EFFECT OF MINERAL OIL. 'ii’A'I‘TY ACIDS, AND LECETHEN CINE VITAMIN D UTELEZATION IN THE.- RAT Time‘s for the Degxee cf M. S. MICHIGAN STATE.- COLLEGE Lawrence \a’fleniine Hank as 1943 g} r» ‘7— -v._ This is to certify that the thesis entitled Effect of Mineral Oil Fatty Acids, and Lecithin on Vitamin D Utilization in the Rat presented by Lawrence Valentine Hankes has been accepted towards fulfilment of the requirements for M. So degree in ChemiStry Qd/W Major professor Date December Q, 1945 EFFECT OF IIHERAL OIL. FATTY ACIDS, AND LEOITBIN OH VITAIIH D UTILIZATION I! THE RAT by LAIRINCI mums: mxaa A THESIS Submitted to the Graduate School of liohigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of .IASTER OF SCIENCE Department of Chemistry 1943 4.. . 'L‘ .vyi‘f V‘s‘r" J Patina”? . ..;].I\ M ’ i; 1.. '._. _| V . ? 4". 3/ 1.»; a! 9R3: i i f H (5214- / ‘ "'i -2.) Acknowledge-out I eieh to thank Dr. Soppert for assistance in planning the problem and in writing this thesis. 155304 Table of Contents I Introduction II Historical III Effect of Mineral Oil on Vitamin D Absorption in the Intestine A B C D Experimental Methods Results Table Discussion IV Effect of Free Fatty Acids and Lecithin on the Absorption of Vitamin D, Subcutaneously A B C D E Experimental Methods Preparations Results Tables Discussion V Summary and Conclusions VI Bibliography (1) (2) (8) (10) (12) (13) (16) (18) (23) (25) (27) (28) (29) Introduction In surveying the literature on the effect of mineral oil on the utilisation of vitamin D, one finds more or less conflicting results. This situation is undoubtedly due to the selection of arbitrary procedures. some of which were far removed from stimulating the ordinary use of mineral oil as a laxative. Therefore, it was decided to carry out further studies in which particular attention vas given to the dosage and the method of administering the mineral oil. The second phase of the thesis deals rith a study of the effect of various fatty acids and lecithin on the uti- lisation of vitamin D from subcutaneously injected oils. It was thought that such additions to the oil.might influ- ence it's diepersibility and thus accelerate the absorption of the vitamin D. -2. Historical Studies of the effect of mineral oil on vitamin D ab- sorption have led to somewhat contradictory conclusions. In 1927, Butcher, Ely and Honeywell, reported that cod li- ver oil mixed with mineral oil promoted calcification in rachitic rats to the same degree as an equal amount of cod liver oil without mineral oil (7). The data they obtained was based on x—ray pictures and the line test develOped by lodellum, Simmonds, Shipley and.Park (3). Almost simultaneously with the above publication. Bur- rows and rarr (6) presented experimental evidence which.conh tradicted the above results. They found that mineral oil, unlike vegetable oil, tends to remove fat soluble vitamins from the food and produces other toxic effects. The proce- dure in the latter experiments involved.mixing the oils in the feed, whereas in the previous work, the oils were fed separately. This difference in procedure may be responsible for the difference in results. The toxic effect of the oil was believed by these investigators to be due to the fact that it acts on the mucus membrane of the intestines by re- moving the fat soluble vitamin reserve. Further information on this subject was published in 1929 by Hawks. Levene, Stucky and.0ser (9). The reported that mineral oil interfered with the utilisation of vitamin D in cod liver oil. Approximately three to four times as much vitamin D was required to produce recaloifioation when mineral oil was substituted for cotton seed oil as a solvent. In the -3- interpretation of their results. they used r-rays and the line test. Later work by Jackson in 1933 (16) agreed essentially with that obtained by Butcher, Ely, and Honeywell. He found that mineral oil administered separately from irradi- ated ergosterol did.not interfere with the utilisation of vitamin D. Jackson's procedure differed from that of pre— vious workers in that he fed the oils to the animals sepa- rately with an eye dropper. The other men incorporated either the vitamin oil or the mineral oil in the feed, and fed.the other oil separately. The difference in the me- thods of procedure may have been responsible for some of the variations in the results. Jackson was the first to adminis- ter a dose of mineral oil that was comparable to a normal hu- man dcse of 30 