British]oumal of Haematology, 1975, 31, 287.

The Three Transcobalamins 1n Myeloproliferative

Disorders and Acute Leukaemia

J. ZITTOUN, R. ZITTOUN,* J. MARQUET AND C. SULTAN

Serví ce Central d' Hématologie-I111111unologie, Hópital Henri Mondar, Creteil, and
*Service d'Hématologie, Hótel Dieu, París, France

(Received 6 March 1975; accepted Jor publícation 25 April 1975)

SuMMARY. The unsaturated vitamin B 12 binding capacity of whole serum (UBBC)

and of the three transcobalamins (TC) has been studied in patients with various
haematological diseascs including myeloproliferativc disorders (MPD) and acute
leukacmia. The binding capacity ofTC I and TC III was increased in MPD; TC I
being particularly high in chronic granulocytic leukacmia (CGL) and TC III espec-
ially raised in polycythaemia rubra vera (PRV) and in infectious leucocytosis. The
binding capacity ofboth TC I and TC III corrclated with blood neutrophil count and
the ratio TC III/TC I was low in CGL and increased in PRV. TC II was increased in
acute myelogenous leukaemia, during remission and blast cell crisis of CGL and
in refractory anaemia with excess of myeloblasts but not in acute lymphoblastic
leukaemia (ALL). TC II correlated inversely with blood neutrophil count. There
is an inverse ratio between TC II and TC I at least in myelogenous leukaemia.
These abnormalities are discussed in relation to granulocyte kinetics. TC III and TC I
reflect probably the total body granulocyte pool and share sorne biochemical and
immunological properties supporting the view that they have a common origin
in the more mature stages of the granulocyte cell line while TC II probably
originates partly in more primitive granulocytes.

Two main vitamin B 12 binding proteins have been identified in normal plasma and serum
(Hall & Finkler, 1963, 1964, 1965). Transcobalamin 1 (TC I), the 'a globulin binder' (Miller &
Sullivan, 1959) carries most of the endogenous vitamin B 12 (Pitney et al, 1954). A closely
related or identical protein has been idcntified in normal human granulocytes (Meyer et al,
1962; Simons & Weber, 1966; Stenman et al, 1968). Transcobalamin 11 (TC II), the 'fJ
binder', takes up most of the vitamin added to serum or plasma in vitro or in vivo (Hall &
Finkler, 1963, 1965). Unsaturated serum vitamin B 1 i-binding capacity (UBBC) is mainly
related to TC II and the function of this protein is probably to transport vitamin B 1 2 absorbed
from the gut to the tissucs (Hall, 1969), though a recent study has shown that vitamin B 12
absorbed from the ileum is taken up by both the vitamin B 1 i-binding proteins (England
et al, 1973). TC 11 is also more active than the other binders in promoting the delivery of

Correspondence: Dr J. Zittoun, Scrvice Central d'Hématologie-Immunologie, H6pital Henri Mondar, Creteil

94010, France.
288 J. Zittoun et al
labelled vitamin B 12 added in vitro to Hela cells (Finkler & Hall, 1967), immature erythro- graph against 0.1 N
cytes (Retief et al, l967b) or PHA transformed lymphocytes (Meyer et al, 1974). The origin were more accurat
of TC II is hypothctical but the decrease in the f3 binder during acute hepatitis in man Unsaturated vitan
(Retief et al, 1969) and the fall of a binder similar to TC II after induced injury ofliver in rats serum for l h at 3
(Tan & Hanscn, 1968), suggest hcpatic synthesis of this protein and recent studies using an (specific activity 5
isolated perfuscd rat liver preparation support this view (Cooksley et al, 1974). However, there visking tubing for •
is also evidence to suggest that TC II is, at least in part, of leucocyte origin (Retief et al, remaining after di
l967a; Carmel & Coltman, 1971) and variations in plasma of TC II concentration that may gamma counter (I
occur in sorne malignant diseases (Hall & Finkler, 1964, 1966; Gottlieb et al, 1966) support
the view that therc is a relation betwcen plasma TC II and leucocytes.
Fractionation oj the
More recently, a third plasma vitamin B 12 binder has been identified in polycythaemia vera
DEAE-cellulose.
(PRV) (Hall & Finklcr, 1969) and in cord plasma (Kumento et al, 1967) which has been
distilled water and
considered to be pathological or embryonic proteins respectively. A third binder has also
and pourcd into co
been found in normal sera (Gizis et al, 1970) but confused for a long time with TC I and TC II
l. 5 ml fractions co
because it shares important properties with them. Fractionation on both DEAE-cellulose and
TC II was elutee
Sephadex G-200 have been used to isolate this third binder (Bloomfield & Scott, 1972). We
collected in tubcs
have identified the three binders in normal serum by a technique comparable to that of Gizis
into tubes 27-40.
& Meyer (1972), using only one column fractionation on DEAE-cellulose with elution of
cluted with 1 M N
the three proteins by discontinuous gradients with buffers of different pH and molarity. We
of the three TC on
have called the third binder 'Transcobalamin III' ('TC III') for convenience.
radioactivity and t
The present investigation demonstrates variations in the serum concentration of the three
binders in acute leukaemia of various cytological types, in myeloproliferative diseases (MPD),
and in infectious leucocytosis, and examines the relationship between these proteins. We
have also attempted to identify the intracellular content of the transcobalamins at the various
stages of maturation of the myeloid cell line and thus to define the origins of the three
binders.
3000

