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Studies on the interaction of vitamin B12: Intrinsic factor and receptors
ARCHIVES
OF
BIOCHEMISTRY
Studies
AND
BIOPHYSICS
on the Interaction and
III. Chemical
and Biological the Stomach, 5. W. COFFEY,
117-123 (1965)
110,
of Vitamin
B12: Intrinsic
Factor
Receptors
Properties
of Vitamin
B,, Combining
Substances
from
Serum, and Ascites Fluid of the Mouse’ HANS J. HANSEN,
AND
0. NEAL
Department of Biochemical Research, Touro Research Institute; and the Nutrition tory of the Departments of Biochemistry and Medicine, Tulane University Neul Orleans, Louisiana Received
November
MILLER and Metabolism LaboraSchool of Medicine
10, 1964
The chemical and biological properties of vitamin Blz combining substances in mouse stomach extracts (MIF), mouse serum (MS), and mouse ascites fluid (AS) were compared. MIF was found to enhance the absorption of vitamin Blz by rings of mouse and guinea pig ileum and to inhibit its absorption by sarcoma-180 (S-180) cells in vitro. 11s and AS, on the other hand, enhanced the absorption of vitamin Blz by S-180 cells but inhibited its absorption by rings of mouse and guinea pig ileum. It was shown that MIF is capable of forming a stable complex with vitamin Blz at pH values from 1.0 to 11.0, while MS and AS are not capable of forming such stable complexes at pH values below 5.0. The complex formed between vitamin Blz and MIF (MIF-vitamin B,?) is not adsorbed by zirconyl phosphate gel (Z-gel) at pH values higher than 5.0; however, the complexes formed between vitamin Bi2 and MS (MSvitamin B,z) or AS (AS-vitamin Bi2) are adsorbed by Z-gel at pH values as high as 7.0. By chromatography on CM-cellulose, it was possible to separate MIF-vitamin B,, from MS-vitamin Bis and AS-vitamin Bip. The latter two complexes behaved identically when chromatographed on CX-cellulose.
It is recognized that, unless given in a massive dose, vitamin B,, must combine with intrinsic factor prior to absorption from the gastrointestinal tract (2). Miller and Hunt’er (3) have demonstrated that hog intrinsic factor concentrates facilitate the uptake of vitamin B,, by slices of rat liver and kidney. It was also shown that receptor proteins in serum (4) and homogenates of rat liver (5) will combine intrinsic factorvitamin B12 complex and thus prevent it’s adsorptiol: by charcoal. Reports (6, 7) have appeared concerning the presence of an irurinsic factor-like substance in blood plasma. Therefore, it was postulated (8) that intrinsic factor may be absorbed into the 1 Supported by the John A. Hartford Foundation, and the U.S. Public Health Service (grant A-l). A preliminary report has appeared (1).
blood stream where it may facilitate the absorption of vitamin Blz by peripheral tissues in a manner analogous t)o its action in bhe gut. Data have been presented which demonstrate that both intrinsic factor and intrinsic factor-vit,amin B1, complex can cross the cell membrane of liver cells of the rat’ following their intravascular administration (9). More recently, data have been presented (10) which suggest t’hat the intrinsic factor-vitamin Blz complex may be absorbed into intestinal epithelial cells where it is converted into some other vitamin Blz-protein complex which is then released into the bloodstream. Results reported below compare some of the properties of the vitamin Bit “binders” found in extracts of mouse stomach (mouse intrinsic factor), in mouse serum, and in 117
118
COFFEY,
HANSEN,
ascites fluid (11) collected from mice bearing sarcoma-180 (S-180) tumor cells. MATERIALS
AND
METHODS
Chemicals. CobaltGO- and Co&r-labeled vitamin B,z were generously provided by E. R. Squibb and Sons. Nonradioactive vitamin Blz was obtained from Merck, Sharp and Dohme. The radioactivity of all samples containing CoGo- or Cos7-vitamin Brz was assayed by means of a center-well gamma scintillation counter. The specific activity of the CoGO-vitamin B1z was 800 counts per minute (cpm) per millimicrogram as determined by means of the counting system described above. Cobaltsr-vitamin B,z was used for those experiments in which the effect of the different binders on the uptake of vitamin Blz by intestinal rings was studied. It had a specific activity of 1830 cpm per millimicrogram. Animals. Swiss mice purchased from the A. R. Schmidt Co., Madison, Wisconsin were used throughout this study, and were maintained on a laboratory chow diet up to the time of sacrifice. The S-180 ascites tumor, which was provided by Dr. Tom McCoy, Noble Foundation, Ardmore, Oklahoma, was transferred every 7 days by intraperitoneal inoculation of stock mice with 0.1 ml of a 10% suspension of tumor cells in 0.9% NaCl. Preparation of vitamin Blz “binders”. Mouse intrinsic factor (MIF) was prepared by the method previously described for rat intrinsic factor (12) from 3 stomachs which were carefully washed with cold 0.9% NaCl and homogenized in 10 ml of icecold distilled water by means of a Waring Blendor. The homogenate was dialyzed with stirring against 500 volumes of cold distilled water for 5 hours and then centrifuged at 8000 g for 10 minutes. The supernatant was decanted from the sedimented pellet and saved as a source of MIF. Ascites fluid was collected as described by Paranchych and Cooper (II), dialyzed against distilled water, and centrifuged at 8000g for 10 minutes; the supernatant was used as a source of ascites fluid binder (AS). Serum prepared from blood collected by heart puncture was used as a source of serum binder (MS). The vitamin B12 binding capacity of each of the solutions (MIF, AS, MS) was determined by the zirconyl phosphate (Z-gel) method of Hansen and Miller (13). This method is based on the observation that Z-gel at a pH of 5.0 will adsorb the complexes formed between vitamin Bls and acceptors such as MIF, MS, and AS but will not adsorb free vitamin Biz. Stock solutions of MIF, AS, and MS were prepared by diluting the above solutions with distilled water so that 1 ml of each of the stock solutions would bind between 6 X 103 and 10 X 10s cpm (S-12 mpg) of CoeO-vitamin B,, to Z-gel at pH 5.0.
