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The absorption of vitamin B12 in animals I. The effect of different binding and intrinsic factors
CLINICA CHIMICA ACT.4
THE I. THE
EFFECT
ABSORPTION
OF VITAMIN
OF DIFFERENT
BINDING
E. S. HOLDSWORTH*
853
B,, IN ANIMALS AND
INTRINSIC
AND &I. E. COATES
National Instifute for Research in Dairy&tag, Shinfield, Reading (Received
FACTORS
(Great Briiain)
April rrth, 1960)
These experiments were begun partly in the hope of devising an animal test to replace or supplement clinical trials of intrinsic factor activity and partly to elucidate the relation, if any, between vitamin B,,-binding and the function of intrinsic factor * *. The most usual source of intrinsic factor preparations for clinical use is the gastro-intestinal tract of the pig. Since feeding experiments have shown that preparations from one species of animal are not necessarily effective in promoting uptake of vitamin B,, in other species’ it seemed logical in the first place to use the pig as a subject for assay. When these attempts proved unsuccessful the rat was chosen as a cheaper and more convenient means of studying the broader aspects of the problem. MATERIALS AND METHODS
A‘%iVCalS
For experiments with pigs, in order to keep down the amounts of test materials needed and the bulk of tissue to be processed, 2-3 week old piglets were chosen in preference to larger animals. They were taken from the sow about an hour before the experiment began. Littermates of both sexes of the Large White breed were obtained from two sources. The animals varied in weight depending on their origin and the size of the litter; thus animals from some litters weighed from 2; to 4 kg and others from 5 to 6 kg, but within one experiment the weights were kept as even as possible. In experiments with rats, adult black-hooded males from our own colony were used. They had been reared on a good stock diet and weighed between 200-300 g. Materials
tested
Vitamin B,, labelled with 5sCo (specific activity 2.5 ,uC/pg) or %o (specific activity 0.9 ,&/,& was used. These samples were mostly gifts from Dr. E. LESTER SMITH, F.R.S. (Glaxo Laboratories Ltd.). Bendogen, a preparation of pig pyloric mucosa clinically-active in pernicious anaemia, was kindly supplied by GEA (Copenhagen} andapurified fraction, also clinically-active, was prepared from it 2. Pig gastric juice was obtained from an &week old pig that had been reared on an artificial milk diet. Under ether anaesthesia a small abdominal incision was made and a ligature placed just above the pylorus. The animal was killed 12 h later and the accumulated * Present address: Biochemistry Department, University of Adelaide, South Australia. ** Throughout this work the term “intrinsic factor” refers to the substance(s) present in the gastro-intestinal tract of any animal which are essential for the proper absorption of vitamin B,,. where reference is made particularly to a material It is prefaced by the term ‘*clinically-active” effective in human pernicious anaemia. Clin. Chim. Acta, 3 (1960) 853.861
Q4
E.S.HOLDSWORTH,M.E.COATES
gastric secretion was removed and filtered. A sample of clinically-active human gastric juice was kindly supplied by Dr. C. C. BOOTH, Postgraduate Medical School, Hammersmith. Fresh sow’s milk was obtained from a lactating sow after injection of posterior pituitary extract, and the sow’s milk binding factor was that described by GREGORY AND HOLDSWORTH~. Crude stomach extracts were prepared from freshly excised stomachs of both pigs and rats. The stomachs were slit open, washed rapidly to remove adhering food, ground with 0.9% NaCl in 0.01 N HCl, dialysed against running tap water, and the resulting extracts freeze-dried. The vitamin B12-protein complex from Ochromonas malhamensis was kindly provided by our colleague Dr. J. PAWELKIEWICZ, and was isolated from the cell contents medium containing G°Co-vitamin B,,. Measurement
of binding
of 0. malhamensis
grown in a
activity
In all the experiments reported here the test materials were added in sufficient quantity exactly to bind the dose of vitamin B1,. Some microbiological measurements of vitamin B,,-binding activity were kindly made for us by our colleague Dr. XI. E. GREGORY. In general, binding activity was measured by determining the amouDi of radioactivity ultrafiltrable from a mixture of B°Co-vitamin B,, and the material being tested. These methods have been described by GREGORY AND HOLDSWORTH *.
Surgical
fwocedure
Pigs were anaesthetized with cyclopropane and rats with ether. This type of anaesthesia was chosen so that the animals recovered consciousness a short time after the operation was completed. An abdominal incision was made, :c expose most of the small intestine, and ligatures were placed at the two extremes of the length of intestine required for experiment. The intestine was cut below an;1 above the proximal and distal ligatures respectively, and a glass cannula was tied mto the upper end of the length under experiment. Warm normal saline (38”) was passed through to remove food and any possible secretion from the upper part of the gut that might influence uptake of vitamin B,,. About I 1 of saline was used in the pig, and about 200 ml in the rat. The cannula was removed and the lower end of the length of intestine was tied off. The experimental dose was delivered mto the upper end by means of a syringe fitted with a blunt-ended needle and a ligature was made as the needle was withdrawn, thus forming a blind length ot intestine enclosing the test dose of vitamin B 1 2 with or without the material under investigation. Where more than one loop was required, the length of intestine was divjdzd into appropriate portions after washing and each was treated separately as described. The gut was replaced and the incision closed, and the animals were allowed to live for 16-20 h. During this time they were kept in a warm room in separate cages and the excreta were collected on filter paper. The animals were supplied with water but no food. At the end of the experiment they were again anaesthetized, the abdomen was reopened and the loop(s) removed. They were then killed and appropriate specimens such as liver, muscle or the whole carcase were taken as required for counting. Prefiaratiolz
qf materials
for counting
4-g samples were convenient for the available equipment, hence when the specific activity was high enough the radioactivity of pieces of tissue was counted CZin.Cltim.
A&,
j (1960)853-861
ABSORPTION
directly.
Thus
processing;
in the rat
the counts
B,,.
OF VITAMIN
experiments
I.
each intestinal
in 4-g samples
855
loop was counted
of liver could be related
without
to the weight of
the whole organ. The pig intestinal loops were digested with 4 N NaOH and 4-ml aliquots taken. Carcases, pieces of muscle and the filter papers on which urine and faeces were collected were ashed at about 700’ and either a portion or the total ash was counted. Counting
methods
Counts
were made in a well-type
activated
NaI
crystal
scintillation
counter.
