Intrinsic Factor-Independent Transport of Vitamin B12 Across the Gut of the Neonatal Rat

Vol. 61, No. 3

GASTROENTEROLOGY

Printed in U.S.A.

Copyright© 1971 by The Williams & Wilkins Co.

INTRINSIC FACTOR-INDEPENDENT TRANSPORT OF VITAMIN B 12 ACROSS THE GUT OF THE NEONATAL RAT NEIL

D.

GALLAGHER,

M .D.,

AND KAREN FoLEY

Repatriation General Hospital, Concord, and Department of Medicine, University of Sydney, Sydney, Australia

When Co 57-vitamin B 12 is fed by stomach tube to neonatal rats the vitamin is present in the wall of the lower jejunum and upper ileum within 30 min. The vitamin appears in the carcass 2 hr after feeding and, by 5 hr, transport from the gut to the carcass approaches a maximum rate. Transport of the vitamin does not require the presence of either gastric intrinsic factor or the vitamin B 12-binding protein in mother's milk. The intestinal transport of the vitamin appears to involve a saturable intermediate. Orally administered vitamin B 12 derivatives interfere with transport. The limited capacity of the transport mechanism contrasts with the ability of the intestinal epithelium to ingest large amounts of the vitamin by pinocytosis. The vitamin which is still present in the intestinal wall after 24 hr, by which time transport to the carcass has ceased, appears to return to the intestinal lumen. The intestinal absorption of vitamin B 12 have shown that the vitamin is bound to in man 1 and in most mammalian species 2 two proteins, one of which has a molecular is dependent on its combination with gas- size comparable with that of IF. 7 The other tric intrinsic factor (IF). The IF-B 12 com- binder presumably assists in the transport lex passes from the stomach to the ileum, of the vitamin within the cell. A third prowhere, in species such as the hamster 3 and tein, transcobalamin II (TCID has also guinea pig, 4· the IF moiety is attached to re- been identified in rat small intestine after ceptors in the free surface membranes of feeding the vitamin. 7 TCII, which is manuthe epithelial cells. The means by which the factured in the liver, 8 is required for the vitamin subsequently enters the cell is transport of the vitamin to the circulation. 9 unknown. Pinocytosis of the IF-B 12 complex Previous studies of the intestinal absorphas been proposed as the mechanism of ab- tion and transport of vitamin B 12 have sorption but there is little supportive evi- been carried out mainly in adult animals. dence. 5 This study is an extension of earlier work The process by which the vitamin is on the absorption of vitamin B 12 in rats transported across the ileal epithelium, dur- in early life. 10• 11 The choice of neonates ing which it becomes separated from IF 6 has three advantages. First, it is known that is also unexplained. Studies of vitamin B 12- vitamin B 12 is ingested by the small intesbinding in the small intestine of adult rats tine of young rats without the intervention of IF. 10 The transport of the vitamin across Received January 11, 1971. Accepted April 28, the intestinal epithelium can therefore be 1971. studied independently of the fate of IF. The Address requests for reprints to: Dr. N. D. Galsecond advantage is that the ileum of the lagher, Medical Professorial Unit, Repatriation Genyoung rat has a well defined phagolysosoeral Hospital, Concord 2139, Australia. 12 14 which is concerned in the This work was supported by the Bushell Trust, the mal system ' National Health and Medical Research Council, and transport of large molecules to the circulathe Postgraduate Medical Foundation of the University tion. 15 Thus, the transport of the vitamin of Sydney. can be considered in relation to these struc332

September 1971

. INTESTINAL TRANSPORT OF VITAMIN B, 2

tures. The third advantage is methodological one. When a )'-emitting isotope, such as Co 5 7 , is administered in the form of Co 5 7 vitamin B 12 , it is possible to recover almost the entire dose using a well counter for detection purposes. Our results show that, while the distal small intestine of suckling rats has an infinite capacity for vitamin B 12 absorption, only small amounts of the vitamin appear in the carcass. When · increasing doses of the vitamin are fed, transport by the small intestine reaches a limiting value. This finding, together with evidence that analogs interfere with the transport of the vitamin, indicates that the small intestine of young rats contains a specific transport system for vitamin B 12 •