AND
MILLER
Effect of pH on the binding of GoGO-vitamin Blz by MIF, MS, and AS. One ml of the stock solutions of MIF, AS, and MS prepared as described above was added to 1 ml of each of the following solutions: HCI (0.2 1V) ; formate buffer (pH = 3.0, p = 0.2); acetate buffer (pH = 5.0, p = 0.2); phosphate buffer (pH = 7.0, p = 0.2) ; bicarbonate buffer (pH = 9.0 or 11.0, p = 0.2), and NaOH (0.2 A;). The final ionic strength of each of the solutions was 0.1, and the pH values ranged from 1 to 14. Fifteen mg of Coeo-vitamin B,, (12,000 cpm) in a volume of 15 ~1 was added with shaking to each of the tubes, followed by the addition of 1 pg of nonradioactive vitamin Bit; and the tubes were allowed to stand at room temperature for 15 minutes. Twelve ml of ammonium acetate2 (pH = 5.0, 0.1 &f) was then added to each tube followed by 1 ml of Z-gel prepared as described eIsewhere (13). The tubes were inverted 12 times by hand and centrifuged at 80009 for 5 minutes, and the supernatant was decanted from the sedimented Z-gel pellet. The pellet was washed once by resuspension in 15 ml of ammonium acetate and re-centrifugation. The amount of CoGo-vitamin Bn bound to the pellet was determined with the gamma scintillation counter. The stability of the complexes between MIF, MS, or AS and CoGo-vitamin B12 was determined by exposing the complexes to a 70-lOO-fold excess of nonradioactive vitamin B12. Instability of the complex would result in an equilibration between the radioactive vitamin Brz and the nonradioactive vitamin Blz. This equilibration would produce a reduction in the amount of CoGO-vitamin Blz bound to Z-gel as compared with a control in which no nonradioactive vitamin B,z was added. Conversely, failure of the presence of a large excess of unlabeled vitamin Blz to dilute the radioactivity of the complex can be interpreted as evidence of stability of the complex. Uptake of vitamin B,z by rings of mouse and guinea pig ileum. The effect of MIF, MS, and AS on the absorption of vitamin Bn by rings of mouse and guinea pig ileum was studied by the method of Boass and Wilson (14). This method involves the use of thin rings of intestinal tissue as an assay system for the study of intrinsic factor-facilitated absorption of vitamin Brx. Rings of mouse intestine were prepared from the lower third of the intestine because preliminary experiments demon2 In most cases the addition of the ammonium acetate brought the pH of the reaction mixture to 5.0. If it did not, the reaction mixture was adjusted to a pH of 5.0 before the Z-gel was added since the in vitro assay for vitamin Brz binding capacity as described by Hansen and Miller (13) is carried out in a medium at pH 5.0.
INTRINSIC
FACTOR.
factor preparation to behave, i.e., it stimulates the uptake of Co6’-vitamin B12 by the intestinal rings. The serum binder and the ascites binder have no stimulatory effect on the uptake of vitamin Bls, and, in fact, inhibit the uptake of vitamin B, by the intestinal rings. Figure 1 shows the results obtained when the binding of COW-vitamin Bl? to MIF, MS, and AS was studied at pH values ranging from 1.0 to 14.0. The MIF binds vitamin Blz, and a stable complex is formed, as determined by the Z-gel method (see Materials and JleLhods), at all pH values between 1.0 and 11.0; the ?rlS and AS form a stable complex with vitamin B,z at pH values between 5.0 and 11.0 with no binding of vitamin B12at pH 1.0 and very little binding at pH 3.0. If the addition of unlabeled vitamin B12 to the incubation mixture is omitted (see Materials and Methods), the curves obtained are identical; thus indicating that once MIF is exposed to a medium with a pH of 14.0 and 31s or AS to media with pH’s of 1.0, 3.0, or 14.0, they are irreversibly altered and lose their ability bo bind vitamin B,, even when the pH is readjusted to 5.0. Highly purified hog in-
strated that this portion of the intestine shows the greatest degree of intrinsic factor-facilitated absorption of vitamin Bls. The lower half of the guinea pig intestine was used for the same reason (15). Rings prepared from three animals were mixed and used randomly for the assays. Approximately 500 mg (wet weight) of intestinal rings was placed in a Warburg flask containing 2.4 ml of Eagle’s minimum essential medium (MEM) (16), 0.1 ml of Cob’-vitamin B,z which contained 30 X lo3 cpm, and 0.5 ml of the appropriate binder having a binding capacity of 25,000 cpm of Cos7-vitamin Blz. The flasks were incubated at 37°C for 2 hours with shaking, after which their contents were transferred to test tubes and the intestinal rings were collected by centrifugation. The rings were washed 3 times with 10 ml of ice-cold saline and their radioactivity was then assayed in the gamma scintillation counter. Chromatography. CM-cellulose was purchased fromMann Research Laboratories. Prior to its use, the CM-cellulose was suspended in 1 M NHaOH at room temperature for 15 minutes. The NHdOH was removed by filtration and the CM-cellulose was washed free of NHdOH with water. RESULTS
The data presented in strate that the mouse (JIIF) does behave in the tems as one would expect
Table I demonstomach extract in vitro assay sysa mouse intrinsic TABLE
OF MIF,
INFLUENCE
MS,
Alouse
stomach
I
AND AS ON THE UPTAKE OF CO~~-VITAMIN MOUSE AND GUINEA PIG ILEUM”
Preparation
Assay system
extract
(h/IIF)
Mouse Guinea
Serum binder
Ascites
fluid
(MS)
binder
(AS)
119
III
ileum pig ileum
Mouse
ileum
Guinea
pig ileum
Mouse Guinea
ileum pig ileum
B12 BY RINGS
OF
Enhancement of Co57 vitamin B,* uptake” (cpm/gm tissue)
Expt. Expt. Expt. Expt. Expt. Expt. Expt. Expt. Expt. Expt. Expt. Expt.
1 2 1 2 1 2 1 2 1 2 1 2
4767 4026 1244 1006 0” 0 0 0 0 0 0 0
a See Materials and Methods for experimental details. b Uptake of Co67-vitamin B12 is reported as uptake above control values. Control values were obtained by including a flask containing the same amount of Cos7-vitamin B,z as experimental flasks but no MIF, MS, or AS. Two experiments were run and both values are reported. c Zero values indicate no uptake above control values. ?vlS and AS actually inhibited the uptake of free COST-vitamin B,t by the intestinal rings.
120
COFFEY,
HANSEN,
AND
MILLER
-
pH 5.0
_____, I - 18 - 16
H o--O
2 $ >O.l t 0.09-
Mouse Stomach extract (MIF) Mouse Serum (MS)
UJ 5 o -I 2 i
/
3
5
7
9
II
13
I5
PH
%
a 6.0
7.0
80
0 x
-12
- IO ,: 0 8
-
-
gg o 0.010
FIG. 1. The effect of pH on the binding of Coeovitamin B,, by MIF, MS, and AS. See Materials and Methods for experimental details.
50
0.08 0.07 0.06 0.05 0.04
- 14?
