The LipidWeb blank

Carnitine, Acylcarnitines and β-Oxidation

Carnitine (L-3-hydroxy-4-aminobutyrobetaine or L-3-hydroxy-4-N-trimethylaminobutanoic acid), and its acyl esters (acylcarnitines) are essential compounds for the catabolism of fatty acids. They are present in animals, plants and some microorganisms, but in animal tissues, carnitine concentrations are relatively high, typically between 0.2 and 6 mmol/kg with most in the heart and skeletal muscle.

Structural formulae for carnitine and acylcarnitines

L-Carnitine can be synthesised de novo in animal cells by a multi-step process with N-trimethyl-lysine derived from protein degradation as the primary precursor and butyrobetaine as an intermediate. However, it is believed that most comes from the diet other than in strict vegetarians, and plasma carnitine levels are positively correlated with the dietary intake. In humans, the major sources of carnitine are meat, fish and dairy products, which can supply 2 to 12 μmol per day per kg of body weight as opposed to 1.2 μmol per day per kg of body weight of endogenous carnitine. The latter is synthesised in the kidney, liver and brain and is transported to other tissues in the circulation before it is taken up by active transport systems. In the kidney, carnitine and butyrobetaine are reabsorbed efficiently so urinary loss is minimized, although excessive amounts can be eliminated when necessary.

1. Carnitine and Fatty Acid Transport into Mitochondria

In mammals, carnitine functions through the reversible esterification of its 3-hydroxyl group with subsequent translocation of the acylcarnitines produced from one cellular compartment to another. Carnitine acyltransferases are the enzymes responsible for the production of acylcarnitines, and these can have differing chain-length specificities, but covering the entire range of acyl chain lengths, depending on the cellular location and metabolic purpose. In consequence, acylcarnitines constitute an appreciable component of the tissue carnitine pool, and tissue and plasma concentrations of carnitine and acylcarnitines are together maintained within relatively narrow limits by a variety of mechanisms.

Fatty acyl-CoA thioesters cannot enter the mitochondrial matrix in animals because they are not able to pass through the inner mitochondrial membrane. Instead, carnitine assists the transport and metabolism of fatty acids into mitochondria, where the enzymes of β-oxidation are located and fatty acids are oxidized as a major source of energy. In so doing, carnitine maintains a balance between free and esterified coenzyme A as an excess of acyl-CoA intermediates is potentially toxic to cells. In addition, carnitine is required to remove any surplus of acyl groups from mitochondria and to export acetyl- and other short-chain acyl groups from peroxisomes via the actions of a short-chain acyl-CoA-specific carnitine acetyltransferase and a medium chain-specific carnitine octanoyltransferase carnitine octanoyltransferase. These activities influence in turn innumerable aspects of carbohydrate and lipid metabolism, including the regulation of insulin secretion by pancreatic β-cells and the determination of tissue insulin sensitivity.

Several enzymes are involved in the various processes that occur. Fatty acids are first activated by being bound to coenzyme A to form highly polar thiol esters, i.e. acyl-CoA, on the outer mitochondrial membrane. As these cannot cross the inner mitochondrial membrane, the acyl group is first transferred to carnitine with formation of acylcarnitines, which can enter the mitochondria with the assistance of specific translocases. The transport system consists of the enzyme carnitine palmitoyltransferase I (CPT-I) present in the mitochondrial outer membrane, which forms a complex with a long-chain acyl-CoA synthetase and the voltage-dependent anion channel. The second transport component is carnitine:acylcarnitine translocase (CACT), an integral inner membrane protein, which forms a complex with carnitine palmitoyltransferase II (CPT-II) located on the matrix side of the inner membrane. The acylcarnitine crosses the inner mitochondrial membrane via a porin channel in exchange for a carnitine molecule in the opposite direction, thus ensuring that the mitochondrial carnitine concentration remains constant. Then, inside the mitochondria, carnitine and acyl-CoA are regenerated from the internatized acylcarnitines, before the acyl-CoA is catabolized in two-carbons units by β-oxidation by the mechanism described below with production of acetyl-CoA in normal circumstances.

