Review
Acylcarnitines: Role in brain

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Abstract

l-carnitine is present in mammalian cells as free carnitine and acylcarnitines. The acylcarnitine profile has been shown to be useful in identifying inborn errors of metabolism and to be altered under different metabolic conditions. While carnitine’s most widely known function is its involvement in β-oxidation of fatty acids, it may also have other roles in metabolism. The importance of acylcarnitines in tissues with high rates of β-oxidation such as heart and muscle is intuitive. However, acylcarnitine and carnitine supplementation have resulted in beneficial effects in the treatment of various neurological diseases, even though fat is not the major fuel for brain. Recent data indicate new, multifactorial roles for acylcarnitines in neuroprotection. Brain acylcarnitines can function in synthesizing lipids, altering and stabilizing membrane composition, modulating genes and proteins, improving mitochondrial function, increasing antioxidant activity, and enhancing cholinergic neurotransmission. Currently a relatively small subset of acylcarnitines is usually investigated. More research is needed on the use of acylcarnitines in the treatment of neurological diseases using a list of acylcarnitines encompassing a wide range of these molecules. In summary, carnitine is not merely a cofactor in β-oxidation, but rather it has many known and yet to be discovered functions in physiology.

Introduction

l-Carnitine (trimethylamino-β-hydroxybutyrate) (LC) is present in cells and tissues as both free carnitine and acylcarnitines, including acetyl-l-carnitine (ALC). LC is a naturally occurring, endogenous compound in all mammalian species and its most widely known function is as an important transporter of long-chain fatty acids into mitochondria for β-oxidation. Humans obtain carnitine from their diet, predominately from meat and dairy, and through endogenous biosynthesis. LC is synthesized in vivo from l-lysine and l-methionine, mostly in liver and kidney [1], [2].

Under normal conditions, carnitine palmitoyltransferase 1 (CPT 1) catalyzes the transfer of acyl groups from acyl-Coenzyme A (acyl-CoA) to carnitine to produce acylcarnitines and free coenzyme A (CoA). This reaction is one of the first highly regulated steps that is common to both pathways of fatty acid oxidation and ketogenesis [3], [4] (Fig. 1). Therefore, the critical substrates carnitine and acylcarnitines are important in understanding the metabolic pathway associated with ketosis. While carnitine is an important metabolite involved in β-oxidation of fatty acids, it may also be important in transporting other metabolites due to its ability to form esters with many carboxylic acids. The free hydroxyl group, shown in Fig. 2, can be enzymatically esterified to activated acetate groups or to another activated carboxylic acid including fatty acids of all chain lengths, to form acylcarnitines (examples of which are shown in Fig. 3). Carnitine acetyltransferase (CAT) catalyzes the synthesis of short-chain acylcarnitines, specifically ALC, and is located on the inner mitochondrial membrane as well as in microsomes and peroxisomes [5], [6]. ALC and other acylcarnitines can be transported across the inner mitochondrial membrane by carnitine–acylcarnitine translocase and transported out of the mitochondria into the cytosol. It is evident, then, that acylcarnitines are activated molecules that are transportable throughout the body, delivering various acyl groups for a wide range of functions.

LC has an amphiphilic structure, making it very mobile throughout the cell. The free hydroxyl group has the potential for many different molecules to attach, creating a wide array of possible acylcarnitines. The ability to esterify and transport metabolites throughout the body distinguishes LC as a unique metabolite and suggests the acylcarnitine profile may be a useful indicator of metabolic changes, particularly related to disease states. In addition, this wide array of possibilities also leads to a broad range of structures that are very different both chemically and metabolically. For instance, ALC is a small, water-soluble molecule that is easily transportable and may be used to deliver acetyl groups to a variety of locations. Yet a long-chain acylcarnitine, such as palmitoyl-l-carnitine (PLC), needs a transporter to cross the plasma membrane and, therefore, may be more restricted in its actions. As a result, changes in individual acylcarnitines may imply changes in specific metabolic pathways. Monitoring specific acylcarnitines should lead to a better understanding of mechanisms of disease and allow for better design of treatment regimens. The acylcarnitine profile has been shown to be useful in identifying inborn errors of metabolism in neonatal screening using tandem mass spectrometry (MS/MS) based analysis. Important examples are fatty acid oxidation defects, such as long-chain acyl-CoA dehydrogenase deficiency (VLCAD) and disorders of organic acid metabolism, such as propionyl-CoA carboxylase deficiency [7], [8], [9]. Recently metabolomics methodology has been used to identify patients with methylmalonic and propionic acidemia [10].

Inborn errors of metabolism can lead to a build-up of toxic metabolites and can be fatal or result in serious health problems early in life. For this reason, early comprehensive neonatal screening is used to detect abnormalities to avoid major physical and neurological effects [11]. Mass spectrometry-based analysis is used to diagnose inborn errors of metabolism in newborns by identifying and quantifying specific metabolites [12], [13]. Newborn screening by MS/MS has identified compounds and provided early treatment to infants with disorders of mitochondrial β-oxidation, organic acidemias, disorders of the urea cycle, and rare disorders of metabolism before the onset of symptoms. For example, after the application of tandem mass spectrometry was introduced clinically, infants with short-chain acyl-CoA dehydrogenase deficiency (SCAD) and isobutyryl-CoA dehydrogenase deficiency have been identified based on elevated butyryl-carnitine/isobutyryl-carnitine concentrations in newborn blood spots [14], even though two out of three infants remained asymptomatic at the time of diagnosis. Age-related variations in acylcarnitine and free carnitine concentrations have been observed and should be taken into consideration when diagnosing and managing inborn errors of metabolism [12], [15]. Inborn errors of metabolism detected by acylcarnitine profile analysis adapted from Rinaldo and coworkers [16] and the corresponding acylcarnitine changes are listed in Table 1.

