Q» THE VITAMIN D CONTENT OF BURBOT LIVER OIL AND CARP, LAKE HERRING AND SUCKER OFFAL OILS Thai: for the Dogrco of M. S. MlCHlGAN STATE COLLEGE Elizabeth Ann Musser 1.944 THE VITAMIN D CONTENT OF BURBOT LIVER OIL AND CARP, LAKE HERRING, AND SUCKER OFEAL OILS *** by ELIZABETH AETN flSSER A THESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of MASTER OF SCIENCE Department of Foods and Nutrition School of Home Beonomics 1944 THES‘Q ACKNOWLEDGMENT ~ A. The experimentor wishes to express her appreciation to Dr. Thelma Porter, Head of the Department of Foods and Nutrition, for her helpfulness and advice; to Dr. Carl Heppert, Professor in Chemistry, for furnishing; the animals used in this study and forhis interest and c00peration; and to Dr. Philip Schaible for furnishing the fish oils which were assayed. 162582 TABLE OF CONTENTS CHAPTER I. INTRODUCTION . . . Purpose . . . . Theoretical value . Practical value. . Necessity of vitamin To fishing industry Descriptions of fish II. SURVEY OF LITERATURE Burbot liver oil .< Carp oil . . . . III. EXPERIMENTAL PROCEDURE Preparation of oils Sources . . . Extraction . . For assay. . . Care of animals . Preliminary . During assay period Line test. . .. . Statistical analysis IV. RESULTS AHD DISCUSSION Results . . . . Weight changes . Food intake . . Healing . . . 10 ll 11 11 ll 12 CHAPTER PAGE Discussion 0 o o o o o o o o o o o o o 14 Comparison with results published in literature . 14 Comparison with data about the vitamin D content Of other fiSh o o o o o o o o o o o 15 Factors affecting the vitamin D content of fish oils. . . . . . _. . . . . . . 16 Possible uses for the oils . . . . . . . . 17 V. SULDARY'AND CONCLVSIONS. . . . . . . . . . . l9 BIBLIOGP ‘HY . . . . . . . . . . . . . . . . 20 AEPENDIXJ o o o o o o o o o o o o o o o o o 23 LIST OF TABLES TABLE PAGE 1. AVERAGE WEIGHT GAINS AND FOOD INTAKE FOR RACHITIC ANIMALS FED FISH OIL SUPPLEMENTS DURIHG A 10 DAY ASSAY PERIOD. . . . . . . . . . . . . 12 II. AVERAGE HEALING SHOWN BY LINE TESTS OF RACHITIC ANIKALS FED FISH OIL SUPPLEHEHTS DURING A 10 DAY AS SAY PA'IPIOD o o o o o o o o o o o o o o 13 LIST OF FIGURES PLATE PAGE PLATE I. REPRESENTATIVE DEGREES OF HEALING IN RACHITIC RATS ON FISH OIL SUPPLEMENTS FOR 10 DAYS . . . 13 Figure 1. Negative control . . . . . . . 13 Figure 2. Positive control: 0.104 gm. oil . . 13 Figure 3. Burbot liver oil: 0.104 gm. oil . . 13 Figure 4. Lake herring offal oil: 0.104 gm. oil 13 Figure 5. Burbot liver oil: 0.052 gm. oil . . 13 Figure 6. Lake herring offal oil! 0.052 gm. oil 13 Figure 7. Carp offal 0118 0.104 gm. oil . . . 13 Figure 8. Sucker offal oil: 0.104 gm. oil . . 13 THE VITAMIN D CONTENT OF BURBOT LIVER OIL AND CARP, TAKE HERRING, AND sucm OFFAL OILS CHAPTER I INTRODUCTION The purpose of this experiment was to determine the vitamin D content of the oils from four freshawater fish which abound in the waters of the Great Lakes. The oils tested were the liver oil of the burbot (Lota L. maculosa), and the offal oils of carp (Cyprinus carpio), lake herring, (Leucichthys artedi), and sucker (Cotostomus c. commer- ugggii). It is of theoretical interest and perhaps of practical value to determine the vitamin D content of these oils. Previous investigations have shown that fish oils vary greatly in their content of vitamin D, so that data concerning different species are of theoretical interest. This is especially true in regard to fresh- water fish oils, since few of them have been tested. There would be a practical value to the study if it were found that these four freshawater fish oils contained an amount of vitamin D which would make them practicable substitutes for the foreign supplies of oils which have become scarce because of the war. Vitamin D has been found to be necessary for human beings, animals and poultry as an anti-rachitic factor. Since McCollum and his coaworkers (1922a) first distinguished between the anti-rachitic factor and vitamin A in fats, considerable interest has been shown in the cause of the condition known as rickets, and the sources of vitamin D which cure or prevent it. 2 The exact manner in which vitamin D functions in this respect is not known. Shohl and Wolbach (1936) have studied experimental rickets in rats, and the latter author has drawn the following conclusions concerning bone histology. In normal bone, there is a continuous proliferation of cartilage cells upon the epiphyseal side, and a simultaneous degeneration of the matured cartilage cells on the diaphyseal side. In the space left by the degeneration of the cartilage cells, capillaries and osteoblasts deposit the bony matrix. In rickets, the degeneration of the cartilage cells does not occur, so that there is no invasion of the cell by the capillaries and osteoblasts to form bone. Osteoid material is deposited around the capil- laries of the diaphysis, and since the proliferation of cartilage cells continues, the width of the cartilage increases, enlargement and swelling of the joints occur. When vitamin D is given, degenerated cartilage cells again appear on the diaphyseal side and calcification is renewed. Thus the necessity of vitamin D for the normal development of bone in the animal body is evident. Guy (1923) has traced the use of cod liver oil to cure rickets through.many centuries, and because fish oils still constitute one of the most important sources of vitamin D, new oils containing it may be important. Fishermen in general would be aided if the value of the catch of burbot, carp, lake herring and sucker could be raised. According to the U. s. Department of the Interior, Fish and Wildlife Service (1940), the total catch of these fish from the Great Lakes, and the commercial value of each were as follows: burbot, 488,000 pounds valued at $5,984; carp, 5,998,000 pounds sold at $147,553; lake herring, 22,480,000 pounds valued at $486,256; and sucker, 4,398,700 pounds valued at $122,403. An increase 3 in the catch of the burbot which is referred to by Jordan (1908) as destruc- tive to other more valuable fish, and the utilization of the waste of the carp, herring and sucker should raise the commercial value of the catches. Descriptions of the fish from.which the test oils were obtained have been given by Jordan (1908), and by Jordan and Evermann, (1923). The burbot, (Lota L. maculosa), also called the ling or lawyer fish is a long fish with imbedded scales. It may average from two to three feet in length. The range of the fish is from the waters of New England through the Great Lakes to the Yukon. It is unusual because it is the only freshawater fish which is related to the cod. Because its food is the young of other species which are considered more palatable by human beings, the burbot is considered a nuisance. The burbot itself is not valued as food. The carp (Cyprinus carpio), is a native of the rivers of China, from 'whence it was imported to Eur0pe some three hundred years ago, and then brought to this country. It is a dull, sluggish fish of brownish color ‘which prefers tranquil waters. It is very hardy, and sometimes reaches a weight of thirty to forty pounds. Insects and vegetable matter make up its food. The carp is used as food in some parts of this country, although it is more commonly eaten in China and EurOpe. The lake herring (Leucichthys artedi), is also known as the cisco. The fish has an elliptical form.with compressed sides and loosely inserted, silvery scales. It is found in great numbers in the Great Lakes, and is one of the most important of the American species of fish. The flesh is considered fair eating. The sucker, (Cotostomus c. commersonii), is a small olivaceous colored fish which is found in almost every stream east of the Rocky 4 Mountains. The body is rather stout, averaging from six to eighteen inches in length. The source of food is insects and small aquatic animals . Hereafter, for convenience of expression, the fish oils which ‘were tested will be designated by the names of borbot liver oil, carp offal oil, lake herring offal oil, and sucker offal oil. CHAPTER II SURVEY OF LITERATURE Previous studies of the vitamin D content of the oils of the four fish, burbot, carp, lake herring and sucker, have been lbnited to burbot liver oil and carp oil. Burbot Liver Oil. The first mention of the vitamin D content of burbot liver oil was made by LcCollum, Simmonds, Becker and Shipley (1922) in a statement that the oil promoted healing in rachitic rats. Clow and Marlott (1929) in a more detailed study of the oil, estimated the potency as eight times that of cod liver oil. Branion (1931), (1934), reported a potency of two to three times that of medicinal cod liver oil, while Nelson, Tolle and Jamieson (1932) found it two to four times as potent as cod liver oil. The estimation of 400 International Units per gram of burbot liver oil was made by Holmes, Tripp and Saterfield (1941). In addition to these experiments in which the oil was tested on rachitic rats, Steenbock, Kletzien and Halpin (1932) and Haman and Steenbock (1936) report that burbot liver oil is as effective as cod liver oil for chickens. A preliminary report by Myers (1937) in.which he treated infants with burbot liver oil indicates that it is an efficient anti-rachitic substance for children. .EEEEHQEA‘ The only experimental work done on carp oil was carried on by Hess, Bills, Weinstock, Honeywell and Rankin (1928). As a result of a study of the relationShip of the anti-rachitic factor to reproduction of fish, they found that both the milt and roe of carp had anti-rachitic prOperties similar to those of the cod, and that the ovary contained even more vitamin D than the liver. CHAPTER III EXPERINENTAL PROCEDURE PREPARATION OF OILS Sources. The oils used in this study were furnished by Dr. P. J. Schaible of the Agricultural Chemistry Experiment Station. The burbot liver oil was a sample obtained in August, 1943, from Mr. Vogelheim, a fisherman of Rogers City, Michigan. He had caught the burbot in April and had rendered the fat from the livers over an Open flame. The oil was kept refrigerated until used. The carp, lake herring and sucker offal oils were extracted from the fish offal which included the viscera, heads and all waste except the scales. The carp offal oil was expressed in January, l944,from the offal of fresh, impounded fish.which were kept alive until eviscerated. The lake herring and sucker offal oils were rendered in late January and mid April,respectively, from.the frozen offal of fish caught during the winter. The oils from the latter two were extracted about two months after the fish had been caught. Extraction.2£_test oils. The offal was autoclaved at fifteen pounds steam pressure for one-half hour, then the tissue fluids and oils were expressed. Cylinders containing the tissue fluids and oils were placed in the refrigerator overnight. The next morning, the oils were removed by pipette, heated on a steam bath, then centrifuged and the oil pipetted off. Next, the oil was filtered to remove any tissue fluid or water which might remain. The oils were then kept refrigerated at 10°C., and prepared for the assays as needed. Preparation g: oils for assgy. New U. S. PharmacOpeia Reference cod liver oil containing 115 I. U. of vitamin D per gram was obtained for use as a staniard. In preliminary assays upon rachitic rats, it was found that two units per day for six days caused a complete narrow line of healing designated as two plus in the metaphyseal cartilage of the distal end of the radius and ulna. The standard cod liver oil was prepared by'weighing 1,7392 gm. of oil on an analytical balance into a 10 cc. volumetric flask, and diluting to 10 cc. with cottonseed oil (wesson Oil). This amount of oil