FACTGRS AFFECTING THE FOaMA'I‘IOfl OF mm A FROM B-CAROTENE IN VITRO USING WODENUM AND OTHER TISSUES PROM DIFFERENT ANIMALS Thesis for I!“ Dogm of Ph. D. MICHIGAN STATE UNIVERSITY Bandeau Siverama Roddy I963 1H ESI. LIBRARY Michigan State University This is to certify that the thesis entitled FACTORS AFFECTING THE FORMATION OF VITAMIN A FROM B-CAROTENE IN VITRO USING DUODENUM AND OTHER TISSUES FROM DIFFEfiENT ANIMALS presented 9 Bandaru Sivarama Reddy has been accepted towards fulfillment of the requirements for PhoDo degree in Dairy J /V flew . fMajor professor ./ - I Date May 13, 1963 0-169 FACTORS AFFECTING THE FORMATION OF VITAMIN A FROM B-CAROTENE IN VITRO USING DUODENUM AND OTHER TISSUES FROM DIFFERENT ANIMALS BY Bandaru Sivarama Reddy AN ABSTRACT OF A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy I963 ABSTRACT FACTORS AFFECTING THE FORMATION OF VITAMIN A FROM B-CAROTENE IN VITRO USING DUODENUM AND OTHER TISSUES FROM DIFFERENT ANIMALS By Bandaru Sivarama Reddy The study herein was conducted to obtain whether an in 31352 system modified from several investigators was satis- factory for studying vitamin A formation from B-carotene using duodenum and other tissue homogenates from different animals. When fresh duodenal homogenates from different animals were incubated with aqueous carotene suspension and 0.25M sucrose for 3 hours at 3700, a considerable amount of vitamin A was formed. Much more vitamin A was formed when 0.25M sucrose was used than when any of the more commonly used buffers were employed. The formation of small amounts of vitamin A were more readily detected and measured when the saponification was performed on the incubated mixture. The conversion process appeared to be a rapid reaction. The tissue homogenized under nitrogen was more effective in vitamin A formation than the tissues homogenized under oxy— gen. The conversion process studied was apparently enzymic. The extent of vitamin A formation was greatly reduced when the homogenates were kept one hour or more before an addi- tion of the carotene suspension. Conversion was greatly reduced by heat, KEN, urea, N03 or iodoacetate but not appreciably reduced by 802, NaCl or CUE. 2 Identification of vitamin A formed by this system was carried out on chromatographed tissue extract by various methods such as absorption spectra, changes in spectra after irradiation, maleic anhydride method, conversion to anhydrovitamin A and paper chromatography. The small intestine especially the duodenum and liver were found to contain the most activity for conversion of carotene to vitamin A in all animal species tested. Many tissues were found to possess the ability to form small amounts of vitamin A from carotene. The decrease in activity of vitamin A formation pro- duced by N03 or NO} was somewhat specific. Large amounts of N03 added to the in vitro system produced a marked in- hibition of vitamin A formation whereas small amounts pro- duced little or no effect. The extent of conversion was decreaSed by using duodenal homogenates from animals fed N03 or NOE. Duodenal homogenates from hypothyroid and thyroidect- omized animals considerably reduced the formation of vita- min A from B-carotene as compared to the duodenal homogen- ates from control and hyperthyroid animals, whereas no difference was observed between the duodenal homogenates from control and hyperthyroid animals. Addition of high levels of'L-thyroxine or L-triiodothyronine to the in vitro system reduced the extent and efficiency of vitamin A for- mation. The inhibitory effect of 3 5' 3"L-triiodothyronine 3 on vitamin A formation was reduced to certain extent by the addition of isomers of thyroxine and triiodothyronine. Little or no vitamin A was formed when carotene was incubated with hemolyzed blood or sedimented blood cells. Vitamin A formation was markedly reduced when these frac- tions were incubated with carotene and duodenal homogenates. The inhibitory factor was located in the sedimented blood cells. When the duodenal homogenates were centrifuged at 5000 X0, 10,000 KB and 25,000 XG and the resulting supernatant and sedimented fractions were incubated with carotene, the supernatant fraction from a 5000 X0 preparation contained more activity than that in the sedimented fractions from 15,000 KB and 25,000 XG preparation. FACTORS AFFECTING THE FORMATION OF VITAMIN A FROM B-CAROTENE IN VITRO USING DUODENUM.AND OTHER TISSUES FROM DIFFERENT ANIMALS BY Bandaru Sivarama Reddy A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1963 ACKNOWLEDGEMENTS The author wishes to express his gratitude to Dr. J. W} Thomas for his advice and guidance throughout the course of the investigation and for critically reading this manuscript. The author is also grateful for the critical reading and helpful suggestions of Dr. H. D. Hafs, Dr. J. Meites, and Dr. C. J. Pollard. The encouragement and suggestions of Dr. L. D. Brown and Dr. R. S. Emery are also deeply appre- ciated. The c00peration extended by the staff and the grad- uate colleagues of dairy cattle nutrition section is also acknowledged. The author is indebted to Dr. C. A. Lassiter for the appointment of research assistantship thereby making this investigation possible. The author wishes to thank his wife, Subhashini, for her unselfish help and encouragement during the preparation of this thesis. TABLE OF CONTENTS INTRODUCTION ........ . ..................... ' ........... REVIEW OF LITERATURE ................................. In vivo conversion of B-carotene into vitamin A. Conversion of carotene to vitamin A by the organs and body fluids other than intes- tine and liver.... ............ .. ........... Products formed when animals were given radio— active B-carotene .......................... In vitro conversion of carotene to vitamin A.... Effect of nitrate and nitrite on the conversion of carotene to vitamin A ................... Importance of bile acids on the conversion of carotene to vitamin A ..... ..... ..... . ...... Importance of tocopherol on the utilization of carotene and vitamin A .................. Interrelationships between thyroid status and vitamin A .......................... ........ The effect of the thyroid gland on the con- version of carotene to vitamin A ........... Mechanism of conversion of B-carotene to vitamin A... ............................... EXPERIMENTAL PROCEDURE ............................... Animals, diets and treatments ................... Reagents and solutions .......................... Procedure ....................................... In vitro experiments using tissue homogenates.... ....................... In vivo conversion of B-carotene to Vltamln A using intestinal lOOpS ...... Page 15 18 23 26 29 36 42 48 54 54 59 62 62 69 iv Page Estimation of carotene ..................... 7l Estimation of vitamin A .................... 71 Determination of carotene and vitamin A in plasma ........................... 82 Determination of carotene and vitamin A in liver ............................ 83 RESULTS .............................................. 85 Preliminary experiments ......................... 85 Isolation and identification of vitamin A derivatives from incubated material ........ 98 Formation of vitamin A by various organs ........ 123 Effect of N03 ion on vitamin A formation in vitro and in vivo ....................... l30 Thyroid status and vitamin A formation .......... 148 Effect of ions and enzyme inhibitors ............ 162 Effect of blood and its fractions on vitamin A formation ........................ 168 Vitamin A formation by cell fractions of duodenal homogenates ................... .... l76 DISCUSSION ........................................... 180 SUMMARY .............................................. 202 LITERATURE CITED ..................................... 207 APPENDIX ............................................. 224 TABLE 10 11 12 LIST OF TABLES Page Incubation of duodenal homogenates with different levels of various chemicals ......... 67 Extent and variation in formation of vitamin Aefter incubating carotene with duodenal homogenates (Experiments 1 & 2) ............... 86 Effect of altering carotene to tissue ratio on amount of vitamin A formed ................. 89 Effect of incubation time on the extent of formation of vitamin A and the recovery of carotene using duodenal homogenates from non-depleted animals .......................... 91 Influence of various buffers as incubating media on the formation of vitamin A from carotene ...................................... 93 Effect of heating duodenal homogenates on vitamin A formation from carotene ........ ..... 96 Absorbancy of Carr-Price reaction product on successive portions eluted from a ’deactivated alumina column ..... . ............ .. 99 Percent of initial potency of samples with and without maleic anhydride treatment at different intervals and "R" values ............ 112 Vitamin A formation from carotene by various tissue homogenates from vitamin A depleted rabbits, pigs, rats and chicks compared to non- depleted rats and sheep. ................. 125 Efficiency of vitamin A formation by various tissue homogenates expressed as % carotene unrecovered.. ..... ..... ...... . ................ 127 Vitamin A formation from carotene by various tissues homogenates from depleted calves (Experiments 16 & 21) ..... ... ................. 129 Effect of adding N03 on vitamin A formation from B- carotene using intestinal 100ps in vivo and in vitro... .................. . ....... 131 TABLE 13 14 15 16 17 18 19 20 21 22 Effect of adding different levels of N03 to the incubation media on vitamin A formation from B-carotene by duodenal homogenates from normal animals ............... Effect of adding different levels of N03 to the incubation media on vitamin A formation from B- carotene by intestinal homogenates from depleted animals ............. Comparison of the inhibitory effect of N03 and N0§ on vitamin A formation from B-caro— tene by duodenal homogenates .................. Effect of dietary N03 on carotene and vitamin A in blood plasma and liver of rabbits fed two different diets.. ......................... Extent of formation of vitamin A from B- carotene by duodenal homogenates of animals fed deficient or normal ration with or with- out added nitrate or nitrite .................. Effect of adding different levels of N05 to the incubation media on vitamin A forma- tion from B-carotene by intestinal homogen- ates from rabbits fed N03 ................. .... Effect of adding different levels of N03 to the incubating media on vitamin A formation from B-carotene by duodenal homogenates from calves and heifers fed N03 or N0§ ............. Effect of adding high levels of L-thyroxine (L-Tq) and L—triiodothyronine (L-T3) on vitamin A formation from B-carotene by duodenal homogenates for a carotene depleted calf (Experiment 8) ........................... Effect of adding low levels of L- -T4 and L- -T3 on vitamin A formation from carotene by duodenal homogenates from steers (Experiments 38 & 39) ...................................... Effect of adding isomers of L- -T4 and L- -T3 and combinations of them with 3 5' 3’ - L- -T3 on vitamin A formation from B- carotene by duod- enal and liver homogenates from depleted calf (Experiment 42) ............................... vi Page 133 134 137 139 142 145 147 149 151 152 TABLE 23 24 25 26 27 28 29 3O 31 32 33 Effect of thyroid status of calves and rabbits on vitamin A formation from B- carotene by duodenal homogenates (Experiments 22 - 27, 29 - 32) ................ Effect of adding L-Tq or L-T3 with or without nitrate on vitamin A formation by duodenal homogenates from control, hyperthyroid and hypothyroid calves and rabbits ................ Effect of adding L-T or L-T3 with or without nitrate on the effiClency of vitamin A forma- tion from B-carotene by duodenal homogenates from control, hyperthyroid and hypothyroid calves and rabbits ............................ Effect of adding different amino acids with and without the combination of L-thyroxine on vitamin A formation from B-carotene by duodenal homogenates from steers (Experi- ments 55 & 56) ............... . ................ Formation of vitamin A from B—carotene when various ions were added to duodenal homogen ates .......................................... Formation of vitamin A from B-carotene when .Various ions were added to duodenal homogen- ates from calves fed nitrate or thiouracil.... Effect of various inhibitors on vitamin A formation from B-carotene by duodenal homog- enates from a heifer (Experiment 54) .......... Effect of dl-alpha tocopherol on vitamin A formation from B-carotene by duodenal and liver homogenates ............................. Formation of vitamin A after incubating various blood fractions with the carotene suspension .................................... Effect of incubating different fractions of blood with duodenal homogenates on the for- mation of vitamin A from carotene ............. Effect of incubating different fractions of blood and centrifuged sedimented cells and supernatant fluid with duodenal homogenates on the formation of vitamin A from B-carotene. vii Page 154 156 157 161 163 164 166 159 171 173 175 viii TABLE Page 34 Vitamin A formation from B-carotene by centrifuged fractions of duodenal homogenates ................................... 177 FIGURE 10 ix LIST OF FIGURES Page A flow sheet of methods for the isolation and identification of vitamin A derivatives from extracts of incubations with B-carotene sus- pension and intestinal homogenates in vitro... 75 Effect of altering carotene to tissue ratio on vitamin A formation........................ 91a Absorption spectra of vitamin A alcohol frac- tion from ether extract of incubated mixture containing duodenal homogenates and B-caro- tene (ether extract was chromatographed on deactivated alumina).......................... 101 Absorption spectra of vitamin A alcohol frac- tion in petroleum ether chromatographed from ether extracts of incubated mixture..... ...... 104 Absorption spectra of vitamin A alcohol frac- tion before and after ultraviolet irradiation. 107 Absorption spectra of Carr-Price reaction products of tissue extract before and after chromatographic separation on deactivated~ alumina................... ....... .... ....... .. 110 Absorption spectra of Carr-Price reaction product of maleic anhydride treated and untreated vitamin A alcohol fraction.......... 114 Absorption spectra of vitamin A alcohol frac- tion in benzene before and after conversion to anhydrovitamin A ....... ...... ....... ....... 117 "Difference" absorption spectra ov anhydro- vitamin A made from tissue extract and from standard vitamin A ............. ............... 119 Absorption spectra of Carr-Price reaction products of vitamin A alcohol fraction before and after conversion to anhydrovitamin A...................................... ....... 121 TABLE II III IV LIST OF APPENDIX TABLES Purpose of various experiments conducted and tissues used .............................. Analysis of variance of data on the effect of adding isomers of L- -T4 and L— T and com— bination of them with 3 5' 3' L- -T3 on vita- min A formation from B- carotene ............... Analysis of variance of data on the effect of adding L- T4 or L- T with or without nitrate on vitamin A Formation by duodenal homogenates from control, hyperthyroid and hypothyroid animals ........................... Analysis of variance of data on the effect of adding different amino acids with and without combination of L- -T4 on vitamin A formation from B- carotene ..................... Page 224.1' 25 41¢ INTRODUCTION During the last 40 years studies on the absorption of B-carotene and its conversion into vitamin A have occupied a large percentage of the investigations on fat soluable vitamins. Although the conversion of B-carotene to vitamin A is a well established process, the mecha- nism underlying the conversion is not understood at the present time. The formation of vitamin A from B—carotene in_zixg has been well established. Numerous attempts have been made to demonstrate the formation of vitamin A from B-carotene 32.232392 but the results are for the most part conflicting. Conversion of carotene to vitamin A in zit£2_has been claimed on the basis of conventional methods for vitamin A analysis while other investigators have criticized or seri- ously doubted such claims. Feeds and forages containing high levels of nitrate have been implicated as a factor responsible for lowering of vitamin A status of animals receiving adequate levels of carotene. In addition there -is an indication that dietary nitrate can adversely affect the normal functioning of thyroid gland and thereby impair the formation of vita- min A from carotene. Some of these results were somewhat conflicting. It is indeed time consuming to study all of the above controver- sial tepics iE.EiXEfi The principal aim of the studies reported in this thesis was to modify the in_vit£2_media used by various investi- gators and to establish whether B-carotene could be converted into vitamin A in this ig_vit£2 system. It was also decided to isolate and identify any vitamin A thus formed using apprOpriate procedures. With the help of this in vitro system, it was then planned to study the efficiency of vitamin A.formation by various tissues, the effect of nitrate and other chemicals on vitamin.A formation, and the interrela- tionship between thyroid status, nitrate and vitamin A formation. REVIEW OF LITERATURE The conversion of B-carotene to vitamin A, whether by central fis- sion or by B-oxidation, may appear to be a very simple process. Although the conversion was discovered 30 years ago, the mechanism of vitamin A formation from carotenoids has not been elucidated very clearly nor in full detail. Moore (128) was the pioneer in developing the idea that carotene was the precursor of vitamin A. The liver of rats depleted of vitamin A contained little or no blue "Iovibond Units" of vitamin A activity as compared to rats given carotene supplements orally for 16 - 26 days. Using the same procedure with chicks, Capper et al. (34) demonstrated that the carotene was not stored in the liver unchanged but was presum- ably converted into vitamin A. These and other deductive experiments established the view that carotene was the precursor of vitamin A. In vivo conversion of B-carotene into vitamin A. According to the present concepts the small intestine plays a major role in the conversion of B-carotene into vitamin A in laboratory animals as well as in large animals. Early work of Moore (129) was interpreted to indicate that the liver was the organ in which the conversion occurred. The liver oils of rats invariably contained vitamin A at extremely high concentrations when diets containing large amounts of carotene were given for prolonged periods. Small amounts of unconverted pigments were also found in the liver oils of all rats which had received carotene. From this evidence it was deduced that vitamin A was formed in the liver. With the aid of fluorescence microscopy, POpper (145) observed the appearance of vitamin A in the intestine, in the Kupffer cells of the 3 liver, in the epithelial cells of the adrenal cortex, in the tubular and Ieydig cells of the testicle, in the granulosa and theca—lutein cells, and in the cortical stroma of the ovary. Although the informa- tion could have been used to indicate vitamin A formation in the intes- tine or other extrahepatic tissue, no such deductions were made by the author or others. The possibility of conversion of carotene to vitamin A in extra- hepatic tissue was suggested by Sexton et al. (157) in 1946. When the carotene was administered either intravenously or intraperitoneally to deficient rats the growth response was low as compared to that of rats given oral doses. Liver vitamin A was determined spectrophotometrically on petroleum ether extracts. These authors suggested the wall of intes- tine as a possible site of such conversion. Vitamin A first appeared in the intestinal wall 1.5 hours after oral administration of carotene dissolved in oil to vitamin deficient rats (119). Analysis of liver and intestine three hours after dosing showed that they contained a total of 1.6 ug of carotene and 22.8 IU of vitamin A and 13.5 ug of carotene and 40.3 IU of vitamin A respectively. Vitamin A was determined by the Carr-Price reaction. The results of Mattson et al. (119) were presented almost simul- taneously with a note by Thompson et al. (175) who dosed pigs with B- carotene in arachis oil and found an increase in the vitamin A ester in blood plasma 3 - 7 hours later. Vitamin A was detected in the mesen- teric lymph channels and in the wall of the intestine and its contents in greater quantities than in control pigs. Later the British workers made a thorough and comprehensive study of B-carotene disappearance in the small intestine using laboratory as well as large animals. In one study when B-carotene was given by mouth to vitamin A deficient rats, vitamin A appeared within 15 minutes in the wall and contents of the small intestine, whereas the first increase in the blood and in the liver did not occur until 45 - 60 minutes had elapsed (176). Up to two hours after dosing there was more vitamin A in the small intestine than in the liver. Similar results were also found in pigs. Further evi- dence showed the appearance of vitamin A in the portal and systemic circulation of pigs and in the mesenteric lymph of pigs and rats two hours after carotene feeding (173). It was found that the vitamin A appeared almost exclusively in the wall of the intestine, 75% of it in the ester form. No conversion was observed in that part of small intes- tine preceeding the entrance of the common bile duct. These workers separated carotene, vitamin A alcohol and its ester by column chroma- tography from the ether extract and then used the Carr-Price reaction to estimate vitamin A content of the samples. These eXperiments indi- cated that the small intestine was the site of conversion of carotene to vitamin A and that the lymphatic system was a route of transport of the vitamin A so formed. Ball and his co-workers (10) fed retinene (vitamin A aldehyde) and found that it was converted to vitamin A alcohol in the intestinal wall. These results gave added emphasis to the idea that the carotene might be converted into vitamin A in the intestinal wall. Further confirmation of extrahepatic conversion of carotene to vitamin A was advanced by Krause and Pierce (110). They fed carotene to rats without any hepatic circulation and found an increase in serum vitamin A 6 - 8 hours after administration of carotene. 6 Further evidence was obtained from the detailed investigations of Glover et al. (66). They fed depleted rats a diet rich in carotene and subsequently killed them a few hours later. These authors separated vitamin A from unsaponifiable matter by column chromatography and proved its identity by maximum absorption in the antimony trichloride reaction at 617 mu and in the ultraviolet light at 328 mu. When the absorption of carotene was maximal, the concentration of vitamin A present in the intestinal wall was comparable to that in the liver. These results helped to establish the view that the wall of small intestine was the site of transformation of carotene into vitamin A. In goats provided with a thoracic canula, an increase in vitamin A levels of the lymph was noted after feeding carotene, indicating that vitamin.A was formed from carotene in the gut wall and transported to the liver via the lymph (70). This work was further extended and it was demonstrated that the intestine or the intestinal wall was the site for the conversion in conscious rats (4). These authors separated vita- min A from unsaponifiable matter by column chromatography and determined vitamin A by antimony trichloride reaction and also by ultraviolet absorption at 328 mu. Klosterman et al. (100) demonstrated that when lambs were given carotene by mouth or allowed green grass for a short period. an increase of vitamin A in the blood serum occurred. When carotene was present in the intestine, vitamin A was also found in the intestinal wall. These observations coupled with the fact that no measurable amounts of carotene were found in the blood of normal sheep suggests that the carotene was converted into vitamin A during diges- tion and/or absorption rather than by liver. 