British Journal ofhlaematology, 1975,31,493.
Forms of Vitamin BI2in Blood and Bone Marrow in Patients with Pernicious Anaemia TSUKASA ABE,*BERNARD GIBBSAND BERNARD A, COOPER Division of‘Hematology and Clinical Chemistry, Royal Victoria Hospital, and McGill University Clinic, Montreal, Quebec, Canada (Received 20 February 1975; acceptedfor publication
25
March 1975)
SUMMARY. The biologically-active forms of vitamin B, in blood and bone marrow and changes induced in these by injections of cyanocobalamin have been measured in patients with pernicious anaemia. Bone marrow methylcobalamin was low before therapy, atid increased 24 h after therapy. The largest portion of bone marrow vitamin Bl was j’deoxyadenosylcobalamin, and this increased more than did methylcobalamin during the 24 h after injection of cyanocobalamin. A single injection of 1000pg of cyanocobalamin induced about 10times the increase of the intracellular coenzyme forms of vitamin B I 2 in bone marrow than followed injection of IOO pg. Plasma methylcobalamin was extremely low before therapy, and increased only moderately 24 h after therapy; the majority of plasma vitamin B I 2 remaining as cyanocobalamin. In contrast, only a minority of intracellular bone marrow vitamin B1 was cyanocobalamin 24 h after injection of cyanocobalamin. The degree of anaemia did not correlate with bone marrow methylcobalamin, nor did bone marrow cobalamin correlate significantly with cobalamin content of washed blood erythrocytes. Correlation was observed between intra-erythrocyte vitamin Biz content and the degree of anaemia; the correlation being inverse with haemoglobin concentration in the peripheral blood. Inverse correlation also was observed between MCV and erythrocyte folate content. These studies suggest that megaloblastic maturation appears at different concentrations of bone marrow vitamin B1 in different patients, presumably because in vitamin B, deficiency, eventual limitation of normoblastic maturation may be determined by factors such as folate metabolism, vitamin B I 2 binders, and the affinity of vitamin B, ,-dependent enzymes. Although the clinical response to 1000 pg of cyanocobalamin does not differ from that to IOO pg, the concentration of vitamin B, coenzymes in bone marrow cells was proportional to the cyanocobalamin injected.
,
,
,
,
Patients deficient in vitamin B I 2 respond to injection of small quantities of the vitamin, although the quantity required to correct methylmalonaturia often exceeds that required to
* Present address: Tokyo Medical and Dental University, Tokyo, Japan. Correspondence: Dr B. A. Cooper, Hematology Division, Royal Victoria Hospital, 687 Pine Avenue West Montreal, Quebec, Canada H3A IAI. 493
T. Abe, B. Gibbs and B. A. Cooper
494
induce haematological response (Cox & White, 1962). The majority of vitamin B,, in the plasma of normal subjects is methylcobalamin, whereas the majority in tissues is 5’deoxyadenosylcobalamin (adenosylcobalamin) (Linnell et al, 1969, 1974), and whereas the former acts as the coenzyme in conversion of methyltetrahydrofolate to tetrahydrofolate (Chanarin, Ig6ga),the latter functions as coenzyme in conversion of methylmalonylCoA to succinylCoA. We have studied the effect of injections of cyanocobalamin on the distribution of coenzyme forms in plasma bone marrow and peripheral blood erythrocytes of patients deficient in vitamin B12to determine if a differential rate of correction of the deficiency of the different forms of vitamin B, occurs after such therapy.