Standards were counted at the same time and the results expressed as vitamin B,, on the assumption that all the radioactive material was vitamin B,,. Recoveries were usually at least 95%. Counts were made on samples of untreated tissue but proved so low as to be negligible. RESULTS
Expe:periments with pigs In the first experiment IOO mpg %o-vitamin B r2 was placed in a loop of intestine 3 feet long, the upper end being about 9 inches below the pylorus to avoid the area believed to be responsible for the production of “intrinsic factor” in the pig5. One pig received the vitamin alone, another vitamin B,, and Bendogen, while the third received vitamin B,, with a clinically-active fraction, prepared from Bendogen 2. The amounts of vitamin B,,, measured as Wo, appearing in the tissue after zz h are given in Table
I. It is apparent
that
both Bendogen TABLE
ABSORPTION
DURING
22
h
OF
VITAMIN
PREPARATIONS
Dose
Substance
given with
INTO
of
vitamin B,,
vitamin B,,
None Bendogen Purified intrinsic factor *
(mpg) 100
100 100
B,,
WITH
OR
WITHOUT
LOOPS
IN
BABY
Amount left Percentage _mpg in gut loop absorbed muscle(mpg)
67.5 92.6 96.6
fraction
markedly
I
PLACED
INTESTINAL
and its purified
32.5 7.4 3.4
INTRINSIC
FACTOR
PIGS
vitamin B12 in tissues
(IOO g)
~ liver (whole)
0.38 0.30 0.30
8.4 4.2 1.9
blood (JO ml)
excreta (total)
0
2.2
0
0.9
0
0.1
* Holdsworth2.
depressed uptake of vitamin Blz. As this effect might have been peculiar to one part of the intestine only, further experiments were done at different sites in the small intestine and several loops were made in each animal to cover a greater length of gut. The results are summarized in Table II. In every instance the levels of vitamin B,, found in the tissues were concordant with the amount that had disappeared from the loop. It is evident from these results that absorption of the vitamin was greater from the middle and lower parts of the small intestine than from the upper, and that on two out of three occasions the presence of Bendogen depressed the uptake of vitamin B,, at all positions in the intestine. Two fractions prepared from Bendogen, one clinically-active and one with vitamin B,,- binding activity only, both depressed absorption of vitamin B,, to about the same extent as Bendogen. It was somewhat surprising that Bendogen, prepared from the pyloric mucosa Clin. Chim. Acta, 5 (1960) 853-861
Sj6
E. S. HOLDSWORTH. M. E. COATES TABLE
EFFECT
OF INTRINSIC VITAMIN
Total
FACTOR
R 1z FROM
5
5 ft. Lower end 2 ft. above ileo-caecal junction
x
Ift.
20
20
5 x 3ft.
FACTORS
SMALL INTESTINE ______
- AtoRe
40
5 x I ft.
ft. Lower end at ileo-caecal junction
BINDING
ON THE OF THE .._--
ABSORPTION BABY
OF
PIG ~
____.
_
94 of dose of u%tamzxB I2 absorbed +
nzpg vitamin B,, placed i% loop
3 x 4ft.
5 ft. Lower end 2 ft. above ileo-caecal junction
AND
DIFFERENT SITES IN THE ~~. ___--_..
No. and length, of loops
lengtk and position of site examined
12 ft. Upper end 9 in. below pylorns
15
PREPARATIONS
II
20
+ Ben- + purifedt intrinsic dogen factor
+ bindingy factor
+ sozv’s milk t i factor
10
0
2
19 32 33 22 49 52 36 43 47 37 31 25 57 43 50 “7
4
3
20
I
33 44 50 55 49 IO I2
I4 14 23 6 I’3 21 16 14
20
13
IO
IO
ICI
II
‘3
7 ‘7
II
7
* Results given in order from proximal to distal ends of the small intestine. t Holdsworthz. tt Gregory and Holdsworth3. TABLE AMOUNTS -
OF VITAMIN
B,,
IN
-.
Substance given with vitamix B,, None Bendogen Sow’s milk binding factor Fresh sow’s milk
LIVER
AND
MUSCLE
IOO mpg .~
III OF BABY
WO-VITAMIN ___x_..
PlGS
1.6 4.3
0.2
0.7
h
AFTER
AN
ORAL
DOSE
OF
._.~
mpg vitamin Bl,/liver _...-__ Expt. I Em@. 2 Ex$% 3 2.4 2.3
8
B,,
I.0 2.3
-
mpg vitamin B,,/Ioo g muscle --~______ I%@. I Expt. 2 Expt. 3 0.19
0.04
0.01
0.20
0.17
0.11
0.03 0
of the pig, should hinder absorption of vitamin B,, in the same species. Another vitamin B,,-binding substance from the pig, the sow’s milk factor3, was therefore investigated (Table II, experiment with Bendogen) and proved to have an even greater inhibitory effect on vitamin B,, uptake than had Bendogen under the conditions of the experiment. The effect of both of these substances on the absorption of vitamin B 12given by mouth to pigs was tested. Absorption was assessed on the levels of vitamin B,, found in the livers and samples of back muscle taken 8 h after the dose had been given by mouth. Fresh sow’s milk was also tested, should the method of preparation of the sow’s milk binding factor have damaged the vitamin B,,-protein complex, thereby rendering it unavailable to the piglet. Table III summarizes the results. In all three experiments both liver and muscle levels of vitamin B,, indicated that Bendogen given by mouth did not interfere with absorption and may even have
ABSORPTION OF VITAMIN TABLE ABSORPTION
DURING
18
B ra
h OF VITAMIN
PLACED
WITH
INTESTINAL
Dose: mpg vitamin B 12 Eeft in Eoo,O
Substance given wit12 vztamzn B,,
-Tip Middle Lowest
7.7
7.7
LOOPS
B,,. I.
857
IV OR WITHOUT OF THE
INTRINSIC
FACTOR
PREPARATIONS
INTO
RAT
ro m,~g vitamin B,,/loop
Percentage
mpg aztamzn B la found
absorbed
Remainder of carcase
in. tissues
Mzsscle Liveq Spleen Kid- Blood Ecueta (4 g) (wkola) (whole) neys (4 ml] (total) -1.24 0.03 o.og 0.72 0.58 0.02 0.03 0.2s 0.03 0.83 0.01 O..!p?