contained 200 I. U. of vitamin D, so that 0.1 cc. of the diluted solution delivered 2 I. U. The U. S. Pharma- copeia XII (1942) states that not more than 0.1 cc. of oil be given per day, so 0.6 cc. of the diluted solution was dissolved in 5 cc. of ethyl ether and incorporated in the basal rachitic diet. Bills, Honeywell, Wirick and Nussmeier (1931) found that it made no difference whether the supplement was given by mouth or in the food. Therefore the oils were all administered in the food, because of the economy in time and because it was felt that the amount given would be more accurate with only one measurement instead of six. Careful, quantitative procedure was observed so that the amount given each animal was as accurate as possible. With the first group of animals, the dilute oil was prepared with 50 gm. of the basal diet, but this amount was more than some of the animals ate, so that subsequently the oil was incorporated into 40 gm. of basal diet. The food 'was allowed to dry, then was given to each animal ad libitum. The burbot liver oil and the carp, lake herring and sucker offal oils were assayed using 1.7392 gm. of each oil made up to 10 cc. with (cottonseed oil (Wesson Oil). If greater healing was evidenced than that 8 of the positive controls receiving the U. S. P. Reference cod liver oil, the amount of fish oil was cut in half (0.8696 gm.) and diluted to 10 cc. with the cottonseed oil. All oils were dissolved in ether and mixed with either 50 or 40 gm. of the basal diet. This mixture was fed to each rat ad libitum. CARE OF AN IlrIALS Preliminary care. 'When young rats from the stock colony which is maintained for vitamin D assays by the Michigan State College Department of Chemistry reached a'weight of 45 to 50 gm., they were placed on a rachitogenic diet of the following composition: Yellow table corn meal 69% Wheat gluten 25 Brewer's yeast 2 CaCO3 3 NaCl __I_l_ 100% At the end of a three weeks period, the animals were rachitic as signified by a watbling gait and terder, swollen joints. 'Weekly weight gains had been recorded, and any animal gaining less than 20 gm. in the three weeks preliminary period was discarded. The rachitic animals were transported in a covered box to the Home Economics building, and placed upon supplements immediately. .Assay period. In the Home Economics Building, the animals were housed in a room with a northeast exposure. No direct sunlight was allowed to touch animals, and to further protect them from sunlight, a screen was placed in an individual round cage with a raised screen floor. The animals were then grouped according to sex and weight, although litter control was not observed. In each group, one animal serving as a negative control, received no supplement of fish oil; one animal acting as a positive control, was given U. S. P. Reference cod liver oil, and either two or four animals received supplements of the test oils. 'With the first groups of aninmls, 0.6 cc. of the cottonseed oil was given in the same manner as the fish oils, to the negative controls, then was discontinued for subsequent groups since no effect upon healing was evident. The basal rachitOgenic diet with the incorporated oil was fed ad libitum in a small feeding cup until it was all consumed, then the basal diet was furnished until the end of the experimental period. Throughout the assay period, distilled water accessible at all times was given each animal. Care was taken to recover all food drOpping onto the paper below the wire screen, so that each animal would receive the full amount of the diet supplemented with the test oil. Food records were kept to ensure that each animal ate the amount which has been deemed necessary for the assay period by the U. S. PharmacoPeia XII (1942). WeightS‘were recorded on the first, fifth and the tenth day of the test period by weighing on a.trip balance. On the tenth day, after weighing, the animals were sacrificed by killing with chloroform. LINE TEST Preparation gf'bone. The preparation of the bone and the technique of the line test is essentially that originated by McCollum, Simmonds, Shipley and Park (1922b)‘ The distal end of the radii and ulnae were removed from the animal, cleaned of flesh, placed on a thread labelled with the number of the animal, and placed in ethyl alcohol for a period of 48 hours to harden. 10 LEE. The left bone of one d‘ the pairs was removed from the alcohol and rinsed in distilled water. The distal end of the radius and ulna were split apart and each bone was cut longitudinally with a sharp razor blade. The four sections of bone were placed in a 2% (by volume) solution of AgNOs, and exposed to actinic light for approximately one minute on dark days and ten seconds on sunny days. After exposure to light, the sections of the bones were rinsed in distilled water, and placed in a small transparent glass dish and covered with distilled water until reading. Reading 2f lipg 233:. A 12 power magnifying lens was used to enlarge the image of the joint. The degree of rickets was assessed by the scale of the Pharmaceutical Society of London, as given by Coward (1938) with o designating no healing, five intermediate steps signifying various degrees of healing and 6 showing complete healing. A sketch of each bone with the healing evidenced was made for a record. STATISTICAL.ANALYSIS The means of fine weight changes, food intakes, and healing as shown by the line test of each group of controls and test animals were analyzed Ml-Hg-O n1° n2 —_._.