Acylcamitine

Long-chain acyl-CoA esters are then converted to acylcamitine esters by readily reversible reactions with L-camitine catalyzed by carnitine palmitoyltransferase I (CPT I). 

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 ...

The transport is accomplished with the participation of carnitine, which takes up the acyl from acyl-CoA on the outer membrane side. Acylcamitine assisted by carnitine translocase diffuses to the inner side of the membrane to give its acyl to the CoA located in the matrix. The process of reversible acyl transfer between CoA and carnitine on the outer and inner sides of the membrane is effected by the enzyme acyl-CoA-camitine transferase. 

In addition to neutral loss scans, mass spectrometers can be used to detect other compounds in a different manner. Acylcamitines are fatty acid esters of carnitine. The masses of acylcamitines differ by the size of the fatty acid attached to it. The tandem mass spectrometer can detect these selectively as well because they all produce a similar product, in this case an ion rather than a molecule. Because it is an ion, it can be detected by the second mass separation device. The ion has a mass of 85 Da and is common to all acylcamitines. Performing a precursor ion scan of 85 Da (essentially a scan of only molecules that produce the 85 ion) reveals a selective analysis of acylcar-nitines, as shown in Fig. 14.2. Additional scans have been added to more selectively detect basic amino acids, free carnitine, short chain acylcamitines and a hormone, thyroxin (T4) which has amino acid components. 

Second, esterases have broad (or even very broad) and overlapping substrate specificities. For example, carboxylesterase (EC 3.1.1.1) also catalyzes reactions characteristic of a number of other hydrolases. The discovery that individual isoenzymes of carboxylesterases may be identical to or closely related to acylglycerol lipase, acylcamitine hydrolase, and palmitoyl-CoA hydrolase (see Sect. 2.4.3) has increased the confusion surrounding esterase classification [59], Many esterases are able to hydrolyze amides, thiolesters,... 

A number of rat liver carboxylesterases identified by their pI values are listed in Table 2.6 [73] five nonspecific carboxylesterases were purified from rat liver and were characterized according to their p/ values [61]. They appeared to be isoenzymes, since they had similar substrate specificities toward phenyl and naphthyl esters and monooleylglycerol. Subsequent studies, however, revealed different specificities with respect to their physiological substrates. The pI 5.2 and 5.6 enzymes were shown to be acylcamitine hydrolases (EC 3.1.1.28), and a p/ 6.0 enzyme an octanoylglycerol lipase. The p/... 

The physiological functions of carboxylesterases are still partly obscure but these enzymes are probably essential, since their genetic codes have been preserved throughout evolution [84] [96], There is some evidence that microsomal carboxylesterases play an important role in lipid metabolism in the endoplasmic reticulum. Indeed, they are able to hydrolyze acylcamitines, pal-mitoyl-CoA, and mono- and diacylglycerols [74a] [77] [97]. It has been speculated that these hydrolytic activities may facilitate the transfer of fatty acids across the endoplasmic reticulum and/or prevent the accumulation of mem-branolytic natural detergents such as carnitine esters and lysophospholipids. Plasma esterases are possibly also involved in fat absorption. In the rat, an increase in dietary fats was associated with a pronounced increase in the activity of ESI. In the mouse, the infusion of lipids into the duodenum decreased ESI levels in both lymph and serum, whereas an increase in ES2 levels was observed. In the lymph, the levels of ES2 paralleled triglyceride concentrations [92] [98],... 

Other agents (e.g., acylcamitine, azone (l-dodecylazacycloheptan-2-one), dicarbox-ylic acids, and enamine)... 

A method for quantitative acylcamitine profiling in human skin fibroblasts using unlabelled palmitic acid diagnosis of fatty acid oxidation disorders and differentiation between biochemical phenotypes of MCAD deficiency. 

Diagnosis of medium chain acyl-CoA dehydrogenase deficiency by stable isotope dilution analysis of urinary acylglycines retrospective and prospective studies, and comparison of its accuracy to acylcamitine identification by FAB/mass spectrometry. 

MCAD deficiency. Acylcamitines (AC) by tandem mass spectrometry (MS-MS) are useful to monitor dietary treatment. 

Rapid diagnosis of MCAD deficiency quantitative analysis of octanoylcamitine and other acylcamitines in newborn blood spots by tandem mass spectrometry. 

Corr PB, Creer MH, Yamada KA, Saffitz JE, Sobel BE Prophylaxis of early ventricular fibrillation by inhibition of acylcamitine accumulation. J Clin Invest 1989 83 927-936. 