7 Other experiments indicated that the intestine was the major site of formation of vitamin A in calves, goats, sheep and rabbits (103, 104). It was also established that the ingestion of carotene in Tween resulted in an earlier and greater increase in blood carotene and vita- min A than ingestion of carotene in oil using calves, rabbits and rats. Whatever the form or the vehicle of dispersion, carotene injected into calves was converted only to a very limited extent into vitamin A (103). Simultaneously, Thompson et al. (174) and Cheng and Deuel (37) showed that the carotene was converted into vitamin A in the intestine of chicks. The former worker determined vitamin A by the Carr-Price reaction after separating vitamin A by column chromatography and the latter by the Carr-Price reaction on crude extracts. Extensive studies by the Canadian workers demonstrated the conver- sion of carotene to vitamin A 33 1119. with chicks (161, 162, 163). In all these studies vitamin A in the sample was determined by the Carr- Price reaction after saponification, extraction and separation of caro- tene and vitamin A by column chromatography. Sibbald and Olsen (163) found that the vitamin A was formed from carotene in a ligated loop of the duodenum proximal to the entry of the bile and pancreatic ducts of the living chick. However, the relocation of vitamin A so formed was inhibited. It was suggested that this inhibition probably was due to pressure resulting from the secretion of fluid into the duodenal 100p. In another study Sibbald and Hutcheson (161) found increased amounts of vitamin A in the duodenal wall using ligated duodenal 100ps in living chicks. When the blood supply to a duodenal 100p was ligated the for- mation of vitamin A from carotene was prevented. They suggested that the blood supplied some factors for the converting mechanism and that 8 the absence of blood supply caused death of the tissue of the duodenal wall and hence inhibited its power of absorption and/or conversion. It was also shown that the minimum time necessary for the production of vitamin A from carotene injected into ligated duodenal 100p of living vitamin A depleted chicks was 15 minutes (162). Although the intestine has been reported to be the site of conver- sion some doubts still existed whether the actual locus of formation was in the wall or in the lumen or both. De and Sundararajan (43) reported that the whole of the small intestine became practically devoid of carotene in about 24 hours time when B-carotene in oil was adminis- tered to albino rats. The intestinal wall contained small amounts of carotene and also vitamin A. The intestinal lumen, on the other hand, contained large amounts of carotene but no vitamin A in most eXperiments. The vitamin A was estimated from ultraviolet absorption at 328 mu after chromatographic separation of the extracts. These authors claimed that the wall of small intestine appeared to be the site of conversion of carotene to vitamin A when carotene was fed. The actual locus of intestinal conversion of carotene to vitamin A was investigated further. Greenberg (73) used histochemical tech- niques to visualize carotene and vitamin A in the unstained frozen tissue sections of the rat small intestine. After a meal containing carotene the first recognizable vitamin A was seen in 5 to 15 minutes and was first visualized within the intestinal lumen just adjacent to the tips of the villi. subsequently the vitamin A was recognized histo- chemically within the mucosa even though small amounts of unchanged carotene are known to be absorbed (73). The data suggested that the conversion of carotene to vitamin A could occur as an extracellular process. 9 Although, most experiments had shown the small intestine to be a major site of conversion of carotene to vitamin A Belgian workers (105, 106) investigated the possibility that conversion could occur in the liver. Kowalewski and Henrotin (105) injected 25 mg of B-carotene emul- sified in isotonic glucose solution into the portal vein and analyzed liver and blood one hour later. There was some indication of synthesis of vitamin A. In another study, Kowalewski et al. (106) investigated the role of portal and lymphatic circulations in the transport of carotene when administered into the intestine. The increase in both carotene and vitamin A in the blood and liver were greater after carotene had been given in an emulsified form than after it had been given in oil. When carotene was emulsified all of it was absorbed by the portal vein, but when it was given in oil a substantial portion was absorbed by the lymphatic duct. Carotene appeared to be absorbed rapidly into the portal vein when given in an aqueous emulsion. The review of the literature pertaining to the formation of vita- min A from carotene £2.2312, clearly indicated that the wall of the small intestine appeared to be the major site of conversion when the carotene was fed to laboratory as well as to large animals. The lym- phatic system was a route of transport of the vitamin A so formed. Conversion of carotene to vitamin A by the organs and body fluids other than intestine and liver. Although such organs as the intestine and liver could convert B- carotene to vitamin A, early work on the site of conversion of intra- venously administered carotene led to the conclusion that perhaps many tissues had the ability to form vitamin A from carotene. The question deciding the relative importance of the various possible sitesfbr conversion of carotene is still undetermined after many experiments. 10 During recent years, several workers have investigated various aspects of the utilization of intravenously administered carotene by various animal Species. Klosterman et al. (100) found that colloidal carotene injected into the blood stream of sheep was rapidly removed with no increase in blood or liver vitamin A. Eaton et al. (48) reported that in dairy calves both intravenous and oral administration of carotene resulted in higher blood plasma levels of carotene and vitamin A than in the controls. There was no real differences in vitamin A concentration of either liver or lungs in above two groups. Warner and Maynard (183) found that intravenous injections of an aqueous dispersion of carotene increased plasma carotene and vitamin A levels significantly in dairy calves as estimated by independent analytical procedures. An ultraviolet examination of chromatographed, post injection, plasma extracts showed typical vitamin A absorption Spectra. Elliot (49) injected high carotene blood plasma intravenously to dairy calves and found no increase in liver vitamin A levels as com- pared with control calves. These results indicated that carotene in blood plasma was not converted to vitamin A in dairy calves. Church et al. (38) found significant increases in plasma vitamin A after injecting a solubilized aqueous carotene in sheep. Carotene injections into calves caused no significant differences in plasma vitamin A values. The data obtained in these experiments indicated a difference between cattle and sheep in their ability to convert intra- venously injected carotene to vitamin A. The results were contrary 11 to those reported by Klosterman et al. (100) with sheep and Eaton et a1. (48) and Warner and Maynard (183) in dairy calves. One factor involved in this difference may have been the nature of the carotene diapersion. Kon et al. (103) studied this problem further using animal Species (rat or rabbit) whose blood does not contain carotenoids and a third (calf) that usually has large quantities of carotenoids in the blood. These experiments provided evidence for the formation of vitamin A from the injected carotene in rats and rabbits. By contrast, all their experiments with calves were negative in that they provided virtually no evidence that vitamin A was formed from injected carotene. Results indicated that a large part of the carotene that appeared initially in the circulation of rats and rabbits and in the lungs was rapidly destroyed, presumably by oxidative breakdown, with the simultaneous appearance of some vitamin A. These workers shared the view of Bieri and Pollard (20) that vitamin A might be one of the products of an oxidative chain and that its formation need not be confined to any particular site, though the lung might well prove to play an important role even if not a specific part. Schuh et al. (154) demonstrated that intravenously administered carotene was well utilized by dairy calves. This was contrary to the results obtained by Church et al. (38) and Kon et al. (103). Bieri and Pollard (19, 20) intravenously administered lOOO ug of aqueous B-carotene solubilized in Tween 40 to vitamin A deficient rats. Serum vitamin A levels reached a maximum of 134 ug% in six hours. Vitamin A was also found in liver, kidney, small intestine, lung and spleen. It was shown that ligating the bile duct did not interfere ‘O 12 with the conversion. Rats injected with carotene after surgical removal of the small intestine had vitamin A in the serum, liver, and kidney. These findings indicated that vitamin A could be formed from carotene in tissues other than the small intestine. The eXperiments conducted by Bieri (17) showed conclusively that the chicken utilized circulating B-carotene and to a lesser extent cryptoxanthin as a source of vitamin A. It was pr0posed that those provitaminaAAwhich escaped conversion during absorption through the small intestine were still utilizable by the bird. Vitamin A was found in liver, kidney, and serum when carotene was injected intravenously into rats and rabbits. . Intravenously administered carotene emulsion was converted into vitamin A and the conversion was less efficient than that of aqueous carotene dispersion (123, 124, 126). These authors concluded that the conversion of the carotene molecule was initiated in the blood where there was rapid breakdown to an intermediate which was then taken up by the tissues and more slowly converted into vitamin A. Attempts have been made to locate more precisely the site at which conversion of injected carotene occurred. Worker (190) demonstrated that the appearance of vitamin A in blood plasma after injection of a Tween diapersion of carotene wasmno way affected by complete removal of the liver in rabbits, or by complete removal of the liver alone or both liver and viscera in rats. Worker (192) concluded that the ability to convert injected caro- tene was an attribute not of any one tissue or organ but of many or perhaps of all cells. This belief was further supported by the results of preliminary in vitro experiments showing that many tissues including 13 blood possessed the ability to form small amounts of vitamin A upon incubation with carotene in Tween dispersed in physiological saline. The identification of vitamin A was carried out by absorption Spectra and Carr-Price reaction after separating ether extracts on alumina. In further endeavors to directly locate the site at which conver- sion of injected B-carotene into vitamin A occurred, Worker (193) attempted to demonstrate the ig'git£9_conversion by carrying out per- fusion and incubation experiments on a variety of tissue preparations. t was concluded that it was not possible to demonstrate conversion of carotene to vitamin A 22.23332 either in perfused organs such as intestine, kidney, liver or lung of the rat or guinea pig. Furthermore it was not possible to demonstrate in vitro conversion in the blood of the rat, guinea pig or sheep. These results contradicted the prelimi- nary i§_!itgg observations that many tissues including blood possessed the ability to form small amounts of vitamin A (190). Vitamin A was estimated by the SbCl3 method as in the previous observations (48). After conducting further eXperiments with rats, McGillivray (122) suggested a mechanism for formation of vitamin A from carotenoids that were injected intravenously in an aqueous dispersion. The suggestion was made that after intravenous injection a rapid break down of the carotene molecule to retinene occurred through a coupled oxidation with unsaturated lipids, possibly with hemoglobin as a catalyst. Retinene was then removed by the tissues from the oxidizing environment of the blood and reduced through the already established enzyme system to vita- min A. This vitamin A was then released slowly from the tissues into the blood from which it was removed by the liver. 14 Attempts have also been made to study the utilization of intra- muscularly and subcutaneously injected aqueous dispersions of carotene. Tomarelli et al. (177) and Bieri and Sandman (21) administered aqueous dispersions of carotene intramuscularly to rats and found an effective conversion to vitamin A as demonstrated by the resumption of growth in vitamin A deficient animals. Further, Bieri and Sandman (21) showed that approximately 4 - 6 times as much carotene was required parenter- ally as orally for maximum growth and that aqueous carotene was also well utilized when given subcutaneously. Working with chicks, Sibbald and Hutcheson (100) found that the cr0p did not possess the ability to convert B-carotene to vitamin A within a four hour period even though carotene might be absorbed by the cr0p wall in the presence of bile. The metabolism of radioactive B-carotene was studied in the dog heart-lung preparation by Olson et al. (140) who showed that less than 0.05 ug vitamin A was formed from 5 to 20 ug B-carotene.‘ It was con- cluded that the lung was probably not an important organ for conversion of B-carotene into vitamin A. A possible relationship between vitamin A and red blood cells has been studied recently. Pollard and Bieri (144) indicated that a remark- able property of the hemolyzed blood from young animals was its ability to destroy vitamin A and carotene. Rabbit and rat blood were more active in this reapect than the blood of mice, guinea pigs and chicks. The activity resided in the cells and was liberated upon rupture of the cells. The lack of a highly positive correlation between number of reticulocytes and the destructive activity suggested that the system was complex. 15 The information reviewed above clearly indicated that in most trials an aqueous dispersion of carotene administered parenterally was utilized for the formation of vitamin A. On the contrary, carotene in oil was not well utilized. This is one explanation for some of the conflicting reports concerning parenterally administered carotene as a precursor of vitamin A. The suggestion also was offered that the ability to convert carotene to vitamin A was an attribute of all cells. Product formed when animals were given radioactive B-carotene. Since the results obtained in the study of carotene conversion were influenced by physiological variables, radioactive B-carotene has been used to investigate the carotene metabolism to overcome some of the difficulties encountered using unlabeled carotene. Krause et al. (109) found that the feeding of Clh labeled carotene resulted in the deposition of all of the absorbed Clh in the carotene- free, non-saponifiable fraction from the total animal. The highest concentration of Clh was found in the extrahepato-intestinal tissues 63nd the least amount was recovered in the liver. The uptake of the czarotene free, non-saponifiable fraction was higher at the end of 6 .knours than 24 hours. In another study Krause and Saunders (111) demon- ESixrated that the feeding of labeled Clu carotene resulted in the Cieposition of approximately 1.5% of absorbed Clh into liver vitamin A Eijfter 24 hours. They determined vitamin A by the method described by C}ZLover and Redfearn (67) which involved the separation of vitamin A from carotene by column chromatography and measuring extinction at 328 inlll. In the total animal, an average of about 15% 0f the absorbed Clh "‘311t into fatty acids and 40% into nonsaponifiable material. An average (Dcf' about 5% of the absorbed 014 remained to be accounted for. 16 Fishwick and Glover (59) studied the metabolism of uniformly cl“ labeled B-carotene in the rat. The results indicated that the forma— tion of vitamin A resulted from oxidative fission of a terminal double bond in B-carotene, involving at least in part some other system than B—oxidation. Wilmer and Laughland (187) determined the distribution of Cl“ in the tissues of the rat at various intervals up to 28 hours after admin- istration of an oral dose of randomly labeled Clh B-carotene. During early stages of absorption, the activity was high in the intestine and blood. Increased activity was observed in other tissues when the metabolic stages became superimposed upon the absorption process. The adrenal glands exhibited the greatest concentration which was maintained throughout the experimental period of 28 hours. Following the adrenal glands in decreasing order of their activity were hypophysis, liver, intestine, kidneys, lungs, heart, spleen, blood and stomach. Normal and vitamin A deficient animals were given 25 to 50 ug of Clh B-carotene dissolved in oil (182). The radioactive B-carotene (mould be detected neither in the lymph nor in the liver which, however cxontained about 1 to 8% of the total activity given. The radioactivity cxf‘the liver lipids was twice as high as that of vitamin A isolated 'tiierefrom. This could indicate an oxidative degradation of the B- <2Eirotene to acid compounds. Using Clu labeled B-carotene, Olson (138, 139) has attempted a InOre direct study of B-carotene absorption and its conversion to vita- min A by rat intestine ELI}. Egg. The formation of vitamin A proceeded ‘°€211.when the 014 labeled B-carotene was administered by intraduodenal injection or by a stomach tube. ,A peak of radioactivity occurred in 17 the intestinal mucosa at 1 hour while in the liver it plateued at 3 to 5 hours. The separation of B-carotene and vitamin A derivatives were carried out by column chromatography and the identification and quantitative analysis of compounds were carried out by visible and ultraviolet absorption spectra, and the Carr-Price reaction. In the intestinal mucosa, vitamin A ester and carotene accounted for a very large share of the total radioactivity. Much smaller amounts of radio- activity occurred in retinene, vitamin A alcohol and the terminal polar fraction in that order. In the liver. vitamin A ester was found in maximal quantities. It was also found that the upper one-third of the intestine was most active in the vitamin A ester formation and that the middle one-third had little ability to carry out the conversion from B-carotene. These studies presented a new approach to the problem of carotene to vitamin A conversion. The radioactive study clearly indicated that within a 24—hour period, B-carotene was metabolized extensively and appreciable amounts of radioactivity appeared in the non-saponifiable and acidic fractions as well as in liver vitamin A. Only a small portion was oxidized to C0 . The finding that C11+ from labeled B- carotene entered into compounds other than vitamin A supported the suggestion that the carotene was not entirely converted into vitamin A and it might be directed into other metabolic pathways. This finding supported the hypothesis of stepwise breakdown of B-carotene by B- oxidation or otherwise to give one molecule of vitamin A. On the contrary, in other studies,appreciable quantities of other radioactive products, non-polar or acidic, did not accumulate. Since vitamin A was also metabolized in the intestine to a small amount of acidic 18 products (138), the acidic fraction which was alleged to derive from B-oxidation of B-carotene might well have arisen from further oxida- tion of vitamin A and not directly from a cleavage reaction. With further refinements this method has promise of elucidating the mechanism of formation of vitamin A from B-carotene. In vitro conversion of carotene to vitamin A. The conversion of carotene to vitamin A in an i2_yit£g_system has received only limited emphasis. Attempts have also been made to prepare active tissue that will carry out the reaction EE.X£EEE° During the past years claims for ig_zit£2 conversion of carotene to vitamin A using tissues have been made (55, 102, 121, 135, 136, 137, 141, 152, 167, 169, 185, 188) while negative results have been reported by others (3, #3. 46, 53, 149, 193). Olcott and McCann (135) incubated rat liver thoroughly ground with sand, phosphate buffer (pH 7.45) and ethyl laurate in which caro- tene was dissolved. They extracted the incubation mixture with ether and Observed an absorption band at 325 mu after dissolving the dried Isample in CHCl3. It was reported that the agent responsible for trans- formation appeared to be an enzyme, provisionally called carotenase since it was destroyed by heat. Pariente and Ralli (141) attempted to prepare carotenase from the livers of normal dogs and of dogs on a vitamin A deficient diet. They succeeded only once out of four trials. A liver extract was made ‘using phOSphate buffer at pH 7.4 saturated with toluene. The extract teas filtered and the filtrate was incubated with colloidal carotene for 36 hours at '3"PC. They estimated vitamin A in the ether extracts with E3bCl3. It was claimed that carotenase was active in a solution of 19 pH 7.4 but inactive in solutions of unfavourable acidity or alkalinity and that it was destroyed by cold. They concluded that in solutions of pH 7.4 carotenase maintained its activity for a considerable period of time if kept at 37°C. Euler and Klussman (55) obtained evidence for the formation of vitamin A by a positive antimony tricloride reaction when liver tissue from cows was incubated with colloidal carotene. Some unusual evidence was reported by Wilson et al. (188) who fed carotene to rabbits anddropped pieces of liver in the melted paraffin after sacrificing the animals. These authors claimed that there was a disappearance of carotene and an appearance of vitamin A after auto- lyzing for 28 days. Wiese et al. (185) presented evidence that the i2_vitro trans- formation of carotene to vitamin A occurred when the small intestine of vitamin A deficient rats were incubated in Ringer-Iocke solution for 3 hours at 37°C. In these experiments, the incubated mixture was saponified, extracted and the vitamin A determined by the Carr- Price reaction. In eXperiments with calf tissues Stallcup and Herman (107) demon- strated conversion of colloidal carotene into vitamin A using minced liver preparations and isolated intestinal 100ps after incubation for 3 hours at 37°C. Vitamin A was determined from the ether extracts by the Carr-Price reaction. McGillivray (121) incubated sections of intestines of sheep with carotene in Ringer-Locke solution maintained at 37°C and the formed product identified as vitamin A by colorimetric and spect0photometric methods. 