MATERIALS AND METHODS

Subjects. 30 normal adult subjects, chosen from healthy volw1teers to a blood transfusion
centre were used as controls. Eight samples of cord serum were also examined. Patients
studied presented with various haematological diseases or abnormalities (Table I): chronic ~ 2000
u

granulocytic leukaemia (CGL: 39), myelosclerosis with myeloid metaplasia (MM: 14),
polycythaemia vera (PRV: 34), secondary polycythaemia (SP: 9), infectious leucocytosis
(IL: 5), acute non-lymphoblastic leukaemia (ANLL: 18), acute lymphoblastic leukaemia
(ALL: 7), chronic myelomonocytic leukaemia (CMML: 6), and refractory anaemia with 1000
excess of myeloblasts (RAEM: 8). Diagnosis was made by standard cytological, cytochemical
and cytogenetic methods.
Blood was collected without anticoagulant to avoid TC modifications induced by heparin
or EDT A (England et al, 1973; Scott et al, 1974). Serum was separated within 2 h of venous
blood collection and stored at - 2oº C. There was no difference in the UBBC or the distri- o
bution of the TCs in serum tested fresh and after storage.
Endogeno11s vitamin B 12 • This was assayed microbiologically using Lactobacillus leischmannii. f1G I.Pattern
The lactic acid produced by the growth of the micro-organism was measured with a potentio- change of buffe1
 Transcobalamins in Myeloproliferative Disorders 289
mature erythro- graph against 0.1 N sodium hydroxide after 48 h culture at 37º C. The results with this method
974). The origin were more accurate and constant than with turbidimetric or radiodilution methods.
hepatitis in man Unsaturated vitamin B 12 -binding capacity. UBBC was determined by incubating 1 ml of
f
rry o~liver _in rats serum for 1 h at 37º C with 0.1 ml of 58 CoB 12 ata concentration between 5 and IO ng
· stud1es usmg an (specific activity 5-50 µCi /µg) and excess vitamin B 12 was removed by dialysis in 8/ 32
. However, there visking tubing for 18 h at 4º C against a K-Na 2 phosphate buffer (0.02 M,pH 6.3). Thevolume
igin (Retief et al, remaining after dialysis was measured and the radioactivity determined in an automatic
1tration that may gamma counter (Packard) and expressed in pg 58 Co B 12 .
al, 1966) support

~ycythaemia vera
Fractionation oj the Transcobalamins
which has been DEAE-cellulose. DEAE-cellulose was treated with 0.5 N HCl and 0.5 N Na OH, washed with
distilled water and thc cellulose equilibrated for 24 h with 0.02 M phosphate buffer pH 6.3
~ binder has also
h TC I and TC II and pourcd into columns (25 cmx 1.4 cm). Serum was applied to the top of thc column and
!AE-cellulose and 1.5 mi fractions collected at a flow ratc of 1 ml/ min.
Scott, 1972). We TC II was eluted with 0.04 M K-Na 2 phosphatc buffer pH 5.9 (Gizis & Meyer, 1972), and
e to that of Gizis collected in tubcs 6- 24. 'TC III' was cluted with 0.06 M K-Na 2 phosphate buffer pH 6.3
1with elution of into tubes 27- 40. This buffer clutes the /3 binder (Bloomficld & Scott, 1972). TC 1 was
hd molarity. W e eluted with 1 M NaCl (Retief et al, 1967a) into tubcs 47-60. Fig 1 shows the elution pattern
of the three TC on DEAE-cellulose. The results wcre expressed as a percentagc of the applied
tion of the three radioactivity and thc rccovery calculatcd. In ali cases recovery was at least 85- 90'.Y,; .
~ diseases (MPD),
[se proteins. W e
rns at the various
------0·04M, pH 5·9
ins of the three

