Clinical Science and Molecrilar Medicine (1974) 46, 163-172.
TISSUE DISTRIBUTION OF COENZYME A N D OTHER
FORMS OF VITAMIN B , , I N CONTROL SUBJECTS A N D
PATIENTS WITH PERNICIOUS A N A E M I A
J. C. L I N N E L L , A. V. H O F F B R A N D , H . A-A. HUSSEIN,
I R E N E J . WISE AND D. M. MATTHEWS
Department of Experimental Chemical Pathology, Westminster Medical School,
London, and Department of Haematology, Royal Postgraduate Medical School,
London
(Received 27 June 1973)
SUMMARY
1. Methylcobalamin (Me-B, ,), adenosylcobalamin (Ado-B ,), hydroxocobalamin
(OH-B,,) and cyanocobalamin (CN-Bl2) have been estimated by a chromatographicbioautographic technique in plasma, erythrocytes, leucocytes and bone marrow
from normal subjects, hospital controls and patients with untreated pernicious
anaemia.
2. Estimates of concentrations of cobalamins have also been obtained in bile,
cerebrospinalfluid, liver biopsies and in autopsy samples of liver, kidney, spleen, brain
and pituitary.
3. In normal and control subjects, Ado-B,, predominated in all samples except
plasma, in which Me-B,, was the predominant form. Me-B,,, Ado-B,,, OH-B12and
CN-B,, were found in normal erythrocytes, leucocytes and bone marrow and the
proportion of each cobalamin was fairly similar in all these tissues. In liver, kidney,
spleen, brain and pituitary, the proportions of the cobalamins were more variable.
No CN-B,, was detected in these organs.
4. In untreated pernicious anaemia, Me-B,, was disproportionately reduced in
plasma, but not in erythrocytes, leucocytes or bone marrow. There was a small
increase in the proportion of CN-B,, in plasma, blood cells and bone marrow in
untreated pernicious anaemia.
Key words : tissue vitamin B,, coenzymes, vitamin B,,, pernicious anaemia.
In man, two cobalamins are known to have coenzyme functions. 5-Deoxyadenosylcobalamin
(Ado-B,) is required for the isomerization of methylmalonyl-coenzyme A to succinyl-coenzyme A (Weissbach & Taylor, 1968) and methylcobalamin (Me-B,,) is involved with folate
in the methylation of homocysteine to methionine (Woods, Foster & Guest, 1965; Weissbach
& Taylor, 1968). Vitamin B,, deficiency leads to megaloblastic anaemia, which is thought to be
Correspondence: Professor D . M. Matthews, Department of Experimental Chemical Pathology, Vincent
Square Laboratories of Westminster Hospital, 124 Vauxhall Bridge Road, London SWlV 2RH.
B
163
J. C.Linnell et al.
164
due to a defect in DNA synthesis, and evidence is now accumulating (Metz, Kelly, Swett,
Waxman & Herbert, 1968; Corcino, Waxman & Herbert, 1970; Van der Weyden, Cooper &
Firkin, 1973) which suggests that methylcobalamin plays an important r61e in DNA synthesis
in human cells. Despite intensive investigation of vitamin B,, metabolism, however, little is
known about the distribution of the different cobalamins in health or in disease.
Total vitamin B,, has been estimated in a number of tissues (Ross & Mollin, 1957; Halsted,
Carroll & Rubert, 1959; Hsu, Kawin, Minor & Mitchell, 1966; Rappazzo, Salmi & Hall,
1970) and the results show that much of the vitamin B,, in the body is in the liver. Tracer
studies with labelled cyanocobalamin (CN-B,,) indicate that after an oral or parenteral dose
most of the radioactivity accumulates in the liver (Adams, 1962; Doscherholmen & Hagen,
1962), although a variable proportion is detectable in other tissues. An enzymic assay has been
developed from the diol-dehydrase reaction (Abeles, Myers & Smith, 1966) for estimating
Ado-B,, and its results indicate that 40-70% of the total vitamin B I Zin liver is present in this
form. More recently the sensitivity of this assay has been increased (Turner & Mervyn, 1971)
but it has not yet been successfully applied to plasma. No enzymic assay has yet been found
suitable for estimating Me-B but a qualitative chromatographic and bioautographic method
has shown that a small proportion of the total vitamin B,, in normal liver is present as Me-B,,
(Lindstrand, 1964). As far as we are aware, nothing is known about the distribution of individual cobalamins in other tissues.