2O Rosenberg and Sobel (152) estimated vitamin A in extracts of intestinal tissue by difference in absorption at 328 mu before and after ultraviolet irradiation. Subtraction of extinction of the postirradiation spectrum from the extinction of the preirradiation spectrum gave a curve which closely resembled that of pure vitamin A. Employing this method the conversion of carotene to vitamin A in the isolated intestinal 100ps of rats was demonstrated i§_zit£2_by incu- bating at 4500 for one hour. A small, but definite amount of conversion of carotene to vita- min A was obtained when preparations of chick duodenum or liver homogenates were incubated with the aqueous suSpension of carotene in Tween 60 (136). The two tissues would appear to be active as enzyme sources. The amount of vitamin A found in the liver prepara- tions was actually greater than the duodenal preparations. Japanese workers (102, 169) made an extensive study ip_zit£2 on metabolic pathways of carotene to vitamin A. Suzuki et al. (169) prepared intestinal homogenates from rats by grinding in a mortor with 0.25 M.sucrose solution and incubated this mixture with an aqueous dispersion of B-carotene. One compound formed was identified as vita- min A ester using paper chromatography. In another study Koilumi et al. (102) used intestinal homogenates of vitamin Bl deficient rats and found that B-carotene was convertible to vitamin A ester in the rats receiving thiamine as well as in rats that were slightly thiamine deficient, but complete failure of the conversion was observed in the severely deficient animals. The additive effects of various coenzymes revealed that the addition of FAD o DPN or DPN e cytochrome C contrib- uted to the metabolic conversion of B-carotene to vitamin A, whereas 21 other combinations of coenzymes gave negative results. It was also demonstrated that FAD + DPN stimulated the 12.13332 formation of vitamin A from B-carotene even in B1 deficient rat intestinal homo- genates. Olson (137) incubated longitudinally cut sections of washed rat intestine in vitro in Krebs-Ringer bicarbonate solution with glycocho- late, under 95% O2 - 5% 002 at 38°C for one hour. After one hour, the intestine was washed, homogenized in CHC13 - CH30H mixture. The hexane extract was chromatographed on alumina and vitamin A deriva- tives determined by ultraviolet absorption Spectra and Carr-Price reac- tion. About 2 - 4 ug vitamin A ester was formed in each section. In spite of all these positive results, many negative results have also been reported. Ahmed (3) failed to observe the conversion of carotene to vitamin A by rat liver lg vitro. Perfusion of an isolated organ also gave negative results. Euler and Euler (53) using shark liver could not obtain vitamin A from carotene after incubation £2.233393 Rea and Drummond (49) were unable to confirm the work of Olcott and McCann (135). They were unable to effect the conversion by incu- bating carotene with liver tissues from vitamin A deficient rats and cats. ttempts to extract the enzyme carotenase were not successful. Drummond and MCWalter (46) claimed that they were again unsuccess- ful with liver incubations, even when the conditions were made more physiological by allowing rabbits to absorb carotene into their livers just before they were killed. De and Sundararajan (43) were unable 22 to demonstrate in the rat any increase in vitamin A after incubating whole intestine with carotene. Workers (193) had made an extensive study using tissues from the rat, guinea pig, and sheep. He claimed that considerable attention was paid to detail and extreme care was taken at all times to ensure that the results obtained were not limited in any way by poor technique. Despite those precautions, he found that it was not possible to demon- strate conversion of carotene into vitamin A 12.22239, either in the perfused intact animal or in tissue sliced or homogenates of abdominal wall, intestine, kidney, liver or lung or in isolated perfused organs (intestine, kidney, liver or lung) of the rat or guinea pig or sheep. It was concluded on the basis of the results reported that either the enzyme involved must be extremely sensitive to non-physiological condi- tions or that the necessary cofactors, perhaps supplied from the circu- lation, were not present in isolated tissues in sufficient concentrations. In some of the above reviewed cases, vitamin A was estimated solely by the antimony trichloride reaction without previous chromatography, the validity of which would appear open to question (167, 185). Others estimated vitamin A by the difference in absorption at 325 mu before and after ultraviolet irradiation, the validity of which may be questioned (152). In contrast to these reservations McGillivray (121) identified vitamin A from ip_!it£g trials by colorimetrically and Spectrophotom- etrically. However, in many cases negative results have been reported. Hence it is apparent from this review that results are somewhat conflict- ing and logical doubt does exist about the alleged i2 vitro formation of vitamin A. 23 Effect of nitrate and nitrite on the conversion of carotene to vitamin A. The effect of nitrate and nitrite on the apparent conversion of carotene to vitamin A have been studied ip_ziyg_using various Species of animals, but the results were for the most part conflicting. Smith et al. (166) reported that the rats fed corn silage contain- ing one percent of KNOB on a dry matter basis showed a reduced level of vitamin A in the plasma and liver and interpreted this to indicate a reduction in the rats of conversion of carotene to vitamin A. In one study, Illinois investigators (93) found that dietary nitrate caused a rapid depletion of vitamin A in steers. In another similar study, Illinois workers (165) found that dietary nitrate exerted no sig- nificant effect upon liver vitamin A. Plasma vitamin A changed in the animals fed hay, hay o nitrate, silage and silage , nitrate from 30 ug% to 20, 18, 17, and 16 ug%, respectively. Dietary nitrate had no effect upon hemoglobin, methemoglobin, hemocrit nor upon plasma or liver contents of vitamin A in sheep. Goodrich et al. (69) found that the effect of sodium nitrate on plasma vitamin A levels in sheep was uncertain, but liver vitamin A stores were significantly lowered by feeding of rations containing 3% sodium nitrate. O'Dell et al. (134) found that dietary nitrate at the level of 0.3% of diet caused a rapid depletion of liver vitamin A in rats. After eight weeks xerophthalmia was noticed in these animals. Holst et al. (86) added nitrite to sheep rations in the form of equal molar mixtures of sodium and posassium nitrite at the levels of 0.1 to 0.75%. Liver vitamin A was low in those animals fed N02. 24 Emerick and Olson (52) found that feeding of 0.5% NaNOQ, but not 3.0% NaNO3 resulted in redhction in the amount of vitamin A stored in liver when vitamin A was administered orally, but not when administered by subcutaneous injection. They suggested this nffnbte might be due to the detrimental action of nitrite on preformed vitamin A within the digestive tract. Both nitrite and nitrate significantly lowered the liver storage of vitamin A with the greatest effect resulting from nitrite feeding. They proposed two possible mechanisms for the effect of nitrite on the liver vitamin A storage. The nitrite ion had an effect on thyroid function which was necessary for conversion of caro- tene to vitamin A. The nitrate effect was also the result of a lowered absorption of carotene. Pugh et al. (146) conducted 12 zit£9_experiments to study carotene and vitamin A destruction by nitrite. Incubations were performed at 37°C for four hours using aqueous dispersions of B-carotene and KN02 having molar ratios ranging from 1 z 0.5 to 1 : 100 and with pH ranging from 1 to 7. Destruction of carotene was greatest at pH 1 to 3 followed by an abrupt decrease to lesser destruction at pH 5 to 7. The amount of B-carotene destroyed increased rapidly as the molar ratio of nitrite to carotene increased. Weichenthal et al. (184) found that feeding 1% KNO3 in the diet appeared to have no effect on plasma vitamin A levels. Hale et a1. (75, 76) studied the effect of ration, energy level and nitrate on liver vitamin A depletion in fattening steers fed a low carotene ration. A high TDN (71.3%) ration resulted in a significantly larger vitamin A depletion than did a low TDN (54.3%lration. The 25 addition of 1% KNO3 in the diet reduced liver vitamin A stores, but the reduction was not significant. The possible interrelationship between nitrate, vitamin A and thyroid gland or iodine has been studied lg vivo. Bloomfield et al. (24) injected 3 and 100 no of carrier-free I131 to rats and sheep, respectively, which were fed a nitrate containing diet for one week. They found that 0.31 and 0.92% dietary nitrate when consumed by rats and sheep, respectively, could affect the normal iodine metabolism of the thyroid gland and recommended that animal rations suspected of containing nitrates be supplemented with adequate amounts of iodine and vitamin A. Yadav et al. (196) fed groups of rats on four vitamin A deficient rations containing low iodine (0.2 ug/ g), 10w iodine-nitrate (1.5% of KNO3), adequate iodine (2 ug/ g) and adequate iodine-nitrate until they were deficient and found that the incidence of xerophthalmia was 45.5, 75.0, 0.00 and 33.0% respectively. The liver vitamin A after a depletion period was highest in the group fed adequate iodine and lowest in the group fed the iodine-nitrate diet. The thyroid weights were increased in the groups fed nitrate. Bloomfield et al. (25) fed a diet containing 1.5% KNO3 to sheep 6 days prior to injection of 100 uc 1131. The thyroidal uptake of 1131 was increased in this group. They Speculated that dietary nitrate induced an increase in pituitary TSH. In another study, Bloomfield et al. (26) found that rats main- 31 uptake tained in a normal environment showed a decreased thyroidal I1 after 7 hours on a 2.5% nitrate supplemented diet but were able to overcome this effect after 2 weeks. The rats could not compensate for 26 a nitrate load under cold stress. Although the information reviewed above is somewhat conflicting, there appears to be some kind of relation- ship between vitamin A, dietary nitrate, and thyroid function. Most results indicated that the addition of N05 to the ration or feeding of rations having a high nitrate content had an adverse effect upon the animal's vitamin A status. The decreased storage of vitamin A in the liver and low serum levels of vitamin A from orally administered carotene in the presence of dietary NO§ or N05 suggested a detrimental action of N03 or N05 on provitamin A. The conclusion that nitrate ion has an effect on thyroid function and that a normal functioning thyroid gland is necessary for conversion of carotene to vitamin A suggested a possible mechanism for the effect of N03 on vitamin A storage. Importance of bile acids on the conversion of carotene to vitamin A. There are many complicating factors, both chemical and physiolog- ical, which will either enhance or inhibit the formation of vitamin A from carotene i2_ziyg_as well as ig’xitgg. One of the most important natural substances which aid the absorption process is bile. The bile and its salts have high emulsifying pr0perties. This could be important in the utilization of B-carotene inside the intestine. Drummond and McWalter (47) recognized the importance of bile for the absorption of carotene and showed that carotene could form a complex with desoxycholic acid. Altschule (5) showed microscopic evidence of epithelial lesions of vitamin A deficiency in infants with severe protracted jaundice due to congenital atresia of the bile ducts in spite of the fact that they were receiving diets adequate in vitamin A. Conclusions were drawn that 27 the deficiency occurred as a result of a failure to absorb vitamin A from the gastro-intestinal tract due to the absence of bile. Using vaginal smear as the criterion for the conversion of caro- tene to vitamin A, Greaves and Schmidt (71) demonstrated that irrespec- tive of the channel used in administering the carotene, little or no vitamin A was formed in the vitamin A deficient icteric rats. In some attempts, carotene could be made effective by the simultaneous admin- istration of sodium desoxycholate or sodium glycodesoxycholate. An interesting eXperiment was conducted by Bernhard et al. (15) who showed that the absorption of vitamin A through the thoracic lymph duct was lowest when bile was withdrawn from the bile duct as compared to control rats. The administration of natural bile of rats through the fistula increased the absorption of vitamin A. The bile pigments greatly retarded the oxidation of vitamin A ip_vitro. Vahouny et al. (180) studied the comparative effects of various bile acids on the intestinal absorption of cholesterol. Cholanic, lithocholic and desoxycholic acids with 0, l and 2-hydroxy1 radicals, respectively, produced no increase in lymph cholesterol over control groups. Conjugated bile salts, glycocholate and taurocholate gave comparable and significant elevations in total lymph cholesterol. Cholic acid with two free hydroxyl groups and an unconjugated carboxyl radical was the most effective bile acid in promoting cholesterol absorption from the intestine. It is possible that these results may be applied to the absorption of carotene and vitamin A. A study on the requirement for bile acids in the intestinal conver- sion of carotene to vitamin A in vivo and in vitro has been made by Olson (137, 138, 139). The eXperiments ig_vivo indicated that ligation 28 of bile duct prevented vitamin A formation in washed duodenal 100ps, but that the addition of either rat bile or sodium glycocholate to B-carotene suspensions allowed the conversion to occur. It was also found that cholic acid was somewhat less effective and 5% deoxycholic acid destroyed the mucosa and allowed no vitamin A formation. Under normal conditions the upper two-thirds of small intestint formed more vitamin A than the lower third of the small intestine. The addition of glycocholate to a B-carotene suspension stimulated vitamin A formation in all portions of intestine. In addition, when longitudinally cut sections of washed rat intestine were incubated ip_zit£2_with glycocholate, vitamin A ester formation also took place. On the other hand, the formation ig'vitro of vitamin A ester from Tween solutions of Clu-vitamin A alcohol or Clu- retinene did not require glycocholate. Bile did not function merely as a general emulsifying agent in this case, but possessed a more Specific function which was due mainly to the cholanic acid structure and was enhanced by conjugation (138). However, Sibbald and Olsen (163) found sodium glycocholate neither improved nor retarded the rate of conversion at a level of 22/100 ml of solution. It is evident that the conjugated bile salts do not solely act as emulsifying agents, but also function in a more specific manner. It seems likely that bile salts primarily enhance B-carotene absorption leading to vitamin A formation. Further, it appears that specific types of bile salts having 3 hydroxyl groups such as taurocholate and glycocho- late were involved in stimulation of vitamin A formation more than other types of bile salts as indicated by i3 vitro eXperiments conducted by Olson (137). 29 The impogtance of t0c0pherol on the utilization of carotene and vitamin A. One of the earliest and most frequent pr0posals for the mechanism of action of tocopherol has been that it served primarily as an intra- cellular antioxidant, preventing the oxidation of.compounds such as unsaturated fats, vitamin A and ascorbic acid. The t0c0pherols are now recognized to be the most important natural stabilizing compound for vitamin A and carotene. Synergistic action of tocopherols on vitamin A deposition in the tissues has also been known for Some time (131). There was also the suggestion that the tocopherols increased the efficiency of utilization of carotene. In opposition to this suggestion, there was also another idea that small doses of t000pherols had little effect on the utilization of carotene whereas larger doses decreased the storage of vitamin A (91). Moore (131) was the first to demonstrate the synergistic action of t0c0pherols on the deposition of vitamin A in the tissues. Rats fed a diet deficient in vitamin E had much lower liver reserves of vitamin A than control animals receiving the same diet supplemented by wheat germ oil concentrate or dl- alpha tocopherol. The mechanism by which t000pherols potentiated the action of vita- min A was not entirely clear. Davies and Moore (42) found that the reserves of vitamin A were used up much more rapidly in rats restricted to a diet deficient in both vitamins A and E, than in control animals given d1 - alpha tocopherol. Prolonged deficiency of vitamin E led to a secondary deficiency of vitamin A as indicated by the disappearance of this vitamin from the liver. They concluded that increased storage of vitamin A accompanying the administration of t000pherol was probably to be ascribed to its antioxidant action. 30 Sherman (164) demonstrated that carotene administered to vitamin A deficient rats was destroyed in the gastro-intestinal tract by the simultaneous feeding of unsaturated fatty acid esters. The gastro— intestinal destruction of carotene was prevented by feeding alpha tocopherol. It appeared to them that in the absence of alpha t0c0ph- erol there was a physiological antagonism between unsaturated fatty acids and carotene which resulted in the inefficient utilization of carotene. Quackenbush (147) believed that in promoting a biological response to carotene the tocopherol functioned as an antioxidant in the gestro- intestinal tract rather than as a compound regulating some phase of metabolism in the tissues. Daily supplements of 5 ug of carotene in ethyl linolate failed to produce growth in rats deficient in vitamin A but when a distillate from soybean oil was given simultaneously growth resulted. The protective factor in the distillate was apparently tocopherol since an equivalent amount synthetic alpha t0c0pherol and soybean t0c0pherol produced a similar response. The synergistic action of vitamin E on both carotene and vitamin A was confirmed by Guggenheim (74) who found that the daily administra- tion of vitamin E in combination with 100 IU of vitamin A increased the liver storage of vitamin A. Vitamin E increased both utilization of vitamin A and carotene and the fecal excretion of carotene. Apparently vitamin E acted by protecting carotene and vitamin A against oxidation in the intestine. The sparing and synergistic action of vitamin E were referred to as covitamin E activities by Hickman and co-workers (79, 80, 81). 31 Natural vitamin E (mixed t0c0pherols) enhanced the growth promoting power of vitamin A alcohol, vitamin A acetate, and USP reference 011. The vitamin A activity of carotene was markedly influenced by the tocoph- erol intake of the experimental rats. Approximately 0.5 mg of natural mixed t0c0pherols was the Optimum daily dose with which to demonstrate the sparing action of vitamin E on carotene. Synergism was largely lost when vitamin E and A were administered on alternate days. Furthermore, the "covitamin E" action was lost when the tocopherol was given parenter- ally, while vitamin A was given orally. The data indicated that sparing action was due to repression of oxidation in and/or near the gastrointes- tinal tract. The results obtained by Rao (148) supported the evidence shown by Hickman et al. (79, 80, 81). The wide variations in growth response of rats to 1 ug of B-carotene in ground nut, olive and coconut oils were reduced when pure alpha tocopherol was added to oils low in t0c0pherol. The covitamin E action on vitamin A was confirmed by lemley et al. (113) who introduced another concept. The favorable effect of t0c0pherol on the storage of vitamin A in the liver of the rat was diminished by extending the supplementing period from three days to three months and by raising the dietary intake of vitamin A. The "covitamin E" activity of tocopherols might have been more pronounced if smaller quantities of vitamin A had been employed. The storage of vitamin A in the liver of rats was increased when tocopherols were given with vitamin A for more than 3 months. The results of these workers were in contrast to the concept of intestinal action of the t0c0pherol as pr0posed by Hickman et a1. (79). 32 An interesting observation was made by Tomarelli and Gyorgy (178) who found that rice bran extracts contained a factor which could further support the action of t0c0pherol by increasing the growth reSponse with small does of carotene. The rice bran extract was effective in that it delayed destruction of tocopherol in a linoleic acid solution. Although there was considerable evidence that alpha t0c0pher01 increased the storage of liver vitamin A and potentiated the action vitamin A, some workers did not find any beneficial effect of alpha tocopherol (9, 61, 98). In 1940, Bacharach (9) was unable to demonstrate any difference between the vitamin A content of the livers of rats receiving vitamin E as tocopherol in normal curative or pr0phylactic doses and those of negative control rats receiving no supplement. Fraps and Meinke (61) determined the relative values of carotene in foods as measured by storage of vitamin A in livers of rats. These investigators did not find any increase in storage of vitamin A by addi- tion of t0c0pherol in rats. KBmmerer et al. (98) noted that alpha t000pherol fed with spinach did not increase the utilization of carotene for liver storage of vita- min A in rats. Embree (50) listed the reasons for these divergent results. The laboratory animals on fortified diets which contained several percent fresh or hydrogenated vegetable oils already contained more t0c0pherols than most human diets or the diets of many farm animals. Addition of more vitamin E to such an experimental diet was, for most purposes, superfluous. He was critical of the diet used by Kemmerer et al. (98) 33 because both test and control diets already contained enough vitamin A to afford optimum utilization of the carotene. Herbert and Morgan (77) reported that the addition of 0.5 mg alpha t0c0pherol daily to the diet of partililzy depleted rats receiv- ing 35 to 129 ug of vitamin A daily for 14 or 28 days produced no significant change in the liver stores of vitamin A. Addition of 0.5 ug alpha t0c0pher01 supplement daily to the diet of similar rats given 24 to 17 ug of carotene in oil daily for 14 or 28 days produced a significant increase in liver vitamin A. Above these levels no effect was found. The vitamin A content of livers was determined by the Carr- Price reaction. Although there seemed to be considerable evidence in favor of t0c0pherol increasing the efficiency of utilization of carotene and enhancing its value as a source of vitamin A, there were some reports which indicated that large doses of tocopherols have detrimental effect on vitamin A storage. Johnson and Baumann (91) found that alpha t0c0pherol did not inter- fere with the storage of vitamin A when the vitamin itself was fed, but the stores of vitamin A due to B-carotene were lowered significantly when 5 to 10 mg of alpha tocopherol were fed with carotene. Tocopherol injected intraperitoneally also appeared to interfere somewhat with the utilization of ingested carotene, but tocopherol fed eight hours after the carotene failed to interfere with the storage of vitamin A. In another study, Swick and Baumann (171) found that high doses of alpha t0c0pherol and alpha t000pheryl acetate diminished the storage of vita- min A in the livers and kidneys of rats fed moderate amounts of B- carotene. 