milliliters. This dosage equivalent was worked.out by Jackson in 1937 (13), and his results were con- firmed by Rowntree in the same year (In). The most recent work on this problem was that done in 1930 by Smith and.Spector (18). They incorporated the min- eral oil in the basal ration at five and ten per cent levels. Therefore. the mineral oil was proportional to the food in- take and lubrication was continuous. According to their cal- oulations. there was a daily ingestion of .5 milliliter of mineral oil. They fed .05 milliliter of cod liver oil sepan rately daily with a pipette during a ten day period. The amount of cod liver oil was such that they obtained a line with a +2.“ healing. -M— In all work previous to that of Smith and Specter. the animals were kept on the diet too long to establish whether or not there were differences in utilisation. This was es- pecially true in the work by Butcher. Ely and Honeywell (7). They held the animals on the vitamin D supplement for twenty-one days. One would expect that unless the mineral oil interfered completely with vitamin D utilisation, heal- ing during this period might be considerable with or without the mineral oil. Beginning with Jackson's work. the official line test procedure was used and interpreted according to a scale developed by Bills (12), which in turn was based on the original description of the D line by ncCollum, Simmons. Shipley and Park (3). In the experiments previously mentioned. there was no attempt made to conform to customary human practice in the use of vitamin D and mineral oil. Therefore. particular care was taken in the present studies to simulate as closely as possible the ordinary methods of taking mineral oil and.vitap min D preparations. The second part of this study deals with the effect of fatty acids and lecithin on vitamin D absorption by the sub- cutaneous route. There is very little to be found in the literature concerning the assimulation of oils injected into the body. In 1925. Burrows and Johnston published some ex. tensive work on the effect of subcutaneous injected oils on body tissues (fl). They injected corn oil in such a way that it formed one large droplet which subsequently broke up into numerous drops of various sisee, each drop then becoming ~5- firmly encapsulated, so that a.multiple cystic tumor was formed. The oil had no stimulating action on the growth of the cells, but acted only to disrupt the normal or- ganisation of the tissue. and to build densely cellular tissue relatively free from blood vessels and intercellup lar substances. These men pointed out that this action delayed the absorption of the oil. Subsequent histolo- gical inspection of tissues revealed that the drops were cut off from the surrounding fibrous tissue by a layer of cells. All of the tissue was filled with cells migrating toward the oil. Vith the movement of the cells toward the oil drap, tiny droplets of oil were continuously break- ing off and.moving out into the tissue. In a few of the animals used, the workers did report that the oil was com- pletely absorbed and the tumor collapsed, whereupon. the cells returned to a normal state. It might be expected that the assimilation of the oil could probably be accel- erated or assisted, if the oil injected was more dispersed or rendered.more easily dispersable by the addition of other agents such as fatty acids. There is also the pos- sibility that the essential nature of a fatty acid may exert an influence. The tissues might show a.preference to take up vitamin D from oils containing the essential acid. In 1930. Burr and Burr (ll) found that rats suffering from a low fat disease were not benifited by the ingestion of saturated acids (lauric and myristic), but were cured by linoleic acid. In 1932. Burr. Burr and.niller (15) added -6-- further information to this subject in that they found cleic acid was among those ineffective in curing the dia- sease, whereas linolenic acid behaved similar to linoleic acid. rm. dependency of the body on outside sources for certain fatty acids might give an increased assimilation of the vitamin D oils containing these acids, in spite of the fact that there is a disturbance caused by the presence ,cf the oil as demonstrated by Burrows and Johnston. If the non-essential acids gave an increase in the vitamin D ab- sorption, the increase might be said to be due to a simple physico-chemical phenomenon. that is, greater dispersi- bility due to soap formation. A greater increase