3000

lood transfusion
~mined. Patients

~
ble 1) : chronic ~ 2000
u
asía (MM: 14),
us leucocytosis
astic leukaemia
y anaemia wi th 1000
al, cytochemical

uced by heparin
111 2 h of venous
3C or the distri- o 20 30 40 50
Tu bes

illus leischma1111ii. F1G I. Pattern of elution of the threc transcobalamins on DEAE cellulosc. Dashed arrows show
with a potentio- change of buffer.
 J. Zittoun et al
Gel filtration on Sephadex G-200. Whole serum labelled with 57 CoB 12 (specific activity
roo µCi/µg) was filtered in a Sephadex G-200 column (roo cm x 2.5 cm). In additional experi-
ments serum was first fra~tionated on DEAE-cellulose and after concentration each TC was
studied on Sephadex G-200 to determine the mol wt (Andrews, 1966) . A graph was plotted of DEAE cellulose colum
log mol wt of three reference proteins (y globulin, bovine albumin andlysozyme), against their
elution volumes: log mol wt = J(Ve)! V0 where Ve = elution volume of the unknown or Molecular weights (Se
mol wt markcr and V 0 = the void volume determined by blue Dextran 2000 M. Electrophoretic mobili
Immunological reactic
Cellulose Acetate Electrophoresis Delivery to PHA tran
This was pcrformed in 0.04 M veronal buffer, pH 9.2. After migration either of the labelled lymphocytes
serum or of each separated TC, the strips wcrc cut in narrow pieces and radioactivity counted.

Immunological Studies whole serum exh

Immunoelectrophoresis was performed by the micro-method of Scheidegger (1955) and senting 69% of th
imnmnodiffusion by the Ouchterlony technique (1962). The radioactive lines were revealed After separation, '
by autoradiography after cxposure on an X-ray film for 2 months. The antisera used were a had a mol wt of :
commercial horse antihuman serum (Red Cross, Amsterdam) and an anti-TC I rabbit serum showed cx 1 cx 2 mo
prepared in thc laboratory from neutrophils isolated on plasmagel and albumin bovine the fast y region.
gradients and containing only TC I. strated on cellulo
Immunological
Functional Properties prccipitin line wi
The delivery of vitamin B 1 2 to cells by each of the binders was tested by the method of isolated on DEAl
Hoffbrand et al (1973). About 30 mi of serum wassaturated with 5-ro ng of 57 CoB 12 per mi was eluted on G
(specific activity roo µCi /µg) and dialyscd, thcn fractionated on DEAE-cellulose. The three mol wt of 37 ooc
binder pcaks were cither conccntratcd by ultrafiltration and lyophilized or immediately Labellcd vitam
frozen at - 8oº C. Lymphocytes were isolated from a normal blood donor after passage on grcater cxtent th
columns of nylon fibres and incubatcd for 72 h at 37º C with phytohaemagglutinin (PHA), uptake from tota
autologous plasma and TC 199 medium (Meyer et al, 1974). The transformed lymphocytes U ptake from TC
were then washed with TC 199, incubated for l hin TC 199 at 37º C with each of the three
TC or with whole normal labelled human serum, at a concentration of bound labelled B12 Endogwous Vitam
(500 pg) in each of the incubations. After washing three times with phosphate-buffered-NaCI, Disorders
the radioactivity of the button of cells was counted in an automatic gamma counter. These results a
active phase of e
L ysozyme M easurement thc othcr MPD,
Serum lysozyme concentration was measured with the turbidimetric method of Parry B 12 was often w
et al (1965), slightly modified (Zittoun & Zittoun, 1975)- Values in 30 normal sera were in MPD, particu
l I.5 ± 4 µg /ml.
also increased in
to MPD.
Binding due t
RESULTS n:n-ú: \on m: bh
Properties of the Time Vi tamin B 1 2 Bi11ders stages of the di
Discontinuous stepwise elution of serum on DEAE ccllulose has identified three distinct increased to a le
peaks for the vitamin B 12 binders (Fig l). These three binders have clear-cut physicochemical, The binding ca
immunological and functional properties (Table I). After gel filtration on Sephadex G-200 leukaemia, the d
 Transcobalamins in Myeloproliferative Disorders 291
12 (specific activity TABLE l. The main properties of the three binders
additional experi-
¡ation each TC was TC I TC II Third binder 'TC III'
raph was plotted of
DEAE cellulose column chromatography Na Cl, l M 0.04 M phosphate 0.06 M phosphate
zyme), against their buffer, pH 5.9 buffer, pH 6.3
ff the unknown or Molecular wcights (Sephadex G-200) II S 000- 12 0000 37 000 140 000
2000 M.
Electrophorctic mobilities a2
CX1, p p, fast y
Immunological reactions (with anti TC I) Reaction No reaction Reaction
Delivery to PHA transformed
ther of the labelled lymphocytes P oor Good Intermedia te
ioactivity counted.

