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Vitamin B12 Uptake by Human Small Bowel Homogenate and its Enhancement by Intrinsic Factor
Vol. 56, No.3 Printed in U.S.A.
GASTROENTEROLOGY
Copyright © 1969 by The Williams & Wilkins Co.
VITAMIN BI2 UPTAKE BY HUMAN SMALL BOWEL HOMOGENATE AND ITS ENHANCEMENT BY INTRINSIC FACTOR RALPH CARMEL, M.D., ARTHUR H. ROSENBERG, M.D., KAM-SENG LAU, M.D., RICHARD R. STREIFF, M.D., AND VICTOR HERBERT, M.D.
Department of Medicine, Mount Sinai School of Medicine of the City University of New York, and the Thorndike Memorial Laboratory, Boston City Hospital, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts
Mucosal homogenates of human ileum were demonstrated to have enhanced vitamin BI2 uptake in the presence of normal human gastric juice or hog intrinsic factor concentrate. This process was demonstrated to require Ca++ or Mg++ and to be optimal at pH 6.6 and above. The enhancement was blocked by antibody to intrinsic factor. Pernicious anemia gastric juice and normal and pernicious anemia serum did not enhance BI2 uptake. In fact, these substances diminished nonspecific uptake of the vitamin, presumably by binding it and rendering it unadsorbable. "Enhancement" by saliva appeared to be an artifact due to the presence of BI2 binders precipitable by centrifugation. Human jejunal mucosa homogenate uptake was not enhanced by intrinsic factor, supporting the concept that the active process of vitamin BI2 uptake is localized to the ileum. Nonspecific uptake of vitamin B 12 , unrelated to intrinsic factor, appeared to be uniform in jejunal and ileal mucosa homogenates, and to be unaffected by pH or cations. Poor vitamin BI2 uptake in some but not other ileal homogenates from patients with regional ileitis suggests that the ileal homogenate technique may be useful in evaluating in vivo function of intestinal receptors for the intrinsic factor-vitamin BI2 complex. This awaits further studies, correlating this technique with in vivo absorption studies. Vitamin BI2 absorption takes place by two mechanisms. I The first is passive and nonspecific, taking place along the entire length of the intestine. 2 The second is the Received June 17, 1968. Accepted September 9,1968. Address requests for reprints to: Dr. Victor Herbert, Nutritional Anemias Division, Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029. This work was supported by United States Public Health Service Grants AM 13358 and AM 11048, and Clinical Research Center Grant FR-71 , United States Public Health Service. Dr. Herbert is recipient of Career Scientist Award 1-435 from the Health Research Council of the City of New York. Dr. Carmel was a United States Public Health Service Postdoctoral Research Fellow (5-F2-AM-32, 518-02), National Institutes of Arthritis and Metabolic Diseases, and is currently at Wilford Hall 548
active mechanism which is the specific one mediated by intrinsic factor. The marked observable difference between these two mechanisms has formed the basis of in vitro techniques for study of United States Air Force Hospital, Lackland Air Force Base, Texas. Dr. Rosenberg was a United States Public Health Service Postdoctoral Fellow (1-F2-AM-24, 310-01). Dr. Lau was a World Health Organization Research Fellow in Hematology, and is currently Head, Department of Pathology, University of Malaya, Kuala Lumpur, Malaysia. Dr. Streiff is currently Assistant Professor of Medicine, University of Florida, Gainesville, Florida. The authors are indebted to Melody Lee, Le Teng Go, and Leona Bandel for their invaluable technical assistance, and to Dr. Henry Janowitz and the Division of Gastroenterology for their help in securing samples for study.
VITAMIN
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the relation of intrinsic factor to vitamin Bl2 adsorption to the mucosal cell, the first step in the absorptive process. These techniques have been utilized on various animalliver 3 - o and intestinal,-14 preparations. Studies with human liver have also been reported. 5o ' l 5 , 10 The purposes of the present study are to establish with human intestinal homogenates various conditions required for vitamin Bl2 uptake, to help explain various clinical observations on the process, and to establish a basis for further study of the intestinal phase of the process by this technique.
Materials and Methods Human ileum was obtained at surgery from patients undergoing resection for carcinoma of the ascending colon, ulcerative colitis, or regional enteritis. Specimens from patients with carcinoma of the colon, or, in a few instances, ulcerative colitis, were referred to as normal ileum. Jejunum was obtained from patients undergoing gastroenterostomy for ulcer disease. Immediately after resection, the intestinal segment, usually measuring 1 to 6 inches, was placed in cold (4 C) 0.85% NaCI (saline). The mucosal surface was washed with cold saline until the effluent was clear. Mucosal scrapings were obtained by exposing and scraping the mucosal surface, or by running a spatula along the serosal surface and, with firm pressure, expressing mucosal tissue as toothpaste is expressed from a tube. The scrapings thus obtained were placed in 50 ml of cold saline and homogenized in a Waring Blendor for 15 sec. The homogenate (0.5 to 5.0 g wet weight) was divided into aliquots and centrifuged for 10 min at 2000 rpm at 4 C, The pellets, washed twice with 5 ml of cold saline, were stored at - 20 C until used . The ability of the homogenate to function in our assay system 'was maintained on such storage for as much as 1 year. Once thawed, specimens were used or discarded. Human gastric juices were obtained from normal subjects, as well as from subjects with pernicious anemia or other conditions, and were depepsinized and neutralized. IJ Intrinsic factor . content was assayed by the method of Gottlieb et al. I With the one exception noted below, only specimens containing intrinsic factor content of at least 80 '( of the total vitamin BI2binding capacity were used . 0
