‘ K i . I 1 x M I'. I 1 1 1 l 1 \HH '\ f _:L T — ,7i , , ,7 _._, —— Afil,’ ‘- ’_._=:-g >71; 77 . AA STUDY. OF FAT ABSORPWON {N THE ALBINO RAT. Thank for fhe Degree of M. S. MiCHlGAN STATE COLLEGE Samuel Juhn Mussel“ 1931 mmnmtfi-m- '1 . V T 1 ’ ” 3'3 4 4 Y é . \ . , ‘ . E ‘I‘ 2":11153211 31.4.3 gm Umvm my g .1 ‘nr-w.: Hm 'fmw 293 _(_)1084 7014 ' ‘ ' 5 IIIIIIIIIIIII IIIIII IIIIIIIII II I; A STUDY OF FAT ABSORPTION IN THE ALBINO RAT By SAMUEL JOHN MUSSER A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Chemistry 1951 A CKNO‘W LEDGMENTS The assistance, advice and guidance of Dr. Carl A. Happert have been greatly appreciated and have proved in- valuable during the course of these investigations. In addition, Mr. Leo Klever. of the Vitamin Assay Laboratory. has been exceedingly helpful. and has provided excellent instruction in the care and handling of animals. The author wishes to eXpress his sincere gratitude. A .TUDY C? F}? ABSORFTION IN TfiE ALBINO R\T BY Samuel John Auseer A14 A3 arms? Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the recuiremente for the degree of m44r‘a CF 3CIEfiCE Department of Chemistry Year l)5l Approved 0 a W I I A fiTUDY C”? F-‘xT a‘ISE-Eié‘Tlll’I‘I IN TIE 9.1431130 R .T The manner in which fatty materials are absorbed in the small intestine has long been the subject of investigation. For many years, the concept that fat is completely hydrolyzed to free fatty acids and glycerol has been accepted as a probable reeuisite before intestinal absorption. In recent years, however, considerable evi- dence has accumulated in cup ort of the theory that fat may be ab- aorbed without hydrolysis in the form of particles or globules. In view of these two osposing theories, an investigation was started with the hOpe that evidence could be obtained in support of one of the two current concepts. In the event fat is absorbed in the form of a particle or glo— bule, it was felt that a foreign material dissolved in the dietary fat would therefore be carried along and deposited in the adipose tissues of test animals. Four different substances were chosen . (hexyl blue. a rat-soluble dye, ergosterol, cholesterol and mineral oil). Each. being resistant to saponification, would be measurable in the uneaponifiable fraction of depot fat. Experiment I demonstrated that a fat-soluble dye, hexyl blue. when dissolved in dietary fat. was laid down in the adipose tissues, staining them a dark blue. In order to determine whether or not the presence of fat in the diet was necessary for the dye absorption, a fat-free diet was devised ( the dye dissolved in alcohol and evaporated on sugar}. No great difference in absorption was observed. \ ' “Ki 'J‘t} ‘-‘."~" Uni cor thr ext rac act of lev ma“ of I fail 'lin fed 1 and 1 {root calcij Fraser or .it; Experiment II used ergosterol as a tracer of fat absorotion. Unirradisted ergosterol was dissolved in lard, butterfat, and/or corn oil and fed to adult male and female albino rats for a period of three woeke. The adipose tissue of the animals was renoved, saoonified, extracted and the unsaoonifiable fraction was irradiated and fed to rachitic rats. The ”line test“ was used as an index of Vitamin 0 activity. No activity was observed attributable to the absorption of ergosterol dissolved in the dietary fat. In a similar manner, Experiment 121 used ergosterol, at a higher level, as an index of fat absorption. The irradiated unsaponifiable ma‘ter of feces of animals fed ergosterol effected complete healing of rachitic rate. ergosterol,diesolved in lard at a level of a: , failed to be absorbed in measurable amounts, as evidenced by the 'line test". A mixture of ergosterol and mineral oil, dissolved in lard, was fed to test animals. As in the preceding experiments, adipose tissue and liver was removed, saponified, extracted and the unsaponifiable fraction was irradiated and fed to rachitic animals. Broad lines of calcification were observed by the "line test”, indicating that the presence of mineral oil permitted some absorption of ergosterol to take place. Insufficient evidence was accumulated to warrant exclusive support of either of the two previously mentioned theories of fat absorption. TA BLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . l HISTORICAL . . . . . . . . . . . . . . . 4 Absorption in. Particulate Form . . . . . . . 6 EXPERIMENTAL STUDIES . . . . . . . . . . 25 MethodolOgy . . . . . . . . . . . . . . 26 Materials and Equipment . . . . . . . . . . 30 Special Diets . . . . . . . . . . . . . . 30 Experiment I . . . . . . . . . . . . . . 31 Experiment II . . . . . . . . . . . . . 33 Experiment III . . . . . . . . . . . . . 37 Table! . . . . . . . . . . . . . . . . 42 Table II . . . . . . . . . . . . . . . 44 Table III . . . . . . . . . . . . . . . 45 Table IV . . . . . . . . . . . . . . . 46 DISCUSSION . . . . . . . . . . . . . . . 48 SUMMARY . . . . . . . . . . . . . . . . 55 BIBLIOGRAPHY..............59 INTRODUCTION INTRODUCTION In order that the animal body be supplied with energy requisite for the support of life. food materials from without the body must be introduced. Some of the more simple materials (water, oxygen, inorganic salts and simple sugars) may readily pass through the guarding cell membrane of the digestive tissues with little change, preceding conversion into materials fundamentally necessary for the processes of life. More complex materials (protein, carbohydrate, and presum- ably. lipidl) are believed to undergo drastic revision in the digestive tract before assimilation and utilization. This process. termed “hydrolysis,” is a function of the specific enzyme systems present in the digestive juices of the alimen— tary tract, and results in the breakdown of complex molecules into smaller molecules. Because the nomenclature of fatty substances in the past has been in a state of confusion it has been necessary to evaluate the meaning of terms. but in general the original terminology of the particular author has been used. The terms "lipidH and "fat” have been interpreted to mean all naturally occurring substances of a fatty nature (including the unsaponifi— able matter). The term "neutral fat" has been understood as triglycerides of fatty acids. The most noticeable consequence of this breakdown is the transformation of insoluble material into a soluble form, a prerequisite for absorption through the walls of the small intestine. Such a mechanism has been generally accepted for the three classes of foods: protein, carbohydrate and lipid, which yield upon hydrolysis: amino acids. hexoses, fatty acids and glycerol, respectively. Absorption from the small intestine takes place through the epithelial cells of the villi by two distinct pathways. One, the portal system, is by way of the capillaries; absorbed ma- terials pass through the intestinal veins into the portal vein, enter the liver and eventually reach the general circulation through the inferior vena cava. The other, the lymphatic sys~ tem, absorbs food material through the lacteals (lymph vessels), forming chyle (if lipid has been ingested). which is pumped by rhythmic contractions into the lymphatic tributaries to the thoracic duct, thence into the bloodstream at the junction of the jugular and subclavian veins (1). The lymphatic system is of particular importance with regard to the absorption of lipids. Some 60 per cent of absorbed fat (triglyceride) can be collected from the lymph of the thoracic duct, the remaining 40 per cent has been supposed by many investigators to be absorbed through the portal system. The exact method of transfer of fatty ma- terial from the epithelial cells to the lacteals (the processes underlying the absorption of fat) has long been the subject of debate. Many theories of the absorption of neutral fat (triglyc- eride) have deveIOped, but mainly the controversy has been centered about two theories. One is the lipolytic theory, in which the triglyceride is hydrolyzed by lipolytic enzymes to free fatty acids and glycerol in the intestine before passage into the epithelial cells of the villi, with