L-Carnitine in Autism Spectrum Disorder: Comparison
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L-carnitine plays an important role in the functioning of the central nervous system, and especially in the mitochondrial metabolism of fatty acids. Altered carnitine metabolism, abnormal fatty acid metabolism in patients with autism spectrum disorder (ASD) has been documented. ASD is a complex heterogeneous neurodevelopmental condition that is usually diagnosed in early childhood. Patients with ASD require careful classification as this heterogeneous clinical category may include patients with an intellectual disability or high functioning, epilepsy, language impairments, or associated Mendelian genetic conditions. L-carnitine participates in the long-chain oxidation of fatty acids in the brain, stimulates acetylcholine synthesis (donor of the acyl groups), stimulates expression of growth-associated protein-43, prevents cell apoptosis and neuron damage and stimulates neurotransmission.

  • autism spectrum disorder (ASD)
  • biomarkers
  • L-carnitine
  • mitochondria

1. Physiological Properties of L-Carnitine and Acetyl-L-Carnitine

L-carnitine (2-hydroxy-4-trimethylammonium butyrate) is a small (162 Da) polar compound that exists as a double ion in physiological pH. Intracellular and extracellular carnitine may be present as either non-esterified free carnitine (FC) or as esters of short-, medium-, or long-chain organic and fatty acids. LC plays many important roles in the intracellular functions of the body, with its most important role as a contributor to cellular energy metabolism. The primary functions of FC and acylcarnitines (AC), mainly acetyl-L-carnitine (ALC) in humans, are the transport of long-chain fatty acids (FAs) to the mitochondrial matrix and maintaining the mitochondrial homeostasis of coenzyme A (CoA) during mitochondrial oxidation of these acids [49][1]. CoA is necessary for activation and oxidation of the FAs from adipose tissue for ATP synthesis in the mitochondria. FAs oxidation reduces glucose oxidation in the tissues where glucose is not an essential fuel. Fatty acids are a very efficient source of human energy. The amount of energy from the total oxidation of FAs is 37.7 kJ/g, compared to 16.7 kJ/g from protein or carbohydrates. Carnitine also participates in the detoxication processes of toxic exogenous compounds (e.g., some xenobiotics, including ampicillin, valproic acid, and salicylic acid), which are excreted by the kidneys when they are combined with carnitine [9][2]. The next most important role of carnitine is contributing to the catabolism of branched-chain ketoacids derived from branched-chain amino acids (valine, leucine, and isoleucine). L-carnitine also inhibits free radicals production and demonstrates antioxidant action [9][2].
LC has antioxidant effects and optimizes the functions of the complex of mitochondrial enzymes [50][3]. Carnitine also significantly increases dopamine levels in the cortex, hippocampus, and striatum of the rat brain [51][4].

1.1. L-Carnitine Content in Food

Red meat is the richest source of L-carnitine in adults and milk in infants and children [7][5], whereas plants contain only traces of carnitine [52][6].
The standard healthy human diet meets about three-quarters of the requirement for L-carnitine; the remaining one-quarter is synthesized in the human body from lysine (creating carnitine carbon skeleton) and methionine (origin of N-methyl groups) with the participation of ascorbic acid, niacin, pyridoxine, and Fe+2 [53][7] in the liver, kidneys, brain [10][8], and placenta [54][9]. Nutritional carnitine is actively (sodium-dependent) and passively transported from the intestinal content into enterocytes, with 54–86% bioavailability, depending on the amount of carnitine in the meal. In vegetarians adapted to diets with low carnitine, concentration and bioavailability are higher, around 66–86%; in individuals who prefer red meat in carnitine-rich diets, the bioavailability is around 54–72% [55][10]. The bioavailability of carnitine from diet supplements is much lower than from the general diet, reaching only 14–18%. The organism maintains carnitine homeostasis; with the increase in carnitine consumption, there is a decrease in carnitine absorption [56][11]. Absorbed carnitine appears mainly in the portal circulation; it is passively secreted into the bile, mainly in the form of acylcarnitine esters (greater than 66% of total carnitine) and long-chain acylcarnitine esters, amounting to 30–50% of the total bile carnitine [56][11]. Kidneys play the main role in carnitine and its ester homeostasis [22][12]. Carnitine is not metabolized in the human organism, but it undergoes filtration in renal tubules and is almost totally (98–99%) reabsorbed in the renal tubules. Transporter OCTN2, named CT1 (carnitine transporter 1), plays a key role in renal carnitine reabsorption in symport with two sodium cations. Renal OCTN2 activity is inhibited by short- and long-chain acylcarnitines [57][13].

