Chapter 11
L-Carnitine as a Detoxicant of Nonmetabolizable Acyl Coenzyme A
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L. L. Bieber, Z. H. Huang, and D. A. Gage Department of Biochemistry, Michigan State University, East Lansing, MI 48824-1319
Liver contains short-chain, medium-chain, and/or long-chain carnitine acyltransferases associated with mitochondria, peroxisomes, and microsomes. These data, plus the demonstration that elevated levels of acylcarnitines can occur in human urine due to impaired metabolism of specific acyl-CoAs, strongly support roles for L-carnitine in both modulat ing the acyl-CoA:CoA ratio and being an acceptor of specific non metabolizable acyl-CoAs. Heretofore, an overlooked aspect of this metabolic problem has been the impact and implications of the inhibitory effects of acyl-CoAs, such as palmitoyl-CoA, on the short-chain carnitine acyltransferase activity. Herein, we show that palmitoyl-CoA is a potent inhibitor of carnitine acetyltransferase and that a secondary carnitine deficiency associated with a renal loss of carnitine can result in urinary excretion of specificα-ketoacids that are substrates for the branched-chain α-keto acid dehydrogenase, pyruvate dehydrogenase, and α-ketoglutarate dehydrogenase. These latter data indicate mitochondrial carnitine deficiency can result in inhibition of mitochondrialα-ketoacid dehydrogenase activity.
Although the pioneering studies of Bremer (1) and Fritz (2) concomitantly established a role for L-camitine in the mitochondrial 0-oxidation of long-chain fatty acids, multiple roles for L-carnitine in intermediary metabolism are strongly supported by data showing: 1) tissues such as liver contain a family of carnitine acyltransferases with overlapping acyl-CoA specificities, 2) peroxisomes, endoplasmic reticulum, and mitochondria all contain shortchain, medium-chain, and long-chain carnitine acyltransferase activity, and 3) the mediumchain/long-chain enzyme is a different protein in each organelle (3,4). Each of the abovementioned organelles contain at least two enzymes with broad acyl-CoA specificities capable of reversibly catalyzing the conversion of Q to greater than acyl-CoAs to acylcarnitines as shown below. Although three different enzyme types, CAT, COT, and CPT, are shown, it should be recognized that COT and CPT use the same substrates. The medium-chain/long-chain carnitine acyltransferase was designated a COT because of its kinetic preference for medium-chain acyl-CoAs, while others refer to the enzymes as CPT on the basis of functional considerations.
0097-6156/96/0643-0140$15.00/0 © 1996 American Chemical Society In Biomarkers for Agrochemicals and Toxic Substances; Blancato, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
11. BIEBER ETAL.
^Carnitine as Detoxicant of Nonmetabolizable Acyl-CoA
Reactions Catalyzed 1.
CAT (Carnitine Acyltransferase) Short-Chain Acyl-CoA +« * Short-Chain Acylcarnitine L-carnitine + CoASH
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2.
COT (Carnitine Octanoyltransferase) Medium-Chain/Long-Chain Acyl-CoA +« * Medium-Chain/Long-Chain Acylcarnitine L-carnitine + CoASH
3.
CPT (Carnitine Palmitoyltransferase) Ix>ng-Chain/Medium-Chain Acyl-CoA +« * Medium-Chain/Long-Chain Acylcarnitine L-carnitine + CoASH
The majority of our studies with CAT have focused on its roles 1) in catalyzing the reaction in which L-carnitine serves as an acceptor for non-metabolizable acyl-CoA derivatives and 2) in modulating the acyl-CoA:CoA ratios intracellularly. Support for such roles has comefromstudies on human disease states which produce secondary carnitine deficiency (5-6) andfrommitochondrial studies documenting a direct effect of carnitine on specific acyl-CoA:CoA ratios (9,10). However, the potential inhibitory effect of specific acyl-CoAs on the activity of carnitine acyltransferases that do not use the specific acyl-CoA as a substrate has received little experimental attention. Herein, we show that palmitoylCoA strongly inhibits CAT and that a secondary carnitine deficiency due to renal carnitine loss can promote excessive excretion of specific a-keto acids. Assays CAT was assayed spectrally at room temperature in a 260 /d reaction mature containing 50 mM potassium phosphate buffer, pH 7.4, 200 /iM dithiopyridine, 98 /iM acetyl-CoA, and 1 p\ of dialyzed commercial pigeon breast muscle CAT, diluted 1:10 in potassium phosphate buffer. Reactions were normally started by addition of 19.5 mM L-carnitine and the change in optical density at 324 nm monitored (full-scale = 1 OD unit, charge speed 2 cm/minute). For figure 1, 10 /d of palmitoyl CoA, final concentration 98 /iM, was added where indicated. For figures 2 and 3, the enzyme was preincubated with palmitoyl-CoA and acetyl-CoA for 1 minute and the reaction initiated with L-carnitine. Palmitoyl-CoA concentration was varied as shown in the graphs. Assays were performed at room temperature, 25°C. A minimum of three separate runs were done for each curve. Extracts for determination of free and total carnitine were prepared as described (11), and free carnitine was quantitated by the radiochemical method of Cederblad and Lindstedt (12). Total carnitine was assayed as described above, after alkaline hydrolysis. Effect of Carnitine on Short-chain and a-Keto Add Metabolism It is well established, from investigations of secondary carnitine deficiency produced by some organic acid acidurias, that L-carnitine can serve as an acyl acceptor for some nonmetabolizable acyl moieties (5,60; in humans, the urinary acylcarnitine profile can be used
In Biomarkers for Agrochemicals and Toxic Substances; Blancato, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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BIOMARKERS FOR AGROCHEMICALS AND TOXIC SUBSTANCES
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