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Free Intrinsic Factor in the Small Intestine in Man
Vol. 70, No.5
70:704-706, 1976 Copyright © 1976 by The Williams & Wilkins Co.
GASTROENTEROLOGY
Printed in U.S. A .
FREE INTRINSIC FACTOR IN THE SMALL INTESTINE IN MAN R. KAPADIA, M.D., V. I. MATHAN, M.D., PH.D., AND S. J. BAKER, M.D., F.R.A.C.P.
CYRUS
Wellcom e Research Unit, Christian Medical College Hospital, Vellore, Tamil Nadu, India
Human jejunal and ileal contents and ileostomy effluents were examined for the presence of free intrinsic factor. This was detected in 9 out of 10 jejunal and three out of five ileal aspirates and in one of three ileostomy effluents studied. The intrinsic factor in the ileal effluent was shown to be physiologically active. The presence of free intrinsic factor in the small intestine has considerable physiological significance in maintaining the enterohepatic circulation of vitamin B I2 . It would also permit the absorption of any free vitamin BI2 produced by ileal bacteria. It is well known that in vitro free intrinsic factor (IF) is gradually inactivated by pepsin and chymotrypsin. I, 2 Little is known, however, of the in vivo fate of IF that is not bound to vitamin BI2 in its transit down the small intestine. The present study was undertaken to determine whether free IF could be detected in the small intestine of man, and if so, whether it was physiologically active.
Subjects and Methods Six volunteer control subjects, with no clinical or laboratory evidence of gastrointestinal disease, and 4 subjects with tropical sprue as evidenced by the presence of chronic diarrhea, steatorrhea, d-xylose, and vitamin BIZ malabsorption, were studied. After an overnight fast the subjects were intubated with a fine radioopaque polyvinyl tube, and aspirates were obtained from the first loop of jejunum. In 6 subjects (4 control and 2 with tropical sprue) the tube was left in situ, and the subjects were given only water or lime juice to drink until the tube reached the ileum (220 to 250 cm from the incisor teeth) where further aspirates were obtained. All the aspirates were stored at -70 0 C until studied. Three further subjects with normal vitamin BI2 absorption who had had defunctioning ileostomies performed for amebic perforation of the colon were also investigated. Two months after antiamebic treatment, ileostomy effluents were collected for several days, the contents of the ileostomy bag being frequently emptied and stored at _20 0 C. The effluents, combined in 24-hour pools, were homogenized with a known volume Received July 11, 1975. Accepted October 26, 1975. Address requests for reprints to: S. J. Baker, M.D., Wellcome Research Unit, Christian Medical College Hospital, Vellore 632004, Tamil Nadu, India. Dr. Kapadia 's present address is: Yale University School of Medicine, Veterans Administration Hospital, Medical SVC, West Spring Street, West Haven, Connecticut 06516. The authors wish to thank Mr. S. P . Swaminathan for technical help. The Wellcome Trust is in collaboration with the World Health Organization.
704
of cold 0.15 M saline and centrifuged at 12,000 x g for 1 hr at 4 0 C, and the supernatant was stored at -20 0 C. Measurement of free IF content was carried out using zirconium phosphate gel at pH 6.25 and the binding IF antibody, as described by Jacob and O'Brien 3 , or using hemoglobin-coated charcoal and the blocking IF antibody according to the method of Gottlieb et al. 4 Both methods gave similar results. To investigate the physiological competence of the IF detected in the ileal effluent of one patient, further studies were carried out. An aliquot of one day's effluent was adjusted to pH 3 and autoclaved at 5 lb pressure for 20 min to release vitamin B 12 bound to IF. 5 The vitamin BI2 content was then measured by microbiological assay using Euglena gracilis Z strain as the test organism. 6 The effluent contained 1.6 ng of vitamin BI2 per ml. To a larger aliquot of the same effluent, 57 Co-Iabeled vitamin BI2 (obtained from the Bhabha Atomic Research Centre, Trombay) was added in slight excess, and the unbound vitamin was removed by dialysis. 7 This material was then lyophilized and stored at - 20 0 C until further use. The material was subsequently reconstituted with distilled water, and the amount of IF vitamin BI2 complex was measured. 3 Ninety milliliters, containing 0.82 JJ.g of IF 57CO vitamin BI2 complex, were given to each of 2 volunteers with vitamin BI2 malaborption due to total gastric atrophy. Both volunteers gave their informed consent to the procedure. The absorption of the labeled vitamin was determined by measuring the rise in serum radioactivity at 8 hr after the doseS and by whole body counting in a moving bed shadow shield detector.