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Saliva was obtained from various subjects by expectoration, occasionally with the aid of Parafilm chewing. Secretion of H substance was determined with Ulex europaeus. l " Samples were stored at - 20 C. Antibody to human intrinsic factor was obtained from patients with pernicious anemia. Presence of the antibody was determined as reported elsewhere, I; and the samples were stored at - 20 C. Hog intrinsic factor concentrate was purchased from the National Formulary of the American Pharmaceutical Association, Washington, D.C. Co 5 , -labeled vitamin BI2 (Co5 ' B I2) of specific activity 10 IlC per Ilg was usually used ; in some experiments CO " BI 2 of specific activity 5.9 IlC per Ilg was used, as well as Co""Bl2 (kindly supplied by Dr. Elmer Alpert of Merck, Sharpe and Dohme Research Laboratories, West Point, Pa.) of specific activity 1.283 IlC per Ilg. All were diluted with saline to a concentration of 10 ng per ml. Rabbit anti-hog intrinsic factor concentrate antiserum was obtained from rabbits immunized with hog intrinsic factor concentrate as previously described . I" The presence of antibody was assayed as with human serum. For 30 min 0.2 to 0.5 ml of Coa' Bl2 was pre-incubated at room temperature with 0 .05 to 0.1 ml of gastric juice, made up to a total volume of 1.0 ml with Krebs Ringer-Tris (KRT) buffer, pH 7.4. Volumes used were such that the Bl2 binding capacity of the specimen equaled, or slightly exceeded, the amount of CO " BI 2 present. Controls with saline substituted for the gastric juice were used with each specimen assayed. On occasion, saliva or serum was used in place of the gastric juice, Gut pellets were thawed, weighed, suspended in cold KRT buffer, and homogenized using a motor-driven Teflon pestle with a fitted glass tube immersed in ice, The homogenate in buffer was constantly shaken to ensure uniformity of suspension, and equal 3-ml aliquots were added to each gastric juice-Co5 ' B l2 sample and to control samples, (Each tube in all of our experiments thus contained 25 to 100 mg of gut homogenate; best results were obtained when at least 60 mg was used. Samples of 15 mg proved too small to produce significant results.) As additional controls, gut homogenate was left out of the system in samples otherwise prepared identically to the test samples, and run concurrently in each assay, The samples were agitated at 90 cycles per min for 90 min at 37 C in a Dubnoff
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Metabolic Shaker. Subsequently, 30-min agitation at room temperature was substituted when it was shown to produce even better results. The reaction was terminated by centrifugating at 2000 rpm for 10 min at 4 C. The precipitate was washed with 5 ml, and then with 3 ml, cold 0.85% NaCI-1O, mM CaCle. Radioactivity of the pellet was determined in a well-type scintillation counter, for a minimum of 10,000 counts. The counts were converted to picograms of Co;'B I ,. Each specimen was run in duplicates and differences between these were generally less than 10%. Ratios of CO·"B I2 uptake by the test specimens (S) over uptake by the saline control (C) were determined (S IC) . In experiments of cation dependence, CaCI, and MgSo4 were omitted in preparing the KRT buffer, and saline, rather than salineCaCh, washes were used. The effect of ethylenediaminetetraacetate (EDTA) was studied using 40 mg sodium versenate (Riker) per sample, diluting it 1: 20 in KRT buffer from which CaCI, and MgS0 4 had been omitted, and adjusting the final pH of the specimen to 7.4 with 0.5 N NaOH. In studying pH dependence , the buffers used consisted of 9 parts Krebs Ringer solution' " brought to appropriate pH with 1 part 0.05 M Tris-acid maleateNaOH buffer."
Results Uptake by Normal Ileal Mucosa Homogenate In the absence of intrinsic factor there was some vitamin BI2 uptake by ileal homogenate. This represented nonspecific uptake and ranged from 10 to 20 pg, oc-
casionally being as much as 35 pg, BI2 per 100 mg homogenate. No more than 4 pg of Co;'B I2 was spun down when buffer alone instead of homogenate suspension was added. As demonstrated by the SIC ratios in figure 1, normal gastric juice markedly enhanced vitamin BI2 uptake. The magnitude of the SIC ratio seemed to correlate primarily with the amount of gut homogenate used, the smaller ratios being seen with the smaller pellets. A given pellet of a single gut homogenate produced comparable results with different intrinsic factor-containing gastric juices. Significant enhancement was seen with gastric juices of normal persons and of patients with duodenal ulcer, tropical sprue, or iron deficiency. While these specimens all had high intrinsic factor content, even a specimen from a patient with tropical sprue where only 40% of the binding capacity was due to intrinsic factor markedly enhanced vitamin BI2 uptake. Hog intrinsic factor concentrate had an identical effect, although perhaps of not so great a magnitude as the human gastric juices. In marked contrast, pernicious anemia gastric juice not only did not increase uptake, but actually inhibited it. This was true with specimens from persons who secreted H substance as well as those who did not. Sera from normal and pernicious anemia subjects also resulted in reduced BI2 uptake by the ileal homogenate (fig. 1).
12
10 x ONLY 40% OF THE 8,2 BINDING CAPACITY OF THIS GASTRIC JUICE WAS DUE TO
S
C (
INTRINSIC FACTOR.
6
TEST SPECIMEN) SALINE CONTROL 4
2 I
------- -- ----------- - - ----- -------- ---- - - ---------- NORMAL GASTRIC
HOG INTRINSIC
PERNICIOUS ANEMIA
JUICE
FACTOR CONCENTRATE
GASTRIC JUICE
SERUM
FIG. 1. Vitamin BI2 uptake by normal ileal mucosa homogenate. (Points represent different mucosa specimens.)