A chromatographic-bioautographic method has recently been described for separating and
estimating the individual cobalamins in plasma and tissues, and values for plasma have been
reported in normal subjects and patients with pernicious anaemia and other conditions in
which the distribution of individual cobalamins is disturbed, including certain types of optic
atrophy (Linnell, Mackenzie & Matthews, 1969a; Linnell, Mackenzie, Wilson & Matthews,
1969b; Linnell, Hussein & Matthews, 1970; Linnell, Hoffbrand, Peters & Matthews, 1971).
Preliminary studies showed that there were considerable differences in cobalamin distribution
between plasma and the cellular tissues (Linnell, 1972). Values have now been obtained for
erythrocytes, leucocytes, bile, liver, bone marrow and cerebrospinal fluid from healthy volunteers and hospital controls, and from other control tissues taken at autopsy. These values
are reported here together with those for cobalamins in plasma, erythrocytes, leucocytes and
bone marrow from patients with untreated pernicious anaemia.
Some of the results have already been reported in brief (Linnell, Hoffbrand, Hussein,
Matthews & Wise, 1973).
,,
MATERIALS A N D METHODS
Patients and control subjects
Normal subjects were healthy adult volunteers (medical and laboratory staff, mean age
29.6 years). Hospital controls were in-patients with conditions in which there was no evidence
of a disturbance of vitamin Blz metabolism. Samples of bone marrow and cerebrospinal fluid
were portions of samples obtained for diagnostic purposes. Samples of bile were aspirated
from gall-bladders removed at operation for gall-stones. The diagnosis of pernicious anaemia
was made on the basis of a megaloblastic marrow, subnormal serum vitamin B,, level, pentagastrin-fast achlorhydria and malabsorption of radioactive vitamin B corrected by intrinsic
factor. In each case the anaemia responded satisfactorily to vitamin B,, therapy.
,
Tissue cobalamins
165
Collection of samples
Heparinized venous blood and sternal bone marrow aspirates were taken in the usual manner
but, to prevent photolytic conversion of the cobalamin coenzymes and CN-B12 into hydroxocobalamin (OH-B,,), syringes and sample containers were covered with aluminium foil. All
stages of the method up to and including the chromatography were carried out by red light or
in darkness.
Leucocytes. An aliquot (10 ml) of blood was mixed with an equal volume of Dextraven-110
(6% dextrans, molecular weight approximately 110 000 in NaCl(l54 mmol/l); Fison's Pharmaceuticals, Loughborough, Leics.) in a 20 ml syringe. The syringe was left in a vertical position
with the tip uppermost for 45 min when sedimentation of the erythrocytes was complete. A 21
gauge x 40 mm hypodermic needle was bent through an angle of 130" and fitted to the syringe.
The upper layer of dextran and leucocytes was then expelled into a conical 15 ml centrifuge
tube and the cells were centrifuged down (10 min at 1500g). After aspiration of the dextran, the
cell pellet was resuspended in water (1 ml) to lyse residual erythrocytes. After 30 s, saline (10
ml) was added to the tube and the cells were again centrifuged down. Finally, the saline was
aspirated and the walls of the tube were carefully wiped with paper tissue to remove residual
droplets. The tube was then wrapped in foil and weighed. The cells were lysed in water (4 ml)
and stored at -20°C in a clean tube. The dry centrifuge tube plus foil wrapping was re-weighed
to allow the weight of cells to be calculated.
Plasma and erythrocytes. Heparinized blood was centrifuged (10 min at 2000 g) and the
plasma separated into a clean tube and stored at -20°C. The leucocyte layer was then
aspirated and rejected; the lower erythrocyte layer was stored at -20°C until required.
