INDUSTRIAL AND ENGINEERING CHEMISTRY
December. 1930
1361
Vitamins A and D in Fish Oils' E. M . Nelson and John Rue1 Manning P R O T R I N AND
NUTRITION DIVISION,
B U R E l U OR C H E X I S T R Y A X D SOILS, A N D D I V I S I O N OF
FISHEXY INDUSTRIES, BUREAU OF FISHERIES,
WASHINGTON,
D.
c.
Tuna and sardine oils were found to be as good as, was omitted and that o n e or better than, cod-liver oil in vitamin D potency. sixteenth of the yeast used fish oils other than fish Menhaden and salmon oils are also important potential had been i r r a d i a t e d with liver oils in this counsources of this vitamin. Salmon oil proved to be as ultra-violet light and its vitry during 1929 was more potent in vitamin A as the poorer grades of cod-liver than 12,000,000 gallons (6). tamin D potency was known oil used for medicinal purposes. These results are imThe six most important oils to be more than adequate to portant commercially because they point the way provide a sufficient quantity are s a r d i n e , 2 m e n h a d e n , towards a potential domestic source of vitamin-bearing of that vitamin in the diet. Alaska h e r r i n g , s a l m o n , fish oils, whereas at present the United States is forced The test period was 35 days. Maine herring, and tuna, and to import about 90 per cent of the cod-liver oil used in the annual p r o d u c t i o n of The animals were weighed twice a week, and the conthese oils ranges from apanimal feeding. dition of the eyes was r e proximately 6,500,000 gallons for sardine to 60,000 gallons for tuna. At present these oils corded a t each weighing. The first tests were carried out on thirty-two animals are used principally in the soap industry and as drying oils in the paint industry, and also to some extent for leather sizing divided into eight groups comparable as to sex and as far as possible with respect to parentage. Two groups were and tempering steel. Studies on some of these oils that have been made within used as controls, one receiving the basal diet only and the the last six years show that they contain varying amounts other 10 mg. per rat daily of a medicinal cod-liver oil of of vitamins A and D ( I to 6, 7, 8, IO, 14). In view of the known high vitamin A potency. The rats in the remaining importance of these vitamins in the feeding of farm animals, groups received 10 mg. each daily of the respective oils to poultry particularly, and the fact that the quantity of fish be tested. The oils were diluted with peanut oil so that body oils produced can be greatly increased and the quality 0.1 cc. of oil carried the desired daily dose. The growth responses of the animals are shown in Chart I. improved, a more thorough study of these oils seemed desirable, The Bureau of Chemistry and Soils of the United It is apparent from these results that, of the oils tested, States Department of Agriculture and the Bureau of Fisheries salmon oil is the only one that falls in the same class as codof the United States Department of Commerce cooperated liver oils. The fact that the groups on tuna and sardine oil survived appreciably longer than the controls and did not in these investigations. Rather than make the preliminary tests on carefully pre- develop severe ophthalmia with the same degree of rapidity pared oils that would show the maximum vitamin potency indicated that these oils were not entirely devoid of vitamin for each of the species, i t was decided to use commercial grades A. The fact that the negative controls survived longer than of oil which would be considered typical of the material now those on menhaden oil is attributed in part to the manner available on the market so that the results would be capable of selecting the animals for the various groups. All the of immediate application. The oils were obtained from animals in one litter were started on curative tests on the concerns engaged in fish-oil production, and the origin and same day. The animals showing the least degree of ophthalmethod of manufacture of each were known. The method mia