the column temperature maintained at 50 °C (Waters ACQUITY, Milford, MA, USA). The individual acylcarnitines were eluted using a gradient at a flow rate of 0.5 mL/min. This involved a 2-min run with 80% mobile phase A (0.001 formic acid in water) and 20% mobile phase B (acetonitrile), followed by a 5-min linear gradient of mobile phase B (20 to 30%), and concluding with 8 min of mobile phase B at 80%. The mass spectrometer featured an electrospray source that operated in the negative ion mode using the multiple reactions monitoring (MRM). The raw UPLC-MS data obtained in MRM mode were analyzed utilizing TargetLynx Application Manager version 4.1 from Waters Corp. (Milford, MA, USA) to derive the calibration equations and the quantitative concentration of each acylcarnitine in the samples.
The liquid chromatographic tandem mass spectrometric (LC-MS/MS) analyses were conducted on an Agilent Series system from Agilent Technologies (CA, USA), which was coupled with a 4000 Q-TRAP instrument from AB SCIEX (Foster City, CA, USA). The chromatographic separation was executed using a reverse-phase approach with mobile phases comprised of 0.1% (v/v) formic acid in bi-distilled water and 0.1% (v/v) formic acid in acetonitrile. The elution was carried out in a gradient mode following three different elution schemes as described in [25] . Acylcarnitines, including acetylcarnitine (AC2), propionylcarnitine (AC3), and others, were analyzed using standardized protocols for non-derivatized methods employing the NeoBase kit from Perki-nElmer (MA, USA). In brief, a single 5-mm DBS punch was placed in each well of a 96-well assay plate, and 100 μL of an extraction solution containing internal standards for acylcarnitines was added to each well. The plate was incubated at 45 °C for 45 min with a shaker set at 700 rpm. Subsequently, the supernatant was transferred to a new plate, followed by centrifugation at 1000 rpm for 5 min. Each well received 1 μL for injection into a C18 column (1.7 µm, 100 mm × 2.1 mm internal dimensions) of an ultra-performance liquid chromatography system with the column temperature maintained at 50 °C (Waters ACQUITY, Milford, MA, USA). The individual acylcarnitines were eluted using a gradient at a flow rate of 0.5 mL/min. This involved a 2-min run with 80% mobile phase A (0.001 formic acid in water) and 20% mobile phase B (acetonitrile), followed by a 5-min linear gradient of mobile phase B (20 to 30%), and concluding with 8 min of mobile phase B at 80%. The mass spectrometer featured an electrospray source that operated in the negative ion mode using the multiple reactions monitoring (MRM). The raw UPLC-MS data obtained in MRM mode were analyzed utilizing TargetLynx Application Manager version 4.1 from Waters Corp. (Milford, MA, USA) to derive the calibration equations and the quantitative concentration of each acylcarnitine in the samples.
Triglyceride concentrations in follicular fluid samples were determined using a colorimetric assay kit (Cayman Chemical, Ann Harbor, MI, USA) according to kit instructions. The 96-well, non-treated microplate was read at 540-nm absorbance on a Synergy 2 microplate reader (Biotek, Agilent, Santa Clara, CA, USA). All samples were assayed on a single plate. The intra-assay coefficient of variation was 1.35%, and the minimal detectable concentration was 1 mg/dL. Concentrations of non-esterified free fatty acids and insulin in follicular fluid were determined by a reference laboratory (Clinical Pathology Laboratory, Cornell University Animal Health Diagnostic Center, Ithaca, NY). Analyses of follicular fluid acylcarnitine profiles were conducted by a reference laboratory (University of Colorado Anschutz Medical Campus School of Medicine Metabolomics Core, Aurora, CO, USA) as previously described92 (see Appendix A.2 for the detailed method).
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More about "Acylcarnitine"
fatty acids, mitochondrial membrane, carnitine, metabolic disorders, fatty acid oxidation defects, carnitine deficiency, energy metabolism, signaling pathways, therapeutic interventions, Formic acid, Acquity UPLC system, MassLynx 4.1 operating system, Trace GC Ultra, Xcalibur 2.2, L-carnitine, TQD mass spectrometer, Triglyceride Quantification Kit, NEFA-HR kit