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What is a Carnitine? |
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| Carnitine is a compound that plays an important role in transporting fatty acids into the inner mitochondrial matrix to be used in fatty acid metabolism. Such is done by accepting acyl groups from acyl-CoA, yielding acylcarnitine and free CoASH. It also serves to remove cellular waste from the mitochondria. Carnitine is capable of this type of transport because it has the ability to esterify with the carboxyl group of a molecule to be transported. |
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| For each unique carboxylic acid that carnitine bonds with, it forms a unique acylcarnitine species. Nomenclature for acylcarnitines is based on the molecule to which carnitine has bonded. Taken from the example below. Carnitine can esterify with a molecule of propionic acid to form propionyl-carnitine. Other examples of acylcarnitine include palmitoyl-carnitine, from palmitic acid, linoleoyl-carnitine, from linoleic acid and arachidonoyl-carnitine, from arachidonic acid. |
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| What Does This Mean? |
| Abnormal levels of specific acylcarnitines are associated with certain inborn errors of metabolism or metabolic disorders. Acylcarnitine profiles are frequently used to screen for inborn errors of metabolism, and are most often used in newborns (Rashed 1995). They have been shown to be both sensitive and cost effective (Wiley 1999). While the acylcarnitine profile has proven to be a valuable tool, there are still many questions remaining to be answered. For instance, how do concentrations of acylcarnitines differ between people? How does diet or medications affect concentrations of acylcarnitines? Can acylcarnitines be used for more than detection of metabolic disorders? Are there potential benefits associated with carnitine supplementation in humans? |
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How Are Acylcarnitines Detected? |
| The acylcarnitine assay is an analytical technique used by our team to isolate carnitine species present in various compartments throughout the body (i.e. RBCs, plasma, and organ tissues). The first step of the assay is to release carnitine from the cellular components of the sample. For example, a sample of red blood cells must be first hemolyzed through addition of water. This process will disrupt cellular membranes and in doing so will release the carnitine present in these cells. Next, the cellular constituents are precipitated out of solution using an acetonitrile/methanol mixture solvent. Subsequent centrifugation isolates the cellular proteins and membranes in the form of a pellet which is discarded. The next stage in the assay involves a technique of separating carnitine from other soluble ions based on charge, using cation-exchange solid phase extraction. This technique works by holding the positively-charged quaternary ammonium group of carnitine to a negatively charged column. The carnitines held in the column are then eluted into a separate vial which is evaporated using nitrogen gas. The resultant sample will contain purified acyl and free carnitines which are analyzed through mass spec. analysis to generate an acylcarnitine profile of the tissue. |
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The Carnitinome Database and Future Plans |
| The Carnitine team uses the data generated through the acylcarnitine assay to answer important questions about carnitine’s role in the body. Analysis of the concentrations of different acylcarnitine species provides a glimpse to how carnitine interacts with fatty acid or acyl-groups present in different tissues throughout the body. Collectively, the study of all the acylcarnitine species in a biological context has been termed the Carnitinome. The Carnitinome Database is a place where the Carnitine Team houses all available information on each identified acylcarnitine species. These data include the molecular formula, weight, and structure for each individual acylcarnitine species. It also includes characteristic fragmentation patterns for each species used to identify the molecule using mass spectrometry. Currently, the Carnitinome database is being updated with all potential acylcarnitine species which are derived from a database of biologically relevant carboxylic acids. These carboxylic acids range from simple fatty acids to complex drug metabolites. In the future, we hope to apply the derived MS fragmentation patterns for the molecules in the Carnitinome, to identify all of the various acylcarnitine species found throughout the body. |
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Carnitine and Ketogenic Therapy |
| The Carnitine team plays an essential role at the General Clinical Research Center (GCRC) in Shands hospital with our prospective Ketogenic Therapy study. While the KetoGator Team is more focused on improving patient care and education, the Carnitine Team is more concerned with the unknown mechanistic action of Ketogenic Therapy. Currently, we are focusing on obtaining an acylcarnitine profile from our patients’ blood samples; we believe that carnitine plays a critical role in metabolism, therefore a complete profile will help in answering the plethora of questions we have concerning this dietary treatment. Members attend clinic every Tuesday and work in the lab to separate blood samples into RBC and plasma samples, which are then taken back to our lab and later assayed. |
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A Piglet Model of Ketogenic Therapy |
Concurrent with the GCRC, the Carnitine Team performs piglet experiments. The piglet has been shown to be an excellent animal model for studying carnitine in human neonates, specifically because of similarities in metabolism, physiology and tissue carnitine concentrations between the two species. Using the piglet, we are studying the effects of Ketogenic Therapy on blood and organ acylcarnitine concentrations. Under ketotic conditions (such as during starvation or on a high fat diet), glucose is not readily available. Organs like the brain rely on glucose as a primary energy source. It was originally proposed that ketone bodies produced under these conditions were the key molecules in the mechanism of the diet; however, various studies have not been able to confirm this conclusively. Our lab hypothesizes that acylcarnitines, especially acetyl-carnitine, can be used as a major energy substrate for the brain in a ketotic state. To test this, we are feeding piglets either a diet that mimics a ketogenic diet at a 4:1 ratio of fat:(protein + carbohydrate) or a control diet at a 0.26:1 ratio. Based on analysis of the changes in the acylcarnitine profile in the blood and organs of these animals, we hope to elucidate a mechanism behind Ketogenic Therapy. |
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| References |
- Rashed MS, Ozand PT, Bucknall MP, Little D, Diagnosis of inborn errors of metabolism from blood spots by acylcarnitines and amino acids profiling using automated electrospray tandem mass spectrometry; Pediatr Res. 1995 Sep;38(3):324-31.
- Wiley V, Carpenter K, Wilcken B, Newborn screening with tandem mass spectrometry: 12 months' experience in NSW Australia; Acta Paediatr Suppl. 1999 Dec;88(432):48-51
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