were put in the negative control group, as that seemed to of obtaining oil from fish or fish offal is much the same, irre- be the best test of the suitability of the animals for the assays. spective of the kind of fish used. The material is subjected To obtain a more accurate comparison between cod-liver to cooking, with or without steam pressure, after which the and salmon oils, three comparable groups (six rats each) oil is expressed, usually by the so-called continuous screw were fed the following daily doses: 5 mg. of cod-liver oil and press. The oil is separated from water in settling tanks, 10 and 20 mg. of salmon oil. The growth response with the and in some cases stearin is afterward removed by chilling. cod-liver oil was intermediate between the two levels of salmon oil (Chart 11). The average gain on 5 mg. of codVitamin A Tests liver oil was 30 grams, whereas it was 22 and 41 grams, Ktamin A tests were carried out on albino rats. Storage respectively, on the two levels of salmon oil. I n the first of the vitamin in the test animals was controlled by the tests the average gain on cod-liver oil was 50 grams and on following technic developed in the Protein and Nutrition salmon oil, 23 grams. Therefore, salmon oil had one-third Division ( l a ) . The stock animals receive a diet rich in vita- the vitamin A potency of the cod-liver oil used for comparison. min A. Litters of young are reduced to six. When the I n order to determine if sardine, Maine herring, Alaska young are 17 days old, mother and young are put upon an herring, menhaden, and tuna oils contained any appreciable A-free diet. When the young weigh 40 to 45 grams they quantities of vitamin A , these oils were fed a t a level of 100 are weaned. By following this procedure most of the ani- mg. per day to five groups of rats containing four animals mals showed distinct symptoms of ophthalmia in 18 to 23 each. One group of negative controls was included in this days from the date of weaning. series. To conserve space the growth curves are not preCurative tests for vitamin A were conducted, following sented. The writers were fortunate in choosing the level essentially the technic described in a previous publication of oil fed, in that they could not have chosen a level that from this laboratory ( I S ) . The only difference in the diet would have given more information about all the oils with used in those experiments was that irradiated cholesterol as little experimentation. Sardine, Alaska herring, and tuna 1 Received June 12, 1930. Presented before the Division of Biological oils were all found to be about one-tenth as potent as codChemistry at the 79th Meeting of the American Chemical Society, Atlanta, liver oil. Maine herring contained about one-half as much Ga., April 7 to 11, 1930. * T h e word "sardine" as used in this paper refers to the California A as Alaska herring oil, and we were unable to demonstrate any vitamin A in menhaden oil. pilchard.
HE total production of
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Chart I-Growth Response of R a t s Fed 10 mg. Daily of Oils Indicated in Curative V i t a m i n A Tests The short vertical line intersecting the growth curves indicates the point at which the rats were segregated and curative measures instituted. Salmon oil is shown to contain a large amount of vitamin A, but apparently the other oils do not compare favorably with cod-liver oil as a source of this vitamin. The uniform response or failure of these animals shows them to be admirably suited for vitamin A tests in spite of the early depletion of stored vitamin. Rat 13 on cod-liver oil was the only animal permanently cured of ophthalmia.
Chart 11-In Curative T e s t s Salmon oil is shown to have approximately one-third the vitamin A potency of a high-grade medicinal cod-liver oil. The short vertical line intersecting the growth curve indicates the point at which oil feeding was begun. There were no cures of ophthalmia on these levels of oil, but all the animals showed some improvement.