————. O ———-——— “1* n2 for significance according to the formula: t 3 (as)? (2 >2 (g )2 inwhich 3- 2x- "'— ny- 1% fiz- .3: 111 + n2 f n3 - 3 CHAPTER IV RESULTS AVD DISCUSSION RESULTS 'Weight changes. Table I, which gives the mean weight changes of all groups of animals, shows that in all except one, there was a slight increase in weight ranging from 0.1 gm. gain made by the positive control group for the burbot liver oil to 7.02 gm. gained by the negative control group for the carp offal oil. The one group losing weight was that of the positive control animals for the sucker offal oil group which showed a mean loss of 1.6 gm. No significant differences in weight gains or losses were found between any one set of animals receiving the test oil and the group of controls for that test oil. The means of weight gains were small, because one group of twenty-one rats was composed of animals which were smaller, very nervous and less sturdy. Of the animals in this group, eighteen lost weight, with one remaining the same for the ten day period. The amounts lost were under 5 grams, so that the results were used. Justi- fication for this was that four negative controls lost up to 5 grams and showed no healing. Other investigators have found that small losses do not affect the degree of healing. Coward and Key (1933) have found that a loss of 5 gm. in weight does not affect healing, and Shipley, Kinney and McCollum (1924) observed that healing did not occur when there were even larger losses of weight. Food Intake. The U. S. PharmacoPeia XII (1942) states that a min- imum.of 28 grams of food should be consumed in an eight day test period. In this study, a ten day period was used, so that the requirement would be 35 grams. From Table I it can be seen that the food intake averaged from 40.3 gm. sm.N so.N m s.ee on.» we.H u NH.N 00.0 o HHo oz mo.N we.o » m.oe om.N NH.H m oe.H- eOH.o m HHo eopHH ecu em.» so.H . e.He ow.N ms.o c om.o eoH.o HH HHo Hseoo eoaoem mm.o mm.H u N.se ms.e Ne.H H NN.N 00.0 HH HHo oz mm.e He.H u e.ee sN.N ew.o m mH.o eoH.o HH HHo eeeHH coo pH.» No.0 u e.ee oo.N NH.H u om.o Nmo.o NH HHo Hommo wowpuos smog os.N os.N m N.em mN.H ow.H m No.s oo.o 0H HHc oz No.HH Ne.m » N.Hm os.m ow.H + eo.m eOH.o OH HHo toeHH ecu ms.m ee.H + n.em oH.m cm.o w NH.© eOH.o NH HHo Hseeo ease mo.e HH.N u e.se me.m oN.H H oo.H oo.o HH HHo oz oe.m Os.H a s.me MN.N NN.o u 0H.o .*eOH.o NH HHo eoeHH eoo mo.s HN.N H N.Ne ON.N *mm.o « oe.o Nmo.o NH HHo eceHH poeiem 0:50 0690 CEO n ‘1 g" excuse owsdno toapom vmow doom mo onpoH Hawfios Mo owodno ma sopflw massage tom eoHecHeoe eooe .oee ooHoeHeoe .eg..oe4 HHo .p.e4 mo .oz HHo eeHa eteeceem eteeeeem Q Cflfiopflp mo .D .H NH ** GdoE caposzpflad Mo hopao pastnspm * Qonmm M4 H amda l2 consumed by the group given cod liver oil in the sucker offal oil test group, to 59.2 gm. eaten by the negative control group of the animals given carp offal oil. The groups of animals previously mentioned lowered the mean of all groups in which they were included, since the food intake for this group was much closer to the minimum.amount. No significant differences in food intake were found between the controls and the group given the test oil. Healing. Preliminary assaying of the burbot liver oil on six rachitic rats gave evidence that when equal amounts of cod liver oil and burbot liver oil were administered, there was more vitamin D in the burbot liver oil than in the cod liver oil, since the test oil showed a mean healing of 4.50 i 0.23 in contrast to 2.67 f 0.42 shown by the positive controls. The differ- ence in healing noted is pictured in Figures 2 and 3 of Plate I. Since thiS'was a significant difference, the amount of burbot liver oil given was cut in half. Table II shows the results of the assay of twelve animals at this level, with the animals getting the burbot liver oil showing a mean healing of 3.00 t 0.24 in comparison to a healing of 2.86 i 0.20 for the group of positive controls. A representative picture of the healing given by this level of burbot liver oil is shown in Figure 5 of Plate I. There was no significant difference between these means, so that may be assumed that this sample of burbot liver oil contained twice as much vitamin D as the U. S. P. Reference oil, or approximately 230 I. U. per gram. The first group of animals given carp offal oil in the same amount as the cod liver oil given the positive controls showed a mean healing of 2.33 in contrast to 2.67 for the animals receiving cod liver oil. This difference was not statistically significant, therefore six more animals .Edaw pom Q mafidywb .0 .H mHH memefispeoo HHo ao>aa woo wedtmdpw oesomomom .m .m .0 mo .0 .H NH ** some new uopao vasondpm * 00.0 00.0 M 00.0 00.0 0 HH0 02 m¢.0 ma.0 m em.m ¢0H.0 m Hfio ambwa woo mHH Hm.0 mH.o & mm.m eOH.o Ha Hflo Hammo aoMosm 00.0 00.0 m 00.0 00.0 HH HH0 03 ¢>.0 mN.0 + ms.m «0H.0 Ha Hflo no>HH too OQN sH.H em.o u mm.N Nmo.o NH Hao Hsmmo woflapog oxoq 00.0 00.0 m 00.0 00.0 oH HHo oz 00.0 Hm.0 o H©.N ¢0H.0 0H Hflo mopfla e00 hHH ho.o NH.o u Ne.N eoH.o NH HHo Hoeeo ease 00.0 00.0 m oo.o oo.o HH Hao oz 00.0 0m.0 o mm.m ** ¢0H.0 NH HHo mobfla too 0mm mm.0 em.0 u 00.m mmo.0 NH ado hobfla poppfim .80 pom .50 Hwo pmop wdaadon me Q sweepw> mo odes mo pwop onHH toflaom poop mo .D .H Coflpdwboe 59 Stone ms ow oopfiw mHoEwms tom optfiakoampd easecspm weflaso: .o>< Hwo .p.84 .0: Hfio swam monmm wemm< wda 0H ¢ cawmbm memmw.qmabm qu mmHm 0mm mqmfiaud OHBHmodm m0 HH mqm< .em eoH.o "Hflo Hemao poxonw m mazmflm $2 w Est 4:: a . OI . \ .em eOH.o "HHo Hemao meHeeo: oxen v oasmflm .em eoH.o "Hflo ao>fla pomasm, m opsmfim wwqa OH mom w .1 pig-2.4] Err», ...,.h. m90m OHBHmUwH 2H meaaum he mmmmuma m>HBxezm .em Nmo,o “HHo Hmmmo mcflahom exam 0 mpzmflm 2% i \ as v . £7 eases llo‘ r Hao eoeHH coo .em eOH.n "Homecoo opwpfimom w mpsmflm —1"' .7, . a , wvfi\./ \‘Qx. _ a a; .2/ n“ 5—: a T e afi / as 2 u. I 2 e _ ,2 :5 a: {a .P .H MBfiH pO£pzm m opsmfim &%Zé§&§ , . a .HH0 oz "Hoapcoo o>apcwwz H opsaflm 13 'were given supplements of the oil. As Table II shows, the results of the twelve rats tested were that the carp offal oil gave a mean value of 2.58 i 0.18 healing while the positive control group averaged 2.61 f 0.21 healing. Figure 7, Plate