3-Methylglutarylcarnitine: a biomarker of mitochondrial dysfunction

Abstract

The acylcarnitines comprise a wide range of acyl groups linked via an ester bond to the hydroxyl group of l-carnitine. Mass spectrometry methods are capable of measuring the relative abundance of hundreds of acylcarnitines in a single drop of blood. As such, acylcarnitines can serve as sensitive biomarkers of disease. For certain acylcarnitines, however, their biochemical origin, and biomedical significance, remain unclear. One such example is 3-methylglutaryl (3MG) carnitine (C5-3M-DC). Whereas 3MG carnitine levels are normally very low, elevated levels are detected in discrete inborn errors of metabolism (IEM) as well as different forms of heart disease. Moreover, acute injury, including γ radiation exposure, paraquat poisoning, and traumatic brain injury manifest elevated levels of 3MG carnitine in blood and/or urine. Recent evidence indicates that two distinct biosynthetic routes to 3MG carnitine exist. The first, caused by an inherited deficiency in the leucine catabolism pathway enzyme, 3-hydroxy-3-methylglutaryl (HMG) CoA lyase, leads to a buildup of trans-3-methylglutaconyl (3MGC) CoA. Reduction of the double bond in trans-3MGC CoA generates 3MG CoA, which is then converted to 3MG carnitine by carnitine acyltransferase. This route, however, cannot explain why 3MG carnitine levels increase in IEMs that do not affect leucine metabolism or various chronic and acute disease states. In these cases, disease-related defects in aerobic energy metabolism result in diversion of acetyl CoA to trans-3MGC CoA. Once formed, trans-3MGC CoA is reduced to 3MG CoA and esterified to form 3MG carnitine. Thus, 3MG carnitine, represents a potential biomarker of disease processes associated with compromised mitochondrial energy metabolism.

1.1. Introduction

Acylcarnitines are esters arising from conjugation of a broad range of acyl chains to the hydroxyl group of l-carnitine (l-ß-hydroxy-4-N-trimethylaminobutyric acid). Carnitine is well known to play a prominent role in transport of long-chain fatty acids from cytosol to mitochondria for ß-oxidation [1]. The occurrence of elevated levels of specific acylcarnitines in different bodily fluids generally implies the existence of metabolic dysfunction wherein specific metabolites accumulate to an extent that requires their excretion. When such metabolites possess a CoA thioester linked carboxylic moiety, they are candidates for conversion to the corresponding acylcarnitine [2]. This process effectively prevents depletion of the intra-mitochondrial CoA pool and provides a means whereby excess, or unusable short-chain acyl moieties, are transported out of mitochondria. These acylcarnitines are then exported from the cell and can be detected in blood plasma and urine. Changes in the relative abundance of specific acylcarnitines reflect different disease processes and, therefore, serve as biomarkers [3]. Mass spectrometry-based analysis of bloodspot acylcarnitine metabolites is widely used in newborn screening for inborn errors of metabolism (IEM) [4, 5]. Thus, acylcarnitines represent an important category of analytes in targeted and non-targeted metabolomics studies. For example, glutaric acidemia type-I is an IEM caused by a deficiency in the lysine / hydroxylysine / tryptophan degradation pathway enzyme, glutaryl CoA dehydrogenase. As glutaryl CoA levels rise in mitochondria, it becomes a substrate for carnitine acyl transferase (CAT)-mediated conversion to glutarylcarnitine, and, once formed, this short-chain acylcarnitine is destined for export from the cell. The topic of the present review is 3-methylglutaryl (3MG) carnitine, a unique acylcarnitine whose corresponding CoA derivative, 3MG CoA, is not part of any known metabolic pathway. Insofar as 3MG CoA is a CAT substrate, accumulation of 3MG carnitine is indicative of aberrant metabolic processes. Whereas it is well known that 3MG carnitine levels rise in IEMs that affect the leucine catabolism pathway enzyme, 3-hydroxy-3-methylglutaryl (HMG) CoA lyase, its occurrence in disorders unrelated to this pathway indicates an alternate biosynthetic route to this compound exists. In the present review, evidence is provided that compromised mitochondrial energy metabolism associated with IEMs, chronic disease or acute injury can result in elevated levels of 3MG carnitine. As such, this unusual acylcarnitine represents a potentially valuable biomarker.