Results Free IF was found in 9 out of 10 of upper jejunal samples, and in four out of six ileal samples. One normal subject did not have free IF in the jejunal juice, and the same normal subject and one patient with sprue had no free IF in the ileal juice (fig. 1). One of the three ileostomy effluents also contained free IF. Analysis of several 24-hr collections of ileostomy effluent from this patient showed that an average of 4.3 JJ.g units of free IF were excreted per day.
FREE INTRINSIC FACTOR IN SMALL INTESTINE
May 1976 8 E
"~
o
7
c
:J
c
6
c::
5
0
IU
« 4
0
LL
0
Ol
0
U (/)
z
c::
I-
~
0
• •
3 0
•
2 0
w w
•
0 0
c::
LL
0 JEJUNUM
ILEUM
ILEOSTOMY
FIG. 1. Free IF concentration in jejunal (0) and ileal (e) aspirates and in ileostomy effluents (-). TABLE
1. Results of vitamin B12 absorption tests in 2 volunteers with
total gastric atrophy Patient R Whole body retention
Serum
dose/liter, %
/lg
dose/liter,_ %
/lg
0.01 0.50
Nil ND b
0.01 0.52
0.05 ND b
0.6 c
0.65
1.2 c
0.46
Serum
1 ILg B12 alone 1 ILg B12 + 40mg hogIFa 0.82 ILg B12 + ileostomy fluid
Patient N Whole body retention
a IF, intrinsic factor. b ND, not done. c Result expressed as percentage of a 1-ILg dose for sake of comparison with other tests.
One of the 2 subjects with gastric atrophy secreted no measurable IF, and the other, 30-ng units ofIF in the 1st hr following histamine stimulation (0.04 mg per kg of body weight) of gastric secretion. Both were unable to absorb vitamin B12 when the test dose was given alone, but had a normal rise in plasma radioactivity when exogenous IF was supplied with the test dose. Both the subjects also absorbed the vitamin added to the ileal effluent (table 1). Discussion Okuda and Sasayama 9 instilled radioactive vitamin B 12 into the human small intestine either following intubation or at laparotomy. They demonstrated that some of the vitamin was absorbed even when put in the lower ileum and suggested that this demonstrated the presence of free IF in the intestinal lumen. The present study shows, by direct assay, that their supposition was correct and that free IF may often be found in the lumen of both jejunum and ileum and at times even in the effluent from an ileostomy. When radioactive vitamin B12 was added to the ileal effluent containing free IF, and the resultant mixture
705
was administered to 2 .mbjects who lacked endogenous IF, the labeled vitamin was well absorbed. It is theoretically possible that the absorption of the labeled vitamin could have been the result of exchange 10 of the labeled vitamin with preexisting unlabeled vitamin B12 IF complex in the effluent. However, since the amount of effluent used contained only 0.14 1J,g of BI2-IF complex, this cannot explain the observed findings. It is therefore clear that the free IF in the ileostomy effluent was physiologically active in promoting vitamin B12 absorption. In the intestinal aspirates it was only possible to measure the concentration of IF, and this gives no indication of the total amount passing through the ileum per day. However, in one subject with an ileostomy of 24-hr output amounted to 4.3 1J,g units, which is severalfold more than is needed to promote the absorption of the minimum daily requirement of vitamin B12.11 It should be noted that the assay procedures used measured only free IF, and variable amounts of IF may still have been present bound to nonradioactive vitamin B 12 , and thus remained undetected. The presence of free IF in the intestinal lumen is of considerable physiological importance. Grasbeck et al 12 demonstrated that when radioactive vitamin B12 is given parenterally, some of the vitamin B12 is excreted