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Enterohepatic circulation of cobalamin in the nonhuman primate
GASTROENTEROLOGY
1981;81:773-P
Enterohepatic Circulation of Cobalamin in the Nonhuman Primate RALPH GREEN, DONALD VAN TONDER, MICHAEL
W. JACOBSEN, SUSAN V. C. KEW, and JACK METZ
Departments of Clinical Research and Biochemistry, Scripps Clinic and Research Foundation, La Jolla, California; and Departments of Hematology and Medicine, School of Pathology, University of the Witwatersrand and The South African Institute for Medical Research, Johannesburg, South Africa
Biliary excretion of cobalamin was studied in baboons after intravenous injection of 57Co-radiolabeled cyanocobalamin. Radioactivity appeared in bile after 20 min, and up to 4.4% of the dose was recovered in 6 h. It was estimated that 4 pg cobalamin traverses the baboon biliary tract each day. These studies in the nonhuman primate confirm that there
is considerable biliary excretion of cobalamin and that enterohepatic circulation of the vitamin is important for maintenance of normal cobalamin balance. The biliary tract appears to be a major route of vitamin B,, (co b a 1amin) recirculation. In humans the amount of cobalamin that enters the bile each day (1,~) considerably exceeds daily body loss (3) or minimal requirements (4) of the vitamin. Biliary cobalamin must therefore be efficiently reabsorbed for conservation of the vitamin. An enterohepatic circulation for the vitamin has indeed been suggested from studies in humans (1,2) and other animals (5-7). The significance of an enterohepatic circulation for cobalamin is not known, but it clearly has bearing on maintenance of normal cobalamin balance and the pathogenesis of cobalamin deficiency. This is illustrated by the observation that cobalamin deficiency develops more rapidly in patients after total gastrectomy than in strict vegans who exclude in-
take of the vitamin through dietary practices. The explanation is that a failure to absorb cobalamin not only precludes its assimilation from the diet, but also impairs reabsorption of the vitamin from the bile. It is based on the assumption that, like the absorption of dietary cobalamin, reabsorption of biliary cobalamin also requires intrinsic factor. We have studied baboons to gain further information on the normal biliary excretion and enterohepatic circulation of cobalamin in primates.
Materials and Methods Animals Adult male chacma baboons, Papio urisinus, cages and weighing 25-40 kg were housed in individual ration biscuits (Epic Oil Mills, were fed Epol-balanced Newtown, Johannesburg, South Africa) supplemented with eggs, fresh fruit, and vegetables.
Radioactive
Materials
and Detectors
[TolCyanocobalamin (IO&300 &i/pg) was obtained from the Radiochemical Centre (Amersham, Buckinghamshire, England). Radioactivity determinations were carried out on small volume samples (< 5.0 ml) in standard gamma spectrometers (Packard Autogamma, models 3002 and 3330).
Received November 24, 1980. Accepted May 12, 1981. Address requests for reprints to: Ralph Green, M.D., Department of Clinical Research, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, California 92037. This work was supported by the National Institutes of Health Grant NS 13714 (R. Green) and by grants from the South African Atomic Energy Board and the Medical Research Council of South Africa. The authors are indebted to Sister 0. Hoffman Devisme for surgical assistance and to Mr. B. Fratscher for animal care. 0 1981 by the American Gastroenterological Association OOlS-5085/61/100773-O4$02.50
Surgical Preparation For surgical procedures baboons were anesthetized after an overnight fast with intramuscular phencyclidine hydrochloride (Sernylan, Park Davis and Co., London, England),
2.0 mg/kg body wt. Intramuscular atropine, body wt, was given. Anesthesia was maintained using intravenous pentobarbitone sodium (Sagatal, May and Baker Ltd., Dagenham, England), 5-10 mg/kg
0.015 mg/kg
774
GASTROENTEROLOGY
GREEN ET AL.
:
0.075
k p
0.050
Biliary
P 0.02 1
Figure 1. Pattern of radioactivity excreted in the bile of a baboon following an intravenous dose of 0.04 pg [57Co]cyanocobalamin bound to plasma. body wt, and fluid replacement was given as isotonic saline containing 5% (wt/vol) dextrose by continuous intravenous infusion at the rate of 20-40 ml/h.