34 A daily dose of 2 mg of t0c0pherol significantly diminished the efficiency of utilization of B-carotene (30). High and co-workers (83) reported that other antioxidents might also possess a variable effect on carotene metabolism. In rats large amounts of tertiary butylhydroquinone and octylhydroquinone decreased the utilization of carotene for tissue deposition of vitamin A, whereas small amounts of octyhydroquinone increased vitamin A deposition. large amounts of octylhydroquinone were more effective than small amounts in inhibiting the oxidative decomposition of carotene in vitro. Large amounts of vitamin E were likewise effective. These investigators postulated that nonspecific impairment of oxidative processes occurred in and near the alimentary tract when large amounts of these substances were administered with carotene, thus interfering with the enzymatic conversion of carotene to vitamin A. In further work High et al. (82) found that large amounts (10 mg/ day) of alpha t0c0pheryl acetate were without effect on the utilization of preformed vitamin A and did not interfere with the absorption of carotene in the alimentary tract. On the other hand, alpha tocopheryl acetate protected carotene markedly against oxidative decomposition 12 21252, These experiments supported the view that the locus of anti- oxidants as well as vitamin E was in the intestinal wall or at the site of conversion of carotene to vitamin A, and that the mode of action was either directly or indirectly involved in the enzymatic conversion of carotene to vitamin A. An interesting observation was made by Dicks et al. (44). Calves partially depleted of their t000pherol and vitamin A stores were fed alpha t0c0pherol ranging from 1 to 25 mg per 100 pounds body weight per 35 day and three levels of vitamin A (10, 100 or 1000 ug) for 4 weeks. The tocopherol resulted in an increase in utilization of vitamin A at 1000 ug level, a decrease in utilization at 100 ug level and an inappreciable change at 10 ug level. Reports also have appeared on the effect of tocopherol on the utilization of intravenously administered carotene. McGillivray and Worker (125) demonstrated that simultaneous intravenous administration of tocopherol and carotene in rats inhibited the conversion of caro- tene into vitamin A. Even as little as 1 mg alpha tocopheryl acetate decreased the amounts of both vitamin A alcohol and ester forms in the liver. It was possible that the inhibitory effect of tocopherol on the mechanism of conversion was apparent only when t000pherol and caro- tene were administered simultaneously. Further investigations by McGillivray and Worker (126) showed that high levels of alpha t0c0pherol in the blood and tissues inhibited the conversion of carotene to vitamin A. On the basis of results obtained with intravenous injections of t0c0pherol previously to, simul- taneously to and subsequently to intravenous administration of carotene, these authors concluded that carotene underwent a rapid breakdown pos- sibly in the blood to an imtermediate which was then more slowly converted to vitamin A. The diverse information reviewed above appeared to be puzzling. The results of different investigators could have been influ- enced by the severity of the deficiency of vitamin E to which the animals were exposed. Similar results could not be eXpected when t0c0pherol was required to correct a deficiency as when tocopherol was given to rePinforce levels which were already adequate in the diet. Also the Lazrge doses of t0c0pherol could inhibit one of the steps from carotene 36 to vitamin A formation, while in smaller doses tocopherol could enhance the formation of vitamin A from carotene. Small doses of t0c0pherol could prevent some destructive oxidation of carotene. Interrelationships between thyroid status and vitamin A. During the past thirty years a considerable volume of literature has accumulated on the interrelationships between vitamin A and thyroid status. Most observations were from eXperimental studies although some clinical observations have also been reported. Some evidence indicated that the thyroid gland regulated the conversion of carotene to vitamin A. Claims were made that the thyroid gland influenced the absorption of carotene. The possible relationships between vitamin A and thyroid were first indicated by clinical studies. Wold (189), in 1932, collected specimens of human liver from 957 cases at autopsy and found that there appeared to be some relationship between certain chronic diseases and low vitamin A status. logaras and Drummond (116) studied the influence of thyroid and vitamin A on metabolic rate and found that the thyroxine treatment increased the storage of vitamin A as compared to control pigs. Clinical investigations (130) showed that high reserves of vitamin A were found in ex0phthalmic goiter, although the small number of cases made the authenticity of these results questionable. Johnson and Baumann (90) depleted rats of vitamin A and at the same time brought them into either hypo-or hyperthyroid state. These rats were fed vitamin A daily for 15 days. Within the dosage limits employed the ability to store preformed vitamin A was approximately the same in hypo'or hyperthyroid rats as in normal rats. 37 Heimer et al. (78) intensively studied the effect of thyroid hormone on the storage of vitamin A and found that vitamin A storage in the liver of rats on a vitamin A free diet was highest in thyroid- ectomized animals, intermediate in the thyroxine supplemented animals, and lowest in the controls. From this it appeared that the storage was affected in some way by the thyroid hormone. The greater the thyroid activity the lower was the storage of vitamin A. Morgan and White (133) found that when 1.68 mg of vitamin A was fed to rats for A2 days there was no significant difference in total vitamin A per liver between controls and animals fed dessicated thyroid gland. Bamji and Sundaresan (11) studied the effect of hypothyroidism and hyperthyroidism on the absorption of retinal (vitamin A aldehyde) from the intestine of rats and on the level of liver vitamin A. The rate of absorption of retinal was faster in hyperthyroid rats than in controls. Hyperthyroid rats were observed to have more vitamin A in livers if analyzed two hours after the last dose of retinal, whereas they stored less vitamin A if analyzed five days after the last dose. This was probably due to greater utilization. Conversely, the hypothyroid rats had consistently higher liver reserves of vitamin A than the controls whether analyzed two hours or five days after the last dose of retinal, probably due to poorer utilization of their reserves of vitamin A. Reciprocal relationship or antagonism between the metabolism of vitamin A and thyroxine was proposed by many research workers. Fasold and Peters (57) concluded that hypervitaminosis A in rats could be prevented or curved by thyroxine injection. Conversely a solution of vitamin A in arachis oil or arachis oil alone prevented 38 the toxic action of thyroxine and permitted the storage in the liver of both carotene and vitamin A but not normal amounts of fat and glycogen. Sherwood et al. (159) demonstrated that cod liver oil produced a depletion of colloid in the thyroid of rats but had no effect when its vitamin A content was removed by heat and oxidation. Abelin (l, 2) found that the ingestion of a vitamin A preparation by rats decreased the rise in basal metabolism resulting from thyroid administration. The action of excess doses of thyroid in lowering the growth rate was also partially counteracted by administration of vitamin A. The latter also increased muscle glycogen. The thyroxine and vita- min A were antagonistic because of their Opposite influences on lipid and carbohydrate metabolism. Sure and Buchanan (168) confirmed the results of Abelin (l, 2) and also demonstrated that a diet containing 50% dried skim-milk powder furnished a sufficient amount of the vitamin B-complex, some of which are antithyrogenic in experimental hyperthyroidism. However such a ration containing 10% butterfat and supplemented with four drops of cod liver oil per animal per day did not provide a sufficient amount of vitamin A to counteract the rapid catabolism produced by oral adminis- tration of thyroxine. Greaves and Schmidt (72) reported that the antagonism between these two compounds was confined to the central nervous system. The vitamin A requirement of the rat might be increased by the administration of thyroxine or dessicated thyroid tissue. In thyroidectomized rats the need for vitamin A was decreased. Schulze and Hundhausen (155) showed that large amounts of vitamin A produced an antithyroid action. Histological examination of the 39 thyroid glands of the rats showed a somewhat increased activity for the rats deprived of vitamin A. The results also indicated a low thyroid stimulating hormone (TSH) in the pituitary gland of rats receiving large amounts of vitamin A and a high content of TSH in the pituitary glands of the rats deprived of vitamin A. Coplan and Samson (MO) found that the thyroid glands of both male and female rats on vitamin A deficient diet showed hyperplasia of epithelial cells similar to that observed in rats on an iodine defi- cient diet for a short period. The degeneration of the epithelial cells seemed to be a Specific effect of vitamin A deficiency. Logan (115) found that patients with both cretinism and some form of malignent disease had a I131 uptake ranging from 21.2 to 68.2% After treatment with 50,000 IU vitamin A per day for three weeks the uptake was reduced from lh.8 to 32.8%. The mechanism of antagonism was studied by Belasco and Murlin (lb) who reported that vitamin A antagonized the active thyroid principle, possibly by inhibiting thyroid function since vitamin A depressed thyroid tissue respiration in_vitro but not that of liver or kidney. The vitamin A was able to take up the available iodine in body tissues by virtue of its double bonds. The organic iodide thus formed could antagonize hyperthyroidism similar to Other inorganic or organic iodine compounds exclusive of thyroxine or thyroglobulin. Thus iodinated vitamin A might effect the storage of colloid either by acting on the anterior pituitary gland or on the thyroid gland directly and conse- quently causes a lower oxygen uptake of the thyroid. ho Working on energy metabolism, Sadhu and Brody (153) demonstrated that excess vitamin A depressed metabolic rate and reduced thyroid size of normal, thiouracil treated and thyroxine treated rats. The effect of thyroid activity on the metabolic requirement for vitamin A was also investigated. COOper et a1. (39) determined the growth response to suboptimal levels of vitamin A in chicks fed thyro- protein or thiouracil. They found that hyperthyroid chicks showed smaller gains than normal at all levels of vitamin A fed (25, 50, 75 and 100 IU per day). The hypothyroid chicks were heaviest at the 25 and 50 IU levels. However hypothyroid chicks did not increase in weight as the levels of vitamin A increased. According to Blaizot and Benac (23) the average oxygen consumption was 10 to 25% greater in the vitamin A deprived rats than in normal rats. The increase in oxygen consumption was an earlier sign of defi- ciency than cessation of growth or xerOphthalmia. This indicated that thyroxine aggravated the effects of avitaminosis. Frapes et al. (60) showed in pigs that insufficient (0.0 IU/lb ration) and excessive (6A00 IU/lb ration) intakes of vitamin A lowered the rate of secretion of thyroxine. The effect of vitamin A on thyroid secretion was thought to be direct and not a reflection of its effect on growth. The evidence which was reviewed has indicated that there was antagonism or at least some kind of interaction between vitamin A and thyroid gland activity or thyroxine. However Baumann and Moore (13) showed that injected thyroxine failed to counteract the hypervitaminosis A in rats receiving excess dietary vitamin A. Animals receiving both thyroxine and excess vitamin A1 A consumed less food, lost weight more rapidly and died earlier than those receiving either agent alone. The injection of thyroxine alone produced a temporary decrease in food intake followed by a marked increase. They did not find any evidence for a specific antagonism between thyroxine and vitamin A. Sheets and Struck (158) found that the effect of small or massive doses of vitamin A on the metabolic rate of rats was questionable. Vitamin A did nOt show any effect in animals fed thyroid powder or in thyroidectomized animals. A very interesting finding was reported by Hochstadt and Malkiel (81+) who found that the inhibitory factor was not the vitamin A and could be separated from the vitamin A; Ether extracts prepared from blood of normal individuals were tested for vitamin A and also for their power to inhibit the protective action of thyroxine in acetonitrile poisoned mice. Little of the latter activity but some vitamin A was found in the serum extracted for a one hour period. After longer extrac- tion periods the extracts contained no vitamin A which was presumably oxidized but had considerable power to inhibit thyroxine. A review of all the evidence indicates that there is some kind of interrelationship, possibily antagonistic,between thyroid status and vitamin A in animals. It is interesting to note that vitamin A can decrease the metabolic regulatory action of thyroxine. Although this activity forms a strong case for specific antagonism, the degree of Specificity of this influence remains undefined. However there was higher reserves of liver vitamin A in hypothyroidism due to poorer utilization of the liver vitamin A reserves whereas in hyperthyroidism A2 there were lower reserves of liver vitamin A due to efficient utiliza- tion of-liver vitamin A reserves. The effect of the thyroid gland on the conversion of carotene to vitamin A. There seems to be some evidence that the thyroid gland is directly or indirectly concerned in the conversion of carotene to vitamin A. Numerous reports have been appearing on this subject and several of them are in disagreement. As early as 1907 VonNoorden (181) suggested that the thyroid gland might be involved in carotene metabolism. He observed that carotenemia might be associated with certain metabolic disorders. Fasold and Heidemann (56) made an interesting observation that after thyroidectomy the carotene content of goat milk increased and the vitamin A content was decreased although goat milk normally contained vitamin A but low carotene. This was attributed to inability to trans- form carotene into vitamin A in the absence of the thyroid gland. Drill and Truant (A5) demonstrated that in rats fed vitamin A free diets supplements of carotene prevented the occular changes character- istic of vitamin A deficiency in control rats but failed to prevent xerophthalmia in similar thyroidectomized rats. This indicated that the carotene was not being utilized in the animals. Daily injections of thyroxine to the animals allowed them to survive, gain weight and to be cured of their xerophthalmia. Johnson and Baumann (90) found that when carotene was fed to the hyperthyroid animals they accumulated largerstores of vitamin A than normal rats receiving equivalent amounts of carotene. However rats receiving thiourea or thiouracil stored very little vitamin A. The administration of thyroxine neutralized the effects of both thiourea 1+3 and thiouracil and increased the ability of animals to convert caro- tene to vitamin A. It was suggested that altered carotene metabolism associated with thyroid dysfunction was not due to changes in the basal metabolic rate per se, but was brought about by some other physio- logical action Of the thyroid gland. Kelly and Day (97) fed rats a vitamin A deficient diet plus 0.5% of thiouracil or 0.6% thyroglobulin. All animals were given B-carotene in wheat germ oil as a single dose. The thyroid status affected the conversion of carotene to vitamin A as indicated by the content of vitamin A found in the liver. The investigators pr0posed that the amount of vitamin A present in the tissues may result from two Opposite effects of thiouracil: l) impairment of carotene conversion to vitamin A and 2) increased retention of vitamin A in the liver once it was deposited there. Cama et a1. (33) confirmed the observation reported by Johnson and Baumann (90) and Kelly and Day (97). In addition these investi- gators also confirmed the results of Worker (191) that thyroid activity had little influence on the conversion of intravenously administered carotene to vitamin A. Kowlewski et al. (107, 108) studied the effect of thyroidectomy and of thyroxine on the utilization Of carotene administered intrave- nously. These investigators found that the thyroidectomy increased and prolonged the rise of carotene in plasma and suppressed the increase of plasma vitamin A in normal and thyroid treated dogs. When obser— vations on thyroidectomized dogs were repeated after adequate treatment with thyroxine carotene disappeared from the serum and vitamin A increased temporarily as in normal animals. ML Swick et al. (172) noted a very slight increase in liver stores of vitamin A in hyperthyroid animals as compared to controls. Depleted pigs were given either thiouracil or iodinated casein and their subse- quent responses to carotene or vitamin A determined. Essentially similar stores Of vitamin A were found in the livers and kidneys of the three groups with a suggestion of increased stores in hyperthyroid animals that were free from diarrhea. Further evidence for an unequivocal relationship between thyroid status and the ability Of the organism to convert B-carotene to vita- min A came from the reports of Serif and Brevik (156). Liver store of vitamin A was increased in hyperthyroid rats and decreased in hypothyroid rats as compared to control animals. This data indicated‘ that butyl h-hydroxy - 3, 5-diiodobenzene (BDH) functioned as an inhib- itor of the process whereby thyroxine was degraded to an active hormone presumably triiodothyronine. Thus BDH could inhibit the action of exogenous thyroxine but not the effects of exogenous triiodothyronine. This led to the conclusion that the effective form of thyroid hormone was triiodothyronine and that thyroxine per se possessed no activity on the conversion of carotene to vitamin A. Further evidence that the thyroid hormone stimulated the formation of vitamin A from carotene was shown by Kaplansky and Balaba (95) using an in vitro system. Iodinated casein acted as a catalyst in vitro by increasing conversion at pH 7.3. If the iodinated casein was heated, it lost this property. Iowry and Lowry (117) and McGillivray (120) were unable to confirm this report. 1+5 Although most of the above reports have added support to the hypo- thesis that the thyroid gland influenced the conversion of B-carotene to vitamin A, several other reports have been appeared with opposite results. Remington et al. (150) reported that oral dosages of carotene were equally as effective as vitamin A in curing xerOphthalmia in thyroidectomized rats. Using growth as the criteria Wiese and his co-workers (186) found that although the extent of maximum growth after vitamin A or carotene feeding was markedly depressed by hypothyroidism, the point of 50% response was unaltered either with the vitamin A or carotene feeding. This data indicated that low levels of carotene and vitamin A were about equally well utilized by hypothyroid rats. Similarly, Bieri and Schultze (22) could not demonstrate any effect of thiouracil on vitamin A concentration in the serum, liver and kidneys of the rats fed or injected with aqueous despersions of carotene. Arnrich and Morgan (8) found that administration of small doses of carotene produced greater amounts of vitamin A in the livers Of the hypothyroid rats than in the normal controls. Extending the studies to dogs, Arnrich (7) found similar increases in liver vitamin A concen- trations in normal and thiouracil treated young mature dogs following the ingestion Of carotene. However thiouracil administration caused a rise in concentration of blOOd vitamin A as compared with the level found in normal controls. Worker (191) showed that thyroidectomy or hyperthyroidism had no direct effect on the conversion of intravenously administered carotene 46 to vitamin A as Observed by similar blood levels and liver storage of vitamin A in treated groups. Recently Boguth and Sari (27) observed that the conver- sion of carotene to vitamin A was somewhat independent of thyroid status. Hypophysectomized and vitamin A depleted rats given 100 ug B-carotene daily recovered from symptoms of deficiency in five days. Horvat and Merril (87) administered 0.4 mg of 3 5'3' — triiOdO-D-thyronine (D-T3) daily for up to 21 days to patients with myxedema and found a decreased level of serum caroten— oids. Vitamin A levels were not consistently affected. Thy- roidectomized rats deprived of vitamin A 5-10 days received 45 ug D-T3 daily per Kg. body weight and 515 ug of carotene per day by mouth for four days before they were slaughtered. This treatment did not affect storage of vitamin A in liver. About 20% of the carotene was recovered in the feces with or without D-T3. These results indicated no evidence that D-T3 enhanced vitamin A formation from B—carotene. In these experiments the experimental period was rather short and previous dietary history may not have been adequately standardized. There is also considerable evidence to show that the thyroid hormone has a significant role in the intestinal absorption of carotene. The results of some experiments have suggested that the thyroxine increased and thiouracil decreased the efficiency of absorption. Cama and Goodwin (32) showed that thiouracil inhibited 47 and desiccated thyroid stimulated the absorption of B— carotene from the intestinal tract in rats. Fecal excre— tion of carotene was decreased 18% in rats given desiccated thyroid compared to controls. Administration of thioura— cil increased excretion an average of 31%. Desiccated thy— roid counteracted the inhibition of Thiouracil when admin- istered together with Thiouracil. Chanda et a1 (35) reported that digestibility of caro- tene by cows and goats was markedly increased by thyroxine and reduced by thiouracil injections. Further evidence that the thyroid might influence the absorption of carotene was shown by Chanda and Owen (36). When cows were deprived of carotene for 15 days the milk yields were not affected but the concentration of vitamin A and carotene in the milk decreased. ‘When a carotene containing diet was fed the concentrations of both carotene and vitamin A increased at rates which were enhanced by thyroxine and diminished by Thiouracil. The effects of discontinuingthiouracil were similar to those of giving thyroxine. These conflicting reports reviewed demonstrate that the thyroid gland either directly or indirectly has some effect on carotene and vitamin A metabolism. But the degree and specificity of this influence was uncertain. The different results obtained by all these investigators were not read— ily understood. Most probably, the conditions used were not identical. It is very difficult to completely thy— rOidectomize animals and a small piece of thyroid gland 48 remaining inside would be sufficient to make the experi- mental results erroneous. Other possible explanations for Obtaining conflicting results may be the medium in which carotene was dispersed since this influences the carotene absorptiOn and different properties of thyroxine and triiodothyronine have different activities in normal ani- mals of different strains and species. Mechanism of conversion of B—carotene to vitamin A. The mechanism of vitamin A formation is not very well understood. There are two hypothesis on the mechanism of the conversion of B-carotene to vitamin A; the direct cen— tral cleavage hypothesis to yield two molecules of vitamin A (96) and the B-oxidation mechanism to yield one molecule of vitamin A (67). Mbst of the studies to date were based on measurements such as growth, liver vitamin A, blood or lymph vitamin A and nutritional balance. It is evident that these results can be affected by several variables such as stability and rate of absorption, transport, turnover, etc. To overcome this variability Cl“ labeled B-carotene has recently been used in an effort to more directly study the mechanism Of conversion (138). Kuhn et al. (112) and Hunter (89) supported the theory of central fission based on biological activity of the isomers of carotene. B—carotene which could theoritically yield two molecules of vitamin A, was twice as effective for-growth promotion as alpha and gamma carotenes which 49 could yield only one molecule of vitamin A. Lutein, zeaxan— thia and violaxanthin had no growth promoting activity. Koehn (lOl) investigated the relative biological potencies of pure B—carotene, vitamin A alcohol and ace— tate for the rat under identical dietary conditions in the presence of adequate alpha tocopherol. Similar growth of vitamin A depleted rats was obtained when receiving daily supplements of either 1 ug of B-carotene or vitamin A alcohol. Similar results were obtained by feeding B-caro- tene and vitamin A acetate at stoichiometrically equivalent levels. The date indicated that B-carotene was quantita— tively converted into vitamin A and supported the theory that the conversion of B—carotene into vitamin A.in vivo was by central cleavage to form two molecules of vitamin A and not by an oxidative reaction that inactivates one half of the carotene molecule. Johnson and coworkers (92) showed that B-carotene was about two-thirds as active as vitamin A on a weight for weight basis in the chick as Observed from growth responses when fed by dropper with 0.5 mg of alpha tocopherol daily. Evidence supporting the central fission theory also came from the studies of Burns et al. (30). In 1937, Holmes and Corbet (85) crystallized vitamin A in the form of pale yellow needles from the liver oils of three different species of fish and found that its biological pOtency for growth was that of B-carotene on a weight for ‘WEight basis. Mead et al. (127) confirmed these results by 50 using a biological assay method. Since the potency of vi— tamin A alcohol and B-carotene was 3181 and 1670 IU/ mg reapectively, vitamin A itself was twice as active as B— carotene on weight for weight basis. In another nutritional study it was shown by Hume (88) that B-carotene seemed to be no more effective, mole for mole, than vitamin A. The investigator emphasized that if 0.3 ug of vitamin A has the same activity as 0.6 ug of B- carotene then the molecule of B—carotene does not split into two molecules of vitamin A as was long supposed. Fur- ther, Zechemister et al. (196) applying resonance theory suggested that the central double bond of a conjugated system will be more stable than a terminal one and hence less susceptible to attack. The theory might be applied to B-carotene. Von Euler et al. (54) showed that B-apo—SLcarotenal was biologically active which provided support for the terminal oxidation theory. Glover and Redfearn (67) prepared possible intermedi— ates Of B-carotene by stepwise degradation from one end such as B-apO-S, B—apo-ld, and B-apo—lé—carotenals and administered them to vitamin A deficient rats. All were transformed to vitamin A. These results indicated that the carotenals were oxidized by a type of B—oxidation to form ‘Vitamin A aldehyde. These investigators suggested that the provitamins A were first formed into vitamin A aldehyde by Stepwise oxidation and was then reduced to vitamin A alcohol. 