1.1.1. Classification and nomenclature of acylcarnitines

An important aspect of metabolomics-derived acylcarnitine data concerns their classification and nomenclature. According to Dambrova et al [2], acylcarnitines can be organized into nine categories, depending on the length and type of the acyl group. These categories include: 1) short-chain; 2) medium-chain; 3) long-chain; 4) very long-chain; 5) hydroxy; 6) branched-chain; 7) unsaturated; 8) dicarboxylic; and 9) miscellaneous, respectively. Short-chain acylcarnitines possess acyl groups two to five carbons in length, medium-chain acylcarnitines contain acyl groups with six to thirteen carbons, long-chain acylcarnitines have acyl groups with fourteen to twenty-one carbons and very long-chain acylcarnitines possess acyl groups with more than twenty-two carbons. A commonly used abbreviation scheme for acylcarnitines includes the acyl chain carbon chain length (e.g., C6, C8, or C10), the presence and number of unsaturated bonds (e.g., linoleoylcarnitine = C18:2), the presence of a hydroxyl group (e.g., 3-hydroxyisovalerylcarnitine = C5-OH), methyl branches (e.g., 2-methylbutyrylcarnitine = C4-2M), or dicarboxylic moieties (e.g., glutarylcarnitine = C5-DC). As seen below, however, caution should be exercised when using abbreviations to identify certain acylcarnitines because the abbreviation used may not fully distinguish between unique molecular weight isomers. Considering the large number of acylcarnitines detected in modern metabolomics studies, individual metabolites are oftentimes clustered into groups or factors [6, 7]. Given the considerable complexity of untargeted metabolomics data sets, this approach can enhance understanding of pathway perturbations that may be responsible for differences observed between control and disease subjects.

1.2. 3MG carnitine

Insofar as modern techniques are capable of identifying hundreds, if not thousands, of metabolites, deciphering the significance of a disease-associated change in a particular acylcarnitine can be daunting, especially if the metabolite in question arises from a nontraditional biochemical route. Such is the case with 3MG carnitine, which has alternately been abbreviated as “C6-DC”, “C5-M-DC”, or “C5-3M-DC”. 3MG carnitine is a methyl-branched, dicarboxylic, short-chain acylcarnitine. Given that its acyl chain is five carbons in length with a methyl branch at carbon three, this acylcarnitine is most accurately abbreviated as C5-3M-DC. Using an accurate abbreviation is important for 3MG carnitine because its molecular weight (289.32 Da) is identical with that of the straight-chain dicarboxylic acylcarnitine, adipoylcarnitine (C6-DC) (Figure 1). As such, these distinct acylcarnitines are susceptible to misidentification and, potentially, misinterpretation of metabolomics data. To avoid confusion, the term C6-DC should only be used to abbreviate adipoylcarnitine. In practice, some effort is required to distinguish between these two metabolites, which in many cases, is essential for appropriate data interpretation. For example, Kraus et al [8] employed liquid chromatography-mass spectrometry analysis of chemically synthesized internal standards to accurately identify these analytes. Others have simply designated the presence of two species (C6-DC #1 and C6-DC #2) to indicate the presence of two distinct acylcarnitines with the same molecular weight in a given sample [9].

Figure 1. Structure of 3-methylglutarylcarnitine (C5-3M-DC) and adipoylcarnitine (C6-DC).

These terminally carboxylated acylcarnitines have an identical molecular weight (289.32 Da) but differ in chemical structure.

2.1. Acylcarnitine transport

All acylcarnitines are produced by the activity of CAT enzymes in reactions that convert acyl CoA substrates into the corresponding acylcarnitine plus free CoA. After long-chain acyl CoAs enter the cell, they must be converted to the corresponding acylcarnitine prior to their entry into mitochondria via the carnitine-acylcarnitine translocase (CACT) [10]. In the case of palmitoyl CoA, for example, carnitine palmitoyltransferase-I generates palmitoylcarnitine plus free CoA in the cytosol. Following translocation to the mitochondrial matrix, carnitine palmitoyltransferase-II converts palmitoylcarnitine back into palmitoyl CoA and free l-carnitine, preceding metabolism of palmitoyl CoA via the fatty acid ß-oxidation pathway (Figure 2A). By contrast, short-chain acylcarnitines (including 3MG carnitine) are produced in the mitochondrial matrix and exported to the cytosol (Figure 2B). Among the three human CAT enzymes (i.e., carnitine palmitoyltransferase, carnitine octanoyltransferase, and carnitine acetyltransferase; [11]), studies by Violante et al [12] provide support for the hypothesis that carnitine acetyltransferase is the CAT enzyme responsible for conversion of 3MG CoA to 3MG carnitine, although this has yet to be confirmed experimentally. The process of 3MG carnitine export from the mitochondrial matrix to the intermembrane space appears to be analogous to export of acetylcarnitine (C2) and occurs in antiport with carnitine via CACT (also known as the SLC25A20 transporter). By mediating net short-chain acylcarnitine efflux from the mitochondrial matrix, CACT functions in the opposite direction to its role in long-chain acylcarnitine import [13]. Subsequent transit of 3MG carnitine (C5-3M-DC) from the intermembrane space to the cytosol most likely occurs via a multimeric porin channel. Upon reaching the cytosol, 3MG carnitine can then escape to the plasma compartment via the organic cation/carnitine transporter novel type 2 (OCT2N), a specific sodium / l-carnitine co-transporter [14]. Because short-chain acylcarnitines are not converted back to the corresponding acyl CoA in cytosol, continued export of short-chain acylcarnitines (with coincident l-carnitine cotransport into mitochondria) is anticipated to deplete the pool of extra-mitochondrial l-carnitine. Although most l-carnitine is derived from the diet, certain tissues (e.g., brain, liver, and kidney) have the capacity to synthesize this compound [15]. Dietary supplements of l-carnitine are available and may enhance formation of short-chain acylcarnitines in IEMs known to generate these compounds [16].

