STUDIES ON THE ABSORPTION OF VITAMIN B12 IF. DISTRIBUTION OF RADIOACTIVE VITAMINE B12 IN THE TISSUES OF ALBINO RATS

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1 THE JOURNAL of VITAMINOLOGY 8, (1962) STUDIES ON THE ABSORPTION OF VITAMIN B12 IF. DISTRIBUTION OF RADIOACTIVE VITAMINE B12 IN THE TISSUES OF ALBINO RATS SHIGEO UKYO The First Division, Department of Jute nail Medicine, Faculty of Medicine, Kyoto University, Sakyo, Kyoto (Received February 3, 1962) Ever since the Castle's original postulation concerning the factors involved in the etiology of Addisonian pernicious anemia (1), a great number of clinical and experi mental studies have been performed to elucidate the mechanism by which intrinsic factor (IF) stimulates the intestinal absorption of vitamin B12 (B12), but the mecha nism of B12 absorption still remains unclarified. More recently, rats have become very valuable as experimental animals in the study of the problems of B12 absorption. Miller et al. (2) have shown that hog IF increases the uptake of Co60-B12 by rat liver slices. This work has been confirmed by other investigators (3-5). Mean while, since Chow et al. (6, 7) and others (8, 9) reported the enhancing effect of D-sorbitol on B12 absorption, a great interest in this agent was aroused. However, the effect of D-glucosamine, one of the hexosamines contained in gastric juice, pos sibly having IF activity, on B12 absorption has never been studied. A large body of researches demonstrate that IF preparation does not always exert its activity unless the IF is species-specific. So the effects of lyophilized saline extract of rat gastric mucosa and of D-glucosamine along with those of hog IF and D-sorbitol on B12 absorption were studied in this investigation. For the purpose of this investiga tion, Co60- or Co58-B12 was given with or without the above-mentioned agents into the three sites of rat gastrointestinal lumen, and the tissue distribution and excre tion of B12 were studied. In addition to this, the tissue distribution of Co60-B12 or Co58-B12 following intravenous injection was studied as well in the rats. This study was made to investigate the effects of the above described agents on the tissue dis tribution of B12 after they were introduced with B12 into the blood stream. In other words, these studies were made to experimentally elucidate the mechanism of intestinal absorption of B Experimental Animal EXPERIMENTAL Methods and Materials Male albino rats of Wistar strain, weighing 200 to 250g, were each kept in an individual metabolic cage and fed on a normal diet prior to use. The animals were divided into 5 groups (2 to 5 in each group): (a) B12-group, receiving B12 alone, 90

2 Vol. 8 ABSORPTION OF VITAMIN B12. IV 91 (b) hog IF group, receiving BI2 together with hog IF, (c) D-sorbitol group, (d) D glucosamine group and (e) rat gastric mucosa group. 2. Materials Radioactive Vitamin B12-Co60-1 and Co58-1B12 of high specific activity (0.98 and 2.84ƒÊc/ƒÊg, respectively) were used. Radioactive B12 was kept in a screw capped bottle and at -20 until used. 2. D-Sorbi tol, D-Glucosamine and Lyophilized Saline Extract of Rat Gastric Mucosa D-Sorbitol2 and D-glucosamine2 as well as lyophilized extract of rat gastric mucosa (RGM) were kept in a glass bottle and at room temperature before use. They were dissolved in radioactive Bit solution. RGM was prepared from rat gastric mucosa by lyophilizing with saline solution, and kept in a wax-sealed test tube at -20 before use. Hog Intrinsic Factor Concentrate-As hog intrinsic factor concentrate (IF), "Bifacton"3 was used. It was also kept at -20 in a screw-capped bottle prior to use. 3. Methods Co60- or Co58-B12, 100mƒÊg, was given with or without various agents to the rats under anesthesia with ether after overnight fasting according to the following experimental procedure into the stomach by a polyvinyl tube, and into the intestinal lumen by a syringe after laparotomy. The administration into the intestinal lumen was made at the upper part of the jejunum and the lower part of the ileum in order to investigate the site of B12 absorption. Hog IF group received 12mg of hog IF, D-sorbitol group 120mg of D-sorbitol, D-glucosamine group 120mg of D-glucosamine and rat gastric mucosa group 5mg of the lyophilized saline extract of RGM together with 100mƒÊg of radioactive B12. Fecal and Urinary Excretion-During 24 or 120 hours following the admin istration, feces and urine were collected separately as far as possible. In this series of experiment, non-radioactive B12 was not given as a flushing dose. An aliquot of 5ml of the collected urine was pipetted into a test tube and its radioactivity was counted for 5 minutes above the background on the top of a well-type scintillation counter as described elsewhere (10). To the collected feces 20ml of water was added and the mixture was subjected to a complete hemogenization by means of a domestic electric mixer. An aliquot, 5ml, of the homogenate was quantitatively pipetted into a test tube. Tissue Distibution of B12 after Placing the Vitamin in Three Sites of Gastrointestinal Lumen-Twenty-four or 120 hours after Ben administration, the rats were sacrificed and the abdomen was opened. The organs such as the liver, spleen, kidneys, bone marrow of the unilateral femur, and small intestine were taken out. These organs and the contents of the small intestine were rinsed with 1 Kindly supplied through the courtesy of Dr. Nathaniel Ritter, Merck and Co., U. S. A. 2 Pur chased from Takeda Chemicals Co., Ltd., Osaka. 3 Generously furnished by Dr. Kenneth Thompson, Grganon Inc., U. S. A,