Top Middle Lowest ._~ 19 23 23 7 12 I2
2.89 I .63
None
Rat r S.I
8.8
8.8
Rat stomach extract
Rat 2 9.3
Rat r 5.4 Rat 2 4.7
5.0 5.3
7.2 7.1
46 53
50 47
28
0.11
0.61
a.?1
0.11
I.37 I.43
0.02
0.09
0.68 0.67
O.Ij
29
0.02
0.66
9.35
8.4
8.8
9.0
16
I2
IO
0
0.29
0.03
0.31
0
0.78
1.28
9.9
10.0
9.4
0
6
0.02
lost
0.07
0.0g
0.02
0.82
0.72
Bendagen Purified intrinsic factor
T
enhanced it slightly. Both the sow’s milk binding factor and the fresh milk depressed absorption very markedly below that of the vitamin given alone. Experiments
with rats
In all the experiments with intestinal loops the greater part of the small intestine was used, from the point where the duodenum emerges from beneath the colon down to the ileo-caecal junction. It was divided into three roughly equal lengths and IO mpg 6oCo-vitamin B,, was placed into each loop with or without preparations of intrinsic or binding factors. The resuIts of the first experiment, in which rat stomach extract, Bendogen or the clinically-active fraction prepared from Bendogen were given with the vitamin B,,, are shown in Table IV. Rat stomach extract markedly increased uptake of the vitamin at all sites of the intestine, whereas both preparations TABLE EFFECT
OF DIFFERENT
BINDING
FROM
AND
DIFFERENT
INTRINSIC SITES
V
FACTOR
PREPARATIONS
OF THE SMALL
INTESTINE
ON UPTAKE OF THE
B,,
OF VITAMIN
RAT
Dose: 10 m,lg vitamin B,,/loop Percentage of dose absorbed No.
Substance givelz with vitamin B,,
of rats
LT$$ler loop
8
None
Rat stomach extract Pig stomach extract Human gastric juice Pig gastric juice Bendogen Purified intrinsic factor Purified intrinsic factor digested with pepsin Purified intrinsic factor digested with pepsin and trypsin Ochromonas protein
-
* Significantly B,, alone. 1 Significantly alone.
different different
<
P <
0.01)
Lowest loop
15 39* 3
13 42* 2
IO
16 7
26t 5
I I I
4 4 9
4 7 9
6
I 4
9 14
12
6 I 4 2
25*
(P < 0.001) from absorption (O.OOI
Middle loop
2
II
iu corresponding
from absorption
3 16
; 12 I2
loops of vitamin
in lowest loop of vitamin B,,
Clin. Chim. Acta, 5 (1960) 853-861
E. S. HOLDSWORTH, M. E. COATES
858
from the pig depressed uptake below that of the free vitamin. In later experiments a number of other materials likely to influence absorption of vitamin Br2 were tested, and the results with these are summarized in Table V. The data concerning the free vitamin, rat stomach extract, human gastric juice and Ochromonas protein were submitted to a variance analysis, from which it was calculated that rat stomach extract very significantly increased absorption at all sites of the small intestine. Human gastric juice had no effect in the upper two loops, but in the lowest loop uptake of increased. Ochromonas protein was without effect at vitamin B i2 was significantly any site. Once again all the preparations from the pig inhibited absorption of vitamin B,,, and digestion of the purified intrinsic factor with pepsin, or with pepsin followed by trypsin, did not remove the inhibition. There was an indication that, as in the pig, uptake of vitamin B,, was greater from the middle and lower portions of the intestine. Examination by Student’s t test showed that absorption from the upper loop was significantly less than from either the middle (P < 0.01) or lowest (P < 0.001) loop. Some of the intrinsic factor preparations were given by mouth with vitamin B,, to a few intact rats. After 4 h the entire gastro-intestinal tract was removed and the remaining carcase was ashed and counted, to determine the amount of vitamin B,, absorbed. The results are given in Table VI. Rat stomach extract did not improve absorption over that of the free vitamin, presumably because under the conditions of this experiment an endogenous supply of intrinsic factor was available to the animals. Bendogen and its purified fraction inhibited uptake but human gastric juice did not. The data regarding the radioactivity determined in the excreta collected in the course of the experiments with intestinal loops are summarized in Table 1’11. This TABLE
VI
EFFECT OF INTRINSICFACTORPREPARATIONSON ABSORPTION GIVEN
Figures given are percentage
BY
MOUTH
absorption
TO
Substance
Experiment Experiment
I 2
34 22 40 32
Mean
AND
EXCRETION WITHOUT
OF
BINDING
4o 19 31 30
Substance givex with vitamin B,,
None Rat stomach extract Human gastric juice Bendogen Purified intrinsic factor Ochromonas protein Pig gastric juice Pig stomach extract
B,,
VITAMIN OR
given with vitamin
Human gastric juice
INTRINSIC
No. of rats IO
8 4 2
4 3 2 I
FACTOR
Purified
i&&sic
factor
77 II 15 14
‘5
‘5
VII
18 h
DURING
B,,
B,,
Bendogez
27 26 27 TABLE
URINARY
B,,
VITAMIN
by one rat of a dose of 30 m,ug vitamin
Rat stomach extract
lVO?W
OF
RATS
FOLLOWING
PREPARATIONS
mpg vitamin B,, absorbed
A
DOSE
INTO
OF
INTESTINAL
30
m/@
PLACED
LOOPS
m,uugvitamin B,, Amount
IN
WITH RATS
excreted
ix excreta
_&k%ztabsoYbed
3.98
1.06
26.6
10.38 4.84 2.58 2.59 2.44 1.61 I.43
0.86 0.83 0.86 0.94 0.91 0.97 0.74
8.3 17.1 33.3 36.3 37.3 60.2 51.7
’
Ioo
Clin. Chins. Acta, 5 (1960) 553-861
ABSORPTIONOF VITAMIN B,,. I.
859
radioactivity must have been largely in the urine since the ligature at the ileo-caecal junction would have prevented any of the dose of vitamin B,, reaching the lower gut except by absorption and re-excretion. It appears from these figures that when vitamin B 12 had been well-absorbed it was also better retained in the body and conversely, when uptake of vitamin B,, had been depressed relatively more had been excreted. DISCUSSION It is somewhat surprising that Bendogen, which is prepared from pig pyloric mucosa and effectively promotes absorption of vitamin B,, in human pernicious anaemia, should inhibit uptake of the vitamin when placed directly into the small intestine of the baby pig. When given by mouth to intact pigs of the same age it did not inhibit, and on occasion enhanced, absorption of vitamin B,,. The possibility that the animals were too young for their absorptive mechanism to be properly developed must be admitted, but seems unlikely when other digestive processes are considered. For example, assimilation of carbohydrate by piglets is normal within a few days after birth6 and although proteolytic activity is small during the first two weeks it develops rapidly’. Furthermore, our results resemble those of HEINRICH s, who found that in a young adult pig a preparation of intrinsic factor inhibited absorption of vitamin B,, when the two substances were introduced directly by cannula into the duodenum from which the pylorus and stomach had been separated; the same preparation promoted absorption when given by mouth. A possible explanation of our results and those of HEINRICH is that during production of the intrinsic factor preparations some alteration of the protein occurred, rendering them ineffective unless passed through the stomach. In this event a fresh saline extract of pig stomach might be expected to promote absorption of vitamin B,,, as rat stomach extract does in the rat. On the only occasion on which we were able to investigate this, the fresh extract of pig stomach did not promote any greater absorption of vitamin B,, from intestinal loops than did Bendogen. Unfortunately, however, the control pig which received vitamin B,, alone died during the course of the experiment, hence no great reliance can be placed on this result. Even more surprising is our finding that vitamin B,, given with sow’s milk binding factor or fresh sow’s milk was unavailable to the baby pig, either from intestinal loops or by mouth. The vitamin B,, naturally present in sow’s milk occurs in a bound form, and in addition there is in the milk a very considerable excess of binding activity3. In the work reported here, this excess binding activity was saturated with the labelled vitamin B,, in order to prepare a material suitable for testing. Unless this procedure leads to a complex which differs from the naturally-occurring one, our findings raise considerable problems regarding the requirement of the suckling pig, and possibly other mammals, for vitamin B,,. The results of the experiments with rats confirmed our previous findings@ that the uptake of vitamin B,, from intestinal loops was enhanced by extract of rat stomach but inhibited by Bendogen and a clinically-active fraction prepared from it. Digestion of this fraction with pepsin, or with pepsin followed by trypsin, did not remove the inhibition. It seems likely that the inhibitory effect of Bendogen in the rat was due to its origin rather than to any alteration in the process of manufacture, since fresh pig stomach extract, unlike a similar preparation from rat stomach, deClin. Chim. Acta, 5 (1960) 853-861
E. S. HOLDSWORTH. M. E. COATES
860 pressed
absorption
of vitamin
B,,
to the same extent
as Bendogen.