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Heating normal gastric juice at 100 C for 30 min (which destroys intrinsic factor activity) prior to testing it eliminated uptake enhancement, reducing an SIC ratio of 6.3 to 0.9. Effect of Antibody to Intrinsic Factor
As shown in figure 2, intrinsic factor effect was eliminated when the normal ~astric juice ":".as incubated, prior to addition of COo' B12 or ileal homogenate, with antibody-containing serum from a patient with pernicious anemia, or if hog intrinsic factor concentrate was similarly pre incubated with rabbit anti-hog intrinsic factor concentrate antiserum . A similar effect was noted when rabbit anti-hog intrinsic factor concentrate antiserum was 9
8 7 6
4 5
C
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VITAMIN B" UPTAKE
3 2
used with human gastric juice. In contrast, sera without antibody had no effect. That the antibody acts on the gastric juice binding of B12, rather than on the gut itself, was shown by lack of effect of antibody incubated for 30 min at room temperature with the ileal homogenate (which was then washed with KRT) prior to addition of gastric juice and CO o' B12. Uptake by Jejunal Mucosa Homogenate
In contrast to ileal uptake, jejunal uptake of vitamin B12 was not enhanced by normal gastric juice (fig. 3). Similarly, pernicious anemia gastric juice had no effect. Nonspecific uptake, as reflected by vitamin B12 uptake in the saline controls, did not differ significantly from that seen with ileal homogenates. Uptake by Abnormal Ileal Mucosa Homogenates
With ileum from various patients with regional ileitis, intrinsic factor enhancement of vitamin B1 2 uptake was demonstrated in some cases (fig. 3). No enhancement was seen generally with those ileal specimens which had appeared grossly abnormal. In one case, however, two specimens from the same patient, one appearing normal to the naked eye and the other abnormal, were tested and both showed no intrinsic factor enhancement. Effect of pH
FIG.' 2. Effect of antibody to intrinsic factor on vitamin B12 uptake by normal ileal mucosa homogenate. (Points represent different mucosa specimens.) Homogenate pre incubated with human anti-intrinsic factor antiserum for 30 min at room temperature prior to incubation with normal human gastric juice and Co57 B" . SIC, test specimen/saline control ratio; NGJ, normal human gastric juice; AB, human anti-intrinsic factor antiserum; RIFC, hog intrinsic factor concentrate; and anti-RIFe, rabbit anti-hog intrinsic factor concentrate antiserum.
The pH of our experimental system was 7.2. To determine pH effect on uptake by normal ileal homogenate, a pH range of 4.8 to 9.0 was studied. As shown in table 1, intrinsic factor effect was minimal or absent at pH 5.5 and absent below that level. Maximal effect was noted at pH 6.6 and above. Nonspecific uptake was not affected by pH change. Effect of Ca++ and Mg++
The intrinsic factor effect of gastric juice was shown to require divalent cation . Omission of both Ca++ and Mg++ abolished intrinsic factor enhancement (table
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Vol. 56, No . 3
7
6 5 4 5
C
• NORMAL GASTRIC JUICE x GUT SPECIMENS FROM SAME SUBJECT; ONE ( SIC 1.2) APPEARING NORMAL. ANO THE OTHER (SIC 0.4) ABNORMAL TO THE NAKED EYE -PERNICIOUS ANEMIA GASTRIC JUICE
3
• •
•
ILEITIS ILEUM
NORMAL JEJUNUM
•
•
FIG. 3. Vitamin B12 uptake by ileal mucosa homogenate from patients with regional ileitis, and by normal jejunal mucosa homogenate. SIC, test specimen/saline control ratio.
2). This was not seen when Mg++ alone was omitted from the system. When Ca++ alone was left out, intrinsic factor effect was abolished with three of four normal gastric juices tested. The one exception demonstrated reduced, albeit still significant, intrinsic factor effect. This gastric juice was then further tested with sodium versenate (EDT A), which abolished its effect entirely (table 3). Addition of equimolar amounts of either CaCl 2 or MgS0 4 to the EDT A re-established intrinsic factor effect. In no instances was there artifactual precipitation of Co 6 'B12 • In previous experiments, EDT A was shown not to interfere with binding of Co 6 ' B12 by gastric juice. Nonspecific uptake of B12 was not affected by either omission of cations or by addition of EDT A.
Effect of Saliva At pH 7.2, only three of eight saliva specimens increased B12 "uptake" by ileum homogenate, with an average SIC of 5.2. Two of these were from normal blood group A secretors of H substance, and one was from a blood group 0 nonsecretor with pernicious anemia. The 5 other subjects studied, regardless of secretor status or presence of pernicious anemia, gave negative results (average SIC of 1.4). The three "positive" saliva specimens, however, produced identically large B1 2
1. Effect of pH on intrinsic factor-mediated vitamin BJ2 uptake by normal ileal mucosa homogenate
TABLE
Experiment 2
Experiment 1
pH
SIC""
4.9 5.5 7.0 7.8 8.5
0.1 0.1 3.5 4.7 4.3
pH
Sica
4.8 5.5 6.6 7.5 9.0
0.5 2.1 6.3 3.7 5.2
a Expressed as test specimen to saline control ratio.
2. Effect of Ca++ and M g++ lack on intrinsic factor-mediated vitamin B12 uptake by normal ileal mucosa homogenate a
TABLE
Base line
3.8 4.9 6.0 9.6 8.3 8.1
Absence of Ca++ and Mg++
1.3 1.4
Absence 01 Ca++
Absence of Mg++
4.0 1.5 1.3 1.8
6.4 10.2
"Expressed as test specimen to saline control ratio.
