Erucic Acid Metabolism by Rat Heart Preparations CHAK-KI CHENG and SHRI V. PANDE, Laboratory of Intermediary Metabolism, Clinical Research Institute of Montreal, Montreal, Quebec, Canada H2W 1 R7
Minn., and P-L Biochemicals, Milwaukee, Wisc., respectively. Erucic acid, erucic anhydride, and erucylchloride were purchased from NuoChekPrep, Elysian, Minn. [1-14C] Palmitic acid was purchased from Amersham/Searle, Arlington Heights, Ill.; L-carnitine hydrochloride was from General Biochemicals, Chargrin Falls, Ohio; DL-carnitine ( c a r b o x y l - 1 4 C ) h y d r o c h l o ride was from ICN Pharmaceutical, Cleveland, Ohio, and other biochemicals were from Sigma, St. Louis, Mo., or P-L Biochemicals. L-Palmitylcarnitine was synthesized as previously described (7).
ABSTRACT
Rat heart preparations metabolized erucic acid at much slower rates than palmitic acid. This applied for activation reaction, for the conversion of acyl-CoA to acylcarnitine, and for the utilization of acyl group for oxidation. As compared to palmityl-CoA, erucyl-CoA exhibited a lower affinity for carnitine palmityltransferase (EC 2.3.1.23), the respective apparent Michaelis constants were 43 and 83 /aM. Presence of erucyl-CoA or erucylcarnitine slowed the mitochondrial oxidation of palmityl groups apparently because of the slower oxidation of erucyl groups. However, presence of erucate did not inhibit the activation of palmitate. Heart mitochondria obtained from rats fed rapeseed oil (50 cal %) or corn oil diet for 3 days showed similar abilities for the coupled oxidation of various substrates and similar carnitine palmityltransferase activities. Thus, a suggestion of gross m i t o c h o n d r i a l malfunction following rapeseed oil consumption was not confirmed.
Synthesis of Erucyl-S-CoA and L-Erucylcarnitine
INTRODUCTION
Erucic acid (cis-13-docosenoic acid) is a constituent of rapeseed oil, an edible oil used for production of margarine, shortenings, and salad oils (1). Use of rapeseed oil as a dietary component is of concern, because a high intake of this fat produces myocardial abnormalities in various animal species investigated (2). Feeding rapeseed oil to rats soon causes an accumulation of cardiac lipid, mostly as triglycerides, and necrotic lesions appear in the hearts after a prolonged feeding period (3). The significant factor involved in the pathogenicity of rapeseed oil appears to be the erucic acid (4). We have studied the myocardial metabolism of erucate to elucidate the mechanisms of its pathophysiological effects at the biochemical level. Some reports on the oxidation of erucic acid in heart mitochondria have appeared recently (5,6). MATERIALS AND METHODS Reagents
Palmitic acid and palmityl-CoA were obtained from the Horrnel Institute, Austin,
Erucyl-S-CoA was prepared according to Stadtman (8), except that erucic anhydride was dissolved in tetrahydrofuran and the reaction was allowed to proceed at 50 C, since, at 0 C, no detectable synthesis occurred. Purification of the erucyl-CoA (precipitated by acidification of the reaction mixture to pH 1 with HC1) involved removal of tetrahydrofuran by evaporation and washing four times with ether/petroleum ether (1:1). Spectral analysis of the product (in water, pH 6) indicated the ratio of molar absorptivity at 232 nm (thiolester bond) to that at 260 nm (adenine ring) to be 0.53 which is characteristic of acyl-CoA esters. Lerucylcarnitine was synthesized from erucylchloride and L-carnitine hydrochloride based upon the procedure of A1-Arif and Blecher (9), except that the erucylcarnitine was extracted with butanol after the ether washings; it was 74% pure as determined by ester group analysis (10). Animal and Diets
Eight week old male Wistar rats, obtained from Bio Breeding Laboratories of Canada, Ltd., were housed individually in metal cages with water and diets available ad libitum. Two groups of 4 rats were fed diets containing 50 cal % rapeseed oil or corn oil for a period of 3 days. The diets contained, as a percentage by wt, 30% casein, 35% cornstarch, 1% vitamin mixture, 4% salt mixture (11), 5% alphacel, and 25% of rapeseed or corn oil. The rapeseed oil contained 34.3% erucic acid, and it was a product of Cooperative Vegetable Oil Ltd., Altona, Canada. Corn oil and other dietary ingredients, including salt and vitamin mixtures, were obtained from Nutritional Biochemicals,
335
C.-K. CHENG AND S.V. PANDE
336 TABLE I
Activation of Erucic and Palmitic Acids by Rat Heart Homogenate a Palmitate (mM)
Erueate (mM)
1 to 6 -----2.0 2.0 2.0
--0.15 0.20 0.25 0.50 0.15 0.20 0.25
Hydroxamate formed (nmoles/min per mg protein)
ascertained that when identical enzyme incubat i o n c o n d i t i o n s were e m p l o y e d , t h e s p e c t r o p h o t o m e t r i c m e t h o d ( 1 2 ) gave t h e same results as those obtained by the radioactive procedure (13). RESULTS AND DISCUSSION