,
MATERIALS AND METHODS Serum and erythrocyte folate levels were determined as described previously (Baker ct a/, 1959). Serum vitamin B,, concentrations were determined with the Z strain of Euglena grucilis (Hutner et al, rgj6) and withE. coli as described.below. The different forms of vitamin B,, were fractionated as described by Linnell et al(196g) using Eastman 6061 silica gel thin layer sheets for thin layer chromatography, and E. coli ATCC 10799 for bioautography. The use of these thin layer sheets facilitated the separation of hydroxocobalamin and 5’deoxyadenosylcobalamin, the former remaining at the point of application during ascending chromatography in secondary-butanol-amonia-water while the latter migrated 10-1 5 cm. Standards of methylcobalamin and j’deoxyadenosylcobalaminwere supplied by Dr Kiyoshi Kawabe; cyanocobalarnin was purchased commercially, and hydroxocobalamin was prepared &om cyanocobalamin by illumination under acid conditions. Desalting of extracted samples utilized a Torbal desalting apparatus. Samples of blood or bone marrow were taken into a heparinizedsyringe,and shielded from light immedigtely. All subsequent extraction was done in a dark room with illuminations limited to a red safe light. Plasma was separated immediately from cells, for the extraction of vitamin Blz. Suspensions of bone marrow cells were obtained by mixing I volume of 20% dextran T15o with 45 volumes of bone marrow aspirate, and leaving the mixture to sediment for 30 min at 4°C. The supernatant was collected, mixed, and allowed to sediment again. The second supernatant was centrifuged at 180 g, the cell pellet washed thrice with ice-cold Krebs Ringer phosphate solution at pH 7.4, and resuspended in phosphate-buffered saline at pH 7.4 for cell counting and extraction of vitamin B1,. Erythrocytes were collected from the sediment after the settling procedure, washed thrice with cold Krebs-Ringer phosphate and resuspended in buffered saline. Median nucleated cell count in washed erythrocytes was less than 100 per pl whereas median erythrocyte count was 3 . 7 ~1 0 ’ per ~ 1. Median nucleated cell count in washed bone marrow preparations was 14.5x 109 per 1. with 2.3 x 10’ erythrocytes. In most instances when bone marrow preparations were assayed for vitamin Bl 2, washed erythrocyte preparations also were assayed. The changes observed after cyanocobalamin therapy in bone marrow preparations were not observed in preparations of washed erythrocytes. Before extraction of cobalamins, washed cells in suspension were homogenized by sonication for z min. Methyldonate excretion was measured by gas-liquid chromatography as described previously (Gibbs el al, 1972)in urine collected for 24 h after feeding 5 g of L-valine.
Vitamin BIZin Blood and Bone Marrow
495
Patients Studied (Table I) Nine patients with pernicious anaemia were studied during therapy for megaloblastic anaemia. The four men and five women studied ranged in age from 59 to 74 years (median 66 years). Three patients without deficiency of vitamin B 1 2also were studied: D.W., a 65year-old man, B.H., a So-year-old woman, and L.S., a Io-year-old child, all of whom were investigated for the possibility of deficiency of vitamin B, and found not to have this. B.H. and L.S. had received injections of cyanocobalamin several months and I week before study, respective1y. TABLE I. Details of patients studied Patiertt
WBC (lo9//.)
E.F.
13.2
J.R. R.S. A.P. K.M.
12.6
4.1 3.9
12.2
6.6
6.7
A.C.
N.C. J.M. P.F.
13.7
I44 142
24
3.2
68
30
11.1
9.0
129
5.2
1.8
2.5
3 .o 5.4 6.1
98 6 161
29 16 36
9.2
23
I0
321
50
76
14.0 > 14.0 > 14.0 17.0 14.2 > 14.0 3.7 11.1 > 14.0
I RESULTS
Fractionation of the vitamin B12 forms in samples of plasma from the six patients with pernicious anaemia revealed little methylcobalamin in plasma; the majority of the vitamin B12 chromatographing as 5 ’deoxyadenosylcobalaniin. These data are similar to those reported previously (Linnell et a / , 1969, 1974). Samples of bone marrow obtained from the patients revealed the distribution of vitamin B, forms listed in the ‘Before B, 2 ’ columns of Tables I1 and 111. Pretreatment samples contained 0-118 ng of methylcobalamin per 1. of nucleated cells (median 2 0 ) , and 104-397 ng per 1. of S’deoxyadenosylcobalamin (median 206). Mixed venous blood cells contained both methylcobalamin and S’deoxyadenosylcobalamin,but this was affected considerably by the content of leucocytes. In four specimens in which leucocytes were selectively removed by repeated differential centrifugation to less than IOO per PI, methylcobalamin ranged from o to 85 ng per 1. oferythrocytes (median 8) and S’deoxyadenosylcobalaniin from 3 I to 84 ng/l. (median 75) (data not shown in Tables). Intramuscular injection of cyanocobalamin induced a striking increase of vitamin B, in the washed cells of the bone marrow (Tables I1 and 111). The increase of both methylcobalamin and s’deoxyadenosylcobalamin in bone marrow was about 10times as great after a single injection of 1000 pg of cyanocobalaniin than after IOO pg. Some cyanocobalamin was found in bone marrow cells of some of the patients after therapy, but this represented a minority of the biologically-active cobalamins present. Determination of vitamin B, forms in plasma after therapy was less reliable because of the large dilution required to
T. Abe, B. Gibbs arid B. A. Cooper
496
prevent the cyanocobalamin spot from obscuring the methylcobalamin and j’deoxyadenosylcobalamin but data were adequate to demonstrate that in most patients methylcobalamin did not increase to normal levels of greater than roo ng/l. TABLE 11. Effect of IOO pg of cyanocobalamin on cobalamins in marrow and plasma
Cobalartrins 24 k a&r B , I (n,q//.)
