Decomposition of linoleate hydroperoxides: Precursors of oxidative dimers

Chem. Phys. Lipids 4 (1970) 197-202 © North-Holland Publ. Co., Amsterdam.

DECOMPOSITION

OF LINOLEATE HYDROPEROXIDES:

P R E C U R S O R S OF O X I D A T I V E D I M E R S * T. L. MOUNTS, D. J. McWEENY,* C. D. EVANS and H. J. DUTTON Northern Regional Research Laboratory,* Peoria, Illinois 61604, U.S.A.

When hydroperoxides are thermally decomposed in the presence of unsaturated fatty acids, dimers are formed in amounts equivalent to the hydroperoxide present. The origin of these dimers has not been shown by any definitive experiment. Radioactively (14C) labeled materials now have been used to determine whether (l) two molecules of hydroperoxide react, (2) a hydroperoxide and an unoxidized fatty acid molecule form the dimer, or (3) two molecules of unoxidized fatty acid dimerize with hydroperoxide catalysis. Methyl linoleate hydroperoxide and unoxidized methyl linoleate were reacted in experiments, first with the 14C-labeled hydroperoxide and then with 14C-labeled ester. The radiotracer experiments indicate that the dimer is formed from a molecule of the hydroperoxide and a molecule of the fatty ester.

Introduction The free r a d i c a l a t t a c k o f oxygen on u n s a t u r a t e d fatty acids d u r i n g fat a u t o x i d a t i o n p r o d u c e s c o n j u g a t e d fatty h y d r o p e r o x i d e s l - 4 ) . Several investigators 5,6) have studied the thermal d e c o m p o s i t i o n o f these h y d r o p e r o x i d e s in the presence o f u n o x i d i z e d fatty acids and r e p o r t the f o r m a t i o n o f a variety o f scission p r o d u c t s a n d h i g h - m o l e c u l a r - w e i g h t m a t e r i a l principally d i m e r i c in nature. These dimers have been suggested as p r o d u c t s which m a y significantly c o n t r i b u t e to flavor reversion o f s o y b e a n oil 7). W h e n h y d r o p e r o x i d e s are t h e r m a l l y d e c o m p o s e d in the presence o f u n s a t u r a t e d fatty esters, the a m o u n t o f d i m e r f o r m e d is a p p r o x i m a t e l y equivalent to the a m o u n t o f h y d r o p e r o x i d e d e c o m p o s e d 5). This o b s e r v a t i o n could indicate t h a t the d i m e r is f o r m e d by the interaction o f two h y d r o p e r o x i d e molecules, Such a c o n c e p t is in line with the existing data. But when h y d r o p e r o x i d e s d e c o m p o s e t h e r m a l l y at low c o n c e n t r a t i o n in a reactive m e d i u m , like the methyl esters o f linoleic acid, then the possibility o f h y d r o p e r o x i d e - m e t h y l linoleate i n t e r a c t i o n should be e x a m i n e d closely when c o n s i d e r i n g r e a c t i o n mechanisms. * Presented at International Food Technologists Meeting, Philadelphia, Pennsylvania, May 19-23, 1968. * Present address: Ministry of Agriculture, Fisheries and Food, Norwich, England. Work conducted in part as Kellogg Foundation Fellow. *t This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture. 197

198

T . L . M O U N T S ET AL.

To date there has been no evidence that hydroperoxide-methyl linoleate interaction is significant; use of radioactive tracers provides a relatively simply way of assessing the extent to which this mechanism is involved in dimer formation. Methods

Two complementary isotopic experiments were performed, each in duplicate. In the first, methyl linoleate was the labeled constituent; in the second, the radioactive constituent was the methyl linoleate hydroperoxide (MLHP). When the specific activity of the starting material is compared with that of the dimer produced during the reaction, the proportion of that constituent which is incorporated into the dimer can be determined.