Bone marrow. The marrow samples were centrifuged (10 min at 2000 g ) in a Wintrobe haematocrit tube and the fat and plasma layers were removed with a Pasteur pipette. The marrow
cell layer, which was paler than the underlying erythrocytes, was then carefully aspirated and
washed with saline into a 15 ml conical centrifuge tube. After centrifuging, the saline was
removed and the walls of the tube were dried with paper tissue to leave a moist cell pellet. The
tube was covered with foil and weighed. The cells were then re-suspended in distilled water (4
ml) and stored at -20°C in a clean tube. Finally, the empty centrifuge tube plus foil was reweighed and the weight of cells was calculated.
Bile. Samples were taken with a foil-covered syringe and stored at -20°C in a foil-covered
tube until assayed.
CerebrospinalJluid (CSF). Samples were taken for diagnostic purposes from a patient with
untreated pernicious anaemia and from another subject found to be haematologically normal.
Both CSF samples had a normal cell count and protein concentration. An aliquot of each was
retained for the estimation of cobalamins. Exposure to white light was avoided and the samples
were stored at -20°C in a foil-wrapped tube until required.
Other tissues. Autopsy specimens were taken not more than 12 h after death from cases of
fatal coronary thrombosis with no evidence of neoplastic disease or haematological abnormality. A small sample (0.1-0.5 g) was cut from the centre of each piece of tissue in the darkroom and weighed wrapped in a tared square of foil. The tissue was homogenized in water
(10 ml) in a Potter-Elvehjem homogenizer and the homogenate stored frozen until analysis.
Portions of liver biopsies obtained for diagnostic purposes were retained for the estimation of
cobalamins. The biopsy specimen (5-10 mg) was weighed in a tared square of foil and then
homogenized in water (5 mi). The homogenate was stored at -20°C until analysis.
J. C.Linnell et al.
166
Estimation of total vitamin B I Zand cobalamins
Aliquots (0.5 or 1.0 ml) of plasma, erythrocytes, bile and the tissue homogenates were
measured with an Eppendorf micropipette and extracted by heating with an acetate-cyanide
buffer containing 12 g of NaOH, 45 ml of acetic acid and 40 mg of KCN, made up to 1 litre and
the pH adjusted to 4.5.Total vitamin B,, was estimated by radioisotopic assay (Matthews,
Gunasegaram & Linnell, 1967) but this method proved insufficiently sensitive for the available
quantities of leucocytes and bone marrow, which were very small, and for these a microbiological method employing Euglena gracilis z strain with papain extraction was used (Anderson,
1965). Further aliquots of tissue homogenates were extracted with hot ethanol, as for plasma,
and the cobalamins estimated by a chromatographic-bioautographic method with one- or
two-dimensional chromatography. Details of the method have already been fully described
(Linnell et al., 196913, 1970, 1971).
Briefly the technique involves ethanol extraction of the sample followed by concentration of
the cobalamins as an aqueous extract. The cobalamins are separated by one- or two-dimensional thin-layer chromatography and located bioautographically by over-layering the chromatograni with agar inoculated with a vitamin B,,-responsive strain of Escherichia coli and a
growth indicator (a tetrazolium salt). After incubation, red zones appear on the bioautogram
in positions corresponding to the separated cobalamins and these are quantified by
photometric scanning and comparison with standard curves constructed for each cobalamin.
All four cobalamins are separable by the two-dimensional technique but with one-dimensional
chromatography adenosylcobalamin and hydroxocobalamin are imperfectly resolved and
estimated together. The mixture is referred to as Ado-OH BIZ.
RESULTS
Normal subjects and hospital controls
Plasma. The results of estimation of plasma cobalamins in twenty-two normal subjects and
hospital controls are shown in Table 1. The separation of cobalamins in one normal subject is
illustrated in Fig. 1. Values for total vitamin BIZ were slightly lower than those reported
previously in seventy-three control subjects (Linnell et al., 1971). Me-B,, was the predominant
cobalamin in all subjects. Analysis of seven samples by the two-dimensional technique showed
that these contained almost three times as much Ado-B,, (mean 22.8%) as OH-B,,.