3 92 UKYO 1962 saline solution repeatedly, and the organs except for the liver were then subjected to heating and acid digestion with concentrated H2SO4. Prior to acid digestion, the extirpated small intestine was cut into three segments, each equal in length, and each was placed into a test tube. The spleen, the kidneys, and the bone marrow were likewise transferred into the test tubes and subjected to acid digestion. The liver was uniformly homogenized with water by an electric homogenizer to the final volume of 20ml. The homogenized liver specimens were poured into 4 test tubes, each containing 5ml. Tissue Distribution of B12 Following Intravenous Administration-One hundred mƒêg of Co60- or Co58-B12 was injected into the tail vein of the rats with or without the agents aforementioned. Tissue distribution 24 or 120 hours after the administration was studied in the similar way. Counting-The counting of radioactivity in the excreta and in the organs was made for 5 minutes above the background on the top of a well-type scintillation counter as described before (10). The results were expressed as mƒêg by calculating from the percentage of the given dose. 4. Technical Considerations Study of Physiologic Oral Dose of B12 Pertinent to Rats Used As described in the first report (10), the percentage of fecal or urinary radioactivity varies in man from dose to dose given. So a preliminary study was made on the relation of doses of 50, 100 and 200mƒÊg of Co60-B12 given into the stomach with urinary excretion following an intramuscular flushing dose of 100ƒÊg of non-radioactive B12. As a result, the greatest urinary excretion occurred at the dose of 100mg of Co60-B12, ranging from 4.05 to 4.65mƒÊg, with a mean of 4.39m/Ag (Table I.) Based on this result, 100mƒÊg of radioactive B12 was given as a test dose throughout the study. TABLE I Sequence of Various Doses of Co60-B12 to Urinary Excretion in Rats a Mean } standard deviation Recovery of Administrated B12-In order to investigate the validity of the values obtained in this study, the radioactivity of the carcasses of four rats was also counted, two carcasses 24 hours and the other two 120 hours after administration.

4 Vol. 8 ABSORPTION OF VITAMIN B12. IV 93 The recovery of administered B12 ranged, as shown in Fig. 1, from 77.2 to 101.9%, demon strating that the carcasses con tained relatively large amounts of B12 administered. In the two carcasses of poor recovery, unrecovered radioactivity seems to be contained in the blood and body fluid which were discarded or lost inevitably alluring the opertion of the samples. At any event, this result suggests that the data presented in this paper can FIG. 1 Recovery of Administered Radioactive B12 U, urine; L, liver; K, kidneys. provide a reasonably well established ground, on which discussions can be made and conclusions can be drawn from. RESULTS 1. Fecal Excretion of Placed in Three Sites of Gastrointestinal Lumen Fecal Excretion in the Rats Sacrificed 24 Hours after Administration-In general, as shown in Fig. 2, it was observed that the lower the site of administra tion, the greater the fecal excretion of B12, regardless of administering B12 with or without the agents described above. It could be noted, however, that ap proximately 50% of the dose was found to be absorbed even when B12 alone was introduced into the lower part of the ileum. The addition of hog IF resulted in increased fecal excretion, irrespective of the site of administra tion, showing that hog IF inhibits B12 absorption from the small intestine of the rats presumably because of species difference. D-Sorbitol did not always decrease fecal excretion, although it FIG. 2 Fecal Excretion of Radioactive B12 when Given into Three Sites of Gas trointestinal Tract IF, hog intrinsic factor "Bifacton" RGM, dialyzed, lyophilized saline extract of rat gastric mucosa, appeared to decrease to some extent, when given together with B12 into the stomach. No definite effect of D-glucos amine was found. Lyophilized saline extract of rat gastric mucosa rather tended to increase the excretion. Fecal Excretion in the Rats Sacrificed 120 Hours after Administration The trend was again observed that the lower the site of administration, the more the fecal excretion. However, no definite effect was observed with D-sorbitol, D-glu cosamine and with the extract of RGM after 120 hours. Hog IF increased the ex cretion also after 120 hours.