51 Fazakerley and Glover (58) found that B-apO-carotenoic acids were biologically active for stimulating growth in vitamin A deficient rats. Glover (64) an exponent Of the stepwise oxidation theory slightly modified the original theory and concluded that there is an oxidative attack at more than one position in the chain. The true nature of the conversion process is far from clear, but the work with synthetic B—apo-carotenoids has eliminated the normal B—oxidation system as originally being responsible. Oxidative enzymes are probably respons— ible for carotene degradation. Most of the above studies were conducted with intact animals, and consequently many of the deductions about the absorption and cleavage of B—carotene were based by necess- ity On Observations which were indirectly related to the problem being studied. This was particularly true of bio- logical methods such as measurements based on growth, vita- min A levels in liver, blood or lymph and nutritional balance. .All of these were affected by several variables such as relative stability, absorption rate, transport, uptake by tissues, rate of turn over and metabolism etc. Hence it is not surprising that considerable controversy exists regarding the influence of various factors on B- carotene cleavage, and that no definite conclusion can be forthcoming from this method of attack. Further evidence for stepwise oxidation theory was 52 provided by Suzuki et al. (169). Studies on metabolic con— version Of B-carotene to vitamin A in vitro by rat tissue homogenates revealed that there were two metabolic interme- diates, one with an absorption maxima 296 mu in ethanol which was identified as B-ionone, and the second with an absorption maxima at 365 mu in petroleum ether which was identified as a vitamin A aldehyde-protein complex. The work attempted by Olson (139,140) represented a new approach to the problem of carotene to vitamin A conversion. He used C14 labeled B-carotene. Although these experiments were not conclusive, the central cleavage mechanism.was favored as the major pathway for B—carotene metabolism in the intestinal mucosa. Appreciable amounts of acidic and polar products which would be expected from stepwise cleavage in the mucosa were not found. (The information reviewed above clearly indicated that the true nature of the conversion process is still far from clear. Glover and his colleagues (64,142) found that B— apo—carotenals and B—apo~carotenoic acids, which possessed but one B-ionone ring, were biologically active in stimu- lating the growth of deficient rats and that radioactive acidic products as well as vitamin A were present 24 hr after radioactive B-carotene was fed to rats. These authors favored the B-oxidation theory for vitamin A forma- tion. Olson (138) who favored central fission theory showed that appreciable quantities of acidic or non polar 53 products did not accumulate in his studies with rats. The possibility exists that the acidic fractions which appeared during B~carotene cleavage might well arise from further oxidation of vitamin A and not from the direct cleavage re— action since vitamin A was also metabolized in the intes— tine to form a small amount of acidic products. The two hypotheses are somewhat contradictory to each other, and the present information is not adequate enough to delineate the pathway for vitamin A formation. 54 EXPERIMENTAL PROCEDURE Animals, diets and treatments Dairy cattle, sheep, rabbits, rats, swine and chicks were used in these experiments. Tissues were obtained from cattle, rabbits, rats, chickens and pigs fed experimental diets and from cattle, sheep and rabbits fed normal diets. All types of dairy cattle used in the experiments. The calves fed experimental diets were reared to 7 days of age under a normal situation. At 7 days of age a mixture of 0.7 lb dried skim milk plus 0.6 lb dried whey reconstituted with water per day per 100 1b of body weight was fed. ‘When the calves attained two seeks of age, they were given free access to carotene dificient diet which was made up of dried beet pulp, soybean flakes, soybean meal, oats and barley, mineral mixture and vitamin D. The substitute milk feeding was discontinued when calves were two to three months of age. Hay was never fed. All eight calves grew at a normal rate and care was taken to prevent drastic depletion of their vitamin A stores. Tissues from these calves were used to study carotene conversion. Some of these calves and other normal calves were fed NaNO3 at the rate of 1% of diet (experiment 11) three weeks prior to slaughtering. In the nitrite studies (experiment 6), one Jersey and lone Holstein yearling heifer were each fed nitrite and one 55 Jersey heifer was kept as control. ,All three received high concentrate diet low in carotene and KN02 was added to the diet of the two heifers in gradually increasing levels to reach the rate of 13 gm per 100 1b body weight. These ani- mals were depleted Of the vitamin A reserves to the extent that they had slight papilledema. Calves used in the experiments to study interrelation- ships between thyroid status and nitrate on carotene con- version were divided into control, hyperthyroid and hypo- thyroid groups with two in each group. All calves received a normal calf ration of hay, grain and milk throughout the experimental period. In addition the hyper — and hypothy— roid groups received by capsule l g of thyroprotein and 10 g of thiouracil daily, respectively (Experiments: 22, 23, 24, 25, 26 and 27). iOther cattle used in these studies received normal rations and were considered to be non-depleted. Sheep used in these experiments were maintained on var- ious barn dried hays as the only feed for several months prior to slaughtering and carotene intake was assumed to be normal (ExperimentslB, 14, and 43). Rabbits of the Dutch Belted breed were generally main— tained on a normal diet consisting of commercial rabbit pellets fed ad libitum. However, in experiment 9 they were transferred from this diet to alfalfa hay and then to a low carotene diet consisting of spelt plus trace mineralized salt and calcium phosphate. After some time on this diet 56 the rabbits were divided into three dietary groups; (1) control group (spelt), (2) control diet plus 2% N03, and (3) control diet plus 0.3% N03. At the same time two extra rabbits were fed the normal diet. Three groups of three rabbits each were used in experi- ment 12. Group one was maintained on normal diet consisting of commercial rabbit pellets, group two received this normal diet plus 2% N03 and group three was fed normal diet plus 0.5% N03. The N03 used was in the form of NaNOé. Some of the other rabbits used in this study were de- pleted of their vitamin A reserves by feeding rolled oats, mineralized salt and calcium phosphate (Experiments: 36, 37, 57, 61 and 63). Rabbits used to study interrelationship between thyroid status and nitrate on carotene conversion (Experiments: 29, 30, 31 and 32) were reared on the commercial rabbit pellet diet. They were divided into four groups of three each. They were designated as (1) control, (2) hypothyroid, (3) hyperthyroid and (4) thyroidectomized. The control and thyroidectomized groups were fed the commercial rabbit pellets while the hyper- and hypothyroid groups received this diet plus .03% therprotein or 0.2% thiouracil, re- Spectively. Rats used in these experiments were cross breds Obtained from the Animal Husbandry Department of Michigan State Uni— Versity. Non—depleted rats of both sexes were used for ex— Periment 28 and vitamin A depleted rats Of both sexes were 57 used for experiment 34. The dams of the latter rats used were maintained on a normal diet. After littering the pups received dams' milk until weaning and when ready to eat solid food their diet was changed to a vitamin A deficient diet which contained the following composition: Ground milo 68.9% Soybean oilmeal, solvent (50% C.P.) 28.0% Dicalcium phosphate 1.8% Calcium carbonate 0.6% B-vitamins, Dawes Forfiee (2—4—9—90) 0.1% Dawes vitamin B12 supplement (6 mg/lb) 0.2% Trace mineralized salt 0.4% Irradiated yeast 9F(9000IU of vitamin Dz/gm) 5.0% The B-vitamin, Dawes Forbee (2-4-9—90) contained the follow— ing: Riboflavin 2.00 g Niacin 9.00 g Choline chloride 90.00 g Soybean meal added to make up to one pound. Under these conditions there was little storage of vitamin .A in the liver. The intestine was completely devoid of Vitamin A. Berkshire young pigs (Experiments 40, 41) were of both Sexes and were obtained from the piggery of the Animan Hus— bandry Department, Michigan State University. They were rearednormally until six weeks of age when they were then 58 kept on vitamin A deficient diet. At the time of slaughter— ing they were eight weeks old. The vitamin A deficient diet had the following composition: Ground milo ' 78.338% Soybean oil meal (50% C.P.) 19.000% Dicalcium phosphate 0.700% Lime stone 1.100% Trace mineralized salt high in zinc 0.500% B—vitamins, Dawes Forbee (2-4-9-90) 0.100% Irradiated yeast 9F(9000 IU vitamin Dz/g) 2.000% Dawes 312 supplement (6 mg/lb) 0.150% Antibiotic, protrap 0.100% Zinc oxide 0.012% Chicks (Experiment 35) were cockerels Obained from the Poultry Department, Michigan State University.' The day old Cobb's strain White Rock Chicks were obtained from hens fed the usual breeder mash and had a normal carryover of vita- min A.from the dam. The birds were then fed the following diet which was adequate in all known nutrients except vita~ min A: Milo 60.0% Soybean oil meal (44% C.P.) 34.0% Lime stone 01.5% Salt (Iodized salt + 10 mg manganese sulphate) 0.5% Vitamin premix 2.0% The vitamin premix contained the following: 59 Riboflavin 200.0 mg DL — Calcium pantothenate 500.0 mg Niacin 1250.0 mg Choline chloride ' 2000.0 mg vitamin B12 0.5 mg Procaine pencillin 200.0 mg Vitamin D3 40,000 IU Soybean meal 2.0 lb The experimental diet was fed ad libitum along with fresh tap water for four weeks. Reagents and solutions .All organic solvents were redistilled before using. ACS grade petroleum ether (bp. 30-60) and acetone were distilled over KOH pellets with the first and last 10% discarded. Chloroform was purified by distillation with the first and last portions discarded as above.' Ethyl alcohol, 95%, used in these experiments was aldehyde free. Care was taken to use only pure anhydrous granular NagSOq which does not ab- sorb vitamin A. Ethyl ether used here met all ACS specifi— cations for purity. Benzene was redistilled to make it moisture free. Other chemicals such as KOH, SbCl3, etc., were reagent grade commercial products. Deactivated.Alumina .Alumina for chromatographic purposes was Merck's re- agent grade chromatographic A1203 (No. 71707). Deactivated alumina was prepared according to the method of Olson (138) 60 with modification. One hundred g of A1203 was shaken with 6 m1 of water in 500 ml of petroleum ether for 3 hr at room temperature. The material was filtered with suction and the alumina was spread on a filter paper and dried by gentle raking with a glass rod for 15 - 30 min. This was immedi- ately stored_in a tightly closed amber colored bottle. Care was taken not to over dry the material since this made it too active. E1uting_solutions. The eluting solutions used in the chromatographic separation of compounds formed after incubating with B— carotene were as follows: 1. Petroleum ether, 2. 3% acetone in petroleum ether, and 3. 8% acetone in petroleum ether. Antimony trichloride (Carr-Price) reagent. The SbCl3 reagent was made by the method described by Parrish (142) with slight modification. Chloroform was added to 100 gm of reagent grade SbC13 to make 500 m1. To this 15 m1 of acetic anhydride was added, the mixture re— fluxed for one-half hour, cooled, filtered and stored in a tightly stOppered amber colored bottle. The solution had to be clear and free from cloudiness. Splution for removing antimony trichloride from cuvettes. The cuvettes were washed first with 40% Hcl, then with detergent solution, distilled water and finally with alco— hol. 61 Vitamin A standard solution. Vitamin A acetate dissolved in cottonseed oil in a gelatin capsule with a potency Of 30 mg vitamin A g/Oil and also pure vitamin A alcohol Obtained from Hoffman-Roche CO., Inc. were used to make a standard curve after dissolving in CHCl3. Carotene standard solution. Pure 100% B-carotene was dissolved in petroleum ether and used to prepare the standard curve. Carotene dispersion for incubations. The B—carotene used in the incubation experiments was 100% pure purchased from Eastman Kodak CO. A suspension of carotene was prepared by adding a weighed amount of B-caro— tene to 50 m1 of distilled water, shaken well and the agi— tating by passing nitrogen into it; then 5 m1 of Tween 80 was added and the mixture was shaken vigorously to make a uniform suspension. Five grams of sodium glycocholate was dissolved in the suspension by stirring with a glass rod. Then the suspension was diluted to 100 ml with distilled water. All the Operations were carried out in a closed room where there was no chance of direct light entering. Caro— tene suspensions were always made immediately before use and any remaining suspension discarded. Physiological buffers. All physiological buffers used in these experiments were made in the conventional manner. Krebs—Ringer solution was prepared as described by Umbreit et al. (179). Phosphate 62 buffer, Tris buffer and veronal buffer were made as described by Gomori (68). To simplyfy the preparation and handling of the solutions, it was found convenient to make up the concentrated stock solutions of all ingredients which were stored at 50 C. ‘When needed the ingredients were mixed and diluted to make up the buffer at the required PH and used immediately. The 0.25'M sucrose solution and 0.9% normal saline solution were made the evening before use and stored at 5° C. Hormones. ‘ I-and D—thyroxine, and I- and D—triiodothyronene were donated by Smith Kline & French Labs; 3 5’3’- triiodothy- ronine was donated by'Warner-Lambert Research Institute. All the solutions of required concentration were made just before use or on the previous night and stored at 5° C. Proceduree All experiments conducted and their main purposes are shown in Appendix table 1. In vitro experiments using tissue homogenates. Experimental animals used in this study were fasted overnight before slaughtering. Thus, when slaughtered less solid material was present in the small intestine and wash- ing of lumen was facilitated. Some tissues were also Ob- tained from various animals at the slaughter house and pre- vious treatment of these animals was not always ascertained. In all cases the animals were thoroughly bled out, the ab— dominal cavity was Opened immediately and the duodenum and 63 the organs were removed as quickly as possible. The duodenum was washed and completely flushed once with cold 0.9% physiological saline solution. The same procedure was followed when other portions of the intestinal tract were used. Flushing out with saline solution removed most of the intestinal contents. When the other organs were used they were removed immediately and rinsed in cold 0.9% physiological saline solution. There was lot of fat and fascia adhering to the outer wall of intestines from calves, heifers, sheep and pigs and these materials were separated from the tissue with a sissors. The lumen of the duodenum (or intestine) was Opened by splitting it lengthwise before they were out into small pieces to facilitate homogeniza— tion. Other organs were cut into small pieces prior to homogenization in a waring blender with freshly prepared cold 0.25 M sucrose solution. .A 2:1 ratio of tissue weight to solution volume was used except in unusual cases. All the above operations were carried out as rapidly as poss— ible. In our experience, speed of Operation was an essential factor. Aliquots of homogenate each weighing 8 to 12 g were placed in low actinic 250 ml flasks, contain— ing 2 m1 of 0.25 M sucrose solution and in some trials 2 m1 of various experimental chemical solutions. ‘When the other organs were used, they were homogenized in the same manner and similarly used. The samples were then incubated for three hours at 38° C in an incubator. Incubation time were varied from 5 min to 4 hours to study 64 the influence of incubation times on vitamin A formation and carotene disappearanCe (Experiment 44, 45, 46). ,All the procedures were conducted in the dark or in dim light. Incubation of different portions of the duodenum In experiments 17 and 18, calf duodenum was split length wise after rinsing and flushing with 0.9% saline and one portion homogenized. The mucosa was separated from the muscular layer for the other half and these two portions were homogenized separately. These three portions were then incubated with a carotene dispersion as described earlier. Incubation of heated duodenal homogenates In experiment 11, the flasks containing calf duodenal homogenates and 2 ml of 0.25'M sucrose were placed in boil— ing water for 1, 2 and 4 min. Internal temperatures were not Observed. ,A similar procedure was followed using sheep duOdenal homogenates in experiment 13, and steer duodenal homogenates in experiment 56. However, in the last two ex— periments the flasks were removed from the bath at 1, 2 and 4 min after the temperature of the contents in the flasks reached 90°C. Then the heated homogenates were cooled to 20°C and incubated with the carotene dispersion as usual. Incubation Of blood and its components with or without duodenal homogenates Blood from heifers or steers was drawn from the jugular vein either into test tubes containing anticoagulant such as sodium citrate or oxalate or into test tubes containing no anticoagulant. From this serum, plasma and sedimented 65 cells were separated. Hemolyzed whole blood was made by vigorously shaking whole blood with distilled water for 1 min. An aqueous dispersion of carotene was incubated for 3 hr with 2 ml each of whole blood, hemolyzed whole blood, sedemented cells, plasma and serum in the low actinic flasks at 33°C as usual. In certain experiments tissue homogenates were incubated with carotene dispersion and 2 ml each of the above blood components separately. In some experiments (NO. 57, 58, 59) the sedimented portion was mixedeith equal volume of distilled water, centrifuged at 27,000XG and the sediment and supernatant of each tube ranging in volume from 0.25 to 2 ml were incubated separately with the carotene dispersion and the tissue ho- mogenates. In other experiments the sediment was further diluted with an equal volume of distilled water, centri— fuged at 27,000 KB and the sediment and supernatent of each ranging in volume from 1 to 2 ml were incubated as above. Incubation of duodenal homogenates with different levels of various chemicals Various solutions were prepared just before use or made the previous evening and stored at 5°C for use the next day. The different levels of various ions and compounds used in these experiments are given in Table 1 and will be evident in the section on Results. Incubation Of duodenal homogenates using different buffers This experiment was designed to compare a 0.9% NaCl solution, a phosphate buffer at PH 7.4, a Krebs-Ringer 66 solution, a Tris buffer at PH 7.2, a veronal buffer at PH 7.4 and a 0.25 M sucrose solution as the incubation media. After flushing theduodenum.with 0.9% NaCl solution, the tissue was homogenized and incubated with each of the buffers separately as previously described for 0.25'M sucrose. Separation of activity by differential centrifugation of homogenates An attempt was made to concentrate the activity from the homogenates of liver and duodenum of depleted rabbits by differential centrifugation. The tissue under investi— gation was homogenized with cold 0.25 M sucrose solution. A 1:1.5 ratio of tissue weight to solution volume was used. The homogenate was transferred into three centrifuging tubes in equal portions and centrifuged for 15 min at 5°C at 5000 X0, 10,000 KC, and 25,000 XG, respectively. The supernatant fluid was separated carefully from the sediment and these two fractions were used as enzyme sources. Then the incubation was carried out using either 1 to 4 m1 of supernatant fluid or 1 to 4 3 sediment, 2 ml 0.25 M sucrose and 2 ml aqueous dispersion of carotene at 38°C for three hours as usual. The total volume of incubation mixture was always kept constant for each degree of centrifugation. lpcubation of isolated intestinal loops In this study the incubation of isolated intestinal loops obtained from rabbits was carried out in vitro to in- Vestigate the effect of the adding nitrate solution directly into the loOp on the formation of vitamin A from carotene. TABLE 1 67 Incubation of duodenal homogenates with different levels of various chemicals Experiment Compounds or Amount added/ no. ions used 3. tissue 3-6, 8-15, 20 N03 0.2 to 1006 u moles 3-5 N05 54 to 536 ' " 1o,11,21,25-27 s02, cog, NaCl 50 to 500 " 22-27, 38, 39 '42 >25-27 25, 27 27 42 55, 56 54 24, 54 24, 54 54, 55 54 L-Thyroxine, L-Tri- iodothyronina D-Thyroxine, L-Tri- iodothyronine Fresh thyroid gland Fresh adrenal gland Thiouracil 3 5'3'-L1riiodothyronine DL-Alanine, DL-Proline, L-Tryptophan p-Chloromercuric benzoste Iodoacetate, KCN Urea I p-Dinitrophenol Antimycin, Hydroxylamine * Final concentration in the flask 0000001 to 1.0 Ug 001 " 1 s 1 s l to 10 mg 10 ug 2 x 10"2 M* 10‘5 to 10‘3 M* 10'5 to 10"3 M* 4 to 8 M* 10’5 to 10'3 M* 10“5 to 10"3 n* 68 The rabbits were killed and the abdominal cavity was opened immediately. The small intestines to be used were quickly removed and placed in the cold 0.9% NaCl solution. The con- tents of small intestine were flushed once with cold 0.9% NaCl solution. The intestinal portion to be used was weighed rapidly on a balance and one end of it was tied off. The 1 ml of aqueous carotene dispersion and 1 ml of 0.25 M sucrose solution were injected into the lumen and the other end tied. These loops were placed in low actinic flasks containing 0.25 M sucrose solution and incubated for 3 hr at 38°C. The control loops not containing carotene suspension were incubated simultaneously. ‘When effect of nitrate was studied 3 sections of equal length were made from the same intestine by ligating at 4 places. The follow- ing combinations were injected into the three loops: Loopl- —~1 m1 of 0.25 M sucrose only, Loop2- - 1 m1 of 0.25 M sucrose