I illustrates the healing Shown by animals receiving the carp offal oil. The results of the assay showed that there is no statistically significant difference between the means of the two oils, so that it may be concl ded that this sample of carp offal contains approximately 115 I. U. of vitamin D per gram. When an equal amount of lake herring offal oil and U. S. P. Reference cod liver oil were each tested on a preliminary group of eight animals, the rats receiving the test oil evidenced an average healing of 3.25 t 0.33, while the positive controls registered a mean healing of 2.33 t 0.21. The degree of healing is shown in Figure 4, Plate I. This difference proved to be slightly significant in favor of the animals receiving the lake herring offal oil, consequently, a group of twelve animals were given one-half as much lake herring offal oil as cod liver oil. Table II shows that the results of this assay were that these animals showed a mean healing of 2.35 i 0.34 in contrast to 2.76 i 0.23 evidenced by the positive control animals. A representative drawing of the healing shown at this level is given in Figure 6, Plate I. Although this difference was not statistically significant, the degree of healing shown, 2.35, was less than the 2.76 given by the cod liver oil, and the degree of variation, i O.34,was the largest shown by any group, so that it seems safe to conclude that the lake herring offal oil apparently contains at least 200 I. U. of vitamin D per gram. 14 A group of three animals given sucker offal oil in the same amount as the cod liver oil fed the positive controls showed a mean healing of 2.3 in comparison to 2.4 for the positive controls, so nine more animals were given supplements of the test oil. One animal of this group was discarded because of a very low food intake, so that the results of the assay on eleven animals as given in Table II is that they showed a mean healing of 2.68 t 0.18, while the positive control animals evidenced a healing of 2.84 f 0.19. Figure 8 in Plate I shows a typical picture of the healing shown by the animals given sucker offal oil. The difference in the means of healing shown by the animals receiving the test oil was not significantly different from that shown by the animals receiving cod liver oil, so that this sample of sucker offal oil contains about the same amount of vitamin D as the standard cod liver oil or 115 I. U. per gram. From the results of the line test on rachitic animals, it was found that the sample of burbot liver oil assayed contained 230 I. U. of vitamin D per gram of oil, While the sample of lake herring offal oil tested contained at least 200 I. U. of vitamin D per gram. The samples of carp offal oil and sucker offal oil each contained approximately 115 I. U. of vitamin D per gram. DISCUSSION Comparison with results published in_1iterature. The results of this study in which the vitamin D content of burbot liver oil is about twice that of standard cod liver oil, compares with those of Branion (1934), and Nelson, Tolle and Jamieson (1932). This is lower than that of the oils tested by Clow and Harlott (1929), and Holmes, Tripp and Satterfield 15 (1941), for the former found the oil eight times as potent as cod liver oil, and the latter state that it contains 400 I. U. per gram, or is almost four times as potent as the standard cod liver oil. It may be that the burbot liver oil assayed in this experiment did not have a higher vitamin D content because it was rendered over an Open flame which may not be the method best suited to retaining the vitamin D originally present. Compgrison.with data 23 the vitamin 2 content 39 other fish. It is of interest to find how the vitamin D content of burbot liver oil, and the carp, lake herring and sucker offal oils compare with that of other fresh- water fish oils. This subject has not received as much investigation as the vitamin D content of the salt water fish. It was found by Bills (1927) that the oil of the fatty tissues of muddy catfish caught in the Ohio River gave a healing rated as 40 in comparison to 100 as shown by cod liver oil. The liver oil of perch from Lake Erie was assayed by Holmes, Tripp and Satterfield (1941), and found to contain about 750 U. S. P. units per gram. An anonymous article, "Fish liver as a vitamin Rich food" (1925) appearing in the Wisconsin Agricultural Experimental Station Bulletin, states that the canned livers of Lake Michigan fish had healed rickets in rats. Burbot and Whitefish livers rated higher than lake trout. No data were given. Amoung the salt water fish, the range of vitamin D content varies from.none found in the gray sole and sturgeon by Bills (1927) to 54,000 I. U. per gram of blue fin tuna liver oil, as assayed by Morgan, Kimmel and Davidson (1939) and 70,000 I. U. per gram of liver oil found in the same species of fish by Holmes, Tripp and batterfield (1941). or the ocean fish, Bills, et o) 16 a1 (1935) have stated that three-fourths of all liver oils are more potent than cod in vitamin D. Factors affecting the vitamin 2 content of fish oils. The amount of vitamin D in fish oils varies with the species. Bills and coaworkers (1935) found that the highest concentration of the vitamin is present in the livers of the higher families of fish, such as the percomorphi which include the mackerel, tuna, sea basses and swordfish, whereas the families of lower orders are poor in D. The fish tested in this study rank in the middle of the families of fish, except for the burbot which is in the lowest order of the last family. Thus it might be expected that if the liver oils of carp, lake herring, and sucker were tested, they might rank higher than that of the burbot. The part of the body of the fish from which the oil is extracted also affects the content of vitamin D in the oil. One of the most detailed studies of this subject has been made by Pugsley (1942). He tested the potency of oils from the body, liver and intestines, and found that in the pilchard the vitamin D content of the body oil was 54 I. U. per gram and of the liver, 200 I. U. per gram. The herring contained 33 I. U. per gram of vitamin D in the body oil and 250 I. U. in the liver. The same author and his assistants (1942) found that in mackerel the liver contained 1035 I. U. of vitamin D per gram of oil, while the intestines, body oil and offal oil contained respectively, 76, 20, and 51 I. U. per gram of oil. Aschehaug, Kringstad, and Lunde (1939) report that the potency of mackerel liver was 11,300 I. U. per 100 gm. of product, while the flesh rated only 85 I. U. for the same amount. 