5 94 UKYO Urinary Excretion of B Introduced into the Three Sites of Rat Gastrointes tinal Lumen Urinary Excretion in the Rats Sacrificed 24 Hours after Administration- As shown in Figs. 3 and 4, no significant amount of B12 was excreted in the urine, whether B12 was given alone or with various agents, or whether it was introduced into the stomach, the jejunum or the ileum. This may be due to the procedure of this investigation that non-radioactive B12 was not injected parenterally as a flushing dose. FIG. 3 Tissue Distribution of Radioactive Rig 24 Hours after Administration FIG. 4 Tissue Distribution of Ra dioactive B12 24 Hours after Admin istration Urinary Excretion in the Rats Sacrificed 120 Hours after Administration- After 120 hours, however, urinary excretion was significantly increased more or less, as shown in Figs. 5 and 6. It was interesting to note that lyophilized saline extract of RGM increased the excretion when introduced with B12 into the jejunum, up to as much amount as excreted when B12 was given alone into the stomach, but did not increase when the extract was given into the ileum. The fact that urinary excretion occurred after 120 hours suggests that the excretion without giving a flushing dose may occur later than after giving the dose. Moreover, no positive FIG. 5 Tissue Distribution of Radioactive B Hours after Administration FIG. 6 Tissue Distribution of Radioactive B Hours after Administration

6 Vol. 8 ABSORPTION OF VITAMIN B12. IV 95 correlation was found between the urinary excretion and site of B12 administration. But outstanding was the finding that the addition of hog IF gave rise to the decrease in urinary excretion 120 as well as 24 hours after administration, showing inhibitory effect of hog IF on B12 absorption in the rat. 3. Tissue Distribution of Radioactive B12 Absorbed from Intestine after In troducing into Three Sites of Gastrointestinal Lumen Tissue distribution of absorbed B12 in the rats sacrificed 24 hours after the dose is shown in Figs. 3 and 4. Generally speaking, when B12 was introduced into the lower part of the ileum, B12 uptakes by various organs were low. It was of interest that the absorbed B12 was preferentially taken up by the kidneys and the liver, especially by the former. The distribution in the small intestine was less than in the liver. Among the three segments of the intestine, the middle part was found to contain the greatest amount of B12 as compared with the proximal and distal parts. No significant amount of B12 was found in the spleen and bone marrow. Hog IF depressed the distribution of B12 in the organs as well, showing the inhibitory effect on B12 absorption, whereas the other agents remarkably enhanced the distribution as compared with that after the administration of B12 alone, Similar results were obtained in the tissue of the rats sacrificed 120 hours after the dose, as illustrated in Figs. 5 and G. O f special interest is that the distribution of B12 in the kidneys were higher than in the liver after 24 hours, while, after 120 hours, the distribu tion in the liver was increased and that in the kidney was decreased. 4. Tissue Distribution of Radioactive B12 Following intravenous Injection In this series of study, five groups of albino rats (2 to 3 in each group) were given intravenously 100mƒÊg of Co60- or Co58-B12 with or without various agents. Another five groups were given the similar dose for the study of tissue distribution in the rats to be sacrificed 120 hours after the injection. It can be noted, as shown in FIG. 7 Tissue Distribution of Radioactive B12 Following Intravenous Injection IF, intrinsic factor; RGM, lyophilized saline extract of rat gastric mucosa, DISCUSSION Fig. 7, that the simultaneous administra tion of B12 and hog IF resulted in in creased B12 uptake by the liver with concomitant decrease in B12 distribution in other tissues, both 24 and 120 hours after the injection. In contrast, the addition of either lyophilized saline ex tract of rat gastric mucosa, D-sorbitol, or n-glucosamine gave rise to higher B12 uptake by the kidneys after 24 and 120 hours, but did not result in the increased uptake by the liver. In addition, it was found that the higher distribution in the kidneys as observed 24 hours after the injection tended to gradually decrease 120 hours after the injection. In the present study, it was noted that the higher the site of B12 administration, the