1.2. Health Risks from High Amounts of L-Carnitine

Consumed, but not absorbed, carnitine is degraded, mainly in intestinal microorganisms to trimethylamine (TMA), which is a non-toxic substance with a very unpleasant fish smell; then, it reaches the liver where it is transformed to toxic trimethylamine N-oxide (TMNO, TMAO), which is excreted with urine, and γ-buterobetaine is eliminated mainly in feces. In humans, TMNO in urine accounts for 8–39% and γ-buterobetaine in feces to 0.1–8% of the total dietary carnitine [58][14]. TMA is produced in the intestines not only from L-carnitine but also from betaine, buterobetaine (GBB), choline, and other compounds containing choline, which are present in the typical diet (red meat, eggs, poultry, fish, and dairy products). Previously, TMA and TMNO were considered non-toxic substances, but recently they have been considered potentially carcinogenic agents because of possible transformation to N-nitrosodimethylamine (NDMA). Current research has proven that TMA and TMNO are compounds that favor the occurrence of cardiovascular disease and atherosclerosis and inhibit reverse cholesterol transport [59][15]. However, clinical implications of TMNO in the central nervous system have not yet been documented, but TMNO is present at detectable levels in the cerebrospinal fluid (CSF). In the small tested groups of subjects, TMNO levels in CSF were apparently unrelated to the diagnosed neurological disorders such as Alzheimer’s disease (AD) [60][16]. Recently, increased concentration of TMNO in the plasma of ASD patients was reported, which correlated with an intensification of ASD symptoms. Plasma levels of TMNO, choline, and betaine were higher in ASD patients than in healthy people. Therefore, Ithe author was suggested that TMNO is a useful biomarker of ASD [61][17]. Elevated L-carnitine levels increase the risk of disease if TMNO levels are also elevated. A diet rich in animal products (containing carnitine and choline) changes the intestinal microflora by increasing the synthesis of TMAO. Additionally, another clinicwal studys reported that disturbed TMNO metabolism and increased TMNO concentration in the serum/plasma were connected with renal diseases, cardiovascular diseases, diabetes type 2, and neurological disturbances [62][18].