Discussion
The salmon oil examined is only about one-third as rich in vitamin A as the cod-liver oil used for comparison, but Of the seventy-four animals used, in only one was ophthalmia definitely and permanently cured. That rat re- medicinal cod-liver oils have been tested in this laboratory ceived 10 mg. of cod-liver oil per day. All animals which which contain no more vitamin A than the sample of salmon oil ( I S ) . received enough vitamin A to promote a rapid growth reThe fish oils studied have not been prepared from the sponse showed some improvement of the eye condition. Growth failures were invariably accompanied by increasing standpoint of vitamin preservation. It is very probable that the vitamin A content of these oils could be substantially severity of ophthalmia. increased by using as much precaution as obtains in the modern manufacture of cod-liver oil. Vitamin D Tests The experience of this laboratory in testing cod-liver oils I n the vitamin D tests the McCollum “line test” (11) for the Food, Drug, and Insecticide Administration leads to as modified by Steenbock and Black (16) was used. Rats the belief that the cod-liver oil used is an oil of average vitaapproximately 4 weeks old and weighing 60 to 65 grams min D content and serves as a fair standard of comparison. were put upon Steenbock’s diet 2965. I n 23 to 25 days the I n so far as the present work duplicates that of others, oil to be tested was added to the diet. After 10 days on the the results are in general agreement. The writers are, theretest diet the animals were killed and their bones examined fore, of the opinion that the oils they used are fairly reprefor healing of the rachitic metaphysis. The healing effect of sentative of the quality of oils that are immediately available. the oils was compared with that of the same medicinal cod- Although the present market for the oils studied does not liver oil used in the vitamin A tests. Preliminary tests were justify increased production, the supply could be greatly f i s t conducted to determine the approximate vitamin D increased, as much fish waste is now discarded’ without atpotency of each of the oils. The level of each of the oils tempting to recover the oil. The use of these oils in animal necessary to produce the same amount of healing as 0.1 per nutrition would make possible profitable utilization of more cent of cod-liver oil in the diet fed to litter mates was then of these by-products and thereby eliminate a large amount determined. The results can be summarized by comparing of economic waste. the oils examined with cod-liver oil. Although this paper deals only with fish oils, the results If we assign to the vitamin D value of cod-liver oil the are applicable to some extent to fish meal and canned fish as arbitrary figure of 100, the oils tested would have the follow- some of these products contain oil which is similar in vitaing vitamin D values: min content to the oils examined. A study of those products is a part of the cooperative program now under way. T u n a . . ................................... 125 Sardine ................................... 100 Inasmuch as the poultry industry has found it practical Menhaden ................................. 75 Salmon.. ................................. 50 to make wide use of cod-liver oil and ultra-violet irradiaAlaska herring.. ........................... 30 tion as sources of vitamin D (9), any new potential sources Maine herring.. ........................... 15
December, 1930
INDUSTRIAL AND ENGINEERING CHEMISTRY
of vitamin D are of considerable commercial importance to this industry, in particular, and likewise offer possibilities of usefulness to other animal industries. At current prices some of the oils tested are cheaper sources of vitamins than cod-liver oil. If the manufacturers of fish oils improve methods of production so as to conserve vitamins, an even greater advantage may accrue to all fishery and agricultural industries concerned. Literature Cited (1) Amundson, Allardyce, and Biely, Sci. Agr. (Canada), 9, 594 (1929). (2) Bills, J . B i d . Chem., 72, 751 (1927).
1363
(3) Brocklesby and Denstedt, Can. Chem. Met., 14, 29 (1930). (4) Davis and Beach, Calif. Agr. Expt. Sta., Bull. 412, 1 (1926). (5) Davis and Beach, Poultry Sci., 7, 216 (1928). (6) Fiedler, Bur. of Fisheries, Doc., Fishery Industries, 1929, in press. (7) Holmes and Pigott, Boston M e d . Surg. J . , 193, 726 (1925). (8) Kik and McCollum, A m . J . H y g . , 8, 671 (1928). (9) Manning, Bur. Fisheries, Doc. 1066 (1930). (10) Maynard and Miller, Cornell Univ. Agr. Expt. Sta., Memoir 108, 14 (1927). (11) McCollum, Simmonds, Shipley, and Park, Proc. SOL.Exptl. Biol. Med., 19, 123 (1921-1922). (12) Nelson, Science, 68, 212 (1928). (13) Nelson and Jones, J . Bioi. Chem., 80, 215 (1928). (14) Schmidt-Nielsen, Signe and Sigval, Biochem. J., 23, 1153 (1929). (15) Steenbock and Black, J . Bioi. Chem., 64, 263 (1925).
Sizing b y Elutriation of Fine Ore-Dressing Products’ A. M. Gaudin, J. 0. Groh, and H. B. Henderson MONTANA SCHOOL
OF
MINES, BUTTE,
MONT.