Cobafattnins 6Pjbre El (rig//.)
Patient
E.F.
Tissue Marrow Plasma Marrow Plasma Marrow Plasma Marrow Plasma Marrow Plasma Marrow Plasma
124
J:R.
I79
R.S.
402
A.P.
457
K.M.
278
Median Median
203
50
Me
CN
j’dc.0
8 9 16.7
8 4
84
0
I1
0
o 0
83 32 98.75 65. 57.7 59 62.2
0
25 1.2
18
0
25.8
o o
33
0
6 0
S’deO
0
62
492
0
0
300
0
0
436 8 41
43 257
I73
12
376
653 18 568 175
230
609 95
365 44
I54
231
I000
75 568 75
0 0
o 16.4
o o
12.5
25.1
o
486 44 77
4 14.7 36
4
21
0
25
0
85.2 64
0
77
43
0
12
300
0
OH
CN
Me
OH
I2
0 0 0
0 0
TABLE III. Effect of 1000 pg of cyanocobalamin on marrow and plasma cobalamins ~~
Cobalanrins 24 /i ajer BI2(tag/!.)
Cobalamins 6 C f . r ~BI2(ng//.)
Patient A.C.
N.C. J.M. P.F. Median
D.W. B.H. L.S.
Tissue Marrow Plasma Marrow Plasma &ROW
Plasma Plasma Marrow Plasma Plasma Plasma Plasma
Me
CN
S‘deO
OH
Me
CN
S’deO
OH
63 3
0 0
a22
0
2398
tr
0
25
o 750
4802
12s
0 0
0
0
0
0
8 I9
0
363 63 190 8
0 0
0
0
4
1156 126 38 1156
0
0
’
o
0
0
0
I931
0
3122
2-52
0
2537
375 1931 226
0 0 0
0 0
25 222
0 0
13
0
74
0
32
2780
81 33 600
0
32
0
17 400
85
0.
63 187 750
750 750
150
1716
Ioooo
0
250
25
200
0
2250
187 525
375
250
38 tr
Methylrnalonatc Excretion Mcthylmaloiiate excretion after valiiie loading was followed in seven patients (Table IV). Although there was a tendency for methylnialonate excretion to decrease more rapidly after 1000than after Ioo,ug ofcyanocobalaniiii, this was not observed in all patients. No correlation
Vitamin B 1 2 in Blood and Bone Marrow
497
was found between j’deoxyadenosylcobalamin levels in fasting or post-treatment plasma and the rate of decrease of methylmalonate excretion (P> 0.1).
Correlation between Vitamin B , Content ofPlasma and Cells and Anaemia Significant correlation was not found between plasma vitamin BIZand either the vitamin B l z content of washed erythrocytes (r = 0.347, t = 1.30; P>o.z), or bone marrow cells (r = 0.75, t = 2.53, P = 0.05); nor was significant correlation found between erythrocyte vitamin B, and bone marrow vitamin B I 2 (r = 0.80, t = 2.67, P = 0.06). If more patients had been studied, the last two correlations may have been significant. Significant correlation was not observed between the level of methylcobalamin in plasma, washed erythrocyte or bone marrow cells and the degree of anaemia. Total vitamin BIZin washed erythrocytes correlated statistically and negatively with the admission haemoglobin level (r = - 0.837, t = 3.416, P P > 0.3), but significant correlation was observed between erythrocyte folate and the mean erythrocyte volume (MCV) when the patient first was seen (r = - 0.669, t = 4.02, P< 0.01).Serum and erythrocyte folate did not correlate significantly in this small sample (r = 0.478, t = 1.752, P>o.z). The correlation between MCV and erythrocyte folate is consistent with the well-known influence of folate status on anaemia in subjects deficient in vitamin B12 . This statistical relationship was found to be dependent on the inclusion of the two patients with MCV 91f l ; no significant relationship was detected between erythrocyte folate concentration and MCV of the other patients in the group. Thus, although the erythrocyte folate concentration of the two patients without significant macrocytosis was greater than thar of those with macrocytosis, the degree of macrocytosis did not correlate with the erythrocyte folate.
