Carnitine

Carnitine is a quaternary ammonium compound involved in metabolism in most mammals, plants, and some bacteria. In support of energy metabolism, carnitine transports long-chain fatty acids from the cytosol into mitochondria to be oxidized for free energy production, and also participates in removing products of metabolism from cells. Given its key metabolic roles, carnitine is concentrated in tissues like skeletal and cardiac muscle that metabolize fatty acids as an energy source. Generally individuals, including strict vegetarians, synthesize enough L-carnitine in vivo.
Carnitine exists as one of two stereoisomers: the two enantiomers -carnitine and -carnitine. Both are biologically active, but only -carnitine naturally occurs in animals, and -carnitine is toxic as it inhibits the activity of the -form. At room temperature, pure carnitine is a whiteish powder, and a water-soluble zwitterion with relatively low toxicity. Derived from amino acids, carnitine was first extracted from meat extracts in 1905, leading to its name from Latin, "caro/carnis" or flesh.
Some individuals with genetic or medical disorders cannot make enough carnitine, requiring dietary supplementation. Despite common carnitine supplement consumption among athletes for improved exercise performance or recovery, there is insufficient high-quality clinical evidence to indicate it provides any benefit.

Biological role

The primary biological functions of carnitine in humans include the following:

fatty acid transport across the mitochondrial membrane by forming long-chain acylcarnitine esters which are shuttled into the mitochondria, where they undergo β-oxidation to produce ATP, the cell's main energy currency;
acetyl-CoA and coenzyme A stabilization by transferring acetyl groups for maintaining metabolic flexibility and energy production, particularly during fasting or exercise;
detoxification of acyl groups by forming acylcarnitine, which is then excreted to prevent the accumulation of potentially toxic fatty acyl intermediates;
regulation of cellular metabolism by participating in the conversion and utilization of different fuel sources, enabling cells to switch between carbohydrate and fatty acid metabolism as needed;
antioxidant action to protect cells from oxidative stress and damage.
Biochemistry

Chemical properties

Carnitine is a zwitterion, meaning it has both positive and negative charges in its structure. In an aqueous solution, L-carnitine is freely soluble and its ionizable groups, COO⁻ and N⁺₃, are over 90% dissociated at physiological pH for humans.

Biosynthesis and metabolism

Physiological effects in humans

As an example of normal biosynthesis of carnitine in humans, a person would produce 11–34 mg of carnitine per day. Adults eating mixed diets of red meat and other animal products ingest some 60–180 mg of carnitine per day, while vegans consume about 10–12 mg per day. Most carnitine obtained from the diet is absorbed in the small intestine before entering the blood. The total body content of carnitine is about in a person weighing, with nearly all of it contained within skeletal muscle cells. Carnitine metabolizes at rates of about 400 μmol per day, an amount less than 1% of total body stores.

Biosynthesis in eukaryotes

Many eukaryotes have the ability to synthesize carnitine, including humans. Humans synthesize carnitine from the substrate TML, which is in turn derived from the methylation of the amino acid lysine. TML is then hydroxylated into hydroxytrimethyllysine by trimethyllysine dioxygenase, requiring the presence of ascorbic acid and iron. HTML is then cleaved by HTML aldolase, yielding 4-trimethylaminobutyraldehyde and glycine. TMABA is then dehydrogenated into gamma-butyrobetaine in an NAD⁺-dependent reaction, catalyzed by TMABA dehydrogenase. Gamma-butyrobetaine is then hydroxylated by gamma butyrobetaine hydroxylase into -carnitine, requiring iron in the form of Fe²⁺.

Fatty acid transport

Carnitine is involved in transporting fatty acids across the mitochondrial membrane, by forming a long chain acylcarnitine ester and being transported by carnitine palmitoyltransferase I and carnitine palmitoyltransferase II.

Acetyl-CoA stabilization

Carnitine plays a role in stabilizing acetyl-CoA and coenzyme A levels through the ability to receive or give an acetyl group.

Tissue distribution of carnitine-biosynthetic enzymes in humans

The tissue distribution of carnitine-biosynthetic enzymes in humans indicates TMLD to be active in the liver, heart, muscle, brain and highest in the kidneys. HTMLA activity is found primarily in the liver. The rate of TMABA oxidation is greatest in the liver, with considerable activity also in the kidneys.

