Determination of carnitine, its short chain acyl esters and metabolic precursors trimethyllysine and γ-butyrobetaine by quasi-solid phase extraction and MS/MS detection

Hirche, Frank; Fischer, Matthias; Keller, Janine; Eder, Klaus

doi:10.1016/j.jchromb.2009.05.048

Cited by 45 publications

(36 citation statements)

References 22 publications

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“…Most of the carnitine is found free in the plasma of humans and the levels are directly correlating with dietary intake (31), however during fasting carnitine uptake into cells is increased resulting in increased β-oxidation (32). Endogenous production of carnitine is based on conversion of the amino acid lysine to TML through methylation of the ε-amino group and subsequent release of TML upon protein degradation (33, 34). TML is further oxidized to γ-butyrobetaine, which is hydroxylated in the liver and kidney to form carnitine (33).…”

Section: Discussionmentioning

confidence: 99%

“…Endogenous production of carnitine is based on conversion of the amino acid lysine to TML through methylation of the ε-amino group and subsequent release of TML upon protein degradation (33, 34). TML is further oxidized to γ-butyrobetaine, which is hydroxylated in the liver and kidney to form carnitine (33). Inside the mitochondria, the carnitine conjugate acetylcarnitine regulates the levels of acetyl-CoA (35), which is essential for the balance between fatty acid and carbohydrate metabolism (30).…”

Section: Discussionmentioning

confidence: 99%

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Development of a Metabolomic Radiation Signature in Urine from Patients Undergoing Total Body Irradiation

et al. 2014

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The emergence of the threat of radiological terrorism and other radiological incidents has led to the need for development of fast, accurate and noninvasive methods for detection of radiation exposure. The purpose of this study was to extend radiation metabolomic biomarker discovery to humans, as previous studies have focused on mice. Urine was collected from patients undergoing total body irradiation at Memorial Sloan-Kettering Cancer Center prior to hematopoietic stem cell transplantation at 4–6 h postirradiation (a single dose of 1.25 Gy) and 24 h (three fractions of 1.25 Gy each). Global metabolomic profiling was obtained through analysis with ultra performance liquid chromatography coupled to time-of-flight mass spectrometry (TOFMS). Prior to further analyses, each sample was normalized to its respective creatinine level. Statistical analysis was conducted by the nonparametric Kolmogorov-Smirnov test and the Fisher’s exact test and markers were validated against pure standards. Seven markers showed distinct differences between pre- and post-exposure samples. Of those, trimethyl-l-lysine and the carnitine conjugates acetylcarnitine, decanoylcarnitine and octanoylcarnitine play an important role in the transportation of fatty acids across mitochondria for subsequent fatty acid β-oxidation. The remaining metabolites, hypoxanthine, xanthine and uric acid are the final products of the purine catabolism pathway, and high levels of excretion have been associated with increased oxidative stress and radiation induced DNA damage. Further analysis revealed sex differences in the patterns of excretion of the markers, demonstrating that generation of a sex-specific metabolomic signature will be informative and can provide a quick and reliable assessment of individuals in a radiological scenario. This is the first radiation metabolomics study in human urine laying the foundation for the use of metabolomics in biodosimetry and providing confidence in biomarker identification based on the overlap between animal models and humans.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Development of a Metabolomic Radiation Signature in Urine from Patients Undergoing Total Body Irradiation

et al. 2014

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“…Although no commercial kit (analogous to those for amino acid analysis) exists for acylcarnitine analysis, many groups have published second-tier methods that are, to varying degrees, more selective and quantitatively rigorous than tandem MS “profiles” [21,22,23,24]. We developed second-tier methods for the quantification of carnitine beginning 40 years ago, using radioactively labeled acetyl-CoA and measuring the production of radiolabeled acetylcarnitine [25,26].…”

Section: Introductionmentioning

confidence: 99%

Quantitative acylcarnitine determination by UHPLC-MS/MS — Going beyond tandem MS acylcarnitine “profiles”