5
IO TUBE
I5 NUMBER
-
6
-
4
-
2
20
FIG. 3. Chromatography of MIF-CoeO-vitamin B 12 on CM-cellulose. A column of CM-cellulose (2 X 15 cm) was prepared (see i!!faterials and Methods) and washed with 0.1 M ammonium acetate (pH = 5.0) until the pH of the solvent flowing from the column was 5.0. Two ml of MIF having a Coeo-vitamin B12 binding capacity of 87 X lo3 cpm/ml (determined by Z-gel method) was mixed with sufficient Coeovitamin Blz to completely saturate the binder. Excess CoeO-vitamin Blz was removed by dialysis against 0.1 M ammonium acetate (pH = 5.0), and the dialyzed material was applied to the column. Material was eluted from the column by the stepwise washing of the column with 0.1 M ammonium acetate (pH = 5.0), 0.1 M ammonium acetate (pH = 6.0), 0.1 M phosphate buffer (pH = 7.0), and 0.1 M phosphate buffer (pH = 8.0). Ten-ml fractions were collected, and a 4-ml aliquot of each fraction was assayed for radioactivity.
pH of Z-gel FIG. 2. The effect of pH on the binding of MIFCo60-vitamin Bi2, MS-CoGO-vitamin Blz, and ASCorn-vitamin Biz to Z-gel. Complexes of Cocavitamin Bi2 withMIF, MS, and AS were formed by adding 10 X lo3 cpm (12 mpg) of Co@-vitamin Blz to 1 ml of the stock solutions of MIF, MS, and AS in a series of tubes containing a total volume of I2 ml and having the pH values indicated on the abscissa (0.1 M phosphate buffers). One ml of Z-gel having the same pH as the buffer in the tube containing the Coeo-vitamin Biz complex was added to each tube and the tube was shaken. The Z-gel was collected by centrifugation, washed, and assayed for radioactivity. Control tubes containing the same amount of CoGO-vitamin Bit but no MIF, MS, or AS were run at each pH value. In the absence of MIF, MS, or AS the Z-gel never bound more than 2% (266 cpm) of the added Co’o-vitamin EL.
FIG. 4. Chromatography of AS-Coso-vitamin Biz on CM-cellulose. See legend of Fig. 3 for experimental details.
INTRINSIC
FACTOR.
121
III
trinsic factor, rat intrinsic factor, and Z-gel at pH values of 5.0, 6.0, and 7.0, but hamster intrinsic factor gave results identical not at 8.0. Figures 3-6 show the results obtained with those obtained with MIF (17). B12, MS-CoGoSince the complexes formed between when MIF-Cow-vitamin vitamin Blz and MIF, MS, and AS were all vitamin Blz, and AS-Co@‘-vitamin B1zwere stable at pH values ranging from 5 to 11 chromatographed on columns of CM-cellu(Fig. l), it was possible to study the binding lose. As Fig. 3 shows, the MIF-Coeoof these complexes to Z-gel at a number of vitamin Blz does not attach to a column of different pH values. As can be seen (Fig. 2) CM-cellulose that has been equilibrated the MIF-C060-vitannn B12complex binds to with 0.1 M ammonium acetate at pH 5.0; Z-gel at pH 5.0 but not at higher pH values. however, the ASCo60-vitamin Blz (Fig. 4) On the other hand, the complexes of MS does attach to the CM-cellulose under these and AS with CoGo-vitamin B,, bind to conditions and is not eluted until the column
-6
1.0 g
0.9
$j
ae
z
0.7
-OPTICAL
DENSITY
-5
RADIOACTIVITY -4 6
z z 0.6 w ; 0.5 4 0 a4 ii 0
3;
0 0
-2
a3 I
0.2 0. I
0 5
0
IO
15
20
25
30
40
35
TUBE
45
50
6.04
5.0+pH
PH
7.04
Bls and MS-CoGO-vitamin
PH B.0-d J 9
t E’ i
60
NUMBER
FIG. 5. Chromatography of a mixture of AS-CocO-vitamin Bit on CM-cellulose. See legend of Fig. 3 for details.
pH
55
1.6
-
OPTICAL
DENSITY
RADIOACTIVITY
1.4
z 1.2 Gi 2 1.0 iii 2 0.B U Ib
0.6 0.4 0.2
IO
20
30
50
40 TUBE
60
70
NUMBER
FIG. 6. Chromatography of a mixture of MIF-Cos’J-vitamin B1, on CM-cellulose. See legend of Fig. 3 for details.
Btz and MS-Co6c-vitamin
122
COFFEY,
HANSEN,
is washed with 0.1 M phosphate buffer (pH 7.0). Co-chromatography of MS-Cowvitamin Blz and AS-CoGo-vitamin B,, on CM-cellulose (Fig. 5) resulted in the appearance of only one band of radioactivity which eluted from the column at the same position as AS-CoGo-vitamin B,, had eluted when chromatographed alone. Figure 6 demonstrates a clear separation of MIFTABLE
II
INFLUENCE OF MIF, MS, AND THE UPTAKE OF CO~~-VITAMIN BY S-180 CELLS5 Time
0
30 min
1 hour
2 hours
ASb
MS’
MIFb
+
-
-
+ f + -
+ + + + -
+ + + +
AS
ON
B12
Enhancement of Cc+’ vitamin B12 uptakeC (cpm)
121d 100 08 282 295 Oe 380 449 06 473 641 0’
a S-180 ascites cells were grown for 7 days in Swiss mice and then collected as previously described (11). The cells were washed twice with 0.9yG NaCl, and a 25ojo suspension of the cells in MEM (16) was prepared. The cells were then incubated at 37°C for various periods of time in a medium containing 0.5 ml of the cell suspension; 0.5 ml of either AS, MS, or MIF; 4.0 ml of MEM; and 0.1 ml of Co6°-vitamin Blz (25 mpg, 20 X lo* cpm). At the end of the incubation period, the cells were collected by centrifugation, washed 3 times by resuspension in MEM and re-centrifugation, and then the packed cell volume assayed for radioactivity. b The solutions of AS, MS, and MIF were diluted with MEM so that 0.5 ml of each solution would bind 15,000 cpm. c Control tubes containing the same amount of CoGa-vitamin Blz but no AS, MS, or MIF were run for each time interval. Uptake of CoBo-vitamin Blz is reported as uptake above control values. d Zero time values were obtained by adding Coeo-vitamin Blo and immediately centrifuging. e The zero indicates that MIF did not stimulate the uptake of vitamin B12 by the S-180 cells; in fact, MIF was found to inhibit the uptake of free vitamin B,.J by the S-180 cells.