Methods 57

Cobalt -labeled vitamin B 12 of specific activity, 50 to 200 me per mg, was obtained from the Radiochemical Center, Amersham, United Kingdom. Unlabeled vitamin was obtained in red crystalline form from Pierre!, s.p.a., Milan, Italy. It was made up in 0.15 M sodium chloride solution as required. Analogs of vitamin B,. were supplied by Dr. L. Mervyn, Glaxo Research, Greenford, Middlesex, United Kingdom. They were prepared by substituting one of the propionamide side chains of the corrin nucleus with methylamide, ethylamide or anilide radicals, or by conversion to a mixture of monobasic acids. Intrinsic factor was prepared from the mucosal scrapings of the glandular portion of adult rat stomachs which were homogenized in icecold distilled water. The supernatant fluid, which was collected after centrifugation of the whole homogenate at 6000 rpm for 10 min at 4 C, was used as the source of intrinsic factor . The vitamin B 12-binding capacity of the supernatant was measured by a charcoal-coated albumin method. 16 Porcine intrinsic factor concentrate was obtained from Nutritional Biochemicals Corporation, Cleveland, Ohio. Suckling animals of either sex were removed from their mothers, dosed by gavage, and then returned to the nest until the experiment was completed. This latter procedure was discontinued when it was found that the animals could be maintained apart from their mothers for periods up to 24 hr without affecting the absorption of the vitamin. The young were then kept in a box which was warmed to 30 C with

333

an electric mantle for the duration of the experiment. Where variations in this experimental procedure took place, reference is made under "Results". Each animal received a dose of labeled vitamin from a polyethylene tube which was passed without anesthesia into the stomach. A measured volume of solution containing Co 5 7B 1 2 was then delivered from a tuberculin syringe. At the conclusion of the experiments the animals were killed by decapitation. The gastrointestinal tract from the cardioesophageal junction to the rectum was removed. The stomach, colon, and washed small intestine were each placed in separate counting vials. The luminal contents which were obtained after washing the small intestine with 40 ml of 0.15 M sodium chloride solution were collected separately in 20-ml and 2 X 10-ml fractions. Theremainder of the animal or carcass was then divided into portions. The organ samples and the intestinal washings were assayed for radioactivity in a NuclearChicago model 186 well counter with a thallium iodide crystal. The dose of Co 57 -B 12 which each animal received was determined by calculating the total radioactivity in the individual samples, after the subtraction of background radioactivity of 600 counts per min. In the majority of experiments each animal received a dose containing 60,000 to 100,000 counts per min of Co 57 -B, 2 In experiments with 15 rats, 98 % of the administered radioactivity was recovered from the whole animal and the intestinal washings. As the variation in counting rate owing to differences in sample size was less than 5%, a correction factor was not applied. Absorption refers to the amount of radioactive vitamin which left the intestinal lumen and was recovered from the wall of the small intestine and the carcass. Transport refers to the amount of radioactivity which was recovered from the carcass.

Results Absorption and transport of varying doses of Co 57-B 12 • The efficiency of absorption of the vitamin in nonfasting, 8- to 10-day-old rats did not alter when the dose was increased from 1 m,ug to 10 ,ug (fig. 1). Approximately 90% of the dose left the intestinal lumen in a 5-hr period. The rate of transport of the vitamin reached a maximum in the experiments with a 50-m,ug dose. A linear relationship was obtained when the data relating absorption and

