ANTIOXIDANTS AND THE AUTOXIDATION OF FATS*

BY H. A. MATTILL

(From the Biochemical Laboratory, State University of Iowa, Iowa City)

(Received for publication, October 21, 1930)

The control of autoxidation reactions by means of antioxidants and prooxidants (promoters) is increasingly useful in the industries. Instances are too numerous to mention. Investigations in the oil and rubber industries, in particular, have revealed a great variety of substances that may be employed to modify reaction velocity or to prevent deterioration of the product through oxida- tion or polymerization. Moureu and Dufraisse (1) were the first to propose an inclusive theory which should account for the effec- tiveness of almost infinitesimally small amounts of material in retarding or accelerating the processes of oxidation. In a recent review of autoxidation, Milas (2) has proposed an electronic explanation for these chain reactions in which molecular oxygen, autoxidizable materials, accelerators, and inhibitors take part. These reactions are characterized by a latent or induction period followed by active oxidation at the gradually increasing rate of an autocatalytic reaction.

The observations reported in this paper were made with the hope of securing some understanding of the nature and role of antioxygenic substances in edible fats. The production of sterility in rats by rearing them on synthetic rations containing butter fat and lard or cod liver oil (3) or on milk powder diets containing these unsaturated animal fats (4) was presumptive evidence that butter fat contained little vitamin E. The incorrectness of this idea was demonstrated by the reproductive adequacy of milk powder rations not containing lard or cod liver oil, by the destruc-

* Reported in part before The Thirteenth International Physiological Congress, at Boston, August, 1929 (Mattill, H. A., and Mattill, H. I., Am. J. Physiol., 90, 447 (1929)).

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142 Antioxygenic Substances of Fats

tive effect of these and certain other fats upon vitamin E (5), and by the antioxygenic effect of wheat germ oil upon the autoxidation of these fats (6). The case for autoxidative destruction was further strengthened by the relation of oxidation to the vitamin E content of milk powders (7) and by observations that iron salts shortened the induction period (6) and hastened the disappear- ance of vitamins A and E from rations which originally possessed them (8-11). Additional evidence comes from the fair degree of parallelism between the ease of autoxidation of fat mixtures and the reproductive performance of rats on diets containing them (12).

Aside from its content of vitamin E, therefore, wheat germ oil possesses a constituent which has antioxygenic power. Corn oil also contains such a substance (13) and the superior keeping qualities of vegetable oils as compared with animal fats of equal or even less unsaturation may be ascribed to the presence of these oxidation inhibitors. They are found in the non-saponifiable portion of the oils (13) and work on their isolation and further st,udy is in progress.

Certain hydroxyl derivatives of aromatic compounds have long been known as inhibitors of oxidation and inasmuch as the sterols are hydroxy aromatic derivatives some information as to the nature and structure of the antioxidizing sterols should be gained from a study of the behavior of hydroxy aromatic compounds of known constitution. The relation of inhibitor to autoxidant is complex and in these studies the latter has been limited to lard, cod liver oil, or a mixture of them; the fact that a given substance may be useful as an antioxidant in one connection is not a guarantee of its universal applicability.

EXPERIMENTAL

Since the effect of an antioxidant is confined largely to the latent or induction period, before active absorption of oxygen has begun, some means of measuring this interval, without reference to the further progress of oxygen absorption, sufficed for these tests. The apparatus used was a slight modification of that described by Greenbank and Holm (14), for determining the thermal oxidation induction period. Erlenmeyer flasks containing 5 or 10 gm. of the fat were immersed in a calcium chloride solution maintained at 70”. The flasks were connected to open manometers through a

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H. A. Mattill

mercury seal, the air in the flasks being displaced by oxygen in a slow stream through a side tube. Platinum contacts in the manom- eters were connected to signal magnets writing on a slow kymo- graph, the upper of the two platinum wires in a manometer being just above the surface of the mercury. By means of half-hourly contacts from a clock the first diminution in volume due to ab- sorption of oxygen was thus recorded. The further progress of oxygen absorption could be observed in the continued rise of the mercury column, but as Milas has pointed out (15) too many variables are involved to make this a satisfactory quantitative method. Some of his objections may apply even to the measure- ment of the induction period, notably the possibility of the forma- Con of a surface film, since the mixture was not stirred. However in these fats polymerization is unlikely at 70”. The oxygen was not washed nor were any measures taken to remove water from the fats; moreuniform results could be expected if the fats were not dehydrated since water retards autoxidation.

