CARNITINE

1905 Carnitine was discovered in meat extract.

1927 The correct structure of carnitine was proposed.

1948 A new vitamin B_T was discovered, essential for growth of the yellow mealworm. The T was derived from the Latin name of this species, Tenebrio mollitor.

1952 Vitamin B_T was identified as carnitine.

1955 Discovery of the enzyme carnitine acetyltransferase (CRAT).

1958 I.B. Fritz discovered that carnitine stimulates long-chain fatty acid oxidation by mitochondria.

1963 Discovery of the enzyme carnitine palmitoyltransferase (CPT)

1973 First papers about patients with inborn errors in carnitine metabolism: muscular carnitine deficiency and CPT deficiency.

1975 Discovery of carnitine acylcarnitine translocase (CACT).

1977 Discovery of the inhibition of CPT-I by malonyl-CoA.

1977 Discovery of CPT-II deficiency with normal CPT-I, and the presence of CPT-I and not CPT-II in erythrocytes.

1982 Discovery of a carnitine transport defect as the cause of primary systemic carnitine deficiency.

1987 Discovery of the localization of CPT-I in the outer mitochondrial membrane.

1988-today Gene analysis of enzymes and transporters of the carnitine system in humans, patients with inborn errors and (transgene) animals. This biochemistry at the DNA-level has not been reviewed here.

1991 Discovery of fatal CPT-II deficiency.

The carnitine system consists of L-carnitine and acylcarnitine esters and the enzymes and transport proteins required for their metabolism and transport, including carnitine acyltransferases, mitochondrial carnitine acylcarnitine translocases, plasma membrane carnitine importers, and the carnitine biosynthesis pathway from lysine and methionine. In contrast to humans, bacteria possess enzymes to catabolize carnitine. Intestinal bacteria degrade most of the orally supplemented carnitine [2].

Acylcarnitine esters are formed from the CoASH esters of acetate, propionate, butyrate, medium-chain, long-chain and very-long-chain fatty acids. The highest possible chain length communicating with the carnitine system is not precisely known, but it is likely, although not proven with certainty, that physiological important fatty acids such as arachidonic acid (C20:5, n-6) and docosahexaenoic acid (C22:6, n-3) are transported as acylcarnitines to the membranes where they are needed. In blood reside carnitine esters up to C18, but in the tissues higher acylcarnitines were encountered [6]. Also most substituted-, branched-chain- and dicarboxylic acyl-CoA esters are converted into acylcarnitines, but at a (much) slower rate than the straight-chain esters.

The carnitine system was first discovered in mitochondria. They were found to contain carnitine acetyltransferase (CRAT), firmly bound to the inner face of the inner mitochondrial membrane and four CPT isoenzymes, named CPT-IA in liver, CPT-IB muscle and most other cells, CPT-IC in brain and testis [7] and CPT-II. The CPT-I enzymes are all localized in the outer mitochondrial membrane, with their catalytic [8]and regulatory [9,10] domains for malonyl-CoA inhibition facing to the cytosolic side, while CPT II is localized as CRAT. Peroxisomes contain also CRAT, a membrane carnitine octanoyltransferase (CROT) inhibited by malonyl-CoA, and another soluble carnitine acyltransferase not inhibited by malonyl-CoA[4]. So it is clear that ac(et)yl transport is not confined to the mitochondria. Also endo- and sarcoplasmatic reticulum, the nuclear membrane and the plasma membrane contain carnitine acyltransferases [4].

The high affinity transporter for carnitine is named OCTN2, a neutral organic cation transporter [14-16], which is stimulated by NaCl [2,17]. This transporter is not fully specific for carnitine, but transports also other drugs. OCTN2 is localized in the plasma membrane of muscle, heart, and the brushborders of small intestine [18] and kidney, and many more cells. OCTN3 had an intermediate affinity for carnitine, is not stimulated by Na⁺ and predominantly expressed in the testis [15]. OCNT1 has such a low affinity for carnitine that it cannot act at physiological carnitine levels [19].