498
T.Abe, B. Gibbs and B. A. Cooper
DISCUSSION Bone marrow cells are not completely depleted of vitamin B, forms in most patients with vitamin R , 2deficient megaloblastic anaemia, although in some patients methylcobalamin concentration is extremely low. The levels of methylcobalamin determined in these cells may be presumed to have been inadequate for effective function of methylfolate transferase, the methylcobalamin-dependent step of folate metabolism. The variations in methylcobalamin concentration in these cells may have been due to vitamin B, in granulocyte precursors rather than erythroid precursors, or may reflect the availability of intracellular vitamin B, for metabolism. It is of interest that the concentration of 5'deoxyadenosylcobalamin in bone marrow cells obtained from most of the patients before therapy was greater than the methylcobalamin concentration 24 h after injection of Ioopg of cyanocobalamin, suggesting that if methylfolate transferase is the critical stepin megaloblastic maturation the total vitamin R , content of megaloblastic cells would be abquate for normoblastic maturation if S'deoxyadenosylcobalamin were readily converted to methylcobalamin. The analysis of cobalamins in our patients before treatment revealed results very similar to those reported by Linnell et al ( 1974). Although numerous reports indicate that the haematologic response to IOO pg of cyanocobalamin is complete and equal to that after injections of larger quantities of cyanocobalamin (Ungley, 1949; Chanarin, 1969a, b), the concentration of coenzyme forms of vitamin B I Z in bone marrow cells was much greater after injehtion of Iooopg than of Ioopg of cyanocobalamin. The bone marrow cells appeared to be cxtremely efficient in converting cyanocobalamin to coenzyme forms, since even 24 h after injection the majority of plasma vitamin H I was cyanocobalamin whereas a minority of bone marrow vitamin B1 was in this form. The low concentration of methyl- and j'deoxyadenosylcobalamin in plasma probably wcre inadequate to explain the large accumulation of intracellular coenzyme forms of vitamin B, in marrow from these coenzyme forms in plasma. Our failure to identify a relationship between the concentration of coenzyme forms of vitamin B I Zin plasma, bone marrow or erythrocytes, and the degree of anaemia suggests that thc absolute concentration of these is not the limiting factor in determining anaemia, but that other factors such as affinity of vitamin B I Zfor intracellular binders, and enzymes, and the folate content of bone marrow cells may determine the intracellular vitamin B1 concentration at which megaloblastic maturation will appear. The negative correlation between erythrocyte vitamin R , content and haemoglobin concentration may reflect the age of the erythrocyte population rather than erythrocyte production, but data are not available to test this. Erythrocyte vitamin B, correlates crudely with serum vitamin B, (Biggs ct al, 1964, but correlation is too weak to be observed among patients deficient in vitamin H , 2 . We did not observe significant correlation between plasma cobalamins and cobalamin concentration in bone marrow or peripheral blood erythrocytes. Correlation might have been observed between bone marrow cobalamins and erythrocyte cobalamins if a larger group of patients had been studied. ACKNOWLEDGMENT
We are grateful to Dr Kiyoshi Kawabe, Director, Quality Control Laboratory, Esai Conipany Ltd, Tokyo, for the gift of materials.
Vitamin B , in Blood and Bone Marrow
499
REFERENCES
V., FRANK,O., PASHER, I., BAKER, H., HERBERT, HUTNER, S.H., WASSEWAN, L.R. & SOBOTKA, H. (1959) A microbiological method for detecting folic acid deficiency in man. Clinical Chemistry, 5, 275. BIGGS,J.C., MASON,S.L.A. & SPRAY,G.H. (1964) Vitamin-B activity in red cells. British Journal of Haematology, 10. 36. CHANARIN, 1. (196ga) The Megaloblastic Anaemias, pp 923327. Blackwell Scientific Publications, Oxford. CHANARIN, I. (1969b) The Megaloblastic Anaemias, pp 961-965. Blackwell Scientific Publications, Oxford. Cox, E.V. & WHITE,A.M. (1962) Methylmalonic acid excretion : an index of vitamin-B deficiency. Lancet, ii, 853. GIBBS, B.F., ITIABA, K., MAMER, O.A., CRAWHALL, J.C. & COOPER, B.A. (1972) A rapid method for the analysis of urinary methylmalonic acid. Clinica Chimica Acta, 38, 447.
HUTNER, S.H., BACH,M.K. &Ross, G.I.M. (1956) A sugar-containing basal medium for vitamin B 2assay with Euglena: application to body fluids. Journal ofProtozoology, 3, IOI. LINNELL, J.C., HOFFBRAND, A.V., HUSSEIN, H.A.-A., WISE,I.J. & MATTXEWS, D.M. (1974) Tissue distribution of coenzyme and other forms of vitamin BIZ in control subjects and patients with pernicious anaemia. Clinical Science and Molecular Medicine, 46s 163. LINNELL, J.C., MAcKmzm, H.M., WILSON, J. & MATTHEWS, D.M. (1969) Patterns ofplasma cobalamins in control subjects and in cases of vitamin BI2 deficiency. Journal .f Clinical Pathology, 22, 545. UNGLEY,C.C. (1949) Vitamin BIZ in pernicious anaemia: parenteral administration. British Medical Journal, ii, 1370.