AND MILLER
Co60-vitamin Blz from MS-CoGo-vitamin Blz when the two are co-chromatographed on CM-cellulose. Paranchych and Cooper (11) have shown that vitamin B1:! bound to mouse ascites fluid substances is taken up by Ehrlich ascites tumor cells in vitro while free vitamin Blz is not. Table II shows the results obtained when the effect of AS, MS, and MIF upon the absorption of CoGo-vitamin Blz by S-180 cells in vitro was studied. As can be seen, both AS and MS stimulate the uptake of vitamin Blz, but MIF has no stimulatory effect. DISCUSSION
It appears that one is justified in equat,ing the mouse stomach extract described in this paper to mouse intrinsic factor in view of the stimulatory effect of the stomach extract on the uptake of vitamin B1z by rings of mouse and guinea pig ileum (Table I). Assay systems based on the uptake of vitamin B,, by intestinal sacs or rings have been used previously several times to measure intrinsic factor activity in a number of species (14, 18). The guinea pig system was included because it has been reported that guinea pig ileum is responsive to intrinsic factor from many species. It was also demonstrated that the assay systems are specific for mouse stomach extracts (intrinsic factor) and that they do not respond to other vitamin Blz “binders” such as those found in serum and ascites fluid. As would be expected, vitamin Blz “binders” not processing intrinsic factor activity (e.g., serum binder and ascites fluid binder) actually inhibited the uptake of vitamin B1, by the intestinal rings by competing for the vitamin B,z. Data presented demonstrate that the vitamin Blz “binder” in mouse serum (MS) has properties different from those of the “binder” present in mouse stomach extracts (MIF) and similar to those of the “binder” present in mouse ascites fluid (AS). MIF is not denatured upon exposure to a pH of 1.0, and in fact forms a stable complex with vitamin Blz under these conditions. MS and AS, on the other hand, are denatured upon exposure to a pH of 1.0 or 3.0 and do not form a stable complex with vitamin B1z until
the pH of the medium
All 3 “binders”
is 5.0 or above.
are denatured at a pH of
INTRINSIC
14.0. MIF-vitamin Blz appears to have a lower isoelectric point than MS-vitamin Blz or AS-vitamin Blz (Fig. 2) since MIFvitamin Bls binds t’o the negatively charged Z-gel at a pH of 5.0 but not at higher values of pH, while MS-vitamin Bls and ASvit’amin Blz bind t’o Z-gel at pH values up to 7.0. Chromatographic data obtained by column chromatography on CM-cellulose (Figs. 3-6) further substantiate the belief that MIF-vitamin B12has a lower isoelectric point than the other complexes. Since a mixture of MS-vitamin B,, and AS-vitamin Blz elutes as a single band from CM-cellulose, and since both MS and AS stimulate the uptake of vitamin Blz by S-180 cells in vitro, these two “binders” seem to be quite similar if not identical. MIFvitamin B,z is easily separated from MSvitamin B12 and AS-vitamin Blz on CMcellulose, and MIF has no stimulatory effect 011 vitamin B,, absorption by S-180 cells; it thus appears that MIF differs considerably from MS and AS. The evidence presented in this paper demonstrates that 11s is not simply MIF which has been absorbed from the gut; however, it does not preclude the possibility that MS is a modified form of MIF which has been somehow altered during passage through the intest’inal wall. The methods used in this study measure the amount of free “binder” t,hat is present in serum and will not detect any “binder” that is saturated with vitamin Blz, e.g., MIF-vitamin Blz which had been absorbed from the gut and which was present in serum in an undissociable form. Rosenthal et al. (20) have shown that there are two forms of bound vitamin B12 in the serum of animals of several species that have received an oral dose of radioactive vitamin Blz. These workers also demonstrated two forms of bound vitamin B,, when the vitamin was added to serum in vitro (Rosenthal and co-workers did not study mouse serum). The data also appear to be consistent with the mechanism of absorption of vitamin B1? as recently proposed by Boass and Wilson (10). According to these workers, the vitamin Blp-intrinsic factor complex is absorbed into intestinal epithelial cells where it is converted into some other vitamin Blz-
FACTOR.
123
III
protein complex which is then released into both lymphatic and blood capillaries. It is possible that the vitamin Blz-intrinsic factor complex is dissociated by a releasing enzyme (21) at the basement membrane of the intestinal cells where the free vitamin B,, then becomes attached to the “binder” which has been shown to be present in serum. The MS-vitamin B12 might then be the “other vitamin B12-protein complex” of Boass and Wilson. Further work is needed to clarify these points and is now in progress in this laboratory. REFERENCES
5. 6. 7.
8. 9.
10. 11. 12. 13. 14. 15. 16. 17.
HANSEN, H. J., COFFEY, J. W., AND MILLER, 0. N., Federation Proc. 23, 187 (1964). CASTLE, W. B., Gastroenterology 37, 377 (1959). MILLER, 0. N., AND HUNTER, F. M., Proc. Sot. Exptl. Biol. Med. 96, 39 (1957). MILLER, 0. N., r2rch. Biochem. Biophys. 72, 8 (1957). MILLER, 0. N., AND HUNTER, F. M., Proc. Sot. Exptl. Biol. Med. 97, 863 (1958). HERBERT, V., Sm. J. Clin. Nutr. 7,433 (1959). HERBERT, V., AND SPAET, T. H., Am. J. Physioz. 196, 194 (1958). MILLER, 0. N., Bull. Tulane Univer. Med. Fat. 16, 115 (1957). MILLER, 0. N., RANEY, J. L., HANSEN, H. J., AND TRONCALE, F. J., Arch. Biochem. Biophys. 100, 223 (1963). BOASS, A., AND WILSON, T. H., Am. J. Physiol. 207, 27 (1964). PARANCHYCH, W., AND COOPER, B.A., Biochim. Biophys. Acta 60, 393 (1962). ELLENBOGEN, L., AND HIGHLEY, D. R., Proc. Sot. Exptl. Biol. Med. 113, 229 (1963). HANSEN, H. J., AND MILLER, 0. N., ilnal. Biothem. 7, 129 (1964). BOASS, A., AND WILSON, T., Am J. Physiol. 204, 97 (1963). SULLIVAN, L., HERBERT, V., AND CASTLE, W. B., J. Clin. Invest. 42, 1443 (1963). EAGLE, H., Science 130,432 (1959). HANSEN, H. J. COFFEY, J. W., AND MILLER, 0. N., Unpublished data.
18. WOLFF,R., AND N&BET, p., in “Vitamin B,~ and Intrinsic Factor” (H. C. Heinrich, ed.), p. 514. Ferdinand Enke, Stuttgart, (1962). 19. WILSON, T., AND STRAUSS, E., Am. J. Physiol. 19’7, 926 (1959). 20. ROSENTHAL, H. L., HILL, R. C., AND HAESSLER, I., Federation Proc. 23, 187, (1964). 21. HERBERT, V., COOPER, B. A., AND CASTLE, W. B., Proc. Sot. Exptl. Biol. Med. 110, 315 (1962).
