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Gut, 1981, 22, 617-622
Subcellular localisation of vitamin B12 during
absorption in the guinea-pig ileum*
W J JENKINS,t R EMPSON, D P JEWELL, AND K B TAYLOR
From the Academic Department of Medicine, Royal Free Hospital, London
SUMMARY The subcellular distribution of vitamin B12 was studied during its absorption in the
guinea-pig ileum. Animals were fed either (57Co) or (,'5Co) cyanocobalamin and killed two or
four hours later. At two hours labelled cyanocobalamin was concentrated in brush border and
lysosomal fractions of ileal homogenates, with some remaining in the sample layer. In contrast,
at four hours labelled cyanocobalamin was concentrated predominantly in the cytosol with much
smaller peaks in the brush border and lysosomal fractions. These findings are consistent with
vitamin B12 absorption by receptor-mediated endocytosis. It is suggested that, after binding at
the brush border, vitamin B12 is first sequestrated within lysosomes, and then released into the
cytosol, from where it leaves the cell to enter the portal blood.
During the absorption of vitamin B12 (B12) in the
ileum, intrinsic factor-vitamin B12 (IF-B12) complexes bind to specific receptors on the. microvillous membranes of ileal enterocytes.1 There is
then a significant delay of about two hours before
B12 appears in the portal blood.2 Neither the
cause for this delay, nor the pathway that B,2
takes in its passage across the enterocyte is
known. Two hypotheses have been advanced. As
IF has not been found in portal blood,4 both involve cleavage of the IF-B,2 complex. Wilson5
first suggested that the IF-B12 complex is absorbed
by pinocytosis, and that cleavage occurs during
this process. It has since been shown that in many
mammalian cells pinocytic vesicles fuse with primary lysosomes to form secondary lysosomes.6
Lysosomes contain powerful hydrolytic enzymes,
and these might cleave the IF-B12 complex, and
so release B12 for transport into plasma. The other
hypothesis is that of MacKenzie and Donaldson,7
who suggested that the IF-B12 complex is split to
an 'active fragment' of the B12 molecule by brush
border hydrolases, and that this smaller fragment
is then transported into the cell, where B12 is resynthesised. However, since advancing this hypothesis, Donaldson has reported evidence that
intrinsic factor may be taken up by isolated ileal
enterocytes. 8
Previous studies in which subcellular fractionation techniques were used to investigate B12 absorption in the ileum have not provided evidence
which supports either view, but have apparently
demonstrated concentration of absorbed B12 in the
mitochondria and microsomes of ileal enterocytes.9 10 This paper reports further subcellular
fractionation studies performed by isopycnic centrifugation of guinea-pig ileal homogenates after
the gastric administration of (57Co) or (`8Co)
cyanocobalamin.
Methods
ANIMALS
Non-pregnant female guinea-pigs (Hartley strain)
weighing between 500 and 600 g were used in all
studies. After an overnight fast, they were lightly
anaesthetised with halothane to permit peroral
gastric intubation, and 0.25 ml cyanocobalamin
solution radiola'belled with 57Co or 58Co was introduced into the stomach. This was followed by
1.5 ml water to wash out the tube contents. In the
first set of experiments six guinea-pigs were given
10 ng (57Co) cyanocobalamin (S.A. 8.14 MBq/,g)
at the start of the study, and the animals were
*Presented in part to the British Society of Gastroenterology Meeting, killed by a blow on the head two hours later. In
Reading, 1980.
the second set of experiments another six guineatAddress for correspondence: Dr W J Jenkins, Academic Depart- pigs were given 10 ng (57Co) cyanocobalamin as
ment of Medicine, Royal Free Hospital, London NW3 2QG.
before, and then 60 ng (5M,Co) cyanocobalamin
Received for publication 3 February 1981
617
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Jenkins. Empson, Jewell, and Taylor
618
(S.A. 1.33 MBq/,g) at two hours. They were
killed at four hours.
HOMOGENISATION
One hundred to one hundred and fifty milligram
portions of ileum 5 cm proximal to the ileocaecal
valve were excised immediately after death and
cut into small pieces. They were homogenised by
12 strokes of a loose-fitting (type A) pestle in a
glass Dounce homogeniser (Kontes Glass Co.,
Vineland, NJ, USA) in 4 ml 0.25 M sucrose containing 3 mM HEPES pH 7.4 (SH medium) at
40C. The homogenate was centrifuged at 600 g
for 10 minutes to remove nuclei, intact cells, and
debris. The pellet was resuspended in another
2 ml SH medium by three strokes of the type A
pestle, and centrifuged again.