pluS; 1 m1 aqueous carotene dispersion, LOOp3- - the above combination as in loop 2 plus 0.5 m1_NO§ solution. The intestinal sections were then placed in low actinic flasks containing about 50 m1 of 0.25 M sucrose solution and incubated at 380C for 3 hr. After incubation, the sec- tions were removed from the flasks and loops were separated by cutting with seissors. The sucrose solution in the flask was discarded. The loops with contents were then homogenized 69 with 20 m1 of l N KOH in alcohol and saponification was carried out at 60°C for 20 min. The usual procedure of ex- traction and determination of carotene and vitamin A was performed as described on page 70. In vivo conversion of B-carotene to vitamin A using intestinal loops The experimental procedure followed was essentially that of Olson (138) with some modifications. The rabbits were fasted overnight before the Operation, Prior to per- forming laparotomy the hair on the abdominal region at the site of operation was clipped with scissors or with clippers. The animals were anesthetized by giving subcutaneous in- jection of 1.6 g of urethane per Kg body weight dissolved in 0.85% saline solution. The laparotomy was performed by a ventral midline incision of about 2 in. The blunt end of a forceps was used to expose the small intestine and three 100ps were formed by ligating near the pyloric end and distally at three more places. The three loops thus formed were approximately of equal length. Double ligatures were made between the segments used. Injections of 1 m1 of 0.25 M sucrose and 0.5 m1 Of the carotene dispersion were made into the first loOp and‘the above combination plus 0.5 ml of nitrate were made into the third loop. Sucrose alone was injected into second loop which served as control to estimate the endogenous carotene and vitamin A. .After injection the small intestine was returned to its original position within the abdominal cavity. The‘muscular layer 70 was sutured with silk thread and the incision on the skin was closed with small wound clips. The animals were then returned to their respective cages. One and one-half hours later the rabbits were slaughtered and the intestinal 100ps were removed and externally rinsed in 0.9% NaCl solution. The three loops with contents were then homogenized separ- ately with 20 m1 of l N KOH in 95% alcohol and saponified at 60°C for 20 min. Following this, the conventional pro- cedure of extraction and determination of carotene and vi- tamin A was carried out as described below. Saponification and extraction After incubation the reaction was stopped by adding 20 ml of l N KOH in 95% ethanol and placing the flask in an oven at 60 to 65°C for 20 min. This mixture was cooled to room temperature and extracted once very thoroughly with a 50 ml of petroleum ether by intermittent shaking during a period of 30 min. From this stage on the Operation was carried out in a cold room. The ether layer was decanted into a 150 m1 beaker and washed with 10 - 20 m1 of distilled water to free it from alkali. The ether layer was then transferred into an Erlemeyer flask containing 5 g of anhydrous Nazsou and agitated gently to remove traces of water. The mixture was usually kept for a short time in a dark cold room before aliquots were taken for carotene and vitamin A quantitation and characterization. With each in— cubation trial a sample of carotene in duplicate without added tissue was carried through the entire procedure except 71 that the sample was kept at room temperature. A sample of homogenate without added carotene was also carried through the entire procedure. These values were used as the amount of carotene added and as the amount of endogenous carotene and vitamin A present in the tissue. Endogenous levels of vitamin A and carotene were determined by the method of Davies (41) with appropriate modifications. Estimation of carotene A standard carotene curve was made using serial dilu- tions of B-carotene in petroleum ether and immediately measuring absorbancy at 440 mu with the Beckman* model B spectrOphotometer. This same aliquot was then evaporated under nitrogen and used to determine the amount of antimony trichloride reaction product due to carotene as described below. The procedure was performed on every control sample of carotene. ' Estimation of vitamin A Vitamin A was estimated by the Carr-Price reaction using two different standard vitamin A preparations. With all experimental samples the same aliquot used to determine carotene was used to estimate vitamin A. After reading ab- sorbancy at 440 mu the solution was evaporated under a stream of nitrogen keeping the test tube warm. The tube was cooled and 0.6 ml of CHCl3 added to the residue. Two ml of SbCl3 reagent was added quickly and the absorbancy was read at 620 mu in a Beckman model B spectrOphotometer 4 72 within 8 to 10 sec after adding the SbCl3 reagent. Under these conditions reproducibility was good and the color was measured very near its peak intensity. The clear blue color produced was without turbidity and faded very rapidly. This is typical of vitamin A in the Carr—Price reaction. Carotene produced a blue color that faded more slowly than vitamin A. The oxidation products Of carotene have been reported to produce a nonfading grey to blue color which is not characteristic of vitamin A in the Carr-Price reaction (193). Vitamin A values obtained by the Carr-Price reaction were always corrected for the carotene present. Chromatographic separation of carotene and vitamin A derivatives Some information indicates that the Carr-Price reaction is relatively non-specific for vitamin A and certain caro- tenoid oxidation products are known to have a much greater specific SbCl3 — 620 mu absorption than carotene (132). This makes it desirable to characterize the individual com- pounds in the extract of the incubated media after chroma- tographic separation. Many methods were available for the separation of carotene and vitamin A derivatives. Chroma- tographic separation Of the lipids in the ether extract was performed using the procedure outlined by Olson (138) with some modifications. The chromatographic column was prepared in a 10 x 300 mm chromatogram tube by pouring a suspension of 10 g Of 73 deactivated alumina in petroleum ether into the tube. It was packed by gravity with slight tapping of the tube. It was desirable to form the column with one pouring to avoid air bubbles. The petroleum ether was alloWed to run out and the whole column was again washed with 10 ml of petrol- eum ether. Meanwhile, the lipid extract was evaporated to a small volume in the oven at 50 - 60°C until a viscous oily resi- due was formed. At this phase it was removed from the oven and final evaporation was carried out under nitrogen. Fi- nally it was diluted to 1 ml with petroleum ether. The concentrated ether extract was added to the column just before the top of the column ran dry. Five ml of petroleum ether was then slowly added. ‘When the solvent had just about all drained into the column the eluting solutions were added. Successive 10 ml eluate fractions were collected into clean test tubes. B-carotene was eluted with 80 m1 petroleum ether; vitamin A ester with an addi- tional 70 ml of petroleum ether; vitamin A aldehyde with 60 ml of 3% acetone in petroleum ether and vitamin A alcohol with 80 m1 of 8% acetone in petroleum ether. No suction was applied to the columns at any stage and the eluents were allowed to percolate slowly through it. The column was examined regularly with the ultraviolet lamp when tissue extracts were chromatographed to observe the flour- escence of the vitamin A alcohol band. Complete elution Of 74 vitamin A alcohol was indicated when the last ml of eluate collected showed no vitamin A florescence. The carotene eluate was colored and all others were colorless. Immedi- ately after the fractions were collected all the tubes were stored at 5°C for further identification and quantitative analysis. The identification and quantitative analysis of products from the column fraction were performed in several ways as outlined in Fig. l and below. CansPrice Reaction The Carr-Price reaction was employed on all 10 m1 fractions collected in elution tubes in the same order as collected. The fraction in each tube was evaporated under nitrogen in the dark and the residue immediately dissolved in 0.6 ml of CHC13. The Carr-Price reaction is somewhat non-specific on any crude extract but when performed on chromatographed products where carotene and vitamin A deriv- atives have been separated this reaction can be used to quantitatively and qualitatively indicate the presence of the above products. Absorption spectrum Another method employed to identify and to quantitate the carotene and vitamin A derivatives was by pooling the carotene fractions, the vitamin A ester fractions, the vitamin A aldehyde fractions and the vitamin A alcohol fractions separated on the column and measuring the ultra- Petroleum ether extract Wash with H20 Dry with Na2S04 \/ Concentration of the ether extract l Paper chromatography 75 Evaporate under N2 F ' I Chromatography Circular Ascending on deactivated Al O 2 3 r I ' B-carotene Vitamin A Vitamin A Vitamin A ester aldehyde alcohol I I I .l’ Carr-Price Absorption Irradi- Maleic Anhydro- reaction spectra ation anhydride vitamin A Figure l A flow sheet of methods for the isolation and identification of vitamin A derivatives from extracts of incubations with B-carotene sus- pension and intestinal homogenates in vitro. ..‘ 76 violet absorption spectra on one aliquot and the Carr-Price reaction on another aliquot. The split pooled aliquots of carotene and vitamin A derivatives were evaporated under nitrogen and diluted to 3-4 ml with petroleum ether for measurement of absorption spectra. Simultaneously, known concentrations of standard vitamin A alcohol were similarly prepared. Ultraviolet absorption spectra on appropriate samples were measured using a Beckman model DU SpectrOphotometer and an automatic recording Beckman Model DK—2 spectrOphoto- meter. The absorption maxima of B-carotene, vitamin A ester, vitamin A aldehyde and vitamin A alcohol in petrol- eum ether have been reported to be at 450 mu, 324 mu, 365 mu, respectively (31). Irradiation of vitamin A alcohol MOdification of the destructive irradiation technique of Bessey et a1 (16) was used on an eluted aliquot of vita- min A alcohol and on standard vitamin A alcohol of a known concentration. Five ml of the pooled vitamin A alcohol sample and 5 ml Of standard vitamin A alcohol were taken in separate clean test tubes. The absorption spectra were measured on both samples. The solutions were placed in test tubes and exposed to an ultraviolet light source for 36 hr in a dark room. One sample from a tissue extract and one pure vitamin A alcohol sample were also irradiated. A green fhxrescence was seen from both solutions during the 77 early period of irradtation. After irradiation they were remade to the 5 m1 mark and the absorption spectrum again determined. This technique was used on two different samples at two different times. Absorption spectrum of blue color_produced by Carr-Price reaction Further characterization of vitamin A alcohol fraction was carried out by measuring the absorption spectra of the blue color produced by the Carr-Price reaction. An aliquot of the eluate using 8% acetone in petroleum ether was evap- orated under nitrogen and the residue was immediately dis- solved in 1 m1 CHC13. The direct absorption spectra was measured in the Beckmann DK-2 spectrOphotometer using this CHC13 solution and 2 ml of Carr-Price reagent in the region of 500-800 mu. The complete absorption curve was rapidly recorded so that 620 mu peak was reached soon after the time offlmaximum color development. The absorption spectra of the Carr-Price reaction product was measured on the un- chromatographed original ether extract from the incubation mixture and on the standard vitamin A alcohol. Maleic anhydride reaction method Further characterization of vitamin A alcohol fraction was carried out by the maleic anhydride reaction method slightly modified from that outlined by Robeson and Baxter (151). The principle of this procedure was based on the fact that maleic anhydride reacts with the conjugated double 78 bonds in vitamin A to form a product which gives no blue color with SbCl3. The Speed of this reaction varies among the several isomers. An aliquot of pooled vitamin A alco- hol fractions was evaporated under nitrogen the residue immediately dissolved in 5 ml of benzene and transferred into a 10 ml volumetric flask. The flask was filled to the mark with 10% maleic anhydride in benzene. The mixture was Shaken well and stored in the dark at 20°C. The Carr-Price reaction was performed on a 0.6 ml aliquot at 4, 8, 12 and 16 hr after addition of maleic anhydride. A sample of the same solution which had not been treated with maleic an— hydride and a sample Of 10% maleic anhydride in benzene alone were also analyzed by the same times. From this, the percentage of initial vitamin A potency was calculated. The recovery value "R" was obtained by determining the content of vitamin A after a predetermined developing time in a solution with and without the addition of maleic anhydride (151). At the end of 48 hours the maleic anhydride treated and untreated samples were evaporated under nitrogen and dissolved in 1 m1 of CHCl3. The absorption spectra pro- duced by the Carr-Price reagent with the maleic anhydride treated sample and with the untreated sample were determined using a Beckmann model DK—2 spectrophotometer. Identification of vitamin A by conversion to anhydrovitamin A Embree (51) has pointed out that vitamin A treated with dry alcoholic HCl formed anhydrovitamin A; a compound which 79 had characteristic absorption bands at 350, 368, and 389 mu and produced a blue color with SbCl3. The formation of anhydrovitamin A is less subject to interference and mis- interpretation than are other vitamin A derivatives. The method of Budowski and Bondi (29) was used to form anhydro- vitamin A from vitamin A alcohol. An aliquot of chromatographed vitamin A alcohol frac- tion from a composite tissue extract and standard vitamin A alcohol in petroleum ether were evaporated separately under nitrogen and dissolved each in 3 m1 of redistilled benzene.' These served as stock solutions. Toluene - p - sulfonic acid in benzene was used as a catalyst to prepare anhydrovitamin A. Toluene - p - sulfonic acid weighing 15 mg was refluxed with 100 m1 of redistilled benzene until dissolved. Then 10 ml of solvent was dis- tilled Off to evaporate any moisture present. The solution was allowed to cool, readjusted to 100 ml volume with ben- zene and used as the catalyst or dehydrating agent. One ml of the above stock solutions was mixed with 4 ml of the catalyst solution. After 1 min the catalyst was neutralized by shaking with l g of dry Na903. The solution was allowed to settle. The absorption spectra was then measured in a Beckmann DK-2 spectrophotometer in the region 340 - 500 mu against standard vitamin A alcohol. The read- ings thus Obtained represented the changes in absorbancy caused by dehydration. Also the absorption spectra of the 80 anhydrO-derivative and the original vitamin A alcohol were measured against benzene for both the standard and the ex- perimental sample. After measuring absorption Spectra these samples were evaporated under nitrogen and dissolved in 1 ml of CHCl3. The absorption spectra of the Carr-Price reaction product was measured immediately. Standard vitamin A alcohol was treated by the same procedure. Identification of vitamin A by paper chromatography In order to more adequately characterize the products formed, the ether extract of the incubated media was sub- jected to two different paper chromatographic procedures. Paper partition chromatography. This procedure was similar to that of Suzuki (170) with minor modifications. In order to separate small amounts of different forms of vitamin A and carotene from the ether extract (unsapanifi— able portion) the technique of one dimentional ascending paper partition chromatography was employed. The strips of chromatographic paper (Whatman No. 54) was impregnated with a Ca(H2POh)2 solution (100 g per liter) at room temperature in a clean glass tray. After three hours, the salt solu- tion was decanted and a 2N NHgOH solution was poured on the wet filter paper in a glass tray. The paper was soaked for One-half hour. Then the paper was thoroughly washed for one-half hour with distilled water or until it did not give a basic reaction or an odor of ammonia. A sample of the ether extract from a tissue-carotene incubation was evapor- 81 ated under nitrogen and separate drOps of the viscous con- centrate thus obtained were applied on the paper strips. Standards were run simultaneously. The atmosphere in the chromatographic tank was satur- ated with the vapor of the developing solvent (petroleum ether). The strips were then developed by the ascending technique with petroleum ether as solvent for 2 to 3 hours. The strips were dried in a hot air oven and then sprayed with antimony trichloride to detect colored areas. Circular paper chromatography. This chromatographic technique was described by Giri and Rao (62,63) and Mahadevan et al. (118) and was used with slight modifica— tions. This technique separated vitamin A alcohol and caro- tene fairly well. Circular Whatman No. 1 filter paper (24 cm) was first impregnated with 10% paraffin oil in petroleum ether and then dried for 24 hr at room temperature to remove the solvent. A small circle of about 4 cm diameter was drawn with a lead pencil and a slit cut at the.Center. In order to irrigate the chromatogram with solvent, a detach- able tail was made using a 0.5 x 4 cm strip of filter paper and this was introduced into the slit. The solvent used for developing the chromatogram was butanol - acetic acid - water (4:1:5). The viScous concentrate from ether extracts and known compounds were spotted separately at different places on the circle drawn with the pencil. The filter paper with its tail end was kept in a pyrex dish in such a 82 way that the tail was immersed in developing solvent con- tained in a small beaker which was kept underneath the paper in the center of a pyrex dish. The outer of perimeter of the filter paper rested on the rim of the pyrex dish. The entire apparatus was kept in a chromatographic tank which was already saturated with the solvent. The chromatogram was allowed to develop in the dark for 12 hr. Frequent ex- aminations were made using an ultraviolet lamp. Determination of carotene and vitamin A in plasma Carotene and vitamin A in the blood were determined by a modification of the method described by Kimble (99). Usually 10 ml of plasma (or serum) was placed in a thick walled test tube. An equal volume of 95% ethanol was added and the mixture was well shaken. Then 10 m1 of petroleum ether (b.p 30-60°C) was added, the tube corked and the con- tents shaken for 5 - 8 min on a mechanical test tube shaker. The corks used were encased with aluminum foil SO that sol- vent would not extract extraneous material from the corks. The tubes were centrifuged for 1 min at 480 XG to Obtain good separation of petroleum ether from the alcohol plasma mixture or allowed to stand at 40°C and 5 ml of petroleum ether layer was then pipetted into a cuvette or into a standardized test tube for reading the carotene concentra- tion. ‘When pipetting care was taken not to touch the sides of the test tube with the pipette tip. The concentration of carotene was measured in the Beckmann B spectrOphotometer 83 at 440 mu with petroleum ether as a blank. Then the petro— leum ether extract in the cuvette was then evaporated under nitrogen and dissolved with 0.6 ml CHC13. Measurement of vitamin A was made by the antimony tricthride reaction in the Beckmann model B spectrOphotometer as described else— where. Determination of carotene and vitamin A in liver. The usual alkali digestion method as described by Davies (41) with some modification was used to determine carotene and vitamin A in the liver. Five g of liver pref- erably Obtained from several different areas were weighed into a beaker. The liver sample was minced well with scissors and 10 m1 of 5% KOH added. This was transferred into an Erlemeyer flask and the mixture digested on a steam bath for 30 min. The digested solution was cooled to room temperature transferred to a separating funnel and the Erlemeyer flask was rinsed with 5 ml distilled water. Then 5 ml of ethanol was added and the mixture was shaken. To this 40 ml of peroxide free ethyl ether were added and the material was shaken for 1 min. The layers were allowed to separate and the lower layer was drawn into another separ- atory funnel and reextracted with 10 ml of ethyl ether as before. The first extract was washed first by shaking with 5 m1 of water and then gently shaken with 50 m1 of water. The same wash water was used to wash the second extract. The two extracts were combined. Two more washings were 84 performed first by using 10 ml of wash solutions made of from 1 g Of KOH, 10 ml ethanol and 90 ml distilled water. The second 10 ml of wash solution was made up of 1 m1 con- centrated HCl and 100 ml ethanol diluted to 1 liter with distilled water. The extract was dried by passing through a layer of anhydrous NaZSOQ and diluted to 50 ml with ethyl ether. Five m1 of this ether extract was transferred into a cuvette and the concentration Of carotene and vitamin A were measured in the same way as already described. 85 RESULTS PRELIMINARY EXPERIMENTS The preliminary experiments were conducted to study whether an incubating media modified from several investi- gators (121, 137, 169, 193) was satisfactory for studying vitamin A formation in vitro. In these trials when fresh duodenal homogenates Of rabbits and Holstein cow were incu- bated a considerable amount of vitamin A was formed. The extent of vitamin A formation in nine samples of duodenal homogenates from animals of these two species is shown in Table 2. Vitamin A formed per gram tissue presented in the table was corrected for endogenous vitamin A. The amount of vitamin A formed was about 14 and 12 times the endogenous level in rabbits and cow, respectively. Vitamin A formed per flask was about 11 ug when duodenal homogenated from rabbits were used and about 7 ug when duodenal homogenate from cowvwere used. The amount of carotene unrecovered was similar in both trials. The extent of vitamin A formed was more in tissue from rabbits than in tissue from the cow. Abcnn:4 and 2.6% of unrecovered carotene was found as vita- A expressed on a weight basis. The results clearly indi- cated that 0.25 M sucrose solution plus an aqueous carotene suspension maintained physiological conditions well enough to allow formation of measurable amountsof vitamin A in vitro. The homogenates from cow duodenum contained some TABLE 2 Extent and variation in formation of vitamin A after incubating carotene with duodenal homogenates (Experiment numbers 1 and 2) Tissue source Carotene used/ Carotene Vitamin A formed and item 3 tissue unrecovered per 3 tissue per ug car otene un- recovered ug 2 ug M.ug Rabbit Av. of 9 samples 112 36.41 1.63 40.09 St. error 0.26 0 16 1 28 Coef. of variation 7.00 28.90 28.70 Holstein cow Av. of 9 samples 112 36.41 1.06 25.74 St. error 0.24 0.02 0.47 Coef. of variation 5.90 15.00 16.00 Av - Average; St. - Standard; Coef. a Coefficient 87 pieces of fat and fascia that were not homogenized into fine particles but the rabbit tissue had a more homogenous appearance. However, the coefficients of variation were less for the tissues from the cow. The Variation and standard error were not excessive, but in all trials dupli- cate samples were incubated simultaneously and the average value used. Effect of addition of glycocholate and saponification of tissue In studying B-carotene cleavage by longitudinally cut sections of washed rat intestines in vitro, Olson (137) observed that sodium glycocholate and perhaps other bile salts were an absolute requirement for vitamin A formation. In the present study 5% sodium glycocholate was incorpor- ated into the aqueous carotene suspension in 5% Tween 80 and incubated with rabbit duodenal homogenates and with duodenal 100ps at 38°C for 3 hr. The addition of glyco- cholate to a carotene suspension increased vitamin A for- mation to 119% of control when duodenal homogenates were used. These results somewhat confirmed the observations of Olson (137). When duodenal loops were used the addition of glycocholate was without any beneficial effect. The lack of any beneficial effect by the addition of sodium glyco- cholate to the duodenal loops may be explained on the basis that the duodenal 100ps already contained some bile. In general an aqueous carotene suspension containing 5% sodium glycocholate and 5% Tween 80 were employed for all subsequent 88 work. In two experiments (No. 10, 11), the effectiveness of petroleum ether to extract vitamin A from the incubated tissue following saponification was compared to incubated tissue not saponified using duodenal homogenates from calves. The results indicated that the extraction of vitamin A was not complete in the non-saponified sample. Only 54 to 66% of the vitamin A extracted following saponification was found by extracting the non-saponified samples. The saponi- fication procedure left only small particles of residue in the mixture, whereas when not saponified the mixture con- tained large masses of tissue. It appeared desirable to saponify all samples in order to more thoroughly extract the vitamin A formed. The vitamin A ester was probably hydrolyzed to vitamin A alcohol by saponification. For all results given saponification was employed prior to ether extraction. Effect of altering carotene to tissue ratio and incubation time The manner in which the ratio of carotene to tissue in- fluenced vitamin A formation was studied. Samples of calf duodenal homogenates were incubated for 3 hours with vari- ous levels of carotene. A summary of two such experiments are presented in Table 3. The results clearly showed that large amounts of carotene added to homogenates would in— crease the total amount of vitamin A formed. Vitamin A formed was corrected for endogenous vitamin A. Vitamin A 89 mm 3 Effect of altering carotene to tissue ratio on amount of vitamin A formed L Tissue xpt. Carotene incubated] Vita-1n A for-ed source no. 3 tissue per 3 tissue As 1 of carotene ' unrecovered ug us 1 Calf fed 10 0 0.03* --- deficient diet . 