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lNDUSTRIAL AND ENGINEERING CHEMISTRY
accuracy with which 2,6-dichlorophenolindophenolmeasures either vitamin C or ascorbic acid. Attention may be called, however, to recent claims (1) of another substance in the antiscorbutic fraction quite different in constitution and in physiological activity from ascorbic acid. Studies in this connection will be pursued here to include other vegetables and, if a procedure is found to secure a reducing effect sufficiently large and Sufficiently controllable, to determine biologically whether increased reducing effect is accompanied by an equivalent antiscorbutic effect. The fact that a reducing value to 2,6-dichlorophenolindophenol is produced in raw succulent pea juice but not in cabbage juice deserves further consideration with reference to the fact that ascorbic acid is produced in the germination of pea seeds.
VOL. 29, NO. 2
Literature Cited (1) Armentano, L., Bents&th,A., BBres, T., Ruszny&k, Iatvan, and Szent-Gyorgyi, A., Deut. med. Wochschr., 62, 1326 (1936). (2) Bezsaonoff, M., c m p t . rend., 172, 92 (1921). (3) Kertesz, 2.I., Dearborn, R. B., and Mack, G . L., J . Biol. Chem., 116, 717 (1936). (4) Kohman, E. F.,Eddy, W. H., and Carlsson, Victoria, IND.ENQ. CHEM.,16, 1261 (1924). (6) Kohman, E. F., Eddy, W. H., and Gurin, C. Z., Ibid., 23, 808 (1931). (6) Kohman, E. F.,and Sanborn, N. H., Ibid., 26, 773 (1934). (7) Natl. Canners Assoc., Research Lab., Canner, 68. Pt. 2 , Convention No., 187 (1929). (8) Szent-Gyiirgyi, A., J . B ~ O Chem., Z. 90, 385 (1931). RECEIVED January 18, 1937.
Vitamin D Content of Menhaden ,Fish Oil W.C. SUPPLEE University of Maryland, College Park, Md.
T
HE tremendous requirements of the feed industry for vitamin D supplements give significance to evidence of new sources of vitamin D which offer possibilities of being more economical than those now utiliied. Menhaden oil, a product of American fisheries, is ordinarily marketed for industrial purposes at a much lower price than that of cod liver oil and sardine oil sold to the feed industry as sources of vitamin D. The potential value of menhaden fish oil as a source of vitamin D has been indicated by the work of several investigators (2-6). Supplee and Lee (6) tested samples of commercial menhaden oil by the chick assay method and found them to be as potent in vitamin D as ordinary grades of cod liver oil now used for poultry feeding. The present paper gives the results of the assay of two samples of menhaden oil speoially prepared by the author and fully substantiates previous work indicating the value of this product as a vitamin D carrier.
Menhaden fish oil tested by the chick assay method was found to be relatively high in vitamin D content. A sample of oil from so-called thin fish was found to be at least twice as potent as a sample of oil from medium fat fish from the same catch. Oil from thin fish gave a bone ash of 45.84 per cent when fed at a per cent level, and medium fish oil gave a value of 45.91 per cent at a l/4 per cent level. The tentative 4-week chick assay method of the Association of Official Agricultural Chemists was used.
Biological Assay The Association of Official Agricultural Chemists’ tentative chick assay method for vitamin D carriers was used in this study (1). The method in detail as applied in this work is as follows : Groups of one-day-old white Leghorn chickens are placed in screened-bottom biological cages. One group is reserved for negative control purposes, and one or more additional groups are reserved for each material to be assayed. Distilled water is kept before the chicks at all times. A sufficient quantity of a basal rachitic ration to provide for the entire feeding period is prepared as shown in Table I.
Preparation of Samples Live fish were taken from the net and sorted roughly into two sizes-small or “thin” fish which yield a very small quantity of oil, and larger, medium fat fish. A suitable quantity of each of these two types was cooked immediately with steam for about 8 minutes to loosen the tissues and then pressed thoroughly to express the oil. The oily juice thus obtained was allowed to stand in large glass cylinders until the oil separated cleanly. The oil was drawn off, strained through absorbent cotton, and finally filtered through paper. The purified oil~l,which were of high quality (extremely low in free fatty acids, light in color, and practically odorless) were stored in glass bottles in an electric refrigerator until Mted.