subsequent resynthesis into the triglyceride for transport via the lacteals. The sec- and general concept is that neutral fat, as the triglyceride, is absorbed as such without complete, or indeed, very little hydrolysis. Because of the extreme complexity of the problem, and in view of the many divergent paths and ingenious methods used to approach it. the efforts of any one experimenter can hardly expect to do more than supplement the accumulation of evidence that may support one of the two current concepts. That has been the purpose of this study. HISTORICAL HISTORICAL The study of the mechanism of lipid absorption has a historical background of many varied and diverse approaches. In the early part of the twentieth century, many inves- tigators supported the theory of (Pfluger (2) that lipid material is absorbed in the form of soaps (metallic salts of fatty acids). He accounted for the presence of neutral fats (triglycerides) in the lacteals by postulating a resynthesis of the triglyceride in- side the epithelial cells of the villi. Others, in agreement with Munk (3) were of the opinion that lipid material was ab- sorbed through the intestine in the form of a fine emulsion, formed with the aid of bile salts. Fundamentally, the question is whether lipids, which are insoluble in water, are changed into water-soluble soaps and glycerol before absorption or whether they enter the epi- thelial cells as globules of neutral fat. The first Opinion is based on the hydrolytic action of lipase. The second is based on the assumption that the lipid content of the epithelial cell wall acts as a solvent fo: neutral fat or that the globules of fatty materials pass between the epithelial cells directly into the lacteals. In essence. the controversy is the same today; evidence in support of each will be presented. Absorption in Particulate Form One of the earlier possibilities which stimulated investi- gation was the concept that leucocytes might possibly serve as intermediate carriers of lipid material from the epithelial cells to the lacteals. Clark and Clark (4) in microscopic studies of the lymphatic endothelium in 213.9 showed that upon injection of lipid material (olive oil, cream. and/or egg yolk) into the tail of frog larvae (tadpoles), the lymphatics send out sprouts toward the globule. Leucocytes responded quickly to the injected sub- stances by migrating toward it in large numbers and actively engulfing the lipid material. Their observations indicated an emulsification of the engulfed lipid. Transfer of the lipid from the leucocyte to the lymphatics appeared to be accompanied by a change to a more soluble. more absorbable form. 7 Histological studies by Leach (5) produced no confirmatory evidence, however, that leucocytes are active in lipid absorption. One of the primary methods of study of the absorption mechanism has been the addition of unsaponifiable matter. such as hydrocarbons. to fat. and the mixture fed to test animals. Bradley and Gasser (6) fed an emulsified mixture of olive oil and petroleum oil to dogs. The thoracic lymph which they obtained by fistula contained both oils in approximately the same relative prOportions as in the emulsion fed. Such a phenomenon suggested to them a mechanical absorption of drop- lets of fatty material and hydrocarbon oil mixtures. In I924, Gage and Fish (7) introduced a new tool in the study of fat absorption. namely the dark field microscOpe. Ex- amination of thoracic lymph and circulatory blood by dark field illumination under the high power of the microsc0pe indicated the presence of absorbed fat as minute particles, approximately half to one micron in diameter. These investigators suggested the term "chylomicrons" for the minute particles. Microsc0pic counting of the chylomicrons after fat in- gestion has since been a valuable index of absorption. Working with the blood of humans. the authors determined that the chylomicrons were indeed fatty material. The following evi- dence was opse rved: 1. Neither protein nor carbohydrate foods gave rise to chylomicrons in the blood. 2. Ingestion of fat always provoked an observable in- crease. 3. A chyle emulsion was determined to be fat, because: a) b) C) d) f) Particles rose to the top of the fluid. They left a grease spot on paper. Osmic acid, a selective stain for fat. responded with a characteristic black stain. Fat soluble dyes. such as Sudan IV. were char- acteristically soluble. A typical iodine absorption of fat was observed. Chyle emulsions showed a refractive index of fat. The observations of the above authOrs caused them to believe that the particulate matter observed in blood was a re— sult of absorption of unhydrolyzed fat in globule form. A few years later. Mellanby (8). in studies of the diges— tion and absorption of fats, asserted that lipase is not found in 9 the cat's intestine, thereby necessitating the absorption of un— hydolyzed fat. He further stated that bile salts are necessary fOr absorption of neutral fat. Liquid petroleum oil was found not to be absorbed. Paradoxically. Channon‘ and Collinson (9. 10) found that the addition of unsaponifiable material (liquid paraffin) to the diet of cats produces an increase in the unsaponifiable fraction of the liver. They suggested that this depends on solubility in bile salts. Later experiments by Channon and Devine (ll), after feeding n—hexadecane to cats, showed tissue absorption. The hydrocarbon was isolated chemically pure from omentum and perirenal fat. muscle and skin. In 1938. Frazer (12) introduced a new theory of fat absorption which supports the original work of Munk. although in modified form. This concept. called the ”Partition Theory." is stated as follows: ”Fat is absorbed both as fat and as fatty acid. Fat is absorbed by the lacteal—lymphstic route. short- circuiting the liver and passes directly to the fat depot. Fatty acids. however. are absorbed by capillariesudirectly to the liver via the portal vein. " He remarked that if the unsaponifiable 10 material incorporated (such as Vitamin A) is soluble in bile salts. then it would pass to the liver. Triglycerides, insolu- ble in bile salts. would pass unchanged to fat depots. Frazer's publications have dominated the literature during the past few years in the field of fat absorption. His original Partition TheOry has been extended by his own work and by the work of others; not, however, without dispute. Wotton and Zwemer (13) have taken photomicrOgraphs of epithelial cells and have studied their ability to take in fat in visible particulate form by the use of diets containing a fat soluble dye. Sudan IV. Their observations indicated that fat droplets pass through the cuticular border of intestinal columnar cells, traverse the cells and leave at the basement membrane into the marginal capillary. Droplets. thus absorbed. pass directly to the liver by the pOrtal system. Some of the fat in the villi passes via the central lacteal to the thoracic duct and through the heart into the capillaries of the lung. Fat of high or low melting point, of plant or animal Origin made no essen- tial difference in the histolOgical picture. Mineral oil did not pass through the columnar cells. ll Frazer (14). in a later investigation, fed adult white rats by stomach tube. feeding Sudan IV-stained solutions of olive oil and. as a comparison. redistilled oleic acid and glycerol. He remarked: "If the lipolytic hypothesis of V’erzar and McDougall is valid. one would expect fatty acid and glycerol to behave during and after absorption in a manner similar to neutral fat." The comparable analyses were made by: an es- timation of the fat administered and the amount remaining in the intestine; chylomicrOgraph observations, investigating the passage of fat along various pathways (simple observation suf- ficed for the lacteals, while chylomicrOgraphs were used for changes in systemic or pOrtal blood fat); histological studies using standard frozen sections of intestines for examination of intestinal cells. The following observations were made: 1. Neutral fat absorption gave rise to large globules in the intestinal cells, whereas fatty acid absorption showed a fine brown granular deposit. 