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(3) Bitting, A. W.,U. S . Dept. Agr., B U T . Chem. Bull. 196, ( 191 6). (4) Bitting, K. G., K’atl. Canners’ Assoc., Bull. 11 (1917). (5) Culpepper, C. W., and Moon, H . A , , U. S. Dept. Agr.. Tech. Bull. 462, 1-23 (1935). (6) Diehl, H . C., Pentser, W. T., Berry, J. A, and Asbury, C. E., Western Canner and Packer, 26, No. 5, 31-33; No. 6, 39-41; NO. 7, 33-35; NO. 8 , 43-44 (1934). (7) Fellers, C. R., Young, R. E., Isham, P. D., and Clague, J. A., Proc. Am. SOC.Hort. Sci., 31, Suppl. 145-51 (1934); Mass. Agr. Expt. Sta., Bull. 305 (1934). (8) Fyler, H. M., and Manchesian, J. T., Hilgardia, 11, 295-314 (1938). (9) Joslyn, M. A,, and Marsh, G. L., Fruit Products J . , 11, 327-31 (1932). (10) Joslyn, M. A . , and Marsh, G. L., Western Canner and Packer, 30, NO.5, 21-22; NO.7, 35-37; NO.8 , 37-40 (1938).
(17) (18) (19) (20) (21)
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Joslyn, M. A. et al., IND.ENG.C H E Y . , 28, 595 (1936): 30, 1068 (1938); 31, 751 (1939). Magoon, C. A., and Culpepper, C. W., U. S. Dept. -\gr., Bull. 1265, 1-47 (1924). Morse. F. W., Mass. Agr. Expt. Sta., BdZ. 171 (1916). Ibid., 172, 297-307 (1917). Pentaer, W.T., Perry, R. L., Hanna, G. C., Wiant, J . S., a n a ilsbury, C. E . , Calif. Agr. Expt. Sta., Bull. 600, 1-46 (1936). Stewart, E . D., Food I n d . , 1. 705-10 (1928). Thornton, N. C., Contrib. Boyce Thompson Inut., 8, 2 5 4 0 (1936); 9, 1 3 7 4 8 (1937). Tressler, D. K., and Evers, C. F.. “Freezing Preservation of Fruits, Fruit Juices and Vegetables”, pp. 236-7, New York, -4vi Pub. Co., 1936. Wassin, G. F., S. Dak. Agr. Expt. Sta., Bull. 261 (1931). Wiegand, E. H., Oreg. Agr. Expt Sta., Circ. 116, 1-12 (1936). Working, E. B., Aria. Agr. Expt. S t a . , Tech. Bull. 5,87-124(1924).
Vitamin A Destruction in Fish Liver Oils RELATION TO E. J. SIMONS, L. 0. BUXTON, .4ND H. B. COLRlAN Nopco Vitamin Laboratories, Harrison, N. J.