SUBCELLULAR FRACTIONATION
Two millilitres of the combined postnuclear supernatants were layered on to a 20 ml preformed
linear gradient of Percoll (Pharmacia (Great
Britain) Ltd, Hounslow, England) in SH medium
extending from a density of 1.025 to 1.15 g/ml.
The gradients were centrifuged at 65000 g for
30 minutes in an MSE 3 swing-out rotor (Measuring and Scientific Equipment, Crawley, Sussex,
UK) at 40C. Linearity of the gradients was preserved during this centrifugation and was reproducible. After tube piercing, 15 fractions were
collected by upward displacement with 60%
sucrose (w/v) into tared tubes. After weighing,
the density of the fractions was determined in-
percentage recoveries (sum of gradient fractions
against original postnuclear supernatant) are given
in addition. Pooling and averaging of distributions
were performed as described by Leighton et al.14
This method requires conversion of all the histograms to the same preset volume intervals, and
causes some loss in resolution.
Results
Figure 1 shows the subcellular distribution of 57Co
in guinea-pig ileal homogenates two hours after
intragastric (57Co) cyanocobalamin. Although
radioactivity is widely distributed within the
gradient, it appears to be concentrated in three
peaks, which correspond to the distribution of
marker enzymes for the brush border and lysosomes with some activity remaining in the sample
layer. This may represent radioactivity present in
the cytosol in vivo, or may be due to release of
(57Co) cyanocobalamin from organelles damaged
during homogenisation. However, the latter possibility is unlikely to be very important, as lysosomal
latency, which is an indirect and sensitive measure
directly by refractometry.
ASSAYS
The following enzymes were assayed in the
gradient fractions and used as markers for subcellular organelles: alkaline phosphatase for the
brush border11; lactate dehydrogenase for the
cytosol12; 18-N-acetyl-D-glucosaminidase for lysosomesll; and glutamate dehydrogenase for mitochondria.1:1 Gradient fractions were counted for
">7Co and ',8Co in an LKB-Wallac 1280 Ultrogamma counter (LKB Produkter-AB, Bromma,
Sweden) using mode 2 for double labelled samples
wih automatic background subtraction.
PRESENTATION OF RESULTS
All results are presented in the form of frequencyvolume histograms. The calculations and plots
were done by computer. Frequency is defined as
the proportion of the total recovered activity found
in the individual fraction divided by the fractional
volume. All histograms are normalised, and the
Fig. 1 Subcellular fractionation of postnuclear
supernatant from guinea-pig ileal homogenate two
hours after intragastric (57Co) B12,. Graph shows
frequency-volume histograms for (57Co) B12' alkaline
phosphatase, /3-N-acetyl-D-glucosaminidase, glutamate
dehydrogenase, and represent averaged results from
six experiments. Frequency (mean+SD) is defined
as the fraction of total recovered activity present in
the individual fraction divided by the fractional
volume. Percentage recoveries (mean+SD); (57Co)
B122, 965; alkaline phosphatase, 103±10;
/3-N-acetyl-D-glucosaminidase, 76+8; glutamate
dehydrogenase, 83±6; lactate dehydrogenase, 63+5.
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Subcellular localisation of vitamin B,, during absorption in the guinea-pig ileum
of lysosomal integrity,"1 and thus organelle
damage, was high (70.7-+-3.3%, mean+±SD) in our
studies.
As radioactivity might have become associated
with subcellular oganelles during cell disruption
and the subsequent centrifugation, an experiment was performed to investigate this possibility.
Ileum from a control guinea-pig was homogenised
in the presence of 10 ng (57Co) cyanocobalamin,
and then fractionated as above. Figure 2 shows
that virtually all the radioactivity remains in the
sample layer, and there is no evidence of concentration in any subcellular organelle. The same
result was obtained when human intrinsic factor
was added with (57Co) cyanocobalamin.