Preparation and isolation of methyl linoleate hydroperoxide Unlabeled or a4C-labeled methyl linoleate (carboxy labeled) was oxidized by bubbling oxygen through it until the peroxide content estimated iodometrically by the method of Fugger et al. 8) reached 600 to 800 meq/g. M L H P was separated from the unreacted ester by the solvent-extraction procedure of Zilch et al. 9) and finally purified by chromatography on silicic acidmethanol column with 2~o methanol in benzene followed by diethyl ether as eluting solvents1°). The elution pattern of M L H P was determined by weighing the residual material after evaporation of the solvent from frozen eluate fractions in vacuo or by liquid scintillation counting of 0.1-ml aliquots from eluate fractions in 10 ml of toluene scintillation solvent (4 g 2,5-diphenyloxazole (PPO)/liter).

Dimerization procedure About 5~o of M L H P with methyl linoleate was decomposed by heating at 210°C for 10 rain. Nitrogen was bubbled through the reaction mixture before and during heating.

Separation of dimer fraction The dimerized reaction mixture was saponified with alcoholic potassium hydroxide11), and after acidification with hydrochloric acid and dilution with water, the acidic components were extracted twice with petroleum ether. Extracts were washed twice with distilled water, dried over anhydrous sodium sulfate, and evaporated at room temperature in a stream of nitrogen. The residue was dissolved in benzene and chromatographed on a silicic acid-methanol column by the method of Evans et al.12). Fractions of approximately 10 ml volume were collected. Aliquots from the chromatographic fractions were taken for microtitra-

PRECURSORS OF OXIDATIVE DIMERS

100II

Monomer

I 5o

199

MeLo-1-14C+ MeLoHP

1000

i

~,~

500 ~.

10

N

. . . . . . . . .

0.3~ ~ 02k Ii

I ,-L..,'

\~""'--

L I00

Fig. 1.

100 ~

.._

/

Oi~mer X

Polar Material-J50 ~

................ i J 200 300 Volume Effluent

l 400

Chromatographic separation of oxidative dimer acids from methyl linoleate-l-14Cmethyl linoleate hydroperoxide. Radioactivity . . . . . Titration . . . . . . . 10

[

1000

i Monomer

MeLo+ MeLo-1-14CHP

|1 || II I| || II || I|

1 I

B

o

500

--5 E

Dimer ~

,

100

Fig. 2.

200 300. VolumeEffluent

Polar Material

400

G,I

500

Chromatographic separation of oxidative dimer acids from methyl linoleate-methyl linoleate-l-14C hydroperoxide. Radioactivity . Titration . . . . . . .

tion (aliquots 0.1 to 2.0 ml volume) a n d for 14C assay by liquid scintillation c o u n t i n g (aliquots 0.01 to 1.0 ml volume). The elution curves p l o t t e d f r o m t i t r a t i o n d e t e r m i n a t i o n s served as the basis for c o m b i n i n g fractions to isolate the reaction p r o d u c t s .

200

T.L. MOUNTS ET AL.

Specific activity determinations Combined fractions were evaporated and the weight was determined. After the residue was dissolved in a known volume of petroleum ether, the total activity was measured by liquid scintillation counting. Milligram specific activity is determined by dividing the total activity by the weight of solids. Results

Elution curves obtained by plotting the radioactivity and titration data

TABLE 1

Analysis of starting sample and products formed during hydroperoxide decomposition Reactants (by weight) Sample

Monomer

Dimer

Polar material

(%)

(%)

(%)

(Vo)

93.5

6.5

90.7

7.6

1.7

94.7

5.3

90.5

7.4

2.1

Ester

Hydroperoxide

(%) Methyl linoleate-14C + methyl linoleate hydroperoxide Methyl linoleate + methyl linoleate hydroperoxide-14C

Products (by titration)

TABLE 2 Radiochemical analysis of starting sample and products formed during hydroperoxide decomposition Labeled reactant Sample Specific Relative* activity (/~c/mg) activity Methyl linoleate-14C ÷ methyl linoleate hydroperoxide Methyl linoleate + methyl linoleate hydroperoxide-14C

Reaction products Monomer .............. Specific Relative* activity (ltc/mg) activity

Dimer Specific Relative* activity (/~c/mg) activity

0.063

1.00

0.0655

1.04

0.0347

0.53

0.127

1.00

0.0045

0.04

0.0624

0.49

* Relative activity -- specific activity of product/specific activity reactant.