Common
Bile Duct Cannulation
The abdomen was opened, and the hepatic and common bile ducts were identified and gently stripped of their serosal coats. A blunt-tipped polythene catheter was inserted through a longitudinal incision made in the hepatic duct immediately proximal to the point of entry of the cystic duct and passed upwards a short distance towards the liver. The catheter was connected to a fraction collector, and bile was collected in l&ml aliquots for periods of 4-6 h.
Blood Sampling Blood specimens were obtained from arm or leg veins via plastic disposable syringes and needles.
Laboratory
Methods
Biliary cobalamin was measured using a radioisotope dilution method (8). To exclude any effect of bile on the cobalamin assay, varying amounts of cyanocobalamin were added to specimens of bile. Recoveries (9!!103%) fell within the range of error encountered with this method. Determination of the unsaturated cobalamin binding capacity (UCBC) of serum and bile were carried out according to the methods of Gottlieb et al. (9).
Preparation and Injection [5’ColCobalamin
81, No.
4
Results
0.100
B
Vol.
Table 1. Biliary Excretion of Radioactivity in 8 Baboons Following Intravenous Injection of rCo]Cyanocobalamin
Experiment
The UCBC of baboon plasma was measured, and 25 ml was saturated with [S7Co]cyanocobalamin. After incubation at 37% for 60 min, hemoglobin-coated charcoal was added to remove unbound radioactivity (lo), and the 57Co-labeled plasma was sterilized by Seitz filtration before injection. The amount of [?o]cyanocobalamin used
counted for calculation of the injected radioactivity. Bile draining through the common bile duct cannula was collected in I.&ml fractions for radioactivity measurement.
of Cobalamin
The mean cobalamin concentration of bile obtained from 8 normal baboons before the injection of [S’Colcyanocobalamin was 15.7 (range 3.82-40.2) ng/ ml. Bile flow-rates varied from 0.1 to 0.9 ml/min between animals and at different times in the same animal. From measured volumes collected over 4-6 h and assuming that this was representative of the physiologic rate of bile flow, the estimated daily rate of bile secretion was calculated to be 256 (range 116879) ml in the eight baboons studied. An average minimum of 4.02 pg of cobalamin (256 ml X 15.7 ng/ ml) was estimated to pass through the baboon biliary tract each day. The time taken for radioactivity to appear in bile following intravenous injection of plasma-bound [57Co]cyanocobalamin ranged from 20 to 74 (mean 37) min, and peak radioactivity was reached between 166 and 342 (mean 240) min. The excretion of [?Zo]radioactivity was variable but continuous over the observation period. A representative pattern for one animal is shown in Figure 1. The total radioactivity excreted in the bile during the period of collection was expressed as a percentage of the dose injected (Table 1). The mean cumulative excretion of [“‘Colcobalamin was 2.22% of the injected dose over the time from the first appearance of radioactivity to the end of the collection period (132-346 min). The mean calculated percentage of the dose excreted per hour was 0.55%. The mean UCBC of baboon bile was 4.22 (range 0.68-17.2) ng/ml.
of Plasma-Bound
ranged from 0.02 to 0.04 pg, depending on its specific activity and the plasma UCBC. A portion of the dose was
Excretion
Time of collection” (min)
Cumulative fraction of dose excreted (W)
Rate of [57Co]cyanocobalamin excretion (W per hour)
4.37 2.42
0.92 0.42
3
265 346 232
1.45
0.38
4
296
1.36
0.28
5
180
0.84
0.28
6
262
2.72
0.82
7
264
2.64
0.60
8
132
1.95
0.89
Mean
2.22
0.55
a From time of first appearance
of radioactivity
1
2
ment.