with the essential acids could be said to be due to the fact that the body shows a preference for these acids, indicated by a more rapid utilisation of the vitamin D. If all of the effects obtained were negative, the idea of cell function interference prepcsed by Burrows and Johnston could be ap- plied in interpreting the results (11»). Because of the quantitative nature of the vitamin D line tests. vitamin D containing oils were used to deter- mine the rate of assimilation of the injected oils. EFFECT or MINERAL OIL, FATTY ACIDS, ilD LECITHIN ON VITAMIN D‘UTILIZATIOI II RATS Effect of Hineral Oil on Vitamin D Absorption in the Intestine -s-. Experimental.lethcds Preparation g£_Animals In these tests, three to four week old rats weighing “5-60 grams were used. The animals were placed in pairs in wire cages and fed a diet that was a slight variation of Steenbock's basal rachitogenic diet (5). The Steenbock diet was modified by the substitution of table cornmeal for ground whole yellow corn and the introduction of oatmeal and brewers yeast to compensate for the losses in food value occasioned by the use of the refined product. The animals were maintained on this diet for a period of three weeks, at the end of which time they had developed the de- sired degree of rickets. Conduct g£_gggg, The rachitic animals were placed in individual cages and fed supplements containing a known number of units of vitamin D. The supplement was incorporated uniformly in 35 grams of feed. The animals were placed on the vitamin D supplemented ration and were fed mineral oil by means of a small syringe. In the initial experiments, the syringe was provided with a,blunt needle to prevent injury to the ani- mals' throats. However. it was found that some throat in- juries were caused due to unayoidable movement of animals. Consequently. a small piece of number ten catheter tubing was rounded off at one end and slipped over the blunt needle. The rubber tubing was attached.in such a way that it pro- truded over the end of the needle about one quarter of an inch to provide a little flexibility when inserting it. The #9" tube could he slipped down the animal's throat a small dis- tance without any harm being done. and by having the tube in the animal's throat, the animal was unable to regurgi- tate the oil as happened frequently previous to the adop- tion of this technique. The mineral oil was fed every morning, different vol- umes being fed to different groups. In addition to varying 'the amount of oil fed, several levels of vitamin D were also used. . Luann At the end of a ten day period, the animals were killed by ethsrisation. the fore-paws removed with a pair of seis- sors, and.the distal ends of the radii and ulnae with.about one-quarter of an inch of the shaft retained for the line test. The flesh was stripped from the bones, and the bones placed in 95fi'ethyl alcohol for a.period of at least twen- ty-four hours. Then according to Shipley's procedure, the bones were halved. immersed in 2‘ silver nitrate for about two minutes. and.the freshly cut surfaces exposed to light. This technique revealed the calcification induced by the vitamin D in the rachitic metaphysis and the extent of cal- cification was evaluated by using a.modification of Bill's scale. Results f In the initial experiments, supplements of 2.7 and 5.k U.S.P. Units of vitamin D were employed. These doses were obtained by diluting a U.S.P. standard reference oil with lesson (cotton seed) oil. The administration of one milliliter of mineral oil daily proved to be excessive be- cause after five days, the rats lost considerable weight and showed very soft stools. It was then found that this dosage corresponded to twice that prescribed for human usage so that for subsequent studies, smaller dosages were employed. A second series of animals was used with the same num- ber of units of vitamin in the feed. Three levels of min- eral oil were used in this phase of the study, namely three—fourths, one-half, and.one—quarter milliliter daily. All of the animals on the three-quarter milliliter level lost weight. and a few of them died before the tests were completed. These animals were unsatisfactory for the line tests, because animals losing weight are always rejected in official vitamin D assays. lost of the animals on the one-half and one-quarter milliliter levels gained weight and were. therefore, satisfactory for the test. The line tests showed definite interference with the assimilation of vitamin D. the amount of interference varying directly with the amount of mineral oil administered. The half milliliter level used corresponds to a 30 milliliter dose for humans. This amount still seemed excessive for the rate, so that subsequent studies were carried out using one-quarter and -11.