in the bile. They further showed that the fecal excretion of the label was less than the biliary excretion and postulated an enterohepatic circulation of the vitamin. The present demonstration of free IF in the lumen of the small intestine establishes the existence of one of the essential conditions which would enable the vitamin excreted in the bile to be reabsorbed in the ileum, thus maintaining the enterohepatic circulation. It is well known that bacteria in the large intestine of man are able to synthesize vitamin B 12 , but since little vitamin B12 is absorbed from the large intestine, this is usually considered to be unavailable to the host. 9, 13 Studies of bacterial flora of the small intestine have shown that the ileum even of normal southern Indian subjects contains large numbers of fecal type organisms. 14 If any of these make vitamin B12 which is liberated into the fluid phase in the intestinal lumen, it could combine with the free IF and thus be available for absorption by the host. This may well be an important reason for the low prevalence of frank vitamin B12 deficiency in the largely vegetarian population of southern India. 15 REFERENCES 1. Castle WB, Ham TH: Observations on the etiologic relationship of achylia gastric a to pernicious anaemia. V. Further evidence for essential participation of intrinsic factor in hematopoietic responses to mixtures of beef muscle and gastric juice and to hog stomach mucosa. JAMA 107:1456-1463, 1936 2. Griisbeck R: Influence of specific group inhibitors and enzymes on intrinsic factor activity. Acta Physiol Scand 45:88-95, 1959 3. Jacob E, O'Brien H: A simple assay of intrinsic factor-vitamin B12 complex employing the binding intrinsic factor antibody. J Clin Pathol 25:320-325, 1972 4. Gottlieb C, Lau KS, Wasserman LR, et al: Rapid charcoal assay for intrinsic factor (IF), gastric juice unsaturated B12 binding ca-
706
5. 6.
7. 8.
9.
KAPADIA ET AL.
pacity, antibody to intrinsic factor and serum unsaturated B .. binding capacity. Blood 25:875-884, 1965 Spray GH: Estimation of intrinsic factor and detection of intrinsic factor antibodies using nonradioactive cyanocobalamin and microbiological assay. J Clin Pathol 20:689-692, 1967 Hutner SH, Bach MK, Ross GIM: Sugar containing basal medium for vitamin B12 assay with Euglena: application to body fluids. J Protozool 3:101-112, 1956 Bird OD, Hoevet B: The vitamin B 12 -binding power of proteins. J BioI Chern 190:181-189, 1951 Mathan VI, Swarnabai S, Baker SJ : Intestinal absorption of radioactive vitamin B 12 : a comparison of plasma, faecal and urinary tests. Indian J Med Res 61 :714-723, 1973 Okuda K, Sasayama K: Intestinal distribution of intrinsic factor and vitamin B12 absorption. Am J Physiol 208:14-17, 1965
Vol . 70, No . .'j
10. Donaldson RM, Katz HJ: Exchange between free and gastric juice bound cyanocobalamin . J Clin Invest 42:534-545, 1963 11. Sullivan LW, Herbert V: Studies on the minimum daily requirement for vitamin BIZ. New Engl J Med 272:340-346, 1965 12. Grasbeck R, Nyberg W, Reizenstein P: Biliary and fecal vitamin B12 excretion in man: an isotope study. Proc Soc Exp BioI Med 97:780-784, 1958 13. Girdwood RH: Intestinal content in pernicious anemia of factors for growth of Streptococcus faecalis and Lactobacillus leichmannii. Blood 5:1009-1016, 1950 14. Bhat P, Shanthakumari S, Rajan D, et al: Bacterial flora of the gastrointestinal tract in southern Indian control subjects and patients with tropical sprue . Gastroenterology 62:11-21, 1972 15. Baker SJ: Human vitamin B12 deficiency. World Rev Nutr Diet 8:62- 126, 1967
70:704-706, 1976 Copyright © 1976 by The Williams & Wilkins Co.