2. Neutral fat absorption was accompanied by milky lac- teals. unnoticeable in fatty acid absorption. 12 3. Neutral fat could be traced to fat depots; when mod- erate doses were fed. it was not deposited in the liver. Fatty acid. however. deposited in the liver and did not appear in the fat depots. 4. Neutral fat absorption resulted in a systemic lipemia with little change in the portal blood. Fatty acid absorption caused a marked portal blood lipemia with little change in the systemic blood. In a simultaneous experiment. Frazer (15) fed groups of animals (albino rats) neutral fat; the control animals were fed the same fat with an excess of active lipase. Characteristic milky lacteals were observed in the first group of animals, whereas the second group (with added lipase) was typical of fatty acid feeding. A second experiment was made, in which a mixture of olive oil and a 1/20.) solution of sodium cetyl sulfate (a lipol- ysis inhibitOr) was fed to experimental animals. The rats, in which lipolysis had been inhibited, absorbed as much fat as the control animals. Samples of intestinal contents were examined, as a check for complete inhibition. with no increase in fatty acids noted after six hours. The conclusions of this work tend 13 to show absorption without hydrolysis, although sodium cetyl sulfate is a surface active compound and a good emulsifying agent. Frazer suggested that lipolysis is not necessary for triglyceride absorption. but that it determines the fate of ab- scrbed fatty material and provides fatty acid for soap and phospholipid formation. In Order that unhydrolyzed fat be absorbed from the in- testine. Frazer. Schulman and Stewart (16) believed that it must pass through the membrane of the intestine either in a state of molecular dispersion. as a diffusible complex, Or in a state of fine emulsion. They found no evidence for the first two conditions to show that triglyceride fat can be dis- persed in an aqueous medium by any known intestinal agents. Bile salts formed an association with fatty acid. but no complex- forming substance had been found that renders triglycerides diffusible. The third state. that of a fine emulsion. was demonstrated by examination of the chyle of rats fed olive oil. The chyle was found to consist of finely dispersed, negatively charged globules of olive oil with an average diameter of less than half a micron. This approximated the size of both the particles seen in the l4 intestinal cells and the chylomicrons found in the blood at the height of fat absorption. In suppOrt of the necessity of emulsification, the absorp- tion of paraffin. commonly believed unabsorbable. because of its inability to form a diffusible complex with bile salts, was demonstrated to be equal to that of olive oil if it were in a state of fine emulsification. Non-emulsified paraffin oil was not absorbed because of its inability to provide its own emul- sifying agent. In 3313 experiments revealed that a bile salt/oleic acid/ monOglyceride system was effective in producing spontaneous emulsification. fine dispersion and good stability at all points of the intestinal pH range of 6.0 to 8.5. Evidence for the above system. necessary for spontaneous emulsification. was found in an in w study of Frazer and Sammons (17) which demonstrated that after a five hour diges- tion of olive oil with pancreatic lipase. the hydrolysis products were fatty acid. diglyceride and monoglyceride; no free glycerol c0uld be found. The various factors which control the extent of hydrolysis, in y’ijrg, of fat by lipase were enumerated by Frazer (18). They 15 consisted of: length of experimental period, relative amounts of fat and enzyme. degree of oil dispersion, presence of ac- tivators (bile salts, albumin. calcium oleate) pH of the contin— uous phase and accumulation of cleavage products. Chylomicron studies indicated that after five hours. a peak was reached in fat absorption (90 per cent). Frazer felt. in view of this fact. that if the end products of hydrolysis were fatty acid and glyc- erides. it was obvious that a significant portion of the fat must have been absorbed as unhydrolyzed or partially hydrolyzed triglycerides. In order to accomplish an increase in lipolysis in _v_i_t_r__g after five hours, it was necessary to raise the pH to 8.5. This may correspond to the change in the intestinal pI-I which occurs in the lower end of the ileum (l9). PhotomicrOgraphs of intestinal cells by Frazer (l9) indi- cated that choline is effective in dispersing fat particles. This was demonstrated by feeding (by gastric tube) mixtures of olive oil and water, and olive oil and choline chloride to animals. Frazer, French and Sagrott (21) observed that the pas- sage of particulate fat through the outer membrane of the cell was dependent on the size and charge of the molecule and was 16 related to the normal function of the adrenal cortex. probably owing to its effect on water and electrolyte metabolism. Such passage may have been affected by absence or presence of choline in the diet. The transport of fatty acids through the cell did not appear to be markedly affected by these factors. Evidence in support of Frazer's Partition Theory has been reported by Becker. Meyer and Necheles (22). In clinical Chylomicron studies of fat absorption in youth and old age (human subjects). a definite decrease in the rate of fat absci'p— tion was demonstrated. The addition of lipase or Tween 80 (an emulsifying agent) caused a definite decrease in chylomicron count. At all times. however. the more aged group showed a higher degree of unsplit fat in the blood as well as a noticeable delay in reaching the peak of chylomicron counts. These ob- servations were explained by the fact that aging shows a sig- nificant decrease in pancreatic lipase. Absorption Following Lipolysis The original theory of the absorption of fat suggested by Pflliger, as mentioned before. in which complete hydrolysis occurs, has been supported by many relatively recent investigations. 17 The majority of workers. however. have realized that the for- mation of soap and the acid or neutral pH level of the upper intestine are incompatible. It is well knmvn that an alkaline reaction is rarely. if ever. found in the small intestine; gen- erally the higher levels are obviously acid and the lower levels less acid (23). A recent unavailable report by Schmidt—Nielsen (24) con- tradicts this belief. Bloor (25), in attempting to demonstrate saponification of fatty acids before absorption in the intestine. used the car- bohydrate ester of a fatty acid as a tracer. He fed isomannid dilaurate to dogs and collected the chyle. which he examined for Optical activity. The isomannid dilaurate was selected because of its strong Optical activity (lost upon saponification). low melting point. ability to emulsify and good utilization by the test animal. The subsequent Observation, the lack of Optical activity in the ether extract of the chyle. was interpreted as negative absOrption of the unchanged ester. Vérzar (26) agreed in part with Pfluger, insofar as the total hydrolysis of neutral fat by intestinal lipase was concerned. In Order to circumvent the inability of soaps to remain stable in 18 acid solution. he presented evidence to show that the presence of bile salts are important aids in the absorption of fat. He illustrated the fact that both taurocholic and glycocholic acids combine in an aqueous emulsion with fatty acids (palmitic. stearic and oleic acids) which dissolved readily and clearly. even at a pH Of 6.25 and subsequently passed through parch— ment membranes. Nephelometric measurements of the solutions of bile acids with fatty acids indicated a molecular relationship. At higher concentration, aggregates occurred. Diffusion experi- ments showed that in very dilute solution, nearly all the fatty acid was diffusible; in concentrated solution. only part diffused. Fine emulsions of neutral fat did not diffuse at