- one-eighth milliliter doses of mineral oil daily. In the next series, the animals were fed supplements containing 2.7 and.5.¥ units of vitamin D. One group was fed one-quarter of a milliliter of oil and.the other one-eighth of a milliliter. Further evidence was obtained to indicate that there is an increased interference with the higher level of mineral oil ingestion. Additional experiments were carried out to determine what the effect of using a higher intake of vitamin D might be. The amount of vitamin D in the feed was raised to 8.1 units, and.mineral oil fed at one-quarter and one-eighth.milliliter levels. The results indicated as before that increased injection of mineral oil interferes to a greater extent with the vitamin D absorption. Another method of conducting the test was to incor- porate both the vitamin D and mineral oil at various levels in the basal rachitogenic diet. The mineral oil was in- cluded in the feed at levels of one, two and three milli- liters per 50 grams of ration, and the vitamin D.kept con- stant at 6.7 0.8.P. Units. This was the method used by .Hawk, Levene, Stucky and Oser (9). The results obtained with these animals were similar to those found.by using the previous technique. The results of all of these tests are summarised in Table I. -12- Table I The Effect of Varying Doses of Mineral Oil on the Responses of Rachitic Rats to neasured Intakes of Vitamin D 7117?? Mineral ll. of 011 3:3,“ 2?:- s'z::5° 3:2.“ jam. “firms“ 118 2.7 .15 10 days 5 None i/s 5.1; 1.00 10 days 2 Hone 1/8 ' 8.1 1.87 10 days it Done 1/4 2.7 .29 10 days 12 None llh 5A .72 10 days 11 lone 1]“ 8.1 1.50 10 days 1|. None 1/2 2.7 .60 - 10 days 5 lone 1/2 5.1!- .70 10 days 5 None None 6.7 1.60 10 days 4 1 m1. None 6.7 .75 10 days 1+ 2 ml. None 6.7 .25 10 days !I- 3 ml. None 6.7 2.12 10 days It None -13- Discussion The results of these experiments agree with those ob- tained by Burrows and.rarr in 1929 (6), who observed that the ingestion of mineral oil limits the assimilation of the fat soluble vitamins from the intestinal tract. As men- tioned previously, Smith and specter (18) made similar ob- servations. It is rather reasonable to expect some decrease in the assimilation of fat soluble vitamins during periods when mineral oil is ingested. These vitamins are soluble in mineral oil, and in as much as the latter is indigestible and not assimilated itself, the oil would.obvicus1y prevent the effective extraction of such vitamins by the digestive Juices- That this interference with the assimilation of fat soluble vitamins is considerable, although not complete, is indicated by the fact that some utilisation of vitamin D can be demonstrated when mineral oil containing this vitamin is fed to rachitic rats. Mineral oil exerts an effect also, be- cause it coats the walls of the tract and interferes with the normal contact of digested food with the absorbing tissues of the digestive tract. It may be of interest to mention here that at least one distributor of mineral oil preparations has taken note of the interference with the assimilation of vitamin A. A certain amount of vitamin A is added to the mineral oil product in order to compensate for the estimated loss occasioned by the use of the oil. lith regard to the conflicting results which are to be found in the literature on this subject these may be easily «~110- accounted for. The length of the experimental period is of considerable importance. If one uses a fairly long period of feeding both vitamin D and mineral oil it is possible to get complete healing in rachitic rats with fairly small doses of vitamin D. This is clearly indicated by the results obtained in the present studies in which the experimental period was varied from 5 to 15 days. Differences in line test responses. which were fairly conspicuous in ten day ani- mals. were no longer evident in fifteen day animals. ”HOT 0! lllml. OIL, FATTY ACIDS, AND LEQITEIII 0N VITAHIII D UTILIZATIOU In RATS Effect of Free Fatty Acids and Lecithin on the Absorption of Vitamin D, Subcutaneously «~16- Egperimental nethods Standard rate such as previously described were used in these tests. The solutions of vitamin D for injection