Function of carnitine in mitochondrial long-chain fatty acid oxidation

This is a greatly simplified account of the process, and a number of enzymes are involved both in the transport and β-oxidation aspects. In fact, at least 25 proteins are required some of which organized into at least three functional subdomains, one associated with the outer mitochondrial membrane, one with the inner mitochondrial membrane and the other in the matrix. In addition, there are three isoforms of CPT I, each present in specific tissues: CPT IA in liver and kidney, CPT IB in heart and skeletal muscle and CPT IC in the brain. While CPT IA and CPT IB are the main enzymes involved in the transfer of long-chain fatty acids into mitochondria for oxidation, the lesser known CPT IC may be a sensor of lipid metabolism in neurons. Malonyl-CoA generated by acetyl-CoA carboxylase binds with high affinity to each of these isoforms and is important for the regulation of the transfer of fatty acids into the mitochondrial matrix and thence their oxidation. Carnitine palmitoyltransferases are also present in peroxisomes where some similar reactions may occur.

2.  β-Oxidation

The main pathway for the degradation of fatty acids is mitochondrial fatty acid β-oxidation, a key metabolic pathway for energy homoeostasis in issues such as the liver, heart and skeletal muscle. During fasting, this is of special importance as most tissues other than the brain can utilize fatty acids directly to generate energy. In addition, the liver can use this mechanism to convert fatty acids into ketone bodies as an additional source of energy for all tissues including the brain.

Four enzymes are involved in the cycle of β-oxidation of a long-chain fatty acid as its Coenzyme A ester. The first step consists in the formation of a double bond between the C2 and C3 by the enzyme acyl-CoA-dehydrogenase to produce trans-Δ2-enoyl-CoA. In the process, flavin adenine dinucleotide (FAD) is reduced to FADH2. Next, the trans-2 double bond is hydrated stereospecifically by an enoyl-CoA hydratase to form L-3-hydroxyacyl-CoA, which is in turn oxidized by a hydroxyacyl-CoA dehydrogenase to produce 3-ketoacyl-CoA while nicotinamide adenine dinucleotide (NAD) is reduced to NADH. The cycle is completed when the keto intermediate is cleaved between C2 and C3 by thiolysis by a 3-ketoacyl-CoA thiolase to produce acetyl-CoA and the CoA ester of a fatty acid two carbons shorter than the original. A new cycle commences and the reaction continues until all the fatty acid is converted to acetyl-CoA units.

Beta-oxidation of long-chain fatty acids

Different isoforms of these enzymes of β-oxidation exist with affinities for fatty acids of different chain lengths, including four acyl-CoA dehydrogenases. Thus, for efficient oxidation to occur, these isoforms must function cooperatively. A further feature of interest is that the last three enzymes involved with a specificity for long-chain fatty acids form a trifunctional enzyme complex on the inner mitochondrial membrane.

Additional enzymes are required for the oxidation of odd-chain and unsaturated fatty acids. Propionyl-CoA is a product of the former but it can be metabolized to succinyl-CoA, which can then enter the citric acid cycle.

The β-oxidation cycle is interrupted when a cis-double bond in position 3 is reached, and three additional enzymes are required before the process can be completed. For example with linoleoyl-CoA, three cycles of β-oxidation cycle yield a 3c,6c-12:2 intermediate, which must be isomerized by an enoyl-CoA isomerase to form 2t,6c-12:2-CoA. This can now undergo one cycle of β-oxidation to yield 4c-10:1-CoA, which is acted upon by an acyl-CoA dehydrogenase to produce 2t,4c-10:2-CoA and this in turn is the substrate for an NADPH-dependent 2,4-dienoyl-CoA reductase to form 3t-10:1-CoA. After further reaction with an enoyl-CoA isomerase to yield 2t-10:1-CoA, four cycles of β-oxidation can occur with acetyl-CoA as the final product.

Beta oxidation of linoleoyl-CoA

Finally, the acetyl-CoA groups are used directly for the generation of energy, for example by the tricarboxylic acid cycle, or they are converted to acetylcarnitine via the action of carnitine acetyltransferase for transport out of the mitochondria.

To complete the picture, β-oxidation of fatty acid also occurs in peroxisomes in animals, and this is believed to be especially important for very-long-chain and methyl-branched fatty acids. The enzymes involved are very different from those in mitochondria, and for example, acyl-CoA oxidase, the first enzyme in peroxisome β-oxidation transfers hydrogen to oxygen to produce H2O2 rather than FADH2. However, the fatty acyl-CoA intermediates formed are the same in peroxisomes and mitochondria. Some fatty acids with methyl branches are not amenable to β-oxidation, but they can be degraded by α-oxidation in peroxisomes. Carnitine and acylcarnitines are not required for these processes.