CT2, carnitine transporter 2, a novel carnitine transporter, is expressed at the luminal membrane of the epididymal epithelium [20]. OCTN2 is also present in these cells, but in the basolateral membrane [21]. The carnitine transporter in the basolateral membranes of the mouse kidney and small intestine, has also been characterized. It is another high affinity transporter, not stimulated by Na⁺ [22].

Most tissues are able to produce deoxycarnitine [24, 25], but the last step is in humans only present in liver, kidney, brain and testes. Only liver and kidney are able to export carnitine to the blood. In healthy adults this biosynthetic pathway may provide enough carnitine to compensate for the urinary loss. In babies it is not enough to meet the demands of the growing body [2, 24]. The carnitine system is regulated via peroxisome proliferator-activated receptors [26] and hormones such as insulin, glucagon, norepinephrine, thyroid hormone, dopamine and androgens [3].

Long-chain fatty acids that are used as energy source, are activated by the palmitoyl-CoA synthetase localized on the outer surface of the outer mitochondrial membrane [27, 28], and converted by CPT-I at the same site [8-10].

There are many acyl-CoA synthetases, differing in acyl-specificity from acetyl-CoA to lignoceroyl-CoA, and in localization [29-32]. Acyl-CoA synthetases are the gateway to mitochondrial or peroxisomal ß-oxidation, or synthesis of triacylglycerols, phospholipids and other acylated products, while the CoA esters and acylated proteins are also acting in cell signaling [33].

In other organs than the liver, the mitochondrial oxidation of fatty acids is firmly linked to the Krebs cycle.

It is generally thought that medium-chain fatty acids do not need carnitine for transport to the matrix. That is true for the liver, but not for (rat) muscle and heart [36]. Another believe is that most carnitine is needed to enable acyl-transport, but experiments with rat muscle mitochondria revealed that the bulk of carnitine was needed to liberate CoASH from the produced acetyl-CoA, because carnitine could be replaced by L-malate [3].

2.-Carnitine stimulates mitochondrial metabolism by decreasing acyl-CoA and increasing CoASH. Removal of long-chain acyl-CoA de-inhibits adenine nucleotide translocator, glutamate dehydrogenase, CRAT etc. Removal of acetyl-CoA de-inhibits pyruvate dehydrogenase, which leads to decreased lactate levels and increased glucose oxidation [3]. Removal of branched-chain acyl-CoA in muscle de-inhibit branched-chain acyl-CoA dehydrogenases. Branched-chain-acylcarnitines are further oxidized in liver [37].

3.-Carnitine decreases oxidative stress and inhibits apoptosis by preventing ceramide formation from palmitate [38] and opening of the permeability transition pore [39]. It inhibits caspases [40].

4.-Carnitine and also acetylcarnitine protects against poisoning by (neuro)toxins [41-45] and corrects mitochondrial, lipid and neurotransmitter receptor abnormalities in aging brain and heart [46-50].

5.-Carnitine stabilizes membranes, abolishes Ca²⁺-overload and stimulates the microcirculation in ischemia [51-54].

6.-Carnitine is involved in membrane repair by reacylation, a process occurring in all cells from erythrocytes to neurons [55,56].

7.-Carnitine restores erythrocyte fluidity, enabling passage through capillaries [57]

8.-Carnitine stimulates the immune response and decreases inflammatory cytokines [58-62].

9.-Carnitine promotes maturation of the fetus [63], lungs [64] and sperm [65-66].

10.-Carnitine is involved in acetyl-delivery to choline, aspartate, lipids, histones, and proteins. Acetylation is an important post-translational modification of proteins.

11.-Carnitine is involved in membrane acylation and G-protein signaling [67].

12.-Carnitine transforms, with little electrical stimulation, fibre type II to I in dog muscle [68].

The defects in the carnitine acyltransferases found until now are confined to the mitochondria. As with several disorders in mitochondrial (very-) long- chain fatty acid oxidation, the mild form of CPT-II affect mainly muscle, with exercise-induced myoglobinuria as presenting symptom. The severe forms of CPT-II and CACT defects are fatal multi-organ disorders with cardiomyopathy and low carnitine levels in tissues and blood. CPT-I liver deficiency affects liver and causes a defective hepatic production of glucose and ketone bodies. As may be expected blood carnitine is not decreased [35].