,
Forms of Vitamin BI2in Blood and Bone Marrow in Patients with Pernicious Anaemia TSUKASA ABE,*BERNARD GIBBSAND BERNARD A, COOPER Division of‘Hematology and Clinical Chemistry, Royal Victoria Hospital, and McGill University Clinic, Montreal, Quebec, Canada (Received 20 February 1975; acceptedfor publication
25
March 1975)
SUMMARY. The biologically-active forms of vitamin B, in blood and bone marrow and changes induced in these by injections of cyanocobalamin have been measured in patients with pernicious anaemia. Bone marrow methylcobalamin was low before therapy, atid increased 24 h after therapy. The largest portion of bone marrow vitamin Bl was j’deoxyadenosylcobalamin, and this increased more than did methylcobalamin during the 24 h after injection of cyanocobalamin. A single injection of 1000pg of cyanocobalamin induced about 10times the increase of the intracellular coenzyme forms of vitamin B I 2 in bone marrow than followed injection of IOO pg. Plasma methylcobalamin was extremely low before therapy, and increased only moderately 24 h after therapy; the majority of plasma vitamin B I 2 remaining as cyanocobalamin. In contrast, only a minority of intracellular bone marrow vitamin B1 was cyanocobalamin 24 h after injection of cyanocobalamin. The degree of anaemia did not correlate with bone marrow methylcobalamin, nor did bone marrow cobalamin correlate significantly with cobalamin content of washed blood erythrocytes. Correlation was observed between intra-erythrocyte vitamin Biz content and the degree of anaemia; the correlation being inverse with haemoglobin concentration in the peripheral blood. Inverse correlation also was observed between MCV and erythrocyte folate content. These studies suggest that megaloblastic maturation appears at different concentrations of bone marrow vitamin B1 in different patients, presumably because in vitamin B, deficiency, eventual limitation of normoblastic maturation may be determined by factors such as folate metabolism, vitamin B I 2 binders, and the affinity of vitamin B, ,-dependent enzymes. Although the clinical response to 1000 pg of cyanocobalamin does not differ from that to IOO pg, the concentration of vitamin B, coenzymes in bone marrow cells was proportional to the cyanocobalamin injected.
,
,
,
,
Patients deficient in vitamin B I 2 respond to injection of small quantities of the vitamin, although the quantity required to correct methylmalonaturia often exceeds that required to
* Present address: Tokyo Medical and Dental University, Tokyo, Japan. Correspondence: Dr B. A. Cooper, Hematology Division, Royal Victoria Hospital, 687 Pine Avenue West Montreal, Quebec, Canada H3A IAI. 493
T. Abe, B. Gibbs and B. A. Cooper
494
induce haematological response (Cox & White, 1962). The majority of vitamin B,, in the plasma of normal subjects is methylcobalamin, whereas the majority in tissues is 5’deoxyadenosylcobalamin (adenosylcobalamin) (Linnell et al, 1969, 1974), and whereas the former acts as the coenzyme in conversion of methyltetrahydrofolate to tetrahydrofolate (Chanarin, Ig6ga),the latter functions as coenzyme in conversion of methylmalonylCoA to succinylCoA. We have studied the effect of injections of cyanocobalamin on the distribution of coenzyme forms in plasma bone marrow and peripheral blood erythrocytes of patients deficient in vitamin B12to determine if a differential rate of correction of the deficiency of the different forms of vitamin B, occurs after such therapy.