GASTROENTEROLOGY
Printed in U.S. A .
FREE INTRINSIC FACTOR IN THE SMALL INTESTINE IN MAN R. KAPADIA, M.D., V. I. MATHAN, M.D., PH.D., AND S. J. BAKER, M.D., F.R.A.C.P.
CYRUS
Wellcom e Research Unit, Christian Medical College Hospital, Vellore, Tamil Nadu, India
Human jejunal and ileal contents and ileostomy effluents were examined for the presence of free intrinsic factor. This was detected in 9 out of 10 jejunal and three out of five ileal aspirates and in one of three ileostomy effluents studied. The intrinsic factor in the ileal effluent was shown to be physiologically active. The presence of free intrinsic factor in the small intestine has considerable physiological significance in maintaining the enterohepatic circulation of vitamin B I2 . It would also permit the absorption of any free vitamin BI2 produced by ileal bacteria. It is well known that in vitro free intrinsic factor (IF) is gradually inactivated by pepsin and chymotrypsin. I, 2 Little is known, however, of the in vivo fate of IF that is not bound to vitamin BI2 in its transit down the small intestine. The present study was undertaken to determine whether free IF could be detected in the small intestine of man, and if so, whether it was physiologically active.
Subjects and Methods Six volunteer control subjects, with no clinical or laboratory evidence of gastrointestinal disease, and 4 subjects with tropical sprue as evidenced by the presence of chronic diarrhea, steatorrhea, d-xylose, and vitamin BIZ malabsorption, were studied. After an overnight fast the subjects were intubated with a fine radioopaque polyvinyl tube, and aspirates were obtained from the first loop of jejunum. In 6 subjects (4 control and 2 with tropical sprue) the tube was left in situ, and the subjects were given only water or lime juice to drink until the tube reached the ileum (220 to 250 cm from the incisor teeth) where further aspirates were obtained. All the aspirates were stored at -70 0 C until studied. Three further subjects with normal vitamin BI2 absorption who had had defunctioning ileostomies performed for amebic perforation of the colon were also investigated. Two months after antiamebic treatment, ileostomy effluents were collected for several days, the contents of the ileostomy bag being frequently emptied and stored at _20 0 C. The effluents, combined in 24-hour pools, were homogenized with a known volume Received July 11, 1975. Accepted October 26, 1975. Address requests for reprints to: S. J. Baker, M.D., Wellcome Research Unit, Christian Medical College Hospital, Vellore 632004, Tamil Nadu, India. Dr. Kapadia 's present address is: Yale University School of Medicine, Veterans Administration Hospital, Medical SVC, West Spring Street, West Haven, Connecticut 06516. The authors wish to thank Mr. S. P . Swaminathan for technical help. The Wellcome Trust is in collaboration with the World Health Organization.
704
of cold 0.15 M saline and centrifuged at 12,000 x g for 1 hr at 4 0 C, and the supernatant was stored at -20 0 C. Measurement of free IF content was carried out using zirconium phosphate gel at pH 6.25 and the binding IF antibody, as described by Jacob and O'Brien 3 , or using hemoglobin-coated charcoal and the blocking IF antibody according to the method of Gottlieb et al. 4 Both methods gave similar results. To investigate the physiological competence of the IF detected in the ileal effluent of one patient, further studies were carried out. An aliquot of one day's effluent was adjusted to pH 3 and autoclaved at 5 lb pressure for 20 min to release vitamin B 12 bound to IF. 5 The vitamin BI2 content was then measured by microbiological assay using Euglena gracilis Z strain as the test organism. 6 The effluent contained 1.6 ng of vitamin BI2 per ml. To a larger aliquot of the same effluent, 57 Co-Iabeled vitamin BI2 (obtained from the Bhabha Atomic Research Centre, Trombay) was added in slight excess, and the unbound vitamin was removed by dialysis. 7 This material was then lyophilized and stored at - 20 0 C until further use. The material was subsequently reconstituted with distilled water, and the amount of IF vitamin BI2 complex was measured. 3 Ninety milliliters, containing 0.82 JJ.g of IF 57CO vitamin BI2 complex, were given to each of 2 volunteers with vitamin BI2 malaborption due to total gastric atrophy. Both volunteers gave their informed consent to the procedure. The absorption of the labeled vitamin was determined by measuring the rise in serum radioactivity at 8 hr after the doseS and by whole body counting in a moving bed shadow shield detector.