GALLAGHER AND FOLEY

334

transport were plotted in double reciprocal fashion. 17 Site of absorption and transport. In experiments in which the absorption of varying doses of Co 57 -B 1 2 was studied, the small intestine from the pylorus to the ileocecal valve was divided into four segments, numbered from 1 to 4 (table 1). When the dose of the vitamin was 10 J.Lg or greater, the site of its absorption could be recognized by the reddish discoloration of the wall of the second, third, and fourth segments. The predilection of the vitamin for the third segment was evident as early as 1 hr after feeding in the experiments with a 100-m,ug dose (table 2). Rate of absorption and transport. Eightto 10-day-old rats which had been fasted overnight were killed at intervals up to 5 hr after the administration of doses of Co 57 -B 12 ranging from 10 to 100 mJ.Lg. The rate of absorption, which was expressed as a percentage of the total dose, was similar for each of the four doses reaching a maximum within 3 to 5 hr. In contrast, the rate at which individual doses left the intestine differed from one another. Transport of the vitamin from the gut wall to the carcass was not detected until 2 hr after feeding, although absorption commenced within 30 min (fig. 2) . 100

0

Ab~orption

Vol. 61, No. 3

Effect of milk protein on the intestinal transport of vitamin B 12 • The initial studies were performed in 8- to 10-day-old rats which suckled up to and during the time of the experiment. In order to exclude the possibility that milk protein was involved in 1. Distribution of total radioactivity in wall of the small intestine 5 hr after feeding different doses of Co " -B ,,a

TABLE

No. of in testin al segm ent Dose

c·

m,g

10' 10' 10' 10• a

6 1 0 0

48 62 74 53

28 5 4 4

18 32 22 43

Results are mean value of four experiments.

2. Distribution of total radioactivity in the wall of the sm all intestine at different times after feeding 100 m!l{J of Co 51 -B, ,0

TABLE

No. of intestin al segment

Tim e after

feeding c

hr

2 3 5

5 8 2

15 16 20 12

74 63 70 72

6 13 8 15

a Results are expressed as mean value of four experiments.

~ Transpor t 80

20

10

20

50

80

100

"'

IO!XXl

Dose I mpg. l

FIG. 1. Effect of the dose on the absorption and transport of Co"-B 12 • Each value is the mean of four to six experiments.

Hours

Fie. 2. Rate of absorption and transport of four different doses of Co"-B 12 • Results are expressed as the mean of four to six experiments.

September 1971

INTESTINAL TRANSPORT OF VITAMIN B, 2

335

the transport of the vitamin across the gut, analogs, or 10 ,ug of the monobasic acid transport was compared in unfasted ani- and anilide analogs. The animals in each mals and in their littermates which had group were killed 5 hr after feeding. The been fasted for 24 hr (table 3). The results absorption of the vitamin was similar in of these experiments indicated that milk the five experimental groups but a smaller protein was not required . for either the ab- amount of the vitamin left the intestine in the groups which received the analogs. The sorption or the transport of the vitamin. In the event that the period of fasting latter values only are shown in table 5. In was inadequate, additional experiments the experiments with the monobasic acid were carried out in newborn rats which and anilide analogs, it was necessary to were taken from their mother within min- use a larger dose in order to demonstrate utes of delivery. Considerable difficulty inhibition of transport. Duration of intestinal transport of was experienced in feeding the dose to animals of this size (5 g) but a mean of 36% Co 57-B 12 • Groups of four 8- to 10-day-old of a 100-m,ug dose was transported from rats were given an oral dose of 100 m,ug the intestine in two experiments. These of the vitamin. They were then returned experiments thus provide further evidence to their mothers and were killed at interthat the transport of the vitamin occurred vals up to 3 days. The accumulation of the vitamin in the carcass did not increase beindependently of milk protein. The effect of endogenous and exogenous yond 24 hr when as much as 20% of the abIF on intestinal transport. The vitamin B 12 - sorbed dose remained in the wall of the binding capacity of whole stomach homog- small intestine (fig. 3). Intestinal excretion of an oral dose of enates was measured · in newborn rats which were from the same litter as those Co 57-B 12 • The retention of the vitamin in which were dosed with 100 m,ug of the wall of the small intestine after the time Co 57 -B 12, in order to .compare the quant- when transport had ceased suggested that ity of vitamin leaving the intestine with the the vitamin was eventually excreted into the amount of endogenous intrinsic factor. intestinal lumen. Experiments were perAssay revealed that there was less than formed with a 100-,ug dose to test this possi2 m,ug U of vitamin B 12-binding material bility. With a dose of this size it was possible in a pooled homogenate from 4 rats. This value is too low to implicate the animal's TABLE 3. Effect of fasting for 24 hr on the 57absorption and transport of a 100-m~ dose of Co -B,,a own IF in the transport of the vitamin. The effect of IF on the absorption and Fasted Fed transport of the vitamin was also studied in 89 ± 4 (7) 87 ± 6 (6) 10-day-old animals. Sufficient rat or por- Absorption (mJLg) . 23 ± 5 (7) 24 ± 7 (6) Transport (mJLg) . cine IF was added to bind the 10 m,ug of Co 5 7 -B 12 which each animal received. a Results are shown as the mean ± standard deviaPorcine IF was used in the knowledge that tion, with the number of observations in parentheses. it fails to enhance the absorption of the vitamin in adult rats. 18 The results of feed- TABLE 4. Effect of the addition of IF• on the absorping IF-B 12 and free B 12 to 10-day-old, tion and transport of a 10-m~ dose of fasting animals are shown in table 4. The Co"-B,,O values in the table represent the pooled reIF + B, Free B12 sults which were obtained with the two sources of intrinsic factor. No difference Absorption 91.1 ± 3.4 (8) 89. 0 ± 4.9 (8) was observed in either absorption or trans(% dose) port when the vitamin was administered in Transport 45.0 ± 12.7 (8) 50.9 ± 5.6 (8) the bound form. (% dose) Effect of orally administered analogs of vitamin B 12 on its transport. The animals • IF, intrinsic factor. were fed 100 m,ug of Co 57-B 12 and either ' Results are shown as the mean ± standard devia1 ,ug of the methylamide or ethylamide tion, with the number of observations in parentheses.