Figure 2. Depiction of carnitine-mediated import of long-chain fatty acids and export of 3MG carnitine.

Panel A) Following fatty acid transport protein (FATP)-mediated long-chain fatty acid entry to a target cell, acyl CoA synthetase (ACS) catalyzes formation of the corresponding long-chain fatty acyl CoA (e.g., palmitoyl CoA). Subsequently, palmitoyl CoA is converted to palmitoylcarnitine through the action of carnitine palmitoyl transferase I (CPT I). Palmitoylcarnitine then passes into the mitochondrial intermembrane space through a porin channel and crosses the inner mitochondrial membrane via the carnitine-acylcarnitine translocase (CACT). In the matrix, carnitine palmitoyl transferase II (CPT II) converts pamitoylcarnitine back to palmitoyl CoA, preceding its entry into the ß-oxidation pathway. Subsequent metabolism of product acetyl CoA molecules via the tricarboxylic acid (TCA) cycle is shown along with reduced coenzymes (NADH and FADH₂) delivering electrons to the electron transport chain (ETC). This process, in turn, leads to proton pumping into the confined inner cristae space, generating the proton motive force that drives ATP synthesis via Complex V. Panel B depicts the situation that occurs in various IEMs and disease states wherein aberrant aerobic metabolism impedes entry of acetyl CoA to the TCA cycle, resulting in diversion of this metabolite to acetoacetyl CoA. Subsequent condensation of acetoacetyl CoA with another acetyl CoA generates HMG CoA, which is then dehydrated to form trans-3MGC CoA. This leucine catabolism pathway intermediate is reduced to 3MG CoA and, subsequently, converted to 3MG carnitine by carnitine acetyltransferase (CAT). 3MG carnitine is then exported from the matrix space via CACT, in antiport with free carnitine, and escapes to the cytosol through a porin channel. Finally, 3MG carnitine is transported out of the cell and into the blood plasma via the OCTN2 transporter. MICOS = mitochondrial contact site and organizing system; OPA = membrane-associated dynamin like GTPases involved in maintenance of cristae structure; CoQ = coenzyme Q; Cyt c = cytochrome c. I, II, III etc. refer to individual electron transport chain complexes. Figure prepared using the BioRender program.

When prevailing metabolic conditions lead to production of 3MG CoA in mitochondria, its carbon skeleton cannot be metabolized further. Instead, 3MG CoA is subject to one of three alternate fates, each of which effectively protect the pool of free CoA in the mitochondrial matrix (Figure 3). First, it can react with l-carnitine to form 3MG carnitine plus free CoA in a reaction catalyzed by a specific CAT enzyme. Second, 3MG CoA can undergo a series of non-enzymatic chemical reactions, yielding 3MG acid [17, 18]. As a terminally carboxylated short-chain acyl CoA, 3MG CoA is susceptible to intramolecular cyclization, forming 3MG anhydride and free CoA as products. 3MG anhydride is a reactive chemical species that, upon hydrolysis, produces 3MG acid, a dead-end organic acid destined for excretion in urine. The third potential fate of 3MG CoA also involves cyclic 3MG anhydride formation. Instead of hydrolytic cleavage, however, the anhydride reacts with protein lysine side chain amino groups to covalently “3MGylate” protein substrates [17]. The proportion of the 3MG anhydride pool that undergoes hydrolysis to 3MG acid versus that which 3MGylates substrate proteins is currently not known. By the same token, the relative proportion of the 3MG CoA pool that is converted to 3MG carnitine versus that which undergoes intramolecular cyclization is also not known, although l-carnitine availability is anticipated to be a determinant of 3MG CoA’s fate. With respect to protein 3MGylation, once formed, this covalent adduct can be enzymatically removed through the action of the NAD⁺-dependent enzyme, sirtuin 4 [19]. Once complete, this reaction is expected to yield 3MG acid via non-enzymatic hydrolytic cleavage [20].

Figure 3. Alternate fates of 3-methylglutaryl CoA.

In pathway (1) 3MG CoA is esterified by CAT, forming 3MG carnitine. Alternatively, as depicted in pathway (2), 3MG CoA is susceptible to intramolecular cyclization, forming 3MG anhydride. Once formed, 3MG anhydride has two alternate fates: 2A) hydrolysis to 3MG acid or 2B) reaction with protein lysine side chain amino groups to covalently modify substrate proteins (protein 3MGylation). 3MG moieties on proteins are removed through the activity of SIRT4 leading to the release of 2′-O-3MG-ADP-ribose and a deacylated protein. 2′-O-3MG-ADP-ribose is hydrolyzed to form 3MG acid. The end products of these reactions, 3MG acid and 3MG carnitine, are excreted in the urine. CAT = carnitine acyltransferase; SIRT4 = sirtuin 4.