17-19 1.2 1.9 1.7 1.0 20.3 20.7 21.0
aActivation of fatty acids was followed by hydroxamate formation (16). Reaction mixtures in a final volume of 500 vliter contained: 50 ~moles Tris-HCl (pH 7.4), 250 #moles NH2OH, 2 ~amoles MgC12, 7.5 #moles ATP, 0.24 /~mole CoA, 2.5/~moles dithiothreitol, and fatty acids as shown. Reaction was initiated by the addition of 180 ttg protein from rat heart homogenate. Incubations were for 60 min at 37 C. Hydroxamate color was extracted with 0.5 ml of 1 : 1 0 diluted (in ethanol) Hill reagent (16). Cleveland, Ohio. T h e v i t a m i n m i x t u r e p r o v i d e d (in m g / 1 0 0 g diet): v i t a m i n A c o n c e n t r a t e ( 2 0 0 u n i t s / m g ) , 4.5; v i t a m i n D c o n c e n t r a t e ( 4 0 0 u n i t s / m g ) , 0 . 2 5 ; a - t o c o p h e r o l , 5.0; a s c o r b i c acid, 4 5 . 0 ; i n o s i t o l , 5.0; c h o l i n e c h l o r i d e , 7 5 . 0 ; m e n a d i o n e , 2 . 2 5 ; p - a m i n o b e n z o i c acid, 5.0; niacin, 4.5; riboflavin, 1.0; p y r i d o x i n e h y d r o chloride, 1.0; t h i a m i n e h y d r o c h l o r i d e , 1.0; calc i u m p a n t o t h e n a t e , 3.0; b i o t i n , 0 . 0 2 0 ; folic acid, 0 . 0 9 0 ; a n d v i t a m i n B-12, 0 . 0 0 1 3 5 . Other Methods
H o m o g e n a t e o f rat h e a r t was p r e p a r e d in 0.25 M sucrose using a P o l y t r o n h o m o g e n i z e r . M e t h o d s for t h e i s o l a t i o n o f m i t o c h o n d r i a f r o m h e a r t w i t h Nagarse a n d p r o t e i n e s t i m a t i o n were as p r e v i o u s l y d e s c r i b e d (7). Details o f e n z y m e assay s y s t e m s a n d c o n d i t i o n s for m i t o c h o n d r i a l o x i d a t i o n s are given in t h e legends to figures a n d tables. F o r c a r n i t i n e p a l m i t y l t r a n s f e r a s e assay, we
T h e a m o u n t o f m y o c a r d i a l free f a t t y acids increases c o n s i d e r a b l y i n rats given d i e t a r y r a p e s e e d oil (14). A d m i n i s t r a t i o n o f 14 C-erucic acid has s h o w n a c c u m u l a t i o n o f e r u c a t e i n t h e free f a t t y acid f r a c t i o n o f rat liver (15). T h e s e o b s e r v a t i o n s suggested t h a t erucic acid a c c u m u lation might be a consequence of the limiting ability for t h e a c t i v a t i o n o f e r u c a t e , a n d t h e results o f o u r e x p e r i m e n t s s u p p o r t this possibility. T h u s , u n d e r o p t i m a l c o n d i t i o n s ( d e t e r m i n e d s e p a r a t e l y ) , h o m o g e n a t e s of rat h e a r t a c t i v a t e d e r u c a t e at o n l y 10% o f t h e r a t e o f p a l m i t a t e a c t i v a t i o n ( T a b l e I). Because o f t h e m u c h slower a c t i v a t i o n o f e r u c a t e , it was of i n t e r e s t t o d e t e r m i n e if its p r e s e n c e w o u l d inhibit the activation of other long chain fatty acids. This is t o b e e x p e c t e d if t h e same e n z y m e is i n v o l v e d in t h e a c t i v a t i o n of d i f f e r e n t l o n g c h a i n f a t t y acids. In line w i t h this, o u r p r e v i o u s e x p e r i m e n t s h a d s h o w n t h a t t h e p r e s e n c e of slowly a c t i v a t e d p h y t a n a t e i n h i b i t e d t h e activat i o n o f p a l m i t a t e (17). H o w e v e r , n o e v i d e n c e for a n i n h i b i t i o n o f p a l m i t a t e a c t i v a t i o n b y erucate could be obtained; instead, under the c o n d i t i o n s o f h y d r o x a m a t e assay ( T a b l e I), t h e presence of erucate and palmitate together s h o w e d a n a d d i t i v e effect u p o n h y d r o x a m a t e f o r m a t i o n t h a t was seen c o n s i s t e n t l y in several different experiments. T h e c o n c e n t r a t i o n s of e r u c a t e e m p l o y e d in t h e e x p e r i m e n t o f T a b l e I were m u c h less t h a n t h o s e o f p a l m i t a t e , b e c a u s e excess o f e r u c a t e itself was i n h i b i t o r y f o r a c t i v a t i o n . Using radioactive assay ( 1 8 ) , it was possible t o e x a m i n e t h e e f f e c t of e r u c a t e u p o n 1 4 C - p a l m i t a t e a c t i v a t i o n
TABLE II Effect of Erucate upon Activation of Paimitate a [ 1-14 C ] Palmitate
Experiment
(#M) l
II
3 3 3 3 1 1
1
Erucate (#M) --3 10 50 --5
20
Palmityl-CoA formed (pmoles) 27 ,27 2'7 18 21 20 1'7
aActivation was measured by the radioactive assay procedure (18) in a final volume of 400 /~liter. Reactions were started by the addition of 2 gg protein from rat heart homogenate and terminated by adding 250 tlliter 0.5 M H2SO 4 after 5 min incubation at 37 C. LIPIDS, VOL. 10, NO. 6
ERUCIC ACID METABOLISM Milo,
~5
Mito,
e._a,
'.'v" "~"
20
337
t~lmilyl-CoA X
!
,.s~, iN. Tn/at" . ~ e,~s~-c*~ \
=;.o \.