all, even in the presence of glycocholic acid. Vérzar states that "only this action of glycocholic and taurocholic acid on the fatty acid makes the absorption of fats possible and explains it under physiolog- ical conditions!" Further experimentation and interpretation Of the work of other investigators led Vérsar (27) to believe that the action of bile salts was due to hydrotrOpism (a term describing the property by which one substance can bring an insoluble substance 19 into solution in water). His understanding of the relationship was that when there is a high fatty acid concentration. the solu- tion is of a poly-disperse nature and that the hydrotrOpic effect is brought about by the formation of a ring or shell of several bile salt molecules around each fatty acid molecule. in such a way that the water soluble group is Oriented outwardly. With low concentrations Of fatty acid. the complex is made up Of a single fatty acid molecule surrounded by several bile sale mole- cules. Because these particles can pass through diffusion mem- branes. they can be regarded as genuine molecularly dispersed systems. Quantitative relationships between the amount of bile acids necessarily excreted in the bile for the adequate absorp- tion of an arbitrary amount of fatty acid have indicated that approximately 240 grams of glycocholic acid would be needed. for the absorption of 50 grams of fat. It is Obvious that such an amount (assuming that the bile salt comprises approximately 2 per cent of the bile) is out of the question, in view of the fact that the normal secretion of bile in man is approximately only 500 ml. daily (28). Vérzar stated that. Obviously. there is another factOr in fat absorption. 20 The theOry. supported vigorously by Vérzar (29). that the resynthesis of neutral fat from fatty acids in the intestinal mucosa through the intermediate production of phospholipids, was first introduced by Sinclair (30). He demonstrated a dis- tinct change in the composition of the phospholipid fatty acids of the intestinal mucosa during the absorption of fat. This change was observed after cats were fed a fat of pronounced characteristics. when the effects On the amount and nature of phosphorylation in the intestinal mucosa were recorded. The degree of unsaturation. measured by the Iodine number. was considered the most convenient measurement for the particular fatty acid used. Artom and co~workers (31) injected and/or fed Orally sodium phosphate using radioactive £332. After extraction of the phospholipids of different Organs. they found a preponder- ance in the intestinal mucosa. liver and kidney. They explained their results as a phosphatide fOrmation in the intestinal mucosa and. especially. as a direct participation of the phospholipids in fat resynthesis in the intestine. They suggested a complete synthesis in the mucosa. not merely a substitution of fatty acid radicals. Zl Ackermann (32.) tested the absorption of lecithin from the frog intestine and confirmed the fact that it is split before absorption. The fatty acids released are synthesized into neutral fat. Bellini and Cera (35) reported that during the absorption of neutral fat the phosphatase activity is higher in the albino rat. However. Barnes. Miller and Burr (34) followed the in- corporation of a tagged fatty acid into both the phospholipid and neutral fat fractions of the intestinal mucosa of rats during fat absorption and could show no apparent parallelism between the rate of fatty acid incorporated into mucosa phospholipid and transport. Their interpretation was based on data obtained from the readily observable spectral absorptions of methyl esters of the conjugated fatty acids found in corn oil. Winter and Crandall (35). by the use of venous fistulae in normal unanesthesized dogs. obtained simultaneous blood samples from the portal and hepatic veins and from the femoral artery before and after fat absorption. The blood samples were analyzed for free fatty acids. No significant differences could be found to support the views of Frazer and his co-workers to the effect that fatty acids are removed through the portal systems. 22 In additional eXperiments. using radioactive phosphorus. Artom and Swanson (36) demonstrated the direct absorption of intact phospholipids. Labeled phospholipids (prepared from the liver of animals injected with P32) were fed by stomach tube to rats. The radioactive phosphorus content in plasma and liver was determined after three hours. They reported that some Of the phospholipid was. no doubt. split in the intestine. but that there was no doubt of the absOrption of some as the intact molecule. Further studies of the relationship of phospholipids to fat absorption were made by Zilversmit. Chaikoff and Enteman (37). They found. using dOgs as the experimental animal. that neither the amount nor the turnover of the phospholipid of the mucosa Or villi of the small intestine were affected by the ab- sOrption of cream. com oil or the fatty acids of com oil. Favarger (38). in recent studies. fed elaidic acid to dOgs and measured its incorporation in intestinal phospholipids at the end of six and a half hours. It is of interest. quali- tatively. that his results indicate that the phospholipids of the epithelial layer of the intestinal mucosa consistently contained 23 a higher concentration of elaidic acid than did those of the deeper layers. Bloom. Chaikoff. Reinhardt and Dauben (39) have re— . l4 . . . . . cently reported. using C synthesized palmitic aC1d (1n the form of a triglyceride) as a tracer. that 96% of the labeled fatty acid in lymph was in nonphospholipid form. The synthe- sized triglyceride was fed by intragastric intubation and the lymph collected by cannulae. Previous work of these investigators (40). in experiments _ , l4 . . . l4 . . . in which C labeled palmitic ac1d and C labeled tripalmitin were fed to rats. indicated that equal percentages of absorbed l4 . . . C in the chyle were recovered. They considered this as evidence against Frazer's Partition Theory. Reiser and Bryson (41) have made very similar obser- vations. By feeding known free fatty acids and triglycerides. they found that the route of absorption of free fatty acids and triglycerides is the same. In view of the exceedingly great volume of past research directed toward solving the problem of fat absorption. the back- ground material presented in this paper has necessarily been far from complete. An attempt has been made. however. to 24 present a review of the past investigations which have culmi- nated in the present controversy. EXPERIMENTAL STUDIES EXPLRIMEN TA L STUDIES MethodOIOgy In order to investigate the problem of lipid absorption. an approach was decided upon which utilized the unsaponifiable fraction of animal depot lipid, assuming changes due to exper— imental dietary influence. as an index of the assimilation mech- ani sm . Extraction of unsaponifiable material. The unsaponifi- able matter of animal depot fat was obtained in accordance with the official method outlined in ”Official and Tentative Methods of Analysis of the Association of Official Agricultural Chem- ists” (42). The procedure was as folIOws: 1. Five grams (5.00) of sample was weighed and trans- ferred to a flat-bottomed flask. Thirty ml. of 95% alcohol was added tOgether with 5 ml. of a 59% aqueous potassium hydroxide solution. The mixture was boiled under reflux for one hour. 2. The saponified material was transferred to an ex- traction funnel. The transfer was completed with first warm Z7 and then cold water until the total volume was approximately 80 ml. 3. The saponification flask was rinsed with 50 ml. of petroleum benzene and the contents added to the extraction funnel which had been previously cooled to room temperature. 4. The extraction cylinder and its contents were shaken for exactly one minute and allowed to separate into two distinct layers. 5. The extraction procedure was repeated 4 times with 50 ml. of petroleum benzene. The petroleum ether layer was then washed by shaking with a 10% solution of alcohol. Three washings were made using 25 ml. of the alcoholic solution. 