Fatty acylcarnitine

FIGURE 25.16 Regulation of fatty acid synthesis and fatty acid oxidation are conpled as shown. Malonyl-CoA, produced during fatty acid synthesis, inhibits the uptake of fatty acylcarnitine (and thus fatty acid oxidation) by mitochondria. When fatty acyl CoA levels rise, fatty acid synthesis is inhibited and fatty acid oxidation activity increases. Rising citrate levels (which reflect an abundance of acetyl-CoA) similarly signal the initiation of fatty acid synthesis. 

The mitochondrial inner membrane is impermeable to fatty acyl-CoA. To be transported into mitochondria, the acyl moiety must first be esterified to L-carnitine (hereafter referred to simply as carnitine) to form the corresponding fatty acylcarnitine. This reaction is catalyzed by carnitine palmitoyltransferase I (CPTI) localized... 

A specific transport protein, the carnitine-acylcarnitine translocase, moves the fatty acylcarnitine into the mitochondrial matrix while returning carnitine from the matrix to the cytoplasm. Once inside the mitochondria, another enzyme, carnitine palmitoyltransferase II (CPT II), located on the matrix side of the mitochondrial inner membrane, catalyzes the reconversion of fatty acylcarnitine to fatty acyl-CoA. Intramitochondrial fatty acyl-CoA then undergoes (3-oxidation to generate acetyl-CoA.Acetyl-CoA can enter the Kreb s cycle for complete oxidation or, in the liver, be used for the synthesis of acetoacetate and P-hydroxybutyrate (ketone bodies). 

In LCAD deficiency, fatty acylcarnitines accumulate in the blood. Those containing 14 carbons predominate. However, these do not appear in the urine. 

TRANSPORT OF FATTY ACYLCARNITINE ACROSS THE LIVER MICROSOMAL MEMBRANE... 

Several additional points should be made. First, although oxygen esters usually have lower group-transfer potentials than thiol esters, the O—acyl bonds in acylcarnitines have high group-transfer potentials, and the transesterification reactions mediated by the acyl transferases have equilibrium constants close to 1. Second, note that eukaryotic cells maintain separate pools of CoA in the mitochondria and in the cytosol. The cytosolic pool is utilized principally in fatty acid biosynthesis (Chapter 25), and the mitochondrial pool is important in the oxidation of fatty acids and pyruvate, as well as some amino acids. 

Figure 3. Mitochondrial fatty acid oxidation. Long-chain fatty acids are converted to their CoA-esters as described in the text, and their fatty-acyl-groups transferred to CoA in the matrix by the concerted action of CPT 1, the acylcarnitine/carnitine exchange carrier and CPT (A) as described in the text. Medium-chain and short-chain fatty acids (Cg or less) diffuse directly into the matrix where they are converted to their acyl-CoA esters by a acyl-CoA synthase. The mechanism of p-oxidation is shown below (B). Each cycle of P-oxidation removes -CH2-CH2- as an acetyl unit until the fatty acids are completely converted to acetyl-CoA. The enzymes catalyzing each stage of P-oxidation have different but overlapping specificities. In muscle mitochondria, most acetyl-CoA is oxidized to CO2 and H2O by the citrate cycle (Figure 4) some is converted to acylcamitine by carnitine acetyltransferase (associated with the inner face of the inner membrane) and exported from the matrix. Some acetyl-CoA (if in excess) is hydrolyzed to acetate and CoASH by acetyl-CoA hydrolase in the matrix. Enzymes ...

Figure 22-1. Role of carnitine in the transport of long-chain fatty acids through the inner mitochondrial membrane. Long-chain acyl-CoA cannot pass through the inner mitochondrial membrane, but its metabolic product, acylcarnitine, can. 

This in fact is what happened. Tandem MS has clearly been shown to be the only technology to screen for disorders of fatty acid oxidation and could also detect many disorders of organic acid metabolism. Tandem MS has the ability to detect both compound classes (amino acids and acylcarnitines) and after demonstrating that both classes could be prepared in the same manner, the MS/MS analysis of blood spots for newborn screening applications was born. 