15
IllfO,
c ._a,,
\
k" \
"\\ %
10 Palmit~l uli/Ixat/oo volt 914.6nmoM/mJn/mg
5 Km#8~d~
0
1
I
I
I
I
2
4
6
8
I0
I
([ru#l - CoAl
x I ~ (#il "l)
10.0
T. 5 Zx I~
I/
5.0
2.5
0
I
I
I
I
I
2
4 G 8 /0 I x IO0 ( ~ - I ) (Palmit#l - CoA l
FIG. 1. Lineweaver-Burk plots of carnitine palmityltransferase activities against concentrations of (A) erucyl-CoA and (B) palmityl-CoA. The assay was according to Bieber, et al., (12) in a final volume of 200 taliter with 10 tag protein from rat heart rnitochondria. L(-)-carnitine (final concentration 1.1 mM) was used to initiate the reaction at 28 C; control curets were without carnitine. V is expressed as nmoles of CoA formed/min per mg protein. at below saturating concentrations of palmitate. Table II shows that even under these conditions a three- to fivefold molar excess of erucate did ndt affect palmityl-CoA formation. Some inhibition was evident at higher levels of erucic acid, but this might have been due to the known (19) unspecific inhibition of fatty acylCoA synthetase (EC 6.2.1.3) by an excess of long chain unsaturated fatty acids. Following activation, utilization of fatty acids for oxidation requires conversion of fatty acyl-CoA to the acylcarnitine ester. Therefore, the ability of erucyl CoA to serve as a substrate for mitochondrial carnitine palmityltransferase (EC 2.3.1.23) was examined, and, for compari-
gr.r ~ uf/l|zot/on ram 9 8.5 nmoleImlnlml
l9 COA utllLsalMa roM 9 IG S . n m o M l ~ A n l
FIG. 2. Oxidation of palmityl-CoA and erucyl-CoA by rat heart mitochondria (mito.). Oxygen uptake was followed polarographieally with a Clark oxygen electrode (7). To 1.7 ml medium (0.23 M mannitol, 70 mM sucrose, 20 mM Tris-HCl, 20 tam EDTA, 5 mM Pi, 2 mM L-carnitine, 5 mM ADP, 1 mM matate, pH 7.2) saturated with air at 28 C was added freshly isolated mitochondria (0.5 mg of protein) from rat heart. Other additions were made as shown. Numerals below the tracings refer to the rate of oxygen consumption in natoms/min/mg protein. son, similar experiments were carried out with palmityl-CoA as well. Results of kinetic experiments (Fig. 1) showed that the apparent Km values with erucyl-CoA and palmityl-CoA were 83 /aM and 43 /~M, respectively; and the corresponding Vmax values as nmoles of CoA formed/min/mg protein were 38 and 59. Thus, compared to palmityl-CoA, erucyl-CoA was in every way a poorer substrate for the carnitine palmityltransferase of rat heart mitochondria. Figure 2 shows that, while palmityl-CoA, in the presence of carnitine, increased the oxygen consumption rate of heart mitochondria by 290% (from 80 to 312 natoms oxygen/min/mg protein, Fig. 2, curve A), erucyl-CoA increased the oxygen consumption rate by only 100% (Fig. 2, curve B) and a mixture of both acyl-CoA esters increased oxygen uptake to a rate between those of the individual CoA esters (curve C, Fig. 2). Likewise, the utilization rate of equimolar acyl-CoA mixture was an average of the rates seen with individual CoA esters. Thus, slower oxidation of erucyl-CoA diminished the concurrent oxidation of palmityl groups, perhaps by competing for the same enzyme system. Qualitatively, our results with acyl-CoA esters are similar to those reported recently by Christophersen and Bremer (6) and Swarttouw (5), except that, under the conditions of our experiments, the stimulation of oxygen consumption rate by erucyl-CoA approached one-third of that seen with palmitylCoA. In the experiments of Christophersen and Bremer (6), the oxygen uptake rate stimulated by erucylcarnitine was only 10% of that with patmitylcarnitine. In our experiments, carnitine LIPIDS, VOL. 10, NO. 6
C.-K. CHENG AND S.V. PANDE
338
TABLE III Effect of Dietary Erucic Acid upon Oxidative Metabolism of Rat Heart Mitochondria a
Substrate
Diet Rapeseed oil
Corn oil
Probability b
Rate of 02 uptake c in the presence of ADP and malate Pyruvate (8 ~umoles) Glutamate (10/~moles) Palmityl-CoA + carnitine Erucyl-CoA + carnitine
Palmityl-CoA Erucyl-CoA
416 306 147 66
+ 30 d • 28 • 4 • 5
437 • 9 358 • 5 162 • 17 71 • 8 Carnitine palmityltransferase activity e 49 4- 1 49 • 1 25 • 1 24 • 0
NS NS NS NS
NS NS
aRats were fed diets containing 50 cal % rapeseed oil (34.3% erucic acid content) or corn oil for 3 days. Each group had four rats. bCalculated according to Student's t-test. NS = not significant (P > 0.05). CMeasured as described previously (7) and in legend to Fig. 2 (in natoms/min per mg protein). dMean -+ standard error of the mean. eAssayed by the radioactive method (13) at 37 C; activity expressed as nmoles of 14Cacylcarnitine formed/min/mg protein. esters o f e r u c a t e a n d p a l m i t a t e gave results i n d i s t i n g u i s h a b l e f r o m t h o s e seen w i t h corres p o n d i n g esters of C o A plus c a r n i t i n e , a n d t h e s e results were n o t m o d i f i e d b y t h e s u b s t i t u t i o n o f adenosine diphosphate (ADP) by 2,4-dinitrophenol (data not shown). H o u t s m u l l e r , et al., ( 1 4 ) r e p o r t e d t h a t rapeseed oil feeding t o rats r e s u l t e d in m a r k e d i m p a i r m e n t o f t h e ability t o oxidize various s u b s t r a t e s . Because t h e o x i d a t i o n rates o f normal m i t o c h o n d r i a o b t a i n e d b y t h e s e investigat o r s were e x t r e m e l y l o w as c o m p a r e d t o w h a t we o b s e r v e in o u r e x p e r i m e n t s , t h e d i e t a r y e x p e r i m e n t s have b e e n r e p e