401 2.47 1.7 134 1.29 2.6 67 0.71 ' 2.4 34 0.43 309 17 0.23 3.5 Calf fed 11 0 0.01* --< 11 N03 ' 291 1.69 2.8 98 0.64 3.1 49 0.49 4.1 24 0.26 3.4 12 0.14 4.2 * - Endogenous level of vitamin A in the tissue 90 formed per g tissue ranged from 2.4 ug down to 0.23 ug, when carotene incubated ranged from 401 to 17 ug/g tissue. The efficiency of vitamin A formation expressed as per cent of carotene unrecovered tended to increaSe as the level of carotene decreased in the incubating media. Similar results were obtained in experiment 11 using tissue from a calf fed the carotene deficient diet. These results indicated that when comparing the amount of vitamin A formed between dif- ferent experiments the carotene to tissue ratio in the homogenates must be considered. The curves in Fig. 2 indicated that formation of vita- min A was practically linear up to about 40 - 60 ug caro- tene/g tissue. At higher carotene levels the vitamin A formation continued to increase with the amount of incubated carotene but at a greatly reduced rate. The results of previous experiments showed that 30 - 40% of carotene incubated could not be recovered and dis- appeared from the incubating mixture. This suggested that during incubation a considerable amount of carotene was destroyed. At the same time it was possible that a small quantity of vitamin A could also be destroyed. Moreover, an incubation time which was Optimum for maximum recovery of vitamin A with the least destruction of carotene appeared to be desirable. Therefore the effect of incubation time on formation of vitamin A was studied. Duodenal homogenates from calf and cows were incubated for periods varying from TABLE 4 Effect of incubation time on the extent of formation of vitamin A and the recovery of carotene using duodenal homogenates from non-depleted animals Tissue Incubation Carotene Carotene Vitamin A formed source time incubated] .recovered pcrggfitissue as Z of and 3 tissue activity carotene Expt. no. . unrecovered min. ug 1 ug Z Z Calf 0 0.0 -- 0.03* --- ---- 44 30 14.1 86 0.47 107 23.2 60 ” 78 0.45 102 14.3 120 " 76 0.44 100 12.7 180 ” 67 0.44 100 9.5 240 ” 41 0.43 98 5.1 * COW 0 ago -- 0.07 --- ---- 45,46 ** ' 5 10.2 74 0.10 24 3.9 15 12.6 68 0.18 46 4.4 30 " 63 0.39 102 8.9 45 ” 56 0.37 98 7.5 60 " ' 58 0.37 97 7.6 120 ” 52 0.38 100 6.8 130 .. ‘ 50 0.38 100 6.6 240 " 44 0.35 92 5.2 * - Endogenous vitamin A 1evel in the tissue ** - Expt. 46 only, others average of 45 and 46 VITAMIN A FORMED (uq per gram tissue) 91a 0 J 4 . . . . ' I I I I 1 s O 80' ISO 240 320 . 400 480 CAROTENE: TISSUE RATIOIug carotene per gram tissue) . Figure 2. Effect of altering carotene to tissue ratio on vitamin A formation A--Tissue from calf fed deficient mation B--Tissue from calf fed 1% N05 ration 92 5 to 240 min at 38°C. Results are summarized in Table 4. The results indicated that even after 5 min of incubation a small amount of Vitamin A was formed (Experiment 45). Maximum vitamin A formation was attained in 30 min and de- creased very slightly thereafter. In experiment 44, using calf duodenal homogenates, the recovery of carotene was 86% at 30 min incubation and then decreased progressively to 41% at 240 min. Similar results were noted with tissues from a cow where the recovery was 74% at 5 min and 44% at 240 min. The efficiency of vitamin A formed (expressed as per cent of carotene unrecovered) also progressively de- creased from 5 to 240 min of incubation. The conversion process appears to be a very rapid reaction since vitamin A was formed after 5 min of incubation. The results also indicated that increasing incubation time beyond 30 min did not have any appreciable effect on the amount of vitamin A formation. Efficiency of various buffers The usefulness of 0.25 M sucrose, 0.9% NaCl, Krebs- Ringer solution, phosphate buffer PH 7.2, veronal buffer PH 7.4 as diluting and incubating media for the formation of vitamin A from B-carotene were compared. The results of such experiments are presented in Table 5. Sucrose was most effective and veronal buffer was least effective. Effici- ency of vitamin A was also maximal when 0.25 M sucrose so- lution was used and minimum when veronal buffer was used. Influence of various buffers as incubating media on the formation of vitamin A from carotene TABLE 5 Tissue source Buffers used Carotene. per 3 tissue Vitamian_formed as Z of caro- and Expt. no. incubated/ tene unrecov- ered ug ug Z calf 0 0008* --- 44 0.25 M sucrose 14 0.44 9.5 0.9% NaCl ” 0.14 2.7 Krebs-Ringer " 0.18 3.1 Phosphate buffer " 0.17 3.0 Veronal buffer ” 0.08 1.3 cow 0 0021* "' 47, 48 0.25 M sucrose 14 0.45 9.2 0.9% NaCl ” 0.16 3.0 Krebs-Ringer ” 0.35 7.2 Phosphate buffer ” 0.15 2.8 Veronal buffer " 0.06 1.1 Tris buffer ” 0.10 1.5 * - EndOgenous level of vitamin A in the tissue 94 The data indicated that 0.25 M sucrose did not function merely as an agent which supplied essential nutrients and Optimum PH for the reaction to proceed in vitro, but prob- ably had a more specific action. The data indicated that of the several buffers tried a 0.25 M sucrose solution was superior. Effect of homogenizing tissue in waring blender of in an omni mixer and using nitrogen or oxygen An experiment was conducted to study whether homogen- izing tissue either in a waring blender or in an Omni Mixer* effected vitamin A formation. Duodenum and liver were each homogenized separately and incubated with B-carotene at 38°C. The results showed that homogenizing tissue in an» Omni Mixer with powdered glass beads reduced vitamin A for— mation to 60% compared to the tissue that was homogenized in a Waring blender. The decrease in activity by homogen- izing in an Omni Mixer may have been due to an effect of the powdered glass beads. It may be possible that homogen- ization with glass beads in an Omni Mixer might have des- troyed nuclei, mitochondria and/or other particulates since powdered glass beads at high speed usually disrupt these subcellular particles (28). Homogenization without powdered glass beads in the waring blender may have minimized this disruption. It appears possible that in vitro formation of *Serval Omni Mixer Homogenizer 95 vitamin A from B-carotene was maximum when the destruction of nuclei, mitochondria and other cell particulates was minimal. In another study, rabbit intestinal tissue was homogen- ized separately in the waring blender as usual and in an Omni Mixer under oxygen and nitrogen. In this procedure, the homogenizing liquid and head space in the tightly en- closed Omni Mixer was saturated with the gas before homog- enization. These' homogenates were then incubated with the carotene suspension at 38°C and the extent of vitamin A formation measured. Tissues homogenized in an Omni Mixer under nitrogen formed more vitamin A than the same tissue homogenized under oxygen. The extent of vitamin A forma- tion and the recovery of carotene were increased under the influence of nitrogen as compared to oxygen. It was also observed that the extent of vitamin A formation was more with the tissue homogenized in the waring blender than with the tissue homogenized in an Omni Mixer under oxygen. These results indicated destruction of carotene and vitamin A occurred under the influence of oxygen probably due to oxidation. These effects were less pronounced under nitro- gen. Effect of heating and other treatments of tissue The results of heating the homogenates are summarized in Table 6. In experiment No. 60 using steer duodenal homogenates, the activity (vitamin A formed/g) was reduced \O O‘\ TABLE 6 Effect of heating duodenal homogenates on vitamin A formation from carotene . Vitamin A formed Tissue Heating Temperature Carotene Carotene per 3 Activity source time observed incubated recovered tissue and exbt. _'no. min. °C ug 1 ug Z Calf fed 0 -- 24 67 0.26 100 17. N03 diet 1 -- " 60 0.13 49 11 2 -- ” 59 0.14 55 4 ' -- " 57 . 0.14 52 Steer 0 -- 15 63 0.53 100 38 I 1 90 " 43 0.06 11 2 ” " 46 0.03 6 3 " " 46 0.04 8 5 ” " 46 0.03 6 Sheep 0 -- 31 68 0.36 100 13 l 90 " 56 0.06 17 . 2 " " 57 0.05 14 4 ” ” 54 0.05 14 97 from 100% down to between 11.3 and 5.7%. This procedure reduced the activity to 14 - 17% for sheep tissue. When the heated duodenal homogenates from the calf in experiment No. 11 were incubated, the activity was reduced to about 50% of unheated homogenates. These values were higher than those for experiments No. 11 and 13. The temperature of the homogenates was not determined in experiment No. 11. This may account for the high activity even after heating the homogenates. In all cases carotene recovery ranged from 43 to 60% in heated samples and 63 - 68% in unheated samples. In another trial (Experiment 33), duodenal tissue was obtained from the pigs which were subjected to the usual procedure of scalding the entire body surface on the Open flame. Interval between slaughtering and homogenization of the duodenum was more than one hour. When these duodenal homogenates were incubated the activity was nil. In the light of these results it is clear that heating of the homogenates inactivated the enzyme or factors which were responsible for conversion of carotene to vitamin A. In another trial, the homogenates were left standing at room temperature (1 22°C) for l, 2 and 3 hours before adding carotene and incubating. This procedure reduced the activity to 25, 8 and 8% of the control value, respectively. In some preliminary experiments when the interval between slaughtering and homogenization of the tissue was too long 98 (i one hour) the formation of vitamin A from carotene was nil. These results indicated that the time between slaughtering animals and incubating homogenized tissue was very important in in vitro studies. ISOLATION AND IDENTIFICATION OF VITAMIN A DERIVATIVES FROM INCUBATED MATERIAL The Carr-Price reaction is relatively non-specific for determining vitamin A since certain carotenoids and their oxidation products give some blue color with SbC13 (132). Some oxidation products of carotenoids which give interfer- ing reactions with SbCl3, produce a non-fading grey to blue color (193). The ether extract that was extracted from the incubated mixture gave typical vitamin A blue color that faded in a few seconds in all Carr-Price determinations. .The ether extract from the incubated tissUe was sub- jected to column chromatography on deactivated A1203 and portions of eluate were used for identification in various ways. The results using this eluate are given below. Carr-Price reaction on column eluates The Carr-Price reaction was employed on all successive 10 ml fractions collected. The results are shown in Table 7. The fractions in tubes No. l - 8, 9 - l6, l7 - 21 and 22 - 29 should contain B-carotene, vitamin A ester, alde- hyde and alcohol, respectively, since the standard B-caro- tene, vitamin A ester, aldehyde and alcohol were eluted in this order. The Carr-Price reaction products were princi- TABLE 7 99 Absorbancy of Carr-Price reaction product on successive portions eluted from a deactivated alumina column Tube no. Optical density Tube no. Optical density . 620 mu 620 mu 1 0.000 16 0.000 2 0.010 17 7 0.000 3 0.010 18 0.005 4 0.020 19 0.005 5 0.050 20 0.000 6 0.025 21 0.000 7 0.015 22 0.060 3 0.005 23 ' 0.150 9 0.000 24 0.050 10 0.000 25 0.420 11 0.005 26 ‘ 0.695 12 0.010 27 0.810 13 0.005 23 0.120 14 0.000 29 ' 0.040 15 0.000 100 pally found in tubes No. 22 - 29 which contained vitamin A alcohol. The Carr-Price reaction Optical density exhibited by carotene fractions was very low (tubes No. l - 8). Little or no vitamin A activity was found in the ester and aldehyde fractions in tubes NO. 9 - l6 and 17 - 21, respectively. The major components of tubes No. l - 8 and 22 - 29 were tenta- tively identified as B-carotene and vitamin A alcohol re- spectively. These results also indicated that any vitamin A ester formed was hydrolyzed to vitamin A alcohol by the saponification process and that any vitamin A aldehyde that may have been formed was small or was destroyed by saponi— fication. Absorption spectra of B-carotene and vitamin A derivatives after separation on deactivated alumina The absorption spectra of each of the four pooled fractions (above) were measured in Beckmann MOdel DU spec- trOphotometer. 'Hhe B-carotene fraction exhibited an ab— sorption maximum at 449 mu. The ester fraction did not con- tain vitamin A since the incubation mixture was saponified. Thus no absorption spectra were found for this fraction. Similarly no spectra was noted for the aldehyde fraction. Ultraviolet absorption spectra of the vitamin A alcohol fraction and standard vitamin A alcohol in petroleum ether are shown in Figure 3. Both vitamin A alcohol fraction and standard vitamin A alcohol eXhibited a pronounced peak at 325 mu. This absorption maxima was similar to that published ABSORBAN CY |.0 0.8 0.6 0.4 0.2 0.0 270 101 1b a a a A V 290 3l0 330 350 370 390 WAVE LENGTH , m A: Figure 3. Absorption spectra of vitamin A alcohol fraction from ether extract of incubated mixture containing duodenal homogenates and B- carotene (ether extract was chromatographed on deactivated alumina). A--Vitamin A alcohol fraction B--Standard vitamin A alcohol I v v 102 for vitamin A alcohol (31) suggesting that the ether ex- tract of incubated mixture contained vitamin A alcohol. A definative experiment (No. 62) was conducted using homogenates from rabbit intestine and muscle to isolate and identify vitamin A formed from B-carotene by various methods. The ether extracts of the incubated tissue with and without carotene added were subjected to column chromatography on deactivated alumina, and fractions collected as described above. The ultraviolet absorption spectra of the vitamin A alcohol fraction was measured using a Beckmann Model DK-2 recording spectrophotometer, on the extracts of muscle and duodenal tissue incubated with and without carotene. A standard vitamin A alcohol was also used. These absorption curves are shown in Figure 4. Standard vitamin A alcohol and vitamin A alcohol fraction from the duodenum-carotene incubation mixture gave characteristic spectra with absorp- tion maxima at 325 mu. The absorption spectra obtained on the vitamin A alcohol fraction from muscle and from duodenum (endogenous level) and from the muscle - carotene incubation mixture did not have the characteristic absorption of vita- min A. Vitamin A if present was in too low a concentration to be detected in these samples. Irradiation of the vitamin A alcohol fraction In this experiment no attempt was made to quantitative- ly estimate the vitamin A alcohol fraction. Although this procedure has been used to determine serum vitamin A (16) 103 Figure 4. Absorption spectra of vitamin A alcohol fractions in petroleum ether chromatographed from ether ex- tracts Of incubated mixture. A--Duodenum + B-carotene B—-Standard vitamin A alcohol C--Duodenum (endogenous) D--Muscle + B-carotene E--Muscle (endogenous) % TRANSMISSION L I'— 4 + A 270 280 290 300 310 320 330 340 01 : : WAVE LENGTH, mu Figure 4 105 Bieri and Pollard (18) showed that this method was of little value for vitamin A in tissue extracts. The irra— diation technique was employed here to help identify vita- min A alcohol in the chromatographed ether extract from an incubated mixture. Figure 5 shows the ultraviolet absorp- tion spectra of vitamin A alcohol fraction and standard vitamin A alcohol before and after irradiation with ultra- violet light for 36 hr. Before irradiation both solutions had a characteristic absorption spectra with the pronounced peak at 325 mu. After irradiation the characteristic ab- sorption spectra was not present. The extract from another incubated mixture was chromatographed and the vitamin A alcohol fraction and standard vitamin A alcohol were irra- diated for 24 hr. This procedure destroyed 75% of the vitamin A in both solutions. The results of this procedure furnished further in- direct evidence that the product formed by incubation of carotene suspension with duodenal homogenates was actually vitamin A and that none was formed when muscle was similarly incubated. Absorption spectra of Carr-Price reaction products. The absorption spectra of the Carr-Price reaction products was measured in a Beckmann DK-2 spectrophotometer. This was performed on a composite tissue extract before and after column chromatography and a standard vitamin A alcohol. It was necessary to add SbCl3 reagent quickly and have a sufficient Figure 5. 106 Absorption spectra of vitamin A alcohol fraction before and after ultraviolet irradiation. Vitamin A alcohol fraction extract before irradiation Vitamin A alcohol fraction extract after irradiation Standard vitamin A alcohol Standard vitamin A alcohol from tissue from tissue before irradiation after irradiation AB SORBANCY L4 . 0.8 7 0.6 ‘- 0.4 ‘ 0.0 ‘ 107 /\\ 270 290 3| 0 330 350 370 390 WAVE LENGTH, mu Figure 5 108 concentration of vitamin A present. Figure 6 shows that the absorption spectra of these 3 products had a maximum absorption at 620 mu. Another observation made was that the blue color produced with all samples faded rather quickly which is characteristic of the reaction between vitamin A and the Carr-Price reagent. The ether extract which was not subjected to column chromatography produced an absorption curve similar to that of standard vitamin A alcohol but with a lower and somewhat broader maximum ab— sorption curve. In another trial, vitamin A was estimated in the ether extract of an incubated mixture by the Carr- Price reaction and also by measuring extinction at 325 mu after chromatographing this same sample. Vitamin A present per ml of ether extract was 0.14 ug when the vitamin A was determined by the Carr-Price reaction and 0.16 ug when it was determined by measuring extinction at 325 mu. Similar results were obtained when vitamin A was estimated by the Carr-Price reaction and by measuring extinction at 325 mu on a concentrated vitamin A alcohol fraction. Vitamin A present per ml of the concentrated vitamin A alcohol frac— tion was 0.804 ug as determined by the former method and 0.780 ug by the later method. These results indicated that the vitamin A determinations made on the crude or on the chromatographed ether extracts from tissue incubations by the Carr-Price reaction actually represented vitamin A present. Any value obtained by the Carr-Price reaction has 109 Figure 6 Absorption spectra of Carr-Price reaction products of tissue extract before and after chromatographic separation on deactivated alumina. A--Tissue extract B--Vitamin A alcohol fraction after chromatographing C--Vitamin A alcohol fraction after chromatographing "/6 TRANSMISSION loo 4 90 8 go .. 