Unlike 3MG acid, 3MG carnitine is readily detected by mass spectrometry (MS/MS) or liquid chromatography (LC)-MS analysis of blood or urine samples. An advantage of this method is that it does not require pre-analysis workup because biological fluids can be analyzed directly. Moreover, 3MG carnitine is commercially available for use as a standard. By contrast, 3MG acid is normally detected in urine by gas chromatography-mass spectrometry analysis. This method requires extraction and trimethylsilane derivatization prior to chromatography. Thus, whereas it is anticipated that elevated levels of 3MG carnitine will likely be accompanied by a parallel increase in 3MG acid, two distinct methods of analysis are required to assess both species. Thus, studies using only one method (e.g., MS/MS analysis of acylcarnitines present in a blood sample) will not provide information on 3MG acid levels. Although mass spectrometry is adequate for most acylcarnitine analytes, the propensity of 3MG CoA to undergo a non-enzymatic intramolecular cyclization reaction that yields the organic acid end product, 3MG acid, implies that the pool of 3MG-containing compounds generated in various mitochondrial disorders is likely to include both 3MG carnitine and 3MG acid.

3.1. HMG CoA lyase deficiency leads to 3MG carnitine production

A known metabolic source of 3MG CoA in mitochondria is the branched-chain amino acid leucine. During leucine catabolism, the pathway intermediate trans-3-methylglutaconyl (3MGC) CoA is formed. Normally, this intermediate is rapidly hydrated by 3MGC CoA hydratase (AUH), forming HMG CoA which is then converted to acetoacetate and acetyl CoA by HMG CoA lyase (HMGCL; Figure 4). Importantly, however, IEMs that lead to HMGCL deficiency are associated with urinary excretion of 3MG carnitine [21, 22]. In HMGCL deficiency, in addition to 3MG carnitine, characteristic organic acids, including 3MGC acid and 3MG acid, are also excreted in urine [22, 23]. Thus, when leucine catabolism is blocked by a deficiency in HMGCL, pathway intermediates, including trans-3MGC CoA, build up. Although the enzyme responsible has yet to be identified, it has been proposed that 3MG CoA is generated in one step by reduction of the double bond in trans-3MGC CoA [19, 21]. As described above, once formed, 3MG CoA then serves as a substrate for CAT-mediated formation of 3MG carnitine. Regarding the “reductase” enzyme involved, it is noteworthy that mitochondria possess a strongly oxidizing environment and few, if any, double bond reductions occur in this organelle. Thus, exactly how trans-3MGC CoA is reduced to 3MG CoA, remains a matter of conjecture. In addition, it is curious that, in HMGCL deficiency, neither HMG CoA nor trans-3MGC CoA are efficiently converted to the corresponding acylcarnitine [21], even though large quantities of the corresponding organic acids are produced. It is conceivable that the CAT enzyme responsible for 3MG carnitine formation displays a preference for saturated short-chain terminally carboxylated acyl CoAs (e.g., 3MG CoA) versus the hydroxylated species, HMG CoA, or the unsaturated species, trans-3MGC CoA.

Figure 4. Formation of 3MG carnitine in IEMs affecting HMG CoA lyase.

When leucine catabolism is blocked by a deficiency in HMG CoA lyase (HMGCL), the pathway intermediate, trans-3MGC CoA, accumulates. Reduction of the double bond in trans-3MGC CoA produces 3MG CoA which is then esterified by CAT to form 3MG carnitine (green hatched box). Notably, trans-3MGC CoA does not undergo esterification to form 3MGC carnitine (red hatched box). 3MCCCase = 3-methylcrontonyl CoA carboxylase; AUH = 3-methylglutaconyl CoA hydratase; CAT = carnitine acyltransferase.