In addition to identifying inborn errors of metabolism, the acylcarnitine profile may also be useful in identifying other metabolic perturbations. In healthy neonates, cord blood concentrations of total acylcarnitines strongly correlated to birth weight, and lower umbilical artery pH caused accumulation of long-chain acylcarnitines [12]. The acylcarnitine profile may additionally be a useful parameter for identifying perinatal asphyxia and other metabolic disturbances in utero.

Outside of newborn screening, other disease states and alterations in metabolism may be detected through monitoring of the acylcarnitine profile. Ulcerative colitis is a disorder involving chronic inflammation of the colonic mucosa for which the etiology and pathogenesis are unknown. Alterations in short-chain fatty acid metabolism have been identified in patients with this disease [17]. Furthermore, celiac disease is an autoimmune disorder derived from gluten intolerance. Bene and coworkers found a significant difference in the acylcarnitine profile in plasma of adult patients with ulcerative colitis and patients with celiac disease compared to controls with no change to free carnitine levels [18], [19]. Comparison of the plasma acylcarnitine profile of diabetic patients to non-diabetic controls found a 300% increase of acylcarnitines with a chain length of 10–12 carbons indicating incomplete long-chain fatty acid β-oxidation [20]. In these patients ALC increased and propionyl-l-carnitine decreased as glycosylated hemoglobin increased.

The metabolome and the targeted carnitinome or acylcarnitine profile are becoming increasingly popular tools. Non-targeted metabolomics is being used to create a more comprehensive metabolic profile of the plasma that allows the investigator to assay thousands of metabolites and identify significantly different metabolic features [10]. Many labs have evaluated the utility of acylcarnitine profiles in the diagnosis of inborn errors of metabolism, some of which were reviewed by Pasquali and coworkers in 2006 [21]. Supplementation of specific acylcarnitines, furthermore, may lead to benefits for particular disease states. For example, propionyl-l-carnitine supplementation may improve general fatigue [3] and may protect heart health [22], while PLC supplementation may regulate lipid esterification [23].

Concentrations of carnitine and acylcarnitines change under altered dietary conditions. During starvation and after eating a high-fat diet, the proportion of carnitine that is acetylated in liver and kidney significantly increases and, oppositely, a high carbohydrate diet causes very low levels of ALC in liver [24]. In humans, there appears to be a delayed decrease in plasma LC and a rapid increase in both long- and short-chain acylcarnitines during fasting or diabetic ketosis [25], [26], [27]. While plasma levels do not directly correlate with cerebral levels, in neonatal rats starvation led to a significant increase in mean brain acylcarnitine concentration compared to control rats, with almost all of the increase attributed to short-chain acylcarnitines [28]. The authors concluded that carnitine and its relative esters may be redistributed to the brain during fasting and the brain may use them for energy production or, possibly, for the delivery of acetyl groups.

Plasma acylcarnitine levels were analyzed in 1–7 year old children after fasting and ingestion of sunflower oil, which is made up largely of linoleic acid (66%), a polyunsaturated fatty acid (PUFA), and oleic acid (21%), a monounsaturated fatty acid (MUFA). Under both conditions there was an increase in all plasma straight-chain acylcarnitines and ALC was the largest contributor to the increase in total esterified carnitine with an almost 4-fold increase after fasting [29]. These and other changes to carnitine concentrations are summarized in Table 2.

Section snippets

Carnitine and acylcarnitines in brain

The importance of carnitine in brain is emphasized by carnitine deficiency symptoms, many of which involve major deleterious effects in brain. Because the brain is highly reliant on oxidative metabolism, impairment of fatty acid metabolism and energy production due to lack of carnitine leads to metabolic encephalopathy. Structurally, the astrocyte swells and mitochondria are expanded in nerve cells under carnitine deprivation [30]. In addition, LC is taken up by neuronal cells through a Na+-

Energy metabolism and membranes

The roles of long-chain acylcarnitines, specifically PLC, in brain have been studied and appear to involve interaction with membranes, acylation of lipids, as well as protein interaction. Mainly due to the amphiphillic nature of PLC, it can react on the surface of membranes and influence membrane fluidity and the activity of membrane enzymes and transporters [46], [47], [48], [49]. PLC has been shown to be involved in phospholipid and fatty acid turnover in rat fetal neurons [50], [51]. PLC

Acetyl-carnitine (ALC): role in brain

ALC is present in relatively high levels in the brain [61] and it is highest in the hypothalamus, [33] where the level of the ALC synthesizing enzyme, CAT, is also high. ALC can readily cross the blood–brain barrier [62], so supplementing with this compound could feasibly affect brain metabolism. Injection of ALC in rats led to reduced oxidation of glucose and increased glycogen synthesis in brain [63]. Changes in the activities of specific enzymes involved in the tricarboxylic acid (TCA)

Conclusions and future directions

Much of the research on carnitine and its esterified derivatives, such as ALC and PLC, has centered on the role of these molecules in metabolism. There is a great deal of evidence in the literature, however, to support new and multifactorial roles for these compounds in neuroprotection. ALC can provide high-energy acetyl groups to metabolic pathways to improve the overall energy status of the brain and to alter the biosynthesis patterns of some neurotransmitters. The acyl groups of

Acknowledgements

The authors express appreciation to the countless students and colleagues in our laboratory who have enhanced our current understanding of carnitine.

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