17 The time of year in which the fish are caught also affects the amount of vitamin D in the oils. Hess, Bills and Honeywell (1929) first found that the amount of vitamin D varied inversely with the amount of oil in the liver. Later Bills, Imboden and Wallenmeyer (1934) found that in halibut liver, the total oil content rose slowly from January to June, then increased suddenly until it was doubled in August, then slowly declined from.August to January. The vitamin D content varied inversely with the oil content, for it was highest in January. Pugsley (1939) found the potency higher in July and August than in September and October 'when the yield of oil was higher. Supplee (1937) found that smaller, thinner fish contained more vitamin D, so that early summer catches were more potent. Since all of the fish oils tested in the present experiment were caught in the winter and very early spring months, it is probable that the vitamin D found present may be near the maximum quantity which might be found in the type of oil tested. The method of extraction of the oils may affect the vitamin D content. Morgan, Kimmel and Davison (1939) tested several oils prepared from the same catch of sardines, but extracted in various ways. The best method for retaining the highest amounts of vitamin D was to steam auto- clave the fish, press out the oils, then centrifuge to remove any other liquid present. This method of extraction was used on the carp offal, lake herring and sucker offal oils, so that it is felt that in these oils there was as little loss of the vitamin as is possible with known methods. Possible uses for the oils. Since all of the test oils fulfill the standard set by the U. S. Pharmac0peiea XII (1942) as far as the vitamin D 18 content is concerned, it is possible that the oils could be used commer- cially as a source of vitamin D. They migit all be considered as a source of vitamin D for human consumption, since burbot liver oil already has been used successfully by Myers (1931) as an anti-rachitic agent. There is a possibility that the others might be quite well used for poultry, although they would have to be tested on chickens first to find the efficacy. It was demonstrated by Bills, Masseugale, and Imboden (1934) that the fish oils differ in their anti-rachitic effect when they found that the blue fin tuna liver oil was only one-sixth as effective on chickens as on rats. They felt that this was due to the fact that there are two or more forms of vitamin D present in fish oils. Eamon and Steenbock (1936) reported that burbot liver oil was as effective as cod liver oil for chickens, but further checking would have to be carried on with the other oils. In the use of these oils for poultry, it would be of special interest to test the carp offal oil, because the quantity of the catch is great, the use for them is limited, and the price is very low. CHAPTER V. SUMMARY AHD CONCLUSIONS Oils from burbot liver and from the offal of carp, lake herring, and sucker caught in the winter and early spring months from the Great Lakes were assayed for their vitamin D content. The burbot oil tested was a sample which had been flame rendered while the carp, lake herring, and sucker offal oils were extracted from the viscera and all other waste except the scales by autoclaving, then pressing out the oil, and centri- fuging it to purify it. A total of 96 rachitic animals, 25 negative controls, 24 positive controls and 47 test animals, were used to find the vitamin D content by the use of the line test technique. The following results were obtained; 1. The sample of burbot liver oil contained about twice as much vitamin D as the standard U. S. P. Reference cod liver oil, or about 230 I. U. per-gram of oil. 2. The lake herring offal oil tested contained approximately twice as much vitamin D as the standard U. S. P. Reference cod liver oil or at least 200 I. U. of vitamin D per gram of oil. 3. The samples of carp and sucker offal oils tested contained the same amount of vitamin D as the standard U. S. P. Reference cod liver oil, or approximately 115 I. U. per gram.of oil. BIBLIOGRAPHY BIBLIOGRAPHY Aschehaug, V., E. Kringstad, and G. Lunde. 1939. The vitamin D potency of different fish and fish products. Jour. Soc. Chem. Ind. 58: 220-223. Bills, C. E. 1927. Antiracketic substances. VI. The distribution of vitamin D, with some notes on its possible origin. Jour. Biol. Chem. 72: 751-758. Bills, E. E., E. M. Honeywell, A. H. Wirick, and M. Nussmeier. 1931. A critique of the line test for vitamin D. Jour. Biol. Chem. 90: 619-636. Bills, C. E., M. Imboden, and J. C. Wallenmeyer. 1934. Potency of vitamin A and vitamin D of halibut liver oil, correlated with seasonal variations in the oil content of halibut liver. Jour. Biol. Chem. 105: x (Proc.) Bills, C. E., F. G. McDonald, 0. N. Massengale, M. Imboden, H. Hall, W. D. Bergert, and J. C. wallenmeyer. 1935. A taxonomic study of the distribution of vitamins A and D in one hundred species of fish. Jour. Biol. Chem. 109: Proc. VII. Bills, C. E., 0. N. Massensale, M. Imboden. 