As indicated elsewhere (12) there is a great and almost myste- rious variability in the induction periods of different samples of the same fat, due probably to differences in origin and conditions of manufacture and storage, and it seemed necessary to carry a blank determination with each trial of antioxidants. With proper attention to cleanliness of glassware and uniformity of procedure it was possible to obtain satisfactory repetition of the blank determinations. The induction period of most samples of cod liver oil seemed too short to be accurate under the conditions of measurement; that of good lard is too long for convenience and changes in atmospheric pressure require too frequent correction on the manometers. Such changes have often been disturbing even in the shorter periods. The most satisfactory fat mixture seemed to be 5 gm. of lard and 10 drops (0.32 gm.) of cod liver oil and this mixture has the further advantage of being one of those commonly used in rat feeding experiments. For the usual lard and cod liver oil samples the blank induction periods varied from 6 to 10 hours. Doubtless other methods are available for measuring the induction period of fat oxidation. The successful use of methylene blue (16) in photoelectric work suggests its applicability in thermal studies. It is not claimed that the present procedure has the merits of a rigorous quantitative method. With

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144 Antioxygenic Substances of Fats

certain modifications its accuracy can be increased but for pur- poses of orientation it is simple and reliable. Reasonable agree- ment between two to five and often more tests was always secured before the results were accepted.

The substances to be tested as antioxidants were used in the quantity of 0.02 per cent of the fat mixture. A few of them were sufficiently insoluble in fat that even this small amount could not be taken up completely. The induction period (in hours) with antioxidant divided by the induction period of the blank is designated as the antioxygenic index and Table I contains this index for the substances that have been examined. A figure of 0.8 to 1.3 for this index has been interpreted to mean that the substance has no antioxygenic effect, that is, the difference in induction periods of the fat with and without the added material is too small to be significant. These inactive compounds have been listed separately from the active ones.

Only a few of the substances examined possess the capacity to retard or inhibit the autoxidation of fat. Phenol was ineffective even when 2 per cent was used; m-cresol was likewise inactive and the other cresols and thymol were only very slightly active. Of the dihydroxy derivatives of benzene the ortho and para com- pounds were very powerful, the meta extremely feeble. When the ortho compound contained OCHs in place of one of the hydroxyls (guaiacol) it was only slightly better than the meta compound. Of the trihydroxy benzenes examined, the symmetrical phloro- glucin acted feebly, pyrogallol powerfully. That the hydroxyl group is responsible for antioxygenic action (13) was verified by the inertness of hydroquinone diacetate. Since p-oxybenzoic and salicylic acids were both inert, the two hydroxyl groups must be directly bound to the ring. The fully hydroxylated benzol com- pound, inosite, was inactive.

Of the substances containing two or more benzene rings, cu-naph- thol was more than ten times as effective as the p derivative; fi-naphthoquinone was very efficient while the a! derivative was inert.

Two very recent papers by Tanaka and Nakamura (17) contain data on the iodine number of linseed oil after exposure to oxygen with and without antioxidants. They indicate the supe- riority of (Y- to p-naphthol and the order of effectiveness of the

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H. A. Mattill 145

di- and triphenols is essentially that found in this study. Moureu and Dufraisse (18) found phenol and resorcinol to have very feeble action in connection with a great variety of autoxidizable substances other than unsaturated fatty acids, while pyrocatechol, hydroquinone, and pyrogallol were very effective. Others (19), still earlier, had made like observations. A number of more recent papers (20-25) contain similar information, most of it in agreement with the facts outlined in Table I. Variations in the relative amounts of antioxidant used may explain some of the apparent contradictions. Moureu and Dufraisse (26) state that anti- oxidation is proportional to the amount of antioxidant present but the proportionality may not be uniform for all antioxidants.

Although the relative activity of various phenols as antioxidants has thus been generally known for some time, apparently no one has yet pointed out that this activity is in some manner asso- ciated with the ortho, para configuration and is not possessed by compounds with the meta configuration or its equiva1ent.r The reaction chains which are started when an unsaturated fat takes on oxygen can be broken by the former but not by the latter type of phenols. The course of oxidation, through “moloxides,” peroxides, and ozonides (27) is interrupted by the preferential oxidation of minute amounts of the more readily oxidizable phenols but this explanation becomes less satisfying when one seeks farther.