OF
BIOCHEMISTRY
Studies
AND
BIOPHYSICS
on the Interaction and
III. Chemical
and Biological the Stomach, 5. W. COFFEY,
117-123 (1965)
110,
of Vitamin
B12: Intrinsic
Factor
Receptors
Properties
of Vitamin
B,, Combining
Substances
from
Serum, and Ascites Fluid of the Mouse’ HANS J. HANSEN,
AND
0. NEAL
Department of Biochemical Research, Touro Research Institute; and the Nutrition tory of the Departments of Biochemistry and Medicine, Tulane University Neul Orleans, Louisiana Received
November
MILLER and Metabolism LaboraSchool of Medicine
10, 1964
The chemical and biological properties of vitamin Blz combining substances in mouse stomach extracts (MIF), mouse serum (MS), and mouse ascites fluid (AS) were compared. MIF was found to enhance the absorption of vitamin Blz by rings of mouse and guinea pig ileum and to inhibit its absorption by sarcoma-180 (S-180) cells in vitro. 11s and AS, on the other hand, enhanced the absorption of vitamin Blz by S-180 cells but inhibited its absorption by rings of mouse and guinea pig ileum. It was shown that MIF is capable of forming a stable complex with vitamin Blz at pH values from 1.0 to 11.0, while MS and AS are not capable of forming such stable complexes at pH values below 5.0. The complex formed between vitamin Blz and MIF (MIF-vitamin B,?) is not adsorbed by zirconyl phosphate gel (Z-gel) at pH values higher than 5.0; however, the complexes formed between vitamin Bi2 and MS (MSvitamin B,z) or AS (AS-vitamin Bi2) are adsorbed by Z-gel at pH values as high as 7.0. By chromatography on CM-cellulose, it was possible to separate MIF-vitamin B,, from MS-vitamin Bis and AS-vitamin Bip. The latter two complexes behaved identically when chromatographed on CX-cellulose.
It is recognized that, unless given in a massive dose, vitamin B,, must combine with intrinsic factor prior to absorption from the gastrointestinal tract (2). Miller and Hunt’er (3) have demonstrated that hog intrinsic factor concentrates facilitate the uptake of vitamin B,, by slices of rat liver and kidney. It was also shown that receptor proteins in serum (4) and homogenates of rat liver (5) will combine intrinsic factorvitamin B12 complex and thus prevent it’s adsorptiol: by charcoal. Reports (6, 7) have appeared concerning the presence of an irurinsic factor-like substance in blood plasma. Therefore, it was postulated (8) that intrinsic factor may be absorbed into the 1 Supported by the John A. Hartford Foundation, and the U.S. Public Health Service (grant A-l). A preliminary report has appeared (1).
blood stream where it may facilitate the absorption of vitamin Blz by peripheral tissues in a manner analogous t)o its action in bhe gut. Data have been presented which demonstrate that both intrinsic factor and intrinsic factor-vit,amin B1, complex can cross the cell membrane of liver cells of the rat’ following their intravascular administration (9). More recently, data have been presented (10) which suggest t’hat the intrinsic factor-vitamin Blz complex may be absorbed into intestinal epithelial cells where it is converted into some other vitamin Blz-protein complex which is then released into the bloodstream. Results reported below compare some of the properties of the vitamin Bit “binders” found in extracts of mouse stomach (mouse intrinsic factor), in mouse serum, and in 117
118
COFFEY,
HANSEN,
ascites fluid (11) collected from mice bearing sarcoma-180 (S-180) tumor cells. MATERIALS
AND
METHODS
Chemicals. CobaltGO- and Co&r-labeled vitamin B,z were generously provided by E. R. Squibb and Sons. Nonradioactive vitamin Blz was obtained from Merck, Sharp and Dohme. The radioactivity of all samples containing CoGo- or Cos7-vitamin Brz was assayed by means of a center-well gamma scintillation counter. The specific activity of the CoGO-vitamin B1z was 800 counts per minute (cpm) per millimicrogram as determined by means of the counting system described above. Cobaltsr-vitamin B,z was used for those experiments in which the effect of the different binders on the uptake of vitamin Blz by intestinal rings was studied. It had a specific activity of 1830 cpm per millimicrogram. Animals. Swiss mice purchased from the A. R. Schmidt Co., Madison, Wisconsin were used throughout this study, and were maintained on a laboratory chow diet up to the time of sacrifice. The S-180 ascites tumor, which was provided by Dr. Tom McCoy, Noble Foundation, Ardmore, Oklahoma, was transferred every 7 days by intraperitoneal inoculation of stock mice with 0.1 ml of a 10% suspension of tumor cells in 0.9% NaCl. Preparation of vitamin Blz “binders”. Mouse intrinsic factor (MIF) was prepared by the method previously described for rat intrinsic factor (12) from 3 stomachs which were carefully washed with cold 0.9% NaCl and homogenized in 10 ml of icecold distilled water by means of a Waring Blendor. The homogenate was dialyzed with stirring against 500 volumes of cold distilled water for 5 hours and then centrifuged at 8000 g for 10 minutes. The supernatant was decanted from the sedimented pellet and saved as a source of MIF. Ascites fluid was collected as described by Paranchych and Cooper (II), dialyzed against distilled water, and centrifuged at 8000g for 10 minutes; the supernatant was used as a source of ascites fluid binder (AS). Serum prepared from blood collected by heart puncture was used as a source of serum binder (MS). The vitamin B12 binding capacity of each of the solutions (MIF, AS, MS) was determined by the zirconyl phosphate (Z-gel) method of Hansen and Miller (13). This method is based on the observation that Z-gel at a pH of 5.0 will adsorb the complexes formed between vitamin Bls and acceptors such as MIF, MS, and AS but will not adsorb free vitamin Biz. Stock solutions of MIF, AS, and MS were prepared by diluting the above solutions with distilled water so that 1 ml of each of the stock solutions would bind between 6 X 103 and 10 X 10s cpm (S-12 mpg) of CoeO-vitamin B,, to Z-gel at pH 5.0.
