ANALYTICAL BIOCHEMISTRY 138, 107-111 (1984)

Determination of Malonyl-Coenzyme A in Rat Heart, Kidney, and Liver: A Comparison between Acetyl-Coenzyme A and Butyryl-Coenzyme A

as Fatty Acid Synthase Primers in the Assay Procedure

BALBIR SINGH, JACOB A. STAKKESTAD, JON BREMER, AND BORGAR BORREBAEK

Institute of Medical Biochemistry, University of Oslo, P.O. Box II 12, Blindern, Oslo 3, Norway

Received August 8, 1983

The malonyl-CoA assay was nonlinear at low malonyl-CoA concentrations when labeled acetyl-CoA was used as fatty acid synthase primer. Linearity was obtained with low concentrations of both fatty acid synthase and labeled acetyl-CoA, but then the assay was disturbed by the diluting effect of endogenous acetyl-CoA. The problems of nonlinearity and dilution of radioactivity by endogenous compounds were absent when labeled butyryl-CoA was used as primer. The levels of malonyl-CoA in rat heart, kidney, and liver were determined. The use of butyryl-CoA gave higher values of malonyl-CoA.

KEY WORDS: malonyl-CoA; butyryl-CoA; fatty acid synthase; liver; heart; kidney.

Malonyl-CoA is assumed to be an impor- with [ 1 -14C]butyryl-CoA as primer have been tant regulator of hepatic fatty acid oxidation compared. through its inhibitory action on carnitine acyltransferase (reviewed in (1)). Since the cel- lular concentration of malonyl-CoA is small, a highly sensitive assay procedure is necessary. One method developed by McGarry et al. (2) is based on the malonyl-CoA-dependent in- corporation of radioactive acetyl-CoA into palmitic acid catalyzed by fatty acid synthase in the presence of excess NADPH. A modi- fication of this method has been recommended by Beynen et al. (3). However, we have found that these methods are unreliable for small amounts of malonyl-CoA because of nonlin- earity of the assay. Dilution of the labeled acetyl-CoA by endogenous acetyl-CoA in the samples is also a problem.

Since liver fatty acid synthase shows a pro- nounced preference for butyryl-CoA over ace- tyl-CoA as primer (45) we have investigated whether the malonyl-CoA-dependent incor- poration of radioactive butyryl-CoA into pal- mitic acid would constitute a more sensitive assay method.

In the present work the radiochemical as- says of malonyl-CoA with [3H]acetyl-CoA or

MATERIALS AND METHODS

Animals. Male Wistar rats (200-250 g body wt) were used. The rats had free access to a standard pellet diet and water.

Chemicals. [3H]Acetyl-CoA (850 mCi/ mmol) and Na [ l-14C]butyrate (13.4 mCi/ mmol) were from New England Nuclear Cor- poration, Boston, Massachusetts. Malonyl- CoA and NADPH were from Sigma Chemical Company, St. Louis, Missouri. DEAE-Se- phadex was from Pharmacia Fine Chemicals, Uppsala, Sweden. N,N’-Carbonyldiimidazole and Union Carbide molecular sieve 4A were from Fluka AG, Buchs SG, Switzerland. To remove peroxides, tetrahydrofuran was dis- tilled with lithium aluminum hydride on the day of use.

Preparation offatty acid synthase. This en- zyme was prepared from rat liver according to Nepokroeff et al. (6). The rats were starved for 48 h and then fed a fat-free carbohydrate diet for 48 h. The specific activity of the pre- pared enzyme was 60 mU/mg protein (1 U = 1 pmol palmitate formed/min at 30°C).

107 0003-2697/84 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

108 SINGH ET AL.

Preparation of tissue extracts. Rats were killed by decapitation and tissues (about 1 g) were rapidly excised, freeze-clamped in liquid nitrogen, crushed in a steel mortar, and trans- ferred to homogenizing tubes together with some liquid nitrogen. The homogenizing tubes were then supplied with 1 ml of 3 N perchloric acid, which was immediately frozen above the tissue powder. The tubes were then shaken at - 10°C for 15 min. During this time, the per- chloric acid melted and penetrated down into the tissue powder at this low temperature. Then, 4 ml of ice-cold distilled water was added and the tissue was further homogenized with a motor-driven Teflon pestle at 0°C. The perchloric acid extracts were finally neutralized to pH 6.5 with KOH.