were made up in 10 milliliter flasks using the official re- ference oil as the source of vitamin D, lesson oil as the diluent, mid relatively pure fatty acids and lecithin as special additions. Subcutaneous injections were made by means of a hypodermic needle. To avoid losing any of the so- lution by leakage from the site of injection, the needle was inserted at the base of the hind leg and passed subcutane- ously to a spot on the abdomen opposite the left front leg. This procedure also assured getting the fluid in the same area.in every animal, so that the same tissues would be in- volved in the absorption of the fluid. The fatty acids used in these studies came from a.vari- ety of sources. The lauric and myristic acids were student preparations of a fairly high degree of purity. The cleic and linoleic acids were from a supply of 0.2. grade of com- mercial origin. Linolenic acid.and lecithin were freshly pre- pared from linseed oil and egg yolk respectively; their pre- paration will be described later in the paper. The various compounds were added on the basis of one gram.per hundred.mil- liliters of solution. All solutions used were made up fresh before each experiment. In making up the various solutions, the vitamin D stan- dard, fatty acids and lecithin were weighed in volumetric flasks and made to volume with.Iesson oil. To insure complete solution of the solid acids, namely myristic and lauric, the flasks were placed in an oven at 42 degrees and left there until a clear solution was formed. In the case of lecithin. it was found convenient to prepare a stock solution in los- son oil in order to avoid decomposition. Because of the difficulty in effecting direct solution, a double volume of ether was used. The ether was then allowed to evaporate and the resulting solution used as a.diluent in making up the preparations which were intended to contain lecithin. After injection, the animals were placed in separate wire cages on the basal diet for periods which varied from five to fifteen days. lost of the tests involved the con- ventional ten day period. The bones were examined accorde ing to the usual procedure for the line test. A five day test was used in order to determine whether there might be more noticeable differences in the effect of the various preparations which were injected. As in previous studies, animals which lost weight during the experimental period were discarded. because of the fact that spontaneous heal- ing or calcification is usually observed. This would.ob- viously make it difficult to evaluate any effect induced by the vitamin D. In addition to the other preparations, an emulsion of the vitamin D containing oil was also used for injection. It was prepared.by adding a solution of castile soap to the oil and.paseing the mixture through an emulsifier. The em- ulsion obtained was quite stable and suitable for injection. ~18- Preparations l. Lecithin a) Cadmium.§al§_(Preparation and Purification) In order to make a fairly pure preparation of lecithin. the best features of several methods were com- bined. The yolks of two doaen eggs were stirred into a homogeneous mass by means of a.mechanical stirrer. and then poured into a.beaker of hot 95$ ethyl alcohol. The extract was filtered and the residue on the filter paper extracted several times with additional hot alcohol. The filtrates from all extractions were combined and the so- lution allowed to cool. A cold solution of cadmium chlor- ide in 95$ methyl alcohol (10) was added to the combined extracts. The precipitate was filtered off on a fluted filter paper. washed.with ethyl alcohol a few times and then with ethyl ether (2). The precipitate was suspended in 500 milliliters of pe- troleum ether and then extracted repeatedly with 75-milli- liter portions of 80$ ethyl alcohol. This extraction was repeated fourteen times, and occasionally petroleum ether added to keep the volume of the extract constant (19). This petroleum ether-alcohol extraction gives a better separation of the lecithin from the ether soluble substances. The first alcohol and ether washing removes most of the cephalins and sphingomylins. b) Liberation 229. W 2.1. may. The lecithin-cadmium salt was suspended in chloro- form ("00 ml./100 gms. salt) and the lecithin separated by adding a cold solution of ammoniaoal-methyl alcohol (2). The solution was centrifuged and the liquid siphoned off. The chloroformpammoniarmethyl alcohol treatment was re- peated on the cadmium salt