1.3. Acetyl-L-Carnitine and the Carnitine Transporter OCTN2

L-carnitine is acetylated in the human intestine to active acetyl-L-carnitine (γ-trimethyl-β-acetylbutyre-betaine (ALC)). Since ALC is more easily transferred through intestinal serous membranes than non-acetylated L-carnitine, the intracellular acetylation of carnitine may facilitate its diffusion across the serous membrane. LC and its short-chain fatty esters do not connect with plasma proteins; though blood cells contain LC, the speed of LC distribution between erythrocytes and plasma is very slow [63][19]. In the circulation, about 75% of LC occurs in the free state, 15% as ALC, and the remaining 10% as esters of carnitine with other acids (e.g., propionyl-L-carnitine) [64][20]. In human tissues, L-carnitine is localized mainly in skeletal and cardiac muscles (98%), with only about 1.5% in the liver, kidneys, and brain, and in plasma, about 0.5–1% [7][5]. In the adult brain, about 80% of carnitine exists as FC, 10–15% as ALC and less than 10% as long-chain acylcarnitines [65][21]. LC is absorbed to the cells mainly with the help of organic cation/carnitine transporter 2 (OCTN2), occurring in skeletal muscles, heart, fibroblasts, placenta, renal tubules, and brain (located mainly in the cells of the endothelium of capillaries creating the blood–brain barrier, in astrocytes, and in neurons). OCTN2 is a unique transporter with a dual-mode of transport as both Na+-independent organic cation transporters and Na+-dependent and high-affinity carnitine transporters [57][13]. Defects in the carnitine transporter (OCTN2), which is coded by the SLC22A5 gene, create primary carnitine deficiency, expressed as low urinary carnitine excretion and low blood and tissues carnitine level, which may be a risk factor of ASD. The next protein transporting carnitine to the brain and to its astrocytes is ATB0,+, which is from the family of neurotransmitters depending on sodium and chlorine ions, coded by the SLC6A14 gene and transports all amino acids with the exception of aspartate and glutamate [66,67][22][23]. ATB0,+ may play an important role in the inefficiency of the OCTN2 transporter. It was demonstrated that ATB0,+ exists and functions in the apical membrane of brain endothelial cells, creating a blood–brain barrier [68][24]. Recently, the neutral amino acid transporter coded by the SLC7A5 gene was reported to also be able to transport L-carnitine, connected with the onset of ASD [69][25].
Disturbance of cell homeostasis in brain tissue leads to multidirectional disturbances of biochemical parameters, mostly in the cholinergic system. Acetylcholine plays a key role in cognitive brain functions and participates in the regulation of cognitive processes, attention, and memory. The incorrect expression of cholinergic receptors, mainly nicotinic, was confirmed in people with autism [70][26]. The chemical structures of L-carnitine and its acylcarnitines are comparable to choline and acetylcholine (Ach). Acetyl-L-carnitine positively influences the nervous system because its acyl groups are donated in acetylcholine (Ach) biosynthesis [71][27]. ALC presents neuroprotective action through the improvement of the energy and function of mitochondria, modulation of gene expressions, antioxidative action, and stabilization of cell membranes (stimulates biosynthesis of proteins and phospholipids of cell membranes). It also prevents cellular death and neuronal damage. ALC favors binding glycocorticoids and nerve growth factor (NGF) in the hippocampus [72][28]. Additionally, mitochondrial acylcarnitines supply acyl groups, which are used for the acylation of nuclear histones [73][29]. Acylcarnitines present very high bioavailability compared to L-carnitine because they have a better ability to cross the blood–brain barrier compared to L-carnitine. Therefore, ALC may be administered in much smaller doses than L-carnitine. ALC is recommended to improve central and peripheral nervous system action, improve memory, learning and memorization, increase energy, and improve physical condition and state of mind in therapies of neurodegenerative brain diseases and peripheral neuropathy [74][30]. Carnitine deficiency may contribute to a reduction in mitochondrial copy number and may disturb neurodevelopment, perhaps specifically affecting neurogenesis or synaptic development [75][31].