The use of low concentrations of digitonin during homogenisation has been descrilbed to selectively disrupt lysosomes while leaving mitochondria
intact.'5 Figure 3 shows the effect of adding digitonin (012 mM final concentration) to the SH
medium before homogenising ileum from a guineapig given intragastric (57Co) cyanocobalamin two
hours earlier. The lysosomes have been disrupted,
so that ,B-N-acetyl-D-glucosaminidase activity remains mostly in the sample layer, where the
radioactivity is also localised. In contrast, the
distribution of glutamate dehydrogenase is unchanged from that shown in Fig. 1, indicating that
the mitochondria remain intact. This makes it
unlikely that vitamin B,, is significantly concentrated in the mitochondria of the enterocytes during its absorption in the ileum.
10.
Density
1-12
8.
>
6
c
c
4,
107 v<_
-4,
4
3
_I
0J
Volume
_.
L1
02
Fig. 2 Subcellular fractionation of postnuclear
of ileum from a control guinea-pig
homogenised with (57Co) B12. Graph shows a
frequency-volume histogram for ( S7Co) B,2, recovery
101% (hatched area), and density (broken line).
supernatant
619
n NAGase
0
Volume
1 0
(lysosomes)
Volume
Fig. 3 Subcellular fractionation of postnuclear
supernatant of guinea-pig ileum two hours after
intragastric (57Co) B,2 homogenised with 0.12 mM
digitonin. Graphs show frequency-volume histograms
(with percentage recoveries in parentheses) for
(57Co) B,2 (92), f8-N-acetyl-D-glucosaminidase (87),
glutamate dehydrogenase (84), and lactate
dehydrogenase (66).
In the second set of experiments (57Co) cyanocobalamin was administered to the guinea-pigs as
before, followed two hours later by ('iCo) cyanocobalamin. Figure 4 shows the subcellular
distribution of 57Co and 59Co in ileal homogenates
after another two hours. Although a small amount
of 57Co given first is still found within the gradient,
the majority is now recovered in the sample layer
corresponding to a localisation in the cytosol. In
contrast, the distribution of 51Co given later is
similar to that shown in Fig. 1 for (-57Co) cyanocobalamin, which was also given two hours before
killing in the first set of experiments. The experiments using both 57Co and .1Co labelled cyanocobalamin were repeated using the isotopes in reverse
order. Whichever was given first was concentrated in the cytosol, and the isotope given later
showed major peaks in the brush border and lysosomal fractions.
Discusion
Although we do not have direct evidence that
radioactivity is associated only with cyanocobalamin, previous studies make it very unlikely that a
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620
Jenkins. Empson, Jewell, and Taylor
3
58Co-B12 (2 hours)
c2
a'
L1
0
5.
Co-B12(4hours)
41
U
c
a,
0' 2
1
0' P.
0
Volume
1
Fig. 4 Subcellular fractionation of postnuclear
supernatant of guinea-pig ileal homogenate two hours
after (58Co) B12 and four hours af ter (57Co) B12.
Graphs show frequency-volume histograms for
(58Co) B12 (recovery 95±5%), and (57Co) B12
(recovery 95±4%), and represent averaged data from
six experiments.
significant amount of 57Co or 58Co is detached
from cobalamins under the conditions of these
experiments.16 Our data show that two hours
after feeding (57Co) or (58Co) cyanocobalamin to
fasted guinea-pigs radioactivity is concentrated
not only in the brush borders as might be expected,
but also in lysosomal fractions of ileal homogenates .(Figs. 1 and 4). However, the standard
deviations shown for the distribution of radiolabelled cyanocobalamin are quite large. This is
partly an inevitable consequence of the averaging
process,14 which is necessary to permit presentation of results from several experiments, but
causes some unavoidable loss of resolution. The
other major limitation is that the time for
the administered cyanocobalamin to pass from the
stomach to the distal ileum may vary in individual
guinea-pigs, so that absorption may be at different
stages in different animals even though they were
all killed after the same interval. For this reason,
it was decided not to study the change in subcellular distribution of aibsorbed B12 with time by
using several time points in different animals.
Instead, we gave individual guinea-pigs first one
radiolabelled cyanocobalamin followed after a
set interval by a differently radiolabelled cyanocobalamin, and compared the difference in subcellular distribution of the two isotopes in ileal
enterocytes. It was assumed that transit time from
the stomach to the ileum was the same for both
isotopes in individual guinea-pigs. The results of
such experiments (Fig. 4) clearly show that at
four hours there is much less of the absorbed
(57Co) cyanocobalamin in the brush border and
lysosomal fractions, where it had previously been
concentrated, but there is concentration of radioactivity in the cytosol.