residue. All‘cf the liquors thus obtained were concentrated under vacuum.at 35 to #0 degrees centigrade. The residue was extracted with abso- lute ethyl alcohol to separate lecithin from any contami— nating cephalin. In order to achieve further purification the lecithin was treated with the cadmium chloride-methyl alcohol re- agent and then filtered. The precipitate was extracted several times with anhydrous ethyl ether to remove any im- purities that might still be present (8). Then the preci— pitate was suspended in chloroform and treated with ammoni- acal-methyl alcohol solution to free the lecithin. The so- lution and residue were treated in the same manner as before. and.the resulting combined extracts concentrated under vacu- um (17). The residue was then dissolved in a minimum of ethyl alcohol and poured into anhydrous acetone to precipi- tate the lecithin. The product was finally dried on a watch glass in a vacuum oven at 38 degrees centigrade and stored under nitrogen in a test tube. The test tube was kept in a refrigerator to prevent decomposition. The final treatment with acetic acid as usually performed was omitted, because according to Maltaner, a.pure product may be Obtained with- out it (10). The acetic acid treatment generally yields a brown product, and reduces the yield. ~20- 2. ggnclenic 5239 (l) a) Preparation gg,thg.fiexabrcmigg One hundred twenty grams of linseed oil was mixed with a solution of 50 grams solid sodium hydroxide, 150 ml. water, and.100 ml. of ethyl alcohol. This mixture was stirred thoroughly, and heated such that it boiled without spattering. As the alcohol and water boiled asay, more was added to keep the volumes constant. After about forty minutes of heating, the saponification was complete, and the hot solution poured into about 3000 ml. of a fil- tered saturated.salt (sodium chloride) solution. The pre- cipitated soap was filtered off with suction, and washed twice with 50 m1. of ice-cold distilled water. Three hun- dred grams of soap obtained was put into 600 ml. of glacial acetic acid in a large erlenmeyer flask, which was subse- quently surrounded with fine ice and salt. The erlenmeyer flask was fitted with a.mechanical stirrer and.a cold bro- mine-glacial acetic acid solution added by means of a drop- ping funnel. The solution obtained was allowed to stand over-night in a refrigerator, and the precipitate obtained. filtered off. The product was recrystallised from “00 ml. of benzene, and the recrystallisation repeated until a melting point of 180-182 degrees Centigrade was obtained. In generation 2.1. the 123.22 mm; Twenty-eight grams of the hexabrcmide was mixed with an equal weight of sinc powder. and this mixture dis— solved in anhydrous methyl alcohol. The latter was pre- pared (20) by treating methyl alcohol with.magnesium turnings. and after the initial vigorous reaction had subsided, it was refluxed for two hours. The anhydrous product was then distilled off into a cotton stoppered flask. A part of the anhydrous alcohol was saturated with dry 301, which was prepared by adding concentrated 801 in a dropping funnel to concentrated sulfuric acid. This gas was dried by passing it through a series of bottles containing concentrated sul- furic acid. The bromine-ainc-alcchcl solution was heated for a half hour before the Hal-alcohol solution was added drcpwise over the course of one hour. This mixture was then refluxed over-night. In the morning, the solution was chilled with ice to obtain the maximum yield of the methyl ester. The solution was extracted with ethyl ether, and the other layer drawn off. The ether solution was treated with water to remove the excess methyl alcohol present and then dried with anhyu drous sodium sulfate. Subsequently, the ether was distilled off in an atmosphere of carbon dioxide on a steam bath, and the ester then vacuum distilled in the presence of 002. c) 1.0.0.1922. 2!. 19.11.212.93. 59.1.! Seven grams of the methyl ester were mixed with 70 m1. of a 5f solution of sodium hydroxide in 951» ethyl alcohol, and allowed to stand over-night. To this solution, 70 ml. of dilute sulfuric acid were added to free the lino- 1enic acid. The solution was then shaken and extracted sev- eral times with ether. Residual methyl alcohol was removed from the other solution by several extractions with water. -28- The ether was distilled off in the presence of carbon di- oxide, and the yellow oily residue containing the fatty acid was subsequently distilled under reduced pressure, likewise in a 002 atmosphere. 