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Carnitine Biosynthesis Analysis Service

Carnitine (L-3-hydroxy-4-N,N,N-trimethylaminobutyrate) is an essential metabolite, which has a number of indispensable roles in intermediary metabolism. First, carnitine has an important role in the transport of activated long-chain fatty acids from the cytosol to the mitochondrial matrix, where β-oxidation takes place. Secondly, carnitine is involved in the transfer of the products of peroxisomal β-oxidation, including acetyl-CoA, to the mitochondria for oxidation to CO2 and H2O in the Krebs cycle. Other functions of carnitine include modulation of the acyl-CoA/CoA ratio, storage of energy as acetylcarnitine and the modulation of toxic effects of poorly metabolized acyl groups by excreting them as carnitine esters. Carnitine is present in most, if not all, animal species, and in several microorganisms and plants. Animal tissues contain relatively high amounts of carnitine, varying between 0.2 and 6 µmol.g-1, with the highest concentrations in heart and skeletal muscle. Although animals obtain carnitine primarily from the diet, most mammals are capable of synthesizing carnitine endogenously.

Carnitine Biosynthesis Analysis Service Figure 1. The carnitine biosynthesis pathway

Carnitine is synthesized ultimately from the amino acids lysine and methionine. Lysine provides the carbon backbone of carnitine and the 4-N-methyl groups originate from methionine. In mammals, certain proteins contain N- trimethyl-lysine (TML) residues. N-methylation of these lysine residues occurs as a post-translational event in proteins such as calmodulin, myosin, actin, cytochrome c and histones. This reaction is catalyzed by specific methyltransferases, which use S-adenosylmethionine as a methyl donor. Lysosomal hydrolysis of these proteins results in the release of TML, which is the first metabolite of carnitine biosynthesis. TML is first hydroxylated on the 3-position by TML dioxygenase to yield 3-hydroxy TML (HTML). Aldolytic cleavage of HTML yields 4-trimethylaminobutyraldehyde (TMABA) and glycine, a reaction catalysed by HTML aldolase (HTMLA). Dehydrogenation of TMABA by TMABA dehydrogenase (TMABA-DH) results in the formation of 4-Ntrimethylaminobutyrate (butyrobetaine). In the last step, butyrobetaine is hydroxylated on the 3-position by γ-butyrobetaine dioxygenase (BBD) to yield carnitine.

Carnitine Biosynthesis Analysis ServiceFigure 2 . The carnitine biosynthesis pathway

The tissue distribution of carnitine-biosynthetic enzymes in humans has been investigated. TMLD activity is highest in the kidney, but also present in the liver, heart, muscle and brain. HTMLA activity is found predominantly in the liver. In the other investigated tissues, the HTMLA activity is low. The rate of TMABA oxidation is greatest in the liver, with substantial activity also found in the kidney, but is low in brain, heart and muscle. These results show that all the investigated tissues contain the enzymes necessary to convert TML into butyrobetaine. However, only the kidney, liver and brain are capable of converting butyrobetaine into carnitine. BBD activity is 3–16-fold higher in the kidney than in liver. Activity in the brain only has been reported by Rebouche and Engel, and is 50% of the activity measured in the liver. As in the rat, liver BBD activity is regulated developmentally in humans. In contrast, kidney BBD activity is not age-dependent, since BBD activity is already present at adult levels in newborns. No evidence was found that the activity of the other three enzymes in liver is age-dependent.

Several methods have been described to measure the concentration of the carnitine biosynthesis metabolites in biological fluids and tissues. In Creative Proteomics, we recently developed a fast and easy method to determine the concentrations of the metabolites of the carnitine biosynthesis. Without prior purification, the sample is derivatized with methyl chloroformate, followed by separation of the analytes by reversed-phase ion-pair HPLC using hepta-fluorobutyric acid as an ion-pairing agent, and detection by electrospray tandem MS. With this method, TML, HTML, butyrobetaine and carnitine can be quantified in a single analysis. This new method is highly reproducible, and has a detection limit of 0.25 pmol for each compound.


  • LC-MS/MS


  • Identification and quantification of carnitine biosynthesis metabolites


  • A detailed technical report will be provided at the end of the whole project, including the experiment procedure, MS/MS instrument parameters.

  • Analytes are reported as uM or ug/mg (tissue), and CV's are generally<10%.

  • The name of the analytes, abbreviation, formula, molecular weight and CAS# would also be included in the report.

Carnitine Biosynthesis Metabolites Quantified in This Service

With integrated set of separation, characterization, identification and quantification systems featured with excellent robustness & reproducibility, high and ultra-sensitivity, Creative Proteomics provides reliable, rapid and cost-effective carnitine biosynthesis metabolites targeted metabolomics services.

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