6. The petroleum benzene layer was transferred quan- titatively to a previously weighed petri dish and evaporated on the steam bath under a current of air. 7. The residue remaining was heated in an oven at 100°C and weighed as unsaponifiable matter. Inasmuch as some interpretation of results was haped to be gained from quantitative differences in the amount of unsaponi- fiable matter. conditions were standardized as much as possible. All samples were saponified for the same length of time (1 hour) 28 and during the extraction process. each shaking was timed for one minute . The ”line test"-—-bi010gical assay for Vitamin D Ac— tivity (43). For each sample of unsaponifiable matter extracted from 5 grams of depot fat or liver. 3 21-day old rats were used. The animals were placed on the basal rachitOgenic ra- tion (Diet III) for a period of 3 weeks. At the end of this period. the rachitic rats were transferred to a diet consisting of the same basal rachitOgenic ration plus the unsaponifiable matter. previously irradiated. In the samples undiluted with c0rn oil. the unsaponifiable material was dissolved in ether and divided equally between the 3 rations; the ether was then allowed to evaporate completely before feeding. At the end of 10 days. the test animals were killed and the bone of the left fareleg re- moved. The "line test" was made on the proximal end of the wrist bone. The removed bone was cleaned of adhering tissue and placed in alcohol for at least 24 hours. A longitudinal median section was made through the end of the bone with a sharp scalpel in such a way as to eXpose a plane surface through the junction of the epiphysis and diaphysis. Each section was again sectioned in the same plane and immersed in a 2 per cent 29 aqueous solution of silver nitrate for approximately one minute. The sections were placed in distilled water and allowed to stand exposed to light. The degree of calcification was re- corded and checked by a competent authority. Irradiation of the unsaponifiable material. The extracted unsaponifiable material in the form of a thin film. previously dried and weighed in a new petri dish. was irradiated for 15 minutes using a COOper-Hewitt mercury arc lamp. The distance from the source of the ultraviolet light. 20 inches. and the time of exposure are both in agreement with original experiments performed by Steenbock and Black (44). Experimental animals. The test animals used in these experiments were albino rats of a known strain. long used at Michigan State College in the Vitamin Assay Laboratory. Twenty- one—day old rats. weighing from 45 to 60 grams. were used for the "line test" f0r Vitamin D activity. Young adult rats of approximately 140 to 150 grams in weight were used for the collection of depot lipid as described in Expe riment II. 30 Adult rats of approximately 300 grams in weight were used in Experiments I and II. Mate rials and Equipment Source of Ultraviolet Light: Extraction Equipment StOpcock Grease Fat Soluble Dye Ergo ste rol. crystalline Chemical Reagents COOper-Hewitt mercury arc lamp. 220 volt. Standard laboratory equip- ment used in extraction and refluxing procedures. An ether insoluble stapcock grease was prepared from glycerol and dextrin as recommended by Herrington and Starr (45). Hexyl Blue Easttnan Kodak Company 95% ethanol (Rossville Petroleum benzene (J. T. Baker Chemical Company Special Diets Diet I Stock Diet. Yellow Corn Meal. ground Ground \iheat Whole Milk. powdered Linseed Oil Meal Alfalfa 32.5% 25.0 22.5 20.0 6.0 31 Brewer's Yeast 3.0 Sodium ChIOride 1.0 With modifications as listed in individual experiments. 21g II RachitOgenic Diet. Yellow Corn Meal. ground 69.0% Wheat Gluten 6 20.0 Yeast. Brewer's 4.0 Casein 3.0 Calcium Carbonate 3.0 Sodium Chloride 1.0 Diet III Lipid Free. Sucrose 60.8% Casein (vitamin free) 24.5 Brewer's Yeast 4.9 Salts IV 4.9 Alfalfa 4.9 Expe riment I A preliminary experiment. making use of a fat soluble dye. hexyl blue. demonstrated the fact that the dye. dissolved 32 in fat. was deposited in the adipose tissue of the albino rat. This confirmed the work of many investigators. probably the first of whom were Mendel and Daniels (46). It was felt to be of interest to determine whether or not the presence of fat was necessary for the transport and absorp- tion of the dye. so a lipid—free diet. previously described. was fed. Eight adult rats were fed diets containing the fat-soluble dye. hexyl blue. Four received the dye. 200 mg. per kiIOgram of stock ration. dissolved in corn oil.2 Four animals received the dye in the same proportion. coated. by alcoholic evaporation. on sugar. and incorporated in a lipid-free diet. Two pregnant female rats were fed the dye dissolved in corn oil and incorporated in the stock ration. Observations and resultg. The dye. hexyl blue. was ab— 50rbed readily in the adipose tissue of the animals fed the diet which contained the dye dissolved in corn oil. It was observed that the dye apparently accumulated. Animals killed at the end of one week were stained a light blue. Continued feeding for a Mazola. 33 total period of 4 weeks resulted in very dark staining of fatty tissue. Animals fed on a lipid—free diet. showed a slower rate of staining than did the animals on a high fat diet. This. no doubt. was in part due to the fact that the animals on the high carbohydrate diet consumed less ration. It was apparent. how— ever. that the presence of fat in the diet was not essential for the absorption of the dye. A cursory examination of new born rats (bOrn from females previously fed the dye) failed to show the presence of the dye. indicating that the dye failed to cross the placental membrane. Almost immediately. however. the typical blue color became evident throughout the bodies of the suckling young rats. indicating passage through the milk. Experiment II In view of the theory that lipid absorption may occur in particle or globule form. it is lOgical to believe that such par- ticles may act as carriers for material dissolved in the fatty substance. In this event. the lipid. within which the material is dissolved, would act as a protective barrier against any 34 mechanism which would ordinarily provide for the removal of the foreign material. Therefore. an investigation was initiated in which pure unirradiated ergosterol. dissolved in fat. was chosen as the measurable foreign agent. The following exper- iment resulted: Twenty-four young adult albino rats were selected for the experiment. Twelve animals were divided into groups of 4 (2 males and 2 females) and each group was fed a diet con- sisting of stock ration incremented by 10% of a particular fat in which 200 mg. (0.2 per cent) of pure crystalline ergosterol was dissolved. The 3 fats used were butterfat. lard. and corn oil. The remaining twelve control animals received the same stock ration with the individual fat added. but without the ergosterol. After a 24-hour starvation period in order to reduce fat reserves. the animals were fed for a period of 3 weeks. during which time the average. approximate food consumption per animal was 240 grams. (230 grams per female. 250 grams per male.) Good weight gains during this period were recorded. At the end of 3 weeks. all animals were sacrificed and the animal 35 fat was removed from depots (subcutaneous. mesentary. genital). The liver of each animal was saved for subsequent examination. I-‘ive grams of the depot fat from each group of 2 ani— mals (male or female) was saponified by the procedure already outlined. the unsaponifiable matter extracted. dried. weighed. and irradiated with ultraviolet light. The unsaponifiable material. after irradiation. was then fed to rachitic animals. testing fOr an increase in Vitamin D activity over control animals by the "line test." Several levels were fed to the rachitic animals (the unsaponifiable diluted in a c0rn oil medium) because of the uncertainty of the biolOgical potency of the unsaponifiable. The same method was used for 5 grams of the livers extracted from each group of 2 animals. Representative samples made up of 5 grams of corn oil. containing 10 mgs. of pure unirradiated ergosterol were sub- jected to the saponification procedure. These samples were irradiated after saponification and extraction for 2 different lev- els of time. 15 minutes and 30 minutes. and fed to rachitic animals. (See Table III.) 36 In recapitulation. the method was as follows: Animals fed ergosterol dissolved in fat Depot fat removed. 