The importance of β-oxidation of fatty acids is seen from the fact that the process generates twice as much energy (39 KJ g-1) as can be obtained from glucose (15 KJ g-1). Energy is produced at each stage of the process. Thus, step-wise shortening of acyl-CoA generates one molecule of FADH2 and NADH for every two-carbon unit released, while each acetyl-CoA molecule yields 3 molecules of NADH, 1 molecule of FADH2 and 1 of GTP via the tricarboxylic acid cycle. In total, the degradation of palmitic acid produces approximately 130 molecules of ATP.

3.  Carnitine and Health

Deficiencies in any of the enzymes involved in the metabolism of carnitine and acylcarnitines can cause an accumulation of acyl-CoA of specific chain-lengths, and these can have toxic effects if they are not removed by formation of acylcarnitines. As the acylation state of carnitine in the plasma reflects the composition of the cytosolic acylcarnitine pool, this serves as a diagnostic marker for the equilibrium between acyl-CoA and acylcarnitine species. In consequence, unusual acylcarnitines may be identified in biological fluids at very much higher concentrations than in healthy individuals, and the chain lengths can be indicative of particular enzymic disorders. For example, acylcarnitines produced as products of incomplete mitochondrial fatty acid oxidation have been detected in obesity, type 2 diabetes, cardiovascular disease and encephalopathy. Often the disorders result in underproduction of acetyl-CoA and dysfunction of the Krebs cycle. As carnitine palmitoyltransferase isoforms are over-expressed in certain cancers, they are seen as potential drug targets.

Scottish thistleSeveral inherited metabolic diseases can be identified from the presence of acylcarnitines in the blood and urine of neonates, and from their chain-length profile, the point of the breakdown in the β-oxidation pathway and the disease involved can be recognized. The clinical manifestations vary from multi-organ failure in the neonate with a fatal outcome to late-onset symptoms associated with significant disabilities. Similarly, patients with peroxisomal biogenesis disorders, such as Zellweger syndrome, or with acidemias have abnormal profiles of circulating acylcarnitines. As β-oxidation is especially important for energy production in breast cancer, targeting this pathway is considered to be a potential strategy for cancer treatment.

While the potential of L-carnitine and its esters as therapeutic agents is controversial, there is no doubt that it is life saving in patients with certain rare genetic disorders of carnitine metabolism. L-Carnitine deficiency is often seen in chronic hemodialysis patients, and in consequence it has been termed a "conditional vitamin". Carnitine is important to lipid metabolism in brain, where fatty acid oxidation is less significant though still relevant. In this tissue, acylcarnitines function in the synthesis of lipids and thence regulate membrane compositions. They also modify the activity of genes and proteins and influence neurotransmission.

D-Carnitine does not occur naturally but may be found in some synthetic preparations; it does not participate in the key biological processes but can sometimes interfere with them.

4. Carnitine in Plants and Bacteria

Although it has long been known that carnitine per se is present at very low levels in the tissues of many plant species, including seeds and leaves, it was only recently that the presence of acylcarnitines was demonstrated definitively and their functions are still relatively obscure. However, they have been associated with anabolic pathways of lipid metabolism during development, including the biosynthesis of membrane and storage lipids. They are also reported to enhance the recovery of Arabidopsis thaliana seedlings subjected to salt stress. The yeast Candida albicans can synthesise carnitine. While it is not clear whether this is possible in bacteria, they can acquire it from the environment or from metabolic precursors as it is important for protection against environmental stresses.

5.  Analysis

Acylcarnitines are highly polar molecules, and special precautions are required for extraction and analysis. For example, butanol saturated with water is usually recommended for extracting them from tissues. They are zwitterionic molecules, so tend to elute with phospholipids such as phosphatidylcholine in many chromatographic systems. However, many of the technical problems appear to have been solved (see the reviews cited below). Mass spectrometric methods appear to be especially suited to routine screening of large numbers of samples of biological fluids from neonates, as they permit a considerable degree of automation, both of the analytical steps and of gathering and interpretation of data.

Recommended Reading

Lipid listings Credits/disclaimer Updated: May 3rd, 2017 Author: William W. Christie LipidWeb icon