Preparation of [ l-‘4C]butyryl-CoA. The procedure is a modified version of the method for preparation of CoA-thioesters of fatty acids described by Kawaguchi et al. (7). The puri- fication of CoA-thioesters from free CoA was as described by Moffatt and Khorana (8).

To obtain free [ 1-14C]butyric acid, 160 ~1 of 0.5 N HCI and a small amount of NaCl were added to 30 pmol of Na [ 1 -14C]butyrate. The free butyric acid was extracted four times with 0.5 ml of benzene. The combined extracts were dried overnight with 80 mg of molecular sieve which subsequently was removed by centrifugation. Excess N,N’-carbonyldiimid- azole was added to the benzene solution and the mixture was allowed to react for 30 min at room temperature. The benzene solution was then taken off and the remaining N,N’- carbonyldiimidazole was washed with 2 X 0.5 ml of water-free benzene. The solvent was evaporated and the residue was dissolved in 2 ml of tetrahydrofuran:H20 (2: 1, v/v). This solution was allowed to react with 50 mg of CoA in 0.5 ml of tetrahydrofuran:H20. Im- mediately before the two solutions were com- bined, 100 ~1 of triethylamine was added to the CoA solution. The mixture was left over- night at room temperature under NZ. On the next day the solvent was evaporated, and the residue was dissolved in 2 ml HZ0 and applied to a 1 X 20-cm column of DEAE-Sephadex in the chloride form. Elution was commenced

using a linear lithium chloride gradient (O-O.8 M) in 5 mM HCl. Eight-milliliter frac- tions were collected at the rate of 0.4 ml/min. The combined fractions with butyryl-CoA were vacuum evaporated to a small volume. To remove lithium, the solution was applied to a 1 X 13-cm column of G-25 Sephadex which was eluted with 5 mM HCl. The col- lected fractions containing butyryl-CoA were once again vacuum evaporated and subse- quently dissolved in 2 mM Mes (Cmorphol- ineethanesulfonic acid), pH 5.5. The yield was approximately 4 1% based on the radioactive butyrate used.

Malonyl-CoA assay with [“Hlacetyl-CoA as primer. The procedure of Beynen et al. (3) was used, with the following minor alterations: The incubation medium containing 67 mM

(instead of 6.7 mM) potassium phosphate and indicated concentrations of fatty acid synthase, [3H]acetyl-CoA, and malonyl-CoA was used. The incubation time was reduced to 20 min since we found that the reaction was always completed within 10 min. An internal mal- onyl-CoA standard allowed for correction of the dilution of [3H]acetyl-CoA radioactivity by the endogenous acetyl-CoA present in the samples. With each sample, two incubations were performed, one with and the other with- out addition of a known amount of malonyl- CoA. The content of malonyl-CoA was then calculated according to McGarry et al. (2).

Malonyl-CoA assay with [f4C]butyryl-CoA as primer. The incubation mixture (1.5 ml total volume) contained 0.1-0.2 ml neutral- ized perchloric acid extract (or malonyl-CoA standard), 100 pmol potassium phosphate (pH 6.9, 15 pmol L-cysteine, 0.2 pmol NADPH, 0.075 mU fatty acid synthase, 0.6 mg fatty- acid-free bovine serum albumin, and indicated concentrations of [‘4C]butyryl-CoA. The in- cubation (20 min at 37’C) was started by the addition of fatty acid synthase and stopped by the addition of 1 ml 1.8 N KOH in 70% methanol (V/V) containing 5 pmol each of caprylic, capric, lauric, myristic, palmitic, and stearic acids (as carriers). The mixture was heated to 70°C for 30 min and then acidified with 1 ml 12 N H2S04. Long-chain fatty acids

ASSAY OF MALONYL-COENZYME A 109

were extracted with 2 X 4.5 ml petroleum ether (bp 40-60°C). The extracts were com- bined, washed with 3 ml 0.1 M butyric acid, and evaporated in glass scintillation vials overnight in the open at 4%50°C. The residue was dissolved in 10 ml scintillation fluid and counted. It is important that plastic scintil- lation vials not be used, apparently because the evaporation of residual labeled butyric acid is prevented by adsorption to the plastic, thus giving high radioactive blank values.