5 grams saponified. and unsaponifiable irradiated fed to rachitic animals ”line test” for Vitamin D activity. Observations and results. Saponification and irradiation of the depot fat failed to show {any significant differences be- tween the animals fed ergosterol and the control animals. No differences could be observed between male and female animals in regard to absorption. even though dilution of the unsaponifi- able after irradiation was discontinued and the entire amount fed in equal parts to 3 rachitic animals. (See Table I.) The slight response evidenced by the "line test" when the total unsaponifiable matter from 5 grams of depot fat was fed to the 3 rachitic animals. was not interpreted as indicative of the presence of provitarnin D (ergosterol absOrbed). No stOrage of ergosterol was indicated in the livers of the test animals. Again the slight response to the “line test“ can not be attributed to the presence of ergosterol. (See Table II.) The complete healing effected by the synthetic samples. ergosterol dissolved in corn oil. definitely indicated that the 37 procedure of saponification and irradiation was a sufficiently accurate method of determination and showed that the process did not destroy the Vitamin D activity. (See Table III.) Expe riment III Because no biOIOgical response was obtained in the pre- ceding experiment. a third experiment was inaugurated which still used the unsaponifiable material of depot fat recovered as the index of absorption. Twelve adult animals were selected and placed on the following diets: 1. Animals I and II received a diet consisting of: 900 grs. of stock ration 98 grs. of lard Z grs. of crystalline ergosterol (dissolved in the lard) 2. Animals III and IV received a diet consisting of: 900 grs. of stock ration 100 grs. of lard 3. Animals V and VI received a diet consisting of: 900 grs. of stock ration 88 grs. of lard 38 10 grs. of mineral oil 2 grs. of crystalline ergosterol (dissolved in mineral oil and lard) 4. Animals VII and VIII received a diet consisting of: 900 grs. of stock ration 90 grs. of sugar (as carrier for mineral oil) 10 grs. of mineral oil 5. Animals IX and X received a diet consisting of: 900 grs. of stock ration 99 grs. of lard 1 gr. of cholesterol (dissolved in the lard) 6. Animals XI and XII received a diet consisting of: 900 grs. of stock ration 9-1) grs. of lard 10 grs. of mineral oil (dissolved in the lard) The test animals were fed for a period of one month. during which time normal weight gains were noted. 'At the end of this feeding period. the animals were killed and the depot fat and livers removed. 39 Depot fat of animals which received higher levels of ergosterol was irradiated in a manner similar to that described in Experiment II. with subsequent feeding to rachitic animals. Control samples of unirradiated. unsaponifiable material was also fed to rachitic animals. In addition. fecal matter from ani- mals fed ergosterol was saponified. extracted. irradiated and the unsaponifiable residue fed to rachitic animals. In each experi- ment. negative control animals were used in order to demon- strate that rickets was produced by the basal rachitOgenic diet. (See Table IV.) Observations and results. Depot fat of animals I and II failed to show an absorption of ergosterol. even though the amount of ergosterol dissolved in lard was increased tenfold. Irradiation of the unsaponifiable material extracted from the depot fat made no apparent difference in the amOunt of Vitamin D activity; irradiated and unirradiated samples each showed a very slight but atypical calcification. The irradiated unsaponifiable matter extracted from five grams of liver of Animals I and II showed a definite biolOgical activity. As an interesting comparison. a second sample. 40 unirradiated. showed a very slight line of calcification. not. however. attributable to Vitamin D. b‘aponified and extracted fecal specimens of Animals I and II. upon irradiation effected complete calcification of the bones of the rachitic rats. Both depot fat and liver of Animals V and VI. after saponification. extraction and irradiation showed a positive cal- cification. The line was definite and sufficiently positive to warrant further investigation. These animals were fed ergos— terol and mineral oil dissolved in lard which was incorporated in their diet (Diet No. 3). Saponified. extracted. and irradiated fecal specimens of Animals V and VI also effected complete calcification of the bones of rachitic animals. The depot fat and liver of Animals VII and VIII (origi- nally fed mineral oil, carried on sugar) failed to show a sig— nificant increase in unsaponifiable matter in depot fat or liver (Diet No. 4). No significant increases in total unsaponifiable matter were observed in the depot fat and liver of Animals IX and X. previously fed a diet with cholesterol added (Diet No. 5). 41 Animals XI and X11. received a diet containing mineral oil. but no quantitative increases were observed in the unsaponi- fiable portion of the depot fat and liver. 42 «>43qu .45 N 2.433: 48 4 48 4: .m8 95 :0 58 a saw. a 4m via—«mu: .45 N gram»: 48 4 48 4: .m8 v.2 :0 :80 a .44 s a steamed .44.: N gamma: 4S 4 48 S .wE N4: «3.433444 s4 *3 s 2 uzusmon .45 N . cinema: .45 4 .45 o4 .mE N.o~ nauseousm 2 w a h .3070 Emma .. .9: o.: «3.40345 h .1: 4w : .e4u43 £34» : .m8 2: “£335 a c a m 44048 2494a : .3: «44 4:3 2 a: a 2 44043 3?: $225 .8 Q4. .mE 3.4 4:3 2 4. a m 2.42.3: 48 4 .48 3 .m8 44.2 v.34 a $4 .4 2 032w»: 48 4 45 cm .mE 9.2 4:45 a N .4 4 a 348 3m was «5584‘ 484345 233% 30444; 3.48442 Imfiomaog snow is 4aE4q< a c.4843; .. 33.4. 2.44.. so 333m so Eats .I'I.Itclxr‘|ss. iii; IIII I“. it» )1 3'33... litifoill‘ll. . It'll-I'll.’ a 56, ii I is... xlItslils. {II-Q ti‘i.\'i girl! lilo! AH mo 3033s 0.4a :aoflsoflmgso 2.43;: on page: somsoamom Jam «omen uo madam m Souk s... 493:0qu on Jigsaw 40.3480 .4. I... I'll bill . I Iii- I'll-l 'IIIII 4438 234. : .mE 3.4 45 44.80 54 *3. .4 3 4423 Sun. A4225 so Q4 .mp4 e.4~ 44.0 :80 42 N4 s 44 undoamum ooh ugoa< 4.8335 2.035% vacuums: 3030.? 20m nungz is 444884.. a 44483:, n :33. 2.44: .8 333m so .4433... IIIIIOIIII. i"! as}?! . (All-I‘ll El I'll.‘.'vl .lll I...Il||n|rl...0l|ls l It‘ll. I (I I'll-ll. III. . It: ll. ,1 I D.‘II.|....'\‘!I' ll it‘ll-lull! In}!!! 4334.488 4 mqmfi. 44 .333”; Q 4385? .3 «sugar»; 0.: :aofisgfigso 39:: as been: nousoamom 33>: mo 3:st m Scum s... .63 40.40309; on 33844: 40.34400 s .Illv-I‘Ii ‘1' -‘n‘l’ ll‘ "ll' . 'll'"".s'l|l.l.- Isn‘t!" 4423 2?: : .3. ~54 440 980 a .3 s 3 44943 Emma A : .3: ~42 :0 980 a N4 .4 44 44948 2434. : .3: 44.3 «sarcasm a $4 a 2 .3043 Ewan : .uE v.4: sarcasm a o a m 4643 Emu. : .448 m4: 4:84 a $4 a 2 4433 44434. 4833: do Q4 .3: 44.4.4 4:45 a N .4 4 unconsom ooh «goat. sagas: Iowans-54 3qu> xom hung—A is 444845. a 4.483; .. :23. 2444: so 344.344 so 2303 ii“! I I'IIiII'II" .‘II. B‘s-I'll... I I III! I! :I1 I '11", :II It." III-Ills .t .ll’ll'l I, .11... III-'nv..ltzi 4.4.54 so .34: 4ommamoomm one 35244.3. so meme/44 : HJQ 4.43.3034in >443 304.485 .48 4 .48 3. 834.44.94.44 :0 880 .88 on >4 soflsoflwoio >443 3o4n48°o 48 4 48 3 4.3.44.8: 440 44.80 .88 m4 444 48:34:25 4384384 304.488 48 4 48 ON 434345.43 :0 500 .88 o... 44 3:34:26 44.34.443.84 301485 48 4 48 S c5835 :0 8.80 848 m4 4 ouaomuom pub «54064.44 43535 0 08:. pungz «4.42.45.40.48 04 30> aura noflsflvsu: cadusm o 883$ .. 33.4. 2444: «a 34334 QMHWHZOnjwm I 4.41:0 7: AOdNHmOUfiH I mH4l2 4» 44> .m8 4...: v 8.4 44444. .44 44> 544344843 304.483 3.4. .m8 ~.$4 m 83,44 444 .4 > 4844844428 40 2.44 4.3.34 .4...» .m8 44.4.4 m .4244 444 .4 > 544344433 4.. 