to end of experi-
October
1983
Discussion The relatively high concentration of cobalamin found in baboon bile suggests that in this species, like humans (1,2), the dog (5) and the rat (6) the biliary tract constitutes an important route of cobalamin excretion. Our estimate of the daily excretion of cobalamin in bile is based on several assumptions. The total diversion of bile in our experiments could have interfered with the action of normal choleretics and reduced the volume of bile flow. However, this should have been minimal over the duration of bile collection. It is assumed that any slight change in bile volume would not influence the total amount of cobalamin excreted so that any reduction in bile volume would be associated with an increase in cobalamin concentration of bile. We calculated that the amount of cobalamin that traverses the biliary tract of baboons each day is approximately 4 pg, which is similar to figures of 3-6 pg preThe daily requireviously described in humans (1,2). ment for cobalamin in the baboon is not known but, based on the human requirement of 0.5-1.0 pg (4), it may be estimated that the figure for baboons is approximately 50% of this on a weight-for-weight basis. The daily excretion of cobalamin in baboon bile is therefore considerably greater than the daily requirement. This supports the concept that reabsorption of biliary cobalamin is important for maintenance of normal cobalamin balance, and that in malabsorption, interference with enterohepatic circulation of cobalamin accelerates the rate at which depletion of the vitamin occurs. The appearance of radioactivity in bile within 1 h after intravenous injection of plasma-bound [“‘Colcyanocobalamin indicates that there is a rapid passage of the vitamin from the plasma through liver cells. A saturating amount of radiolabeled cobalamin was used so that all three plasma transcobalamins (TCI, TCII, and TCIII) carried [“‘Colcyanocobalamin. Burger et al. (11) showed that in rabbits, cobalamin attached to the glycoprotein TCIII is cleared from the circulation in minutes.by hepatocytes through the nonspecific mechanism for uptake of asialoglycoproteins (12)and a fraction of the intact TCIII-[“‘Collabeled cobalamin complex then appears in the bile within the next hour. Most of the remaining TCoJcobalamin is released from TCIII in the hepatocyte, and reenters the plasma bound to TCII, the plasma protein responsible for specific receptor-mediated transport of cobalamins (13,14). These workers also suggested that TCIII, which appears to be largely derived from granulocytes (15,16), may play a scavenging role by returning cobalamins released from broken down cells specifically to the liver (11). Most of the endogenous
ENTEROHEPATIC
CIRCULATION
OF COBALAMIN
775
plasma cobalamin is bound to TCI, a glycloprotein very similar to TCIII in size and immunologic properties, but which has a different carbohydrate composition (15). TCI-bound cobalamin is also cleared from the plasma to hepatocytes, but much more slowly (13) at a rate that is probably dependent on intravascular desialation of the glycoprotein. Cobalamin becomes bound to TCII following its absorption in the ileum (13,17). Excretion of cobalamin in bile may therefore constitute a salvage mechanism whereby the fraction of glycoprotein-cobalamin complex that escapes degradation in the hepatocyte can enter the intestinal lumen to be reabsorbed. This reabsorbed biliary cobalamin then becomes bound to TCII in the portal circulation and available for specific receptor-mediated uptake.
References 1. Grasbeck R, Nyberg W, Reizenstein
2.
3. 4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
P. Biliary and fecal vit B1* excretion in man. An isotope study. Proc Sot Exp Biol Med 1958;97:780-4. Ardeman S, Chanarin I, Berry V. Studies on human gastric intrinsic factor. Observations on its possible absorption and enterohepatic circulation. Br J Haematol 1965;11:11-4. Adams JF, Boddy K. Metabolic equilibrium of tracer and natural vitamin B,,. J Lab Clin Med 1968;72:392-6. World Health Organization Technical Report Series No. 452. Requirements of ascorbic acid, vitamin D, vitamin Blz, folate, and iron. Report of a joint FAO/WHO expert group, 1970. Willigan DA, Cronkite EP, Meyer LM, et al. Biliary excretion of Cow labeled vitamin B,, in dogs. Proc Sot Exp Biol Med 1958:SS:81-4. Grasbeck R, Runeberg L, Simons K. Intrinsic factor and radiovitamin B1z excretion in rats. Acta Physiol Stand 1959;47:3704. Booth MA, Spray GH. Vitamin B,, activity in the serum and liver of rats after total gastrectomy. Br J Haematol 1960;6:28895. Green R, Newmark PA, Musso AM, et al. The use of chicken serum for measurement of serum vitamin BX2concentration by radioisotope dilution: description of method and comparison with microbiological assay results. Br J Haematol 1974;27:507-26. Gottlieb C, Lau K-S, Wasserman LR, et al. Rapid charcoal assay for intrinsic factor (IF), gastric juice unsaturated B,, binding-capacity, antibody to IF, and serum unsaturated B,, binding capacity. Blood 1965;25:875-84. Lau K-S, Gottlieb C, Wasserman LR, et al. Measurement of . serum vitamm B,, level using radioisotope dilution and coated charcoal. Blood 1965;26:202-14. Burger RL, Schneider RJ. Mehlman CS, et al. Human plasma R-type vitamin B,,-binding proteins. II. The role of transcobalamin I, transcobalamin III, and the normal granulocyte vitamin B,,-binding protein in the plasma transport of vitamin BIZ. J Biol Chem 1975;250:7707-13. Ashwell G, Morel1 AG. The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv Enzymol 1974;41:99-128. Hall CA, Finkler AE. The dynamics of transcobalamin II. A vitamin BX2 binding substance in plasma. J Lab Clin Med 1965;65:459-68.
