- Service Details
- Case Study
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 is synthesized in the kidney, liver, and brain of organisms. Through circulation, carnitine and acylcarnitines are transported to other tissues to fulfill their roles. Meat, fish, and dairy products are the main sources of carnitine.
The Role 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 mitochondria. Carnitine plays a vital role in transporting fatty acyl-CoA thioesters into mitochondria. Through this process, carnitine maintains the balance between free and acyl-CoA intermediates, which is potentially toxic to cells. Besides, carnitine assists in 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 the mitochondria, by binding to coenzyme A, fatty acids are activated and converted to highly polar acyl-CoA, which can't pass the inner membrane of the mitochondria. Then, the acyl group is transferred from acyl-CoA to carnitine and forms acylcarnitines, which, with the help of translocase, can enter the inner membrane of the mitochondria. And then acyl-CoA is metabolized in two-carbon units through β-oxidation and regenerates acetyl-CoA. Finally, through the 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 the accumulation of acyl-CoA of specific chain lengths, which are toxic to cells if not removed from the body through the formation of acylcarnitines. Acylcarnitine analysis enables the diagnosis of 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' Lipodomics Analytical Services
Creative Proteomics offers highly sensitive and reliable LC-MS platform for the quantification of carnitine and 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.
Quantification of Carnitine and Analogues
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.
How to place an order:
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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.
High-Throughput Analysis of Underivatized Amino Acids and Acylcarnitines in Infant Serum: A Micromethod Based on Stable Isotope Dilution Targeted HILIC-ESI-MS/MS
Journal: J Agric Food Chem
Published: 2023
Abstract:
This study presents the development and validation of a high-throughput HILIC-MRM-MS/MS method for the rapid quantification of underivatized amino acids (AAs), AA derivatives, and acylcarnitines (ACs) in human serum. The method offers comprehensive analysis of AA and AC metabolism along with quantitative metabolite data. For the first time, this method was applied to serum samples from 145 healthy infants aged 3-4 months, demonstrating excellent reproducibility over multiple days of analysis and enabling simultaneous amino acid and acylcarnitine profiling in this age group.
Techniques: Targeted LC-MS/MS quantitative detection technology
Results:
The researchers established a novel targeted LC-MS/MS method for high-throughput analysis of 62 AAs and ACs metabolites in human serum, generating rapid quantitative metabolic energy fingerprints for AAs and ACs. After validation, this method was applied to serum samples from 145 infants aged 3-4 months.
Research Conclusions:
A high-throughput HILIC-MRM-MS/MS method was developed and validated for the rapid quantification of underivatized AAs, AA derivatives, and ACs in human serum. It can simultaneously detect 62 metabolites within a 20-minute run time. This method provides extensive AA and AC metabolic profiles along with quantitative metabolite data.
For the first time, high-throughput, non-derivatized, and highly efficient UHPLC-MRM-MS/MS were used to determine AA and AC concentrations in serum from healthy infants aged 3-4 months.
The method demonstrated excellent reproducibility and minimal technical variability over multiple days of measurement, allowing for individual profiles of AA and AC metabolism in large sample sizes.
Reference
- Wudy SI, Mittermeier-Klessinger VK, Dunkel A, Kleigrewe K, Ensenauer R, Dawid C, Hofmann TF. High-Throughput Analysis of Underivatized Amino Acids and Acylcarnitines in Infant Serum: A Micromethod Based on Stable Isotope Dilution Targeted HILIC-ESI-MS/MS. J Agric Food Chem. 2023 Jun 7;71(22)