whole serum. exhibited two radioactive peaks, one corresponding to mol wt 37 ooo repre-
degger (1955) and senting 69% of the total radioactivity, the other peak with a mean mol wt of about 130 ooo.
ines were revealed After separation, TC I hada mol wt between II5 ooo and 120 ooo, whilst TC II and TC III
Hisera used were a hada mol wt of 37 ooo and 140 ooo respectively. On cellulose acetate electrophoresis, TC 1
C I rabbit serum showed ix 1 ix 2 mobility, TC II migrated in the f3 zone whilst TC III migrated in the f3 zone or
i albumin bovine the fast y region. Mobilities on immunoelectrophoresis were closely similar to those demon-
strated 011 cellulose acetate electrophoresis.
Immu11ological studies showed a el ose similarity betwecn TC I and TC III; both gave a
precipitin line with anti TC I serum, while TC II did not. Treatment of TC I and TC III
by the method of isolated 011 DEAE ccllulose with the anti TC I rcsulted in the formation of a complex which
57
CoB 12 per ml was eluted on G-200 with the void volume. TC II treated in the same conditions kept a
Ilulose. The three mol wt of 37 ooo.
or immediately Labellcd vitami11 B 1 2 was taken up from TC II by PHA transformed lymphocytcs to a
after passage on greater extent than from the othcr binders. Dclivery to cclls was poor with TC I whilst
~gglutinin (PHA), uptake from total scrum was lower than from TC II but greater than from TC III and TC l.
tned lymphocytes Uptake from TC III was slightly more than from TC I.
f each of the three
und labelled B 12 E11doge11011s Vitamill B 1 2 , UBBC and Transcobalamin Binding Capacities in Various Haematological
e-buffered-NaCl, Disorders
a counter. TlleSe results are shown in Table II. Endogenous vitamin B 1 2 was greatly increased in the
active phase of CGL and to a lesser degree in the blastic crisis of CGL, in MM and CMML. In
the other MPD, the increase was moderate, especially in PRV where endogenous vitamin
ethod of Parry B12 was often within the normal rangc. The most significant increase of UBBC was found
ormal sera were in MPD, particularly in CGL, and to a lcsser extent in MM and PRV although UBBC was
also increased in acute non-lymphoblastic leukaemia and in infectious leucocytosis unrelated
toMPD.
Binding due to TC I was especially increased in ali cases of active CGL but was lower in
remission or blastic crisis. The difference of the mean binding capacity of TC I at various
stages of the disease was significantly different (P< 0.001). The binding capacity of TC 1
ed three distinct increased to a lesser degree in MM and in PR V but was normal in secondary polycythaemia.
physicochemical, The binding capacity of TC I was increased in ANLL but was normal in acute lymphoblastic
Sephadex G - 2 00 leukaemia, thedifference being significant (P< 0.05). There was also a moderate TC 1 increase
 N
IO
N
TABLE II. Levels of UBBC, endogenous B 1 , , TC I, TC II and TC III binding capacities in various haematological states