A sample
of
pig gastric juice was equally inhibitory. Uptake of the only bacterial product tested, the vitamin B,,-protein complex from inside the cells of Ochromonas malhamensis, was not distinguishable from that of free vitamin Brz. Human gastric juice did not affect absorption of vitamin B,, in the upper two-thirds of the small intestine, but significantly
enhanced
its uptake
from the lowest third. The reason is not clear, but
this finding should prove useful as the basis of an assay for intrinsic factor activity in human gastric juice. When given by mouth Bendogen and its purified fraction inhibited absorption of vitamin B,,, a result in keeping with our failure to reduce its inhibitory effect in intestinal loops by treatment with pepsin and trypsin, and also with the reported resistance of Bendogen to enzymic digestionl°F1l. Neither rat stomach extract nor human gastric juice increased uptake of vitamin B,, when given by mouth, probably because in the intact rat production of intrinsic adequate to ensure maximal absorption from the dose given. When the values are examined in intestinal
loops with different
for the urinary
excretion
binding and intrinsic
of vitamin
factor preparations
factor B,,
was
placed
it can be
Seen that broadly speaking an inverse ratio existed between the amount excreted and the amount absorbed. Thus although absorption in the presence of rat stomach extract was nearly threefold that of the free vitamin, excretion was slightly less; pig stomach extract and pig gastric juice depressed absorption to less than half that of free vitamin B,, but excretion was not reduced to anything like the same extent, This finding suggests that possibly intrinsic factor exerts an effect not only on the absorption of vitamin B,, but also on its retention by the tissues. The findings of MILLER AND HUNTER 1Band HERBERT~~ that pig intrinsic factor increased the uptake of vitamin B,, by rat tissues i,rz zit~o support the view that intrinsic factor may be concerned with retention of vitamin B,, in the tissues, although under their experimental conditions a homologous source of intrinsic factor was not apparently necessary. In the course of our experiments there were strong indications that the most active site of absorption of vitamin B,, was the middle-to-lower portion of the small intestine. This aspect of the problem is considered in more detail in the accompanying paper.
We are very grateful to Dr. D. TOMLIN of the Physics Department, University of Reading, for the loan of scintillation equipment and assistance with the measurements of radioactivity. SUMMARY I. The absorption of radioactive vitamin B,,, bound to different preparations of binding or intrinsic factors, and given to rats and baby pigs either by mouth or into intestinal loops, was studied. 2. A commercial preparation of clinically-active intrinsic factor from pig pyloric mucosa (Bendogen, GEA) depressed absorption of vitamin B,, from intestinal loops in baby pigs, but slightly enhanced absorption when given by mouth. 3. The vitamin B,,-binding factor from sow’s milk inhibited absorption of vitamin B,, in the pig when given either by mouth or into intestinal loops. Fresh sow’s milk was also inhibitory when given by mouth.
ABSORPTION
4. In the rat Bendogen, inhibited
uptake
of vitamin
OF VITAMIN
fresh extract B,,
B,,.
861
I.
of pig stomach
from intestinal
and pig gastric
loops, but an extract
juice all
of rat stomach
significantly enhanced it. Human gastric juice increased absorption of vitamin B,, in the lower part of the rat small intestine. The binding protein from Ochromonas malhamensis had no effect on absorption. 5. Absorption of vitamin B 12 was depressed by Bendogen but not by rat stomach extract or human gastric juice given by mouth to the rat. 6. The effect of the different binding and intrinsic factors on absorption was the reverse of their effect on excretion, in that a relatively greater proportion of the vitamin
was excreted
proportion
in the urine when its uptake was depressed and a much smaller
was excreted
when uptake
was enhanced.
REFERENCES 1 M. E. COATES, M. E. GREGORY, G. F. HARRISON, K. M. HENRY, E. S. HOLDSWORTH AND S. K. KON, Proc. Nutrition Sot. (Engl. and Scot.), 14 (1955) xiv. 2 E. S. HOLDSWORTH, Biochem. J., 66 (1957) 5gP. 3 M. E. GREGORY AND E. S. HOLDSWORTH, B&hem. J., 5g (1955) 329. 4 M. E. GREGORY AND E. S. HOLDSWORTH, Biochem. J., 66 (1957) 456. 6 H. HENNING AND H. BRUGSH, Deutsch. Med. Wochschr., 57 (1931) 757. 6 R. BRAUDE, A. M. DOLLAR, K. G. MITCHELL AND J. W. G. PORTER, Proc. Nutrition Sot. (Engl. and Scot.), 17 (1958) xv. 7 A. M. DOLLAR, K. G. MITCHELL, J. W. G. PORTER AND D. M. WALKER, Proc. Nutrition Sot. (Engl. and Scot.), 17 (1958) xlix. B H. C. HEINRICH, in H. C. HEINRICH (Ed.), Vitamin B,, und Intrinsic Factor, Ferdinand Enke Verlag, Stuttgart, 1957, p. 213. @ E. S. HOLDSWORTH AND M. E. COATES, Nature, 177 (1956) 701. I0 M. E. GREGORY, E. S. HOLDSWORTH AND M. OTTESEN, Compt. rend. trav. lab. Carlsberg. St?. chim., 30 (1957) ‘47. I1 R. GR~SBECK, Acta Chem. &and., 12 (1958) 142. I2 0. N. MILLER AND F. M. HUNTER, Federation Proc.. 17 (1958) 485. I3 V. HERBERT, Federation Proc., 17 (1958) 440.
