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Cis and trans-octadecenoic acids as precursors of polyunsaturated acids
CIS .AND
ACIDS POLYUNSATURATED ACIDS
TRANS-OCTADECENOIC
AS PRECURSORS
OF
R. T. HOLMANand M. M. MAHFOUZ* The Hormel Institute, University (~ Minnesota, Austin, Minnesota 55912 U,S.A.
INTRODUCTION
The unsaturated fatty acids of vegetable and animal fats are almost exclusively of the cis configuration and the double bonds are located at A9- and A9,12-positions of the dominant monoenes and dienes, respectively. Hydrogenation of vegetable oils in the manufacture of shortenings and margarine produces monounsaturated fatty acids of cis and trans configuration, and the double bonds also migrate along the hydrocarbon chain, producing positional isomers ranging from A6 to AI4 in both cis and trans series. '~' 27, 31 These fats contain significant quantities of trans fatty acid isomers which reach 17% in commercial vegetable oils, a 47% in margarines 22' 2a and 58% in vegetable shortenings, zg, aa These dietary unnatural acids are incorporated into tissue lipids of animals consuming such fats and they are now found in tissues of man ranging as high as 14% of the fatty acids of certain lipids) ~ Trans fatty acids inhibit lipid metabolism in vitro, 3T and they depress the conversion of cis-isomers to the highly unsaturated essential fatty acids, ~ even though they may be incorporated into tissue lipids) ~' a7 Mixed dietary trans acids in partially hydrogenated oil intensify the symptoms of essential fatty acid deficiency. 1° Positional and geometric isomers of unsaturfited acids are metabolized at different rates by isolated enzyme systems, 9' 1~ subcellular particles 7' ~a and organisms) ~ The present study was made to examine how isomerism in octadecenoic adds alters the rates and positions of their desaturation by rat liver microsomes, and to measure the effects of these isomers upon the rates of desaturation of natural substrates. MATERIALS AND M E T H O D S
The optimal concentration of substrate, microsomal protein and the time of incubation for the desaturation of oleic acid were used to compare the substrates. Is The microsomes were separated according to the procedure of Marcel et al. t9 The structures of dienoic acid esters isolated by thin layer chromatography (TLC) were identified by using preparative radio gas chromatography. The double bond positions were determined as described by Privett and Nickell, z6 by partial reduction of the dienoic acids with hydrazine hydrate without double bond migration to give a mixture of monoenoic and saturated adds. The isolated monoenes were ozonized, and the ozonides were reduced to aldehyde and aldehyde esters by Lindlar's catalyst. 26 The chain lengths of the labeled aldehyde esters were identified by preparative gas chromatography using unlabeled carriers of aldehyde esters with chain lengths from C3 to C1~. is RESULTS AND D I S C U S S I O N
Desaturation of trans.18: l isomers
One of the desaturation products of trans-octadecenoates was cis, trans-octadecadi. enoates in which the cis double bond was introduced by the enzyme. The conversion rate *Present address: Department of Biochemistry, Medical Research Institute, 165-Tarik EI-Horeya, EI-Hadara, Alexandria, Egypt. IS!
152
R.T. Holman and M. M. Mahfouz
/r~n~ IB:l ~ c i s - g , lrans- 15~2
1
~ oa
,3 4 5 6 7 8 g 10 It 12 13 I~. 15 POSITION OF TRANS DOUBLE BOND FIG. 1. Rat~s of &9-desaturation of positionally isomeric trans-octadecenoic acids by rat liver microsomes. The product measured was the trans,cis-18:2.