Minkler

Stoll

Ingalls

et al. 2015

Molecular Genetics and Metabolism

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Tandem MS “profiling” of acylcarnitines and amino acids was conceived as a first-tier screening method, and its application to expanded newborn screening has been enormously successful. However, unlike amino acid screening (which uses amino acid analysis as its second-tier validation of screening results), acylcarnitine “profiling” also assumed the role of second-tier validation, due to the lack of a generally accepted second-tier acylcarnitine determination method. In this report, we present results from the application of our validated UHPLC-MS/MS second-tier method for the quantification of total carnitine, free carnitine, butyrobetaine, and acylcarnitines to patient samples with known diagnoses: malonic acidemia, short-chain acyl-CoA dehydrogenase deficiency (SCADD) or isobutyryl-CoA dehydrogenase deficiency (IBD), 3-methyl-crotonyl carboxylase deficiency (3-MCC) or β-ketothiolase deficiency (BKT), and methylmalonic acidemia (MMA). We demonstrate the assay’s ability to separate constitutional isomers and diastereomeric acylcarnitines and generate values with a high level of accuracy and precision. These capabilities are unavailable when using tandem MS “profiles”. We also show examples of research interest, where separation of acylcarnitine species and accurate and precise acylcarnitine quantification is necessary.

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“…Concentrations of free carnitine and acetyl carnitine in plasma and muscle were determined by tandem mass spectrometry using deuterated carnitine-d 3 as internal standard as described recently in detail [44]. Concentration of total carnitine was calculated as the sum of free and acetyl carnitine.…”

Section: Methodsmentioning

confidence: 99%

Supplemental carnitine affects the microRNA expression profile in skeletal muscle of obese Zucker rats

2014

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BackgroundIn the past, numerous studies revealed that supplementation with carnitine has multiple effects on performance characteristics and gene expression in livestock and model animals. The molecular mechanisms underlying these observations are still largely unknown. Increasing evidence suggests that microRNAs (miRNAs), a class of small non-coding RNA molecules, play an important role in post-transcriptional regulation of gene expression and thereby influencing several physiological and pathological processes. Based on these findings, the aim of the present study was to investigate the influence of carnitine supplementation on the miRNA expression profile in skeletal muscle of obese Zucker rats using miRNA microarray analysis.ResultsObese Zucker rats supplemented with carnitine had higher concentrations of total carnitine in plasma and muscle than obese control rats (P < 0.05). miRNA expression profiling in skeletal muscle revealed a subset of 152 miRNAs out of the total number of miRNAs analysed (259) were identified to be differentially regulated (adjusted P-value < 0.05) by carnitine supplementation. Compared to the obese control group, 111 miRNAs were up-regulated and 41 down-regulated by carnitine supplementation (adjusted P-value < 0.05). 14 of these miRNAs showed a log2 ratio ≥ 0.5 and 7 miRNAs showed a log2 ratio ≤ −0.5 (adjusted P-value < 0.05). After confirmation by qRT-PCR, 11 miRNAs were found to be up-regulated and 6 miRNAs were down-regulated by carnitine supplementation (P < 0.05). Furthermore, a total of 1,446 target genes within the validated miRNAs were revealed using combined three bioinformatic algorithms. Analysis of Gene Ontology (GO) categories and KEGG pathways of the predicted targets revealed that carnitine supplementation regulates miRNAs that target a large set of genes involved in protein-localization and -transport, regulation of transcription and RNA metabolic processes, as well as genes involved in several signal transduction pathways, like ubiquitin-mediated proteolysis and longterm depression, are targeted by the miRNAs regulated by carnitine supplementation.ConclusionThe present study shows for the first time that supplementation of carnitine affects a large set of miRNAs in skeletal muscle of obese Zucker rats suggesting a novel mechanism through which carnitine exerts its multiple effects on gene expression, which were observed during the past.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-512) contains supplementary material, which is available to authorized users.

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Determination of carnitine, its short chain acyl esters and metabolic precursors trimethyllysine and γ-butyrobetaine by quasi-solid phase extraction and MS/MS detection

Cited by 45 publications

References 22 publications

Development of a Metabolomic Radiation Signature in Urine from Patients Undergoing Total Body Irradiation

Development of a Metabolomic Radiation Signature in Urine from Patients Undergoing Total Body Irradiation

Quantitative acylcarnitine determination by UHPLC-MS/MS — Going beyond tandem MS acylcarnitine “profiles”

Supplemental carnitine affects the microRNA expression profile in skeletal muscle of obese Zucker rats

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