1934. Demonstration of the existence of two forms of vitamin D in fish liver oils. Science 803 5960 Bills, C. E., O. N. Massengale, h. Imboden, and H. Hall. 1937. The multiple nature of the vitamin D of fish oils. Jour. of Nutrition, 138 435-4520 Branion, H. K. 1934. The fatawoluble vitamin content of the liver oil of the burbot. Sci. Agric. 15: 1-11. Branion, H. D. 1931. The vitamin content of burbot liver oil. Canadian Chem. and Metal. 15: 214. Glow, B. and A. Larlott. 1929. The antirachitic factor in burbot liver oil. Jour. Ind. and Eng. Chem. 21: 281-282. Coward, K. H. 1938. The biological standardization of the vitamins. London. Bailliere, Tindall mid Cox. Coward, K. H. and K. M. Key. 1933. The degree of accuracy obtainable by the line test in estimations of vitamin D. Biochem. Jour. 27: 451-465. 21 Fish liver a vitamin rich food. 1925. lTis. Agric. Exp. Sta. Bull. 373: 68. Guy, R. A. 1923. The history of cod liver oil as a remedy. Amer. Jour. Dis. Child. 26: 112-1160 Haman, R. W., and H. Steenbock. 1936. The antirachitic effectiveness of vitamin D from various sources. Jour. Biol. Chem. 114: 505-514. 5853, A. F., C. E. Bills, and E. Q. honeywell. 1929. Antirachitic potency in relation to volume of oil in the liver of the cod. Jour. Amer. Med. Assoc. 92: 226-228. Hess, A. G., C. E. Bills, H.'Jeistock, E. Honeywell, and H. Rivkin. 1928. Relation of the antirachitic factor to reproduction in fish. Proc. SOC. Expt'l. B1010 1“led. 25: 652-6530 Holmes, A., F. Tripp, and G. H. Satterfield. 1941. Fish-liver and body oils. Chemical characteristics, physical properties and vitamin content. Ind. Eng. Chem. 33: 944-949. Jordan, D. S. 1908. Fishes. New York: Henry Holt and Co. 789 p. Jordan, D. 3., and B. W. mvermann. 1923. American Food and Game Fishes. .A popular account of all the species found in America north of the Equator, with keys for ready identification, life histories and Inethod of capture. New York: Doubleday, Page and Company, 574 p. LcCollum, E. V., N. Simmonds, J. E. Becker, and P. G. Shipley. 1923. Studies on experimental rickets. XXI. An experimental demonstra- tion of the existence of a vitamin which promotes calcium deposition. Jour. Biol. Chem. 53: 293-312. thollum, E. V., N. Simmonds, P. G. Shipley, and E. A. Park. 1922a. (1) Studies on experimental rickets. XVI. A delicate biological test for calcium-depositing substances. Jour. Biol. Chem. 51: 41-49. In-IcCollum, E. V., N. Simmonds, P. G. Shipley, and 1;. A. Park. 1922b. (2) Studies on experimental rickets. XII. Is there a substance other than fat soluble A associated with certain fats which plays an important role in bone develOpment. Jour. Biol. Chem. 50: 5-30. lJorgan, A. F., L. Kimmel, and B. G. Davison. 1939. Vitamin content of certain Pacific fish oils. Food Research 4: 145-158. byers, T. 1937. Burbot liver oil as an antirachitic (Preliminary study). Journal-Lancet, NeW'5eries 57: 110-111. 22 Nelson, E. M., C. Tolle, G. S. Jamieson. 1932. Chemical and physical pr0perties of burbot-liver oil and its vitamin content. U. 8. Dept. of Commerce: Bureau of Fisheries. Vol. I, Investigational Kept. No. 12: 1-6 Pharmacopeia of the United States of America. Twelfth Decennial Revision. 1942. Laston, Pa. hack Printing Company. Pugsley, L. I. 1942. Vitamin.A and D potencies of oil from body, liver and intestines of pilchard, herring, salmon and gullibee. Jour. Fisheries Research Board Can., 58 428-437. Pugsley, Lo Io, Jo To Kelly, 1"". A. Crandall, and C. A. Morrell. 19420 The vitamins A and D potency of the oils obtained from the liver, intestines, body and offal of shad, and mackerel. Canadian Jour. of Research. 203 167-169. Pugsley, L. I. 1939. The vitamin D potency of British Columbia pil- chard oil as related to yield. Fisheries Research Board Can., Progress Repts., Pacific Station 39: 3-4. Shipley, P. G., E. M. Kinney, and E. V. McCollum. 1924. Studies on experimental rickets. XXV. .A study of the antirachitic effect of certain oils. Jour. Biol. Chem. 59: 177-182. Shohl, A. T., with a note by S. B. 1"tolbach. 1936. Rickets in lC‘iats. XV. The effect of low calciumphigh phosphorus diets at various levels and ratios upon the production of rickets and tetany. Jour. Nutrition 11: 275-291. Steenbock, H., S. W. F. Kletzien, and J. G. Halpin. 1932. The reaction of the chicken to irradiated ergosterol and irradiated yeast as contrasted with the natural vitamin D of fish-liver oils. Jour. Biol. Chem. 97: 249-264. Supplee, W. C. 1937.? Vitamin D content of menhaden fish oil. Indust. and Eng. Chem. (*ndust. Ed.) 29: 190-191. U. 8. Dept. of Interior, Fish and Wildlife Service. 1940. Fishery statistics of the United States. Statistical Digest, No. 4. APPENDIX TA. .h': 1.1.5| I 0 Individual records of rachitic animals receiving no oil used as negative controls for assay of burbot liver oil No. of Initial Final Weight Food Healing rat 'weight weight change intake on line ‘3: test Gm. Gm. Gm. Gm. 168 or“ 67.0 750 8.0 57.0 0 1769 70.0 760 6.0 55.0 0 180$E 66.7 67.5 0.8 50.0 0 226 o” 82.0 87.3 5.3 53.0 0 23257 59.0 61.0 2.0 49.9 0 2379 74.8 70.0 -4.8 38.0 0 2419 69.0 70.0 1.0 48.4 0 2459 64.4 67.0 2.6 46.8 0 249a” 70.0 57.0 -5.0 40.0 0 254a? 60.0 59.0 -l.0 35.0 0 2609? 64.6 67.7 3.1 54.5 0 M 68.0 69.8 1.81 47.96 0 0_ 3.65 7.03 0 1.10 2.11 0 TABLE II. Individual records of rachitic animals receiving 0.104 gm. cod liver oil used as positive controls for assay of burbot liver oil No. of rat 169d” 1732 1777 1819 2270” 23387 238? 242$ 2469 25082 25563 261? I nitial weight Gm. 67.0 76.0 70.0 66.0 64.5 60.0 67.2 58.5 64.5 66.01 Final 'Weight Food weight change intake J==== Gm. Gm. Gm. 70.0 3.0 46.0 80.0 4.0 57.0 70.0 0.0 51.0 71.0 5.0 40.0 64.7 0.2 37.0 61.0 2.0 40.0 73.0 -l.0 49.0 65.0 -0.5 40.0 59.0 -l.0 46.8 63.0 -4.2 40.0 57.0 -1.5 40.0 60.0 -4.5 37.5 66.14 0.13 43.7 2.83 5.9 0.82 1.70 * Healing calculated up to 40 Gm. intake Healing on line test 3.2* 2.86 0.69 0.20 TABLE III. Individual records of rachitic animals receiving 0.052 gm. burbot liver oil No. of rat 182? 