Carnitine is a vitamin-like quaternary ammonium salt, playing an important role in the human energy metabolism by facilitating the transport of long-chained fatty acids across the mitochondrial membranes. An easy, fast, and convenient procedure for the separation of the enantiomers of carnitine and 0-acylcarnitines has been reported on a lab-made teicoplanin-containing CSP [61]. The enantioresolution of carnitine and acetyl carnitine was enhanced when tested on a TAG CSP, prepared in an identical way [45]. Higher a values were reached also in the case of A-40,926 CSP [41]. 

Fatty acyl carnitine is transported via a translocase that transports acylcarnitine into and carnitine out of the mitochondrion (Chapter 7). 

Under physiologic conditions, carnitine is primarily required to shuttle long-chain fatty acids across the inner mitochondrial membrane for FAO and products of peroxisomal /1-oxidation to the mitochondria for further metabolism in the citric acid cycle [40, 43]. Acylcarnitines are formed by conjugating acyl-CoA moieties to carnitine, which in the case of activated long-chain fatty acids is accomplished by CPT type I (CPT-I) [8, 44]. The acyl-group of the activated fatty acid (fatty acyl-CoA) is transferred by CPT-I from the sulfur atom of CoA to the hydroxyl group of carnitine (Fig. 3.2.1). Carnitine acylcarnitine translocase (CACT) then transfers the long-chain acylcarnitines across the inner mitochondrial membrane, where CPT-II reverses the action of CPT-I by the formation of acyl-CoA and release of free un-esterified carnitine. 

While MS-MS allows for unequivocal identification of most metabolites, there are a few exceptions. In particular, the short-chain acylcarnitines of 4 and 5 carbons represent more than one analyte. C4-Acylcarnitine is known to be a mixture of bu-tyrylcarnitine derived from fatty acid metabolism and isobutyrylcarnitine derived from the metabolism of valine (Fig. 3.2.3) [21, 58]. C5-Acylcarnitine is a mixture of isovalerylcarnitine and 2-methylbutyrylcarnitine derived from leucine and isoleucine degradation, respectively (Fig. 3.2.4) [20, 59]. Samples of patients treated with antibiotics containing pivalic acid may contain pivaloylcarnitine another C5 species... 

Ventura FV, Costa CG, Struys EA, et al (1999) Quantitative acylcarnitine profiling in fibroblasts using U-C-13 palmitic acid an improved tool for the diagnosis of fatty acid oxidation defects. Clin Chim Acta281 l-17... 

Shen JJ, Matern D, Millington DS, et al (2000) Acylcarnitines in fibroblasts of patients with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency and other fatty acid oxidation disorders. J Inherit Metab Dis 23 27-44... 

Giak Sim K, Carpenter K, Hammond J, Christodoulou J, Wilcken (2002) Quantitative fibroblast acylcarnitine profiles in mitochondrial fatty acid beta-oxidation defects phenotype/me-tabolite correlations. Mol Genet Metab 76 327-334... 

Okun JG, Kolker S, Schulze A, et al (2002) A method for quantitative acylcarnitine profiling in human skin fibroblasts using unlabelled palmitic acid diagnosis of fatty acid oxidation disorders and differentiation between biochemical phenotypes of MCAD deficiency. Biochim Biophys Acta 1584 91-98... 

Schulze-Bergkamen A, Okun JG, Spiekerkotter U, et al (2005) Quantitative acylcarnitine profiling in peripheral blood mononuclear cells using in vitro loading with palmitic and 2-oxoadipic acids biochemical confirmation of fatty acid oxidation and organic acid disorders. Pediatr Res 58 873-880... 

Schmidt-Sommerfeld E, Penn D, Duran M, et al (1992) Detection and quantitation of acylcarnitines in plasma and blood spots from patients with inborn errors of fatty acid oxidation. Prog Clin Biol Res 375 355-362... 

Browning MF, Larson C, Strauss A, Marsden DL (2005) Normal acylcarnitine levels during confirmation of abnormal newborn screening in long-chain fatty acid oxidation defects. J Inherit Metab Dis 28 545-550... 

Roe DS, Yang BZ, Vianey-Saban C, Struys E, Sweetman L, Roe CR (2006) Differentiation of long-chain fatty acid oxidation disorders using alternative precursors and acylcarnitine profiling in fibroblasts. Mol Genet Metab 87 40-47... 

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