7 96 UKYO 1962 more B12 was absorbed. B12 distribution in three segments of the small intestine of the rats sacrificed 24 hours after the dose is indicative of the site of B12 absorp tion from the intestine. Thus B12 was found to be mainly absorbed from the middle portion of the small intestine. Booth et al. (11) have shown that B12 given to the rats was taken up by the intestine very rapidly and the greatest proportion of B12 was found in the middle portion of the small intestine of the rats sacrificed 1/4 hour and 2 hours after the oral dose. However, an emphasis must be placed on the fact that approximately 50% of the dose could be absorbed after 24 hours even from the distal portion of the ileum, when B12 was introduced alone into the ileum. Regarding the urinary excretion, a significant amount of B12 failed to be detected in 24-hour urine of the rat, while it was detected in 120-hour urine. This fact may indicate that the omission of intramuscular flushing dose resulted in the retardation of urinary excretion. The phenomenon that 120-hour urinary excretion was raised, when B,2 was introduced together with lyophilized saline extract of RGM into the jejunum, up to the amount observed after the administration of B12 alone into the stomach, may be natural, since the extract is species-specific to the rats used. The problem of species difference is of importance. The effect of IF on B12 absorption or tissue distribution varies from animal to animal or from origin to origin from which the IF was obtained. As a matter of fact, it was found in this investigation that the addition of hog IF uniformly inhibited Bit absorption or tissue distribution when introduced into the gastrointestinal lumen. It would be reasonably accepted that this inhibitory effect of hog IF could be attributable to species difference, i.e., hog IF was not species-specific to the rats used. A number of reports concerning the species differences have so far been made. In the rats, the kidneys were found to be the main site of Co60-B12 localiza tion even when urinary excretion was absent (12-14). In man, the greatest per centage of a parenterally injected dose of Co60-B12 was found in the liver (15, 16). In the hamster, guinea pig and mouse, the liver was the chief organ of localization (9). In the present investigation, the kidneys were found to be the chief organ of B12 localization and the liver was the second site of localization when the rats were sacrificed 24 hours after the dose, in spite of the fact that no significant amount of B12 was detected in the 24-hour urine., So it seems probable that the kidneys re present an important site of B12 localization in the rats rather than a mere storage site for further urinary excretion. In this connection, Miller et al. (12) and Ro senthal (17) have made similar conclusions. Furthermore, in view of the fact that the distribution of B12 in the kidneys were decreased 120 hours after the dose, while that in the liver tended to be increased, it appears that (a) these two organs are B12-storage organ in the rat, or (b) the absorbed B12 may be first metabolized in the kidney in some unknown way without being excreted into the urine and then ultimately stored in the liver. As regards the effects of the agents other than hog IF on fecal excretion, no definite effects were observed. Similarly, no definite effects of these agents on urinary excretion could be seen, though the addition of lyophilized saline extract of rat gastric mucosa presented the data to suggest that the extract enhances B12 absorp tion when introduced into the jejunum. However, in the light of tissue distribution after the dose of these agents with B12, they seem to contribute to some extent to

8 Vol. 8 ABSORPTION OF VITAMIN B12. IV 97 the enhancement of B12 absorption. These discrepancies may be accounted for as follows: (a) The failure in collecting feces and urine may be responsible for. actually it was of utmost difficulty to collect them as separately as possible. Some times, the urine washed B12 contained in the feces, and sometimes the feces or urine specimen was lost in part for unknown reasons. (b) The rats rather tended to have diarrhea when these agents were given. This diarrhea may have facilitated the mingling of urine and feces, thus leading to the falsified results. (e) The reason for the fact that lyophilized saline extract of RGM, specific to the rats used, did not necessarily enhance B12 absorption is unknown, but some technical faliure in preparing the extract may be responsible for it. Meanwhile, the study of tissue distribution of B12 24 or 120 hours following intravenous injection did present the evidence that hog IF enhanced the uptake of B12 by the liver with concomitant decrease in B12 uptake by other organs, while it inhibited the absorption of Bit when introduced into the gastrointestinal lumen. This result is in good agreement with the observation by Okuda et al. (14). The addition of the agents other than hog IF resulted in increased B12 uptake by the kidneys, but in decreased B12 uptake by the liver, in contrast to hog IF. In conclusion, the present author feels it to be justified to accept a hypothesis that two mechanisms may possibly exist in the absorption of Bit, i.e., B12 absorp tion itself across the intestinal mucusa and the subsequent uptake of B12 by the organs. Taking this hypothesis into consideration, the effects of various agents used in this study can be reasonably explained as follows: Hog IF has only one activity, and plays a role in the uptake of B12 by the organs, but does not exert any enhanc ing effect on the intestinal absorption itself in the rats. D-Sorbitol and D-glucos amine seem to have the similar activity as hog IF, while lyophilized saline extract of RGM contributes to B12 absorption in the two ways of mechanisms described above. SUMMARY For the purpose of clarifying the mechanism of B12 absorption, Go60- or Go58-B12 was given intravenously or into the three sites of gastrointestinal lumen of albino rats, and tissue distribution of the vitamin was studied. The effects of the agents such as hog IF, D-sorbitol, D-glucosamine, and lyophilized saline extract of rat gastric mucosa were also studied. 1. It was interesting to note that B12 could be absorbed even from the ileum in the rats, 2. The higher the site of B12 administration, the more B12 was absorbed. But, generally speaking, the main site of B12 absorption appeared to be in the middle portion of the small intestine in the rat. 3. It was observed that the chief site of B12 localization was in the kidneys and the liver in the rat, suggesting that these organs may be the B12-storage organs. 4. Hog IF inhibited B12 absorption in the rat, presumably because of species dif ference. Some discussions were made on the problem of species difference as well. However, when hog IF was given intravenously, the agent exceedingly enhanced B12 uptake by the liver with concomitant decrease in the uptake by other organs. 5. In contrast, the agents other than hog IF resulted in increased B12 uptake by