RESULTS

Assay with labeled acetyl-CoA. Figure la shows that with small amounts of malonyl- CoA the formation of long-chain fatty acids extractable with petroleum ether is nonlinear. This figure was obtained with standard so- lution; no tissue extract was involved. How- ever, we have observed that different amounts of the tissue extract in the assay mixture gave different malonyl-CoA values (per ml extract) even when the internal malonyl-CoA standard (see Materials and Methods) was used. This is related to the nonlinearity of the standard curve (Fig. la). Figure la also shows that ad-

dition of CoA to the incubation mixture in amounts which may be present in tissue ex- tracts (9,lO) depresses the incorporation of ra- dioactivity from labeled acetyl-CoA into long- chain fatty acids, and the nonlinearity of the standard curve gets more pronounced.

Figure 1 b shows that the linearity of the standard curve improved with decreasing concentrations of fatty acid synthase in the assay mixture. However, no further improve- ment of the linearity was obtained using en- zyme concentrations less than 50 pU/ml.

Figure lc shows that almost complete lin- earity was obtained when the concentration of acetyl-CoA was lowered to 0.3 PM, i.e., when the malonyl-CoA/acetyl-CoA ratio was increased. With a low concentration of fatty acid synthase and a high malonyl-CoA/acetyl- CoA ratio (curve B in Fig. lc) it was possible to measure less than 50 pmol malonyl-CoA relatively accurately. However, under these conditions the unlabeled acetyl-CoA in bio- logical extracts will dilute the labeled acetyl- CoA several fold. This gives a decreased mal- onyl-CoA/acetyl-CoA ratio which with free CoA present will effect the linearity of the assay to an unknown extent.

Malonyl-CoA Malonyl-CoA Malonyl-CoA (nmol) lnmoll I nmoll

FIG. 1. Standard curves with [‘Hlacetyl-CoA as primer. (a) At different concentrations of added CoA. The incubation mixture (1.5 ml) contained 7.5 nmol [‘Hlacetyl-CoA, 0.075 mU of fatty acid synthase, and the indicated amounts of malonyl-CoA. Curves A, B, C, D, and E were obtained with 8.0, 4.0, 2.0, 1.0, and 0 nmol CoA, respectively. (b) At different concentrations of fatty acid synthase. The incubation mixture (1.5 ml) contained 7.5 nmol [3H]acetyl-CoA and the indicated amounts of malonyl-CoA. Curves A, B, C, D, and E were obtained with 15, 1.5,0.75,0.3, and 0.075 mU of fatty acid synthase, respectively. (c) At low concentrations of acetyl-CoA. The incubation mixture (1.5 ml) contained 0.075 mU of fatty acid synthase and the indicated amounts of malonyl-CoA. Curves A and B were obtained with 0.75 and 0.5 nmol [3H]acetyl-CoA, respectively.

110 SINGH ET AL.

Assay with labeled butyryl-CoA. When la- beled butyryl-CoA was used as primer instead of labeled acetyl-CoA, an almost completely linear standard curve was obtained even in the presence of 5 PM butyryl-CoA (Fig. 2) i.e., under conditions similar to those of curve E in Fig. lb.

Figures 3 and 4 show that butyryl-CoA is the preferred primer with equal concentrations of butyryl-CoA and acetyl-CoA. The incor- poration of radioactive butyryl-CoA into long- chain fatty acids was inhibited only 26% by the presence of an equal amount of nonlabeled acetyl-CoA (Fig. 3), while the incorporation of radioactive acetyl-CoA was inhibited 75% by nonlabeled butyryl-CoA (Fig. 4).

The presence of free CoA did not depress the incorporation of labeled butyryl-CoA or change the linearity of the assay. On the con- trary, free CoA gave a slight stimulation (not shown). This stimulation is probably explained by the requirement of free CoA by fatty acid synthase (9,lO). When the assay with butyryl- CoA was used with the tissue extracts, con- sistent values of malonyl-CoA (per ml extract) which did not vary with the amounts of tissue extracts added to the assay mixture were ob-

Malonyl -CoA ( nmol)

F)G. 2. Standard curve with [‘4C]butyryl-CoA as primer. The incubation mixture (1.5 ml) contained 8 nmol [14C]butyryl-CoA, 0.075 mU of fatty acid synthase, and the indicated amounts of malonyl-CoA. Filled symbols indicate the presence and open symbols the absence of added fatty acid carriers during extraction and evaporation.