3.1.1. Primary 3MGC aciduria

Another rare IEM, involving a deficiency in the leucine pathway enzyme AUH, leads to urinary excretion of organic acids, including 3MGC acid, 3MG acid, and 3-hydroxyisovaleric (3HIV) acid [23]. Together, the organic acid profile resulting from deficiencies in either HMGCL or AUH are considered “primary” 3MGC acidurias [24, 25]. Unlike HMGCL deficiency, however, 3MG carnitine has not been reported in cases of AUH deficiency, suggesting that unique distinguishing features exist between these disorders. Because subjects with deficiencies in HMGCL or AUH are unable to metabolize leucine to completion, pathway intermediates, most notably trans-3MGC CoA, accumulate. Individuals suffering from these disorders excrete large quantities of trans- and cis-3MGC acid, along with much lower amounts of 3MG acid. An important question relates to how trans-3MGC CoA is converted to the corresponding acid. Whereas short-chain, terminally carboxylated acyl CoAs (e.g., glutaryl CoA, succinyl CoA, HMG CoA, 3MG CoA) are prone to non-enzymatic intramolecular cyclization followed by hydrolysis [16], the presence of a trans double bond sterically prevents trans-3MGC CoA from undergoing a similar reaction. However, because trans-3MGC CoA is an intrinsically unstable chemical entity, it is susceptible to isomerization, forming cis-3MGC CoA [19]. Unlike trans-3MGC CoA, cis-3MGC CoA is structurally poised to undergo intramolecular cyclization, forming cis-3MGC anhydride and free CoA (Figure 5). Studies by Jones et al [26] revealed that 3MGC acid isomerizes during gas chromatography such that, when either isomer is subjected to elevated temperatures, a mixture of cis- and trans-3MGC acid is detected. Furthermore, evidence indicates that trans-3MGC CoA displays enhanced susceptibility to isomerization compared to the corresponding acid, with this reaction proceeding under physiological conditions [27]. Following isomerization, intramolecular cyclization and hydrolysis yields the organic acid, cis-3MGC acid. NMR-based studies provide direct evidence for the presence of cis- and trans-3MGC acid in urine of subjects with primary 3MGC aciduria [28, 29]. Moreover, when subjects with IEMs in HMGCL or AUH are challenged with a leucine-rich diet, urinary excretion of 3MGC acid and 3MG acid increases. Thus, in primary 3MGC aciduria, evidence indicates that a deficiency in specific leucine catabolism pathway enzymes leads to a buildup of pathway intermediates, causing trans-3MGC CoA to undergo a series of non-enzymatic chemical reactions that culminate in excretion of 3MGC acid. At the same time, it is evident that some portion of the trans-3MGC CoA pool is reduced to 3MG CoA [30]. As described above, this metabolite can also undergo non-enzymatic intramolecular cyclization and hydrolysis, yielding 3MG acid or serve as a substrate for CAT-mediated formation of 3MG carnitine. Bearing in mind that primary 3MGC aciduria is caused by rare IEMs in specific leucine pathway enzymes, the appearance of 3MG acid / 3MG carnitine in numerous unrelated IEMs, as well as acute and chronic disease states (see below), suggests an alternate metabolic route to 3MG CoA exists.

Figure 5. Non-enzymatic chemical reactions of trans-3MGC CoA.

When trans-3MGC CoA accumulates as a result of either IEMs affecting leucine catabolism enzymes or the acetyl CoA diversion pathway, this metabolite is subject to a series of non-enzymatic chemical reactions including isomerization to cis-3MGC CoA, intramolecular cyclization to cis-3MGC anhydride plus CoA, and hydrolytic cleavage of the cyclic anhydride to yield the organic acid, cis-3MGC acid, which is excreted in urine. In addition to this outcome, cis-3MGC anhydride can react with protein lysine side chain amino groups to covalently 3MGCylate these residues. 3MGCylated proteins may be deacylated by the NAD⁺ requiring enzyme sirtuin 4 (SIRT4), yielding cis-3MGC acid as a product. Non-enzymatic chemical reactions in this process are depicted in italics.