Yamada KA, McHowat J, Yan GX, Donahue K, Peirick J, Richer AG, Corr PB Cellular uncoupling induced by accumulation of long-chain acylcamitine during ischemia. Circ Res 1994 74 83-95. 

Lecluyse EL, Sutton SC, Fix JA. In vitro effects of long-chain acylcamitines on the permeability, transepithelial electrical resistance and morphology of rat colonic mucosa. J Pharmacol Exp Ther 1993 265 955-962. 

As many as 1 in 10,000 persons may inherit such prob-lems.48 50a Tire proteins that may be defective include a plasma membrane carnitine transporter carnitine palmitoyltransferases camitine/acylcamitine trans-locase long-chain, medium-chain, and short-chain acyl-CoA dehydrogenases 2,4-dienoyl-CoA reductase (Eq. 17-1) and long-chain 3-hydroxyacyl-CoA dehydrogenase. Some of these are indicated in Fig. 17-2. 

A Arduini, A Peschechera, S Dottori, AF Sciarroni, F Seraflni, M Calvani. High performance liquid chromatography of long-chain acylcamitine and phospholipids in fatty acid turnover studies. J Lipid Res 37 684-689, 1996. 

Today, many of the older methods used historically in clinical chemistry are being replaced by methods that utilize MS. In 2013, some areas of interest in clinical chemistry include steroid and vitamin D analyses that require a high selectivity and relative sensitivity. For example, the improved selectivity is essential in the analysis of testosterone in infants due to low concentrations. Immunoassays are not sufficiently selective to provide an accurate measurement of the true concentrations of these metabolites. There are many more assays being used or that are now in development in clinical chemistry, but a discussion of this growing use of metabolic screening is too broad and too evolutionary to be included here. Therefore, this chapter focuses on those proteins and metabolites (e.g., amino acids and acylcamitines) that are part of a classic newborn screening panel. 

There are many other isomers in MS/MS analysis, such as several acyl-camitines that share the same mass, such as many of the hydroxyacylcami-tines and dicarboxylic acid acylcamitines. In addition, interfering products may, at times, share a common fragment ion or neutral loss and as a result will be detected in a profile. No method, however, is perfectly selective and a physician reviewing results from a newborn screen, whether analyzed using an immunoassay or MS, should be aware of the fact that in some cases, similar mass compounds may lead to potential sources of error. 

Interpretation of complex metabolic profiles and communication of lab results to clinicians has been one of the greatest challenges in dried blood spot analysis by MS/MS for acylcamitines and amino acids. Following its introduction, MS/MS defined numerous diseases of fatty acid and organic acid metabolism that were uncommonly encountered in a typical practice and not well understood by most pediatricians. Furthermore, even in disorders such as PKU that were well known, the improvements made by MS/MS screening for PKU compared with older technology such as BIA or fluorometry were not well... 

It is important to first note that many classical clinical assays have traditionally measured one metabolite to detect one disease. Consequently, many of the rules of method validation were designed around this premise. MS/MS, as originally designed, detected two classes of compounds, amino acids and acylcamitines, in four to five different MS methods (known as scan modes such as neutral loss, precursor ion, or selected reaction monitoring), for approximately 500 distinct masses, more than 70 known compounds, and 20-30 stable isotope internal standards. How then did one approach such a complicated validation to gain acceptance as a reliable, useful method The answer is quite simple - start simply and compare to what was already established. 

MS/MS not only could compare to existing newborn screening methods but also could potentially improve them. It was therefore easy to add acylcamitine analysis to a screening panel since the extraction and derivatization schemes could be unified as butylesters (acylcamitines were analyzed as methylesters in the diagnostic application). 

FIGURE 6 The concentration of metabolites as measured in a multiplex method such as MS/MS of amino acids and acylcamitines can detect metabolic aberrations that are attributed to disorders with basis in genomics (inherited disease) or are due to environmental or developmental cause (nongenetic). 

Acylcamitines or amino acids may also be important in disease monitoring and treatment or as markers for new therapies, toxicities, etc. In one application using dried plasma spots, carnitine and acylcamitines may be useful in detecting possible carnitine deficiency as a result of kidney dialysis for patients with end-stage renal disease (36,37). A deficiency should result in carnitine supplementation in those patients that cannot replenish their levels fast enough. In fact, this is one of the first pharmaceutical-related applications of screening. The measurement of certain amino acids such as Phe and Tyr and their ratio is also routinely performed to monitor the effectiveness of dietary intervention in patients with PKU. 

---