AND
MILLER
Effect of pH on the binding of GoGO-vitamin Blz by MIF, MS, and AS. One ml of the stock solutions of MIF, AS, and MS prepared as described above was added to 1 ml of each of the following solutions: HCI (0.2 1V) ; formate buffer (pH = 3.0, p = 0.2); acetate buffer (pH = 5.0, p = 0.2); phosphate buffer (pH = 7.0, p = 0.2) ; bicarbonate buffer (pH = 9.0 or 11.0, p = 0.2), and NaOH (0.2 A;). The final ionic strength of each of the solutions was 0.1, and the pH values ranged from 1 to 14. Fifteen mg of Coeo-vitamin B,, (12,000 cpm) in a volume of 15 ~1 was added with shaking to each of the tubes, followed by the addition of 1 pg of nonradioactive vitamin Bit; and the tubes were allowed to stand at room temperature for 15 minutes. Twelve ml of ammonium acetate2 (pH = 5.0, 0.1 &f) was then added to each tube followed by 1 ml of Z-gel prepared as described eIsewhere (13). The tubes were inverted 12 times by hand and centrifuged at 80009 for 5 minutes, and the supernatant was decanted from the sedimented Z-gel pellet. The pellet was washed once by resuspension in 15 ml of ammonium acetate and re-centrifugation. The amount of CoGo-vitamin Bn bound to the pellet was determined with the gamma scintillation counter. The stability of the complexes between MIF, MS, or AS and CoGo-vitamin B12 was determined by exposing the complexes to a 70-lOO-fold excess of nonradioactive vitamin B12. Instability of the complex would result in an equilibration between the radioactive vitamin Brz and the nonradioactive vitamin Blz. This equilibration would produce a reduction in the amount of CoGO-vitamin Blz bound to Z-gel as compared with a control in which no nonradioactive vitamin B,z was added. Conversely, failure of the presence of a large excess of unlabeled vitamin Blz to dilute the radioactivity of the complex can be interpreted as evidence of stability of the complex. Uptake of vitamin B,z by rings of mouse and guinea pig ileum. The effect of MIF, MS, and AS on the absorption of vitamin Bn by rings of mouse and guinea pig ileum was studied by the method of Boass and Wilson (14). This method involves the use of thin rings of intestinal tissue as an assay system for the study of intrinsic factor-facilitated absorption of vitamin Brx. Rings of mouse intestine were prepared from the lower third of the intestine because preliminary experiments demon2 In most cases the addition of the ammonium acetate brought the pH of the reaction mixture to 5.0. If it did not, the reaction mixture was adjusted to a pH of 5.0 before the Z-gel was added since the in vitro assay for vitamin Brz binding capacity as described by Hansen and Miller (13) is carried out in a medium at pH 5.0.
INTRINSIC
FACTOR.
factor preparation to behave, i.e., it stimulates the uptake of Co6’-vitamin B12 by the intestinal rings. The serum binder and the ascites binder have no stimulatory effect on the uptake of vitamin Bls, and, in fact, inhibit the uptake of vitamin B, by the intestinal rings. Figure 1 shows the results obtained when the binding of COW-vitamin Bl? to MIF, MS, and AS was studied at pH values ranging from 1.0 to 14.0. The MIF binds vitamin Blz, and a stable complex is formed, as determined by the Z-gel method (see Materials and JleLhods), at all pH values between 1.0 and 11.0; the ?rlS and AS form a stable complex with vitamin B,z at pH values between 5.0 and 11.0 with no binding of vitamin B12at pH 1.0 and very little binding at pH 3.0. If the addition of unlabeled vitamin B12 to the incubation mixture is omitted (see Materials and Methods), the curves obtained are identical; thus indicating that once MIF is exposed to a medium with a pH of 14.0 and 31s or AS to media with pH’s of 1.0, 3.0, or 14.0, they are irreversibly altered and lose their ability bo bind vitamin B,, even when the pH is readjusted to 5.0. Highly purified hog in-
strated that this portion of the intestine shows the greatest degree of intrinsic factor-facilitated absorption of vitamin Bls. The lower half of the guinea pig intestine was used for the same reason (15). Rings prepared from three animals were mixed and used randomly for the assays. Approximately 500 mg (wet weight) of intestinal rings was placed in a Warburg flask containing 2.4 ml of Eagle’s minimum essential medium (MEM) (16), 0.1 ml of Cob’-vitamin B,z which contained 30 X lo3 cpm, and 0.5 ml of the appropriate binder having a binding capacity of 25,000 cpm of Cos7-vitamin Blz. The flasks were incubated at 37°C for 2 hours with shaking, after which their contents were transferred to test tubes and the intestinal rings were collected by centrifugation. The rings were washed 3 times with 10 ml of ice-cold saline and their radioactivity was then assayed in the gamma scintillation counter. Chromatography. CM-cellulose was purchased fromMann Research Laboratories. Prior to its use, the CM-cellulose was suspended in 1 M NHaOH at room temperature for 15 minutes. The NHdOH was removed by filtration and the CM-cellulose was washed free of NHdOH with water. RESULTS
The data presented in strate that the mouse (JIIF) does behave in the tems as one would expect
Table I demonstomach extract in vitro assay sysa mouse intrinsic TABLE
OF MIF,
INFLUENCE
MS,
Alouse
stomach
I
AND AS ON THE UPTAKE OF CO~~-VITAMIN MOUSE AND GUINEA PIG ILEUM”
Preparation
Assay system
extract
(h/IIF)
Mouse Guinea
Serum binder
Ascites
fluid
(MS)
binder
(AS)
119
III
ileum pig ileum
Mouse
ileum
Guinea
pig ileum
Mouse Guinea
ileum pig ileum
B12 BY RINGS
OF
Enhancement of Co57 vitamin B,* uptake” (cpm/gm tissue)
Expt. Expt. Expt. Expt. Expt. Expt. Expt. Expt. Expt. Expt. Expt. Expt.
1 2 1 2 1 2 1 2 1 2 1 2
4767 4026 1244 1006 0” 0 0 0 0 0 0 0
a See Materials and Methods for experimental details. b Uptake of Co67-vitamin B12 is reported as uptake above control values. Control values were obtained by including a flask containing the same amount of Cos7-vitamin B,z as experimental flasks but no MIF, MS, or AS. Two experiments were run and both values are reported. c Zero values indicate no uptake above control values. ?vlS and AS actually inhibited the uptake of free COST-vitamin B,t by the intestinal rings.
120
COFFEY,
HANSEN,
AND
MILLER
-
pH 5.0
_____, I - 18 - 16
H o--O
2 $ >O.l t 0.09-
Mouse Stomach extract (MIF) Mouse Serum (MS)
UJ 5 o -I 2 i
/
3
5
7
9
II
13
I5
PH
%
a 6.0
7.0
80
0 x
-12
- IO ,: 0 8
-
-
gg o 0.010
FIG. 1. The effect of pH on the binding of Coeovitamin B,, by MIF, MS, and AS. See Materials and Methods for experimental details.
50
0.08 0.07 0.06 0.05 0.04
- 14?