Clin. Chim. Acta, 5 (1960) 853-861
THE I. THE
EFFECT
ABSORPTION
OF VITAMIN
OF DIFFERENT
BINDING
E. S. HOLDSWORTH*
853
B,, IN ANIMALS AND
INTRINSIC
AND &I. E. COATES
National Instifute for Research in Dairy&tag, Shinfield, Reading (Received
FACTORS
(Great Briiain)
April rrth, 1960)
These experiments were begun partly in the hope of devising an animal test to replace or supplement clinical trials of intrinsic factor activity and partly to elucidate the relation, if any, between vitamin B,,-binding and the function of intrinsic factor * *. The most usual source of intrinsic factor preparations for clinical use is the gastro-intestinal tract of the pig. Since feeding experiments have shown that preparations from one species of animal are not necessarily effective in promoting uptake of vitamin B,, in other species’ it seemed logical in the first place to use the pig as a subject for assay. When these attempts proved unsuccessful the rat was chosen as a cheaper and more convenient means of studying the broader aspects of the problem. MATERIALS AND METHODS
A‘%iVCalS
For experiments with pigs, in order to keep down the amounts of test materials needed and the bulk of tissue to be processed, 2-3 week old piglets were chosen in preference to larger animals. They were taken from the sow about an hour before the experiment began. Littermates of both sexes of the Large White breed were obtained from two sources. The animals varied in weight depending on their origin and the size of the litter; thus animals from some litters weighed from 2; to 4 kg and others from 5 to 6 kg, but within one experiment the weights were kept as even as possible. In experiments with rats, adult black-hooded males from our own colony were used. They had been reared on a good stock diet and weighed between 200-300 g. Materials
tested
Vitamin B,, labelled with 5sCo (specific activity 2.5 ,uC/pg) or %o (specific activity 0.9 ,&/,& was used. These samples were mostly gifts from Dr. E. LESTER SMITH, F.R.S. (Glaxo Laboratories Ltd.). Bendogen, a preparation of pig pyloric mucosa clinically-active in pernicious anaemia, was kindly supplied by GEA (Copenhagen} andapurified fraction, also clinically-active, was prepared from it 2. Pig gastric juice was obtained from an &week old pig that had been reared on an artificial milk diet. Under ether anaesthesia a small abdominal incision was made and a ligature placed just above the pylorus. The animal was killed 12 h later and the accumulated * Present address: Biochemistry Department, University of Adelaide, South Australia. ** Throughout this work the term “intrinsic factor” refers to the substance(s) present in the gastro-intestinal tract of any animal which are essential for the proper absorption of vitamin B,,. where reference is made particularly to a material It is prefaced by the term ‘*clinically-active” effective in human pernicious anaemia. Clin. Chim. Acta, 3 (1960) 853.861
Q4
E.S.HOLDSWORTH,M.E.COATES
gastric secretion was removed and filtered. A sample of clinically-active human gastric juice was kindly supplied by Dr. C. C. BOOTH, Postgraduate Medical School, Hammersmith. Fresh sow’s milk was obtained from a lactating sow after injection of posterior pituitary extract, and the sow’s milk binding factor was that described by GREGORY AND HOLDSWORTH~. Crude stomach extracts were prepared from freshly excised stomachs of both pigs and rats. The stomachs were slit open, washed rapidly to remove adhering food, ground with 0.9% NaCl in 0.01 N HCl, dialysed against running tap water, and the resulting extracts freeze-dried. The vitamin B12-protein complex from Ochromonas malhamensis was kindly provided by our colleague Dr. J. PAWELKIEWICZ, and was isolated from the cell contents medium containing G°Co-vitamin B,,. Measurement
of binding
of 0. malhamensis
grown in a
activity
In all the experiments reported here the test materials were added in sufficient quantity exactly to bind the dose of vitamin B1,. Some microbiological measurements of vitamin B,,-binding activity were kindly made for us by our colleague Dr. XI. E. GREGORY. In general, binding activity was measured by determining the amouDi of radioactivity ultrafiltrable from a mixture of B°Co-vitamin B,, and the material being tested. These methods have been described by GREGORY AND HOLDSWORTH *.
Surgical
fwocedure
Pigs were anaesthetized with cyclopropane and rats with ether. This type of anaesthesia was chosen so that the animals recovered consciousness a short time after the operation was completed. An abdominal incision was made, :c expose most of the small intestine, and ligatures were placed at the two extremes of the length of intestine required for experiment. The intestine was cut below an;1 above the proximal and distal ligatures respectively, and a glass cannula was tied mto the upper end of the length under experiment. Warm normal saline (38”) was passed through to remove food and any possible secretion from the upper part of the gut that might influence uptake of vitamin B,,. About I 1 of saline was used in the pig, and about 200 ml in the rat. The cannula was removed and the lower end of the length of intestine was tied off. The experimental dose was delivered mto the upper end by means of a syringe fitted with a blunt-ended needle and a ligature was made as the needle was withdrawn, thus forming a blind length ot intestine enclosing the test dose of vitamin B 1 2 with or without the material under investigation. Where more than one loop was required, the length of intestine was divjdzd into appropriate portions after washing and each was treated separately as described. The gut was replaced and the incision closed, and the animals were allowed to live for 16-20 h. During this time they were kept in a warm room in separate cages and the excreta were collected on filter paper. The animals were supplied with water but no food. At the end of the experiment they were again anaesthetized, the abdomen was reopened and the loop(s) removed. They were then killed and appropriate specimens such as liver, muscle or the whole carcase were taken as required for counting. Prefiaratiolz
qf materials
for counting
4-g samples were convenient for the available equipment, hence when the specific activity was high enough the radioactivity of pieces of tissue was counted CZin.Cltim.
A&,
j (1960)853-861
ABSORPTION
directly.
Thus
processing;
in the rat
the counts
B,,.
OF VITAMIN
experiments
I.
each intestinal
in 4-g samples
855
loop was counted
of liver could be related
without
to the weight of
the whole organ. The pig intestinal loops were digested with 4 N NaOH and 4-ml aliquots taken. Carcases, pieces of muscle and the filter papers on which urine and faeces were collected were ashed at about 700’ and either a portion or the total ash was counted. Counting
methods
Counts
were made in a well-type
activated
NaI
crystal
scintillation
counter.