of trans-octadecenoates to cis, trans-ortrans, cis-octadecadienoates was highest for A4, A6 and A13 acids ranging from 0,263 nmol/mg/min for A4 to 0.402 nmol/mg/min for A13. The conversion rates of A7, A11, A12 and AI4 acids ranged from 0.088 nmol/mg/min for A7 to 0.157 nmol/mg/min for A14. The trans-A8, A9 and A10 acids were not desaturated at measurable rates. The site of desaturation of all the trans 18:1 isomers was the 9-position, in agreement with the results of James ~2 and Pollard et al. 2~ The relative rates of these reactions are shown in Fig. 1. The cis double bond introduces a bend in the molecule which is clearly distinct from the shape of saturated acids. Acyl transferase discriminates between the normal cis isomers and the unnatural trans forms with respect to esterification into phospholipids and triglycerides; a chain containing trans double bond is transferred as if it were a saturated carbon chain. Trans-18:1 acids also resemble stearic acid rather than cis-18:l acids in their slow rates of cholesteryl ester formation by rat liver microsomes, a2 Because trans-18:l acids were found to be desaturated by 9-desaturase, it is not surprising to find that trans-A8, A9 and A10 acids were not desaturated, in agreement with Brenner 2 who found that all trans-linoleic and elaidic acids were not desaturated by rat liver microsomes in vitro, but this disagrees with the report that t9-18:1 was desaturated to c5.t9-18:2 acid when other substrates for this enzyme are lacking. 16 Cis, cis-Diene Products from trans-18: l Isomers Another desaturation product from trans-18:1 acids was identified as cis,cis-octadeca. dienoate. Trans-A4 and A5 acids gave the highest rates ofcis,cis-diene formation. After incubation of t5-18:1, the labeled cis, cis-diene ester recovered from the AgNOa TLC plate showed a gas liquid chromatographic (GLC) retention time identical to the cis,cis-18:2 ester. The free acids obtained from the labeled diene esters were separated by quantitative paper chromatography. ~4 The radioactivity was mainly located with the 14:0 before reduction and with 18:0 after reduction, indicating that it was 18:2 acid. Partial reduction and ozonolysis gave Cs and C9 aldesters. We concluded that t5-18:1 produced c5,c9-18:2 acid. Trans-5-18:l and t4-18:1 were each incubated and the conversion to cis, trans- and to cis, cis-18:2 was measured with time. In the case of t5-18:1, the c,t-18:2 was not detected except during the first 5 rain of incubation (about 3.5%) after which it decreased as the c,c-18:2 increased. For the t4-18:1, during the first 15 rain when the rate of desaturation is high, both c,t-18:2 and c,c-18:2 were detected. After 15 min, the c,t-18:2 decreased slightly and c,c-18:2 increased from 5 to 13.8% indicating that the c,t-18:2 produced during this period was converted to c,c-18:2. The rates of desaturation to cis, cis diene products are shown in Fig. 2. One possible pathway for formation of c,c-18:2 is t- 18:1 --* c-18:1 --* c,c-18: 2. A second possible pathway is t-18:1 -* c,t-18: 2 -~, c,c-18: 2. We found that cis 4-, 5-, 6- and 7-18:1 were not desaturated by rat liver microsomes ~7 and that for the isomers which are desaturated, the c,c-18:2 acids produced do not contain a new cis-9 double bond. These observations indicate that the first pathway is
Cis and trans-octadecenoic acids as precursors
153
1 ?' 3
4 ,5 6 7 8 9 I0 II 12 13 I~- 1,5 POSITION OF" TRANS DOUBLE BOND
FIG. 2. Rates of A9-desaturation and cis-trans isomerization of positionally isomeric transoctadecenoic acids to cis,cis-octadecadienoic acids by rat liver microsomcs.
inoperative, as shown in Fig. 3. The second pathway is confirmed by the presence of cis-9-double bond in all c,c-18:2 acids just as in the c,t-18:2 products. The inserted cis-double bond in each c,c-18:2 has the same position as the trans-double bond in the c,t-18:2 produced from the same substrate acid, indicating that c,t-18:2 is isomerized to yield c,c-18:2 acid without migration of double bond. The CoA esters of six of the t-18:l isomers were prepared and subjected to desaturation. Their rates of desaturation were compared to those of the free acids in the presence of CoA. In each case, the rates of desaturation were the same for the two substrates, and the ratio of cis, trans to cis,cis product was the same for the two substrates indicating that the activation of the isomeric t-18:l acids is not a rate-limiting step in the desaturase system, nor does the activation step influence the isomerization of trans- to cis-double bond. Desaturation of Cis-18:1 Isomers The cis-18:l isomers with the double bond at carbons 4 through 7 were not measurably desaturated. The maximum conversion to 18:2 was obtained for c8- and cg-isomers which gave 3.4 and 3.9~/~ respectively. The rate of desaturation of oleic acid to 6,9-18:2 (0.118 nmol/mg protein/min) is comparable to that obtained by Castuma et al. 6 The cis-18:1 isomers with the double bond at carbons 4, 5, 6 or 7 were not desaturated, and the 10- and 11-isomers were desaturated at low rates. The 8- and 9-isomers were desaturated at relatively higher rates. The c12-18:1 acid was not significantly desaturated by rat liver microsomes, s Our study shows that the position of the double bond in a cis-18:l acid controls which desaturase acts upon it, and that the several cis-18:l isomers are substrates for different desaturases. The c9-18:1 was desaturated by A6-desaturase, in agreement with the previous reports}' 2o. 2~ Acids which have different chain lengths and numbers of double bonds, but have the first double bond at 9-position from the carboxyl group, are desaturated at A6.1' 5.3o The c8-18:1 and c11-18:1 were desaturated by A5-desaturase as are other A8 acids ~ and All acids. ~' 6.3,~ Desaturation of the c11-18:1 isomer to 5,11-18:2 acid (not to 8,11-18:2) confirms the absence of A8-desaturase from the rat liver microsome) ~ The c10-18:1 isomer gave 1.5% conversion to 18:2 acid with 70% of the double bonds at AT,10- and 30% as the A5,10-18:2 acid. Bernert and
cL*X- 18:1
i~,~'
~rons X-18:l
~%~,
ci$X~ci$9*18:~
~n~c/s9-18:2 FIG. 3. Metabolic pathway for desaturation-isomerization oftrans-18:l isomers.