776
GREEN ET AL.
14. Retief FP, Gottlieb CW, Herbert V. Mechanism of vitamin B,, uptake by erythrocytes. J Clin Invest 1966;45:1997-15. 15. Burger RL, Mehlman CS, Allen RH. Human plasma R-type vitamin B1z binding proteins. I. Isolation and characterization of transcobalamin I, transcobalamin III, and the normal granulocyte vitamin B1z binding protein. J Biol Chem 1975; 250:779Ci-17. 16. Scott JM, Bloomfield FJ, Stebbins R, et al. Studies on the deri-
GASTROENTEROLOGY
Vol. 81, No. 4
vation of transcobalamin III from granulocytes. Enhancement by lithium and elimination by fluoride of in vitro increments in vitamin B,, binding capacity. J Clin Invest 1974;53:228-39. 17. Cooper BA, White JJ. Absence of intrinsic factor from human portal plasma during CossB,z absorption in man. Br J Haemato1 1968;14:73-8.
1981;81:773-P
Enterohepatic Circulation of Cobalamin in the Nonhuman Primate RALPH GREEN, DONALD VAN TONDER, MICHAEL
W. JACOBSEN, SUSAN V. C. KEW, and JACK METZ
Departments of Clinical Research and Biochemistry, Scripps Clinic and Research Foundation, La Jolla, California; and Departments of Hematology and Medicine, School of Pathology, University of the Witwatersrand and The South African Institute for Medical Research, Johannesburg, South Africa
Biliary excretion of cobalamin was studied in baboons after intravenous injection of 57Co-radiolabeled cyanocobalamin. Radioactivity appeared in bile after 20 min, and up to 4.4% of the dose was recovered in 6 h. It was estimated that 4 pg cobalamin traverses the baboon biliary tract each day. These studies in the nonhuman primate confirm that there
is considerable biliary excretion of cobalamin and that enterohepatic circulation of the vitamin is important for maintenance of normal cobalamin balance. The biliary tract appears to be a major route of vitamin B,, (co b a 1amin) recirculation. In humans the amount of cobalamin that enters the bile each day (1,~) considerably exceeds daily body loss (3) or minimal requirements (4) of the vitamin. Biliary cobalamin must therefore be efficiently reabsorbed for conservation of the vitamin. An enterohepatic circulation for the vitamin has indeed been suggested from studies in humans (1,2) and other animals (5-7). The significance of an enterohepatic circulation for cobalamin is not known, but it clearly has bearing on maintenance of normal cobalamin balance and the pathogenesis of cobalamin deficiency. This is illustrated by the observation that cobalamin deficiency develops more rapidly in patients after total gastrectomy than in strict vegans who exclude in-
take of the vitamin through dietary practices. The explanation is that a failure to absorb cobalamin not only precludes its assimilation from the diet, but also impairs reabsorption of the vitamin from the bile. It is based on the assumption that, like the absorption of dietary cobalamin, reabsorption of biliary cobalamin also requires intrinsic factor. We have studied baboons to gain further information on the normal biliary excretion and enterohepatic circulation of cobalamin in primates.