TC I TC JI TC III
UBBC Endoge11011s
B,, pg/111/ % pg/11rl % pg/111/ %
Normal controls (30)* l275t 260 320 25.4 805 62.7 150 lI.8
(960-1660) t (170-510) (610-1200) (60-360)
Cord serum (8) 1333 1500 448 (6) 33-5 680 51 210 (6) 15.7
(1010-1830) (340-550) (440-920) (150-390)
Active chronic granulocytic leukaemia 3293 (22)§ 3680 2054 (24) 61.8 843 (1) 26.1 403 (20) 13-4
(CGL) (24) (1800-5450) (100-3270) (550-1490) (120-1000)
CGL remission (6) 20II (2) 637 503 (3) 26.6 1319 (2) 63.6 189 (4) 8
(1600-2750) (300-730) (770-2270) (140-280)
CGL blast crisis (9) 1922 (5) 2776 589 (5) 33 II48 (5) 60.4 109 (2) 6.5 ~
(780-2950) (120-u70) (660-1740) (0-215)
Mycloid metaplasia (MM) (14) 2191 (8) 1560 853 (8) 35.3 1099 (2) 52 271 (12) 12.5
~
e;
¡:;:
(1300-3600) (325-1710) (695-1350) (0-630) ;:::
Polycythaemia vera (PV) (34) 2198 (21)
(1430-4020)
931 621 (15)
(270-1800)
28.8 1045 (7)
(580-1880)
52.2 4II (3 l)
(170-1200)
18.8
"'....
;::_
Secondary polycythaemia (9) 1663 (2) 405 376 24 1104 (2) 65.5 172 (2) 10.5
(1230-2070) (160-540) (740-1530) (50-250)
Infectious leucocytosis (IL) (5) 2122 (3) 396 523 (2) 23.9 1248 (1) 60.3 3 5 l (5) 15.8
(1650-2750) (300-810) (1050-1440) (200-620)
Acute 11011-lymphoblastic leukaemia 2832 (18) 689 596 (10) 21.4 2017 (17) 66.8 225 (II) 7.9
(ANLL) (18) (2060-4500) (240-1010) (u30-3500) (105-460)
Acute lymphoblastic leukaemia (ALL) (7) 1620 (1) 828 346 (1) 20.7 II08 (1) 69.1 165 9.9
(1210-2520) (200-610) (820-1780) (0-310)
Chronic myelomonocytic leukaemia 1776 (4) II57 488 (3) 28.8 II38 (2) 64.4 159 (3) 8.7
(CMML) (6) (1380-2360) (185-1010) (700-1650) (80-310)
Refractory anaemia with excess of myeloblasts 1944 (5) 551 509 (2) 25.3 1365 (5) 65.8 182 (3) 8.8
(RAEM) (8) (1500-2900) (320-1050) (II20-2I40) (90-340)

• No. of cases studied. t Mean. t Rangc. §No. of patients significantly differcnt fron1 normal rncan ± 3 SD.

p_.,.-... ..... !::::!°) ..... p_. .....

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 Transcobalamins in Myeloproliferative Disorders 293
in chronic myelomonocytic leukaemia, in preleukaemic states and in the leucocytosis pro-
duced by infection.
The unsaturated binding capacities of TC II also varied considerably, the most striking
increase being in ANLL where 17 of l 8 patients studied hada mean level significan ti y different
from normal controls (P< o.oor) and from ALL (P< 0.0025). In CGL TC II was normal in
the active phasc of the disease, but increased significantly during rcmission and blastic crisis
(P< 0.05). There was an in verse relationship betwcen the absolutc values of TC 1 and TC II
during the course of CGL.

TABLE III. TC III/TC I ratio in various haematological states

Mean ratio Co111pariso11 with 111ea11

TC III/ TC I nor111al ratio

Normal controls 0.468

CGL in active phasc 0.25 P<O.OOI
CGL in blastic crisis 0.22 P<O.OOI
CGL in rcmission 0.40 NS
MM 0.40 NS
PRV o.686 P<O.OOI
Secondary polycythaemia 0.461 NS
Infectious leucocytosis 0.69 P<O.OI
ANLL 0.39 NS
RAEM 0-42 NS
CMML 0.33 NS
00
~
00