Erytlzrocytes. Erythrocyte cobalamins were estimated in twelve normal subjects and
hospital controls. The values showed considerable differences from those in normal plasma
(Table 1). In all samples Ado-OH BIZpredominated, and, in two samples analysed by the twodimensional technique, more than half of the total vitamin BIZwas present as Ado-B,, (mean
59%) and almost one-third as OH-B,, (mean 30%). CN-B,, formed a higher percentage of the
total vitamin B,, (5.8% k 1.1) than in plasma.
Leucocytes. In leucocytes total vitamin B, was about 10 times the plasma concentration.
Ado-OH B, predominated in all samples and, in five, separate analysis for Ado-B and OHB,, showed that almost half of the total vitamin B,, was Ado-B,, and approximately onequarter was OH-B,,. Me-B,, accounted for about 20% of the total vitamin BIZ.The CN-BIZ
concentration in leucocytes was almost 15 times as great as in erythrocytes.
Bone marrow. Results in bone marrow were fairly similar to those in leucocytes, though
,
,,
Tissue cobalamins
FIG. 1. Plasma cobalamins from a normal subject (two-dimensional chromatography and bioautography). A trace of CN-BI2 is present in this sample. In this and subsequent Figures the
origin is at the lower left-hand corner and marked by a dot.
FIG.2. Cobalamins in a liver biopsy from a control subject (total vitamin B I Z 500 ng/g). The
chromatogram has been overloaded to show up the very small proportion of Me-Bl2 in this tissue.
(Facing p . 166)
J. C. Linnell et al.
FIG.3. Cobalamins in a sample of spleen from a control subject (total vitamin BIZ 56 ng/g). The
relatively high proportion of Me-BI2 can be seen.
FIG.4. Plasma cobalamins from a patient with untreated pernicious anaemia (plasma total vitamin
B I 2 80 pg/nil). Me-B1, is disproportionately reduced. No CN-BI2 is detectable in this sample.
Control 1
Control 2
Control 3
Early
pernicious
anaemia
129602 14-40
6450 L- 1404
13
10
15
14
65
25
PA)
Mean? SEM.
1700k316 13
984+462 15
657290 20
518+243 20
3124.2
23k3.2
251k22
22k3.5
(pg/ml)
Me-Bi2
2742 108
375+ 139
139+40
243 + 202
+
7.8 1.2
8.2k2.2
(pdml)
18400
210
18
20
Bile
Bile
CSF
CSF
150
0.8
700
21
1.4
4
4
10
8
0
1235
0
1.8
0
7
0
10
17.0
85
50
78
14
2.2
7210
25
1.4
109402 1275 85
5091~ 110179
2.1
5.8
79
77
2978L-458 76
1813L-1364 70
159224
129L-29
5.5
9.5
33
66
(%I
4.1
9.4
126L-12
58k7.6
(pg/ml)
Ado-OHBiZ
2.0
9.3
(%)
9255
164
13.4
CN-BI 2
TABLE2. Cobalamins in bile and cerebrospinal fluid (CSF)
38302437
2575 L- 1467
+
202 25
168239
18
3
12
9
22
12
(Pg/d
Total vitamin BIZ
945
210
280
Leucocytes
Controls
Pernicious anaemia
Bone marrow
Controls
Pernicious anaemia
Plasma
Controls
Pernicious anaemia
Erythrocytes
Controls
Pernicious anaemia
No. of
samples
TABLE
1. Blood and bone marrow cobalamins in control subjects and patients with pernicious anaemia
11
39
12
8
4
Q\
c-r
(autopsy 1)
(autopsy 2)
(autopsy 1)
(autopsy 2)
Mean
(autopsy 1)
(autopsy 2)
(autopsy 3)
Mean
(autopsy 2)
Kidney
Spleen
Brain
Pituitary
Mean
Mean
(autopsy 1)
(autopsy 2)
Liver
Mean
(SEM)
(biopsy 1)
(biopsy 2)
(biopsy 3)
(biopsy 4)
(biopsy 5)
(biopsy 6)
(biopsy 7)
Liver
Tissue
47
9
6
3
6
21
25
23
31
36
34
20
23
21.5
11
8
6
7
18
25
14
12.7
(2.6)
29
25
27
30.0
44.6
37.3
21
111
20
86
44
50
52.2
71
25.7
4.3
4.3
11.4
47.8
56.6
55
86
30.0
23.7
26.9
48
57.1
62.4
63.8
61.1
41.4
44.6
43.0
84.3
55.2
69.8
565
243
404
3.0
5.2
4.1
59.9
58.8
55.0
60.0
65.0
67.9
61.1
61.1
(1.6)
635
490
275
423
943
1175
648
656
(117)
1.o
1.o
1.2
1.o
1.2
1.5
1.3
1.2
(0.07)
No cyanocobalamin detected in any sample.