precipitates when tested with jejunal and colonic mucosa, and, indeed, when tested with buffer alone without homogenate. Antibody to intrinsic factor had no effect
March 1969
VITAMIN
3. Effect of sodium versenate (EDTA) on intrinsic factor-mediated vitamin B12 uptake by normal ileal mucosa homogenate
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strable on ileal but not jejunal mucosa homogenates. A previous studl l of vitamin BI 2 uptake along the entire length of small intestine, obtained within 2 hr after death from a patient who died of acute Gastric juice. . . . . . . . . . . . . . . . . . . 2.3 . . . . . . leukemia, showed consistent enhancement by normal gastric juice of vitamin B12 upGastric juice + EDT A. ........ 0.3 take by mucosal homogenates of all segGastric juice + EDTA + Ca++ . .... .. 6.6 Gastric juice + EDTA + Mg++. . . . . . . 5.6 ments from the distal half. Segments from Gastric juice + EDTA + Ca++ + the proximal half of the small intestine Mg++... ..... . . . . . . . . . . . . 4.3 did not demonstrate this effect. We have not been able to repeat this study successa Expressed as test specimen to saline control fully, probably due to the rapid postmorratio. tem autolysis of intestinal mucosa which makes it mandatory that the specimen be on results with ileal or jejunal homogeobtained within 2 hr of death. Similar nate. When the saliva was centrifuged at 4000 autolysis probably explains the better rpm for 15 min and only the supernate results obtained in the present study when used, the positive effect with ileal mucosa incubation was carried out for 30 min at or with buffer alone disappeared (SIC of room temperature instead of 90 min at 0.3). When the pH of the system was 37 C. Intrinsic factor action was shown to be changed to 9.0, it was found that even salivas negative at pH 7.2 produced large dependent on divalent cations, as had been demonstrated in vivo in man, 5. 32 , 33 and in precipitates of vitamin B 12 . vitro in animals ."~' 14, 33 Omitting both Discussion Ca++ and Mg++ from our system abolished In vivo studies in man and both in vitro intrinsic factor effect of all gastric juice. and in vivo animal studies suggest that sion alone produced no change, Ca++ omisvitamin BI2 absorption occurs by two mech- sion did. EDTA completely abolished the anisms, I passive transport and an active intrinsic factor effect of all gastric juice, process which is dependent on intrinsic Adding either Ca++ or Mg++ alone to the factorl , 2 2, 2 3 and is limited to the il- EDT A re-established activity, suggesting eum. 23 - 3 0 The results ofthe present in vitro that either cation was effective. It is probstudy demonstrate the role of intrinsic fac- able that endogenous Ca++ and Mg++ in tor in the uptake of vitamin BI2 by human the gastric juice and gilt homogenate acileal mucosa. counts for the discrepancies in some cation Neither serum, saliva, nor pernicious omission experiments. anemia gastric juice or heated normal gasIntrinsic factor was maximally effective tric juice increase vitamin BI 2 uptake. In in the range of pH 7, minimally effective fact, these substances even interfere with at pH 5.5, and ineffective below that level, passive uptake, probably because they similar to guinea pig ileal homogenates. 13 bind' the vitamin, rendering it unavailIn contrast to the intrinsic factor-mediable to the gut mucosa. Hog intrinsic fac- ated process, the nonspecific uptake of tor concentrate resembles normal human vitamin BI2 does not seem to be affected gastric juice in this system. by pH or divalent cations and to occur in Human auto-antibody to intrinsic factor, jejunal as well as ileal mucosa. obtained from patients with pernicious Enhancement by some salivas containanemia, and anti-hog intrinsic factor con- ing H substance of vitamin BI 2 uptake by centrateantiserum, prepared in rabbits, guinea pig ileal homogenate, which had both block intrinsic factor effect on the been reported to occur independently of gut homogenate. intrinsic factor,2 was not demonstrated The intrinsic factor effect was demon- with human ileal homogenate. Three saTABLE
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CARMEL ET AL.
liva specimens of the eight tested seemingly enhanced vitamin BI2 uptake. However, this effect was artifact, as demonstrated by the equally large vitamin BI2 precipitates with jejunal and colonic as compared with ileal mucosa homogenates, and even on incubation without gut homogenate. Antibody to intrinsic factor had no effect on saliva. In addition, a nonsecreting patient with pernicious anemia was fed Co57BI2 with 250 ml of saliva from two blood group 0 secretors of H substance, in a Schilling test to determine whether the saliva enhanced BI2 absorption. Urinary excretion in a 24-hr period was negligible (1.8%). The precipitates with saliva are probably a manifestation of a precipitable component, possibly mucus. 34 This serves to emphasize the need for a control consisting of the test samples without gut homogenate, or with added EDT A, which would eliminate effect of intrinsic factor, but not effect of artifact. It has been postulated that receptor sites exist on the ileal mucosal cell surface for the intrinsic factor-B l2 complex. 2, 35 , 36 Depressed vitamin BI 2 absorption has been reported in patients with regional ileitis, 36 Our data on ileal homogenates from patients with this disease show that vitamin BI2 uptake is subnormal in some cases, but not in others, and that this need not be related to the gross appearance of the mucosa to the naked eye. This suggests that this technique may be useful in examining the functional status of the receptors on the mucosal cell for the intrinsic factor-Bl2 complex. However, further studies, correlating this technique with in vivo absorption studies, are required.
4.
5.
6.
7.
8.
9.
10.
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15. 16.