9 98 UKYG 1962 the kidneys, not by the liver, when given intravenously. 6. Based on these results obtained, the present author has put forward a hypothe sis concerning the mechanism of B12 absorption. Two mechanisms may possibly exist: B12 absorption itself across the intestinal mucosa and the subsequent uptake of the vitamin by the organs. Finally, according to the hypothesis, some comments were made regarding the effects of various agents used in this study on B12 absorp tion. ACKNOWLEDGEMENTS The author wishes to express his gratitude to Professor Gyoichi Wakisaka for his constant encouragement and kind supervision in this investigation. The author is also deeply indebted to Instructor Haruto Uchino, who have directly undertaken to supervise this study. Grate ful acknowledgement is given, to Dr. Nathaniel Ritter, Merck and Co., U.S.A., for his generous supply of Co58- and Co60-labeled vitamin B12 and also to Dr. Kenneth Thompson, Organon Inc., U.S.A., for his kind supply of hog intrinsic factor preparation, " Bifacton. This study was carried out in part by a grant-in-aid for the Scientific Research from the Educational Ministry of Japan. REFERENCES 1. Castle, W. B., Am, J, Med. Sci, 178, 748 (1929). 2. Miller, O, N,, Raney, J. L., and Hunter, F. M., Proc. Soc. Exptl. Biol, Med. 16, 393 (1957). 3. Latner, A. L., and Raine, L., Biochem, J. 66, 53 (1957), 4. Herbert, V., Proc. Soc. Exptl. Biol. Med. 97, 668 (1958). 5. Herbert, V., J. Gun. Invest. 37, 646 (1958). 6. Chow, B. F., Meier, P., and Free, S. M,, Amer. J Gun. Nutrition 6, 36 (1958). 7. Chow, B. F., Horonick, A., and Okuda, K,, ibid. 4, 434 (1956). 8. Greenberg, S. M., Herdon, J. F,, Rice, E. G., Parmelee, E. T., Gulesich, S. J., and Van - Loon, E. J., Nature 180, 1401 (1957). 9. Okuda, K., Hsu, J. M., and Chow, B. F., J. Nutrition 72, 99 (1960). 10. Ukyo, S., J. Vitaminol. 8, 41, (1962) 11. Booth, C. C., Chanarin I., Anderson, B. B,, and Mollin, D. L., Brit. J. Haematol, 3, 253 (1957). 12. Miller, A,, Gaull, G., Ross, J. F., and Lemon, H. M., Proc. Soc. Exptl. Biol. Med. 93, 33 (1956). 13. Rosenblum, C., Chow, B, F., Condon, G. P., and Yamamoto, R., J. Biol. Chem. 108, 915 (1952). 14. Harte, R. A., Chow, B, F,, and Barrow, L,, J. Nutrition 49, 669 (1953). 15. Glass, G. B. J., Boyd, L. J., and Gellin, G. A. Blood 10, 95 (1955). 16. Gkuda, K., Wider, J. A., and Chow, B. F., J. Lab, and Olin. Vied. 54, 535 (7959). 17. Rosenthal, H. L,, J. Nutrition 68, 297 (1959).