E
DIBLE oils and fats develop rancidity on storage. The most common type of rancidity is due to the action
of air on the unsaturated components of the fat, and its rate of development varies widely with the conditions of storage and the type of oil or fat. I n general, the highly unsaturated fats are most susceptible to atmospheric oxidation. Rancid and oxidized fats have disagreeable flavors and odors and decreased nutritive value. This is particularly true of fish liver oils and is very important when these oils are used for their vitamin content. Fish liver oils are the best natural source of vitamins A and D, and are extensively used to supply these essentials. Thorough studies on the stability of these products are needed. Since vitamin D has been found more stable to atmospheric oxidation than vitamin A, most of the work has been devoted to studies of the stability of vitamin A. Wokes and Willimott (22), Jones (8),Huston et al. (T), Evers (a), Norris and Church ( I C ) , Marcus (IS),Dann (I), MacWalter ( l a ) , Holmes et al. (6),and Fraps and Kemmerer (4) reported wide variations in the stability of vitamin A under different conditions; Fridericia (6) and Powick (15) showed that rancid fats destroy vitamin A and were the first to suggest that the destruction might be due to organic peroxides. Rosenheim and Webster (17) found peroxides in samples of cod liver oil when most of the vitamin A had disappeared. Lease et al. (IO) observed that, if oxidized fat and vitamin A were fed separately but a t the same time, the combination resulted in lower storage of vitamin A in the liver, and they suggested that the preformed peroxides in the fat destroy the vitamin A between the time of feeding and storage in the liver. Whipple (21) reported a drop in vitamin A potency of cod liver oils as the peroxide value increased, and Lowen et al. (11) found that vitamin A in halibut and salmon oils was rapidly destroyed after the termination of the induction
PEROXIDE FORMATION period as judged by the peroxide value. While our work was in progress, Smith (19) reported that vitamin A dissolved in rancid fats containing peroxides was destroyed in the absence of air a t a rate approximately proportional to the peroxide concentration. From the previous work it is difficult to compare the effect of rancidity on the vitamin A content of the various oils since the experimental conditions employed varied widely, and only a limited number of oils was studied. This work was undertaken to compare the oxidative changes of several oils when stored under like conditions.
Materials and Procedure High-grade commercial fish liver oils were selected from large lots so that the samples could be considered representative of the species. The content of free fatty acids was below one per cent in all cases, and the oils were substantially free of moisture and foreign materials. The samples of the U. S. P. reference oil were obtained from the usual source. Several chemical tests have been proposed for measuring oxidative changes in fats. Since the estimation of peroxides is one of the most sensitive and widely used methods (9), it was chosen as the criterion for evaluating the state of oxidation of the oil. Under the usual conditions of storage, oxidation of the oils would have been too slow to complete the tests within a reasonable time; therefore accelerated tests were run. High temperature, light, and catalysts accelerate the oxidation of oils, though these agents were avoided so as t o make the tests more comparable with usual storage conditions. After several trials the following method of exposure during storage was adopted since it satisfied the above conditions and gave reproducible results:
MAY, 1940
INDUSTRIAL AND ENGINEERING CHEMISTRY
Tw-o milliliters of oil were carefully measured into several open vials, 23 mm. in diameter and 27 mm. in height, which had been thoroughly cleaned just prior to use. The samples were set aside in the dark, either in a dust-free cabinetoat room temperature or in a constant-temperature oven at 34.5 * 0.5" C. Care was taken to avoid smearing of oil on the walls of the vials. At recorded intervals three vials of the same lot of oil were removed, and individual determinations were immediately made on the peroxide value and the vitamin A content. Every value was therefore determined in triplicate, and the mean used for graphical representation.
0
tm
/oo
3w
400
HOURS AT 34.5'C.
OF FISHLIVEROILS FIGCRE 1. PEROXIDATIOX
Curve 1 2
3 4
5 6
ti
Oil Swordfish liver
Pollack liver Pollack liver Halibut liver Cod liver U. S. P. reference Halibut liver Dogfish (shark) liver
Vitamin A/Gram 133,000 6,860 9,100 79,000 3,640 3,190 54,000 26,100
The peroxtde values were determined essentially as suggested by Wheeler (20). One-gram samples of oil were dissolved in 25 ml. of a solution of 60 per cent glacial acetic acid and 40 per cent chloroform in a 125-ml. conical beaker. One-half milliliter of a saturated potassium iodide solution was added, and the flask rotated gently for one minute. Fifty milliliters of distilled water were then added, and the liberated iodine was titrated immediately with 0.02 N sodium thiosulfate, using starch as an indicator toward the end of the titration. The results were expressed in millimoles of peroxide per kilogram of oil. The vitamin A content was measured with a medium-sized quartz Bausch & Lomb spectrophotometer equipped with a Hilger rotating sector and a quartz biprism. A hydrogen discharge tube was used as a light source. A conversion factor of 2000 was used for calculation of the vitamin A content. There are objections to this method of estimation, especially as oxidation proceeds, because the absorption at 3280 A. of the oxidized vitamin may not entirely disappear ( 2 , 16, 19), Other components in the oils having some absorption a t 3280 A. may also be present, and their absorption may undergo a change during oxidation. On the other hand, no attempt was made to confirm the findings by biological assay, mainly because, as Lease et al. (IO) have shown, the destruction of the vitamin A by the fatty peroxides would continue during the course of the bioassay.