We found no evidence of concentration of radioactivity in the mitochondria of ileal enterocytes
during the absorption of (57Co) cyanocobalamin.
This contrasts with previous studies,9 10 and may be
due to differences in the fractionation procedures
employed. Peters and Hoffbrand9 demonstrated
that two hours after oral (57Co) cyanocobalamin
radioactivity was concentrated in a combined
mitochondrial and lysosomal fraction obtained
by differential centrifugation of guinea-pig ileal
homogenates. Additional fractionation of the
combined mitochondrial and lysosomal fraction
into partially purified mitochondria and lysosomes showed concentration in the former rather
than the latter. However, no assessment of the
purity of these fractions was made, and the maximum proportion of 57Co in the combined mitochondrial and lysosomal fraction was 31 %. In
contrast, approximately 50% of the total radioactivity was recovered in the soluble fraction at
all times studied from 30 minutes to four hours.
Rosenthal et al.,10 who also performed differential
centrifugation of guinea-pig ileal homogenates,
found that the cytosol contained very little B,2
during early absorption, but reached peak values
four hours after dosage. T'his is similar to our
findings. They also demonstrated transient concentration of B12 in both mitochondrial and microsomal fractions maximal after 90 minutes and suggested that, during absorption, B12 was first
localised there, and then released into the cytosol
in dialysable form. However, no attempt was made
to separate the'lysosomes, which would therefore
have sedimented with the crude mitochondrial
and microsomal fractions which they obtained.
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Subcellular localisation of vitamin B12 during absorption in the guinea-pig ileum
We found that the use of iso-osmotic gradients
of Percoll in sucrose made possible rapid and
reproducible subcellular fractionation of guineapig ileal enterocytes with minimal damage to the
organelles, as demonstrated by the high lysosomal
latency. Figure 3 shows that damaging the lysosomes, in this case 'by treatment with digitonin,
causes release of their contents, which are recovered mainly in the soluble fraction. In previous
studies of vitamin B,2 absorption9 it is possible
that some mechanical disruption of the lysosomes
may have occurred in the multiple centrifugation
and resuspension procedures, and caused (57Co)
cyanocobalamin, which had been contained within
the lysosomes in vivo, to be recovered in the
soluble fraction.
We conclude that during the absorption of
(57Co) cyanocdbalamin it is significantly concentrated in the lysosomes of the ileal enterocytes,
and that concentration in the cytosol occurs later,
so that the period of ileal delay corresponds to the
intralysosomal phase. These conclusions are consistent with the process of receptor-mediated endocytosis, which has been described for the uptake
of many receptor-bound proteins,'7 including the
uptake of transcobalamin II-bound B,2 by rat
kidney'8 and liver,'9 and by human fibroblasts.20
We suggest that, after binding at the brush border,
IF-B,2 is first sequestrated in secondary lysosomes
formed by the fusion of endocytic vesicles with
primary lysosomes, and then B,2 is released into
the cytosol, from where it leaves the cell to enter
the portal blood bound to transcobalamin II.
What happens to IF is unknown. It may be degraded by lysosomal enzymes, which is the fate of
many other transport proteins.'7 This possibility
may be elucidated by future studies with satisfactorily radiolabelled IF. IF appears to be internalised during vitamin B,2 absorption, as studies in
which isolated guinea-pig ileal enterocytes were
incubated with (57Co) cyanocobalamin complexed
with human IF show intracellular 57Co still associated with IF after 45 minutes.8 These results complement our own, and lend support to the suggestion that IF-B,, is absorbed intact in the ileum
by receptor-mediated endocytosis.
This work was supported by the Peter Samuel
Fund of the Royal Free Hospital. We acknowledge
the interest and encouragement of Professor A V
Hoffbrand. KBT was supported by a US Public
Health Service Fogarty Senior International
Fellowship.
621
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Subcellular localisation of vitamin
B12 during absorption in the
guinea-pig ileum.
W J Jenkins, R Empson, D P Jewell and K B Taylor
Gut 1981 22: 617-622
doi: 10.1136/gut.22.8.617
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