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lation of vitamin BI2 uptake in tissue slices by intrinsic factor concentrate. Proc. Soc. Exp. BioI. Med . 96: 39-43. Herbert, V. 1958. Development of a possible in vitro assay for intrinsic factor. Proc. Soc. Exp. BioI. Med. 97: 668-671. Herbert, V. 1959. Studies on the role of intrinsic factor in vitamin B12 absorption, transport and storage. Amer. J. Clin. Nutr. 7: 433- 443. Minard, F. N., and C. R Wagner. 1958. In vitro assay of hog intrinsic factor with rat liver homogenate. Proc. Soc , Exp. BioI. Med. 98: 684686. Herbert, V. 1959. Mechanism of intrinsic factor action in everted sacs of rat small intestine. J . Clin , Invest. 38: 102-109. Herbert, V" and W. B. Castle. 1961. Divalent cation and pH dependence of rat intrinsic factor action in everted sacs and mucosal homogenates of rat small intestine. J. Clin. Invest . 40: 1978- 1983, Boass, A., and T. H. Wilson. 1963. An assay for gastric intrinsic factor. Amer. J. Physiol. 204: 97-100. Okuno, A" and D. Miller. 1962. Kinetics of B12 uptake in preparations of hamster intestine. J. Lab. Clin. Med. 60: 1002-1003. Wilson, T. H., and E . W. Strauss. 1959. Some species difference in the intrinsic factor stimulation of B1 2 uptake by small intestine in vitro. Amer. J. Physiol. 197: 926-928. Strauss, E . W. , T . H .Wilson, and A. Hotchkiss. 1960. Factors controlling B12 uptake by intestinal sacs in vitro. Amer. J. Physiol. 198: 103-107. Sullivan, L. W., V. Herbert, and W. B. Castle. 1963. In vitro assay for human intrinsic factor. J. Clin . Invest . 42: 1443- 1458. Donaldson, R M., I. L. Mackenzie, and J. S. Trier, 1967. Intrinsic factor-mediated attachment of vitamin B12 to brush borders and microvillous membranes of hamster intestine, J , Clin . Invest. 46: 1215-1228. Herbert, V. 1958. In vitro organ specificity of intrinsic factor action. Fed . Proc. 17: 440. Herbert, V. , and T. H. Spaet, 1958. Distribution of intrinsic factor activity. Amer. J. Physiol. 195: 194-196. Gottlieb, C. W., K.-S . Lau, L. R Wasserman, and V, Herbert, 1965. Rapid charcoal assay for intrinsic factor (IF), gastric juice unsaturated B12 binding capacity, antibody to IF, and serum unsaturated B1 2 binding capacity. Blood 25: 875-884. Race, R R, and R Sanger. 1962. Blood groups in man. Ed. 4, p. 233. F. A. Davis Co., Philadelphia.
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GASTROENTEROLOGY
Copyright © 1969 by The Williams & Wilkins Co.
VITAMIN BI2 UPTAKE BY HUMAN SMALL BOWEL HOMOGENATE AND ITS ENHANCEMENT BY INTRINSIC FACTOR RALPH CARMEL, M.D., ARTHUR H. ROSENBERG, M.D., KAM-SENG LAU, M.D., RICHARD R. STREIFF, M.D., AND VICTOR HERBERT, M.D.
Department of Medicine, Mount Sinai School of Medicine of the City University of New York, and the Thorndike Memorial Laboratory, Boston City Hospital, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts
Mucosal homogenates of human ileum were demonstrated to have enhanced vitamin BI2 uptake in the presence of normal human gastric juice or hog intrinsic factor concentrate. This process was demonstrated to require Ca++ or Mg++ and to be optimal at pH 6.6 and above. The enhancement was blocked by antibody to intrinsic factor. Pernicious anemia gastric juice and normal and pernicious anemia serum did not enhance BI2 uptake. In fact, these substances diminished nonspecific uptake of the vitamin, presumably by binding it and rendering it unadsorbable. "Enhancement" by saliva appeared to be an artifact due to the presence of BI2 binders precipitable by centrifugation. Human jejunal mucosa homogenate uptake was not enhanced by intrinsic factor, supporting the concept that the active process of vitamin BI2 uptake is localized to the ileum. Nonspecific uptake of vitamin B 12 , unrelated to intrinsic factor, appeared to be uniform in jejunal and ileal mucosa homogenates, and to be unaffected by pH or cations. Poor vitamin BI2 uptake in some but not other ileal homogenates from patients with regional ileitis suggests that the ileal homogenate technique may be useful in evaluating in vivo function of intestinal receptors for the intrinsic factor-vitamin BI2 complex. This awaits further studies, correlating this technique with in vivo absorption studies. Vitamin BI2 absorption takes place by two mechanisms. I The first is passive and nonspecific, taking place along the entire length of the intestine. 2 The second is the Received June 17, 1968. Accepted September 9,1968. Address requests for reprints to: Dr. Victor Herbert, Nutritional Anemias Division, Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029. This work was supported by United States Public Health Service Grants AM 13358 and AM 11048, and Clinical Research Center Grant FR-71 , United States Public Health Service. Dr. Herbert is recipient of Career Scientist Award 1-435 from the Health Research Council of the City of New York. Dr. Carmel was a United States Public Health Service Postdoctoral Research Fellow (5-F2-AM-32, 518-02), National Institutes of Arthritis and Metabolic Diseases, and is currently at Wilford Hall 548
active mechanism which is the specific one mediated by intrinsic factor. The marked observable difference between these two mechanisms has formed the basis of in vitro techniques for study of United States Air Force Hospital, Lackland Air Force Base, Texas. Dr. Rosenberg was a United States Public Health Service Postdoctoral Fellow (1-F2-AM-24, 310-01). Dr. Lau was a World Health Organization Research Fellow in Hematology, and is currently Head, Department of Pathology, University of Malaya, Kuala Lumpur, Malaysia. Dr. Streiff is currently Assistant Professor of Medicine, University of Florida, Gainesville, Florida. The authors are indebted to Melody Lee, Le Teng Go, and Leona Bandel for their invaluable technical assistance, and to Dr. Henry Janowitz and the Division of Gastroenterology for their help in securing samples for study.
VITAMIN
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the relation of intrinsic factor to vitamin Bl2 adsorption to the mucosal cell, the first step in the absorptive process. These techniques have been utilized on various animalliver 3 - o and intestinal,-14 preparations. Studies with human liver have also been reported. 5o ' l 5 , 10 The purposes of the present study are to establish with human intestinal homogenates various conditions required for vitamin Bl2 uptake, to help explain various clinical observations on the process, and to establish a basis for further study of the intestinal phase of the process by this technique.