2. Role of Carnitine in the Oxidation of Fatty Acids

L-carnitine is important in fatty acid metabolism in the brain, although fatty acid oxidation in nervous tissue is less important than glucose oxidation (in normal conditions, brain tissue does not use fatty acids as energy substrates because glucose is the basic brain fuel) [65][21]. As mentioned earlier, L-carnitine participates in the transport of long-chain acyl groups from the cytoplasm to the mitochondria, thus regulating the concentration of acyl-CoA and CoA in the cytosol and mitochondria and protecting correct cell metabolism [76,77][32][33]. In the mitochondria, activated fatty acids are cut into two carbon fragments (acetyl-coenzyme A (acetyl-CoA)), which are oxidized in the tricarboxylic acids cycle (Krebs cycle). Acid groups not removed from the cellular pool inhibit the oxidation of short-chain fatty acids, which results in the peroxidation of lipids in cellular membranes. The deficiency of free CoA limits the efficiency of the Krebs cycle in activating anaerobic glucose metabolism (which inhibits pyruvate dehydrogenase activity, which is responsible for the conversion of pyruvate to acetyl-CoA in aerobic glucose metabolism in cells of the human organism), causing an increase in the concentration of toxic lactate in tissues [58,78,79][14][34][35]. The brain does not oxidize fatty acids directly but uses ketone bodies derived from acetyl-CoA and acetoacetyl-CoA, which are generated by liver β-oxidation of fatty acids. The mitochondrial membrane is not permeable to acyl-CoA, and fatty acids must be connected with L-carnitine to gain entry to the mitochondria interior. L-carnitine creates high energetic ester bonds with long-chain fatty acids catalyzed by carnitine palmitoyltransferase I (CPT-1, CPT I), which is localized in the internal mitochondrial membrane. Three isoforms of CPT-1: CPT-1A, CPT-1B, and CPT-1C [80][36], were described. CPT-1C is expressed in the liver, brain, kidneys, lungs, spleen, intestines, pancreas, ovaries, and fibroblasts [81][37]. CPT-1B is a muscular isoform that is strongly expressed in the heart, skeletal muscles, and testicles [81][37]. CPT1-C is an isoform specific for neurons, but its function in neuronal metabolism remains controversial [82][38]. Carnitine palmitoyltransferase II (CPT-2, CPT II), localized in the internal mitochondrial membrane, removes carnitine from acylcarnitines and again generates acyl-CoA [81][37]. After transporting fatty acids from the cytoplasm to the mitochondria, L-carnitine returns to the cytoplasm for the next cycle using carnitine-acylcarnitine translocase (CACT), while acetyl-CoA may enter (under aerobic conditions at low concentrations of ATP) β-oxidation, with the final production of acetyl-CoA (Figure 21) [83][39].
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Figure 21. Metabolic roles of carnitine and other substances in the brain. Abbreviations: GLUT, glucose transporters; OCTN2, sodium-dependent carnitine organic cation transporter; CPT I, carnitine palmitoyltransferase I; CPT II, carnitine palmitoyltransferase II; CACT, carnitine-acylcarnitine translocase; CAT, carnitine acetyltransferase; CoASH, coenzyme A; Acetyl-CoA, acetyl coenzyme A; TCA cycle, tricarboxylic acid cycle.
CPT-1 and CPT-2 are mainly engaged in the import of long-chain acyl-CoA, such as palmitoyl-CoA, oleoyl-CoA, and linoleoyl-CoA, into the mitochondria [81][37]. Oxidation medium- (C6–C10) and short-chain (C4–C6) fatty acids seem to be independent of the carnitine shuttle [84][40]. In physiological conditions, long-chain fatty acids are oxidized with the participation of the mitochondrial β-oxidation system, with minimal participation of peroxisomes, where the longest chain fatty acids are oxidized (C ≥ 22). In the peroxisomes, the longest chain FAs are shortened and transported to the mitochondria for β-oxidation and total oxidation to CO2 and H2O [85][41]. Fatty acids may be ω-oxidized with the participation of microsomal oxidase, producing dicarboxylic acids. Fatty acids may also be degraded by β-oxidation in peroxisomes to succinate and acetyl-CoA or completely oxidized after transport to the mitochondria in the process of β-oxidation. In physiological conditions, ω-oxidation is a secondary pathway for the oxidation of fatty acids. During disturbances of β-oxidation, the activity of ω-oxidation may increase, generating an excess of dicarboxylic acids. Dicarboxylic acids are unspecific markers of defects in the oxidation of fatty acids [86][42].
During the incorrect process of fatty acid oxidation, fats released from the fat tissue accumulate in the liver, skeletal muscles, and heart. In the liver, defects in L-carnitine or β-oxidation induce fat deposit and decrease ketone production, which is an alternative source of energy (instead of glucose) for the heart, skeletal muscles, and brain. In the liver, acetyl-CoA allosterically activates pyruvate carboxylase, favoring gluconeogenesis. When fatty acid oxidation is disturbed, fats cannot be used, so glucose is used without regeneration in the cycle of gluconeogenesis because of pyruvate carboxylase inhibition, ketone bodies cannot be produced, glucose level (hypoglycemia) decreases, and brain functioning impairment with loss of consciousness can occur [83][39].