230
35
138
69
81
70
56
63
115
152
134
555
670
440
1060
833
500
705
1450
1730
1060
1048
(161)
TABLE
3. Cobalamins") in normal liver, kidney, spleen, brain and pituitary gland
72
6
46
22
25
20
6
13
29
30
29
85
174
130
414
335
219
275
489
530
398
380
(42.2)
31
17.2
33.3
31.9
27.5
28.6
10.7
19.7
25.2
19.7
22.5
12.7
39.6
26.2
39.1
40.2
43.8
39.0
33.7
30.6
37.6
37.7
(1.6)
Tissue cobalamins
169
total vitamin B,, was higher. In two samples, the complete separation of all four cobalamins
showed that more than half of this zone was Ado-B,, (mean 55%).
Bile and cerebrospinalfluid. In bile, Ado-B,, was the major cobalamin in both samples
(Table 2). In cerebrospinal fluid, Ado-B,, predominated and in addition there were small
proportions of Me-B,, and OH-B,,.
Liver, kidney, spleen, pituitary and brain. Table 3 shows the results in seven liver biopsies from
haematologically normal subjects together with those from autopsy samples of liver, kidney,
spleen, pituitary and brain. Figs. 2 and 3 show the separation of cobalamins in a liver biopsy
and a control sample of spleen respectively. Values for Ado-B,, in liver are in good agreement
with values obtained by the diol-dehydrase assay (Cardinale, Dreyfus, Auld & Abeles, 1969).
Although Me-B,, represented only a small percentage of the total vitamin B,,, the actual
concentration was more than 50 times that in normal plasma. The liver biopsies were found to
have proportionately less Me-B,, and more OH-B,, than the autopsy samples. Although the
reason for this is uncertain, some photolysis of Me-B,, may have occurred in the small liver
biopsies by accidental exposure to white light during sampling.
Ado-B,, was the predominant cobalamin in liver, kidney, spleen and brain, and as a
proportion of the total vitamin B, ,, values for this cobalamin were remarkably constant among
these organs although the actual concentration varied widely. OH-B,, followed a similar
pattern. In contrast the proportion of Me-B,, varied widely between different organs. The
significance of the proportions of the different cobalamins in the various tissues is not known.
Untreated pernicious anaemia
In the present series of patients, values for plasma cobalamins (Table 1) were similar to those
reported earlier (Linnell et al., 1971). The separation of cobalamins in plasma from one patient
is shown in Fig. 4.Me-B,, was reduced disproportionately to a mean value of only 8% of the
normal. In contrast, plasma CN-B,, (mean 8.8%&2.0) was significantly raised (t = 3-8,
P<O.OOl) and in three cases CN-B,, accounted for more than 14% of the total plasma
vitamin B,,.
In the red cells, unlike the plasma, the ratio Me-B,,/Ado-OH B,, was not disturbed. CNB,, was, however, increased.
The alteration of the cobalamin pattern in leucocytes and bone marrow was similar to that in
red cells and each contained a higher proportion of CN-B,, than leucocytes and bone marrow
from normal subjects (0.05 >P>0.02).