R.T. Holman and M. M. Mahfouz
154
AB
c-18:1~ c,c-I~:~
-
i -ioo ~
I
mmmm d
~
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~
7 ~
~,s
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9 I~ II
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~ ~-~tad~Oi~
I~ I~ I~ ~ ~1~ a~Jd~ ~ ~at l i v ~ ~ i ~ r ~ O ~ ,
Sprecher I found that the 10,13-20:2 acid was converted to 7,10'13-20:3 at a very low rate. The relative rates of desaturation are shown in Fig. 4. The most abundant cis-18:1 isomers in hydrogenated soybean oil were reported to be the c9-18:1 (76%) and cl 1-18:1 (10~/o),27 and those isomers which were found not to be desaturated by rat liver mierosomes (A4 to A7) were present in less than 1% of the total cis-18:1 acids. 27 The major cis-18: i acids present in hydrogenated oils are thus potentially metabolized to cis, cis-18:2 acids, which in turn may be converted to higher polyunsaturated acids and perhaps prostaglandin-like compounds. Our study shows that cis. and trans-18:l acids are desaturated differently by rat liver microsomes, whereas the trans-octadecenoic acids and saturated acids are desaturated similarly. This may be a function of the generally straight cylindrical configuration of the trans and saturated acids and the bent configuration of cis-unsaturated acids, as Polyunsaturated Acids
We should expect that all the c,t- and c,c-18:2 acids derived from t-18:1 acids and which have the first double b o n d at A9-position could be further desaturated by A6-desaturase and chain elongated to give all-cis or mixed isomers of 20:3 and 20:4. In fact, Pollard et al. 24 found that t13-18:1 acid gave c9,t13-18:2 which was desaturated again to yield c6,c9,t13-18:3. Thus, t-18:1 isomers present in dietary fats can be the precursors of polyunsaturated fatty acids (PUFA) of unusual structure, which in turn could be precursors of unnatural or unusual series of prostaglandin-like c o m p o u n d s having biological activities different from the c o m m o n ones. In Fig. 5, the principal pathways for formation of P U F A from trans-monoenoic acids are summarized. In each case, only those desaturations which occurred in vitro using rat liver microsomes are included as first steps in the sequences. Subsequent steps are predicted from previous knowledge of chain elongation and desaturation by liver microsomes. In this figure, the location of the trans double b o n d from the original t-18:1 isomer is indicated in boldface type. The trans isomers of 18:1 which occur in measurable amounts in partially hydrogenated fats range from A6 through A15, but the AS, A9 and 6-18:1 -* 6.9-18:2 --" 8,11-20:2 --' 5,8,11-20:3 7-18:1 -" 7,9-18:2 --'*9.11-20:2 ---"6,9, I 1-20:3 8-18:1 9-18:1 10-18:1 11-|8: | ~ 9.11-18:2 --~6.9.11-18:3 -* 8.11.13-20:3 ---,5.8.11.13-20:4 !~-18:1 --~9,12-18:2 --~6,9,12-18:3 --~8,11,14-20:3 --~ 5,8,11,14-20;4 13-18:1 --~9,13-18:2 ~ 6.9,13-18:3 --~8,11,15-20:3 --o 5,8,11,15-20:4 14-18:1 --*9.14-18:2 --~6.9,14-18:3 --*8,11,16-20:3 -* 5,8d 1J6-20:4 15-18:1 --~9.15-18:2--~6.9,15-18:3 ---~8,11,17-20:3 -* 5,8,11,17-20:4 Boldface numbers indicate positions of trans double bonds. FI6. 5. Probable formation of PUFA from trans-18:1 isomers
Cis and trans-octadecenoic acids as precursors
155
c8-18:1 ---~ 5,8-18:2 --~ 7,10-20:2 --, 4,7,10-20: 3 c9-18 : 1 ~ 6,9-18:2 --* 8,11-20:2 --~ 5,8,11-20:3 c10-18 : 1 --~ 7,10-18:2 --~ 9,12-20:2 --~ 6,9,12-20:3 c10-18 : 1 ~ 5,10-18:2 --~ 7,12-20:2 --* 4,7,12-20:3 c11-18:1 --~ 5,11-18:2 --~ 7,13-20:2 --~ 4,7,13-20:3 FIG. 6. Probable formation of PUFA from cis-18:1 isomers