The percentage of vitamin A oxidized in fish liver oils at various peroxide values is independent of the initial concentration of the vitamin. When the percentage of vitamin A oxidized at various peroxide values was determined in a series of oils which had been left exposed to the air, the oils fell into two groups. These groups differ in unsaturation so that this behavior under atmospheric oxidation is probably related to this property. In the oils with higher unsaturation, the percentage of vitamin A oxidized was smaller at various peroxide values than in the other oils at similar peroxide values. Within each group of oils the percentage of vitamin A oxidized is related to the peroxide value of the oil. The higher the peroxide value of an oil becomes on standing, the larger the percentage of vitamin A oxidized. When two samples of oil were stored at different temperatures, less of the vitamin A was oxidized at the lower temperature for the same peroxide value.
hours of storage of the same oils whose rate of peroxide formation was given in Figure 1. Again there is no apparent relation between the oils even when different lots of oil from the same species of fish are compared. However, when the data from Figures 1 and 2 are combined so as to eliminate time as in Figure 3, these same oils fall into two general groups after the peroxide value is somewhat greater than 30. I
Peroxide and Vitamin A Contents Samples of cod, pollack, dogfish (shark), swordfish, and U. S. P. reference oil were set aside and examined at intervals for peroxide and vitamin A content as described above. The results are shown graphically in the figures. Each curve in Figure 1 represents a sample of oil where the peroxide value is plotted against hours of storage a t 34.5' C. The oils display divergent rates of peroxide format.ion. Figure 2 represents the percentage destruction of vitamin A against time in
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I
I
ZOO 300 HOURS AT 34.5 C.
FIGURE 2.
VITAMIXA DESTRUCTION IN Fmr LIVER011,s
Group I is comprised of cod, pollack, and U.S.P. reference oils, and group I1 of halibut, dogfish (shark), and swordfish liver oils. In the oils of group I, the percentage of vitamin A oxidized a t various peroxide values is smaller than in the oils
INDUSTRIAL ,4SD ENGINEERING CHEMISTRY
708
of the other group a t similar peroxide values. This segregation is not influenced by the individual resistance of the oils to oxidation, as some of the most stable and unstable oils represented in Figures 1 and 2 occur in the same groups; nor can it be explained by the differences of the vitamin A concentration of the oils, for there is a wide range of vitamin concentration in each group of oils. In Figure 4, where the values for the same oils stored a t room temperature are shown, the same kind of segregation is again evident, although a higher peroxide value is required to destroy a given percentage of vitamin A than was the case in the oils stored a t 34.5’-6.
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f4D fZ0
a
h3
100
80
2
8
6o 40
20
x
0
/O
20 30
.4y3
50
V ~ A M AI Nmsrmym AT
60
70
mm
80
90
TEMR
FIGURE 4. RELATION OF VITAMIXA DESTRUCTIOX AND PEROXIDATION AT ROOM TEMPERATURE
0
f0
20
30
40
50
60
70
80
90
FIGURE3. RELATIONOF VITAMINA DESTRUCTION AND PEROXIDATION AT
34.5’
c.
The division of the oils into groups is probably related to their composition. Although complete analyses are not available, the fatty acid composition of oils from some of these species was reported by Schsnfeld (18) and is shown in Table I with the usual range of iodine values.
the formation of peroxides from the unsaturated glycerides and in a second reaction the peroxides oxidize the vitamin. The division of the oils into two groups corresponds to two different values for the ratio of the rate of peroxide formation to the rate of oxidation of vitamin il by peroxide. Certain factors, although they may alter the rates of the two reactions, do not affect the ratio so that the division of the oils occurs, regardless of their stability. Changes in unsaturation, however, alter the rate of one reaction more than the other and so affect the ratio which, in turn, determines whether the oil falls into group I or 11.