Materials and Methods Human ileum was obtained at surgery from patients undergoing resection for carcinoma of the ascending colon, ulcerative colitis, or regional enteritis. Specimens from patients with carcinoma of the colon, or, in a few instances, ulcerative colitis, were referred to as normal ileum. Jejunum was obtained from patients undergoing gastroenterostomy for ulcer disease. Immediately after resection, the intestinal segment, usually measuring 1 to 6 inches, was placed in cold (4 C) 0.85% NaCI (saline). The mucosal surface was washed with cold saline until the effluent was clear. Mucosal scrapings were obtained by exposing and scraping the mucosal surface, or by running a spatula along the serosal surface and, with firm pressure, expressing mucosal tissue as toothpaste is expressed from a tube. The scrapings thus obtained were placed in 50 ml of cold saline and homogenized in a Waring Blendor for 15 sec. The homogenate (0.5 to 5.0 g wet weight) was divided into aliquots and centrifuged for 10 min at 2000 rpm at 4 C, The pellets, washed twice with 5 ml of cold saline, were stored at - 20 C until used . The ability of the homogenate to function in our assay system 'was maintained on such storage for as much as 1 year. Once thawed, specimens were used or discarded. Human gastric juices were obtained from normal subjects, as well as from subjects with pernicious anemia or other conditions, and were depepsinized and neutralized. IJ Intrinsic factor . content was assayed by the method of Gottlieb et al. I With the one exception noted below, only specimens containing intrinsic factor content of at least 80 '( of the total vitamin BI2binding capacity were used . 0
BI2
UPTAKE
549
Saliva was obtained from various subjects by expectoration, occasionally with the aid of Parafilm chewing. Secretion of H substance was determined with Ulex europaeus. l " Samples were stored at - 20 C. Antibody to human intrinsic factor was obtained from patients with pernicious anemia. Presence of the antibody was determined as reported elsewhere, I; and the samples were stored at - 20 C. Hog intrinsic factor concentrate was purchased from the National Formulary of the American Pharmaceutical Association, Washington, D.C. Co 5 , -labeled vitamin BI2 (Co5 ' B I2) of specific activity 10 IlC per Ilg was usually used ; in some experiments CO " BI 2 of specific activity 5.9 IlC per Ilg was used, as well as Co""Bl2 (kindly supplied by Dr. Elmer Alpert of Merck, Sharpe and Dohme Research Laboratories, West Point, Pa.) of specific activity 1.283 IlC per Ilg. All were diluted with saline to a concentration of 10 ng per ml. Rabbit anti-hog intrinsic factor concentrate antiserum was obtained from rabbits immunized with hog intrinsic factor concentrate as previously described . I" The presence of antibody was assayed as with human serum. For 30 min 0.2 to 0.5 ml of Coa' Bl2 was pre-incubated at room temperature with 0 .05 to 0.1 ml of gastric juice, made up to a total volume of 1.0 ml with Krebs Ringer-Tris (KRT) buffer, pH 7.4. Volumes used were such that the Bl2 binding capacity of the specimen equaled, or slightly exceeded, the amount of CO " BI 2 present. Controls with saline substituted for the gastric juice were used with each specimen assayed. On occasion, saliva or serum was used in place of the gastric juice, Gut pellets were thawed, weighed, suspended in cold KRT buffer, and homogenized using a motor-driven Teflon pestle with a fitted glass tube immersed in ice, The homogenate in buffer was constantly shaken to ensure uniformity of suspension, and equal 3-ml aliquots were added to each gastric juice-Co5 ' B l2 sample and to control samples, (Each tube in all of our experiments thus contained 25 to 100 mg of gut homogenate; best results were obtained when at least 60 mg was used. Samples of 15 mg proved too small to produce significant results.) As additional controls, gut homogenate was left out of the system in samples otherwise prepared identically to the test samples, and run concurrently in each assay, The samples were agitated at 90 cycles per min for 90 min at 37 C in a Dubnoff
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Metabolic Shaker. Subsequently, 30-min agitation at room temperature was substituted when it was shown to produce even better results. The reaction was terminated by centrifugating at 2000 rpm for 10 min at 4 C. The precipitate was washed with 5 ml, and then with 3 ml, cold 0.85% NaCI-1O, mM CaCle. Radioactivity of the pellet was determined in a well-type scintillation counter, for a minimum of 10,000 counts. The counts were converted to picograms of Co;'B I ,. Each specimen was run in duplicates and differences between these were generally less than 10%. Ratios of CO·"B I2 uptake by the test specimens (S) over uptake by the saline control (C) were determined (S IC) . In experiments of cation dependence, CaCI, and MgSo4 were omitted in preparing the KRT buffer, and saline, rather than salineCaCh, washes were used. The effect of ethylenediaminetetraacetate (EDTA) was studied using 40 mg sodium versenate (Riker) per sample, diluting it 1: 20 in KRT buffer from which CaCI, and MgS0 4 had been omitted, and adjusting the final pH of the specimen to 7.4 with 0.5 N NaOH. In studying pH dependence , the buffers used consisted of 9 parts Krebs Ringer solution' " brought to appropriate pH with 1 part 0.05 M Tris-acid maleateNaOH buffer."
Results Uptake by Normal Ileal Mucosa Homogenate In the absence of intrinsic factor there was some vitamin BI2 uptake by ileal homogenate. This represented nonspecific uptake and ranged from 10 to 20 pg, oc-
casionally being as much as 35 pg, BI2 per 100 mg homogenate. No more than 4 pg of Co;'B I2 was spun down when buffer alone instead of homogenate suspension was added. As demonstrated by the SIC ratios in figure 1, normal gastric juice markedly enhanced vitamin BI2 uptake. The magnitude of the SIC ratio seemed to correlate primarily with the amount of gut homogenate used, the smaller ratios being seen with the smaller pellets. A given pellet of a single gut homogenate produced comparable results with different intrinsic factor-containing gastric juices. Significant enhancement was seen with gastric juices of normal persons and of patients with duodenal ulcer, tropical sprue, or iron deficiency. While these specimens all had high intrinsic factor content, even a specimen from a patient with tropical sprue where only 40% of the binding capacity was due to intrinsic factor markedly enhanced vitamin BI2 uptake. Hog intrinsic factor concentrate had an identical effect, although perhaps of not so great a magnitude as the human gastric juices. In marked contrast, pernicious anemia gastric juice not only did not increase uptake, but actually inhibited it. This was true with specimens from persons who secreted H substance as well as those who did not. Sera from normal and pernicious anemia subjects also resulted in reduced BI2 uptake by the ileal homogenate (fig. 1).