a t e d t o d e t e r m i n e if t h e r e p o r t e d m i t o c h o n d r i a l m a l f u n c t i o n is, indeed, a t r u e p a t h o g e n i c e f f e c t o f d i e t a r y erucic acid. G r o u p s o f 4 rats were fed a diet o f 50 cal % r a p e s e e d off or a c o n t r o l diet o f c o r n oil a n d sacrificed 3 days l a t e r w h e n cardiac lipidosis, i n d u c e d b y r a p e s e e d oil diet, is k n o w n t o r e a c h p e a k i n t e n s i t y (2,14). Hearts i s o l a t e d f r o m t h e r a p e s e e d oil-fed rats were visibly pale, u n l i k e those from the control group. A comparison of t h e m i t o c h o n d r i a l o x i d a t i o n rates w i t h various s u b s t r a t e s a n d c a r n i t i n e p a l m i t y l t r a n s f e r a s e activities ( T a b l e III) s h o w e d n o significant differe n c e s b e t w e e n t h e r a p e s e e d a n d c o r n oil-fed rats. R e s p i r a t o r y c o n t r o l a n d A D P / o r a t i o s w i t h p y r u v a t e o r g l u t a m a t e in t h e s e t w o g r o u p s of a n i m a l s were also alike ( d a t a n o t s h o w n ) . T h u s , t h e suggestion (14) of a m a r k e d f u n c t i o n a l d a m a g e in h e a r t m i t o c h o n d r i a due t o u p t a k e o f erucic acid is n o t s u p p o r t e d . LIPIDS, VOL. 10, NO. 6
F r o m o u r results, it a p p e a r s t h a t a c t i v a t i o n m a y c o n s t i t u t e t h e r a t e - l i m i t i n g r e a c t i o n in t h e overall m e t a b o l i s m o f e r u c a t e in heart. Inasm u c h as t h e p r e s e n c e o f e r u c y l - C o A slowed t h e u t i l i z a t i o n of p a l m i t y l - C o A f o r o x i d a t i o n , it is likely t h a t t h e a c c u m u l a t i o n o f a c t i v a t e d f a t t y acids due to slower o x i d a t i o n p r o m o t e s triglyceride s y n t h e s i s as suggested b y C h r i s t o p h e r s e n a n d B r e m e r (6). We c o n s i d e r it p r o b a b l e t h a t triglyceride d e p o s i t i o n in h e a r t , f o l l o w i n g rapeseed oil c o n s u m p t i o n , results, n o t o n l y f r o m i n c r e a s e d s y n t h e s i s , b u t also f r o m s i m u l t a n e o u s i n h i b i t i o n of lipolysis d u e t o t h e a c c u m u l a t i o n o f free erucic acid. It is e s t a b l i s h e d t h a t h i g h c o n c e n t r a t i o n s o f free f a t t y acids i n h i b i t horm o n e - s e n s i t i v e lipase in a d i p o s e tissue ( 2 0 ) a n d r e c e n t evidences suggest t h a t s u c h r e g u l a t o r y m e c h a n i s m s also are p r e s e n t in h e a r t (21). As is k n o w n f o r adipose tissue, cyclic A M P activates lipolysis i n h e a r t as well ( 2 1 ) , a n d it is of i n t e r e s t in this c o n n e c t i o n t h a t i n g e s t i o n of r a p e s e e d oil decreases a d e n y l cyclase activity in h e a r t (22). A l t h o u g h a s h o r t t e r m f e e d i n g o f r a p e s e e d oil (3 days at 50 cal %) did n o t alter m i t o c h o n d r i a l f u n c t i o n to a n y n o t i c e a b l e degree, it is possible t h a t excess free erucic acid e v e n t u a l l y plays a role in t h e d e v e l o p m e n t o f m y o c a r d i a l lesions, seen in rats s u s t a i n e d o n r a p e s e e d oil diets, b e c a u s e e l e v a t e d free f a t t y acid c o n c e n t r a t i o n s h a v e b e e n suggested ( 2 3 ) t o be i n v o l v e d in m y o c a r d i a l necrosis. ACKNOWLEDGMENTS This work was supported by grants from the Medical
339
ERUC1C ACID METABOLISM Research Council of Canada (MA-4264) and the Quebec Heart Foundation. REFERENCES 1. McAnsh, J., JAOCS 50:404 (1973). 2. Abdellatif, A.M.M., Nutr. Rev. 30:2 (1972). 3. Beare-Rogers, J.L., E.A. Nera, and B.M. Craig, Lipids 7:548 (1972). 4. Abdellatif, A.M.M., and R.O. Vies, Nutr. Metabol. 15:219 (1973). 5. Swarttouw, M.A., Biochim. Biophys. Acta 337:13 (1974). 6. Christopbersen, B.O., and J. Bremer, Ibid. 280:506 (1972). 7. Pande, S.V., and M.C. Blanchaer, J. Biol. Chem. 246:402 (1971). 8. Stadtman, E.R., in "Methods in Enzymology," Vol. III, Edited by S.P. Colowick and N.O. Kaplan, Academic Press, New York, N.Y., 1957, p. 931. 9. AI-Arif, A., and M. Blecher, Biochim. Biopbys. A c t a 248:416 (1971). 10. Skidmore, W.D., and C. Entenman, J. Lipid Res. 3:356 (1962). 11. Rogers, Q.R., and A.E. Harper, J. Nutr. 87:267 (1965). 12. Bieber, L.L., T. Abraham, and T. Helmrath, Anal.
Biochem. 50:509 (1972). 13. Pande, S.V., J. Biol. Chem. 246:5384 (1971). 14. Houtsmuller, U.M.T., C.B. Struijk, and A. Van der Beek, Biochirm Biophys. Acta 218:564 (1970). 15. Carroll, K.K., Lipids 1:171 (1966). 16. Pande, S.V., and J.F. Mead, J. Biol. Chem. 243:352 (1968). 17. Pande, S.V., A.W. Siddiqui, and A. Gattereau, Biochim. Biophys. Acta 248:156 (1971). 18. Pande, S.V., Ibid. 270:197 (1972). 19. Pande, S.V., and J.F. Mead, J. Biol. Chem. 243:6180 (1968). 20. Rodbell, M., in "Handbook of Physiology," Section 5, Edited by A.E. Renold and G.F. Cahill, Jr., American Physiological Society, Washington, D.C., 1965, p. 471. 21. Shipp, J.C., L.A. Menahan, M.F. Crass llI, and S.N. Chaudhuri, Rec. Advan. Stud. Cardiac Struct. Metab. 3:179 (1973). 22. Cresteil, T., P. Ketevi, and D. Lapous, C.R. Acad. Sci., Ser. D. 275:1443 (1972). 23. Hoak, J.C., E.D. Warner, and E.W. Connor, in "Recent Advances in Studies on Cardiac Structure and Metabolism," Vol. I, Edited by E. Bajusz and G. Rona, University Park Press, Baltimore, Md., 1972, p. 127.