Materials and Methods Animals Adult male chacma baboons, Papio urisinus, cages and weighing 25-40 kg were housed in individual ration biscuits (Epic Oil Mills, were fed Epol-balanced Newtown, Johannesburg, South Africa) supplemented with eggs, fresh fruit, and vegetables.
Radioactive
Materials
and Detectors
[TolCyanocobalamin (IO&300 &i/pg) was obtained from the Radiochemical Centre (Amersham, Buckinghamshire, England). Radioactivity determinations were carried out on small volume samples (< 5.0 ml) in standard gamma spectrometers (Packard Autogamma, models 3002 and 3330).
Received November 24, 1980. Accepted May 12, 1981. Address requests for reprints to: Ralph Green, M.D., Department of Clinical Research, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, California 92037. This work was supported by the National Institutes of Health Grant NS 13714 (R. Green) and by grants from the South African Atomic Energy Board and the Medical Research Council of South Africa. The authors are indebted to Sister 0. Hoffman Devisme for surgical assistance and to Mr. B. Fratscher for animal care. 0 1981 by the American Gastroenterological Association OOlS-5085/61/100773-O4$02.50
Surgical Preparation For surgical procedures baboons were anesthetized after an overnight fast with intramuscular phencyclidine hydrochloride (Sernylan, Park Davis and Co., London, England),
2.0 mg/kg body wt. Intramuscular atropine, body wt, was given. Anesthesia was maintained using intravenous pentobarbitone sodium (Sagatal, May and Baker Ltd., Dagenham, England), 5-10 mg/kg
0.015 mg/kg
774
GASTROENTEROLOGY
GREEN ET AL.
:
0.075
k p
0.050
Biliary
P 0.02 1
Figure 1. Pattern of radioactivity excreted in the bile of a baboon following an intravenous dose of 0.04 pg [57Co]cyanocobalamin bound to plasma. body wt, and fluid replacement was given as isotonic saline containing 5% (wt/vol) dextrose by continuous intravenous infusion at the rate of 20-40 ml/h.
Common
Bile Duct Cannulation
The abdomen was opened, and the hepatic and common bile ducts were identified and gently stripped of their serosal coats. A blunt-tipped polythene catheter was inserted through a longitudinal incision made in the hepatic duct immediately proximal to the point of entry of the cystic duct and passed upwards a short distance towards the liver. The catheter was connected to a fraction collector, and bile was collected in l&ml aliquots for periods of 4-6 h.
Blood Sampling Blood specimens were obtained from arm or leg veins via plastic disposable syringes and needles.
Laboratory
Methods
Biliary cobalamin was measured using a radioisotope dilution method (8). To exclude any effect of bile on the cobalamin assay, varying amounts of cyanocobalamin were added to specimens of bile. Recoveries (9!!103%) fell within the range of error encountered with this method. Determination of the unsaturated cobalamin binding capacity (UCBC) of serum and bile were carried out according to the methods of Gottlieb et al. (9).
Preparation and Injection [5’ColCobalamin
81, No.
4
Results
0.100
B
Vol.
Table 1. Biliary Excretion of Radioactivity in 8 Baboons Following Intravenous Injection of rCo]Cyanocobalamin
Experiment
The UCBC of baboon plasma was measured, and 25 ml was saturated with [S7Co]cyanocobalamin. After incubation at 37% for 60 min, hemoglobin-coated charcoal was added to remove unbound radioactivity (lo), and the 57Co-labeled plasma was sterilized by Seitz filtration before injection. The amount of [?o]cyanocobalamin used
counted for calculation of the injected radioactivity. Bile draining through the common bile duct cannula was collected in I.&ml fractions for radioactivity measurement.