'The product which distilled over at about 225 degrees Centigrade was sealed under ni— trogen before storing in a refrigerator. Results Preliminary injections of vitamin D containing oils in- dicated that there was some assimilation of the vitamin D‘by the subcutaneous route. Therefore, further tests were made in which the following factors were varied: (1) the amount of vitamin D, (2) the volume of the injected oil, and (3) the length of the test period. This was done to obtain the best conditions for the subsequent studies with fatty acids and lecithin. These exploratory studies also in— oludsd a comparison between the vitamin D containing oil, with and without myristic acid. The ten day test with a one milliliter injection in- dicated that myristic acid accelerated the assimilation of the injected vitamin D. Although the effect was not very great, it was consistent for levels of vitamin D in this volume of oil. Ihen the volume of the injected oil was re- duced to one-quarter of a milliliter, the resulting line tests showed considerable variability. The best condition for comparison appeared to be a half milliliter injection of a solution containing 10.8 units of vitamin D per milli- liter. rairly good results were obtained by injection of a half milliliter of a solution containing 16.2 units per milliliter. However, the differences in the lines were not as noticeable as in the series in which 10.8 units of vitae min D was used. Apparently, a fairly uniform distribution of oil is obtained with half-milliliter injections. The experiments involving the time factor resulted as one would expect-the longer the test period, the stronger ~21}. was the line obtained. For the levels of vitamin D used in these tests, a ten day period revealed the greatest differences in utilisation and was subsequently adopted as the standard procedure. At fifteen days, the differ- ences were less pronounced as may be seen in tables 11 and III. The five day tests gave very low or negative responses, which indicated the animals had not yet assimi- lated enough vitamin D to produce noticeable healing. In general, the addition of a fatty acid to a solution of vitamin D increased the rate of absorption of the vitamin by the subcutaneous route. These increases were relatively large in the case of myristic, cleic, and linolenic acids. The test with the castile soap emulsion also showed slightly stronger responses indicating that previous dispersion of the oil accelerates the assimilation of the vitamin D. Iith the preparations containing lecithin, weaker lines were obtained than without the lecithin. This decrease in utilisation of the vitamin is not readily explained, a1- thcugh it may hays been due to decreased dispersion of the oil in the subcutaneous tissues. All of the tests were car- ried out over a ten day period except those noted in the tables. ' -25- Table II The Effect of the Amount of Vitamin D, the Velume of the Injected Oil and the Duration of the DIperimental Period on the Line Test Responses of Raohitic Rats Units of Vqume o? fTine on 30. of Average D per m1. Injection Acid Used Test Animals D Line 5.“ 1 m1. Done 10 days 3 1.06 8.1 1 .1. none 10 days 7 137 10.8 1 ml. None 10 days 10 1.37 10.8 1/2 Ila Ian. 10 day. 21 069 16.2 1/2 m1. Done 10 days It 1.12 21.6 1/11 s11. Done 10 days 2 .75 32.“ 1]“ ml. lone 10 days .29 8.1 1 nl. Done 5 days 3 .75 8.1 1 .1. Hon. 15 day. a 2.00 32.I| 1/# m1. None 5 days 1.75 32.# l/fi m1. None 15 days a 2.;0 5.0 1 ml. lyristio 10 days 1. K 8.1 1 .1e n’ri'tio 10 “y. 7 Isl 10.8 1 I10 lyrictic 10 day. 10 10 ; 10.8 1/2 m1. lyristic 10 days 21 1.3 16.2 1/2 m1. lyristio 10 days 5 1. 21.6 1 ml. Iyristio 10 days 5 .6 32.11 ill; .1. lyristic 10 days ; 1.55 8.1 1 m1. lyristic 5 days .5 32.1; 1/11 .1. lyristio 5 days a .5 10.8 1/2 n1. lyristic 5 days . -26- Table III The xffect of the Addition of Various Fatty Acids and Lecithin on the Assisilation of Vitamin D from Bubcutsneously Injected Oils A‘s-n. feoithin .