70 «- 60 ‘I' 40 4 30 q» 844% o 110 l l A l U I I v 550 600 650 700 WAVE LENGTH, m A] Figure 6 111 very close estimates of the true vitamin A content in the extract. Maleic anhydride reaction method Although the maleic anhydride reaction has been used to determine the per cent of neovitamin A (151), this method was used in this study to indicate the presence of vitamin A in two different extracts from incubated mixtures. One sample was a composite of several extracts and the other was the extract from the duodenal-carotene incubation in experiment 62. Maleic anhydride reacted rapidly with the vitamin A alcohol fraction and standard vitamin A alcohol in Benzene. The results are shown in Table 8. The percentages of in- itial vitamin A potency after 16 hr were 2 and 94 for maleic anhydride treated and untreated vitamin A alcohol fraction, respectively, and 2.3 and 96 for maleic anhydride treated and untreated standard vitamin A alcohol. The recovery value "R" was determined by the formula (6): 100 X units in treated solution Units in untreated solution The recovery values "R" for the vitamin A alcohol fraction and for the standard vitamin A alcohol were estimated to be 2.2 and 2.4%, respectively. At the end of 48 hours maleic anhydride treated and untreated tissue extracts and vitamin A alcohol solutions were evaporated under nitrogen and dis- solved in 1 ml of CHCl3. The absorption spectra produced TABLE 8 112 Percent of initial potency of samples with and without maleic anhydride trot ment at different intervals and "R" values Source of material Treatment __gours and 1 initial pot ncy R 0 4 8 12 16 Z Chromatographed tissue extract* Treated 100 24.5 16.3 4.1 2.0 2.16 Untreated Standard vitamin A alcohol Treated 100 26.9 15.4 6.9 2.3 2.39 Untreated 100 98.1 96.2 97.0 96.2 Chromatographed 5 ‘ tissue extract* Treated 100 36.5 8.7 5.2 2.6 2.82 Untreated 100 f 95.2 92.0 96,0 92.0 * Two different samples done at different times 113 by the Carr-Price reaction was measured in the Beckmann DK-2 spectrophotometer (Fig. 7). The vitamin A alcohol fraction which was not treated with maleic anhydride had a characteristic absorption spectra and a pronounced peak at 620 mu. The maleic anhydride treated sample gave little or no peak at 620 mu, which indicated the destruction of vitamin A alcohol and formation of a non-chromaphoric add- juct with maleic anhydride. The vitamin A alcohol fraction (representing the edogenous vitamin A from this experiment #62) was subject- ed to the same procedure. Maleic anhydride treated and un- treated samples were reacted with SbCl3 and initial optical densities were 0.008 and 0.009, respectively. After 16 hr of treatment no blue color was produced by these two samples with SbCl3 and the optical density recorded was 0.000. These results indicated that the endogenous level of vitamin A in the duodenum was barely detectible. This extract gave no.peak at 620 mu when the above absorption spectra was measured on Carr-Price reaction product. The results of these experiments suggested that the ex- tract from two different saponified incubation mixtures con- tained vitamin A and that the endogenous duodenal level of vitamin A was practically negligible. Conversion to anhydro vitamin A Ultraviolet absorption spectra of a vitamin A alcohol fraction and standard vitamin A alcohol before and after ABSORBANCY LG |.4 |.2 0.8 0.6 0.4 0.2 0.0 114- A V t 500 540 580 620 660 700 WAVE LENGTH, mu Figure 7. Absorption spectra of Carr-Price reaction product of maleic anhydride treated and untreated vitamin A alcohol fraction. A--Samp1e treated with maleic anhydride B--Sample untreated with maleic anhydride 115 conversion to anhydrovitamin A are shown in Figure 8. The gspectra of the vitamin A alcohol fraction and standard vitamin A alcohol in benzene had maximum at 331 mu. Using benzene as the solvent shifts the absorption nearly 6 — 7 mu compared to petroleum ether as a solvent (29). Anhydro- vitamin A made from the vitamin A alcohol fraction and the standard vitamin A alcohol exhibited absorption maxima at 358, 377 and 399 mu and minima at 364 and 346. All these absorption maxima were consistent with those reported by Budowsky and Bondi (29). The source of the chromatographed vitamin A alcohol fraction used in this trial was a com- posite of ether extracts from several incubation mixtures. Identification of vitamin A alcohol by conversion to anhy- drovitamin A was performed using 2 different composite samples at 2 different times and once using extracts from a given incubation (Experiment 62). ,All 3 gave similar results. The increase in absorbancy or decrease in per cent transmission caused by dehydration was also measured by de- termining absorption spectra of anhydrovitamin A using the standard vitamin A alcohol in benzene in the control cu- vette. Figure 9 shows this absorption spectra of anhydro- vitamin A made from the vitamin A alcohol fraction and from standard vitamin A alcohol. The "difference" absorption spectra of anhydrovitamin A made from the vitamin A alcohol fraction was similar to that made from standard vitamin A alcohol. The "difference" absorption spectra has absorption 116 Figure 8 Absorption spectra of vitamin A alcohol fraction in benzene before and after conversion to anhy- drovitamin A. ’ A--Vitamin A alcohol fraction from ether extract of duodenum and B-carotene incubation mixture B--Standard vitamin A alcohol C--Anhydrovitamin A from Sample A D--Anhydrovitamin A from Sample B 96 TRANSMISSION T l00 i 001- 70 <- SO‘L 404» 30" 300 320 117 A f 340 360 300 400 415 WAVE LENGTH, mu 0 A 4P Figure 8 118 Figure 9 ”Difference" absorption spectra of anhydrovitamin A made from tissue extract and from standard vi- tamin A alcohol. A—eAnhydro derivative from vitamin A alcohol fraction of chromatographed ether extract B--Anhydro derivative from standard vitamin A alcohol % TRANSMISSION so» I 70 1* 60 ‘I 50 d- 40 -~ 30‘ 20 " L '360 J 370 380 390 400 WAVE LENGTH, mu Figure 9 119 120 maxima at 377 and 399 mu. These results were consistent with published values (29). After measuring absorption spectra, the above samples were each evaporated under nitrogen and dissolved in 1 ml CHCl3. The absorption spectra of the Carr-Price reaction products were then measured. Figure 10 shows the charact- eristic absorption spectra obtained with vitamin A alcohol and their anhydro derivatives. In all samples, the maximum absorption was observed at 620 mu which is characteristic of the Carr-Price reaction products with both vitamin A and anhydrovitamin A (31). The conversion of vitamin A in the chromatographed tissue extracts to anhydrovitamin A and its identification offers strong evidence for the formation of vitamin A in the incubated tissue. In experiment 62 ether extracts from the incubated IIiixture of carotene plus intestinal homogenates and from 1:11e incubated mixture of intestinal homogenate alone (:ciesignated as endogenous) were chromatographed separately on deactivated alumina and fractions collected. Vitamin A é'iJLcOhOl fraction was converted to the anhydrovitamin A ‘C1 7. 1 Sheep None 31 68 0.36 199 3:7 13 1 m1 Whole blood ” 45 0.06 i ;17 0.4 1 r 2 m1 ” " 51 0.08 21 0.5 Steer None 15 64 0.43 100 8.1 49,50 1 2 m1 Whole blood* " 32 0.14 35 1.9 2 Sedimented blood " 34 0.14 25 1.1 cells " Hemolyzed blood " 31 0.11 25 1.0 cells ' ” Serum ” 62 0.34 78 6.8 ” Plasma(oxalate) " 61 0.35 82 6.1 ” P1asma(citrate) " 58 0.39 90 6.6 * - Value obtained from Expt. No. 49 17h control by the addition of either serum or plasma but when whole blood, hemolyzed blood or sedimented blood cells were added the efficiency was considerably reduced (p = 0.01). Two attempts were made to wash the sedimented blood cells with water in order to remove the factorCs) respons- ible for the destruction of carotene and for inhibiting the formation of vitamin A. The sedimented blood cells were further centrifuged at 27,000 XG after diluting with an equal volume of water. All these fractions were added to usual incubation. The results of two such trials are pre- sented in Table 33. The carotene destroying system was more pronounced in the sedimented portion than in super- natant solution irrespective of quantity used. There was little difference between the carotene destroying capacity of the supernatant solution, the original sedimented blood cells or the original hemolyzed blood when each was incu- bated with duodenal homogenates. Vitamin A.formed per g of tissue was reduced to about 20% of control using sedi- mented blood cells or hemolyzed blood. Activity was re- duced to 65 - 80% in case of the supernatant solution and to about 57% in case of sediment. The smaller amount of sediment and supernatant had less effect than did the larger amounts of these materials. The efficiency of vita- min A formation was also reduced to about the same degree. All above vitamin A values were corrected for endogenous vitamin A. The results indicated that the factor responsible TABLE 33 175 Effect of incubating different fractions of blood and centrifuged sedimented cells and supernatant fluid with duodenal homogenates on the formation of vitamin A from B-csrotene Tissue Carotene Vitamin A formed source Blood fraction added ncubated7v recovered er 3 tissue as Z of and expt. g tissue fictivity carotene no. unrecov- “g ered Us 2 ug Z Z Heifer None 11 74 0.43 100 15.0 52,53 2 ml sedimented cells 13 44 0.10 20 1.4 ” hemolyzed blood 11 47 0.10 23 1.7 ” plasma " 74 0.36 84 10.2 ” supernatant* " 53 0.28 66 5.7 1 ml ” " 58 0.32 78 8.0 0.5 m1 " 13 49 0.32 65 4.9 0.25 ml ” " 52 ' 0.39 80 6.4 2 m1 sediment* ll 36 0.15 36 2.2 1 m1 ” " 42 0.18 43 3.1 0.5 ml ” 13 36 0.15 31 1.9 0025 m1 II N ‘ A ? 35 0028 57 3.4 fi * - Sedimented cells were mixed with equal volume of water and centrifuged at 27000 ZIG. Supernatant fluid was designated as supernatant and sedimented portion was designated as sediment. 176 for inhibition of vitamin A formation and the carotene des- truction may be found in the sedimented blood cells and that washing these cells with water and further centrifu- gation removed some of the inhibitory effect but not all of it. VITAMIN A FORMATION BY CELL FRACTIONS OF DUODENAL HOMDGENATES Duodenal homogenates were slightly diluted and centri- fuged at 5000 KC and 25,000 XG to obtain information on distribution of the activity that forms vitamin A. The re- sults are shown in Table 34. In experiments 57 and 58 the supernatant from the 5000 X6 fraction contained the most activity for formation of vitamin A from carotene when ex- pressed on dry matter basis. The sediment ofthe 15,000 and 25,000 XG fractions both contained about 89% as much activity as the sediment of the 5000 KC fraction. The su- pernatant portion of the 15,000 XG and 25,000 XG fractions had the least activity of all cell fractions. However even these fractions with the least activity contained more ac- tivity on a dry matter basis than the original homogenates per se. Different results were obtained with cell fractions of rabbit duodenal homogenates in experiment 59. Here only the 15,000 XG centrifugation was performed. Unlike the results obtained in experiment 57 and 58, the supernatant TABLE 34 Vitamin A formation from B-carotene by centrifuged fractions of duodenal homogenates 177 Carotene Tissue source ' Vita in A formed and expt. no. Eorm of tissue incubated] per g per 3 activity 3 tissue or ml D.M. on D.M. ‘ basis Us Us u8 7- Rabbit(dep1ated Homogenates 0 0.06 0.38 --- 57,58 ” 13 p.29 1.93 100 Supernatant 5000 KG 0 0.09 0.87 --- " 35 0.50 5.08 300 Sediment 5000 X G‘ 0 0.08 0,59 --- " 23 0.49 3.57 202 Supernatant 15000 X0 03 0.05 0.57, --- " 33 0.28 2.76 144 Sediment 15000 X(} 0 0.09 0.52 --- " 33 0.69 4.69 261 Supernatant 25000 KG 0 0.02 0.23 --- " 25 p.28 2.81 158 Sediment 25000 KG 0 0.11 0.74 --- " 38 0.78 5.07 268 Rsbbit(dep1eted) Homogenates 0 0.03 0.17 --- 59 " 27 0.67 3.88 100 Supernatant 15000 X G 0 0.03 0.43 «- " 35 0.90 12.89 332 Sediment 15000 X G 0 0.00 0.00 --- " 53 1.11 6.29 162 178 portion from the 15,000 XG preparation contained twice as much activity as the sediment portion. It is difficult to explain the discrepancy. However, if activity was expressed on a volumeteric or fresh weight basis the sediment from the 15,000 XG preparation contained more activity than the supernatant from 15,000 XG preparation in all these exper- iments. The error might be in estimating the dry matter content of cell fractions in experiment 59. At present, this is the only possible explanation that can be given for this discrepancy. Under the conditions of these exper- iments these results indicated that the supernatant mater- ial from the 5000 XG preparation contained the most vitamin A forming activity followed by the sedimented portions from preparation of 15,000 XG and 25,000 XG. The supernatant fractions from the 5000 XG and 25,000 XG preparations con- tained the least activity. An attempt was also made to prepare acetone powder from duodenum, kidney and liver of calves (Experiment 19, 20). The whole tissue was disintegrated in 10 volumes of acetone at ~15°C in a waring blender for 3 minutes in the cold room and the precipitate obtained was allowed to settle for 10 minutes. The supernatant aqueous acetone was decanted. The material was filtered with suction. The precipitate was washed twice by 3 volumes of acetone at ~15°C and once with diethyl ether at -15°C. The acetone powder thus prepared was dried and stored over Ca 012 at 179 -15°C until it was used. The acetone powder was suspended in an equal (w/v) amount of phosphate buffer at PH 7.2 and incubated with the aqueous carotene suspension using a phOsphate buffer as in- cubating media. The results of two such experiments showed that the vitamin A formed per gram acetone powder was very little or negligible. 180 DISCUSSION One of the purposes of this investigation was to es- tablish whether carotene could be converted into vitamin A in an in vitro system. The results indicate that 0.25 M sucrose solution and aqueous carotene suspension maintained physiological conditions well enough to allow the formation of vitamin A from B-carotene in vitro by tissue homogenates. In all experiments the amount of vitamin A formed ranged from 10 to 18 times the endogenous level in duodenum and 8 to 16 times the endogenous level in liver. Even in other tissues such as kidney and large intestine the amount formed after incubation was about 5 times the endogenous level. During the past years, numerous attempts were made to show in vitro formation of vitamin A from B-carotene by the small intestine and liver. As mentioned in the review of literature there were successful and unsuccessful at- tempts to form vitamin A in vitro. In the light of the success observed in this study it is not easy to explain negative results. The difference may have been due to (l) incubating media, (2) better extraction following saponi- fication of incubated media, or (3) preservation of activ- ity in tissue used or other less obvious reasons. Consid- erable effort was made throughout the experiments so that the results obtained would not be attributable to poor technique or open to criticism in some other manner. The 181 extraction procedure employed to isolate the carotene and vitamin A from incubated media were all standard procedures with some minor modifications. The capability of vitamin A formation by intestinal homogenates was largely dependent on the incubating solution used. Much more vitamin A was formed when 0.25 M sucrose was used than when any of the more commonly used buffers were employed. Besides furnish— ing essential nutrients during the in vitro reaction the 0.25 M sucrose solution may have had a more specific action or effect. Other investigators (43, 103, 104, 193) used either phosphate buffer, 0.9% saline or Krebe-Ringer buffer as incubating solutions. Saponification of the incubated mixture allowed a more complete extraction of vitamin A. The formation of small amounts of vitamin A were thus more readily detected and measured. In studies by other investigators (43, 103, 104, 193) saponification was not employed and hence the possi- bility exists that whatever small amounts of vitamin A were formed escaped extraction and detection. The accuracy of determining the efficiency of conver- sion in early experiments may not have been as precise as desirable. Rather high levels of carotene were used in early experiments. Carotene not recovered was measured by difference which has certain inherent inaccuracies. In order that the carotene recovered might be satisfactorily determined, low amounts of carotene were used for incubation 182 so as to make extraction and determination of carotene easier and more accurate. The term "vitamin A formed as percent of carotene unrecovered" was used to express the efficiency of vitamin A formation. Altering carotene to tissue ratio influenced vitamin A formation. These results demonstrated that with low levels of carotene (20 — 40 ug/g tissue) the formation of vitamin A was linear. As level of carotene increased (400 ug/g tissue) vitamin A formation also continued to increase, but with greatly reduced efficiency. Similar results have been obtained in vivo by Olson (138). The observations of various investigators (71, 103, 137) conclusively demonstrated that the bile was necessary for conversion of carotene to vitamin A both in vitro and in vivo. The results of present in vitro studies indicate that the incorporation of sodium glycocholate into the carotene suspension increased the vitamin A formation to over 120% of control using duodenal homogenates. The possi- bility exists that the bile salts (or more specifically sodium glycocholate) did not function merely as emulsify- ing agents (138). When the bile duct was ligated or when the entire intestine was removed the vitamin A formation from intravenously injected carotene suspension in Tween 40 was similar to that in animals not so treated (20). This leads to the idea that bile salts or sodium glyco- cholate do not act solely as emulsifying agents, but might 183 also function in a more specific manner. Olson (138) sug- gested that conjugated bile acids possessed a more specific function which was located mainly in the cholanic acid structure but was enhanced by conjugation. Bernhard et al. (15) showed in vitro that bile greatly retarded oxidation of the vitamin A. On these assumPtions it appeared that the more specific action of conjugated bile salts may be the retardation of vitamin A oxidation and this may be due to the cholanic acid structure. The conversion process appeared to be a very rapid re- action based on the appearance of vitamin A within 5 min after incubating B-carotene with duodenal homogenates. The reaction from B-carotene to vitamin A alcohol or ester may occur so rapidly that intermediary product such as vitamin A aldehyde could not be easily identified. The data indi- cates that increasing the incubation time beyond 30 min did not increase the net amount of vitamin A formation. Maximum vitamin A formation occurred at 30 min and decreased slowly thereafter. This observation may not be attribu- table to a feed back mechanism since the recovery of B— carotene at 30 min was high and the recovery was least at More likely this observation may be attributable 240 min. to the destruction of these compounds after incubating be- yond 30 min. The data indicate that tissue homogenized under nitrogen was more effective in vitamin A formation than the tissue homogenized under oxygen. These results 184 clearly suggest that the oxidative destruction of carotene and vitamin A was less under nitrogen. The conversion process studied was apparently enzymatic. In a preliminary experiment the tissue was obtained about 1 hour after slaughter and the extent of conversion was neg- ligible. The extent of vitamin A formation was greatly re- duced when the homogenates were kept l - 2 hours before addition of the carotene suspension. Further the data clearly indicate that heating homogenates inactivated the enzyme or factors which were responsible for conversion of carotene to vitamin A. Duodenal tissue from the pigs which were subjected to scalding the entire body surface on open flame did not show any activity. This process might have inactivated the enzyme(s) or factor(s). The finding, that the tissue respiratory poison KCN considerably reduced the formation of vitamin A, indicates that cytochrome oxidase system may also be involved in the conversion process. The fact that iodoacetate reduced the formation of vitamin A reveals that some type of alcohol dehydrogenase or some kind of thiol enzyme may be involved in the cleavage of carotene to vitamin A. All the above experimental findings initiate that a type of enzyme is involved in this process. ‘Although these experiments are not entirely conclusive, these results provide considerable evidence for the exis- tence of an enzyme system responsible for vitamin A forma- tion from B-carotene . 185 The positive results obtained on in vitro conversion dfcanflane to vitamin A (167, 185) have been questioned amencxdy the SbCl3 method was used for vitamin A determin- However, Rosenberg and Sobel (152) determined vita- ation. min A formed in vitro by intestines with the ultraviolet irradflnflon.technique proposed by Bessey et al. (16). The applhxufion of this method to intestinal vitamin A deter- mhunfion was shown to be of little use (18). Moore(l32) indicated that the determination of vitamin A in the pres- ence of carotene and its oxidation products requires special and careful techniques. 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Animal Dietary Tissue used Purpose of experiment no. treatment 1 Rabbit Normal Small intestine Extent of conversion 2 Holstein " Duodenum " cow 3 Rabbit " Small intestine Effect of N05, N03 and L-T4 4 Guernsy " Duodenum " bull 5 Rabbit " Small intestine Effect of N05, N05, L-T4 and glycocholate and conversiOn in loops 6 Heifer Deficient Duodenum Extent of conversion and effect of N05 7 'l H + moz II H H H H H 7 Rabbit Normal Small intestine Effect of N03‘ig vivo loops " " " Effect of NO3‘32 vitro 8 Calf Deficient Duodenum Conversion in lower gut, effect of N03, L-T4 and L-T3 9 Rabbit Deficient Small intestine Effect of N03 3 I! "+0.27", N03 H II II Normal I! ll 10 Calf Deficient Duodenum Comparison between saponification and non_saponification, effect of N03, S02, COS and NaCl. Effect of heating homogenates and altering carotene : tissue 11 Calf Deficient " " + 17. N03 12 Rabbit Normal Small intestine Effect of N05 H "+0.57o N03 H H H "+2007o N05 H H 2253 APPENDIX TABLE 1 (Continued) Expt. Animal Dietary Tissue used Purpose of experiment no. species treatment 13- Sheep Normal Duodenum Effect of N05, heating and blood 14' ” ” ” Effect of N03, KCN, urea and iodoacetate 15 Calf ” ” Effect of N0; and aging tissue 16 " Deficient Various tissues Conversion by various; tissues 17,18 ” Normal Duodenum Conversion by portions of duodenum 19,20 ” ” ” Conversion by acetone powder 21 " Deficient Various tissues Effect of 302, C03 and NaCl. Conversion by various tissues 22,25 ” Normal Duodenum Effect of NOS, L-T4, L-T3, h-Ta + N0" and L-T + NO' ‘_ 3 3 3 23,26 " "-I- Thio- " ’9 " uracil 24,27 ” ”+Thyro- " “ protein 28 , Rats Normal Various tissues Conversion by various tissues 29 Rabbits " Small intestine Same as experiment 22 (Thyroide- ctomised) 30 Rabbits " ” ” 31 " "I-Thio- " " uracil 32 " ”+Thyro- " ” protein 33 Pigs Normal Duodenum Extent of conversion 34 Rat Deficient Various tissues Conversion by various tissues 35 Chick ” ” " ’ 36,37 Rabbits ” " ” 38,39 Steer, Normal Duodenum Effect of L-Ta and L-T3 heifer 40,41 Pig Deficient Various tissues Conversion by various tissues and effect of N03 225b APPENDIX TABLE 1 (Continued) liver Expt. Animal Dietary Tissue used Purpose of experiment no. species treatment 42 Calf Deficient Duodenum,liver Effect of D-T4, D-TB, L-T4 L'T3 and 3 5'3. L’T3 43 Sheep Normal Various tissues Conversion by various organs and effect of N03 44 Calf ” Duodenum Effect of varying incubations time and various buffers 45,46 {Cow " ” Effect of varying incubation time 47,48 " ” ” Effect of various buffers 49,50 Steer, ” ” Effect of blood fractions heifer ° 51 Heifer ” ” Incubation of blood fractions and carotene 52,53 Heifer, ” " Effect of washed and centrifuged steer 54 Heifer ” ” Effect of various enzyme inhibitors 55,56 Steer " " Effect of amino acids and L-T4 57,58 Rabbit Deficient Small intestine Conversion by cell fractions 59 60 " ” " Effect of heating and tocopherol 61 ”A ” " Comparison of homogenization in waring blender and omni-mixer 62 ” Normal ” Effect of homogenization under N0 and 02 and identification of vifamin A formed 63 ” Deficient Small intestine, Extent of conversion in extremely deficient animal APPENDIX TABLE II ANALYSIS 0? VARIANCE OF DATA ON THE EFFECT OF ADDING IBOHBRS 0? L924 AND LPT3 AND COMBINATION OF rm ma 3 5'4' L-T3 on VITAMIN A romnou ram s-cmorsuz (run 22). 226 Duodenum Source of D.P. Sum of Mean F Signif. Variation Squares Square Treatment* 1 0.0230 0.0230 12.11 0.01 Thyroid compounds** 4 0.0170 0.0063 2.26 n.a. T X Tc 4 0.0580 0.0145 7.63 0.01 Between cells 9 0.0980 0.0109 5.74 0.01 Error 10 0.0190 0.0019 Total 19 0.1170 Liver Source of D.F. Sum of Mean P Signif. Variation Squares Square Treatment* 1 0.0140 0.0140 9.33 0.05 Thyroid compounds** 4 0.0020 0.0005 0.33 n.s. T X Tc 4 0.0540 0.0135 9.00 0.01 Between cells 9 0.0700 0.0078 5.20 0.01 Error 10 0.0150 0.0015 Total 19 0.085 * With or without adding 3 5'3' LPT3 ** With or without adding L- and D~T4 or L- and Data 227 APPENDIX TABLE III ANALYSIS OF VARIANCE OF DATA ON THE EFFECT OF ADDING L-Ta OR LPT WITH OR WITHOUT NITRATB 0N VITAMIN A FORMATION Y DUODENAL HOMOGENATBS FROM CONTROL HYPERTHYROID AND HYPOTRYROID ANIMALS (TABLE 24). Calves Source of D.F. Sum of Mean F Signif. Variation Squares Square Status 2 0.404 0.2020 12.63 0.01 Thyroxine level 2 0.082 0.0410 2.56 n.s. Nitrate level 2 1.046 0.5230 32.69 0.01 S X T 4 0.019 0.0048 0.30 n.s. T X N 4 0.158 0.0395 2.47 0.05 N X S 4 0.061 0.0153 0.96 n.s. S X T X N 8 0.034 0.0043 0.27 n.s. Error 81 1.300 0.0160 Total 107 2.144 Rabbits Source of D.F. Sum of Mean F Signif. Variation Squares Square Status 3 0.4119 0.1373 274.6 0.01 Thyroxine level 2 0.0014 0.0007 1.4 ass. Nitrate level 1 0.8855 0.8855 1777.1 0.01 8 X T 6 0.0110 0.0018 3.6 n.s. T X N 2 0.0467 0.0234 46.8 0.01 N X S 3 0.1818 0.0606 121.2 0.01 Error 6 0.0030 0.0005 Total 23 1.5413 ANALYSIS OF VARIANCE OF DATA ON THE EFFECT OF ADDING DIFFERENT AMINO ACIDS WITH AND WITHOUT COMBINATION OF L-T4 ON VITAMIN A FORMATION FROM B-CAROTENB (TABLE 26). APPENDIX TABLE IV Vitamin A Formed Per Gram Tissue 228 Source of D.P. Sum of Mean F Signif. Variation Squares Square Amino acids 3 0.0198 0.0061 19.41 0.01 Thyroxine 1 0.0666 0.0666 195.88 0.01 Experiments 1 0.0001 0.0001 0.29 n.s. A X T 3 0.0136 0.0045 13.23 0.01 T X E 1 0.0007 0.0007 2.06 n.s. E X A 3 0.0043 0.0014 4.12 0.05 A X T X E 3 0.0006 0.0002 0.58 n.s. Error 16 0.0054 0.00034 Total 31 0.1111 Vitamin A Formed as Z Carotene Unrecovered Source of D.F. Sum of Mean F Signif. Variation Squares Square Amino acids 3 51.2312 17.0771 35.48 0.01 Thyroxine 1 31.4112 37.4112 77.73 0.01 Experiments 1 14.3112 14.3112 29.73 0.01 A X T 3 1.6713 0.5571 1.16 n.s. T X E 1 2.7613 2.7613 5.74 0.05 E X A 3 7.5013 2.5004 5.20 0.05 A X T X E 3 4.9012 1.6337 3.39 0.05 Error 16 7.7000 0.4813 Total 31 127.4887 ROOM USE 0.5.311 R“ Um ill." llll Ill H A” "I! 3 1293 03178 3370