Acknowledgment The authors wish to thank B. Demarest of the Biophysical Laboratory, National Oil Products Company, for the spectrophotometry required in this investigation.
Literature Cited TABLE I. UNSATURATED FATTY ACIDSAND IODINE VALUESOF FISHLIVEROILS Oil
Group
Cod Pollack Halibut Shark Swordfish
I I I1 I1 I1
YUnsatd. Fatty Acids, %One double More than one bond double bond 20 65 11 73 53 27 40 41 .. ..
Iodine Value
160-170 160-170 130- 145 110-135 130- 145
Cod and pollack oils appear only in group I. The fatty acids of these oils consist of a high percentage of acids with more than one double bond and a low percentage of acids with one double bond. The fatty acids of halibut and dogfish (shark) oils in group I1 consist of a lower percentage of the highly unsaturated acids and a higher percentage of acids with only one double bond. The stability of the oils is not always the greatest in those with lower unsaturation. As Figure 1 shows, some of the most unstable oils occur in group 11,so that factors other than total unsaturation influence the rate of oxidation of these oils but not the division of the oils into the groups shown: the latter occurs irrespective of their stability. This division of the oils into groups may furnish some clue to the mechanism of atmospheric oxidation of vitamin A. If at least two reactions are involved, the following might be considered a reasonable explanation. One reaction may be
(1) Dann, W. J., Biochem. J., 26, 666 (1932). (2) Demarest, B., 2. Vitaminforsch., 9, 20 (1939). (3) Evers, N., Quart. J . Pharm. Pharmcol., 2, 556 (1929). ( 4 ) Fraps, G. S., and Kemmerer, A. R., Texas Agr. Expt. Sta.. Bull. 557, 3 (1937). (5) Fridericia, L. S.,J . Biol. Chem., 62, 471 (1924). (6) Holmes, H . N., Corbet, R . E., and Hartaler, E. R., IND. Eiia. CHEM.,28, 133 (1936). (7) Huston, R. C., Lightbody, H. D., and Ball, C. D., Jr., J . BioE. Chem., 79, 507 (1928). (8) Jones, J. M., and McLachlan, T., Quart. J . Phann., 1, 400 (1929). (9) Lea, C. K.,Dept. Sci. Ind. Researbh (Brit.), Food Invest., Special Rept. 46, 119 (1938). (10) Lease, E. J., Lease, J. G., Weber, J., and Steenbock, H., J. Nutrition, 16, 571 (1938). (11) Lowen, L., Anderson, L., and Harrison, R. W., IKD.EKO.CHEX., 29, 151 (1937). MacWalter, R. J., Biochem. J., 28, 472 (1934). Marcus, J. K., J . B i d . Chem., 90, 507 (1931). Norris, E. R., and Church, A. E., Ibid., 89, 589 (1930). Powick, W.C., J . Agr. Research, 31, 1017 (1925). Robinson, F. A., Biochem. J., 32, 807 (1938). Rosenheim, O., and Webster, T. A , , Lancet, 2, 806 (1926). Shonfeld, H., “Chemie und Technologie der Fette und Fettprodukte”, Vol. 1, p. 92 (1936). Smith, E. L., Biochem. J., 33, 201 (1939). Wheeler, D . H., Oil & Soup, 9, 89 (1932). Whipple, D. V., Ibid., 13, 231 (1936). Wokes, F., and Willimott, S.G., Biochem. J . , 21,419 (1927)I. PRESENTED before the Division of Biological Chemistry at the 97th Meeting of t h e American Chemical Society, Baltimore, Md.