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336

to follow the changes in the amount of the vitamin in the intestinal wall by noting the disappearance of its reddish discoloration as well as by measurement of radioactivity. A total of five litters of 8- to 10-day-old rats was used in these experiments. After the administration of the dose, the animals were returned to their mothers until their removal on successive days. The rate of disappearance of the vitamin from the intestine decreased abruptly after 24 hr, and thereafter at a slower rate (fig. 4). Carcass radioactivity did not increase during this time. In a number of experiments with the same litter, the difference between the amount of radioactivity in the wall of the small intestine of animals killed on successive days was recovered from the small intestinal washings and the colon. It is therefore assumed that the decrease in intestinal 5. Effect of oral administration of B 12 analogs on the transport of a 100-mp.g dose of Co"-B 12•

TABLE

-~- -- -- ---..--------.--------

Amount of analog

Experiment

I'll

Control Ethylamide Methylamide Anilide Monobasic acid

10 10

Co 51 ·B 12 transported across the gut mpg/5 hr

25.5 ± 4.4 (6) 8.8±1.7(6) 8.8 ± 2.1 (5) 6.0 ± 1.9 (5) 8.1 ± 2.1 (5)

a Results are expressed as the mean ± standard deviation, with the number of observations in parentheses.

Vol. 61, No.3

radioactivity after 24 hr was due to the excretion of the vitamin into the intestinal lumen. Discussion Pinocytotic absorption of vitamin B 12 in the infant rat was first proposed by Boass and Wilson 10 who found that the uptake of large amounts of free vitamin by rings of distal small intestine decreased abruptly at a time when morphological evidence of pinocytosis disappea-red. This occurs 18 days after birth at a time when the ileal epithelium view was indistinguishable from that of the adult animal. 15 It has also been observed in electron microscope autoradiographic studies that the highest density of the tritiated vitamin is located in the absorptive vacuoles and in the giant supranuclear vacuole of the ileal epithelium. 19 A smaller amount was present in the cytoplasm and was mainly associated with mitochondria. These findings, together with our observations that millimicrograms of the vitamin are absorbed in vivo as efficiently as micrograms, provide a substantial body of evidence in favor of pinocytotic absorption of the free vitamin in the young rat. The observation that a constant percentage of each of four doses was taken up by the small intestine, per unit time, is also consistent with a mechanism such as pinocytosis. The readiness with which large quantities of the vitamin entered the intestinal epithelium contrasts with the small amounts

80

. J~'.----~--------~

70 60

10

~~--~--~~--~--~~~--~------~ IZ zo 16 M • ~ n Hours

FIG. 3. Duration of transport of a 100-mllg dose of Co"-B 12 from the small intestine to the carcass.