ECEXT developments The possibilities and limitations of sedimentation sample is placed in beakers of in ore-dressing have and elutriation as applied to the sizing of finely ground 800 to 1000 cc. c a p a c i t y brought about finer ore products have been experimentally investigated. (about 50 grams to a beaker); grinding of ore pulps, This This study has led to the design of a new elutriator the b e a k e r s are filled with is particularly true of flotaand to a r&~ement of the usual methods of sizing by liquid to a p r e d e t e r m i n e d tion. Quantitative measure sedimentation. The practical value of acetone as depth (10 em. is convenient); settling medium has been demonstrated. Microthe pulp is stirred thoroughly of fineness of a ground material is inadequately rendered ScOPic inspection and measurement indicate the acand allowed to settle for a by the customary statement curacy of the sizing obtained. given time; the suspension is decanted from the sediment. expressing the percentage of the material passing a 200-mesh or a 325-mesh sieve. I n ref- The settling time depends on the depthof settling and the seterence to flotation products, for instance, it is no more informa- tling rate corresponding to the largest particles desired in the tive to state that 72 per cent of the pulp is minus 200 mesh than overflow. The settling rate is determined by application of it is to describe a gravel as 72 per cent minus 1 em. The size Stokes’ law (7). If all particles started on their settling jourof particles finer than 37 microns (equivalent to the aperture ney from the top of the beaker, one sedimentation would suffice; of the finest sieve cloth made: that of the 400-mesh screen) but as they start from any point between the lip of the beaker and the bottom, repetition of the operation is necessary. I n has to be determined by a method other than screening. The experiments that were undertaken a t the Montana practice from eight to twenty washings are necessary forthesuSchool of Mines during the past year were designed to find pernatant liquid to become substantially clear at the end of the out the effect of particle size on flotation. It was desired sedimentation period. The solids in the combined decanted to segregate particles of different sizes in order to study further suspensions make up the finest grade. After flocculating, this each size group as to chemical composition, state of libera- grade is dried and weighed. The next coarser size in the tion of the various minerals, etc. This automatically ex- sample may be removed by repeating the decanting operations eluded all methods of size estimation on particles finer than with a correctly shortened settling time. Repetition of the 37 microns except methods based on settling of the material sedimentation operation is again necessary to insure fairly in fluid media. clean sorting. If water is the settling liquid, a deflocculating Two methods of sizing by settling in viscous fluids were agent, such as sodium silicate (a), is frequently necessary to used side by side. One method consisted in allowing the disperse the pulp. Sodium silicate was required in varying suspended solids to settle in a still fluid, the other in allowing amounts, rarely in excess of 1 part in 4000 parts of liquid. them to settle against a rising current of fluid. Sizing by All grades but the finest may be separated from the associthe first method is commonly termed “sedimentation;” ated fluid by settling, decanting, and drying. The finest by the second, “elutriation.” product usually requires either the addition of a flocculating Air and water are the common elutriation media. I n sizing agent or a tremendous amount of evaporation. I n the case of fine flotation products air cannot be used as elutriation me- organic liquids evaporation is necessary to recover the valudium, because the products are wet and they contain enough able fluid. I n the case of water flocculation by the joint adextremely fine material to cake on drying. Dispersion in air dition of sodium hydroxide (about 1 gram in 20 liters) and calof caked material is, of course, practically impossible. Be- cium chloride (about l gram in 20 liters) was found satisfacsides water, several organic liquids were tested as prospective tory. Flocculation was followed by decantation of the clear elutriating media. Highly satisfactory results were obtained liquid and drying on a hot plate. with acetone, which is much superior to water in certain Sedimentation in beakers with water as the liquid medium fields of work. is not satisfactory for size-splits coarser than 400 mesh (37 microns) owing to the short time required for settling, nor for Sizing by Sedimentation size-splits finer than 1120 mesh (11.3 microns) because of When a quick analysis is desired, sizing by settling in the loose character of the sediment formed by very fine mabeakers may be employed. Manipulation is as follows: The terial, and the consequence that the sediment is disturbed by decanting the suspended material from the beaker. For 1 Received June 9, 1930.
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