,
MATERIALS AND METHODS Serum and erythrocyte folate levels were determined as described previously (Baker ct a/, 1959). Serum vitamin B,, concentrations were determined with the Z strain of Euglena grucilis (Hutner et al, rgj6) and withE. coli as described.below. The different forms of vitamin B,, were fractionated as described by Linnell et al(196g) using Eastman 6061 silica gel thin layer sheets for thin layer chromatography, and E. coli ATCC 10799 for bioautography. The use of these thin layer sheets facilitated the separation of hydroxocobalamin and 5’deoxyadenosylcobalamin, the former remaining at the point of application during ascending chromatography in secondary-butanol-amonia-water while the latter migrated 10-1 5 cm. Standards of methylcobalamin and j’deoxyadenosylcobalaminwere supplied by Dr Kiyoshi Kawabe; cyanocobalarnin was purchased commercially, and hydroxocobalamin was prepared &om cyanocobalamin by illumination under acid conditions. Desalting of extracted samples utilized a Torbal desalting apparatus. Samples of blood or bone marrow were taken into a heparinizedsyringe,and shielded from light immedigtely. All subsequent extraction was done in a dark room with illuminations limited to a red safe light. Plasma was separated immediately from cells, for the extraction of vitamin Blz. Suspensions of bone marrow cells were obtained by mixing I volume of 20% dextran T15o with 45 volumes of bone marrow aspirate, and leaving the mixture to sediment for 30 min at 4°C. The supernatant was collected, mixed, and allowed to sediment again. The second supernatant was centrifuged at 180 g, the cell pellet washed thrice with ice-cold Krebs Ringer phosphate solution at pH 7.4, and resuspended in phosphate-buffered saline at pH 7.4 for cell counting and extraction of vitamin B1,. Erythrocytes were collected from the sediment after the settling procedure, washed thrice with cold Krebs-Ringer phosphate and resuspended in buffered saline. Median nucleated cell count in washed erythrocytes was less than 100 per pl whereas median erythrocyte count was 3 . 7 ~1 0 ’ per ~ 1. Median nucleated cell count in washed bone marrow preparations was 14.5x 109 per 1. with 2.3 x 10’ erythrocytes. In most instances when bone marrow preparations were assayed for vitamin Bl 2, washed erythrocyte preparations also were assayed. The changes observed after cyanocobalamin therapy in bone marrow preparations were not observed in preparations of washed erythrocytes. Before extraction of cobalamins, washed cells in suspension were homogenized by sonication for z min. Methyldonate excretion was measured by gas-liquid chromatography as described previously (Gibbs el al, 1972)in urine collected for 24 h after feeding 5 g of L-valine.
Vitamin BIZin Blood and Bone Marrow
495
Patients Studied (Table I) Nine patients with pernicious anaemia were studied during therapy for megaloblastic anaemia. The four men and five women studied ranged in age from 59 to 74 years (median 66 years). Three patients without deficiency of vitamin B 1 2also were studied: D.W., a 65year-old man, B.H., a So-year-old woman, and L.S., a Io-year-old child, all of whom were investigated for the possibility of deficiency of vitamin B, and found not to have this. B.H. and L.S. had received injections of cyanocobalamin several months and I week before study, respective1y. TABLE I. Details of patients studied Patiertt
WBC (lo9//.)
E.F.
13.2
J.R. R.S. A.P. K.M.
12.6
4.1 3.9
12.2
6.6
6.7
A.C.
N.C. J.M. P.F.
13.7
I44 142
24
3.2
68
30
11.1
9.0
129
5.2
1.8
2.5
3 .o 5.4 6.1
98 6 161
29 16 36
9.2
23
I0
321
50
76
14.0 > 14.0 > 14.0 17.0 14.2 > 14.0 3.7 11.1 > 14.0
I RESULTS
Fractionation of the vitamin B12 forms in samples of plasma from the six patients with pernicious anaemia revealed little methylcobalamin in plasma; the majority of the vitamin B12 chromatographing as 5 ’deoxyadenosylcobalaniin. These data are similar to those reported previously (Linnell et a / , 1969, 1974). Samples of bone marrow obtained from the patients revealed the distribution of vitamin B, forms listed in the ‘Before B, 2 ’ columns of Tables I1 and 111. Pretreatment samples contained 0-118 ng of methylcobalamin per 1. of nucleated cells (median 2 0 ) , and 104-397 ng per 1. of S’deoxyadenosylcobalamin (median 206). Mixed venous blood cells contained both methylcobalamin and S’deoxyadenosylcobalamin,but this was affected considerably by the content of leucocytes. In four specimens in which leucocytes were selectively removed by repeated differential centrifugation to less than IOO per PI, methylcobalamin ranged from o to 85 ng per 1. oferythrocytes (median 8) and S’deoxyadenosylcobalaniin from 3 I to 84 ng/l. (median 75) (data not shown in Tables). Intramuscular injection of cyanocobalamin induced a striking increase of vitamin B, in the washed cells of the bone marrow (Tables I1 and 111). The increase of both methylcobalamin and s’deoxyadenosylcobalamin in bone marrow was about 10times as great after a single injection of 1000 pg of cyanocobalaniin than after IOO pg. Some cyanocobalamin was found in bone marrow cells of some of the patients after therapy, but this represented a minority of the biologically-active cobalamins present. Determination of vitamin B, forms in plasma after therapy was less reliable because of the large dilution required to
T. Abe, B. Gibbs arid B. A. Cooper
496
prevent the cyanocobalamin spot from obscuring the methylcobalamin and j’deoxyadenosylcobalamin but data were adequate to demonstrate that in most patients methylcobalamin did not increase to normal levels of greater than roo ng/l. TABLE 11. Effect of IOO pg of cyanocobalamin on cobalamins in marrow and plasma
Cobalartrins 24 k a&r B , I (n,q//.)
