Carnitine and acylcarnitines are fundamental compounds for fatty acids metabolism. They widely exist in animals, plants and some microorganisms. Carnitine ranges from 0.2 to 6 mmol/kg in animal tissues, especially high in the heart and skeletal muscle. With methionine and lysine from protein degradation as precursors, L-Carnitine are synthesized in kidney, liver and brain of organisms. Through the circulation, carnitine and acylcarnitines are transported to other tissues to fulfill their roles. Meat, fish and dairy products are the main source of carnitine.
Fatty acids must be oxidized through β-oxidation in mitochondria to generate energy. However, fatty acyl-CoA thioesters can’t directly pass through the inner membrane of mitochondrial. Carnitine plays a vital role in transporting fatty acyl-CoA thioesters into mitochondrial. Through the process, carnitine maintains the balance between free and acyl-CoA intermediates, which is potentially toxic to cells. Besides, carnitine assists the removal of excess acyl groups from mitochondria through the action of carnitine octanoyltransferase.
A number of enzymes are involved in the above-mentioned processes. CPT-I (carnitine palmitoyltransferase I) exists in the mitochondrial outer membrane, CPT-II (carnitine palmitoyltransferase II) locates on the matrix side of the inner membrane and carnitine: acylcarnitine translocase is an integral inner membrane protein. Firstly, on the outer membrane of mitochondrial, by binding to coenzyme A, fatty acids are activated and convert to highly polar acyl-CoA, which can’t pass the inner membrane of mitochondrial. Then, the acyl group is transferred from acyl-CoA to carnitine and form acylcarnitines, which, with the help of translocase, can enter inner membrane of mitochondrial. And then acyl-CoA is metabolized in two-carbon units through β-oxidation and regenerate acetyl-CoA. Finally, through action of carnitine acetyltransferase, acetyl groups are transferred to carnitine and form acetylcarnitine, which can be transported out of the mitochondria.
The deficiencies of any of these enzymes can lead to accumulation of acyl-CoA of specific chain lengths, which are toxic to cells if can’t removed in time from the body through the formation of acylcarnitines. Acylcarnitine analysis enables the diagnosis of for inherited disorders of fatty acid and organic acid metabolism. Though acylcarnitine analysis results are for a specific disorder in some cases, further testing is needed to confirm the diagnosis in most cases.
Creative Proteomics offers highly sensitive and reliable LC-MS platform for the quantification of acylcarnitines. The precursor ion scan mode is used to give a record of the molecular species (derived from specifically methylated acylcarnitines of the sample) which give rise to fragment ions at m/z 99. Stable isotope dilution is methods used for quantification.
Creative Proteomics has established and launched a proven and reliable method for the absolute quantification of carnitine and carnitine analogues, from C0 to C18, for the detailed determination of more than thirty carnitine and carnitine analogues in samples in a single run. The solution employs an ultra performance liquid chromatography-tandem quadrupole mass spectrometry platform using multiple carnitine and carnitine analogues isotopic standards and non-isotopic standards as internal standards, providing a solution for the simultaneous quantitative determination and analysis of multiple carnitine and carnitine analogues in biological samples, significantly improving the accuracy, throughput and stability of qualitative and quantitative determination. The method is applicable to a wide range of biological samples and can meet multiple needs.
workflow
Platform
- LC-MS
Summary
- Identification & Quantification of Acylcarnitines
| Acylcarnitines Quantified in Our Service | ||
|---|---|---|
| 3-Hydroxy-dodecanoyl- | 3-Hydroxymyristoyl- | 3-hydroxyhexadecanoyl- |
| 3-Hydroxy-linoleyl- | 11cis,14cis-Eicosadienoyl- | Acetyl- |
| Arachidoyl- | Arachidonoyl- | Butyryl- |
| cis-4-Decenoyl- | cis-5-Tetradecenoyl- | cis-11-Eicosenoyl- |
| Decanoyl- | Eicosatrienoyl- | Glutaryl- |
| Hexanoyl- (caproyl-) | Lauroyl- | Linoleyl- (linoelaidyl-) |
| L-carnitine | Myristoyl- | Octanoyl- |
| Oleoyl- (Elaidic-, Vaccenyl-) | Palmitoyl- | Palmitoleoyl- |
| Propionyl- | Stearoyl- | Tetradecadienoyl- |
| trans-2-Dodecenoyl- | trans-2-Octenoyl- | Valeryl- |
Sample Requirement
| Sample | Minimum amount | General amount |
|---|---|---|
| Animal and clinical tissue samples | 5-10 mg | 50 mg |
| Blood samples (serum, plasma and whole blood) | 10 μL | 50 μL |
| Urine samples | 10 μL | 50 μL |
| Stool and intestinal contents | 10mg | 25 mg |
| Body fluid samples (cerebrospinal fluid, saliva, etc.) | 5-10 μL | 25 μL |
| Plant tissue samples (roots, stems, leaves flowers and fruits, etc.) | 10 mg | 50 mg |
| Cells and microbial organisms | 1*10⋀5 cells | 1*10⋀6 cells |
| Culture media and fermentation broth | 10 μL | 50 μL |
Report
- A detailed technical report will be provided at the end of the whole project, including the experiment procedure, MS instrument parameters.
- Analytes are reported as uM/ml, while CV's are generally 10%.
- The name of the analytes, abbreviation, formula, molecular weight and CAS# would also be included in the report.
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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 acylcarnitines targeted metabolomics services.