Binding due to TC III was particularly increased in PRV where 3 r of 34 patients had levels
above normal and the mean level was significantly higher than that in secondary poly-
cythaemia (P< o.or). TC III was also increased in infectious leucocytosis and in MPD. The
absolute level of TC III in the active phase of CGL was as high as that in PRV but the level
decreased during remission or blastic crisis. The ratio of TC III to TC 1 was increased in
~
"' PRV (P< o.oor) and in leucocytosis due to infection but was normal in MM, acute
"'
:eo"' leukaemia, preleukaemic states and secondary polycythaemia and was significantly decreased
:G in the active phase of CGL, and in CGL blastic crisis (P< o.oor) (Table III).
~
...¡
$ '§"'
"s>-.
...... Correlations of each TC binding capacity and UBBC with WBC blood neutrophil count
o
""' and serum lysozyme are shown in Tables IV and V. A good overall correlation was found
'§" ..I<
::l
"'
""' between UBBC and total leucocyte count (Table IV) except in patients with active CGL and
~ ~
""
..l<i .~ " in ANLL. UBBC also correlated well with the neutrophil count in the whole group of
::l ..e::
~
~
u
u
o ·~ patients, particularly when patients with CGL, acute leukaemia and preleukaemic states were
e
':o"ª o
s '§"' excluded. However, the best correlation was found between UBBC and the log of the neutro-
o o "e phil count in normal subjects, PRV, secondary polycythaemia and infectious leucocytosis.
~,_ "
ª"s
~
"~
s :::
....;>. ~
u;...<
·~::E
o~
00