OCTN2-deficiency is the same as primary systemic carnitine deficiency and is discussed in the next section about carnitine deficiency.

Defects in OCTN2 causes primary systemic carnitine deficiency presenting with cardiomyopathy and often myopathy and/or hepatic encephalopathy in early childhood, and is a treatable condition. These patients are cured by carnitine supplementation. Before treatment, the carnitine levels in muscle and blood are very low [68-70].

Secondary carnitine deficiency results from three main, sometimes overlapping, causes.

1. A decrease in available carnitine. By diarrhea, malnutrition and inhibition of OCTN2, for instance by acidosis, drugs [71-72] (atropine, cephaloride antibiotics [73], diphteria toxin, edrophonium, valproate [74]), long-chain acylcarnitine [3], or absence of driving NaCl-gradient [2, 17].
2. Loss of carnitine. By diarrhea, inhibition of OCTN2 (see above), in pregnancy [75], lactation , hemodialysis, sepsis [71], skin burn [76], mitochondrial diseases [35, 71], and drugs [71, 72] (benzoate, buvicaine, cisplatin [77], doxorubicin, emetine, lidocaine, pivaloyl antibiotics, pyrimethamine, sulphopiazine, valproate, zidovudine).
3. Decreased carnitine biosynthesis. By malnutrition (deficiency of lysine, methionine, vitamins B3, B6, B12, C, folate, iron), in prematures, neonates, disease of the biosynthetic organs, use of valproate [78].

There are many assays for carnitine. Here follows a selection of 4 commonly used methods.

1. A spectrofotometric method. Free and total (free + acyl-) carnitine are determined after the removal of protein, and without or with an alkaline step to hydrolyze acylcarnitine, by the reaction catalyzed by added carnitine acetyltransferase.



2. A radioactive method is based upon a similar reaction with ¹⁴C-acetyl-CoA and its conversion into ¹⁴C-acetylcarnitine, which is purified on a column. The –SH group of the CoASH formed is oxidized by N-ethylmaleimide [79].
3. In another spectrofotometric method free carnitine is measured with purified bacterial carnitine dehydrogenase and NAD⁺. Acylcarnitine is hydrolyzed with bacterial acylcarnitine hydrolase before the determination of total carnitine [80].
4. The Tandem mass spectrometric method measures carnitine and all individual acylcarnitine species [81, 82]. It is developed for diagnostic screening purposes, as the occurrence of acylcarnitine species in urine or blood is highly suggestive for the presence of inborn errors [81-83], and is of great value in addition to the GC-MS screening for the organic acidurias.

Supplementation

Carnitine supplementation is also advocated for healthy babies who are not consuming milk, pregnant and lactating women, for sporters, in malnutrition and in animal feeding.

Carnitine, acetylcarnitine and propionyl-carnitine are safe and have only few side effects in a minority of the patients, such as diarrhea, sleeplessness [94] or trimethylamine formation [95], the odor of rotten fish in skin and hair. This usually disappears by taking less carnitine, or by reducing the activity of the intestinal bacteria [95] or by supplementation with extra (10 mg/d) riboflavine [96]. Hundreds of patients were treated with acetylcarnitine and propionyl-carnitine, but trimethylamine formation was not observed [97].

In early days carnitine was isolated from animal muscle products and the most pure L-carnitine preparations were liquids. Then DL-carnitine HCl became available in bulk quantities, and later pure L-carnitine.

Initially, carnitine deficient patients were supplemented with DL-carnitine HCl. But after publications that haemodialysed patients got problems after consumption of pure DL-carnitine [103], this compound was banned, and L-carnitine became the preferred drug.

This has been written as a tribute to Professor Willem C Hülsmann (1928-2000) who was the first Director of our Department of Biochemistry of the Medical Faculty Rotterdam, later Erasmus University of Rotterdam from 1968 to 1988. The author Hans R. Scholte retires the end of the month September 2003 as professor of Biochemistry at Erasmus MC-University Medical Center Rotterdam, and thanks dr Ben Tilly and Ton Verkerk for their valuable indispensable help to get this work in cyberspace.