INTESTINAL TRANSPORT OF VITAMIN B, 2

September 1971 100

80

60

\

40

20

DAY 0

·-·

~

CARCASS RAD IOAC TIVITY

·"--..._.

•

·=--=:

FIG. 4. Disappearance of radioactivity from the gut wall compared with accumulation in the carcass after feeding 100 p.g of Co 57 -B 1 2 •

which left the intestine_ In order to be certain that the transport of the vitamin was not due to the vitamin B 12 -binding protein which is present in milk, 20 experiments were also performed in fasting animals. These experiments indicated that neither the absorption nor the transport of the vitamin was dependent on the ingestion of milk. The presence of the vitamin in the carcass of newborn rats which were removed from their mothers before they had suckled provided further evidence that milk protein was not involved in transport_ Absorption of the vitamin independently of milk had previously been shown to occur in a study in which absorption was measured by whole body counting. 21 The experiments with newborn rats exclude the possibility that IF played a part in either transport or absorption, for the amount of vitamin B 12-binding protein in their stomachs was insufficient to bind the quantities which entered the intestinal mucosa. The negligible part played by IF was also demonstrated in the experiments with 10-day-old rats. The values for absorption and transport which were obtained

337

after feeding IF-B 12 did not differ from those in animals which were fed the free vitamin. As neither IF nor milk protein played a part in transport it is probable that these proteins were digested after their entry into the absorptive vacuoles, perhaps by the hydrolases in the closely associated lysosomal structures. 12 - 14 The development of a technique for micropuncture of the supranuclear vacuole could assist in answering this question. While the transport of vitamin B 12 in infant rats is independent of exogenous protein, the binding of the vitamin to protein is a ubiquitous phenomenon_ Binding of the vitamin to a protein in the wall of the small intestine other than IF has already been demonstrated in adult rats. 7 It may be the same protein which we postulate is involved in the ileal transport of the vitamin. Its presence would account for the observation that the rate of transport of the vitamin attained a maximum value. Saturation of a protein carrier is consistent with the finding that the kinetics of the transport mechanism are similar to those observed between an enzyme and its substrate. 22 The reduction in transport which followed the oral administration of analogs of the vitamin supports our contention that a specific protein is required_ The inhibition of transport by simultaneous feeding of the analogs suggests that the carrier protein is located within the wall of the supranuclear vacuole for the transport of macromolecules into the cytoplasm is arrested at this leveL 20 This protein, in common with other specific vitamin B 12-binding proteins, 23 • 24 is probably a glycoprotein_The possibility that it is formed by the Golgi apparatus ought to be considered in view of the evidence that this region contributes to the formation of the wall of the supranuclear vacuole. 13 The persistence of the vitamin in the wall of the small intestine after its transport had ceased suggested that it was eventually shed into the lumen within effete epithelial cells. This was shown indirectly in the experiments in which it was found that the disappearance of the vitamin from the intestinal wall occurred in an exponential fashion. In common with experiments on the intestinal excretion of iron and vita-

GALLAGHER AND FOLEY

338

min B 12 in adult rats, 2 5 • 2 6 these ex periments may provide a measure of the life span of the intestinal epithelium which is increased in young animals. 27 The retention of the vitamin by the epithelial cells suggests that they act as short-lived macrophages once their capacity to transport the vitamin is exceeded.

14. 15.

16.