2280’7 2306? 239? 243? 2479 252d” 25709 263? Initial weight 64.0 62.7 70.5 65.4 61.0 62.5 55.0 56.4 Final weight 67.0 67.7 73.0 67.0 60.0 60.0 51.0 54.0 64.7 'Weight change 3.0 5.0 2.5 1.6 -1.0 -2.5 2.80 0.81 * Healing calculated up to 40 gm. intake Food Healing intake on line test ======:- Gm. 56.0 3 55.0 4 40.0 3 55.0 4 44.8 2 37.2 4.3* 33.9 2.4* 45.5 3 40.0 2 39.0 3 33.0 2.4* 39.0 3 43.2 3.00 7.03 0.82 2.11 0.24 TABLE IV. assay of carp offal oil Individual records of rachitic animals receiving no oil used as negative controls for M No. of Initial Final Height Food Healing rat 'weight weight change intake on line 1 test Gm. Gm. Gm. GD. 1020'7 61.5 69.3 7.8 55.0 0 10607 62.2 75.0 11.8 70.0 0 11007 63.1 80.0 16.9 62.0 0 1180” 66.6 74.0 7.4 63.0 0 1220’7 72.0 83.0 11.0 64.5 0 14802 69.0 70.0 1.0 49.5 0 15007 64.0 74.0 10.0 60.0 0 156? 65.2 60.0 ~5.2 40.0 0 159% 71.5 75.0 3.5 60.0 0 16207 75.0 81.0 6.0 68.0 0 M 67.01 74.13 7.02 59.20 0 c- 5.87 8.52 0 1.86 2.70 0 51 TABLE V. Individual records of rachitic animals receiving 0.104 gm. cod liver oil used as positive controls for assay of carp offal oil. No. of Initial Final 'Weight Food Healing rat weight weight change intake on line Mm — Gm.. Gm. Gm. Gm. 10107 59.0 65.5 6.5 47.5 2 1056'7 62.0 61.0 -1.0 38.0 2.11* 10937 63.0 80.0 17.0 49.0 2 1170’7 66.0 74.0 8.0 60.0 4 12107 69.5 82.5 13.0 76.0 2 14907 61.0 67.0 6.0 40.0 3 1520” 70.0 76.4 6.4 50.0 3 l55¥‘ 62.0 61.0 -1.0 40.0 2 1589- 70.0 71.7 1.7 60.0 3 161? 74.7 74.7 0.0 52.0 3 M 65.72 71.38 5.66 51.25 2.61 27— 5.70 11.02 0.66 cqfi 1.80 3.48 0.21 * Healing calculated up to 40 gm. intake TABLE VI. Individual records of rachitic animals receiving 0.104 gm. carp offal oil No. of Initial Final 'Weight Food Healing rat weight ‘weight change intake on line test Gm. Gm. Gm. Gm. 103?. 59.0 64.0 5.0 50.0 3 1079 61.7 74.0 12.3 58.5 3 111? 65.0 76.0 11.0 60.0 3 11967 68.5 78.0 9.5 60.0 2 12:30" 72.5 80.0 7.5 57.0 2 14607 67.0 73.3 6.3 56.0 3 1476"7 67.0 71.0 4.0 51.5 3 1510” 64.5 70.0 5.5 53.0 3 1540(7 78.0 81.8 3.8 40.0 2 1579? 67.2 70.0 2.8 49.0 3 160$2 73.8 77.7 3.9 58.0 1 1639 76.0 78.5 2.5 59.0 5 M 68.35 74.53 6.18 54.33 2.58 cr- 3.1 5.73 0.65 cr— 0.9 1.66 0.18 TABLE VII. Individual records of rachitic animals receiving no oil used as negative controls for assay of lake herring offal oil ho. of Initial Final Weight Food Healing rat weight weight change intake on line test Gm. Gm. Gm. Gm. 16407 79.0 90.0 11.0 52.0 0 1769 70.0 76.0 6.0 55.0 0 1809 66.7 67.5 0.8 50.0 0 1840’7 60.0 70.0 10.0 46.0 0 22607 82.0 87.3 5.3 53.0 0 2379 74.8 70.0 -4.8 38.0 0 241? 69.0 70.0 1.0 48.4 0 2459 64.4 67.0 2.6 46.8 0 24907 70.0 67.0 -3.0 40.0 0 25407 60.0 59.0 -1.0 35.0 0 260$? 64.6 67.7 3.1 54.5 0 M 69.14 71.95 2.82 47.15 0 cr— 4.75 6.55 0 ($5 1.43 1.98 0 TABLE VIII. Individual records of rachitic animals receiving 0.104 gm. cod liver oil used as positive controls for assay of lake herring offal oil ho. of Initial Final weight Food Healing rat weight weight Chang intake on line test Gm. Gm. Gm. Gm. 173? 76.0 80.0 4.0 57.0 4 177? 70.0 70.0 0.0 51.0 4 1819 66.0 71.0 5.0 40.0 3 1859 61.9 64.0 2.1 50.0 2 22707 64.5 64.7 0.2 37.0 2.16* 238? 74.0 76.0 2.0 49.0 3 2429 65.5 65.0 -0.5 40.0 2 2469 60.0 59.0 -l.0 46.8 2 2500’7 67.2 63.0 -4.2 40.0 3 2556? 58.5 57.0 -l.5 40.0 2 261? 64.5 60.0 -4.5 37.5 3.2* M 66.19 66.37 0.15 44.39 2.76 or- 2.87 6.33 0.74 Chi 0.86 1.91 0.23 * Healing calculated up to 40 gm. intake. TABLE IX. of rachitic animals of lake herring offal oil Individual records receiVing 0.052 gm. 31 * Healing calculated up to 40 gm. intake No. of Initial Final 'Weight Food Healing rat weight weight change intake on line test Gm. Gm. Gm. Gm. 16707 70.0 70.0 0.0 50.0 3 175? 73.0 73.0 0.0 46.0 2 179? 67.0 67.0 0.0 45.0 2 183? 62.2 62.0 -0.2 44.0 4 186? 62.0 74.0 12.0 50.0 5 22907 64.0 65.4 1.4 47.5 1 240? 70.0 71.7 1.7 46.8 2 244? 65.0 67.0 2.0 47.9 2 2489’ 59.5 57.0 -2.5 38.7 1.03* 25107 63.0 60.0 -3.0 40.0 3.08* 2560;7 57.8 53.5 -4.3 35.0 1.14* 2629’ 60.0 60.0 0.0 44.0 2 M 64.46 65.05 0.59 44.68 2.35 Cr— 3.90 3.17 1.17 1.13 0.92 0.34 TABLEIX. receiving no oil used as negative controls for assay of sucker offal oil Individual records of rachitic animals No. of Initial Final Weight Food healing rat weight weight change intake on line test Gm. Gm. Gm. Cm. 2320" 59.0 67.0 8.0 49.9 0 24907 70.0 67.0 -3.0 40.0 0 2540’7 60.0 59.0 —l.0 35.0 0 260? 64.6 67.7 3.1 54.5 0 26607 69.5 73.0 3.5 54.0 0 270? 69.8 72.0 2.2 47.0 0 M 65.5 67.6 2.13 46.71 0 cr— 3.50 7.29 0 ‘?h 1.43 2.97 0 TABLE XI. Individual records of rachitic animals receiving 0.104 gm. cod liver oil used as positive controls for assay of sucker offal oil No. of [Initial Final weight Food Healing rat ' weight 'weight change intake on line . test L Gm. Gm. Gm. Gm. 233:” 59.0 61.0 2.0 40 3 25007 67.2 63.0 -4.2 40 3 2550” 58.5 57.0 -1.5 40 2 26192 64.5 60.0 -4..5 37.5 5.21 2719 66.3 66.5 0.2 44.0 3.0 M 63.1 61.5 -1.6 40.3 2.84 c7t 2.5 2.09 0.42 67m. 1.12 0.93 0.19 * Healing calculated up to 40 gm. intake TABLE XII. Individual records of rachitic animals receiving 0.104 gm. sucker offal oil No. of Initial Final Weight Food Healing rat weight weight change intake on line test Gm. Gm. Gm. Gm. 23407 58.5 62.9 4.4 45.0 2 2350" 58.2 57.0 -1.2 40.0 3 23607 54.5 61.0 6.5 45.8 2 25367 61.0 62.0 1.0 47.5 3 25807 54.0 52.0 -2.0 38.5 4.15. 259cf7 52,5 52.0 -0.5 38.0 3.15* 265‘?2 54.5 53.5 -l.0 40.0 2 26807 65.5 64.0 -1.5 40.0 2 2690r7 60.5 61.5 1.0 45.0 2 2729 61.0 61.0 0.0 38.0 3.16* 273? 60.0 60.0 0.0 40.0 3 M 58.2 58.8 0.60 41.6 2.68 cr- 2.50 3.54 0.61 ”In 0.75 1.07 0.18 * Healing calculated up to 40 gm. intake. fab-‘6 4‘ 1 1‘1]! 11.1 a‘ij‘il‘u‘l In. ’11:“! A033. . I, . MICHIGAN STATE UNIVERSWY L'BRfl-RIES I1 111.11 1 “w 1 |" |H1| kl": N ml I 3 1293 031 3 0378 1 o I