u :;E
Six of 15 cases of CGL in the active phase hada relatively low UBBC in front of the high
WBC count. Of the 18 patients with ANLL seven patients who presented with marked
:;u 8 ::E "'r.r.i
..c:u "et;<
<t: leukopenia had a high UBBC, six with increased lcucocyte counts hada relatively low UBBC
i:; U~ c:i::~
whilst the remainder approximated to the regression line. Four of seven patients with RAEM
had an increased UBBC compared to the regression line.
294 J. Zittoun et al
TABLE IV. Correlations between UBBC, TC I, TC II, TC III and the blood leucocytc Only delivery l
(WBC) or blood ncutrophil (PMN) count using two diffe
whereas previo
No. of cases r p
together.
UBBC/WBC I07 0.52 P<0.001
TCI /WBC I02 0.51 P<0.001 Relation bcttveen
TC II/WBC I02 0.009 NS The results of
TC III/WBC 102 O.I03 NS
count and this i:
TC I/PMN in thc wholc patients I02 0.37 P<O.OOI
TCII/PMN in thc wholc paticnts I02 -0.29 P<0.01 kapa et al, 1971
TCIII PMN in the whole paticnts I02 0.35 P<o.001 UBBC and neu
pool (TBGP).1
of the synthesis
our data, it appt
TABLE V. Correlations bctwccn thc thrce TC and betwecn TC and lysozyme of a decreased !
excess ofUBBC
No. of cases r p or immature) ii:
ditions, the disc1
TC I /TC II in the whole series 168 -0.09 NS ineffective gram
TC I /TC II in myclogcnous leukaemia (chronic and acute) 51 -0.36 P<0.01
TC I /TC III in the whole series 160 0.72 P<0.001
assumed that T<
TC I/serum lysozyme (normal, secondary polycythaemias and from disrupted
infectious leucocytosis) 28 0.65 P<o.oor with the TBGP
TC II /serum lysozyme (samc series) 28 0.45 P<o.05
sky et al, 1971;
TC III /scrum lysozyme (same series) 28 0.50 P<O.OI
lopoiesis where
phils but also f
acute leukaemi
TC I correlated wcll with WBC and with the neutrophil count; but TC III correlated thc 1naturc gra
only with the neutrophil count, whilst TC II did not correlatc with WBC and correlated On thc othe
inverscly with thc neutrophil count (P< 0.01) (Table IV). Thcre was good overall correlation although impo
between TC III and TC I (Table V), but no corrclation between TC I and TC II except an cannot be exp
inverse corrclation in AML and CGL (r = - 0.36; P< 0.01) (Table V). A correlation was also neutrophils (Ha
found betwcen the tllfce TC and serum lysozymc in thc whole group of patients when CGL n1.ay occur sim
and ANLL are excepted (Table V). phagocytic act
TC I reflect to
followed (Rach
DISCUSSION
Identification of the Three Binders The Third Bind
The three binders which we have isolatcd by a smglc column tcchnique are similar to the W e ha ve co
transcobalamins identified by studies of molecular wcight (Hom et al, 1966), elcctro- states with the
phoretic mobility (Hall & Finkler, 1963) and imnrnnological properties (England et al, 1973). capacity of TC
Their functional properties, measured by the ability of each of them to dcliver labelled an increase ofT
vitamin B 12 to PHA transformcd lymphocytes, are also similar to those noted by other The variation
workers (Meyer et al, 1974). It seems unlikely that endogenous vitamin B 12 can significantly TC I, and the
affect the cellular uptakc of 57 CoB 12 added to the binders in 11itro as endogenousvitamin found to a les
B 12 is shared about equally between the three binders in normal serum (Zittoun et al, 1975a). by a disturbe
 Transcobalamins in Myeloproliferative Disorders 295
lcucocytc Only delivery from thc most saturatcd binder, TC III, is likely to be underestirnated. By
using two different phosphate buffers we have been able to separate 'TC III' from TC II
p
whereas previous workcrs who used only onc buffer were probably cluting both bindcrs
together.
l.OOI
l.OOI Relation bctween UBBC, TC I and the Gra1111locyte Ccll Li11e
The results of this study show a good correlation between UBBC and WBC or neutrophil
l.OOI count and this is in agreement with the findings of other workers (Gilbert et al, 1969; Chik-
1.0I kapa et al, 1971). Chikkapa et al (1971) showed that thc best correlation occurs betwecn
1.00!
UBBC and neutrophil count and particularly betwecn TC 1 and the total blood granulocytc
pool (TBGP). The correlation bctwcen TC 1 and neutrophils is in keeping with observations
ofthe synthesis ofTC 1 by granulocytes (Simons & Weber, 1966; Corcino et al, 1970). From
our data, it appears that UBBC is increased in MPD, ANLL and preleukaemic states in spite
rie of a decreased granulocyte count in patients with the latter two conditions. The relative
excess of UBBC in these cases is probably dueto an enlarged granulocyte pool (mature and /
p or immature) in bone marrow with release of TC in to the circulating blood. In these con-
ditions, the discrepancy betwecn a hypcrccllular bone marrow and neutropenia is related to
09 NS
36 P<o.or
ineffective granulopoiesis with intramedullary cell death (Catovsky et al, 1971). It is generally
72 P<O.OOI assumed that TC originates from intact granulocyte cells (Corcino et al, 1970) and lysozyme
from disrupted cells (Fink & Finch, 1968). According to this hypothesis, UBBC correlates
55 P<o.oor
with the TBGP (Chikkapa et al, 1971) and lysozyme with granulocytc turnover rate (Catov-
is P<o.05
)O P<O.OI sky et al, 1971; Hansen, 1973). This does not seem valid in discases with incffective granu-
lopoiesis where there is probably a leakage of the TC from_not only dying maturc neutro-
phils but also from their precursors (Gilbert et al, 1969). Morcover, at least in sorne cases of
acute leukaemia, the relative increase of UBBC may be explained by an expansion of
C III correlated the maturc granulocyte pool (Galbraith et al, 1970).
: and correlated On the other hand, in CGL, the increase of UBBC and TC 1 (Rachmilcwitz et al, 1971),
erall correlation although important, was insufficient comparcd to the high blood lcucocyte count. This
fC II except an cannot be explained, as for serum lysozyme, by a lcngthening of the half life of maturc
·elation was also neutrophils (Hansen et al, 1973; Zittoun & Zittoun, 1975). A qualitative granulocyte defcct
ents whcn CGL may occur similar to the decrease of leucocyte alkaline phosphatase and the reduction of the
phagocytic activity (Zittoun & Berche, 1973) already established. Nevertheless, UBBC and
TC I reflect total body granulocyte pool and allow the efficiency of the chemotherapy to be
followed (Rachmilewitz & Rachmilewitz, 1971).