Since to the physical chemist “it seems at present impossible to give very precise definition to the ideas which we express in the term unsaturation” (28) and since the structure of the benzene ring is still one of the live issues among organic chemists, an ex- tended attempt to explain the observations made would likely be profitless, if not out of place, even if the writer were competent to make it. A few comments may be permissible. Most theories of autoxidation assume that a very few of the molecules of the autoxidant are in a much more reactive form than the rest. In

r Seyewetz and Sisley (Bull. Sot. chim., 31, 672 (1922)) in calling atten- tion to the origin of the term antioxidant which was first proposed by Lumiere and Seyewetz (19), also point out that they (19) associated anti- oxygenic properties with the capacity to act as a photographic developer. The commonly used developers are easily oxidizable ortho and para de- rivatives.

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the recent valuable discussion mentioned above (2) Milas has made the assumption that “in all auto-oxidations . . . the atoms to which the oxygen molecule initially adds may be regarded as making definite contributions of two electrons to it,” forming metastable or “dative” peroxides of high instability and energy content. Furthermore, the possession of unshared or “exposed” electrons, is also a characteristic of inhibitors and “if an inhibitor collided with the dative peroxide, all the excess energy of the latter would be completely absorbed by the molec- ular valence electrons of the former, thus the initiation of new reaction chains is prevented. . .” In his study of electron displacement in the benzene ring Lucas (29), employing the Pauling structure of benzene and its derivatives, has demonstrated that the introduction of a positive group (like OH) makes the p-hydrogen most negative, o- next, and m- least negative. Since phenol, as shown above, is not an antioxidant, we may assume that the electrons even though displaced away from the ring have not been displaced far enough. Lucas does not discuss the effect of the introduction of a second (OH) group in the ortho or para position but this should increase the electron displacement still more, while the influence of such a group in the meta position would tend to be dissipated. If the theory of electron displace- ment in the ring contains the explanation of antioxygenic activity it should be possible to predict the effectiveness of antioxidants (various OH, NHS, Cl, and alkyl derivatives) and conversely the antioxygenic capacity of compounds might throw some light on their electronic structure.

The antioxygenic capacities of CY- and p-naphthol indicate that the former has the character of an ortho compound, the latter that of a meta, a fact which their differences in reactivity have long indicated. A recent excellent review by Obermiller (30) sum- marizes the varied evidence. It is interesting that oc-naphthol is so effective an antioxidant although containing only one hydroxyl group. The introduction of a methyl group into phenol in the ortho position (o-cresol) increased the antioxygenic capacity only very slightly; the presence of another benzene ring joined as it is in this position is much more effective.

The behavior of the quinones is not to be explained at present; it is surprizing that any of them should have antioxygenic activity.

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H. A. Mattill 147

Nor is it evident why quinone and or-naphthoquinone should act so differently. A peculia.rity in the action of quinone should be mentioned. The rate of oxygen absorption, once oxidation has begun, is ordinarily the same, whether an inhibitor is present or not; Yamaguchi (31) has recently reported a similar observation. In the case of quinone a slow absorption of oxygen proceeded for some time before the autocatalytic character of the reaction was manifested. If this behavior indicates the presence of traces of hydroquinone in equilibrium with quinone a similar condition does not exist in the naphthol derivative. The recognized stability of anthraquinone and its derivatives receives further evidence in the lack of antioxygenic activity.

Gossypol and its derivatives (32) were of interest because of the toxic action and the presence of six hydroxyl groups. It is again evident that the OH group is concerned with antioxygenic ca- pacity. In view of the relatively harmless character of hydro- quinone when ingested, the toxicit,y of gossypol can hardly be related to its feeble activity as an inhibitor of oxidation.

The various sterol preparations of animal origin and sitosterol from three different plant sources were all of them inactive. This was to be expected since there is only one hydroxyl, although the presence of some other sufficiently positive group might serve in its place. The antioxygenic sterols of vegetable fats are not precipitable by digitonin and methods for their separation are greatly needed. Estill and McCollum (33) obtained from cod liver oil a compound with LiCl which was evidently antioxygenic since it prevented “salt ophthalmia” in rats on diets containing ferrous sulfate. The fact that a substance with this property could be obtained from a readily autoxidizable animal fat is sig- nificant. Perhaps oxidation in the animal body is in part gov- erned by the presence of traces of inhibiting substances of this kind. Epinephrine is an o-dihydroxy compound, thyroxine is allied to a p-dihydroxy compound.