‘fiher fine on 50. of Average Used Treatment Acid Used Test Animals D Line lone lone Lauric 10 days 7 1.13 None None Oleic 10 days 8 1.16 lone lone Linoleic 10 days 5 1.00 lone None Linoleicc 10 days 6 1.37 11. lone None 10 days 3 .25 None lmulsified None 10 days a 1.00 lone lone Lauric 5 days .u; lone lone Oleic 5 days ll- Neg. lone lone Lincleic 5 days It He . None None Linolehéc 5 days It .51 None hulsified lone 5 days u .102 1f None None 5 days It . lfi lone lyristic 10 days 8 .0§ 1% None Oleic 10 days 8 1.1 1$ lone Linoleic 10 days .3 lf Done Linolehec 10 days .5 1f Done Laurie 10 days .81 1‘ lane Laurio 5 days 4 leg. 1% Done Hyristic 5 days 3 Reg. 11 None Oleic 5 days .13 1% None Linoleic 5 days It leg. ' Injection - 1/2 m1. of oil - 10.8 units of D per m1. -2 7.. Discussion The results obtained in these experiments indicate that the fatty acids in some way accelerate the assimila- tion of vitamin D frem the subcutaneous tissue. This may be due to a greater ease of dispersion of the oil, since it is well known that the presence of free fatty acids in fats er oils greatly facilitates their dispersion or emul- sification. Just what effect the lecithin has on the assimilation of the oil, whether it be physics-chemical er bio-chemical in nature, was not determined. Lecithin does, however, definitely reduce the utilization of the vitamin D. That lecithin or any of the other compounds used do not destroy the vitamin D in the oil was indicated by the fact that when the various oils were incorporated in the basal diet according to the standard method of vitamin D assay, the eXpected response was obtained. There seemed to be no Spe- cific difference between the effects of the saturated and the unsaturated fatty acids, although there were variations to be found among the members of each group. The idea of the oils being dispersed in the tissues due to soap forma- tion is a possible means of explaining the results. How- ever, no studies were made to determine whether or not this idea is tenable. -33- Summary and Conclusions 1. Ihen mineral oil was fed to rachitic rats at a level corresponding to the laxative dose for humans, there was marked interference with the absorption of vitamin D. 2. The addition of certain fatty acids to subcu- taneously injected vitamin D-vegetable oils tends to increase the rate of assimilation of the vitamin D as determined by the line test responses. 3. The addition of lecithin alone or-with.oertain fatty acids to subcutaneously injected vitamin D-vege- table oils tends to retard the assimilation of vita- min D. ! h. Emulsification of a vitamin D containing oil, pregious to injection resulted in more rapid assimilan tion of the vitamin D. 5. The effects of adding fatty acids is probably due to increased dispersion of the injected oils. ‘ 6. lhether or not the presence of lecithin in oils affects the ease of dispersion was not determined in these experiments. It is possible that some other me- chanism may be responsible for the retarding effect of lecithin. 1. 2. 3. 7. -29-- Bibliography Rollet, A. Zur kenntnis der linolsaure Z. Physiol. 0hem., Bd. 62: 410, 1909 Levene, P. and I. Rolf Lecithin J. Biol. Ohem. ll>6: 353, 1921 llcOollum, E., E. 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Minneapolis, 1929 Maltaner, T. A.lote on the Purification of Lecithin. J. Amer. Ohem. 800. 52: 1718, 1930 Burr, 0., and M. Burr 0n the Mature and.nole of the Fatty Acids Essential in Uutrition. J. Biol. Ohem. 86: 537, 1930 Bills, 0., E. Honeywell, A. Iereck, and M. Mesemeier A Oritique of the Line Test for Vitamin D. J. Biol. Ohem. 90: 619, 1931 Jackson, H. The Effect of Mineral 011 Administration Upon the nutritional Economy of the Eat Soluble Vitamins. J. of nutrition n: 171, 1931 Rowntree, J. The Effect of the Use of Mineral Oil Upon the Absorp- tion of Vitamin A. J. of nutrition 3: 3‘5, 1931 Burr, 0., M. Burr, and E. Miller Tatty Acids Necessary in nutrition J. Biol. Chem. 97: 1, 1932 16. 17. 18. 19. 20. -3 lam Jackson, H. The Effect of Mineral Oil Administration Upon the Nutritional Economy of Eat-soluble Vitamins. J. of nutrition 7: 617, 1933 Morrow, C., and I. Sandstrom Lecithin from.Egg Yolk Bio-chem. Lab. Methods, 2nd rs., Pg. 2%, 1935 mith, M. and H. Specter Calcium.and.Phosphorous Metabolism in Rats and Dogs As Influenced by the Injection of Mineral Oil. J. of nutrition 20: 19, 191:0 Pangborn, M. A Note on the Purification of Lecithin J. Biol. Chem. 137: 51:7, 191:1 Feiser, L. Solvents Exper. in Organic Chemistry, 2nd.Ed., 359, l9u1 ‘.‘-. mi, ' ——..' In. I I . v n u I , \ n o 1 . —.I 1 L , . . . I ,. I J . . .. _ I .p _ 0 \ l \ . . .w I fr" 111' "r— :'.':' - .39“... -1.“ .W ’W' ——'" 'T'":".'IC s- WW :r-‘a-r 'V '1 v—' 1..." _ _' l mil-"g- | ! T612.015 0-11:--m - - H241 Hankes 156004 156004 '1 Effect of mineral oil, fatty acids, and lecithin on vitamin D utilim3_ zation in tfié’rat. \\\\\\\\\\\\ \\ \\\\\\\\\\\\\\\\\\\\\\\\\\\\\8\\ \\\\\\\6\\\\\ W 93 02446