Standards were counted at the same time and the results expressed as vitamin B,, on the assumption that all the radioactive material was vitamin B,,. Recoveries were usually at least 95%. Counts were made on samples of untreated tissue but proved so low as to be negligible. RESULTS
Expe:periments with pigs In the first experiment IOO mpg %o-vitamin B r2 was placed in a loop of intestine 3 feet long, the upper end being about 9 inches below the pylorus to avoid the area believed to be responsible for the production of “intrinsic factor” in the pig5. One pig received the vitamin alone, another vitamin B,, and Bendogen, while the third received vitamin B,, with a clinically-active fraction, prepared from Bendogen 2. The amounts of vitamin B,,, measured as Wo, appearing in the tissue after zz h are given in Table
I. It is apparent
that
both Bendogen TABLE
ABSORPTION
DURING
22
h
OF
VITAMIN
PREPARATIONS
Dose
Substance
given with
INTO
of
vitamin B,,
vitamin B,,
None Bendogen Purified intrinsic factor *
(mpg) 100
100 100
B,,
WITH
OR
WITHOUT
LOOPS
IN
BABY
Amount left Percentage _mpg in gut loop absorbed muscle(mpg)
67.5 92.6 96.6
fraction
markedly
I
PLACED
INTESTINAL
and its purified
32.5 7.4 3.4
INTRINSIC
FACTOR
PIGS
vitamin B12 in tissues
(IOO g)
~ liver (whole)
0.38 0.30 0.30
8.4 4.2 1.9
blood (JO ml)
excreta (total)
0
2.2
0
0.9
0
0.1
* Holdsworth2.
depressed uptake of vitamin Blz. As this effect might have been peculiar to one part of the intestine only, further experiments were done at different sites in the small intestine and several loops were made in each animal to cover a greater length of gut. The results are summarized in Table II. In every instance the levels of vitamin B,, found in the tissues were concordant with the amount that had disappeared from the loop. It is evident from these results that absorption of the vitamin was greater from the middle and lower parts of the small intestine than from the upper, and that on two out of three occasions the presence of Bendogen depressed the uptake of vitamin B,, at all positions in the intestine. Two fractions prepared from Bendogen, one clinically-active and one with vitamin B,,- binding activity only, both depressed absorption of vitamin B,, to about the same extent as Bendogen. It was somewhat surprising that Bendogen, prepared from the pyloric mucosa Clin. Chim. Acta, 5 (1960) 853-861
Sj6
E. S. HOLDSWORTH. M. E. COATES TABLE
EFFECT
OF INTRINSIC VITAMIN
Total
FACTOR
R 1z FROM
5
5 ft. Lower end 2 ft. above ileo-caecal junction
x
Ift.
20
20
5 x 3ft.
FACTORS
SMALL INTESTINE ______
- AtoRe
40
5 x I ft.
ft. Lower end at ileo-caecal junction
BINDING
ON THE OF THE .._--
ABSORPTION BABY
OF
PIG ~
____.
_
94 of dose of u%tamzxB I2 absorbed +
nzpg vitamin B,, placed i% loop
3 x 4ft.
5 ft. Lower end 2 ft. above ileo-caecal junction
AND
DIFFERENT SITES IN THE ~~. ___--_..
No. and length, of loops
lengtk and position of site examined
12 ft. Upper end 9 in. below pylorns
15
PREPARATIONS
II
20
+ Ben- + purifedt intrinsic dogen factor
+ bindingy factor
+ sozv’s milk t i factor
10
0
2
19 32 33 22 49 52 36 43 47 37 31 25 57 43 50 “7
4
3
20
I
33 44 50 55 49 IO I2
I4 14 23 6 I’3 21 16 14
20
13
IO
IO
ICI
II
‘3
7 ‘7
II
7
* Results given in order from proximal to distal ends of the small intestine. t Holdsworthz. tt Gregory and Holdsworth3. TABLE AMOUNTS -
OF VITAMIN
B,,
IN
-.
Substance given with vitamix B,, None Bendogen Sow’s milk binding factor Fresh sow’s milk
LIVER
AND
MUSCLE
IOO mpg .~
III OF BABY
WO-VITAMIN ___x_..
PlGS
1.6 4.3
0.2
0.7
h
AFTER
AN
ORAL
DOSE
OF
._.~
mpg vitamin Bl,/liver _...-__ Expt. I Em@. 2 Ex$% 3 2.4 2.3
8
B,,
I.0 2.3
-
mpg vitamin B,,/Ioo g muscle --~______ I%@. I Expt. 2 Expt. 3 0.19
0.04
0.01
0.20
0.17
0.11
0.03 0
of the pig, should hinder absorption of vitamin B,, in the same species. Another vitamin B,,-binding substance from the pig, the sow’s milk factor3, was therefore investigated (Table II, experiment with Bendogen) and proved to have an even greater inhibitory effect on vitamin B,, uptake than had Bendogen under the conditions of the experiment. The effect of both of these substances on the absorption of vitamin B 12given by mouth to pigs was tested. Absorption was assessed on the levels of vitamin B,, found in the livers and samples of back muscle taken 8 h after the dose had been given by mouth. Fresh sow’s milk was also tested, should the method of preparation of the sow’s milk binding factor have damaged the vitamin B,,-protein complex, thereby rendering it unavailable to the piglet. Table III summarizes the results. In all three experiments both liver and muscle levels of vitamin B,, indicated that Bendogen given by mouth did not interfere with absorption and may even have
ABSORPTION OF VITAMIN TABLE ABSORPTION
DURING
18
B ra
h OF VITAMIN
PLACED
WITH
INTESTINAL
Dose: mpg vitamin B 12 Eeft in Eoo,O
Substance given wit12 vztamzn B,,
-Tip Middle Lowest
7.7
7.7
LOOPS
B,,. I.
857
IV OR WITHOUT OF THE
INTRINSIC
FACTOR
PREPARATIONS
INTO
RAT
ro m,~g vitamin B,,/loop
Percentage
mpg aztamzn B la found
absorbed
Remainder of carcase
in. tissues
Mzsscle Liveq Spleen Kid- Blood Ecueta (4 g) (wkola) (whole) neys (4 ml] (total) -1.24 0.03 o.og 0.72 0.58 0.02 0.03 0.2s 0.03 0.83 0.01 O..!p?
Top Middle Lowest ._~ 19 23 23 7 12 I2
2.89 I .63
None
Rat r S.I
8.8
8.8
Rat stomach extract
Rat 2 9.3
Rat r 5.4 Rat 2 4.7
5.0 5.3
7.2 7.1
46 53
50 47
28
0.11
0.61
a.?1
0.11
I.37 I.43
0.02
0.09
0.68 0.67
O.Ij
29
0.02
0.66
9.35
8.4
8.8
9.0
16
I2
IO
0
0.29
0.03
0.31
0
0.78
1.28
9.9
10.0
9.4
0
6
0.02
lost
0.07
0.0g
0.02
0.82
0.72
Bendagen Purified intrinsic factor
T
enhanced it slightly. Both the sow’s milk binding factor and the fresh milk depressed absorption very markedly below that of the vitamin given alone. Experiments
with rats
In all the experiments with intestinal loops the greater part of the small intestine was used, from the point where the duodenum emerges from beneath the colon down to the ileo-caecal junction. It was divided into three roughly equal lengths and IO mpg 6oCo-vitamin B,, was placed into each loop with or without preparations of intrinsic or binding factors. The resuIts of the first experiment, in which rat stomach extract, Bendogen or the clinically-active fraction prepared from Bendogen were given with the vitamin B,,, are shown in Table IV. Rat stomach extract markedly increased uptake of the vitamin at all sites of the intestine, whereas both preparations TABLE EFFECT
OF DIFFERENT
BINDING
FROM
AND
DIFFERENT
INTRINSIC SITES
V
FACTOR
PREPARATIONS
OF THE SMALL
INTESTINE
ON UPTAKE OF THE
B,,
OF VITAMIN
RAT
Dose: 10 m,lg vitamin B,,/loop Percentage of dose absorbed No.