The Third Binder and its Interest in PRV

·e similar to the We have consistently identified thc third binder in normal subjects as well as in pathological
1966), electro- states with the exception of three patients with CGL, MM and ALL. The assay of the binding
and et al, 1973). capacity ofTC III and TC 1 is a useful tool in the diagnosis of PRV which is characterized by
deliver labelled anincrease ofTC III (Hall& Finklcr, 1969), and, according to ourdataof the ratio TC IIl/TCI.
11oted by other The variation of TC III in many pathological conditions does not exactly parallcl that of
:an significantly TC I, and the ratio TC III/TC 1 is decreased in CGL. The increase of this ratio in PRV, also
genous vitarn.in found to a lesser degree in infectious leucocytosis and normal cord blood, could be explained
un et al, 1975a). by a disturbed regulation in the synthesis of both binders by granulocytes, but may also be
 J. Zittoun et al
uptake. Arcliives
related toan enlarged total blood granulocyte pool, or for PRV, toan enlarged storage pool
79.
of mature neutrophils in thc bone marrow. GALBRAITH, P.R., (
( 1970) Pa tterns
Relationship betwce11 Blast Cell Proliferation a11d TC II myelogenous a
The variation in CGL and ANLL of TC 11 scems interesting. In CGL, TC 11 is normal but Blood, 36, 371.
frequcntly increases during remission or in thc course of blastic crisis wherc it is invcrscly GILBERT, H.S., KRA
V. & W ASSERMA
relatcd to TC l. Thcse data confirm the postulation ofHall & Finkler (1966) that a reciproca! content and u
relationship exists bctwcen TC 1 and TC 11. The variations in rcmission and blastic crisis capacity in my1
closely parallel the dccrcasc of mature granulocytes, and for thc last condition, the increased differential diag1
activity. A1111als e
pcrcentage of blast cells. Gizis, E.]., DIETRIC
The highest levels of TC 11 were obscrved in ANLL. Previous obscrvations are contro- (1970) A 57 Co vi
versia!; Chikkapa et al (1971) found a low UBBC in ANLL and considered that myeloblasts eluted by DEA
0.1 M sodium pi
do not synthcsize B 12 binders; TC 11 was found unchanged by Rachmilewitz et al (1971b),
Laboratory and C
but increased by others (Gottlicb et al, 1966; Begley & Hall, 1975). According to one hypo- G1z1s, E.]. & MEYE
thcsis (Rachmilewitz et al, 1972), TC 11 originates only from the liver and the increase of cobalamins on
TC 11 in haematological diseases could be explained by hepatic dama ge and relea se of TC II. Proceedings of the
Medicine, 140, 32
However, it is surprising to sec that the leve] of this binder is norm.al or scarcely increased GOTTLIEB, c.w.,
in diseases characterized by a marked extramedullary haemopoiesis of thc liver such as CGL HERBERT, V. (l!
in the active phase or MM. Moreover, hepatoccllular damage in ANLL is rarely important proteins with dis
to hypo- and hy
and it is probably not higher than in ALL despite the significantly highcr leve! of TC JI turnover. (Abstr
in ANLL. In our opinion, liver damage cannot, by itself, explain the increase of TC II in 45, 1016.
ANLL and an alternative explanation is to be found in leucocyte origin of TC 11. It is HALL, C.A. (1969)
(Annotation). Br
our hypothesis that TC 11 may be synthesized by normal or leukaemic blast cells with
HALL, C.A. & FIN
consequent ovcr production in ANLL. The data observed during the coursc of CGL suggest B 1 2 -binding sub
a balance with TC 1 production in the more mature stagcs of granulocytic line whilst the et Biopliysica Act
increasc of TC 11 in rcfractory anacmia with cxccss of myeloblasts al so argues for a blast cell HALL, C.A. & f¡
port of vitamin
origin of TC 11. This speculation becomes stronger whcn we consider thc ccll contcnt in thc genous leukaem
three TC at each stage of maturation (Zittoun et al, 1975b). HALL, C.A. & FIN
transcobalamin
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SuMMARY.
disorders a
binders; th
'TC II' wh
probably o
The intr
bone man
gradient. 1
myelocyte
'TC II' dec
a 1nax11nm
m.al TC di
to a disord

Abnormalities
discases has hel
is grcatly incri
rclationship be
amounts of vi
from. normal
lcukacmoid re:
phils synthesiz<
binding capac
vitam.in B 12 -b
similar to TC •
and rclcased b
cxplain thc go
found by Chi
chcmothcrapy
Corresponden e>
94010, France.