3.1.2. Secondary 3MGC aciduria

Another category of 3MGC aciduria, termed “secondary” 3MGC aciduria, has emerged from studies of disparate IEMs unrelated to leucine metabolism. In these disorders, although lower amounts of 3MGC acid are excreted, the relative proportion of 3MG acid to 3MGC acid is greater [30, 31]. In all cases of secondary 3MGC aciduria, no defects in leucine catabolism pathway enzymes exist and, unlike primary 3MGC aciduria, leucine loading has little or no effect on the amount of these organic acids excreted. In addition, whereas 3HIV acid excretion is a prominent feature of primary 3MGC aciduria, levels of this organic acid are not elevated in secondary 3MGC aciduria [23]. Given that trans-3MGC CoA is unique to the leucine catabolism pathway, its metabolic origin in secondary 3MGC aciduria has been a matter of conjecture for many years [32, 33]. Because nearly every IEM associated with secondary 3MGC aciduria has a direct, or indirect, effect on mitochondrial energy metabolism, a previously unrecognized aberrant metabolic pathway, termed the “acetyl CoA diversion” pathway, has been described [25]. This pathway is initiated when IEMs negatively affect aerobic energy metabolism, most likely in extra-hepatic tissues. As electron transport chain (ETC) function declines, reduced cofactors (NADH and FADH₂) accumulate in the matrix space, causing feedback inhibition of enzymes that generate these reduced cofactors. When specific TCA cycle enzymes are inhibited by accumulating NADH / FADH₂, cycle activity slows and reduced amounts of acetyl CoA enter the cycle. Instead, acetyl CoA is diverted to trans-3MGC CoA in three enzyme-mediated steps, catalyzed by acetoacetyl CoA thiolase 1, HMG CoA synthase 2, and AUH, respectively [25, 33]; Figures 2B and 6A]. The first enzyme condenses two acetyl CoA into acetoacetyl CoA plus free CoA in a reversible reaction. HMG CoA synthase 2 then catalyzes condensation of acetoacetyl CoA and acetyl CoA, yielding HMG CoA and free CoA. Subsequently, HMG CoA is dehydrated by AUH, forming trans-3MGC CoA and H₂O. This leucine catabolism intermediate cannot proceed further up the pathway because the next reaction, catalyzed by 3-methylcrotonyl CoA carboxylase, is irreversible. The acetyl CoA diversion pathway is fully consistent with known facts about secondary 3MGC aciduria, including the absence of an increase in 3HIV acid excretion and no increase in 3MGC acid excretion upon leucine loading. As trans-3MGC CoA accumulates, some portion of this metabolite pool is reduced to form 3MG CoA which then reacts with CAT to yield 3MG carnitine. As depicted in Figure 5, trans-3MGC CoA generated via the acetyl CoA diversion pathway is also susceptible to non-enzymatic chemical reactions that lead to formation of cis-3MGC acid, an organic acid waste product that is excreted in urine. Thus, in secondary 3MGC aciduria, trans-3MGC acid is derived from acetyl CoA rather than leucine [34]. Although it is anticipated that this pathway does not operate under normal metabolic conditions, over twenty discrete IEMs have been identified that give rise to secondary 3MGC aciduria and, in most cases, 3MG acid is also present [31].

Figure 6. Comparative acetyl CoA metabolism pathways.

Each of the three pathways depicted utilize acetyl CoA acyltransferase (ACAT) 1 or 2 to condense two acetyl CoA molecules into acetoacetyl CoA which is then condensed with another acetyl CoA molecule via the activity of HMG CoA synthase 1 or 2, producing HMG CoA. Panel A) the acetyl CoA diversion pathway wherein 3-methylglutaconyl CoA hydratase (AUH) catalyzes dehydration of HMG CoA to trans-3MGC CoA, a precursor of 3MG carnitine, 3MG acid, and 3MGC acid. In panel B, HMG CoA serves as a substrate for HMG CoA lyase, generating acetyl CoA and acetoacetate, a ketone body. In Panel C, HMG CoA reductase converts HMG CoA into mevalonate, a precursor of sterols, dolichols, and ubiquinone.

Of interest to the present discussion, recent studies indicate that 3MG carnitine is also observed in secondary 3MGC aciduria [35]. Mackay et al reported that an IEM in TMEM70, encoding mitochondrial transmembrane protein 70, which localizes to the inner mitochondrial membrane and functions in assembly of the F₁ and F_o subunits of Complex V, manifests 3MG carnitine excretion along with 3MGC aciduria. Defective TMEM70 function reduces Complex V activity, resulting in increased membrane potential, enhanced reactive oxygen species formation, and diminished ATP production [36]. Consistent with these findings, ultrastructural studies of tissues harboring mutations in TMEM70 indicate aberrant cristae morphology [37]. Thus, in IEMs that affect TMEM70 function, disrupted ETC activity leads to diminished TCA cycle activity and diversion of acetyl CoA to trans-3MGC CoA. Subsequent reduction of the double bond in trans-3MGC CoA yields 3MG CoA which serves as the immediate precursor of 3MG carnitine. The observation that an IEM associated with compromised mitochondrial energy metabolism leads to 3MG carnitine formation [35] suggests that other disease processes will also be characterized by increased levels of this unique acylcarnitine.

Given the concept that 3MG carnitine can arise from acetyl CoA via the acetyl CoA diversion pathway, it is important to consider whether other metabolites or metabolic networks are perturbed in conjunction with 3MG carnitine accumulation. Insofar as mitochondrial dysfunction associated with defective ETC activity is expected to trigger the acetyl CoA diversion pathway, it may be anticipated that a connection exists between 3MG carnitine and its biosynthetic precursors, acetoacetyl CoA, HMG CoA, trans-3MGC CoA and 3MG CoA. Furthermore, it is conceivable that the concentration of certain TCA cycle intermediates could be increased as a result of stalled activity caused by inefficient transfer of electrons from NADH and FADH₂ to the ETC. Another potentially interesting aspect to consider is whether leucine catabolism pathway intermediates may be altered due to the reversed flux of acetyl CoA toward trans-3MGC CoA. In this case, it is conceivable that levels of upstream leucine pathway byproducts, such as 3HIV carnitine or 3-methylcrotonylglycine may increase. However, it is worth noting that the 3MG carbon skeleton is metabolically inert and cannot be metabolized further. Hence, it is destined for excretion, either as 3MG acid or 3MG carnitine.