3. Primary and Secondary L-Carnitine Deficiency

Disturbed L-carnitine metabolism has recently been connected with neurodevelopment disturbances, including autism spectrum disorder. The L-carnitine biosynthetic pathway includes four enzymes: ε-N-trimethyllysine hydroxylase (TMLD), β-hydroxy-ε-N-trimethyllysine aldolase (HTMLA), 4-N-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH), and γ-butyrebetaine dioxygenase (BBD) (Figure 32).
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Figure 32. Carnitine biosynthetic pathway. Abbreviation: enzymes: 1, S-adenosyl methionine: ε-N-lysine methyltransferase; 2, protease (lysosomes); 3, ε-N-trimethyllysine dioxygenase (TMLD*) (mitochondria); 4, β-hydroxy-ε-N-trimethyllysine aldolase (HTMLA) (cytosol); 5, 4-N-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH) (cytosol); 6, γ-butyrobetaine dioxygenase (BBD*) (cytosol); second substrates and products: (a) S-adenosyl methionine (SAM)→ S-adenosyl homocysteine (SAH); (b) 2-ketoglutarate + 02 →succinate + C02; (d) NAD+ → NADH + H+; (e) 2-ketoglutarate + 02 → succinate + C02; coenzymes: (b) vitamin C, iron; (c) pyridoxal phosphate; (e) vitamin C, iron. * genetically determined enzyme deficiency.
The OCTN2 transporter (organic cation/carnitine transporter) transports carnitine to the cells. L-carnitine allows the transport of long-chain fatty acids from the cytosol to the mitochondrial matrix by the mitochondrial carnitine–acylcarnitine cycle, which is composed of three enzymes: carnitine palmitoyltransferase I (CPT I), carnitine-acylcarnitine translocase (CACT), and carnitine palmitoyltransferase II (CPT II) (Figure 21). Therefore, autism may be induced by inborn errors in L-carnitine biosynthesis, the transport of L-carnitine to the mitochondria, or the mitochondrial carnitine–acylcarnitine cycle [98][43]. L-carnitine deficiency creates defects in fatty acid oxidation, which is used to produce energy. Three types of L-carnitine deficiency have been distinguished: primary systemic carnitine deficiency, primary myopathic L-carnitine deficiency, and secondary L-carnitine deficiency. Genetic defects in the enzymes participating in L-carnitine synthesis, defects in the proteins responsible for carnitine transport into cells, or defects in the mitochondrial carnitine–acylcarnitine cycle lead to disturbances in fatty acids metabolism. Recognition of primary carnitine deficiency may be confirmed biochemically by low levels of free carnitine in plasma (<8 μM; reference values: 25–50 μM), caused by lowered renal feedback absorption (<90%) and correct renal function, without abnormality in urinary excretion of organic acids. However, a diagnosis should be confirmed by molecular tests [99][44].
Clinical symptoms of disorders of biosynthesis or carnitine excretion differ but predominantly cover hypoketotic hypoglycemia, myopathy/cardiomyopathy, and liver insufficiency. Symptoms of disturbances in biosynthesis and excretion of carnitine occur because of energy shortages and the accumulation of fatty acids in some organs. Secondary carnitine deficiency may also occur due to other reasons, e.g., malnutrition, poor absorption, valproic acid toxicity, pivalinic acid present in antibiotics, hemodialysis, and increased loss in urine. Carnitine deficiency connected with low plasma carnitine concentration is less harmful than primary carnitine deficiency and may be treated with low doses of carnitine.