of Cobalamin
The mean cobalamin concentration of bile obtained from 8 normal baboons before the injection of [S’Colcyanocobalamin was 15.7 (range 3.82-40.2) ng/ ml. Bile flow-rates varied from 0.1 to 0.9 ml/min between animals and at different times in the same animal. From measured volumes collected over 4-6 h and assuming that this was representative of the physiologic rate of bile flow, the estimated daily rate of bile secretion was calculated to be 256 (range 116879) ml in the eight baboons studied. An average minimum of 4.02 pg of cobalamin (256 ml X 15.7 ng/ ml) was estimated to pass through the baboon biliary tract each day. The time taken for radioactivity to appear in bile following intravenous injection of plasma-bound [57Co]cyanocobalamin ranged from 20 to 74 (mean 37) min, and peak radioactivity was reached between 166 and 342 (mean 240) min. The excretion of [?Zo]radioactivity was variable but continuous over the observation period. A representative pattern for one animal is shown in Figure 1. The total radioactivity excreted in the bile during the period of collection was expressed as a percentage of the dose injected (Table 1). The mean cumulative excretion of [“‘Colcobalamin was 2.22% of the injected dose over the time from the first appearance of radioactivity to the end of the collection period (132-346 min). The mean calculated percentage of the dose excreted per hour was 0.55%. The mean UCBC of baboon bile was 4.22 (range 0.68-17.2) ng/ml.
of Plasma-Bound
ranged from 0.02 to 0.04 pg, depending on its specific activity and the plasma UCBC. A portion of the dose was
Excretion
Time of collection” (min)
Cumulative fraction of dose excreted (W)
Rate of [57Co]cyanocobalamin excretion (W per hour)
4.37 2.42
0.92 0.42
3
265 346 232
1.45
0.38
4
296
1.36
0.28
5
180
0.84
0.28
6
262
2.72
0.82
7
264
2.64
0.60
8
132
1.95
0.89
Mean
2.22
0.55
a From time of first appearance
of radioactivity
1
2
ment.
to end of experi-
October
1983
Discussion The relatively high concentration of cobalamin found in baboon bile suggests that in this species, like humans (1,2), the dog (5) and the rat (6) the biliary tract constitutes an important route of cobalamin excretion. Our estimate of the daily excretion of cobalamin in bile is based on several assumptions. The total diversion of bile in our experiments could have interfered with the action of normal choleretics and reduced the volume of bile flow. However, this should have been minimal over the duration of bile collection. It is assumed that any slight change in bile volume would not influence the total amount of cobalamin excreted so that any reduction in bile volume would be associated with an increase in cobalamin concentration of bile. We calculated that the amount of cobalamin that traverses the biliary tract of baboons each day is approximately 4 pg, which is similar to figures of 3-6 pg preThe daily requireviously described in humans (1,2). ment for cobalamin in the baboon is not known but, based on the human requirement of 0.5-1.0 pg (4), it may be estimated that the figure for baboons is approximately 50% of this on a weight-for-weight basis. The daily excretion of cobalamin in baboon bile is therefore considerably greater than the daily requirement. This supports the concept that reabsorption of biliary cobalamin is important for maintenance of normal cobalamin balance, and that in malabsorption, interference with enterohepatic circulation of cobalamin accelerates the rate at which depletion of the vitamin occurs. The appearance of radioactivity in bile within 1 h after intravenous injection of plasma-bound [“‘Colcyanocobalamin indicates that there is a rapid passage of the vitamin from the plasma through liver cells. A saturating amount of radiolabeled cobalamin was used so that all three plasma transcobalamins (TCI, TCII, and TCIII) carried [“‘Colcyanocobalamin. Burger et al. (11) showed that in rabbits, cobalamin attached to the glycoprotein TCIII is cleared from the circulation in minutes.by hepatocytes through the nonspecific mechanism for uptake of asialoglycoproteins (12)and a fraction of the intact TCIII-[“‘Collabeled cobalamin complex then appears in the bile within the next hour. Most of the remaining TCoJcobalamin is released from TCIII in the hepatocyte, and reenters the plasma bound to TCII, the plasma protein responsible for specific receptor-mediated transport of cobalamins (13,14). These workers also suggested that TCIII, which appears to be largely derived from granulocytes (15,16), may play a scavenging role by returning cobalamins released from broken down cells specifically to the liver (11). Most of the endogenous
ENTEROHEPATIC
CIRCULATION
OF COBALAMIN
775
plasma cobalamin is bound to TCI, a glycloprotein very similar to TCIII in size and immunologic properties, but which has a different carbohydrate composition (15). TCI-bound cobalamin is also cleared from the plasma to hepatocytes, but much more slowly (13) at a rate that is probably dependent on intravascular desialation of the glycoprotein. Cobalamin becomes bound to TCII following its absorption in the ileum (13,17). Excretion of cobalamin in bile may therefore constitute a salvage mechanism whereby the fraction of glycoprotein-cobalamin complex that escapes degradation in the hepatocyte can enter the intestinal lumen to be reabsorbed. This reabsorbed biliary cobalamin then becomes bound to TCII in the portal circulation and available for specific receptor-mediated uptake.