4.1. 3MG carnitine and heart disease

Building upon the seminal reports of Shah et al [6, 7], Kraus and coworkers [8] reported that circulating short-chain terminally carboxylated acylcarnitine metabolites are predictive of cardiovascular disease (CVD) events. These authors analyzed genetics, epigenetics, transcriptomics, and metabolomics in samples from a large CVD cohort to identify novel genetic markers in an effort to better understand the role of metabolites in CVD pathogenesis. Among the metabolites identified, 3MG carnitine levels positively correlated with variation in genes that regulate components of endoplasmic reticulum (ER) stress. Despite the fact that ER stress is an independent predictor of CVD risk, no cause-and-effect relationship between ER stress and increased 3MG carnitine has been established.

In another metabolomics study designed to identify biomarkers in patients with acute coronary syndrome (ACS), Wang et al [38] analyzed urine samples collected from ACS patients and healthy control subjects. Among numerous metabolites examined, 3MG carnitine levels were elevated in patients versus controls. These authors surmised that 3MG carnitine is a byproduct of the fatty acid ß-oxidation pathway, although no evidence or metabolic rationale for this interpretation was provided. In a similar manner, in their study of 68 heart failure patients, Ruiz et al [9] reported significant increases in “C6-DC” content [a mixture of two species, most likely 3MG carnitine (C5-3M-DC) and adipoylcarnitine (C6-DC)], as compared to 72 control subjects. These authors posited that one or both of these acylcarnitines could be a product of peroxisomal ω-oxidation. Likewise, in a study of 273 patients with non-ischemic dilated cardiomyopathy, Verdonschot et al [39] observed a positive correlation between 3MG carnitine (C5-3M-DC / C6-DC) levels and disease severity. These authors proposed that increased C5-3M-DC / C6-DC in symptomatic subjects is related to impaired ketone-body production, consistent with a switch in fuel utilization from fatty acids to ketone bodies. Since ketone body biogenesis is restricted to liver and to a lesser extent, kidney, it is unclear how progression of heart disease impacts ketogenesis.

4.1.1. 3MG carnitine and breast cancer

Moore et al [40] conducted a metabolomics study to investigate the association between body mass index (BMI) and breast cancer risk. In this study, 621 postmenopausal breast cancer case participants and 621 matched control participants were investigated. Metabolomics analysis revealed that 67 of 617 identified metabolites were associated with BMI and that two of these metabolites, 3MG carnitine and the steroid 16α-hydroxydehydroepiandrosterone sulfate, were strongly associated with breast cancer risk. The authors concluded that these metabolites identify metabolic pathways that contribute to breast carcinogenesis as well as the association of BMI with increased postmenopausal breast cancer risk. In the case of 3MG carnitine, Moore et al considered this metabolite to arise from aberrant leucine catabolism [40]. On the basis of evidence presented herein, however, the possibility that the 3MG carbon skeleton arises from the acetyl CoA diversion pathway as a result of mitochondrial dysfunction, should be further explored in this context.

4.1.2. 3MG carnitine levels in acute illness

In a study of paraquat-induced kidney toxicity, Wan et al [41] observed that 3MG carnitine levels were three-fold higher in paraquat-exposed versus control subjects. Given the known effects of paraquat poisoning on mitochondrial function [42], it is reasonable to hypothesize that activation of the acetyl CoA diversion pathway is involved in 3MG carnitine formation in cases of paraquat poisoning.

The appearance of 3MG carnitine in liver, serum, and urine of mice exposed to γ radiation was interpreted by Golla et al [43] to result from radiation-induced effects on ketone body biosynthesis. This potential mechanism appears plausible insofar as the first two steps in the acetyl CoA diversion pathway are shared with the biosynthetic routes to ketone bodies (Figure 6). In other words, if mitochondrial function is adversely affected by exposure to γ radiation in extrahepatic tissues, then excess acetyl CoA can be diverted to trans-3MGC CoA via the acetyl CoA diversion pathway, and some portion of this metabolite pool will be reduced to 3MG CoA, which is then converted to 3MG carnitine by CAT. It is important to note that expression of HMG CoA lyase is far lower in brain and heart tissue versus liver [44] such that, under the prevailing metabolic conditions, HMG CoA is dehydrated to trans-3MGC CoA. Figure 6 depicts similarities between the acetyl CoA diversion pathway, ketone body biogenesis, and first stage of the mevalonate pathway. It is worth noting, however, that the mevalonate pathway occurs in cytosol while ketone body biogenesis is restricted to liver (and kidney) mitochondria. On the other hand, the acetyl CoA diversion pathway, while mimicking ketone body biogenesis up to the point of HMG CoA formation, most likely occurs in metabolically compromised mitochondria of extra-hepatic tissues.