3.1. Systemic Primary L-Carnitine Deficiency

Systemic primary carnitine deficiency (SPCD) is a progressive autosomal disturbance connected with impaired carnitine uptake by plasmatic membranes because of a deficiency in the OCTN2 transporter, which is coded by the SLC22A5 gene (localized on the 5q31 chromosome). Heterozygous or homozygous deficiency of OCTN2 transporters may be an autism risk factor [100,101][45][46]. In early life, SPCD is usually recognized as a metabolic decompensation manifested by hypoketotic hypoglycemia; encephalopathy, frequently connected with liver enlargement; increased serum level of aminotransferases; hyperammonemia; cardiomyopathy; muscular weakness; changed intestinal peristalsis and repeated infections in early life. The symptoms of carnitine deficiency may resemble Rey syndrome and may end with the sudden death of the neonate. The large accumulation of lipids in the skeletal muscles, heart, and liver and lowered carnitine concentration in the above-mentioned organs are found in SPCD. Plasma levels of FC, TC, and AC are very low, so autistic children require carnitine supplementation [102,103][47][48]. The low concentrations of plasma carnitines in patients with SPCD are caused by excessive urinary carnitine excretion because of defective reversible absorption in renal tubules; therefore, the carnitine level in plasma and tissues (heart and skeletal muscles) may drop to below 10% of normal values. In the muscles of patients with SPCD, the total carnitine content may be very low, even less than 5% in comparison to healthy people [102,103,104][47][48][49]. Basic treatment of SPCD patients involves carnitine supplementation, which should be introduced as soon as possible after diagnosis before the appearance of irreversible damage to the internal organs [100][45]. Monocarboxylic acid transporter 9, coded by the SLC16A9 gene, which transports carnitine from renal tubules to plasma, may be important. The deficit in this gene was not reported in SPCD patients, but it was reported in numerous individual polymorphisms, including RS7094971, which is associated with abnormal plasma levels of FC and AC [105][50].
Detection of frequent inborn errors in the biosynthesis of carnitine from trimethyllysine (Figure 21), caused by a deficiency in the activity of the TMLHE gene, may help explain the reasons for dysmorphic autism. TMLHE (conjugated with chromosome X), which codes trimethyllysine dioxygenase (TMLD), the first enzyme on the mitochondrial carnitine synthesis pathway, was detected in men with nondysmorphic autism (frequency 1 in 350 men). However, only about 3% of men with TMLHE gene deficiency developed autism. The risk of nondysmorphic autism connected with TMLHE mutation may be diminished by appropriate carnitine supplementation during the early stages of child brain development [11,12,96,106][51][52][53][54]. Butyrobetaine dioxygenase (BBD) is the next false enzyme participating in carnitine biosynthesis, coded by the BBOX1 gene, which may induce autism [98][43]. Symptoms of carnitine deficiency (small head, delay in speech, tenuous growth, and presence of some dysmorphic traits) in a girl with a homozygous deletion of BBOX1 were described by Rashidi-Nezhad et al. [107][55]. Clinical symptoms of carnitine deficiency were not detected in the presented girl, but laboratory tests showed plasma FC concentration was at the lower limit of the reference values. Acylcarnitine profile was also within the reference ranges, and the free carnitine/acylcarnitine ratio was in the normal range in this girl. TheIt authorwas suggested that dietary carnitine consumption and renal reabsorption were sufficient for correct carnitine homeostasis in the above-described casone [107][55]. Recently, Lee et al. [108][56] showed that expression of BBOX1 is decreased in the mouse model of schizophrenia. They tested 284 people in the Korean population with schizophrenia and 409 healthy people and found that BBOX1 polymorphisms may be connected with increased schizophrenia susceptibility in this population. As mentioned earlier, the mitochondrial carnitine–acylcarnitine cycle is composed of three enzymes: CPT-1, particularly neuronal isoform CPT-1C (localized in the hypothalamus, almond body, and hippocampus, which plays an important role in neurological and neuropsychiatric disorders); CACT; and CPT-2., WThe did not find data ata wasn't found on defects concerning the above-mentioned enzymes in autistic children in the literaturen.