A10 isomers are not desaturated. Thus, PUFA could be expected from 7 isomers. These could give rise to 7 isomers of 20:3 and 5 isomers of 20:4, most of which have unusual double bond positions and all of which contain a trans double bond. Because in the desaturation of t-18:1 isomers, the trans double bond is isomer/zeal to the corresponding cis double bond, all the positional isomers shown in Fig. 5 could also occur in ali-cis configuration. Among these, some are identical to endogenously synthesized PUFA and some are naturally occurring essential fatty acids (EFA). The former are 8,11-20:2 and 5,8,11-20:3 (Mead's acid) which can be derived from oleic acid and which increase significantly in EFA deficiency when endogenous PUFA are synthesized as substitutes for co6 acids. The EFA are 9,12-18:2, 6,9,12-18:3, 8,11,14-20:3 and 5,8,11,14-20:4, all essential fatty acids of the co6 family, in this case derived from t12-18:1 which is a significant component in partially hydrogenated oils. Although few c-18:1 isomers are desaturated and those only at low rates, the 8-, 9-, 10-, and ll-isomers could be expected to yield PUFA (Fig. 6). The c9-18:1 could be converted to 8,11-20:2 and 5,8,11-20:3, both characteristic of EFA deficiency. The 12-18:1, from which co6 acids could be derived, is poorly desaturated. The PUFA predicted from the desaturation of isomeric c- and t-18:1 isomers thus might yield isomers of PUFA including those characteristic of EFA deficiency, those pps__s_essing EFA activity, and those in which double bond positions are unusual or in which a trans bond occurs. The possible conversion of the latter to prostaglandin-like products through oxidative metabolism can also be predicted. However, the rates at which these are formed and the biological activities of the products cannot yet be predicted. Thus the physiological consequences of consumption of partially hydrogenated fats cannot be predicted, but it would appear likely that the trans fatty acids in such fats could be the precursors of new, unusual substances having perhaps unanticipated biological acti,~ities. , Acknowledgement--This study was supported in part by Program Project Grant HL 08214 Program Project Branch, Extramural Programs, National Heart, Lung, and Blood Institute; Grant HL 21513 from the National Institutes of Health; and The Hormel Foundation.
REFERENCES 1. 2. 3. 4. 5.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
BERNERT,J. T. and SPR~CMEa, H. Biochim. Biophys. Acta 398, 354-363 (1975). BRENNER, R. R. Lipids 6, 567-575 (1971). CARPeNTeR, D. L., LEHMANN,J., MASON, B. S. and SLOWR, H. T. J. Am. Oil Chem. Soc. 53, 713-718 (1976). C A ~ P ~ a , D. L. and S~owR, H. T. J. Am. Oil Chem. Soc. 50, 372-376 (1973). CASTUMA,J. C., BR~NNElC,R. R. "and KUN^U, W. Adv. Exp. Med. Biol. 83, 124-134 (1977). CASTUMA,J. C., CATALA,A. and B ~ , ~ R , R. R. J. Lipid Res. 13, 783-789 (1972). Ct~ANG, H. C., JANK~ J., PUSCH, F. arid HOLMAN, R. T. Biochim. biophys. Acta 306, 21-25 (1973). GURR, M. E., ROBINSON, M. P., JAMES,A: T., MORRIS, L. J. and HOWlinG, D. Biochim. biophys. Acta 280, 415-421 (1972). HEIMERMANN,W. H., HOLMAN, R. T., GORDON, D. F., KOWALYSHUN, D. E. and Je'~SEN, R. G. Lipids 8, 45-47 (1973). H I ~ , E. G., JOHNSON, S. and HOLMAN, R. T. J. Nutr. 109, 1759-1765 (1979). HOLMAN, R. T., EGWIM, P, O. and CHRIS~. W. W. J. biol. Chem. 244, 1149-1151 (1969). JAMES,A. T. Adv. Exp. Med. Biol. 83, 51-74 (1977). J~NKI~, H. M., ANO~nSON, L., HOLMAN, R. T., ISMAIL,I. A. and GU'~STONE,F. D. J. Bacteriol. 98, 1026-1029 (1969). KAUFMANN,H. P., SCHNUP,I~USCH, H. and SHOEB, Z. E. Fette Seifen Anstrichm. 62, 1-5 (1959). KU~aM~ROW,F. A. J. Am. Oil Chem. Soc. 51, 255-259 (1974). L~MARCHAL,P. and BOaNENS, M. Bull. Soc. Chim. Biol. 50, 195-217 (1968).