2.44 4.3.5 3» .m8 .3 m 8.4 444 .4 > 48443444043 imam 3r .m8 ed... ~ 42.44 >4 .4 444 4844344428 4444443 3» .448 ~....4 N 8,4 >4 .4 444 544344643 4.43;. 02 .m8 44.4.4 4 .334 44 .4 4 544344423 40 2444 4.344 3.» .448 ~.- 4 82.44 44 .4 4 48443444043 334488 4....» .8. 44.4.4. 4 38,4 44 .4 4 4844344423 444944... oz .m8 ~.m4 4 24.4 44 a 4 45443444943 imam 3» .88 44.4.4 4 8:4 44 .4 4 358044‘ Q 4283:, 033m 4034.542 40.445442 . . . . 4404534494.: ImnomaunD 04538 I 44:08 054: mo magnum uumn 1244.434 3 444335 ‘III.r. - V .11!“ III.’ I" ('4' .1.» I!‘ mHMHQ mDOHZ<4JMUmH§ ZO mdgflfifi I Gmm>ud Q24 HH H4m<8 47 .334an Q 4448344/ no 3044:: v.44— ..nofiaowfigdu Emma: nu v3»: munnomnom .voGEOQNQ usauw m .4. .IIIAI 1.3!)! Iu‘i' 0"... Ills, .u.’ .D. it‘..‘|" .I I‘ll). L‘l'. . . .‘Q’u’I’lu‘! nl‘n'. I'i'lvlllu. I. ..I I I (0‘11! ’lt'l .1 .llll .‘L. llIOIn.. in. A II! t; (I‘ll iliii III. o ‘iil’l’ .448 44.4 .4 33.4 4444 .4 444 .m8 .2. 44 3.4 4444 .4 444 .m8 .4....4 m .3344 x .4 x4 .48 :4 m 8.44 x .4 x4 > Cr 0 :45 4 333m .40 E: .3 844 new 30M4Q .o ”div 44053294.: 04439434: no Z wanna. ducal .49 .H. .‘H: u 3 m MO «Ammoa. u 5 u . < Illil‘llnl 'lln'lllalll ll'ul'f 'i‘ln. till ‘0); ll."} . 1.-.... nl. Ili III. ulnI‘IIU. .l.‘ 44.08.448.04 >4 ”444.45. DISCUSSION DISCUSSION In the foregoing investigations. attempts were made to augment the content of the unsaponifiable material associated with the depot fat of eXperimental animals by additions to the diet. By such a procedure. it was hOped that information would be obtained that would help to explain the mechanism of fat absOrption. The preliminary study of the absorption of a fat-soluble dye failed to Show any striking differences of absOrption in animals fed a diet high in fat and those animals fed a dye relatively free of fat. The rate of absOrption. as evidenced by the degree of staining of adipose tissue, however, was slower in animals fed the dye in a lipid-free diet. The absorption of a fat-soluble dye incorporated in a diet lacking a fat vehicle has been explained by the fact that fat has been found in the intestinal contents of animals fed a fat-free diet. presumably resulting from bacterial synthesis or from cellular debris. Schoenheimer and Rittenburg (47). in one of their clas- sical studies, using fatty acid material labeled with deuterium incorpOrated in diets. found that a large percentage of the fatty 50 material fed was laid down in the fat depots before utilization. Therefore, if particulate fat. with unsaponifiable matter incor- porated. accumulated in the fat depots. some information of this absorption mechanism should result. Although the same investigators. mentioned directly above (48), found that pure ergosterol fed to experimental ani- mals was not absorbed, it was felt that the possible protective action of the fat. in which the plant sterol was dissolved. might aid in its absorption. The results of the investigations in Ex- periment 11 failed to confirm such a possibility. Based on total average food consumption of the test animals. 230 grams of ration consumed by female and 250 grams of ration consumed by male animals, it was calculated that each animal ingested from 45 to 50 mg. of pure crystalline ergosterol. Assuming the total body fat of a 200 gram animal to be approximately 25 grams, a 5 gram sample of the depot fat should have contained an amount of ergosterol. which. upon saponification and irradia- tion, would be far greater than needed for complete healing of rickets. In all of the bones examined for calcification. which were removed from rachitic animals fed undiluted, unsaponifiable 51 matter. a thin high line of calcification was observed. This could not be attributed to any biOIOgical activity due to the ab- sOrption of ergosterol. Neither could it be attributed to the activation of 7-dehydrocholesterol. because unirradiated mater- ial showed the same type of calcification. Presumably, this was caused by failure of the rachitic animals to eat an adequate amount of food. The investigations of Steenbock and Black (44) found that starvation of rachitic animals caused such high lines of calcification. Because of the absence of any biological response as evidenced by Vitamin D activity, no conclusions could be reached with regard to differences in the absorption of the three fats (lard, corn oil and butterfat) used as vehicles for the ergosterol. In Experiment III, the depot fat of animals fed a tenfold increase (as compared to those in Experiment II) of ergosterol, failed to show absOrption. The absence of any Vitamin D ac- tivity after irradiation seems to indicate a selective mechanism in the intestine which removes foreign material before absorp- tion of the fat occurs. PrOponents of the hydrolytic theory would attribute these results to the hydrolysis of the neutral fat. leaving the foreign plant sterol free for elimination. An 52 indication of this possibility has been reported by Irwin. Steenbock and Templin (49) in studies of the absorption rates of various natural fats. In fOrced feeding experiments with subsequent analysis of the contents of the alimentary tract. they found that the amount of unsaponifiable matter present in the fat. influenced the rate of absOrption. A significant difference was observed in the Vitamin D activity of the unsaponifiable matter of the liver of EXperimen- tal Animals I and II. The observation is even more interesting in view of the fact that the unirradiated sample showed no ac- tivity attributable to Vitamin D. Ergosterol. a plant sterol essentially foreign to the animal body. was definitely demonstrated to be eliminated un- changed. lrradiation of the unsaponifiable matter from the feces of test animals fed ergosterol, with subsequent refeeding to rachitic animals. caused complete healing. The excretion by way of the gut of foreign materials in such large amounts was not unexpected. The experiment in which test animals were fed ergosterol and mineral oil. both dissolved in fat. proved interesting by the fact that both the depot fat and liver showed activity. This is 53 of particular note because of the comparatively low amount of unsaponifiable matter obtained from the 5 grams of depot fat. A duplicate sample of the fat was subjected to the saponifica— tion and extraction procedure simultaneously and was quantita— tively comparable. If there is a selective mechanism far the removal of foreign material from fat. the above observations may possibly be interpreted as an interference mechanism. Perhaps the presence of the mineral oil interfered with the normal removal of ergosterol and allowed absorption of the provitamin D. Such a possibility warrants additional investi- gation. Test animals VII and VIII. IX and X, and XI and X11. with mineral oil coated on sugar, cholesterol, and mineral oil alone. fed respectively. failed to show any quantitative increases in the amount of unsaponifiable matter present in depot fat and liver. In view of the particulate theOry of fat absOrption, def- inite increases would be expected. That the study of fat absorption in particulate form is of extreme impartance. is emphasized by recent work of Setala and Ermala (50). They have stressed the importance of lipid chylomicrons in the blood as the transporting agents for 54 water-insoluble carcinogenic hydrocarbons. They believe that because of solubility in fat. the mechanism of carcinOgen ab- sorption is closely allied with the absorption of lipids. The apparent interference mechanism previously ascribed to mineral oil in combination with ergosterol, indicates some evidence in favor of the particulate theOry of fat absorption. In view of these considerations. it is felt that further investigations of the problem are warranted using similar techniques. SUMMARY I. SUMMARY A study of the absOrption of fat containing various amounts of added unsaponifiable matter (hexyl blue, ergosterol. cholesterol. and mineral oil) was made. A fat-soluble dye. hexyl blue. was found to be laid down accumulatively in the adipose tissues of the albino rat. No marked differences in absorption. using a diet high in fat and a diet relatively free of fat. were observed. Unirradiated ergosterol, incorporated in a diet high in fat was found to be eliminated in the feces. Saponification and extraction of the feces yielded unsaponifiable matter. which. upon irradiation, effected complete healing of rachitic rats. In experiments using ergosterol. dissolved in fat, as a tracer of fat absorption. the following observations were made: a. Ergosterol. fed in a high fat diet at a leval of 0.2% of the fat, was not found in depot fat or liver. as evi- denced by a lack of Vitamin D activity after irradia- tion of the unsaponifiable fraction. 