5
IO TUBE
I5 NUMBER
-
6
-
4
-
2
20
FIG. 3. Chromatography of MIF-CoeO-vitamin B 12 on CM-cellulose. A column of CM-cellulose (2 X 15 cm) was prepared (see i!!faterials and Methods) and washed with 0.1 M ammonium acetate (pH = 5.0) until the pH of the solvent flowing from the column was 5.0. Two ml of MIF having a Coeo-vitamin B12 binding capacity of 87 X lo3 cpm/ml (determined by Z-gel method) was mixed with sufficient Coeovitamin Blz to completely saturate the binder. Excess CoeO-vitamin Blz was removed by dialysis against 0.1 M ammonium acetate (pH = 5.0), and the dialyzed material was applied to the column. Material was eluted from the column by the stepwise washing of the column with 0.1 M ammonium acetate (pH = 5.0), 0.1 M ammonium acetate (pH = 6.0), 0.1 M phosphate buffer (pH = 7.0), and 0.1 M phosphate buffer (pH = 8.0). Ten-ml fractions were collected, and a 4-ml aliquot of each fraction was assayed for radioactivity.
pH of Z-gel FIG. 2. The effect of pH on the binding of MIFCo60-vitamin Bi2, MS-CoGO-vitamin Blz, and ASCorn-vitamin Biz to Z-gel. Complexes of Cocavitamin Bi2 withMIF, MS, and AS were formed by adding 10 X lo3 cpm (12 mpg) of Co@-vitamin Blz to 1 ml of the stock solutions of MIF, MS, and AS in a series of tubes containing a total volume of I2 ml and having the pH values indicated on the abscissa (0.1 M phosphate buffers). One ml of Z-gel having the same pH as the buffer in the tube containing the Coeo-vitamin Biz complex was added to each tube and the tube was shaken. The Z-gel was collected by centrifugation, washed, and assayed for radioactivity. Control tubes containing the same amount of CoGO-vitamin Bit but no MIF, MS, or AS were run at each pH value. In the absence of MIF, MS, or AS the Z-gel never bound more than 2% (266 cpm) of the added Co’o-vitamin EL.
FIG. 4. Chromatography of AS-Coso-vitamin Biz on CM-cellulose. See legend of Fig. 3 for experimental details.
INTRINSIC
FACTOR.
121
III
trinsic factor, rat intrinsic factor, and Z-gel at pH values of 5.0, 6.0, and 7.0, but hamster intrinsic factor gave results identical not at 8.0. Figures 3-6 show the results obtained with those obtained with MIF (17). B12, MS-CoGoSince the complexes formed between when MIF-Cow-vitamin vitamin Blz and MIF, MS, and AS were all vitamin Blz, and AS-Co@‘-vitamin B1zwere stable at pH values ranging from 5 to 11 chromatographed on columns of CM-cellu(Fig. l), it was possible to study the binding lose. As Fig. 3 shows, the MIF-Coeoof these complexes to Z-gel at a number of vitamin Blz does not attach to a column of different pH values. As can be seen (Fig. 2) CM-cellulose that has been equilibrated the MIF-C060-vitannn B12complex binds to with 0.1 M ammonium acetate at pH 5.0; Z-gel at pH 5.0 but not at higher pH values. however, the ASCo60-vitamin Blz (Fig. 4) On the other hand, the complexes of MS does attach to the CM-cellulose under these and AS with CoGo-vitamin B,, bind to conditions and is not eluted until the column
-6
1.0 g
0.9
$j
ae
z
0.7
-OPTICAL
DENSITY
-5
RADIOACTIVITY -4 6
z z 0.6 w ; 0.5 4 0 a4 ii 0
3;
0 0
-2
a3 I
0.2 0. I
0 5
0
IO
15
20
25
30
40
35
TUBE
45
50
6.04
5.0+pH
PH
7.04
Bls and MS-CoGO-vitamin
PH B.0-d J 9
t E’ i
60
NUMBER
FIG. 5. Chromatography of a mixture of AS-CocO-vitamin Bit on CM-cellulose. See legend of Fig. 3 for details.
pH
55
1.6
-
OPTICAL
DENSITY
RADIOACTIVITY
1.4
z 1.2 Gi 2 1.0 iii 2 0.B U Ib
0.6 0.4 0.2
IO
20
30
50
40 TUBE
60
70
NUMBER
FIG. 6. Chromatography of a mixture of MIF-Cos’J-vitamin B1, on CM-cellulose. See legend of Fig. 3 for details.
Btz and MS-Co6c-vitamin
122
COFFEY,
HANSEN,
is washed with 0.1 M phosphate buffer (pH 7.0). Co-chromatography of MS-Cowvitamin Blz and AS-CoGo-vitamin B,, on CM-cellulose (Fig. 5) resulted in the appearance of only one band of radioactivity which eluted from the column at the same position as AS-CoGo-vitamin B,, had eluted when chromatographed alone. Figure 6 demonstrates a clear separation of MIFTABLE
II
INFLUENCE OF MIF, MS, AND THE UPTAKE OF CO~~-VITAMIN BY S-180 CELLS5 Time
0
30 min
1 hour
2 hours
ASb
MS’
MIFb
+
-
-
+ f + -
+ + + + -
+ + + +
AS
ON
B12
Enhancement of Cc+’ vitamin B12 uptakeC (cpm)
121d 100 08 282 295 Oe 380 449 06 473 641 0’
a S-180 ascites cells were grown for 7 days in Swiss mice and then collected as previously described (11). The cells were washed twice with 0.9yG NaCl, and a 25ojo suspension of the cells in MEM (16) was prepared. The cells were then incubated at 37°C for various periods of time in a medium containing 0.5 ml of the cell suspension; 0.5 ml of either AS, MS, or MIF; 4.0 ml of MEM; and 0.1 ml of Co6°-vitamin Blz (25 mpg, 20 X lo* cpm). At the end of the incubation period, the cells were collected by centrifugation, washed 3 times by resuspension in MEM and re-centrifugation, and then the packed cell volume assayed for radioactivity. b The solutions of AS, MS, and MIF were diluted with MEM so that 0.5 ml of each solution would bind 15,000 cpm. c Control tubes containing the same amount of CoGa-vitamin Blz but no AS, MS, or MIF were run for each time interval. Uptake of CoBo-vitamin Blz is reported as uptake above control values. d Zero time values were obtained by adding Coeo-vitamin Blo and immediately centrifuging. e The zero indicates that MIF did not stimulate the uptake of vitamin B12 by the S-180 cells; in fact, MIF was found to inhibit the uptake of free vitamin B,.J by the S-180 cells.