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R.T. Holman and M. M. Mahfouz
MAHFOUZ. M. and HOLMAN, R. T. Lipids 15, 63-65 (1980). MAHFOUZ, M. M., VALICEN~, A. J. and HOLMAN, R. T. Biochim. biophys, Acta, accepted for publication. MARCEL, Y. L., CHRISTIANSEN, K. and HOLMAN, R. T. Biochim. biophys. Acta 164, 25-34 (1968). MEAD. J. F. and How~o~q, D. R. 3. biol. Chem. 229, 575-582 11957). ME^D, J. F. and SLATOr~, W. H. J. biol. Chem. 219, 705-709 (1956). OTTErqST~iN. D. M., WITTinG. L. A., WAL~R. G., M^HADEVA~q,V. and PEL~Cr~ N. J. Am. Oil Chem. Soc. 54, 207-209 (1977). PERK~qS. E. G., McCAgTHY, F. P., O'BR~E~q, T. and KUMMEROW, F. A. J. Am. Oil Chem. Soc. 54, 279-281 (1977). POLLED, M., GUNSTOCk, F., MORn~S, L. J. and J ~ s , A. T, cited in Adv. Exp. Med. Biol. 83, 51-74 [1977). P~IV~TT, O S. and BLANK, M. L. J. Am. Oil Chem. Soc. 41, 292-297 (1964). PglvEYr, O S. and NiCKELL, E. C. Lipids 1, 98-103 (1966). REICaWALD-HACKER. J.. ILSEMANN. K, and MUKm.,'R~EE,K. J. Nutr. 109, 1051-1056 (1979). RE1TZ. R., LANO~, W. E. M., CrlglS~E, W. W. and HOLMAN, R. T. J. biol. Chem. 2~3, 2241-2246 (1968). RYLANDER, P. W. J. Am. Oil Chem. Soc. 47, 482-485 (1970). SCSLE~K. H., SAND, D. M. and SE~, N. Biochim. biophys. Acta 84, 361-364 (1964). SCI~OLFmLD.C. R., DAVlSON, V. L. and D~yrolq, H. J. J. Am. Oil Chem. Soc. 44, 648-651 (1967). SC,OUT~S. D. A. Biochemistry 9, 1826-1833 (1970). SGOUT~S, D. and KUMMEROW, F. A. Am. J. Clin. Nutr. 23, 1111-1119 (1970). ULLMAN, D. and SPRECHER, H. Biochim. biophys. Acta 240, 186-197 (1971). VANDF-~qHI:-U'CEL,F. A. J. Am. Chem. Soc, 40, 455-472 {1963). WooD, R. and Cm~MaL~R, F. Lipids 13, 75-84 (1978). WOOD, R., Cm~MnER, F. and W~EGAND, R. J. biol. Chem. 252, 1965-1970 (1977).
ACIDS POLYUNSATURATED ACIDS
TRANS-OCTADECENOIC
AS PRECURSORS
OF
R. T. HOLMANand M. M. MAHFOUZ* The Hormel Institute, University (~ Minnesota, Austin, Minnesota 55912 U,S.A.
INTRODUCTION
The unsaturated fatty acids of vegetable and animal fats are almost exclusively of the cis configuration and the double bonds are located at A9- and A9,12-positions of the dominant monoenes and dienes, respectively. Hydrogenation of vegetable oils in the manufacture of shortenings and margarine produces monounsaturated fatty acids of cis and trans configuration, and the double bonds also migrate along the hydrocarbon chain, producing positional isomers ranging from A6 to AI4 in both cis and trans series. '~' 27, 31 These fats contain significant quantities of trans fatty acid isomers which reach 17% in commercial vegetable oils, a 47% in margarines 22' 2a and 58% in vegetable shortenings, zg, aa These dietary unnatural acids are incorporated into tissue lipids of animals consuming such fats and they are now found in tissues of man ranging as high as 14% of the fatty acids of certain lipids) ~ Trans fatty acids inhibit lipid metabolism in vitro, 3T and they depress the conversion of cis-isomers to the highly unsaturated essential fatty acids, ~ even though they may be incorporated into tissue lipids) ~' a7 Mixed dietary trans acids in partially hydrogenated oil intensify the symptoms of essential fatty acid deficiency. 1° Positional and geometric isomers of unsaturfited acids are metabolized at different rates by isolated enzyme systems, 9' 1~ subcellular particles 7' ~a and organisms) ~ The present study was made to examine how isomerism in octadecenoic adds alters the rates and positions of their desaturation by rat liver microsomes, and to measure the effects of these isomers upon the rates of desaturation of natural substrates. MATERIALS AND M E T H O D S
The optimal concentration of substrate, microsomal protein and the time of incubation for the desaturation of oleic acid were used to compare the substrates. Is The microsomes were separated according to the procedure of Marcel et al. t9 The structures of dienoic acid esters isolated by thin layer chromatography (TLC) were identified by using preparative radio gas chromatography. The double bond positions were determined as described by Privett and Nickell, z6 by partial reduction of the dienoic acids with hydrazine hydrate without double bond migration to give a mixture of monoenoic and saturated adds. The isolated monoenes were ozonized, and the ozonides were reduced to aldehyde and aldehyde esters by Lindlar's catalyst. 26 The chain lengths of the labeled aldehyde esters were identified by preparative gas chromatography using unlabeled carriers of aldehyde esters with chain lengths from C3 to C1~. is RESULTS AND D I S C U S S I O N
Desaturation of trans.18: l isomers
One of the desaturation products of trans-octadecenoates was cis, trans-octadecadi. enoates in which the cis double bond was introduced by the enzyme. The conversion rate *Present address: Department of Biochemistry, Medical Research Institute, 165-Tarik EI-Horeya, EI-Hadara, Alexandria, Egypt. IS!