REFERENCES 1. Castle WB: Factors involved in the absorption of vitamin B 12• Gastroenterology 37:377-383, 1959 2. Glass GBJ: Gastric intrinsic factor and its function in the metabolism of vitamin B 12• Physiol Rev 143:529-849, 1963 3. Donaldson RM , MacKenzie IL, Trier JS: Intrinsic factor-mediated attachment of vitamin B 12 to brush borders and microvillous membranes of hamster intestine. J Clin Invest 46:1215- 1228, 1967 4. Rothenberg SP: Identification of a macromolecular factor in the ileum which binds intrinsic factor and immunologic identification of intrinsic factor in ileal extracts. J Clin Invest 47 :913-923, 1968 5. Wilson TH: Membrane transport of vitamin B 1 ,. Medicine (Bait) 43:669-677, 1964 6. Boass A, Wilson TH : Intestinal absorption of intrinsic factor and B 12-intrinsic factor complex. Amer J Physiol207 :27-32, 1964 7. Frentz GD, Miller ON, Hansen HG: Characterisation of vitamin B 1 , binders in rat intestine after oral administration of rat intrinsic factor-B 12Co 57 complex. Clin Res 14:432, 1966 8. Tan CH, Hansen HJ: Studies on the site of synthesis of transcobalamin II. Proc Soc Exp Bioi Med 127:740-744, 1968 9. Hall CA, Finkler AE: The dynamics of transcobalamin II. A vitamin B 12 binding substance in plasma. J Lab Clin Med 65:459-468, 1965 10. Boass A, Wilson TH: Development of mechanisms for intestinal absorption of vitamin B 1 , in growing rats. AmerJPhysiol204:101- 104, 1963 11. Gallagher ND: Mechanism and site of absorption of vitamin B 12 in suckling rats. Nature (London) 222:877, 1969 12. Vacek Z: Submikroskipicka struktura a cytochemie epitelu tenkeho streva u krysich mladat. Cesk Morf 12:292-301, 1964 13. Cornell CA, Padykula HA: A cytological study of

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intestinal absorption in the suckling rat. Amer J Anat 125:291-316, 1969 Williams RM, Beck F: A histochemical study of gut maturation. J Anat 105:487- 501, 1969 Clark SL: The ingestion of proteins and colloidal material by columnar absorptive cells of the small intestine in suckling rats and mice. J Biophys Biochem Cytol5:41-50, 1959 Gottlieb C, Lau KS , Wasserman LR, eta! : Rapid charcoal assay for intrinsic factor (IF), gastric juice unsaturated binding capacity, antibody to IF, and serum unsaturated B 12 binding capacity. Blood 25: 875-884, 1965 Lineweaver H, Burk D: The determination of enzyme dissociation constants. J Amer Chern Soc 56:658-666, 1934 Wilson TH, Strauss EW: Some species differences in the intrinsic factor stimulation of B 1 , uptake by small intestine in vitro. Amer J Physiol 197:926928, 1959 Graney DO: The uptake of tritiated vitamin B 1 , by intestinal absorptive cells of suckling rats. An electron microscope autoradiographic study. Anat Rec 160:355-366, 1968 Gregory ME, Holdsworth ES: The occurrence of a cyanocobalamin binding protein in milk and the isolation of a cyanocobalamin protein complex from sow's milk. Biochem J 59:292-334, 1955 Williams DL, Spray GH: The absorption of " Co cyanocobalamin by unweaned rats. Brit J Nutr 22:297-301, 1968 Neilands JB, Stump PK: Outlines of Enzyme Chemistry. New York, Wiley and Sons Inc, 1955 p 75-76 Simons, K: Gastric intrinsic factor and other vitamin B 12 transport proteins: chemical and physiologic properties, Progress in Gastroenterology, vol 1. Edited by GBJ Glass. New York, Grune and Stratton, 1968, 195-220 Grasbeck R: Intrinsic factor and transcobalamins with reflection on the general function and evolution of soluble transport proteins. Scand J Clin Lab Invest 19, (suppl95):7-18, 1967 Conrad ME, Crosby WH: Intestinal mucosal mechanisms controlling iron absorption. Blood 22:406-415, 1963 Loehry CA, Creamer B: Vitamin B 12 excretion by rat small intestine. Gut 10:662-664, 1969 Koldovsky 0 , Sunshine P, Kretchmer N: Cellular migration of intestinal epithelia in suckling and weaned rats. Nature (London) 212:1389-1390, 1966