In another example, Jeter et al [45] reported that 3MG carnitine is the only acylcarnitine observed to increase in patients suffering from severe traumatic brain injury, as compared to all other groups studied. Given that traumatic brain injury is associated with mitochondrial dysfunction [46] and depletion of coenzyme Q [47], these data are consistent with activation of the acetyl CoA diversion pathway in brain and formation of trans-3MGC CoA, which is then reduced to 3MG CoA and converted to 3MG carnitine.

Recently, Dillard et al [48] investigated potential metabolic signatures in “nonacute” and “severe” cases of COVID-19 (84 patients total) by profiling their plasma metabolomes. Machine learning and metabolic modeling approaches were then employed to identify potential prognosticators of COVID-19 disease severity. These authors found that 3MG carnitine levels were significantly elevated in cases of severe COVID-19, providing potential mechanistic insight with respect to musculoskeletal degeneration and host metabolic perturbations in response to SARS-CoV-2 infection.

5.1. Summary and conclusions

The present review is focused on an unusual acylcarnitine that is associated with various disease states. 3MG carnitine is generated directly from 3MG CoA in a reaction catalyzed by one or more CAT enzyme. Given that 3MG CoA is not part of any metabolic pathway and its carbon skeleton cannot be metabolized to yield energy, it seems likely that its appearance reflects an aberration of normal metabolic processes. Existing data strongly support the view that 3MG CoA arises directly from reduction of the double bond present in the leucine catabolism intermediate, trans-3MGC CoA. Recently, a novel biosynthetic route to trans-3MGC CoA was described that appears to function in specific inherited mutations that disrupt aerobic energy metabolism as well as chronic disease states and acute injuries. In these maladies, sluggish ETC activity slows oxidation of NADH and FADH₂ generated by metabolic processes, most notably, the TCA cycle. When this occurs, product inhibition of TCA cycle enzymes prevents acetyl CoA entry into the cycle. In extrahepatic tissues, acetyl CoA accumulation leads to its condensation to acetoacetyl CoA followed by condensation of acetoacetyl CoA with acetyl CoA to form HMG CoA. In liver tissue, this process is normally followed by HMGCL-mediated cleavage of HMG CoA, producing acetoacetate and acetyl CoA. Unlike liver, however, HMGCL levels in muscle and brain are quite low, which results in HMG CoA being susceptible to an alternate fate, AUH-mediated dehydration to form trans-3MGC CoA. This leucine pathway intermediate has two potential fates, non-enzymatic conversion to 3MGC acid or reduction to form 3MG CoA. It remains unclear what, if anything, regulates the proportion of trans-3MGC CoA that is converted to 3MGC acid versus that which is reduced to 3MG CoA. Moreover, the nature of the enzyme responsible for reduction of trans-3MGC CoA is not known. As 3MG CoA is generated, though, it reacts with l-carnitine to form 3MG carnitine plus free CoA. Complicating the situation, however, is the fact that 3MG CoA can also undergo non-enzymatic intramolecular cyclization followed by hydrolysis, to form the organic acid, 3MG acid. It is conceivable that the proportion of 3MG carnitine generated is affected by l-carnitine availability, although this has yet to be confirmed experimentally. If so, because 3MG carnitine is expected to be produced under conditions of compromised mitochondrial energy metabolism, then l-carnitine supplementation may provide a means to enhance the amount of 3MG carnitine produced.

Given the broad range of disorders identified that correlate with elevated blood / urine levels of 3MG carnitine, it is important to place information regarding 3MG carnitine detection into perspective with other disease-related phenotypic features, including the presence / absence of other acylcarnitines. Whereas 3MG carnitine levels increase in response to various acute injuries, how the severity of such injuries impact 3MG carnitine levels has yet to be evaluated. Likewise, in chronic diseases, such as CVD, how do 3MG carnitine levels change as a function of disease progression and is there a correlation between 3MG carnitine and disease severity? Answers to these, and other questions, are likely to provide insight into the potential utility of 3MG carnitine as a disease biomarker. Thus, while much work is required, evidence accumulated to date reveals how mitochondrial dysfunction can lead to 3MG carnitine production via a previously unrecognized metabolic route.

Highlights.

• Acylcarnitine’s are a diverse collection of molecules that serve as biomarkers

• 3-methylglutarylcarnitine levels increase in certain inborn errors of metabolism

• Other chronic and acute disease states manifest elevated 3-methylglutarylcarnitine

• A novel pathway to 3-methylglutarylcarnitine (3MG carnitine) is described

• Knowledge of 3MG carnitine’s metabolic origins enhances its utility as a biomarker

Abbreviations:

HMG

3-hydroxy-3-methylglutaryl

3MGC

3-methylglutaconyl

3MG

3-methylglutaryl

IEM

Inborn error of metabolism

CVD

Cardiovascular disease

ACS

Acute coronary syndrome

CAT

Carnitine acyltransferase

CACT

carnitine-acylcarnitine translocase

HMGCL

HMG CoA lyase

AUH

3MGC CoA hydratase

3HIV

3-hydroxyisovaleric