152
R.T. Holman and M. M. Mahfouz
/r~n~ IB:l ~ c i s - g , lrans- 15~2
1
~ oa
,3 4 5 6 7 8 g 10 It 12 13 I~. 15 POSITION OF TRANS DOUBLE BOND FIG. 1. Rat~s of &9-desaturation of positionally isomeric trans-octadecenoic acids by rat liver microsomes. The product measured was the trans,cis-18:2.
of trans-octadecenoates to cis, trans-ortrans, cis-octadecadienoates was highest for A4, A6 and A13 acids ranging from 0,263 nmol/mg/min for A4 to 0.402 nmol/mg/min for A13. The conversion rates of A7, A11, A12 and AI4 acids ranged from 0.088 nmol/mg/min for A7 to 0.157 nmol/mg/min for A14. The trans-A8, A9 and A10 acids were not desaturated at measurable rates. The site of desaturation of all the trans 18:1 isomers was the 9-position, in agreement with the results of James ~2 and Pollard et al. 2~ The relative rates of these reactions are shown in Fig. 1. The cis double bond introduces a bend in the molecule which is clearly distinct from the shape of saturated acids. Acyl transferase discriminates between the normal cis isomers and the unnatural trans forms with respect to esterification into phospholipids and triglycerides; a chain containing trans double bond is transferred as if it were a saturated carbon chain. Trans-18:1 acids also resemble stearic acid rather than cis-18:l acids in their slow rates of cholesteryl ester formation by rat liver microsomes, a2 Because trans-18:l acids were found to be desaturated by 9-desaturase, it is not surprising to find that trans-A8, A9 and A10 acids were not desaturated, in agreement with Brenner 2 who found that all trans-linoleic and elaidic acids were not desaturated by rat liver microsomes in vitro, but this disagrees with the report that t9-18:1 was desaturated to c5.t9-18:2 acid when other substrates for this enzyme are lacking. 16 Cis, cis-Diene Products from trans-18: l Isomers Another desaturation product from trans-18:1 acids was identified as cis,cis-octadeca. dienoate. Trans-A4 and A5 acids gave the highest rates ofcis,cis-diene formation. After incubation of t5-18:1, the labeled cis, cis-diene ester recovered from the AgNOa TLC plate showed a gas liquid chromatographic (GLC) retention time identical to the cis,cis-18:2 ester. The free acids obtained from the labeled diene esters were separated by quantitative paper chromatography. ~4 The radioactivity was mainly located with the 14:0 before reduction and with 18:0 after reduction, indicating that it was 18:2 acid. Partial reduction and ozonolysis gave Cs and C9 aldesters. We concluded that t5-18:1 produced c5,c9-18:2 acid. Trans-5-18:l and t4-18:1 were each incubated and the conversion to cis, trans- and to cis, cis-18:2 was measured with time. In the case of t5-18:1, the c,t-18:2 was not detected except during the first 5 rain of incubation (about 3.5%) after which it decreased as the c,c-18:2 increased. For the t4-18:1, during the first 15 rain when the rate of desaturation is high, both c,t-18:2 and c,c-18:2 were detected. After 15 min, the c,t-18:2 decreased slightly and c,c-18:2 increased from 5 to 13.8% indicating that the c,t-18:2 produced during this period was converted to c,c-18:2. The rates of desaturation to cis, cis diene products are shown in Fig. 2. One possible pathway for formation of c,c-18:2 is t- 18:1 --* c-18:1 --* c,c-18: 2. A second possible pathway is t-18:1 -* c,t-18: 2 -~, c,c-18: 2. We found that cis 4-, 5-, 6- and 7-18:1 were not desaturated by rat liver microsomes ~7 and that for the isomers which are desaturated, the c,c-18:2 acids produced do not contain a new cis-9 double bond. These observations indicate that the first pathway is
Cis and trans-octadecenoic acids as precursors
153
1 ?' 3
4 ,5 6 7 8 9 I0 II 12 13 I~- 1,5 POSITION OF" TRANS DOUBLE BOND
FIG. 2. Rates of A9-desaturation and cis-trans isomerization of positionally isomeric transoctadecenoic acids to cis,cis-octadecadienoic acids by rat liver microsomcs.