In vitro experiments (34) have indicated that carotene is a prooxidant, and its greater effectiveness as vitamin A when ad- ministered in peanut oil as compared with petroleum oil (35) or ethyl oleate (36, 37) is very reasonably explained by the presence of antioxidants in vegetable oils. When this natural protection is not afforded some other inhibitor must be present if the vitamin is

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148 Antioxygenic Substances of Fats

to survive even a short period. In the preparation and manipula- tion of other readily oxidizable materials like phospholipids the addition of traces of an antioxidant would greatly retard un- desirable oxidative changes.

The production of prooxidants by the irradiation of fats with ultra-violet light is well recognized. Irradiation of the mixed sterols of corn oil (13) produced traces of peroxide substance and

TABLE I

A&oxygenic Index* of Substances toward Lard and Cod Liver Oil

Ineffective substances, index of 0.8-1.3 p-Cresol, 1.4-l .7 Phenol, m-cresol o-Cresol, 1.2-l .4 p-Oxybenzoic acid, p-oxybenzaldehyde Thymol, 1.2-1.7 Salicylic acid, salol Hydroquinone, 120 Inosite, t hydroquinone diacetatet Pyrocatechol, >55 Phenolphthalein, phthalic acid Guaiacol, 1.4-1.8 or-Naphthoquinonet Resorcinol, 1.3-1.7 Anthraquinone, 1,5-dihydroxy anthra- Orcinol, 3-5

quinone,t alizarin, t purpurin Quinone, t 8-10 Hexacetyl gossypol,$ hexacetyl apogos- Pyrogallol, >64

SYPOl§ Phloroglucinol, 2.8-3.3 Cholesterol, 11 ergosterol,t, Ij sitosterols, 1 a-Naphthol, 31

acterol, oscodal, ** non-saponifiable P-Naphthol, 1.3-1.9 matter of cod liver oil (specially pre- @-Naphthoquinone, t 23-27 pared) Gossypol, $ 3-5

*This index is the ratio of the induction period (in hours) with anti- oxidant to the induction period of the fat with no addition.

t Eastman Kodak Company. $ National Aniline and Chemical Company, Incorporated. $ Kindness of Dr. E. P. Clark. Ij Both before and after irradiation. y Prepared from corn, wheat, and lettuce. ** Kindness of Dr. Dubin.

recent literature contains several suggestions that sterols are oxidative catalysts especially after irradiation, but in the quantity (0.02 per cent) used in these tests, cholesterol and ergosterol behaved alike both before and after exposure to ultra-viglet light.

The probability that the functions of fat-soluble accessories are not comprised within the commonly known effects of vitamins A, D, and E or that they are not yet properly allocated, is suggested by several observations. Most recent of these (38) is the appear-

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H. A. Mattill 149

ante of muscular dystrophy in rabbits on vitamin E-free diets. The striking discovery that vitamin A rather than vitamin E is concerned with sterility in the male rat (39) we have recently confirmed. The growth-promoting function of vitamin E (40,41) has not been explained nor has the effectiveness of butter fat in the cure of “black tongue” in dogs (42). Progress in the solution of these problems depends on the successful segregation of the factors concerned, among them probably pro- and antioxidants.

SUMMARY

To secure information on the chemical nature of the antioxygenic substances that are found in natural oils and that prevent the autoxidative destruction of fat-soluble vitamins a series of hydroxy aromatic compounds was tested for their capacity, when used in a quantity of 0.02 per cent, to prolong the thermal oxidation induc- tion period of a standard mixture of lard and cod liver oil. The observations indicate that antioxygenic capacity of phenols re- sides in two hydroxyl groups in the ortho or para configuration; when these are in the meta position the compound is inactive. The hydroxyls are ineffective unless attached directly to the ring; the fully hydroxylated inosite is inactive. In the naphthols one hydroxyl group is sufficient and in keeping with its accepted behavior, cY-naphthol has the character of an ortho compound and is much more effective, as an antioxidant, than p-naphthol; quinone is effective and P-naphthoquinone is even more so but the 01 form is entirely inactive.

The relation of these facts to the more recent theories of the electronic structure of the benzene ring and autoxidation is briefly discussed and it is suggested that in the preparation and manipulation of easily autoxidizable substances, the presence of traces of antioxidant will prevent undesirable oxidative changes.

A number of sterols of animal origin and sitosterol from wheat, corn, and lettuce were all inactive.

The existence of pro- and antioxygenic substances among the non-saponifiable constituents of natural fats and oils suggests that some of these may be concerned with the physiological action of the fat-soluble vitamins, and methods for their segregation from the sterols are under investigation.

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H. A. MattillAUTOXIDATION OF FATSANTIOXIDANTS AND THE

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