Cobafattnins 6Pjbre El (rig//.)
Patient
E.F.
Tissue Marrow Plasma Marrow Plasma Marrow Plasma Marrow Plasma Marrow Plasma Marrow Plasma
124
J:R.
I79
R.S.
402
A.P.
457
K.M.
278
Median Median
203
50
Me
CN
j’dc.0
8 9 16.7
8 4
84
0
I1
0
o 0
83 32 98.75 65. 57.7 59 62.2
0
25 1.2
18
0
25.8
o o
33
0
6 0
S’deO
0
62
492
0
0
300
0
0
436 8 41
43 257
I73
12
376
653 18 568 175
230
609 95
365 44
I54
231
I000
75 568 75
0 0
o 16.4
o o
12.5
25.1
o
486 44 77
4 14.7 36
4
21
0
25
0
85.2 64
0
77
43
0
12
300
0
OH
CN
Me
OH
I2
0 0 0
0 0
TABLE III. Effect of 1000 pg of cyanocobalamin on marrow and plasma cobalamins ~~
Cobalanrins 24 /i ajer BI2(tag/!.)
Cobalamins 6 C f . r ~BI2(ng//.)
Patient A.C.
N.C. J.M. P.F. Median
D.W. B.H. L.S.
Tissue Marrow Plasma Marrow Plasma &ROW
Plasma Plasma Marrow Plasma Plasma Plasma Plasma
Me
CN
S‘deO
OH
Me
CN
S’deO
OH
63 3
0 0
a22
0
2398
tr
0
25
o 750
4802
12s
0 0
0
0
0
0
8 I9
0
363 63 190 8
0 0
0
0
4
1156 126 38 1156
0
0
’
o
0
0
0
I931
0
3122
2-52
0
2537
375 1931 226
0 0 0
0 0
25 222
0 0
13
0
74
0
32
2780
81 33 600
0
32
0
17 400
85
0.
63 187 750
750 750
150
1716
Ioooo
0
250
25
200
0
2250
187 525
375
250
38 tr
Methylrnalonatc Excretion Mcthylmaloiiate excretion after valiiie loading was followed in seven patients (Table IV). Although there was a tendency for methylnialonate excretion to decrease more rapidly after 1000than after Ioo,ug ofcyanocobalaniiii, this was not observed in all patients. No correlation
Vitamin B 1 2 in Blood and Bone Marrow
497
was found between j’deoxyadenosylcobalamin levels in fasting or post-treatment plasma and the rate of decrease of methylmalonate excretion (P> 0.1).
Correlation between Vitamin B , Content ofPlasma and Cells and Anaemia Significant correlation was not found between plasma vitamin BIZand either the vitamin B l z content of washed erythrocytes (r = 0.347, t = 1.30; P>o.z), or bone marrow cells (r = 0.75, t = 2.53, P = 0.05); nor was significant correlation found between erythrocyte vitamin B, and bone marrow vitamin B I 2 (r = 0.80, t = 2.67, P = 0.06). If more patients had been studied, the last two correlations may have been significant. Significant correlation was not observed between the level of methylcobalamin in plasma, washed erythrocyte or bone marrow cells and the degree of anaemia. Total vitamin BIZin washed erythrocytes correlated statistically and negatively with the admission haemoglobin level (r = - 0.837, t = 3.416, P P > 0.3), but significant correlation was observed between erythrocyte folate and the mean erythrocyte volume (MCV) when the patient first was seen (r = - 0.669, t = 4.02, P< 0.01).Serum and erythrocyte folate did not correlate significantly in this small sample (r = 0.478, t = 1.752, P>o.z). The correlation between MCV and erythrocyte folate is consistent with the well-known influence of folate status on anaemia in subjects deficient in vitamin B12 . This statistical relationship was found to be dependent on the inclusion of the two patients with MCV 91f l ; no significant relationship was detected between erythrocyte folate concentration and MCV of the other patients in the group. Thus, although the erythrocyte folate concentration of the two patients without significant macrocytosis was greater than thar of those with macrocytosis, the degree of macrocytosis did not correlate with the erythrocyte folate.