inoperative, as shown in Fig. 3. The second pathway is confirmed by the presence of cis-9-double bond in all c,c-18:2 acids just as in the c,t-18:2 products. The inserted cis-double bond in each c,c-18:2 has the same position as the trans-double bond in the c,t-18:2 produced from the same substrate acid, indicating that c,t-18:2 is isomerized to yield c,c-18:2 acid without migration of double bond. The CoA esters of six of the t-18:l isomers were prepared and subjected to desaturation. Their rates of desaturation were compared to those of the free acids in the presence of CoA. In each case, the rates of desaturation were the same for the two substrates, and the ratio of cis, trans to cis,cis product was the same for the two substrates indicating that the activation of the isomeric t-18:l acids is not a rate-limiting step in the desaturase system, nor does the activation step influence the isomerization of trans- to cis-double bond. Desaturation of Cis-18:1 Isomers The cis-18:l isomers with the double bond at carbons 4 through 7 were not measurably desaturated. The maximum conversion to 18:2 was obtained for c8- and cg-isomers which gave 3.4 and 3.9~/~ respectively. The rate of desaturation of oleic acid to 6,9-18:2 (0.118 nmol/mg protein/min) is comparable to that obtained by Castuma et al. 6 The cis-18:1 isomers with the double bond at carbons 4, 5, 6 or 7 were not desaturated, and the 10- and 11-isomers were desaturated at low rates. The 8- and 9-isomers were desaturated at relatively higher rates. The c12-18:1 acid was not significantly desaturated by rat liver microsomes, s Our study shows that the position of the double bond in a cis-18:l acid controls which desaturase acts upon it, and that the several cis-18:l isomers are substrates for different desaturases. The c9-18:1 was desaturated by A6-desaturase, in agreement with the previous reports}' 2o. 2~ Acids which have different chain lengths and numbers of double bonds, but have the first double bond at 9-position from the carboxyl group, are desaturated at A6.1' 5.3o The c8-18:1 and c11-18:1 were desaturated by A5-desaturase as are other A8 acids ~ and All acids. ~' 6.3,~ Desaturation of the c11-18:1 isomer to 5,11-18:2 acid (not to 8,11-18:2) confirms the absence of A8-desaturase from the rat liver microsome) ~ The c10-18:1 isomer gave 1.5% conversion to 18:2 acid with 70% of the double bonds at AT,10- and 30% as the A5,10-18:2 acid. Bernert and
cL*X- 18:1
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~rons X-18:l
~%~,
ci$X~ci$9*18:~
~n~c/s9-18:2 FIG. 3. Metabolic pathway for desaturation-isomerization oftrans-18:l isomers.
R.T. Holman and M. M. Mahfouz
154
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c-18:1~ c,c-I~:~
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i -ioo ~
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Sprecher I found that the 10,13-20:2 acid was converted to 7,10'13-20:3 at a very low rate. The relative rates of desaturation are shown in Fig. 4. The most abundant cis-18:1 isomers in hydrogenated soybean oil were reported to be the c9-18:1 (76%) and cl 1-18:1 (10~/o),27 and those isomers which were found not to be desaturated by rat liver mierosomes (A4 to A7) were present in less than 1% of the total cis-18:1 acids. 27 The major cis-18: i acids present in hydrogenated oils are thus potentially metabolized to cis, cis-18:2 acids, which in turn may be converted to higher polyunsaturated acids and perhaps prostaglandin-like compounds. Our study shows that cis. and trans-18:l acids are desaturated differently by rat liver microsomes, whereas the trans-octadecenoic acids and saturated acids are desaturated similarly. This may be a function of the generally straight cylindrical configuration of the trans and saturated acids and the bent configuration of cis-unsaturated acids, as Polyunsaturated Acids