TABLE I. BASALRACHITIC RATION Ground yellow aorn Pure wheat flour middlings Crude domestio acid-precipitated oasein Calcdm carbonate ( reoipitated) Calcium phosphate grecipitated) Iodized spit (0.02% potassium iodide) Nonirradisted yeast (7% minimum nitrogen)
Parts by weight 69 25 12
1 1 1
1
The test rations are made up by adding the desired percentages of vitamin-D-bearing oil to the basal rachitic ration. Sufficient corn oil is also added to those rations containing the smaller amounts of test oil to bring the total added oil
FEBRUARY, 1937
INDUSTRIAL AND ENGINEERING CHEMISTRY
t o the same level in all cases. The negative control ration is prepared by adding sufficient corn oil to the basal ration to bring the oil level to that of the test rations. The first day the chicks receive only distilled water. On the second day they are given two 15-minute feedings of their respective rations. Beginning the third day, all groups receive their respective rations ad libitum for 28 days. The birds are then killed, and the left tibia is removed, cleaned of adhering tissue, and placed in 95 per cent alcohol until analyzed. The cleaned bones are wrapped individually in filter paper, crushed, extracted for 20 hours with hot 95 per cent ethyl alcohol, and finally for 20 hours with ethyl ether. The fat-free bones are dried, weighed, and ashed in a muffle furnace a t approximately 850' C. for l hour. The ash is weighed. The individual ash percentages are calculated on the basis of the dry fat-free bone, and the average for each group is then computed.
Interpretation of Results Negative control groups fed the basal rachitic ration alone become severely rachitic, showing tibia ash contents of 30 to 34 per cent within 4 weeks. Groups receiving the basal ration supplemented by additions of vitamin-D-carrying materials show ash averages varying from these minimums of 30 to 34 per cent to maximum group averages of 44 to 49 per cent, depending upon the quantity of vitamin D supplied by the supplement. In a test of any material, therefore, if a group average of less than 44 per cent is produced, calcification is considered to have been subnormal and hence the supply of vitamin D to have been insufficient. Group averages above 44 per cent indicate normal calcification and a sufficient supply of vitamin D. The levels chosen for study were 1/8, l/4, and l / 2 per cent, the basal ration being supplemented with these percentages of each of the two oils and fed as described in the method. The results of the bone ash determinations are given in Table I1 as well as the average ash content of the tibia of the negative control group. The oil from the thin fish is seen to have produced normal calcification a t '/E,per cent, the lowest level fed. Oil from the medium fat fish failed to protect at l/s per cent but was entirely adequate for normal calcification at l/4 per cent. Recent work with the method used in this study has shown that the equivalent of approxkately 20 U. S. P. units of
191
vitamin D per 100 grams of basal ration is needed to produce an ash content of 44 per cent or more. On this basis, the potency of the thin fish oil is shown to have approximated at least 160 units per gram, and the medium fish oil potency t o have been somewhat more than 80 units per gram. TABLE 11. TIBIAASH OF CHICKS FEDMENHADFJN OIL AS A SOURCE OF VITAMIN D Oil from Medium Fat Fiah
Oil from Thin Fish l/a%
l/4%
1/4%
'/x%
40.61 39.19 35.71 37.23 39.41 40.18
48.12 44.67 42.29 46.43 46.12 48.62
38.70
45.91
46.04 45.23 46.86 47.47 46.47 41.68 46.26 45.71
$/a%
'/a%
Par cent ash
Group av.
46.76 43.44 46.77 44.36 46.13 47.64 45.76 45.84
45.95 47.69 45.92 46.97 48.73 46.74 45.90 46.84
47.73 45.81 45.82 47.45 48.04 46.31 47.17 46.90
...
...
.
Contro
.. .. .. ... ...... .....
33.67
The definite difference between the two types of oil studied here, in view of the fact that the fish were taken from the same school, strongly suggests the probability that oil from smaller thin fish will consistently exceed fat fish oil in vitamin D. Therefore, if menhaden oil should be utilized as a vitamin D supplement, oil from the early summer catches, when the fish carry less fat, would probably prove preferable.
Acknowledgment The author wishes to express his appreciation to The Standard Products Company, Incorporated, Whitestone, Va., for cooperation in the preparation of the samples used in this work.
Literature Cited Griem, W. B., J . Assoc. Oflcial Agr. Chem., 17, 69 (1934). Halverson, J. 0.. and Dearstyne, R. S.,N. C. Am. Expt. Sta., 46th Ann. Rept., p. 62 (1933). Harrison, R. W., and Pottinger, 6. R., U. S. Bur. Fisheries, Investigational Rept. 4 (1931). Manning, J. R., Nelson, E. R., and Tolle, C. D., Ibid., 3 (1931). Nelson. E. M., and Manning, 22, 1361 - J. R., IND.ENQ.CHEM., (1930).
Supplee, W. C., and Lee, C. F., Univ. Md. Agr. Expt. Sta.,'Bull. 389 (Nov., 1936). RECEIVED January 6,
1937.
ANTHRACITE DIAMOND COLLIERY,SCRANTON, PA. From a painting reproduced by oourtesy of School of Mines, Mineral Gallery, The Pennsylvania State College