[Received December 10, 1974]
LIPIDS, VOL. 10, NO, 6
Minn., and P-L Biochemicals, Milwaukee, Wisc., respectively. Erucic acid, erucic anhydride, and erucylchloride were purchased from NuoChekPrep, Elysian, Minn. [1-14C] Palmitic acid was purchased from Amersham/Searle, Arlington Heights, Ill.; L-carnitine hydrochloride was from General Biochemicals, Chargrin Falls, Ohio; DL-carnitine ( c a r b o x y l - 1 4 C ) h y d r o c h l o ride was from ICN Pharmaceutical, Cleveland, Ohio, and other biochemicals were from Sigma, St. Louis, Mo., or P-L Biochemicals. L-Palmitylcarnitine was synthesized as previously described (7).
ABSTRACT
Rat heart preparations metabolized erucic acid at much slower rates than palmitic acid. This applied for activation reaction, for the conversion of acyl-CoA to acylcarnitine, and for the utilization of acyl group for oxidation. As compared to palmityl-CoA, erucyl-CoA exhibited a lower affinity for carnitine palmityltransferase (EC 2.3.1.23), the respective apparent Michaelis constants were 43 and 83 /aM. Presence of erucyl-CoA or erucylcarnitine slowed the mitochondrial oxidation of palmityl groups apparently because of the slower oxidation of erucyl groups. However, presence of erucate did not inhibit the activation of palmitate. Heart mitochondria obtained from rats fed rapeseed oil (50 cal %) or corn oil diet for 3 days showed similar abilities for the coupled oxidation of various substrates and similar carnitine palmityltransferase activities. Thus, a suggestion of gross m i t o c h o n d r i a l malfunction following rapeseed oil consumption was not confirmed.
Synthesis of Erucyl-S-CoA and L-Erucylcarnitine
INTRODUCTION
Erucic acid (cis-13-docosenoic acid) is a constituent of rapeseed oil, an edible oil used for production of margarine, shortenings, and salad oils (1). Use of rapeseed oil as a dietary component is of concern, because a high intake of this fat produces myocardial abnormalities in various animal species investigated (2). Feeding rapeseed oil to rats soon causes an accumulation of cardiac lipid, mostly as triglycerides, and necrotic lesions appear in the hearts after a prolonged feeding period (3). The significant factor involved in the pathogenicity of rapeseed oil appears to be the erucic acid (4). We have studied the myocardial metabolism of erucate to elucidate the mechanisms of its pathophysiological effects at the biochemical level. Some reports on the oxidation of erucic acid in heart mitochondria have appeared recently (5,6). MATERIALS AND METHODS Reagents
Palmitic acid and palmityl-CoA were obtained from the Horrnel Institute, Austin,
Erucyl-S-CoA was prepared according to Stadtman (8), except that erucic anhydride was dissolved in tetrahydrofuran and the reaction was allowed to proceed at 50 C, since, at 0 C, no detectable synthesis occurred. Purification of the erucyl-CoA (precipitated by acidification of the reaction mixture to pH 1 with HC1) involved removal of tetrahydrofuran by evaporation and washing four times with ether/petroleum ether (1:1). Spectral analysis of the product (in water, pH 6) indicated the ratio of molar absorptivity at 232 nm (thiolester bond) to that at 260 nm (adenine ring) to be 0.53 which is characteristic of acyl-CoA esters. Lerucylcarnitine was synthesized from erucylchloride and L-carnitine hydrochloride based upon the procedure of A1-Arif and Blecher (9), except that the erucylcarnitine was extracted with butanol after the ether washings; it was 74% pure as determined by ester group analysis (10). Animal and Diets
Eight week old male Wistar rats, obtained from Bio Breeding Laboratories of Canada, Ltd., were housed individually in metal cages with water and diets available ad libitum. Two groups of 4 rats were fed diets containing 50 cal % rapeseed oil or corn oil for a period of 3 days. The diets contained, as a percentage by wt, 30% casein, 35% cornstarch, 1% vitamin mixture, 4% salt mixture (11), 5% alphacel, and 25% of rapeseed or corn oil. The rapeseed oil contained 34.3% erucic acid, and it was a product of Cooperative Vegetable Oil Ltd., Altona, Canada. Corn oil and other dietary ingredients, including salt and vitamin mixtures, were obtained from Nutritional Biochemicals,
335
C.-K. CHENG AND S.V. PANDE
336 TABLE I
Activation of Erucic and Palmitic Acids by Rat Heart Homogenate a Palmitate (mM)
Erueate (mM)
1 to 6 -----2.0 2.0 2.0
--0.15 0.20 0.25 0.50 0.15 0.20 0.25
Hydroxamate formed (nmoles/min per mg protein)
ascertained that when identical enzyme incubat i o n c o n d i t i o n s were e m p l o y e d , t h e s p e c t r o p h o t o m e t r i c m e t h o d ( 1 2 ) gave t h e same results as those obtained by the radioactive procedure (13). RESULTS AND DISCUSSION