We should expect that all the c,t- and c,c-18:2 acids derived from t-18:1 acids and which have the first double b o n d at A9-position could be further desaturated by A6-desaturase and chain elongated to give all-cis or mixed isomers of 20:3 and 20:4. In fact, Pollard et al. 24 found that t13-18:1 acid gave c9,t13-18:2 which was desaturated again to yield c6,c9,t13-18:3. Thus, t-18:1 isomers present in dietary fats can be the precursors of polyunsaturated fatty acids (PUFA) of unusual structure, which in turn could be precursors of unnatural or unusual series of prostaglandin-like c o m p o u n d s having biological activities different from the c o m m o n ones. In Fig. 5, the principal pathways for formation of P U F A from trans-monoenoic acids are summarized. In each case, only those desaturations which occurred in vitro using rat liver microsomes are included as first steps in the sequences. Subsequent steps are predicted from previous knowledge of chain elongation and desaturation by liver microsomes. In this figure, the location of the trans double b o n d from the original t-18:1 isomer is indicated in boldface type. The trans isomers of 18:1 which occur in measurable amounts in partially hydrogenated fats range from A6 through A15, but the AS, A9 and 6-18:1 -* 6.9-18:2 --" 8,11-20:2 --' 5,8,11-20:3 7-18:1 -" 7,9-18:2 --'*9.11-20:2 ---"6,9, I 1-20:3 8-18:1 9-18:1 10-18:1 11-|8: | ~ 9.11-18:2 --~6.9.11-18:3 -* 8.11.13-20:3 ---,5.8.11.13-20:4 !~-18:1 --~9,12-18:2 --~6,9,12-18:3 --~8,11,14-20:3 --~ 5,8,11,14-20;4 13-18:1 --~9,13-18:2 ~ 6.9,13-18:3 --~8,11,15-20:3 --o 5,8,11,15-20:4 14-18:1 --*9.14-18:2 --~6.9,14-18:3 --*8,11,16-20:3 -* 5,8d 1J6-20:4 15-18:1 --~9.15-18:2--~6.9,15-18:3 ---~8,11,17-20:3 -* 5,8,11,17-20:4 Boldface numbers indicate positions of trans double bonds. FI6. 5. Probable formation of PUFA from trans-18:1 isomers
Cis and trans-octadecenoic acids as precursors
155
c8-18:1 ---~ 5,8-18:2 --~ 7,10-20:2 --, 4,7,10-20: 3 c9-18 : 1 ~ 6,9-18:2 --* 8,11-20:2 --~ 5,8,11-20:3 c10-18 : 1 --~ 7,10-18:2 --~ 9,12-20:2 --~ 6,9,12-20:3 c10-18 : 1 ~ 5,10-18:2 --~ 7,12-20:2 --* 4,7,12-20:3 c11-18:1 --~ 5,11-18:2 --~ 7,13-20:2 --~ 4,7,13-20:3 FIG. 6. Probable formation of PUFA from cis-18:1 isomers
A10 isomers are not desaturated. Thus, PUFA could be expected from 7 isomers. These could give rise to 7 isomers of 20:3 and 5 isomers of 20:4, most of which have unusual double bond positions and all of which contain a trans double bond. Because in the desaturation of t-18:1 isomers, the trans double bond is isomer/zeal to the corresponding cis double bond, all the positional isomers shown in Fig. 5 could also occur in ali-cis configuration. Among these, some are identical to endogenously synthesized PUFA and some are naturally occurring essential fatty acids (EFA). The former are 8,11-20:2 and 5,8,11-20:3 (Mead's acid) which can be derived from oleic acid and which increase significantly in EFA deficiency when endogenous PUFA are synthesized as substitutes for co6 acids. The EFA are 9,12-18:2, 6,9,12-18:3, 8,11,14-20:3 and 5,8,11,14-20:4, all essential fatty acids of the co6 family, in this case derived from t12-18:1 which is a significant component in partially hydrogenated oils. Although few c-18:1 isomers are desaturated and those only at low rates, the 8-, 9-, 10-, and ll-isomers could be expected to yield PUFA (Fig. 6). The c9-18:1 could be converted to 8,11-20:2 and 5,8,11-20:3, both characteristic of EFA deficiency. The 12-18:1, from which co6 acids could be derived, is poorly desaturated. The PUFA predicted from the desaturation of isomeric c- and t-18:1 isomers thus might yield isomers of PUFA including those characteristic of EFA deficiency, those pps__s_essing EFA activity, and those in which double bond positions are unusual or in which a trans bond occurs. The possible conversion of the latter to prostaglandin-like products through oxidative metabolism can also be predicted. However, the rates at which these are formed and the biological activities of the products cannot yet be predicted. Thus the physiological consequences of consumption of partially hydrogenated fats cannot be predicted, but it would appear likely that the trans fatty acids in such fats could be the precursors of new, unusual substances having perhaps unanticipated biological acti,~ities. , Acknowledgement--This study was supported in part by Program Project Grant HL 08214 Program Project Branch, Extramural Programs, National Heart, Lung, and Blood Institute; Grant HL 21513 from the National Institutes of Health; and The Hormel Foundation.
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