3.2. Secondary L-Carnitine Insufficiency

The clinical consequences of secondary L-carnitine deficiency, even though it occurs more frequently than primary L-carnitine deficiency, are less harmful than those of primary L-carnitine deficiency. A diet poor in L-carnitine (e.g., vegetarian diet), malnutrition, absorption and transport disturbances, increased urinary carnitine excretion, liver diseases (cirrhosis), chronic renal diseases, and administration of some drugs (e.g., antiepileptic drugs, including valproic acid and carbamazepine, phenytoin, phenobarbital, beta-lactam antibiotics, and anticancer drugs) may be reasons for secondary L-carnitine deficiency. The above-listed drugs are excreted in the urine in combination with L-carnitine, thus lowering the carnitine plasma concentration. The above-mentioned drugs may also inhibit OCTN2 transporters, leading to secondary carnitine deficiency [109,110][57][58]. Secondary carnitine deficiency may result from defects in any enzyme participating in the mitochondrial oxidation of fatty acids because excess fatty acids linked to carnitine are excreted into the urine as acylcarnitines. Therefore, with secondary carnitine deficiency, the free carnitine pool is shifted in the direction of acylcarnitines because carnitine is used to eliminate the acyl groups of the accumulated fatty acids resulting from the dysfunction of fatty acids mitochondrial metabolism. Normal or slightly lowered FC levels are present in plasma, but AC levels and AC/FC ratio are increased [111,112][59][60]. Enzymatic defects in fatty acid oxidation may concern very-long-chain fatty acids acyl-CoA dehydrogenase (VLCAD), medium-chain acyl-CoA dehydrogenase (MCAD), long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency (LCHAD), CPT-2, and CACT deficiency, which may cause secondary carnitine deficiency [109,113][57][61]. It was demonstrated that defects in the activity of long-chain acyl-CoA dehydrogenase (LCAD) when oxygenizing long-chain fatty acids or dehydrogenase deficiency may cause autism. Significant increases in the concentration of unsaturated fatty acids C14:1 and C14:2 were reported in ASD patients. The acylcarnitine profile change of ASD patients is similar to the change observed in experimental mice with a deficit in LCAD activity [114][62]. Disturbances in the metabolism of amino acids, e.g., isovaleric acidemia, methylmalonic acidemia, or glutaric aciduria type I, may be another reason for secondary carnitine deficiency [115][63]. In primary carnitine deficiency, the plasma free carnitine concentration is below 5 μM/L, but plasma free carnitine concentration in secondary carnitine deficiency is higher and may amount to a little below 20 μM/L, which is accompanied by a normal tissue concentration of carnitine. Supplementation with carnitine in secondary carnitine deficiency requires small carnitine doses because carnitine levels may be normalized quickly. As carnitine biosynthesis provides only 25% of the carnitine pool, defects in carnitine biosynthesis are not the reason for carnitine deficiency in people with normal renal function and a regularly balanced diet. However, limited consumption of carnitine may cause its deficit, especially in neonates (in parenteral nutrition or during the administration of milk substitutes without carnitine). Additionally, in neonates, endogenic carnitine synthesis is limited because of the immaturity of liver enzymes, particularly γ-butyrobetaine dioxygenase (BBD), and the immaturity of renal canaliculus, which decreases the kidney’s carnitine reabsorption ability [7][5]. Laboratory tests in ASD patients demonstrated a disturbed acylcarnitine profile, which may be potential biomarkers reflecting the existence of acquired mitochondrial disease. The secondary FC deficiency is connected with the increased concentration of AC and AC/FC ratio [111][59].