17-19 1.2 1.9 1.7 1.0 20.3 20.7 21.0
aActivation of fatty acids was followed by hydroxamate formation (16). Reaction mixtures in a final volume of 500 vliter contained: 50 ~moles Tris-HCl (pH 7.4), 250 #moles NH2OH, 2 ~amoles MgC12, 7.5 #moles ATP, 0.24 /~mole CoA, 2.5/~moles dithiothreitol, and fatty acids as shown. Reaction was initiated by the addition of 180 ttg protein from rat heart homogenate. Incubations were for 60 min at 37 C. Hydroxamate color was extracted with 0.5 ml of 1 : 1 0 diluted (in ethanol) Hill reagent (16). Cleveland, Ohio. T h e v i t a m i n m i x t u r e p r o v i d e d (in m g / 1 0 0 g diet): v i t a m i n A c o n c e n t r a t e ( 2 0 0 u n i t s / m g ) , 4.5; v i t a m i n D c o n c e n t r a t e ( 4 0 0 u n i t s / m g ) , 0 . 2 5 ; a - t o c o p h e r o l , 5.0; a s c o r b i c acid, 4 5 . 0 ; i n o s i t o l , 5.0; c h o l i n e c h l o r i d e , 7 5 . 0 ; m e n a d i o n e , 2 . 2 5 ; p - a m i n o b e n z o i c acid, 5.0; niacin, 4.5; riboflavin, 1.0; p y r i d o x i n e h y d r o chloride, 1.0; t h i a m i n e h y d r o c h l o r i d e , 1.0; calc i u m p a n t o t h e n a t e , 3.0; b i o t i n , 0 . 0 2 0 ; folic acid, 0 . 0 9 0 ; a n d v i t a m i n B-12, 0 . 0 0 1 3 5 . Other Methods
H o m o g e n a t e o f rat h e a r t was p r e p a r e d in 0.25 M sucrose using a P o l y t r o n h o m o g e n i z e r . M e t h o d s for t h e i s o l a t i o n o f m i t o c h o n d r i a f r o m h e a r t w i t h Nagarse a n d p r o t e i n e s t i m a t i o n were as p r e v i o u s l y d e s c r i b e d (7). Details o f e n z y m e assay s y s t e m s a n d c o n d i t i o n s for m i t o c h o n d r i a l o x i d a t i o n s are given in t h e legends to figures a n d tables. F o r c a r n i t i n e p a l m i t y l t r a n s f e r a s e assay, we
T h e a m o u n t o f m y o c a r d i a l free f a t t y acids increases c o n s i d e r a b l y i n rats given d i e t a r y r a p e s e e d oil (14). A d m i n i s t r a t i o n o f 14 C-erucic acid has s h o w n a c c u m u l a t i o n o f e r u c a t e i n t h e free f a t t y acid f r a c t i o n o f rat liver (15). T h e s e o b s e r v a t i o n s suggested t h a t erucic acid a c c u m u lation might be a consequence of the limiting ability for t h e a c t i v a t i o n o f e r u c a t e , a n d t h e results o f o u r e x p e r i m e n t s s u p p o r t this possibility. T h u s , u n d e r o p t i m a l c o n d i t i o n s ( d e t e r m i n e d s e p a r a t e l y ) , h o m o g e n a t e s of rat h e a r t a c t i v a t e d e r u c a t e at o n l y 10% o f t h e r a t e o f p a l m i t a t e a c t i v a t i o n ( T a b l e I). Because o f t h e m u c h slower a c t i v a t i o n o f e r u c a t e , it was of i n t e r e s t t o d e t e r m i n e if its p r e s e n c e w o u l d inhibit the activation of other long chain fatty acids. This is t o b e e x p e c t e d if t h e same e n z y m e is i n v o l v e d in t h e a c t i v a t i o n of d i f f e r e n t l o n g c h a i n f a t t y acids. In line w i t h this, o u r p r e v i o u s e x p e r i m e n t s h a d s h o w n t h a t t h e p r e s e n c e of slowly a c t i v a t e d p h y t a n a t e i n h i b i t e d t h e activat i o n o f p a l m i t a t e (17). H o w e v e r , n o e v i d e n c e for a n i n h i b i t i o n o f p a l m i t a t e a c t i v a t i o n b y erucate could be obtained; instead, under the c o n d i t i o n s o f h y d r o x a m a t e assay ( T a b l e I), t h e presence of erucate and palmitate together s h o w e d a n a d d i t i v e effect u p o n h y d r o x a m a t e f o r m a t i o n t h a t was seen c o n s i s t e n t l y in several different experiments. T h e c o n c e n t r a t i o n s of e r u c a t e e m p l o y e d in t h e e x p e r i m e n t o f T a b l e I were m u c h less t h a n t h o s e o f p a l m i t a t e , b e c a u s e excess o f e r u c a t e itself was i n h i b i t o r y f o r a c t i v a t i o n . Using radioactive assay ( 1 8 ) , it was possible t o e x a m i n e t h e e f f e c t of e r u c a t e u p o n 1 4 C - p a l m i t a t e a c t i v a t i o n
TABLE II Effect of Erucate upon Activation of Paimitate a [ 1-14 C ] Palmitate
Experiment
(#M) l
II
3 3 3 3 1 1
1
Erucate (#M) --3 10 50 --5
20
Palmityl-CoA formed (pmoles) 27 ,27 2'7 18 21 20 1'7
aActivation was measured by the radioactive assay procedure (18) in a final volume of 400 /~liter. Reactions were started by the addition of 2 gg protein from rat heart homogenate and terminated by adding 250 tlliter 0.5 M H2SO 4 after 5 min incubation at 37 C. LIPIDS, VOL. 10, NO. 6
ERUCIC ACID METABOLISM Milo,
~5
Mito,
e._a,
'.'v" "~"
20
337
t~lmilyl-CoA X
!
,.s~, iN. Tn/at" . ~ e,~s~-c*~ \
=;.o \.