57 b. Ergosterol. fed in a high fat diet at a level of 2% of the fat. resulted in Vitamin D activity in the irradiated unsaponifiable fraction of the liver. Unirradiated sam- ples showed no such activity. nor did the irradiated unsaponifiable fraction of depot fat. c. A combination of mineral oil and ergosterol (2% of the fat), dissolved in lard was fed to test animals. Saponi- fication and extraction of M depot fat and liver yielded unsaponifiable matter. which. upon irradiation. showed Vitamin D activity. This surprising observa- tion perhaps may be explained by the fact that the presence of mineral oil affects the selective mech- anism in the intestine which ordinarily prevents the absorption of ergosterol. Mineral oil. dissolved in lard or carried on sugar in the absence of fat, when added to the diet. failed to produce a measurable increase in the amount of unsaponifiable matter of depot fat. or livers of test animals. The results of these studies have not contributed sufficient evidence to warrant support of the exclusive Operation of either of the two principal theories of the absorption of fat. 58 They do. however. offer encouragement for further study of the problem. BIBLIOGRAPHY 10. BIBLIOGRAPHY Best. C. H.. and N. B. Taylor. The PhysiOIOgicaI Basis of Medical Practice." 3rd Edition. Williams 8: Wilkins (30.. Baltimore. Maryland. 1943. Rfluger. E.. Pflugers Arch.. Q, 111 (I900). Munk. ”Lehrbuch d Physiol.." 199 (1898). Clark. E.. and E. Clark. A Study of the Reaction of Lymphatic Edothelium and of Leucocytes in the Tad- pole's Tail toward Injected Fat.. Am. J. Anat.. Q. 421 (I917). Leach, E. 1-1.. The Role of Leucocytes in Fat Absorption, J. Physiol.. 33. l (1938). Bradley, H. C., and H. S. Gasser. Intestinal Absorption. J. Biol. Chem., Ll. xx (1912). Gage, S. H.. and P. A. Fish. Fat Digestion, Absorption, and Assimilation in Man and Animals as determined by the Dark-Field MicroscOpe and a Fat—Soluble Dye. Am. J. Anat.. 11. l (l924—-l925). Mellanby. J.. The Digestion and Absorption of Fat; Petro- leum Emulsion in the Small Intestine. J. Physiol., £31, Proc. v and xxxiii (I928). Channon. H. J.. and G. A. Collinson, The BioIOgical Sig- nificance of the Unsaponifiable Matter of Oils, Biochem. J.. _Z_Z. 391 (1928). Ibid.. The Absorption of Liquid Paraffin from the Alimen- tary Tract in the Pig and Rat, _2__._3_. 676 (I929). ll. 12. l3. 14. 15. 16. I7. 18. I9. 20. 21. 22. 61 Channon. H. J.. and J. Devine. Absorption of n—Hexadecane from the Alimentary Trace of the Cat. Biochem. J.. ;_s_. 467 (1934). Frazer, A. 0.. Fat Absorption and Metabolism, Analyst, 23, 308 (1938). Wotton, R. M.. and R. L. Zwemer. Studies on Direct and Visible Ingestion of Fat by Differentiated Body Cells in the Cat. Anat. Record. 1;, 493 (1939). Fraser. A. C.. Differentiations in the Absorption of Olive Oil and Oleic Acid in the Rat. J. Physiol.. 102. 306 (1943). Frazer, A. C.. Lipolysis and Fat Absorption. J. Physiol.. 102. 329 (1943). Fraser. A. C.. J. H. Schulman and H. G. Stewart, Emul- sification of Fat in the Intestine of the Rat and its Relationship to Absorption. J. Physiol.. 103. 306 (1944). Frazer. A. C.. and H. G. Sammons. The Formation of Mono- and Di~Glycerides during the Hydrolysis of Triglyceride by Pancreatic Lipase., Biochem. J., 12. 122 (1945). Fraser, A. C., The Absorption of Triglyceride Fat from the Intestine. Physiol. Rev.. _2_6. 103 (1946). Frazer, A. C.. The Absorption of Fat from the Intestine. Chem. and Ind., 379 (I947). Frazer. A. C.. The Effect of Choline on the Intestinal Ab— sorption of Fat. Nature, 121. 414 (1946). Frazer. A. C.. J. M. French and P. E. Sagratt. The Intes- tinal Cell in Fat Absorption, C. A. 151. 2563 (I951). Becker. G. H.. J. Meyer and H. Necheles. Fat Absorption in Young and Old Age. GastroenterOIOgy, _l_4_l_, 80 (I950). 23. Z4. Z6. Z7. 28. 29. 30. 31. 32. 33. 34. 62 McClendon. J. F.. Acidity Curves of Stomach and Duode~ num. Am. J. Physiol.. §_8_. 191 (1915). Schxnidt—Nielson. K.. "Annual Rev. of Biochem.." Annual Reviews. Inc.. Stanford University. California. 253 (1948). Bloor. W. R.. On Fat Absorption. J. Biol. Chem.. LI. 429, (1912). Vérzar. F.. The Absorption of Fat. Am. J. Physiol., 2Q. 546 (I929). Vérzar. F.. Fat Absorption. Nut. Abstracts and Revs.. 2. 441 (1933). Best. C. H.. and N. B. Taylor. "The PhysiolOgicaI Basis of Medical Practice." 3rd Edition. Williams & Wilkins Co.. Baltimore. Maryland (1943). Sperry. W. M.. "Ann. Rev. of Biochem.." Annual Reviews. Inc.. Stanford University. California. 231 (1939). Sinclair. R. J.. The Role of Phospholipids of the Intestinal Mucosa in Fat Absorption. J. Biol. Chem.. pg. 117 (1929). Artom. C.. Rate of 'Organification' of Phosphorus in Animal Tissues. Nature. 139. 836 (1937). Ackerman. J.. "Ann. Rev. of Biochem.." Annual Reviews. Inc.. Stanford University. California. 172 (1938). Bellini. I... and B. Cera. The Intestinal Phosphatase during the AbsorptiOn of Neutral Fat in Normal and Rickety Rats. C. A.. Q3. 7350 (1940). Barnes. R. H.. E. s. Milleriand G. o. Burr. The Absorp- tion and Transport of Fatty Acid Across the Intes- tinal Mucosa. J. Biol. Chem.. 140. 233 (1941). 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 63 Winter. 1. C.. and L. A. Grandall. The Question of the Portal Absorption of Fatty Acids. J. Biol. Chem.. 140. 97 (1941). Artom. C.. and M. A. Swanson. On The AbsOrption of Phospholipids. J. Biol. Chem.. 175, 871 (1948). Zilversmith. D. B.. I. L. Chaikoff and C. Enteman, Are Phospholipids Obligatory Participants in Fat Trans- port Across the Intestinal Wall? J. Biol. Chem.. _1_Z_2_. 637 (1948). Favarger. P.. La Participation des phospholipides a la resorption des graisses dans les différentes regions de l'intestin de chien. Helv. Physiol et Pharmacol. Acta, 1. 371 (1949). Bloom. B.. I. L. Chaikoff. W. O. Reinhardt and W. G. Dauben. Participation of Phospholipids in Lymphatic Transport of Absorbed Fatty Acids. J. Biol. Chem.. _1__8_9. 261 (1951). Bloom. B.. I. L. Chaikoff. W. O. Reinhardt, C. Enteman and W. G.' Dauben. The Quantitative Significance of the Lymphatic Pathway in Transport of Absorbed Fatty Acid. J. Biol. Chem.. L83. 1 (1950). Reiser. R.. and M. J. Bryson, Route of Absorption of Free Fatty Acids and Triglycerides from the Intestine. J. Biol. Chem.. 182. 87 (1951). Official and Tentative Methods of Analysis. A. O. A. C.. 6th Edition. Washington, D. C.. 504 (1945). McCullum. E. V.. and N. Simmons. A Delicate BiolOgical Test for Calcium-Depositing Substances. J. Biol. Chem.. _5__l_. 41 (1922). Steenbock. H.. and A. Black. The Induction of Growth-Pro- moting and Calcifying PrOperties in Fats and their Unsaponifiable Constituents by Exposure to Light. J. Biol. Chem.. 63. 263 (1925). 45. 46. 47. 48. 49. 50. 64 Herrington. B. L... and M. P. Starr. An Ether—Insoluble Stopcock Lubricant. Ind. Eng. Chem.. Anal Edit.. 14. 62 (1942). Mendel. L. B.. and A. L. Daniels. Behavior of Fat-Soluble Dyes and Stained Fat in the Animal Organism. J. Biol. Chem.. _1_3. 71 (1912). Schoenheimer. R.. and D. Rittenberg, Deuterium as an Indicator in the Study of Intermediate Metabolism. J. Biol. Chem.. 111. 163 (1935). Schoenheimer. R.. New Contributions in Sterol Metabolism. Science. 14. 579 (1931). Irwin. M. C.. H. Steenbock and V. M. Templin. A Tech- nique for Determining the Rate of Absorption of Fats. J. Nutrition. _1__2_. 85 and 103 (1936). Setala. K., and D. Ermala. Chylomicrons as Carriers for Carc0genic Hydrocarbons. Science. 114. 151 (1951). 11111111111111“