4. L-carnitine A Potential Biomarker of Mitochondrial Disturbances

Free carnitine transports long-chain fatty acids from the cytoplasm to the mitochondria as acylcarnitines. Acylcarnitines undergo β-oxidation, producing energy in the mitochondria. There are short-(3–5), medium- (6–12), and long-chain (>12 carbon atoms) fatty acids. Defects in the L-carnitine shuttle (L-carnitine is released from fatty acid and creates acetyl-CoA) in the mitochondria mainly disturb the oxidation of long-chain fatty acids, as a medium- and short-chain fatty acids may directly penetrate mitochondrial membranes without L-carnitine mediation. Abnormal fatty acid oxidation has been observed in ASD: (1) A low-level of free carnitine (main cofactor in the transport of long-and very-long-chain fatty acids from the cytoplasm to the mitochondrial matrix) was observed in ASD children because of lowered mitochondrial β-oxidation of fatty acids. (2) Increases in the concentrations of long- and very-long-chain fatty acids were detected in the serum of ASD children in comparison to the control group, which suggested an excess of unprocessed fatty acids. (3) In ASD patients, a significant increase in the concentration of acylcarnitines was detected. These data suggest the existence of abnormalities in fatty acids metabolism with the participation of L-carnitine [14][64]. It is possible that mitochondrial defects are caused by L-carnitine deficiency and blockade of secondary fatty acid oxidation, as low serum concentrations of free and total carnitine were observed in children with ASD in comparison to the control group (p < 0.001) [116][65]. Children with ASD had considerably lowered concentrations of short- and long-chain acylcarnitines compared with medium-chain acylcarnitines, which suggested the presence of disturbances in L-carnitine circulation and mitochondrial dysfunction [117][66]. The observed carnitine deficiency in ASD patients, accompanying lactate increase in the blood, and the significant increases in alanine and ammonia levels point to mild mitochondrial dysfunction [111,116][59][65]. The results published by Rossignol and Frye indicated the presence of low total carnitine plasma concentrations in up to 90% of the investigated ASD children [38][67].
Accurate and precise determination of free carnitine and individual acylcarnitines became possible after the introduction of new diagnostic techniques such as mass spectrometry. Characteristic acylcarnitines profiles observed in disturbances connected with mitochondrial defects in people with autism spectrum disorder may be promising biomarkers of primary and secondary carnitine deficiencies. Abnormalities in the acylcarnitine profiles in ASD patients, such as abnormally increased acylcarnitine panel C4OH, C14, C16:1, and C16 in 74 (35%) of 213 investigated patients, confirmed the results of Frye et al. [14][64]. Incorrectly increased acylcarnitines concentrations (hydroxybutyryl carnitine, myristoyl carnitine, and enoylpalmitoyl carnitine) were found in 17% of the investigated children with ASD [14][64]. Lv et al. [117][66] reported low concentrations of free carnitine and acylcarnitines (glutarylcarnitine, octylcarnitine, and carnosylcarnitine) in children with ASD in dry blood spots in comparison to healthy children. The diagnostic accuracy, analyzed using the receiver operating characteristic (ROC), showed 93% specificity and 40% sensitivity (area under the curve (AUC) = 0.72) for glutarylcarnitine. Diagnostic accuracy analyzed with ROC showed 80% specificity and 50% sensitivity (AUC = 0.66) for free carnitine and 80% specificity and 50% sensitivity (AUC = 0.66) for carnosylcarnitine. The profiles of carnitine and acyl-carnitines change significantly during the first year of life but remain at the same level between 2 and 15 years of age. Therefore, determining the profiles in a dried blood spot can provide an early indication of higher classification efficiency (sensitivity 72.3%, specificity 72.1%) [48][68]. The lowered amounts of free carnitine and short- and long-chain acylcarnitines in children with ASD suggest potential mitochondrial dysfunction and the pathologic metabolism of fatty acids. Serum concentrations of glutarylcarnitine and carnosylcarnitine may be potential biomarkers in ASD diagnostics, as suggested by Lv et al. [117][66]. Disturbances in carnitine metabolism may be important in the diagnosis of nondysmorphic autism, as suggested by Ratajczak and Sothern [118][69]. The experimental research conducted by MacFabe [119][70] in rats demonstrated changes in the composition of phospholipids during the induction of autistic behavior after intra-intestinal infusion of propionic and butyric acids, which is similar to ASD in people. It appeared that intra-intestinal infusions of propionic acid increased the motor activity of experimental rats. Additionally, propionic acid increased the immune reactivity of monocarboxylate transporter 1 (mainly in the external bag of white matter), which suggested changes in both transport and metabolism of short-chain fatty acids in the brain. Propionic acid and butyric acid (to a lesser degree) in all phospholipid classes lowered the level of all monounsaturated fatty acids, all ω-6 fatty acids, and saturated fatty acids. The general amount of acylcarnitines, long-chain acylcarnitines (C12:24), short-chain acylcarnitines (C2:9), and the ratio of bound to free carnitine increased after experimental infusions of propionic and butyric acids. Evidence indicating the relationship of ASD with processes in the alimentary tract is increasing due to intestinal microflora, intestinal bacterium cell membrane components (lipopolysaccharides), or intestinal bacteria metabolites. Propionic acid produced by Clostridia, Bacteroides, and Desulfovibri, during carbohydrates fermentation or presence in the diet may disturb the functioning of the GABA-ergic system, which plays a significant role in autism pathogenesis [5,95,119][70][71][72]. Lowered concentrations of free and total carnitines in the plasma of autistic children are connected with gastrointestinal symptoms. L-carnitine deficiency, accompanied as mentioned earlier by a slight increase in lactate and significant increases in alanine and ammonia, is a symptom of mitochondrial dysfunction. Determination of blood acylcarnitine profiles may be a biomarker of mitochondrial disturbances in fatty acid oxidation.

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