15
IllfO,
c ._a,,
\
k" \
"\\ %
10 Palmit~l uli/Ixat/oo volt 914.6nmoM/mJn/mg
5 Km#8~d~
0
1
I
I
I
I
2
4
6
8
I0
I
([ru#l - CoAl
x I ~ (#il "l)
10.0
T. 5 Zx I~
I/
5.0
2.5
0
I
I
I
I
I
2
4 G 8 /0 I x IO0 ( ~ - I ) (Palmit#l - CoA l
FIG. 1. Lineweaver-Burk plots of carnitine palmityltransferase activities against concentrations of (A) erucyl-CoA and (B) palmityl-CoA. The assay was according to Bieber, et al., (12) in a final volume of 200 taliter with 10 tag protein from rat heart rnitochondria. L(-)-carnitine (final concentration 1.1 mM) was used to initiate the reaction at 28 C; control curets were without carnitine. V is expressed as nmoles of CoA formed/min per mg protein. at below saturating concentrations of palmitate. Table II shows that even under these conditions a three- to fivefold molar excess of erucate did ndt affect palmityl-CoA formation. Some inhibition was evident at higher levels of erucic acid, but this might have been due to the known (19) unspecific inhibition of fatty acylCoA synthetase (EC 6.2.1.3) by an excess of long chain unsaturated fatty acids. Following activation, utilization of fatty acids for oxidation requires conversion of fatty acyl-CoA to the acylcarnitine ester. Therefore, the ability of erucyl CoA to serve as a substrate for mitochondrial carnitine palmityltransferase (EC 2.3.1.23) was examined, and, for compari-
gr.r ~ uf/l|zot/on ram 9 8.5 nmoleImlnlml
l9 COA utllLsalMa roM 9 IG S . n m o M l ~ A n l
FIG. 2. Oxidation of palmityl-CoA and erucyl-CoA by rat heart mitochondria (mito.). Oxygen uptake was followed polarographieally with a Clark oxygen electrode (7). To 1.7 ml medium (0.23 M mannitol, 70 mM sucrose, 20 mM Tris-HCl, 20 tam EDTA, 5 mM Pi, 2 mM L-carnitine, 5 mM ADP, 1 mM matate, pH 7.2) saturated with air at 28 C was added freshly isolated mitochondria (0.5 mg of protein) from rat heart. Other additions were made as shown. Numerals below the tracings refer to the rate of oxygen consumption in natoms/min/mg protein. son, similar experiments were carried out with palmityl-CoA as well. Results of kinetic experiments (Fig. 1) showed that the apparent Km values with erucyl-CoA and palmityl-CoA were 83 /aM and 43 /~M, respectively; and the corresponding Vmax values as nmoles of CoA formed/min/mg protein were 38 and 59. Thus, compared to palmityl-CoA, erucyl-CoA was in every way a poorer substrate for the carnitine palmityltransferase of rat heart mitochondria. Figure 2 shows that, while palmityl-CoA, in the presence of carnitine, increased the oxygen consumption rate of heart mitochondria by 290% (from 80 to 312 natoms oxygen/min/mg protein, Fig. 2, curve A), erucyl-CoA increased the oxygen consumption rate by only 100% (Fig. 2, curve B) and a mixture of both acyl-CoA esters increased oxygen uptake to a rate between those of the individual CoA esters (curve C, Fig. 2). Likewise, the utilization rate of equimolar acyl-CoA mixture was an average of the rates seen with individual CoA esters. Thus, slower oxidation of erucyl-CoA diminished the concurrent oxidation of palmityl groups, perhaps by competing for the same enzyme system. Qualitatively, our results with acyl-CoA esters are similar to those reported recently by Christophersen and Bremer (6) and Swarttouw (5), except that, under the conditions of our experiments, the stimulation of oxygen consumption rate by erucyl-CoA approached one-third of that seen with palmitylCoA. In the experiments of Christophersen and Bremer (6), the oxygen uptake rate stimulated by erucylcarnitine was only 10% of that with patmitylcarnitine. In our experiments, carnitine LIPIDS, VOL. 10, NO. 6
C.-K. CHENG AND S.V. PANDE
338
TABLE III Effect of Dietary Erucic Acid upon Oxidative Metabolism of Rat Heart Mitochondria a
Substrate
Diet Rapeseed oil
Corn oil
Probability b
Rate of 02 uptake c in the presence of ADP and malate Pyruvate (8 ~umoles) Glutamate (10/~moles) Palmityl-CoA + carnitine Erucyl-CoA + carnitine
Palmityl-CoA Erucyl-CoA
416 306 147 66
+ 30 d • 28 • 4 • 5
437 • 9 358 • 5 162 • 17 71 • 8 Carnitine palmityltransferase activity e 49 4- 1 49 • 1 25 • 1 24 • 0
NS NS NS NS
NS NS
aRats were fed diets containing 50 cal % rapeseed oil (34.3% erucic acid content) or corn oil for 3 days. Each group had four rats. bCalculated according to Student's t-test. NS = not significant (P > 0.05). CMeasured as described previously (7) and in legend to Fig. 2 (in natoms/min per mg protein). dMean -+ standard error of the mean. eAssayed by the radioactive method (13) at 37 C; activity expressed as nmoles of 14Cacylcarnitine formed/min/mg protein. esters o f e r u c a t e a n d p a l m i t a t e gave results i n d i s t i n g u i s h a b l e f r o m t h o s e seen w i t h corres p o n d i n g esters of C o A plus c a r n i t i n e , a n d t h e s e results were n o t m o d i f i e d b y t h e s u b s t i t u t i o n o f adenosine diphosphate (ADP) by 2,4-dinitrophenol (data not shown). H o u t s m u l l e r , et al., ( 1 4 ) r e p o r t e d t h a t rapeseed oil feeding t o rats r e s u l t e d in m a r k e d i m p a i r m e n t o f t h e ability t o oxidize various s u b s t r a t e s . Because t h e o x i d a t i o n rates o f normal m i t o c h o n d r i a o b t a i n e d b y t h e s e investigat o r s were e x t r e m e l y l o w as c o m p a r e d t o w h a t we o b s e r v e in o u r e x p e r i m e n t s , t h e d i e t a r y e x p e r i m e n t s have b e e n r e p e a t e d t o d e t e r m i