PRECURSORS OF OXIDATIVE DIMERS

201

from one experiment of each type are presented in figs. 1 and 2. In each experiment a small amount of polar material formed. Composition analysis of the reactants (by weight) and products (by titration) are given in table 1. Shown in table 2 is the specific activity of the labeled constituent and the reaction products. These results were confirmed in duplicate experiments.

Discussion Dimer formation by hydroperoxide-linoleate interaction is indicated in figs. 1 and 2; radioactive labelling of either reactant leads to incorporation of radioactivity into the dimeric product. If the dimer were formed only from the hydroperoxide or only from the fatty ester, one or the other of the two different experiments would yield dimer having no 14C activity. The extent of ester-hydroperoxide interaction can be determined by considering the specific activities of the dimers formed. The ratio of the dimer specific activity to the specific activity of the starting a4C-labeled compound is a measure of incorporation of that constituent into the dimer. Expressed as relative activity in table 2, the ratio was 0.5 in each experiment, within experimental error. The dimer was, therefore, formed from one molecule of hydroperoxide and one molecule of fatty ester. Williamson 6) proposed that the dimers formed during the thermal decomposition of M L H P in the presence of methyl linoleate arise from the union of two ester radicals. He postulated that the hydroperoxide acted as initiator of the free radical reaction. Later, Frankel et al. 5) suggested that only hydroperoxides are involved since dimers formed during thermal decomposition of isolated hydroperoxides were similar to those formed during decomposition of hydroperoxides in mixtures with fatty esters. Radioactive tracers, as used here, have provided clear, straightforward, and conclusive evidence that in the latter reaction the dimer is formed from the interaction of both the ester and the hydroperoxide.

Summary Methyl linoleate hydroperoxide was prepared both as 14C-labeled and unlabeled compounds. The 14C-labeled hydroperoxide was decomposed (210 °C, 10 min) with unlabeled methyl linoleate; the unlabeled hydroperoxide was likewise decomposed with ~4C-labeled methyl linoleate. Oxidative dimers formed during this reaction were isolated by liquid-liquid partition chromatography and specific activities determined. The specific activity of the dimer, formed when the hydroperoxide was the labeled constituent, was one-half that of the hydroperoxide. When the ester was the labeled constituent, the specific activity of the dimer was one-half that of the ester.

202

T . L . M O U N T S ET AL.

R a d i o c h e m i c a l a n a l y s i s i n d i c a t e t h a t t h e d i m e r is f o r m e d f r o m o n e m o l e c u l e of ester and one molecule of hydroperoxide.

References 1) S. Bergstrom, Arkiv Kemi 21A (1945) 1 2) J. A. Cannon, K. T. Zilch, S. C. Burket and H. J. Dutton, J. Amer. Oil Chem. Soc. 29 (1952) 447 3) O. S. Privett, W. O. Lundberg, N. A. Kahn, W. E. Tolberg and D. H. Wheeler, J. Amer. Oil Chem. Soc. 30 (1953) 61 4) H. H. Sephton and D. A. Sutton, J. Amer. Oil Chem. Soc. 33 (1956) 263 5) E. N. Frankel, C. D. Evans and J. C. Cowan, J. Amer. Oil Chem. Soc. 37 (1960) 418 6) L. Williamson, J. Appl. Chem. (London) 3 (1953) 301 7) C. D. Evans, E. N. Frankel, P. M. Cooney and H. A. Moser, J. Amer. Oil Chem. Soc. 37 (1960) 452 8) J. Fugger, J. A. Cannon, K. T. Zilch and H. J. Dutton, J. Amer. Oil Chem. Soc. 28 (1951) 285 9) K.T. Zilch, H. J. Dutton and J. C. Cowan, J. Amer. Oil Chem. Soc. 29 (1952) 244 10) E. N. Frankel, C. D. Evans, D. G. McConnell and E. P. Jones, J. Amer. Oil Chem. Soc. 38 (1961) 134 11) Official Method Cd 3-25, American Oil Chemists' Society, Chicago, Illinois 12) C. D. Evans, D. G. McConnell, E. N. Frankel and J. C. Cowan, J. Amer. Oil Chem. Soc. 42 (1965) 764