I 0.5

Malonyl -CoA (nmol)

FIG. 3. The inhibition by acetyl-CoA of the incorpo- ration of [r4C]butyryl-CoA into long-chain fatty acids. The incubation mixture (1.5 ml) contained 1 nmol [%]butyryl-CoA, 0.075 mU of fatty acid synthase, and the indicated amounts of malonyl-CoA. Filled symbols indicate the presence and open symbols the absence of 1 nmol acetyl-CoA.

tamed. Also, linearity was obtained when dif- ferent amounts of standard malonyl-CoA were added together with the tissue extract.

Malonyl -CoA (nmol)

FIG. 4. The inhibition by butyryl-CoA of the incor- poration of [‘Hlactyl-CoA into long-chain fatty acids. The incubation mixture (1.5 ml) contained 1 nmol [3H]acetyl- CoA, 0.075 mU of fatty acid synthase, and the indicated amounts of malonyl-CoA. Filled symbols indicate the presence and open symbols the absence of 1 nmol butyryl- CoA.

ASSAY OF MALONYL-COENZYME A 111

Table 1 shows the levels of malonyl-CoA zyme concentration, and their relative con- in rat heart, kidney, and liver, measured with tribution to the total radioactivity would be the two different primers. The values were large when the malonyl-CoA/acetyl-CoA ratio generally higher when labeled butyryl-CoA is low. was used. Malonyl-CoA assay with butyryl-CoA as

primer showed a linear standard curve with DISCUSSION a relatively high level of butyryl-CoA (Fig. 2).

The finding in heart and kidney that the This means that acetyl-CoA in extracts to be

levels of malonyl-CoA are reduced in fasted assayed has very little diluting effect. It was

rats (11) supports the possibility that this therefore unnecessary to calibrate the assay of

compound acts as a regulator of fatty acid each sample by addition of standard malonyl-

oxidation in these tissues. It has previously CoA. Altogether, our assay with butyryl-CoA

been shown that rat heart and kidney camitine as primer constitutes a less elaborate and more

acyltransferases are inhibited by malonyl- reliable procedure for low levels of malonyl-

CoA (12). CoA. With this method we have calculated

The lower values found in tissue extracts that the incorporation of butyryl-CoA into

with acetyl-CoA as primer (Table 1) are most nonvolatile fatty acids was 90% of the theo-

likely due to the nonlinearity of the assay retical value based on the use of six malonyl-

which varies with the content of endogenous CoA to convert one butyryl-CoA to palm&ate.

free CoA and acetyl-CoA in the samples. This ACKNOWLEDGMENTS nonlinearity is probably explained by the ob- servation of Abdinejad et al. (5) that fatty acid

This work was supported by grants from the Nansen Foundation, the Nordic Insulin Fund, and the Norwegian

synthase converts significant amounts of ace- Research Council for Science and the Humanities. Thanks tyl-CoA into short-chain (e.g., butyric acid) as are due to Mr. Jan Cervenka for excellent technical as- well as long-chain fatty acids. The radioactivity sistance.

of short-chain fatty acids is not detected in the assay procedure since they are either not REFERENCES

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6. Nepokroeff, C. M., Iakshmanan, M. R., and Porter, TABLE I

THE MALOIWL-COA CONTENT OF RAT HEART, KIDNEY, AND LIVER’

Malonyl-CoA

Tissue Acetyl-CoA primer Butyryl-CoA primer

Heart 3.68 + 0.30 4.37 -t 0.51 Kidney 0.70 + 0.05 1.15 + 0.07 Liver 4.10 + 0.12 7.60 + 0.27

J. W. (1975)in Methods in Enzymology (Lowen- stein, F. M., ed.), Vol. 35, pp. 37-44, Academic Press, New York.

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’ Values are means + SE from four different rats and 12. Saggerson, E. D., and Carpenter, C. A. (1981) FEBS are presented as nmol/g wet wt. Lett. 129, 229-232.