n e if t h e r e p o r t e d m i t o c h o n d r i a l m a l f u n c t i o n is, indeed, a t r u e p a t h o g e n i c e f f e c t o f d i e t a r y erucic acid. G r o u p s o f 4 rats were fed a diet o f 50 cal % r a p e s e e d off or a c o n t r o l diet o f c o r n oil a n d sacrificed 3 days l a t e r w h e n cardiac lipidosis, i n d u c e d b y r a p e s e e d oil diet, is k n o w n t o r e a c h p e a k i n t e n s i t y (2,14). Hearts i s o l a t e d f r o m t h e r a p e s e e d oil-fed rats were visibly pale, u n l i k e those from the control group. A comparison of t h e m i t o c h o n d r i a l o x i d a t i o n rates w i t h various s u b s t r a t e s a n d c a r n i t i n e p a l m i t y l t r a n s f e r a s e activities ( T a b l e III) s h o w e d n o significant differe n c e s b e t w e e n t h e r a p e s e e d a n d c o r n oil-fed rats. R e s p i r a t o r y c o n t r o l a n d A D P / o r a t i o s w i t h p y r u v a t e o r g l u t a m a t e in t h e s e t w o g r o u p s of a n i m a l s were also alike ( d a t a n o t s h o w n ) . T h u s , t h e suggestion (14) of a m a r k e d f u n c t i o n a l d a m a g e in h e a r t m i t o c h o n d r i a due t o u p t a k e o f erucic acid is n o t s u p p o r t e d . LIPIDS, VOL. 10, NO. 6
F r o m o u r results, it a p p e a r s t h a t a c t i v a t i o n m a y c o n s t i t u t e t h e r a t e - l i m i t i n g r e a c t i o n in t h e overall m e t a b o l i s m o f e r u c a t e in heart. Inasm u c h as t h e p r e s e n c e o f e r u c y l - C o A slowed t h e u t i l i z a t i o n of p a l m i t y l - C o A f o r o x i d a t i o n , it is likely t h a t t h e a c c u m u l a t i o n o f a c t i v a t e d f a t t y acids due to slower o x i d a t i o n p r o m o t e s triglyceride s y n t h e s i s as suggested b y C h r i s t o p h e r s e n a n d B r e m e r (6). We c o n s i d e r it p r o b a b l e t h a t triglyceride d e p o s i t i o n in h e a r t , f o l l o w i n g rapeseed oil c o n s u m p t i o n , results, n o t o n l y f r o m i n c r e a s e d s y n t h e s i s , b u t also f r o m s i m u l t a n e o u s i n h i b i t i o n of lipolysis d u e t o t h e a c c u m u l a t i o n o f free erucic acid. It is e s t a b l i s h e d t h a t h i g h c o n c e n t r a t i o n s o f free f a t t y acids i n h i b i t horm o n e - s e n s i t i v e lipase in a d i p o s e tissue ( 2 0 ) a n d r e c e n t evidences suggest t h a t s u c h r e g u l a t o r y m e c h a n i s m s also are p r e s e n t in h e a r t (21). As is k n o w n f o r adipose tissue, cyclic A M P activates lipolysis i n h e a r t as well ( 2 1 ) , a n d it is of i n t e r e s t in this c o n n e c t i o n t h a t i n g e s t i o n of r a p e s e e d oil decreases a d e n y l cyclase activity in h e a r t (22). A l t h o u g h a s h o r t t e r m f e e d i n g o f r a p e s e e d oil (3 days at 50 cal %) did n o t alter m i t o c h o n d r i a l f u n c t i o n to a n y n o t i c e a b l e degree, it is possible t h a t excess free erucic acid e v e n t u a l l y plays a role in t h e d e v e l o p m e n t o f m y o c a r d i a l lesions, seen in rats s u s t a i n e d o n r a p e s e e d oil diets, b e c a u s e e l e v a t e d free f a t t y acid c o n c e n t r a t i o n s h a v e b e e n suggested ( 2 3 ) t o be i n v o l v e d in m y o c a r d i a l necrosis. ACKNOWLEDGMENTS This work was supported by grants from the Medical
339
ERUC1C ACID METABOLISM Research Council of Canada (MA-4264) and the Quebec Heart Foundation. REFERENCES 1. McAnsh, J., JAOCS 50:404 (1973). 2. Abdellatif, A.M.M., Nutr. Rev. 30:2 (1972). 3. Beare-Rogers, J.L., E.A. Nera, and B.M. Craig, Lipids 7:548 (1972). 4. Abdellatif, A.M.M., and R.O. Vies, Nutr. Metabol. 15:219 (1973). 5. Swarttouw, M.A., Biochim. Biophys. Acta 337:13 (1974). 6. Christopbersen, B.O., and J. Bremer, Ibid. 280:506 (1972). 7. Pande, S.V., and M.C. Blanchaer, J. Biol. Chem. 246:402 (1971). 8. Stadtman, E.R., in "Methods in Enzymology," Vol. III, Edited by S.P. Colowick and N.O. Kaplan, Academic Press, New York, N.Y., 1957, p. 931. 9. AI-Arif, A., and M. Blecher, Biochim. Biopbys. A c t a 248:416 (1971). 10. Skidmore, W.D., and C. Entenman, J. Lipid Res. 3:356 (1962). 11. Rogers, Q.R., and A.E. Harper, J. Nutr. 87:267 (1965). 12. Bieber, L.L., T. Abraham, and T. Helmrath, Anal.
Biochem. 50:509 (1972). 13. Pande, S.V., J. Biol. Chem. 246:5384 (1971). 14. Houtsmuller, U.M.T., C.B. Struijk, and A. Van der Beek, Biochirm Biophys. Acta 218:564 (1970). 15. Carroll, K.K., Lipids 1:171 (1966). 16. Pande, S.V., and J.F. Mead, J. Biol. Chem. 243:352 (1968). 17. Pande, S.V., A.W. Siddiqui, and A. Gattereau, Biochim. Biophys. Acta 248:156 (1971). 18. Pande, S.V., Ibid. 270:197 (1972). 19. Pande, S.V., and J.F. Mead, J. Biol. Chem. 243:6180 (1968). 20. Rodbell, M., in "Handbook of Physiology," Section 5, Edited by A.E. Renold and G.F. Cahill, Jr., American Physiological Society, Washington, D.C., 1965, p. 471. 21. Shipp, J.C., L.A. Menahan, M.F. Crass llI, and S.N. Chaudhuri, Rec. Advan. Stud. Cardiac Struct. Metab. 3:179 (1973). 22. Cresteil, T., P. Ketevi, and D. Lapous, C.R. Acad. Sci., Ser. D. 275:1443 (1972). 23. Hoak, J.C., E.D. Warner, and E.W. Connor, in "Recent Advances in Studies on Cardiac Structure and Metabolism," Vol. I, Edited by E. Bajusz and G. Rona, University Park Press, Baltimore, Md., 1972, p. 127.
[Received December 10, 1974]
LIPIDS, VOL. 10, NO, 6