Results Free IF was found in 9 out of 10 of upper jejunal samples, and in four out of six ileal samples. One normal subject did not have free IF in the jejunal juice, and the same normal subject and one patient with sprue had no free IF in the ileal juice (fig. 1). One of the three ileostomy effluents also contained free IF. Analysis of several 24-hr collections of ileostomy effluent from this patient showed that an average of 4.3 JJ.g units of free IF were excreted per day.
FREE INTRINSIC FACTOR IN SMALL INTESTINE
May 1976 8 E
"~
o
7
c
:J
c
6
c::
5
0
IU
« 4
0
LL
0
Ol
0
U (/)
z
c::
I-
~
0
• •
3 0
•
2 0
w w
•
0 0
c::
LL
0 JEJUNUM
ILEUM
ILEOSTOMY
FIG. 1. Free IF concentration in jejunal (0) and ileal (e) aspirates and in ileostomy effluents (-). TABLE
1. Results of vitamin B12 absorption tests in 2 volunteers with
total gastric atrophy Patient R Whole body retention
Serum
dose/liter, %
/lg
dose/liter,_ %
/lg
0.01 0.50
Nil ND b
0.01 0.52
0.05 ND b
0.6 c
0.65
1.2 c
0.46
Serum
1 ILg B12 alone 1 ILg B12 + 40mg hogIFa 0.82 ILg B12 + ileostomy fluid
Patient N Whole body retention
a IF, intrinsic factor. b ND, not done. c Result expressed as percentage of a 1-ILg dose for sake of comparison with other tests.
One of the 2 subjects with gastric atrophy secreted no measurable IF, and the other, 30-ng units ofIF in the 1st hr following histamine stimulation (0.04 mg per kg of body weight) of gastric secretion. Both were unable to absorb vitamin B12 when the test dose was given alone, but had a normal rise in plasma radioactivity when exogenous IF was supplied with the test dose. Both the subjects also absorbed the vitamin added to the ileal effluent (table 1). Discussion Okuda and Sasayama 9 instilled radioactive vitamin B 12 into the human small intestine either following intubation or at laparotomy. They demonstrated that some of the vitamin was absorbed even when put in the lower ileum and suggested that this demonstrated the presence of free IF in the intestinal lumen. The present study shows, by direct assay, that their supposition was correct and that free IF may often be found in the lumen of both jejunum and ileum and at times even in the effluent from an ileostomy. When radioactive vitamin B12 was added to the ileal effluent containing free IF, and the resultant mixture
705
was administered to 2 .mbjects who lacked endogenous IF, the labeled vitamin was well absorbed. It is theoretically possible that the absorption of the labeled vitamin could have been the result of exchange 10 of the labeled vitamin with preexisting unlabeled vitamin B12 IF complex in the effluent. However, since the amount of effluent used contained only 0.14 1J,g of BI2-IF complex, this cannot explain the observed findings. It is therefore clear that the free IF in the ileostomy effluent was physiologically active in promoting vitamin B12 absorption. In the intestinal aspirates it was only possible to measure the concentration of IF, and this gives no indication of the total amount passing through the ileum per day. However, in one subject with an ileostomy of 24-hr output amounted to 4.3 1J,g units, which is severalfold more than is needed to promote the absorption of the minimum daily requirement of vitamin B12.11 It should be noted that the assay procedures used measured only free IF, and variable amounts of IF may still have been present bound to nonradioactive vitamin B 12 , and thus remained undetected. The presence of free IF in the intestinal lumen is of considerable physiological importance. Grasbeck et al 12 demonstrated that when radioactive vitamin B12 is given parenterally, some