Substance givelz with vitamin B,,
of rats
LT$$ler loop
8
None
Rat stomach extract Pig stomach extract Human gastric juice Pig gastric juice Bendogen Purified intrinsic factor Purified intrinsic factor digested with pepsin Purified intrinsic factor digested with pepsin and trypsin Ochromonas protein
-
* Significantly B,, alone. 1 Significantly alone.
different different
<
P <
0.01)
Lowest loop
15 39* 3
13 42* 2
IO
16 7
26t 5
I I I
4 4 9
4 7 9
6
I 4
9 14
12
6 I 4 2
25*
(P < 0.001) from absorption (O.OOI
Middle loop
2
II
iu corresponding
from absorption
3 16
; 12 I2
loops of vitamin
in lowest loop of vitamin B,,
Clin. Chim. Acta, 5 (1960) 853-861
E. S. HOLDSWORTH, M. E. COATES
858
from the pig depressed uptake below that of the free vitamin. In later experiments a number of other materials likely to influence absorption of vitamin Br2 were tested, and the results with these are summarized in Table V. The data concerning the free vitamin, rat stomach extract, human gastric juice and Ochromonas protein were submitted to a variance analysis, from which it was calculated that rat stomach extract very significantly increased absorption at all sites of the small intestine. Human gastric juice had no effect in the upper two loops, but in the lowest loop uptake of increased. Ochromonas protein was without effect at vitamin B i2 was significantly any site. Once again all the preparations from the pig inhibited absorption of vitamin B,,, and digestion of the purified intrinsic factor with pepsin, or with pepsin followed by trypsin, did not remove the inhibition. There was an indication that, as in the pig, uptake of vitamin B,, was greater from the middle and lower portions of the intestine. Examination by Student’s t test showed that absorption from the upper loop was significantly less than from either the middle (P < 0.01) or lowest (P < 0.001) loop. Some of the intrinsic factor preparations were given by mouth with vitamin B,, to a few intact rats. After 4 h the entire gastro-intestinal tract was removed and the remaining carcase was ashed and counted, to determine the amount of vitamin B,, absorbed. The results are given in Table VI. Rat stomach extract did not improve absorption over that of the free vitamin, presumably because under the conditions of this experiment an endogenous supply of intrinsic factor was available to the animals. Bendogen and its purified fraction inhibited uptake but human gastric juice did not. The data regarding the radioactivity determined in the excreta collected in the course of the experiments with intestinal loops are summarized in Table 1’11. This TABLE
VI
EFFECT OF INTRINSICFACTORPREPARATIONSON ABSORPTION GIVEN
Figures given are percentage
BY
MOUTH
absorption
TO
Substance
Experiment Experiment
I 2
34 22 40 32
Mean
AND
EXCRETION WITHOUT
OF
BINDING
4o 19 31 30
Substance givex with vitamin B,,
None Rat stomach extract Human gastric juice Bendogen Purified intrinsic factor Ochromonas protein Pig gastric juice Pig stomach extract
B,,
VITAMIN OR
given with vitamin
Human gastric juice
INTRINSIC
No. of rats IO
8 4 2
4 3 2 I
FACTOR
Purified
i&&sic
factor
77 II 15 14
‘5
‘5
VII
18 h
DURING
B,,
B,,
Bendogez
27 26 27 TABLE
URINARY
B,,
VITAMIN
by one rat of a dose of 30 m,ug vitamin
Rat stomach extract
lVO?W
OF
RATS
FOLLOWING
PREPARATIONS
mpg vitamin B,, absorbed
A
DOSE
INTO
OF
INTESTINAL
30
m/@
PLACED
LOOPS
m,uugvitamin B,, Amount
IN
WITH RATS
excreted
ix excreta
_&k%ztabsoYbed
3.98
1.06
26.6
10.38 4.84 2.58 2.59 2.44 1.61 I.43
0.86 0.83 0.86 0.94 0.91 0.97 0.74
8.3 17.1 33.3 36.3 37.3 60.2 51.7
’
Ioo
Clin. Chins. Acta, 5 (1960) 553-861
ABSORPTIONOF VITAMIN B,,. I.
859
radioactivity must have been largely in the urine since the ligature at the ileo-caecal junction would have prevented any of the dose of vitamin B,, reaching the lower gut except by absorption and re-excretion. It appears from these figures that when vitamin B 12 had been well-absorbed it was also better retained in the body and conversely, when uptake of vitamin B,, had been depressed relatively more had been excreted. DISCUSSION It is somewhat surprising that Bendogen, which is prepared from pig pyloric mucosa and effectively promotes absorption of vitamin B,, in human pernicious anaemia, should inhibit uptake of the vitamin when placed directly into the small intestine of the baby pig. When given by mouth to intact pigs of the same age it did not inhibit, and on occasion enhanced, absorption of vitamin B,,. The possibility that the animals were too young for their absorptive mechanism to be properly developed must be admitted, but seems unlikely when other digestive processes are considered. For example, assimilation of carbohydrate by piglets is normal within a few days after birth6 and although proteolytic activity is small during the first two weeks it develops rapidly’. Furthermore, our results resemble those of HEINRICH s, who found that in a young adult pig a preparation of intrinsic factor inhibited absorption of vitamin B,, when the two substances were introduced directly by cannula into the duodenum from which the pylorus and stomach had been separated; the same preparation promoted absorption when given by mouth. A possible explanation of our results and those of HEINRICH is that during production of the intrinsic factor preparations some alteration of the protein occurred, rendering them ineffective unless passed through the stomach. In this event a fresh saline extract of pig stomach might be expected to promote absorption of vitamin B,,, as rat stomach extract does in the rat. On the only occasion on which we were able to investigate this, the fresh extract of pig stomach did not promote any greater absorption of vitamin B,, from intestinal loops than did Bendogen. Unfortunately, however, the control pig which received vitamin B,, alone died during the course of the experiment, hence no great reliance can be placed on this result. Even more surprising is our finding that vitamin B,, given with sow’s milk binding factor or fresh sow’s milk was unavailable to the baby pig, either from intestinal loops or by mouth. The vitamin B,, naturally present in sow’s milk occurs in a bound form, and in addition there is in the milk a very considerable excess of binding activity3. In the work reported here, this excess binding activity was saturated with the labelled vitamin B,, in order to prepare a material suitable for testing. Unless this procedure leads to a complex which differs from the naturally-occurring one, our findings raise considerable problems regarding the requirement of the suckling pig, and possibly other mammals, for vitamin B,,. The results of the experiments with rats confirmed our previous findings@ that the uptake of vitamin B,, from intestinal loops was enhanced by extract of rat stomach but inhibited by Bendogen and a clinically-active fraction prepared from it. Digestion of this fraction with pepsin, or with pepsin followed by trypsin, did not remove the inhibition. It seems likely that the inhibitory effect of Bendogen in the rat was due to its origin rather than to any alteration in the process of manufacture, since fresh pig stomach extract, unlike a similar preparation from rat stomach, deClin. Chim. Acta, 5 (1960) 853-861
E. S. HOLDSWORTH. M. E. COATES
860 pressed
absorption
of vitamin
B,,
to the same extent
as Bendogen.
