Carnitine, Acylcarnitines, and β-Oxidation of Fatty Acids

The quaternary ammonium compound and amino acid carnitine (L-3-hydroxy-4-aminobutyrobetaine or L-3-hydroxy-4-N-trimethylaminobutanoic acid) and its acyl esters (acylcarnitines) have many important functions in animals, plants, and some microorganisms. In particular, they are essential for the oxidative catabolism of long-chain fatty acids by shuttling them across the mitochondrial membrane from the cytosol into the mitochondrial matrix for β‑oxidation and cellular energy production, and thence for maintaining energy homeostasis in the human body. In addition to its role in the oxidation of fatty acids, carnitine binds acyl residues resulting from the intermediary metabolism of amino acids and aids their elimination, and it regulates the acyl-CoA/CoA ratio, thereby controlling the activity of several mitochondrial enzymes.

Structural formulae for carnitine and acylcarnitines

1. Carnitine Synthesis and Dietary Uptake

In animal tissues, L-carnitine concentrations are relatively high, typically between 0.2 and 6 mmol/kg with almost all in the heart and skeletal muscle. It is 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 insufficient is produced by this means, and most comes from the diet other than in strict vegetarians. 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 carnitine synthesised endogenously. For some individuals with genetic or medical disorders and for pre-term infants, who cannot make enough, carnitine is a conditionally essential nutrient.

Dietary carnitine is taken up from the intestinal lumen into the enterocyte by an active transport mechanism, but it passes across the serosal membrane into the circulation by simple diffusion. In some laboratory animals, such as the rat and guinea pig much of it is acetylated prior to export from the intestines. Synthesis of carnitine occurs in the kidney, liver and brain, and it is transported to other tissues in the circulation before it is taken up by tissues by tissue-specific plasma membrane carnitine transporters. For example, in the kidney, carnitine and butyrobetaine are reabsorbed efficiently by active transport by a high affinity carnitine transporter termed the 'organic cation transporter novel 2 (OCTN2)', located in the renal brush border membrane. Urinary loss is thereby minimized, although excessive amounts can be eliminated when necessary.

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.

2. Carnitine and Fatty Acid Transport into Mitochondria

The substrates for oxidation and most of the energy production in cells are unesterified fatty acids, which originate from three main sources: exogenous fatty acids that enter cells from the blood, either from the diet or by mobilization from other tissues, those that arise via synthesis de novo from acetyl-CoA, and those released within the cell by the hydrolysis of phospholipids and triacylglycerols. First, they must be transported to the mitochondria where the enzymes of β-oxidation are located and fatty acids are oxidized, with only a minimal contribution from the peroxisomal system for which the preferred substrates are very long-chain fatty acids.

Intracellular unesterified fatty acids must first undergo thio-esterification to coenzyme A, a process catalysed by acyl-CoA synthases and consuming the equivalent of two ATP, with formation of acyl-CoAs, i.e., the activated form of fatty acids. These are usually bound to proteins with characteristic acyl-CoA binding domains and depending upon on the energy status of the cell, they are directed to particular metabolic pathways, for example storage or energy production, by many different proteins including fatty acid binding proteins and sterol carrier protein 2. Long-chain 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, and in so doing, carnitine maintains a balance between free and esterified CoA, as an excess of acyl-CoA intermediates is potentially toxic to cells.

In mammals, carnitine functions through the reversible esterification of its 3-hydroxyl group by fatty acids with subsequent translocation of the acylcarnitines produced from one cellular compartment to another. Carnitine acyltransferases are the enzymes responsible for 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. Thus, carnitine is required to transport fatty acyl groups into mitochondria and to remove any surplus of acyl groups, as well as 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. 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. 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 are required to transport long-chain fatty acids (>C8) into mitochondria. Activation of fatty acids to form highly polar thiol esters, i.e., acyl-CoA, occurs on the outer mitochondrial membrane, but to enable transport across the inner mitochondrial membrane, the acyl group is then 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 internalized 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 many enzymes are involved both in the transport and β-oxidation aspects. In fact, at least 25 proteins are required some of which are 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, for example, 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 - isoform 2 (ACC2) binds with high affinity to each of the carnitine palmitoyltransferase isoforms and is important for the regulation of the transfer of fatty acids into the mitochondrial matrix and thence their oxidation. When the energy demand is high, inhibition of the isoform ACC2 of acetyl-CoA carboxylase increases fatty acid oxidation, while inhibition of the isoform ACC1 decreases fatty acid synthesis. Carnitine palmitoyltransferases are also present in peroxisomes where some similar reactions may occur.

In contrast, short-chain fatty acids at least up to octanoate can permeate the inner mitochondrial membrane in non-esterified form, and they are activated to their CoA-derivatives in the mitochondrial matrix for use in energy-dependent mitochondrial processes.

3.  β-Oxidation of Fatty Acids in Mitochondria

The main pathway for the degradation of fatty acids is mitochondrial fatty acid β-oxidation with acetyl-CoA as the end-product, 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 via acetyl-CoA and acetoacetyl-CoA as an additional source of energy for all tissues, but especially for the brain.