12
10 x ONLY 40% OF THE 8,2 BINDING CAPACITY OF THIS GASTRIC JUICE WAS DUE TO
S
C (
INTRINSIC FACTOR.
6
TEST SPECIMEN) SALINE CONTROL 4
2 I
------- -- ----------- - - ----- -------- ---- - - ---------- NORMAL GASTRIC
HOG INTRINSIC
PERNICIOUS ANEMIA
JUICE
FACTOR CONCENTRATE
GASTRIC JUICE
SERUM
FIG. 1. Vitamin BI2 uptake by normal ileal mucosa homogenate. (Points represent different mucosa specimens.)
March 1969
Heating normal gastric juice at 100 C for 30 min (which destroys intrinsic factor activity) prior to testing it eliminated uptake enhancement, reducing an SIC ratio of 6.3 to 0.9. Effect of Antibody to Intrinsic Factor
As shown in figure 2, intrinsic factor effect was eliminated when the normal ~astric juice ":".as incubated, prior to addition of COo' B12 or ileal homogenate, with antibody-containing serum from a patient with pernicious anemia, or if hog intrinsic factor concentrate was similarly pre incubated with rabbit anti-hog intrinsic factor concentrate antiserum . A similar effect was noted when rabbit anti-hog intrinsic factor concentrate antiserum was 9
8 7 6
4 5
C
551
VITAMIN B" UPTAKE
3 2
used with human gastric juice. In contrast, sera without antibody had no effect. That the antibody acts on the gastric juice binding of B12, rather than on the gut itself, was shown by lack of effect of antibody incubated for 30 min at room temperature with the ileal homogenate (which was then washed with KRT) prior to addition of gastric juice and CO o' B12. Uptake by Jejunal Mucosa Homogenate
In contrast to ileal uptake, jejunal uptake of vitamin B12 was not enhanced by normal gastric juice (fig. 3). Similarly, pernicious anemia gastric juice had no effect. Nonspecific uptake, as reflected by vitamin B12 uptake in the saline controls, did not differ significantly from that seen with ileal homogenates. Uptake by Abnormal Ileal Mucosa Homogenates
With ileum from various patients with regional ileitis, intrinsic factor enhancement of vitamin B1 2 uptake was demonstrated in some cases (fig. 3). No enhancement was seen generally with those ileal specimens which had appeared grossly abnormal. In one case, however, two specimens from the same patient, one appearing normal to the naked eye and the other abnormal, were tested and both showed no intrinsic factor enhancement. Effect of pH
FIG.' 2. Effect of antibody to intrinsic factor on vitamin B12 uptake by normal ileal mucosa homogenate. (Points represent different mucosa specimens.) Homogenate pre incubated with human anti-intrinsic factor antiserum for 30 min at room temperature prior to incubation with normal human gastric juice and Co57 B" . SIC, test specimen/saline control ratio; NGJ, normal human gastric juice; AB, human anti-intrinsic factor antiserum; RIFC, hog intrinsic factor concentrate; and anti-RIFe, rabbit anti-hog intrinsic factor concentrate antiserum.
The pH of our experimental system was 7.2. To determine pH effect on uptake by normal ileal homogenate, a pH range of 4.8 to 9.0 was studied. As shown in table 1, intrinsic factor effect was minimal or absent at pH 5.5 and absent below that level. Maximal effect was noted at pH 6.6 and above. Nonspecific uptake was not affected by pH change. Effect of Ca++ and Mg++
The intrinsic factor effect of gastric juice was shown to require divalent cation . Omission of both Ca++ and Mg++ abolished intrinsic factor enhancement (table
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7
6 5 4 5
C
• NORMAL GASTRIC JUICE x GUT SPECIMENS FROM SAME SUBJECT; ONE ( SIC 1.2) APPEARING NORMAL. ANO THE OTHER (SIC 0.4) ABNORMAL TO THE NAKED EYE -PERNICIOUS ANEMIA GASTRIC JUICE
3
• •
•
ILEITIS ILEUM
NORMAL JEJUNUM
•
•
FIG. 3. Vitamin B12 uptake by ileal mucosa homogenate from patients with regional ileitis, and by normal jejunal mucosa homogenate. SIC, test specimen/saline control ratio.
2). This was not seen when Mg++ alone was omitted from the system. When Ca++ alone was left out, intrinsic factor effect was abolished with three of four normal gastric juices tested. The one exception demonstrated reduced, albeit still significant, intrinsic factor effect. This gastric juice was then further tested with sodium versenate (EDT A), which abolished its effect entirely (table 3). Addition of equimolar amounts of either CaCl 2 or MgS0 4 to the EDT A re-established intrinsic factor effect. In no instances was there artifactual precipitation of Co 6 'B12 • In previous experiments, EDT A was shown not to interfere with binding of Co 6 ' B12 by gastric juice. Nonspecific uptake of B12 was not affected by either omission of cations or by addition of EDT A.
Effect of Saliva At pH 7.2, only three of eight saliva specimens increased B12 "uptake" by ileum homogenate, with an average SIC of 5.2. Two of these were from normal blood group A secretors of H substance, and one was from a blood group 0 nonsecretor with pernicious anemia. The 5 other subjects studied, regardless of secretor status or presence of pernicious anemia, gave negative results (average SIC of 1.4). The three "positive" saliva specimens, however, produced identically large B1 2
1. Effect of pH on intrinsic factor-mediated vitamin BJ2 uptake by normal ileal mucosa homogenate
TABLE
Experiment 2
Experiment 1
pH
SIC""
4.9 5.5 7.0 7.8 8.5
0.1 0.1 3.5 4.7 4.3
pH
Sica
4.8 5.5 6.6 7.5 9.0
0.5 2.1 6.3 3.7 5.2
a Expressed as test specimen to saline control ratio.