A sample
of
pig gastric juice was equally inhibitory. Uptake of the only bacterial product tested, the vitamin B,,-protein complex from inside the cells of Ochromonas malhamensis, was not distinguishable from that of free vitamin Brz. Human gastric juice did not affect absorption of vitamin B,, in the upper two-thirds of the small intestine, but significantly
enhanced
its uptake
from the lowest third. The reason is not clear, but
this finding should prove useful as the basis of an assay for intrinsic factor activity in human gastric juice. When given by mouth Bendogen and its purified fraction inhibited absorption of vitamin B,,, a result in keeping with our failure to reduce its inhibitory effect in intestinal loops by treatment with pepsin and trypsin, and also with the reported resistance of Bendogen to enzymic digestionl°F1l. Neither rat stomach extract nor human gastric juice increased uptake of vitamin B,, when given by mouth, probably because in the intact rat production of intrinsic adequate to ensure maximal absorption from the dose given. When the values are examined in intestinal
loops with different
for the urinary
excretion
binding and intrinsic
of vitamin
factor preparations
factor B,,
was
placed
it can be
Seen that broadly speaking an inverse ratio existed between the amount excreted and the amount absorbed. Thus although absorption in the presence of rat stomach extract was nearly threefold that of the free vitamin, excretion was slightly less; pig stomach extract and pig gastric juice depressed absorption to less than half that of free vitamin B,, but excretion was not reduced to anything like the same extent, This finding suggests that possibly intrinsic factor exerts an effect not only on the absorption of vitamin B,, but also on its retention by the tissues. The findings of MILLER AND HUNTER 1Band HERBERT~~ that pig intrinsic factor increased the uptake of vitamin B,, by rat tissues i,rz zit~o support the view that intrinsic factor may be concerned with retention of vitamin B,, in the tissues, although under their experimental conditions a homologous source of intrinsic factor was not apparently necessary. In the course of our experiments there were strong indications that the most active site of absorption of vitamin B,, was the middle-to-lower portion of the small intestine. This aspect of the problem is considered in more detail in the accompanying paper.
We are very grateful to Dr. D. TOMLIN of the Physics Department, University of Reading, for the loan of scintillation equipment and assistance with the measurements of radioactivity. SUMMARY I. The absorption of radioactive vitamin B,,, bound to different preparations of binding or intrinsic factors, and given to rats and baby pigs either by mouth or into intestinal loops, was studied. 2. A commercial preparation of clinically-active intrinsic factor from pig pyloric mucosa (Bendogen, GEA) depressed absorption of vitamin B,, from intestinal loops in baby pigs, but slightly enhanced absorption when given by mouth. 3. The vitamin B,,-binding factor from sow’s milk inhibited absorption of vitamin B,, in the pig when given either by mouth or into intestinal loops. Fresh sow’s milk was also inhibitory when given by mouth.
ABSORPTION
4. In the rat Bendogen, inhibited
uptake
of vitamin
OF VITAMIN
fresh extract B,,
B,,.
861
I.
of pig stomach
from intestinal
and pig gastric
loops, but an extract
juice all
of rat stomach
significantly enhanced it. Human gastric juice increased absorption of vitamin B,, in the lower part of the rat small intestine. The binding protein from Ochromonas malhamensis had no effect on absorption. 5. Absorption of vitamin B 12 was depressed by Bendogen but not by rat stomach extract or human gastric juice given by mouth to the rat. 6. The effect of the different binding and intrinsic factors on absorption was the reverse of their effect on excretion, in that a relatively greater proportion of the vitamin
was excreted
proportion
in the urine when its uptake was depressed and a much smaller
was excreted
when uptake
was enhanced.
REFERENCES 1 M. E. COATES, M. E. GREGORY, G. F. HARRISON, K. M. HENRY, E. S. HOLDSWORTH AND S. K. KON, Proc. Nutrition Sot. (Engl. and Scot.), 14 (1955) xiv. 2 E. S. HOLDSWORTH, Biochem. J., 66 (1957) 5gP. 3 M. E. GREGORY AND E. S. HOLDSWORTH, B&hem. J., 5g (1955) 329. 4 M. E. GREGORY AND E. S. HOLDSWORTH, Biochem. J., 66 (1957) 456. 6 H. HENNING AND H. BRUGSH, Deutsch. Med. Wochschr., 57 (1931) 757. 6 R. BRAUDE, A. M. DOLLAR, K. G. MITCHELL AND J. W. G. PORTER, Proc. Nutrition Sot. (Engl. and Scot.), 17 (1958) xv. 7 A. M. DOLLAR, K. G. MITCHELL, J. W. G. PORTER AND D. M. WALKER, Proc. Nutrition Sot. (Engl. and Scot.), 17 (1958) xlix. B H. C. HEINRICH, in H. C. HEINRICH (Ed.), Vitamin B,, und Intrinsic Factor, Ferdinand Enke Verlag, Stuttgart, 1957, p. 213. @ E. S. HOLDSWORTH AND M. E. COATES, Nature, 177 (1956) 701. I0 M. E. GREGORY, E. S. HOLDSWORTH AND M. OTTESEN, Compt. rend. trav. lab. Carlsberg. St?. chim., 30 (1957) ‘47. I1 R. GR~SBECK, Acta Chem. &and., 12 (1958) 142. I2 0. N. MILLER AND F. M. HUNTER, Federation Proc.. 17 (1958) 485. I3 V. HERBERT, Federation Proc., 17 (1958) 440.
Clin. Chim. Acta, 5 (1960) 853-861