of the vitamin B12 is excreted in the bile. They further showed that the fecal excretion of the label was less than the biliary excretion and postulated an enterohepatic circulation of the vitamin. The present demonstration of free IF in the lumen of the small intestine establishes the existence of one of the essential conditions which would enable the vitamin excreted in the bile to be reabsorbed in the ileum, thus maintaining the enterohepatic circulation. It is well known that bacteria in the large intestine of man are able to synthesize vitamin B 12 , but since little vitamin B12 is absorbed from the large intestine, this is usually considered to be unavailable to the host. 9, 13 Studies of bacterial flora of the small intestine have shown that the ileum even of normal southern Indian subjects contains large numbers of fecal type organisms. 14 If any of these make vitamin B12 which is liberated into the fluid phase in the intestinal lumen, it could combine with the free IF and thus be available for absorption by the host. This may well be an important reason for the low prevalence of frank vitamin B12 deficiency in the largely vegetarian population of southern India. 15 REFERENCES 1. Castle WB, Ham TH: Observations on the etiologic relationship of achylia gastric a to pernicious anaemia. V. Further evidence for essential participation of intrinsic factor in hematopoietic responses to mixtures of beef muscle and gastric juice and to hog stomach mucosa. JAMA 107:1456-1463, 1936 2. Griisbeck R: Influence of specific group inhibitors and enzymes on intrinsic factor activity. Acta Physiol Scand 45:88-95, 1959 3. Jacob E, O'Brien H: A simple assay of intrinsic factor-vitamin B12 complex employing the binding intrinsic factor antibody. J Clin Pathol 25:320-325, 1972 4. Gottlieb C, Lau KS, Wasserman LR, et al: Rapid charcoal assay for intrinsic factor (IF), gastric juice unsaturated B12 binding ca-
706
5. 6.
7. 8.
9.
KAPADIA ET AL.
pacity, antibody to intrinsic factor and serum unsaturated B .. binding capacity. Blood 25:875-884, 1965 Spray GH: Estimation of intrinsic factor and detection of intrinsic factor antibodies using nonradioactive cyanocobalamin and microbiological assay. J Clin Pathol 20:689-692, 1967 Hutner SH, Bach MK, Ross GIM: Sugar containing basal medium for vitamin B12 assay with Euglena: application to body fluids. J Protozool 3:101-112, 1956 Bird OD, Hoevet B: The vitamin B 12 -binding power of proteins. J BioI Chern 190:181-189, 1951 Mathan VI, Swarnabai S, Baker SJ : Intestinal absorption of radioactive vitamin B 12 : a comparison of plasma, faecal and urinary tests. Indian J Med Res 61 :714-723, 1973 Okuda K, Sasayama K: Intestinal distribution of intrinsic factor and vitamin B12 absorption. Am J Physiol 208:14-17, 1965
Vol . 70, No . .'j
10. Donaldson RM, Katz HJ: Exchange between free and gastric juice bound cyanocobalamin . J Clin Invest 42:534-545, 1963 11. Sullivan LW, Herbert V: Studies on the minimum daily requirement for vitamin BIZ. New Engl J Med 272:340-346, 1965 12. Grasbeck R, Nyberg W, Reizenstein P: Biliary and fecal vitamin B12 excretion in man: an isotope study. Proc Soc Exp BioI Med 97:780-784, 1958 13. Girdwood RH: Intestinal content in pernicious anemia of factors for growth of Streptococcus faecalis and Lactobacillus leichmannii. Blood 5:1009-1016, 1950 14. Bhat P, Shanthakumari S, Rajan D, et al: Bacterial flora of the gastrointestinal tract in southern Indian control subjects and patients with tropical sprue . Gastroenterology 62:11-21, 1972 15. Baker SJ: Human vitamin B12 deficiency. World Rev Nutr Diet 8:62- 126, 1967