498
T.Abe, B. Gibbs and B. A. Cooper
DISCUSSION Bone marrow cells are not completely depleted of vitamin B, forms in most patients with vitamin R , 2deficient megaloblastic anaemia, although in some patients methylcobalamin concentration is extremely low. The levels of methylcobalamin determined in these cells may be presumed to have been inadequate for effective function of methylfolate transferase, the methylcobalamin-dependent step of folate metabolism. The variations in methylcobalamin concentration in these cells may have been due to vitamin B, in granulocyte precursors rather than erythroid precursors, or may reflect the availability of intracellular vitamin B, for metabolism. It is of interest that the concentration of 5'deoxyadenosylcobalamin in bone marrow cells obtained from most of the patients before therapy was greater than the methylcobalamin concentration 24 h after injection of Ioopg of cyanocobalamin, suggesting that if methylfolate transferase is the critical stepin megaloblastic maturation the total vitamin R , content of megaloblastic cells would be abquate for normoblastic maturation if S'deoxyadenosylcobalamin were readily converted to methylcobalamin. The analysis of cobalamins in our patients before treatment revealed results very similar to those reported by Linnell et al ( 1974). Although numerous reports indicate that the haematologic response to IOO pg of cyanocobalamin is complete and equal to that after injections of larger quantities of cyanocobalamin (Ungley, 1949; Chanarin, 1969a, b), the concentration of coenzyme forms of vitamin B I Z in bone marrow cells was much greater after injehtion of Iooopg than of Ioopg of cyanocobalamin. The bone marrow cells appeared to be cxtremely efficient in converting cyanocobalamin to coenzyme forms, since even 24 h after injection the majority of plasma vitamin H I was cyanocobalamin whereas a minority of bone marrow vitamin B1 was in this form. The low concentration of methyl- and j'deoxyadenosylcobalamin in plasma probably wcre inadequate to explain the large accumulation of intracellular coenzyme forms of vitamin B, in marrow from these coenzyme forms in plasma. Our failure to identify a relationship between the concentration of coenzyme forms of vitamin B I Zin plasma, bone marrow or erythrocytes, and the degree of anaemia suggests that thc absolute concentration of these is not the limiting factor in determining anaemia, but that other factors such as affinity of vitamin B I Zfor intracellular binders, and enzymes, and the folate content of bone marrow cells may determine the intracellular vitamin B1 concentration at which megaloblastic maturation will appear. The negative correlation between erythrocyte vitamin R , content and haemoglobin concentration may reflect the age of the erythrocyte population rather than erythrocyte production, but data are not available to test this. Erythrocyte vitamin B, correlates crudely with serum vitamin B, (Biggs ct al, 1964, but correlation is too weak to be observed among patients deficient in vitamin H , 2 . We did not observe significant correlation between plasma cobalamins and cobalamin concentration in bone marrow or peripheral blood erythrocytes. Correlation might have been observed between bone marrow cobalamins and erythrocyte cobalamins if a larger group of patients had been studied. ACKNOWLEDGMENT
We are grateful to Dr Kiyoshi Kawabe, Director, Quality Control Laboratory, Esai Conipany Ltd, Tokyo, for the gift of materials.
Vitamin B , in Blood and Bone Marrow
499
REFERENCES
V., FRANK,O., PASHER, I., BAKER, H., HERBERT, HUTNER, S.H., WASSEWAN, L.R. & SOBOTKA, H. (1959) A microbiological method for detecting folic acid deficiency in man. Clinical Chemistry, 5, 275. BIGGS,J.C., MASON,S.L.A. & SPRAY,G.H. (1964) Vitamin-B activity in red cells. British Journal of Haematology, 10. 36. CHANARIN, 1. (196ga) The Megaloblastic Anaemias, pp 923327. Blackwell Scientific Publications, Oxford. CHANARIN, I. (1969b) The Megaloblastic Anaemias, pp 961-965. Blackwell Scientific Publications, Oxford. Cox, E.V. & WHITE,A.M. (1962) Methylmalonic acid excretion : an index of vitamin-B deficiency. Lancet, ii, 853. GIBBS, B.F., ITIABA, K., MAMER, O.A., CRAWHALL, J.C. & COOPER, B.A. (1972) A rapid method for the analysis of urinary methylmalonic acid. Clinica Chimica Acta, 38, 447.
HUTNER, S.H., BACH,M.K. &Ross, G.I.M. (1956) A sugar-containing basal medium for vitamin B 2assay with Euglena: application to body fluids. Journal ofProtozoology, 3, IOI. LINNELL, J.C., HOFFBRAND, A.V., HUSSEIN, H.A.-A., WISE,I.J. & MATTXEWS, D.M. (1974) Tissue distribution of coenzyme and other forms of vitamin BIZ in control subjects and patients with pernicious anaemia. Clinical Science and Molecular Medicine, 46s 163. LINNELL, J.C., MAcKmzm, H.M., WILSON, J. & MATTHEWS, D.M. (1969) Patterns ofplasma cobalamins in control subjects and in cases of vitamin BI2 deficiency. Journal .f Clinical Pathology, 22, 545. UNGLEY,C.C. (1949) Vitamin BIZ in pernicious anaemia: parenteral administration. British Medical Journal, ii, 1370.
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