AND MILLER
Co60-vitamin Blz from MS-CoGo-vitamin Blz when the two are co-chromatographed on CM-cellulose. Paranchych and Cooper (11) have shown that vitamin B1:! bound to mouse ascites fluid substances is taken up by Ehrlich ascites tumor cells in vitro while free vitamin Blz is not. Table II shows the results obtained when the effect of AS, MS, and MIF upon the absorption of CoGo-vitamin Blz by S-180 cells in vitro was studied. As can be seen, both AS and MS stimulate the uptake of vitamin Blz, but MIF has no stimulatory effect. DISCUSSION
It appears that one is justified in equat,ing the mouse stomach extract described in this paper to mouse intrinsic factor in view of the stimulatory effect of the stomach extract on the uptake of vitamin B1z by rings of mouse and guinea pig ileum (Table I). Assay systems based on the uptake of vitamin B,, by intestinal sacs or rings have been used previously several times to measure intrinsic factor activity in a number of species (14, 18). The guinea pig system was included because it has been reported that guinea pig ileum is responsive to intrinsic factor from many species. It was also demonstrated that the assay systems are specific for mouse stomach extracts (intrinsic factor) and that they do not respond to other vitamin Blz “binders” such as those found in serum and ascites fluid. As would be expected, vitamin Blz “binders” not processing intrinsic factor activity (e.g., serum binder and ascites fluid binder) actually inhibited the uptake of vitamin B1, by the intestinal rings by competing for the vitamin B,z. Data presented demonstrate that the vitamin Blz “binder” in mouse serum (MS) has properties different from those of the “binder” present in mouse stomach extracts (MIF) and similar to those of the “binder” present in mouse ascites fluid (AS). MIF is not denatured upon exposure to a pH of 1.0, and in fact forms a stable complex with vitamin Blz under these conditions. MS and AS, on the other hand, are denatured upon exposure to a pH of 1.0 or 3.0 and do not form a stable complex with vitamin B1z until
the pH of the medium
All 3 “binders”
is 5.0 or above.
are denatured at a pH of
INTRINSIC
14.0. MIF-vitamin Blz appears to have a lower isoelectric point than MS-vitamin Blz or AS-vitamin Blz (Fig. 2) since MIFvitamin Bls binds t’o the negatively charged Z-gel at a pH of 5.0 but not at higher values of pH, while MS-vitamin Bls and ASvit’amin Blz bind t’o Z-gel at pH values up to 7.0. Chromatographic data obtained by column chromatography on CM-cellulose (Figs. 3-6) further substantiate the belief that MIF-vitamin B12has a lower isoelectric point than the other complexes. Since a mixture of MS-vitamin B,, and AS-vitamin Blz elutes as a single band from CM-cellulose, and since both MS and AS stimulate the uptake of vitamin Blz by S-180 cells in vitro, these two “binders” seem to be quite similar if not identical. MIFvitamin B,z is easily separated from MSvitamin B12 and AS-vitamin Blz on CMcellulose, and MIF has no stimulatory effect 011 vitamin B,, absorption by S-180 cells; it thus appears that MIF differs considerably from MS and AS. The evidence presented in this paper demonstrates that 11s is not simply MIF which has been absorbed from the gut; however, it does not preclude the possibility that MS is a modified form of MIF which has been somehow altered during passage through the intest’inal wall. The methods used in this study measure the amount of free “binder” t,hat is present in serum and will not detect any “binder” that is saturated with vitamin Blz, e.g., MIF-vitamin Blz which had been absorbed from the gut and which was present in serum in an undissociable form. Rosenthal et al. (20) have shown that there are two forms of bound vitamin B12 in the serum of animals of several species that have received an oral dose of radioactive vitamin Blz. These workers also demonstrated two forms of bound vitamin B,, when the vitamin was added to serum in vitro (Rosenthal and co-workers did not study mouse serum). The data also appear to be consistent with the mechanism of absorption of vitamin B1? as recently proposed by Boass and Wilson (10). According to these workers, the vitamin Blp-intrinsic factor complex is absorbed into intestinal epithelial cells where it is converted into some other vitamin Blz-
FACTOR.
123
III
protein complex which is then released into both lymphatic and blood capillaries. It is possible that the vitamin Blz-intrinsic factor complex is dissociated by a releasing enzyme (21) at the basement membrane of the intestinal cells where the free vitamin B,, then becomes attached to the “binder” which has been shown to be present in serum. The MS-vitamin B12 might then be the “other vitamin B12-protein complex” of Boass and Wilson. Further work is needed to clarify these points and is now in progress in this laboratory. REFERENCES
5. 6. 7.
8. 9.
10. 11. 12. 13. 14. 15. 16. 17.
HANSEN, H. J., COFFEY, J. W., AND MILLER, 0. N., Federation Proc. 23, 187 (1964). CASTLE, W. B., Gastroenterology 37, 377 (1959). MILLER, 0. N., AND HUNTER, F. M., Proc. Sot. Exptl. Biol. Med. 96, 39 (1957). MILLER, 0. N., r2rch. Biochem. Biophys. 72, 8 (1957). MILLER, 0. N., AND HUNTER, F. M., Proc. Sot. Exptl. Biol. Med. 97, 863 (1958). HERBERT, V., Sm. J. Clin. Nutr. 7,433 (1959). HERBERT, V., AND SPAET, T. H., Am. J. Physioz. 196, 194 (1958). MILLER, 0. N., Bull. Tulane Univer. Med. Fat. 16, 115 (1957). MILLER, 0. N., RANEY, J. L., HANSEN, H. J., AND TRONCALE, F. J., Arch. Biochem. Biophys. 100, 223 (1963). BOASS, A., AND WILSON, T. H., Am. J. Physiol. 207, 27 (1964). PARANCHYCH, W., AND COOPER, B.A., Biochim. Biophys. Acta 60, 393 (1962). ELLENBOGEN, L., AND HIGHLEY, D. R., Proc. Sot. Exptl. Biol. Med. 113, 229 (1963). HANSEN, H. J., AND MILLER, 0. N., ilnal. Biothem. 7, 129 (1964). BOASS, A., AND WILSON, T., Am J. Physiol. 204, 97 (1963). SULLIVAN, L., HERBERT, V., AND CASTLE, W. B., J. Clin. Invest. 42, 1443 (1963). EAGLE, H., Science 130,432 (1959). HANSEN, H. J. COFFEY, J. W., AND MILLER, 0. N., Unpublished data.
18. WOLFF,R., AND N&BET, p., in “Vitamin B,~ and Intrinsic Factor” (H. C. Heinrich, ed.), p. 514. Ferdinand Enke, Stuttgart, (1962). 19. WILSON, T., AND STRAUSS, E., Am. J. Physiol. 19’7, 926 (1959). 20. ROSENTHAL, H. L., HILL, R. C., AND HAESSLER, I., Federation Proc. 23, 187, (1964). 21. HERBERT, V., COOPER, B. A., AND CASTLE, W. B., Proc. Sot. Exptl. Biol. Med. 110, 315 (1962).