2. Effect of Ca++ and M g++ lack on intrinsic factor-mediated vitamin B12 uptake by normal ileal mucosa homogenate a
TABLE
Base line
3.8 4.9 6.0 9.6 8.3 8.1
Absence of Ca++ and Mg++
1.3 1.4
Absence 01 Ca++
Absence of Mg++
4.0 1.5 1.3 1.8
6.4 10.2
"Expressed as test specimen to saline control ratio.
precipitates when tested with jejunal and colonic mucosa, and, indeed, when tested with buffer alone without homogenate. Antibody to intrinsic factor had no effect
March 1969
VITAMIN
3. Effect of sodium versenate (EDTA) on intrinsic factor-mediated vitamin B12 uptake by normal ileal mucosa homogenate
BI2
UPTAKE
553
strable on ileal but not jejunal mucosa homogenates. A previous studl l of vitamin BI 2 uptake along the entire length of small intestine, obtained within 2 hr after death from a patient who died of acute Gastric juice. . . . . . . . . . . . . . . . . . . 2.3 . . . . . . leukemia, showed consistent enhancement by normal gastric juice of vitamin B12 upGastric juice + EDT A. ........ 0.3 take by mucosal homogenates of all segGastric juice + EDTA + Ca++ . .... .. 6.6 Gastric juice + EDTA + Mg++. . . . . . . 5.6 ments from the distal half. Segments from Gastric juice + EDTA + Ca++ + the proximal half of the small intestine Mg++... ..... . . . . . . . . . . . . 4.3 did not demonstrate this effect. We have not been able to repeat this study successa Expressed as test specimen to saline control fully, probably due to the rapid postmorratio. tem autolysis of intestinal mucosa which makes it mandatory that the specimen be on results with ileal or jejunal homogeobtained within 2 hr of death. Similar nate. When the saliva was centrifuged at 4000 autolysis probably explains the better rpm for 15 min and only the supernate results obtained in the present study when used, the positive effect with ileal mucosa incubation was carried out for 30 min at or with buffer alone disappeared (SIC of room temperature instead of 90 min at 0.3). When the pH of the system was 37 C. Intrinsic factor action was shown to be changed to 9.0, it was found that even salivas negative at pH 7.2 produced large dependent on divalent cations, as had been demonstrated in vivo in man, 5. 32 , 33 and in precipitates of vitamin B 12 . vitro in animals ."~' 14, 33 Omitting both Discussion Ca++ and Mg++ from our system abolished In vivo studies in man and both in vitro intrinsic factor effect of all gastric juice. and in vivo animal studies suggest that sion alone produced no change, Ca++ omisvitamin BI2 absorption occurs by two mech- sion did. EDTA completely abolished the anisms, I passive transport and an active intrinsic factor effect of all gastric juice, process which is dependent on intrinsic Adding either Ca++ or Mg++ alone to the factorl , 2 2, 2 3 and is limited to the il- EDT A re-established activity, suggesting eum. 23 - 3 0 The results ofthe present in vitro that either cation was effective. It is probstudy demonstrate the role of intrinsic fac- able that endogenous Ca++ and Mg++ in tor in the uptake of vitamin BI2 by human the gastric juice and gilt homogenate acileal mucosa. counts for the discrepancies in some cation Neither serum, saliva, nor pernicious omission experiments. anemia gastric juice or heated normal gasIntrinsic factor was maximally effective tric juice increase vitamin BI 2 uptake. In in the range of pH 7, minimally effective fact, these substances even interfere with at pH 5.5, and ineffective below that level, passive uptake, probably because they similar to guinea pig ileal homogenates. 13 bind' the vitamin, rendering it unavailIn contrast to the intrinsic factor-mediable to the gut mucosa. Hog intrinsic fac- ated process, the nonspecific uptake of tor concentrate resembles normal human vitamin BI2 does not seem to be affected gastric juice in this system. by pH or divalent cations and to occur in Human auto-antibody to intrinsic factor, jejunal as well as ileal mucosa. obtained from patients with pernicious Enhancement by some salivas containanemia, and anti-hog intrinsic factor con- ing H substance of vitamin BI 2 uptake by centrateantiserum, prepared in rabbits, guinea pig ileal homogenate, which had both block intrinsic factor effect on the been reported to occur independently of gut homogenate. intrinsic factor,2 was not demonstrated The intrinsic factor effect was demon- with human ileal homogenate. Three saTABLE
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CARMEL ET AL.
liva specimens of the eight tested seemingly enhanced vitamin BI2 uptake. However, this effect was artifact, as demonstrated by the equally large vitamin BI2 precipitates with jejunal and colonic as compared with ileal mucosa homogenates, and even on incubation without gut homogenate. Antibody to intrinsic factor had no effect on saliva. In addition, a nonsecreting patient with pernicious anemia was fed Co57BI2 with 250 ml of saliva from two blood group 0 secretors of H substance, in a Schilling test to determine whether the saliva enhanced BI2 absorption. Urinary excretion in a 24-hr period was negligible (1.8%). The precipitates with saliva are probably a manifestation of a precipitable component, possibly mucus. 34 This serves to emphasize the need for a control consisting of the test samples without gut homogenate, or with added EDT A, which would eliminate effect of intrinsic factor, but not effect of artifact. It has been postulated that receptor sites exist on the ileal mucosal cell surface for the intrinsic factor-B l2 complex. 2, 35 , 36 Depressed vitamin BI 2 absorption has been reported in patients with regional ileitis, 36 Our data on ileal homogenates from patients with this disease show that vitamin BI2 uptake is subnormal in some cases, but not in others, and that this need not be related to the gross appearance of the mucosa to the naked eye. This suggests that this technique may be useful in examining the functional status of the receptors on the mucosal cell for the intrinsic factor-Bl2 complex. However, further studies, correlating this technique with in vivo absorption studies, are required.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15. 16.
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19. Kaplan, M. E., R. Zalusky, J . Remington, and V. Herbert. 1963. Immunologic studies with
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