DISCUSSION
Me-B,, is the major cobalamin in normal plasma (Lindstrand & Stahlberg, 1963; Linnell
et al., 1969a, 1971) and in milk (Craft, Matthews & Linnell, 1971). Ado-B,, and OH-B,, are
also present in plasma and milk, and in a minority of samples there may be traces of CN-BIZ.
The present results show that Ado-B,, is the predominant cobalamin in the cellular tissues
analysed. Total vitamin B,, concentration varies from tissue to tissue. Among bone marrow
cells, peripheral leucocytes and mature erythrocytes, total vitamin B,, is highest in the marrow
and lowest in erythrocytes. The proportions of individual cobalamins in erythrocytes, leucocytes and marrow are, however, remarkably constant. Approximately half of the total vitamin
B,, is present as Ado-B,,, one-fifth as Me-B,, and between one-quarter and one-third as
J. C. Linnell et al.
170
OH-B,,. A small proportion of CN-B,, is present in some samples of marrow and blood cells.
In several organs total vitamin B,, levels are very much higher than those in blood or bone
marrow and the cobalamin pattern is less constant in these organs. Of those analysed, liver
contains by far the highest levels of both Ado-B,, and OH-B,,, spleen and brain the lowest.
On the other hand, the highest levels of Me-B,, were found in pituitary and kidney. The liver
was found to contain 5% or less of Me-B,, but this cobalamin accounts for about a quarter of
the total vitamin B,, in kidney. One kidney weighing perhaps 150 g would contain more than
six times the Me-B,, in the total plasma volume, and it may likewise be calculated that in an
adult, the liver, kidney, spleen and brain contain about 50 pg of Me-B,,, i.e. some 2% of the
total cobalamin content of the body. The large proportion of Me-B,, in the spleen might at
first sight be attributed to the high blood content of this organ, but even if the spleen were all
blood it would contain only perhaps 30 ng of Me-B,,. In fact the adult spleen is found to
contain almost 150 times as much. It is of interest that cobalamin levels in the marrow are so
much lower than those in many of the other organs of the body.
One constant finding was the absence of CN-B,, from all samples of liver, kidney, spleen,
brain and pituitary. This contrasted with the situation in blood, bone marrow, bile and CSF
where a small proportion of CN-B,, was detected in many of the normal samples. In untreated
pernicious anaemia the proportion of CN-B,, in blood and bone marrow was increased, and
was greatest in the marrow. An increased proportion of CN-B,, in plasma has also been
reported in association with certain neuro-ophthalmological disorders (Linnell, Wilson &
Matthews, 1969c; Wilson, Linnell & Matthews, 1971).
The present results show that in untreated pernicious anaemia, it is only in the plasma that
Me-B is disproportionately reduced. In cellular tissues the percentage reduction of Me-B, ,,
Ado-B,, and OH-B,, is fairly similar. In view of the dramatic effects of vitamin B,, deficiency
on the normal functioning of the bone marrow, it is perhaps surprising that in pernicious
anaemia the marrow cobalamin levels are not more severely reduced. Vitamin B,, is taken up
preferentially by proliferating primitive cells rather than by mature cells (Schilling & Meyer,
1964; Hoffbrand, Tripp & Das, 1973). The bone marrow in untreated megaloblastic anaemia
contains proportionately far more primitive cells than a normal marrow and it may be that the
marrow cells in pernicious anaemia are in fact much more depleted of vitamin B,, for their
stage of development that is apparent by a direct comparison with normal marrow.
The chromatographic-bioautographic method is the only one at present available for estimation of all four forms of vitamin B,, found in blood and other tissues. Besides providing basic
information about normal cobalamin distribution, it may be expected to be particularly
useful in investigation of inborn errors of vitamin B,, metabolism in which normal plasma
concentrations of total vitamin B,, may mask a gross of disturbance of the pattern of individual cobalamins (Matthews & Linnell, 1971).
,
ACKNOWLEDGMENTS
Our thanks are due to Mr J. 0. Morgan for carrying out the microbiological vitamin B,, assays
and to all those who kindly provided specimens. Financial support from the Wellcome Trust
and the Variety Club of Great Britain is gratefully acknowledged.
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