References 1. Grasbeck R, Nyberg W, Reizenstein
2.
3. 4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
P. Biliary and fecal vit B1* excretion in man. An isotope study. Proc Sot Exp Biol Med 1958;97:780-4. Ardeman S, Chanarin I, Berry V. Studies on human gastric intrinsic factor. Observations on its possible absorption and enterohepatic circulation. Br J Haematol 1965;11:11-4. Adams JF, Boddy K. Metabolic equilibrium of tracer and natural vitamin B,,. J Lab Clin Med 1968;72:392-6. World Health Organization Technical Report Series No. 452. Requirements of ascorbic acid, vitamin D, vitamin Blz, folate, and iron. Report of a joint FAO/WHO expert group, 1970. Willigan DA, Cronkite EP, Meyer LM, et al. Biliary excretion of Cow labeled vitamin B,, in dogs. Proc Sot Exp Biol Med 1958:SS:81-4. Grasbeck R, Runeberg L, Simons K. Intrinsic factor and radiovitamin B1z excretion in rats. Acta Physiol Stand 1959;47:3704. Booth MA, Spray GH. Vitamin B,, activity in the serum and liver of rats after total gastrectomy. Br J Haematol 1960;6:28895. Green R, Newmark PA, Musso AM, et al. The use of chicken serum for measurement of serum vitamin BX2concentration by radioisotope dilution: description of method and comparison with microbiological assay results. Br J Haematol 1974;27:507-26. Gottlieb C, Lau K-S, Wasserman LR, et al. Rapid charcoal assay for intrinsic factor (IF), gastric juice unsaturated B,, binding-capacity, antibody to IF, and serum unsaturated B,, binding capacity. Blood 1965;25:875-84. Lau K-S, Gottlieb C, Wasserman LR, et al. Measurement of . serum vitamm B,, level using radioisotope dilution and coated charcoal. Blood 1965;26:202-14. Burger RL, Schneider RJ. Mehlman CS, et al. Human plasma R-type vitamin B,,-binding proteins. II. The role of transcobalamin I, transcobalamin III, and the normal granulocyte vitamin B,,-binding protein in the plasma transport of vitamin BIZ. J Biol Chem 1975;250:7707-13. Ashwell G, Morel1 AG. The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv Enzymol 1974;41:99-128. Hall CA, Finkler AE. The dynamics of transcobalamin II. A vitamin BX2 binding substance in plasma. J Lab Clin Med 1965;65:459-68.
776
GREEN ET AL.
14. Retief FP, Gottlieb CW, Herbert V. Mechanism of vitamin B,, uptake by erythrocytes. J Clin Invest 1966;45:1997-15. 15. Burger RL, Mehlman CS, Allen RH. Human plasma R-type vitamin B1z binding proteins. I. Isolation and characterization of transcobalamin I, transcobalamin III, and the normal granulocyte vitamin B1z binding protein. J Biol Chem 1975; 250:779Ci-17. 16. Scott JM, Bloomfield FJ, Stebbins R, et al. Studies on the deri-
GASTROENTEROLOGY
Vol. 81, No. 4
vation of transcobalamin III from granulocytes. Enhancement by lithium and elimination by fluoride of in vitro increments in vitamin B,, binding capacity. J Clin Invest 1974;53:228-39. 17. Cooper BA, White JJ. Absence of intrinsic factor from human portal plasma during CossB,z absorption in man. Br J Haemato1 1968;14:73-8.