Carnitine shuttle system

The free-floating fatty acids, released from adipose tissues to the blood, bind to carrier protein molecules known as serum albumin that carry the fatty acids to the cytoplasm of target cells such as the heart, skeletal muscle, and other tissue cells, where they are used for fuel. Before the target cells can use the fatty acids for ATP production and β oxidation, the fatty acids with chain lengths of 14 or more carbons must be activated and subsequently transported into mitochondrial matrix of the cells in three enzymatic reactions of the carnitine shuttle.
The first reaction of the carnitine shuttle is a two-step process catalyzed by a family of isozymes of acyl-CoA synthetase that are found in the outer mitochondrial membrane, where they promote the activation of fatty acids by forming a thioester bond between the fatty acid carboxyl group and the thiol group of coenzyme A to yield a fatty acyl–CoA.
In the first step of the reaction, acyl-CoA synthetase catalyzes the transfer of adenosine monophosphate group from an ATP molecule onto the fatty acid generating a fatty acyl–adenylate intermediate and a pyrophosphate group. The pyrophosphate, formed from the hydrolysis of the two high-energy bonds in ATP, is immediately hydrolyzed to two molecules of P_i by inorganic pyrophosphatase. This reaction is highly exergonic which drives the activation reaction forward and makes it more favorable. In the second step, the thiol group of a cytosolic coenzyme A attacks the acyl-adenylate, displacing AMP to form thioester fatty acyl-CoA.
In the second reaction, acyl-CoA is transiently attached to the hydroxyl group of carnitine to form fatty acylcarnitine. This transesterification is catalyzed by an enzyme found in the outer membrane of the mitochondria known as carnitine acyltransferase 1.
The fatty acylcarnitine ester formed then diffuses across the intermembrane space and enters the matrix by facilitated diffusion through carnitine-acylcarnitine translocase located on the inner mitochondrial membrane. This antiporter returns one molecule of carnitine from the matrix to the intermembrane space for every one molecule of fatty acyl–carnitine that moves into the matrix.
In the third and final reaction of the carnitine shuttle, the fatty acyl group is transferred from fatty acyl-carnitine to coenzyme A, regenerating fatty acyl–CoA and a free carnitine molecule. This reaction takes place in the mitochondrial matrix and is catalyzed by carnitine acyltransferase 2, which is located on the inner face of the inner mitochondrial membrane. The carnitine molecule formed is then shuttled back into the intermembrane space by the same cotransporter while the fatty acyl-CoA enters β-oxidation.

Regulation of fatty acid β oxidation

Balance

The carnitine-mediated entry process is a rate-limiting factor for fatty acid oxidation and is an important point of regulation.

Inhibition

The liver starts actively making triglycerides from excess glucose when it is supplied with glucose that cannot be oxidized or stored as glycogen. This increases the concentration of malonyl-CoA, the first intermediate in fatty acid synthesis, leading to the inhibition of carnitine acyltransferase 1, thereby preventing fatty acid entry into the mitochondrial matrix for β oxidation. This inhibition prevents fatty acid breakdown while synthesis occurs.

Activation

Carnitine shuttle activation occurs due to a need for fatty acid oxidation which is required for energy production. During vigorous muscle contraction or during fasting, ATP concentration decreases and AMP concentration increases leading to the activation of AMP-activated protein kinase. AMPK phosphorylates acetyl-CoA carboxylase, which normally catalyzes malonyl-CoA synthesis. This phosphorylation inhibits acetyl-CoA carboxylase, which in turn lowers the concentration of malonyl-CoA. Lower levels of malonyl-CoA disinhibit carnitine acyltransferase 1, allowing fatty acid import to the mitochondria, ultimately replenishing the supply of ATP.

Transcription factors

is a nuclear receptor that functions as a transcription factor. It acts in muscle, adipose tissue, and liver to turn on a set of genes essential for fatty acid oxidation, including the fatty acid transporters carnitine acyltransferases 1 and 2, the fatty acyl–CoA dehydrogenases for short, medium, long, and very long acyl chains, and related enzymes.
PPARα functions as a transcription factor in two cases; as mentioned before when there is an increased demand for energy from fat catabolism, such as during a fast between meals or long-term starvation. Besides that, the transition from fetal to neonatal metabolism in the heart. In the fetus, fuel sources in the heart muscle are glucose and lactate, but in the neonatal heart, fatty acids are the main fuel that require the PPARα to be activated so it is able in turn to activate the genes essential for fatty acid metabolism in this stage.

Metabolic defects of fatty acid oxidation

More than 20 human genetic defects in fatty acid transport or oxidation have been identified. In case of fatty acid oxidation defects, acyl-carnitines accumulate in mitochondria and are transferred into the cytosol, and then into the blood. Plasma levels of acylcarnitine in newborn infants can be detected in a small blood sample by tandem mass spectrometry.
When β oxidation is defective because of either mutation or deficiency in carnitine, the ω oxidation of fatty acids becomes more important in mammals. The ω oxidation of fatty acids is another pathway for F-A degradation in some species of vertebrates and mammals that occurs in the endoplasmic reticulum of the liver and kidney, it is the oxidation of the ω carbon—the carbon farthest from the carboxyl group.