At its simplest, four enzymes are involved in the cycle of β-oxidation of a long-chain saturated fatty acid as its CoA ester in mitochondria. 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 saturated 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 fatty acids, with propionyl-CoA is a product that can be metabolized to succinyl-CoA, which can then enter the citric acid cycle.

With unsaturated fatty acids, 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

The end-product is acetyl-CoA, and this is used directly for the generation of energy, for example by the tricarboxylic acid cycle, or it is converted to acetylcarnitine via the action of carnitine acetyltransferase for transport out of the mitochondria to be utilized elsewhere.

Feed-back regulation occurs as each of the enzymes of β-oxidation is inhibited by its own fatty acyl-CoA product. The reaction is also regulated allosterically by the ratios of NADH/NAD+ and acetyl-CoA/CoA, and a rise in either ratio is inhibitory. Transcriptional regulators of the enzymes include the peroxisome proliferator-activated receptors (PPARs) and a transcription factor coactivator PGC-1a and involve the retinoid X receptor, but the details of the process depend upon the specific tissue.

Peroxisomal oxidation: To complete the picture, β-oxidation of fatty acids 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. In a further minor reaction, fatty acids can also undergo ω-oxidation with the eventual production of dicarboxylic acids. Carnitine and acylcarnitines are not required for these processes.

Ketone bodies: During periods of fasting and some other physiological conditions, water-soluble molecules containing a ketone group, such as acetoacetate, β‑hydroxybutyrate, and the spontaneous breakdown product of acetoacetate, acetone, are produced from acetyl-coA by the liver after oxidation of fatty acids. These are known as "ketone bodies" and are readily transported into tissues outside the liver and converted back to acetyl-CoA, which then enters the citric acid cycle and is oxidized in the mitochondria for energy. In the brain, they are used also to make acetyl-CoA for fatty acid synthesis. Ketone bodies are produced by the liver from fatty acids and other precursors under conditions of intense gluconeogenesis, especially when the liver glycogen stores have been depleted.

Ketone bodies

Energy produced: 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, stepwise 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 one molecule of palmitic acid produces approximately 130 molecules of ATP. For example, during cold exposure in mammals, thermogenesis is a protective measure against a reduction in ambient temperature. Recent research suggests that cold stimulates adipocytes in white adipose tissue to release unesterified fatty acids that activate the nuclear receptor HNF4α, which is required for acylcarnitine production in the liver. This organ then undergoes a metabolic switch to produce acylcarnitines, which are transported in plasma to brown adipose tissue to serve as a fuel for thermogenesis. At the same time, uptake of acylcarnitines into white adipose tissue and liver is blocked. However, the quantitative contribution of acylcarnitines from the liver to thermogenesis in brown adipose tissue has still to be determined.

4.  Carnitine, β-Oxidation 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 specific 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 thistleInherited defects of fatty acids oxidation are transmitted as autosomal recessive traits in humans, and more than thirty inherited metabolic diseases can be identified from the presence of acylcarnitines in the blood and urine of newborn infants, especially. 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.

In addition to its role in energy metabolism with some antioxidant properties, L-acetyl-carnitine can cross the blood-brain barrier, where it modulates brain neurotransmitters, such as acetylcholine, serotonin and dopamine, and acts on neurotrophic factors by modifying the activity of the relevant genes. It is marketed as a drug to alleviate neuropathic pain, and there are suggestions that it may help with cognitive and mood disorders. Carnitine is important to lipid metabolism in brain in general, where fatty acid oxidation is less significant though still relevant, and there is interest in its use for neuroprotection in disorders that include traumatic and other brain injuries and Alzheimer's disease. Acylcarnitines function in the synthesis of lipids in the brain and thence regulate membrane compositions. In addition, carnitine and its esters are being studied in relation to cardiovascular disease. While the potential of carnitine, acetyl-carnitine, and other esters as therapeutic agents is perhaps controversial, there is no doubt that they are lifesaving in patients with certain rare genetic disorders of carnitine metabolism, for example Primary Carnitine Deficiency (PCD). L-Carnitine deficiency is often seen in chronic haemodialysis patients, and in consequence it has been termed a "conditional vitamin".

5. 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. Free L-carnitine is present in plants in µg g-1 amounts, while the acylcarnitine content is 17 to 38 ng g-1. Although the functions of acylcarnitines are still relatively obscure, they may take part in a carnitine shuttle system in mitochondria analogous to that in animal cells, and they have been associated with anabolic pathways of lipid metabolism during development, including the biosynthesis of membrane and storage lipids. For example, there is some evidence that acylcarnitines are involved in plastidial export of fatty acids synthesised de novo, or in the import of fatty acids into the endoplasmic reticulum for synthesis of specific glycerolipids. 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 in some species.

6.  Analysis

Acylcarnitines are polar molecules that require special precautions for extraction and analysis, and 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 the gathering and interpretation of data.

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Lipid listings Contact/credits/disclaimer Updated: April 20th, 2022 Author: William W. Christie LipidWeb icon