FURTHER STUDIES ON THE VARIOUS HEMOGLOBINS AND THE SERUM
PROTEIN FRACTIONS IN EARLY EMBRYONIC LIFE
ISAC HALBRECHT, M.D., CHAJA KLIBANSKI, M.Sc, HANOCH BRZOZA, PH.D.,
AND MIRIAM LAHAV, M.Sc.
Department of Obstetrics and Gynaecology, Hasharon Hospital, Petah Tikva, Rogoff Clinical Research
Laboratory, Beilinson Hospital, Petah Tikva, and The Central Laboratory of the Kupal Holim
Zamenhof, Tel Aviv, Israel
At the present time, there is relatively little
information with regard to the various stages of
biosynthesis of the proteins of blood serum and
of the normal hemoglobins found during early
embryonic life.
In our previous paper,3 which dealt chiefly
with the description of the new embryonic hemoglobin found in the blood of a 10-week-old
embryo, we reported on the unusual electrophoretio pattern of the serum proteins of this
embryo.
The present paper deals with studies performed on the blood of .15 embryos, ranging in
weight from 18 to 550 Gm. (10 to 20 weeks old),
in order to identify the various fractions of serum
protein and varieties of hemoglobin throughout
the first stages of embryonic life.
METHOD AND RESULTS
Using the method described by Singer and coworkers,6 we found that the blood of the 15
embryos contained 53 to 93 per cent of the alkaliresistant type of hemoglobin. It is interesting to
note that the highest percentage of fetal hemoglobin was observed in the blood of a 4-monthold fetus, whereas the 2 smallest embryos (18
Gm. each) contained only 73 and 83 per cent of
the alkali-resistant type of hemoglobin.
Received, October 14, 1957; revision received,
November 19; accepted for publication December
19.
Dr. Halbrecht is Director of the Department of
Obstetrics and Gynecology, Hasharon Hospital,
Petah Tikva, and Associate Clinical Professor at
the Jerusalem University-Hadassa Medical School.
Mrs. Klibanski is Research Fellow at the Rogoff
Clinical Research Laboratory of the Kupat Holim
Beilinson Hospital. Dr. Brzoza is Head of the
Chemical Department of the Kupat Holim Central Laboratories, Zamenhoff, Tel Aviv. Mrs.
Lahav is Head of the Immunological Department
of the Kupat Holim Central Laboratories, Tel
Aviv.
The electrophoretic examination of the hemoglobin extracted from the erythrocytes of the
embryos was performed in a Thomas electrophoresis apparatus on Whatman No. 1 filter
paper, using a solution of veronal as a buffer,
with an ionic strength of 0.04 at pH 8.6. In a
20-hr. analysis at 150 v. and 1.5 ma. per inch of
filter paper, the slow-moving type of embryonic
hemoglobin that was described in our previous
paper was found in the blood of 11 of the 15
embryos (Fig. 1).
This slow-moving hemoglobin, therefore, can be
definitely regarded as a third normal human
hemoglobin that is present in the erythrocytes of
the human embryo during the first 4 to 5 months
of its intrauterine development. After this first
stage of embryonic maturation, the early type of
the respiratory pigment disappears, and it is
gradually replaced by the other 2 hemoglobins,
which have previously coexisted with it.
The serum proteins were determined by means
of the biuret electrometric method, with the
Kingsley's modification for separating albumin
from globulin. The fetal blood serum is generally
characterized by its low concentration of protein.
The lowest concentrations of total protein, i.e.,
2.9 and 3.4 Gm. per 100 ml., were found in the
blood of the 2 smallest embryos, each 10 weeks
old. The concentration of globulin was 0.4 Gm.
per 100 ml. in both of the serums, and the ratios
of albumin to globulin were, respectively, 6.25
and 7.50. In the 13 older embryos (80 to 550 Gm.),
the concentration of total protein was relatively
constant, the values fluctuating from 4.0 to 4.5
Gm. per 100 ml., whereas much greater differences
were found in the concentrations of globulin,
which varied from a minimum of 0.5 to a maximum of 1.4 Gm. per 100 ml.
The results of the electrophoretic examination
of the embryonic blood serums were of special
interest. In 6 of these serums that were collected
from the smallest embryos, the 7-globulin fraction was absent or almost indistinguishable. On
340
April 1958
H E M O G L O B I N S AND SEKUM
341
PROTEINS
F I G . 1. Comparison of the electrophoretic mobilities of fetal
embryonic (middle), and adult (loioer) hemoglobins.
(upper),
•fe.jW^'.K^^/S
F I G . 2. Elcctrophoretic p a t t e r n of the serum
proteins in a 10-week-old embryo, characterized
by a strong concentration of the specific fetal
protein fraction (A') and the complete lack of yglobulin.
F i o . 3. Elcctrophoretic p a t t e r n of the fractions of serum proteins in a
16-week-old fetus, with the specific fetal protein fraction (A") located between
the albumin and a-1 globulin fractions.
342
HALBKECHT ET
the other hand, a new fraction of protein, located
between the albumin and the a-1 globulin fractions, was found in the blood scrums of all of the
embryos that were from IS to 350 Gm. in weight,
or from 10 to JS weeks old. This fraction could
not be detected thus far in the electrophoretic
patterns of the serum proteins of older fetuses or
full-term, newborn infants. The concentration of
this fraction was greatest in the blood serum of
the 10-week-old embryos, and it gradually
diminished in strength with the increasing age of
the older embryos (Figs. 2 to 5).
Hepatic junction tests. In 13 embryos, 80 to
550 Gm. in weight, we were able to obtain enough
scrum for further examinations and a battery of
so-called hepatic function tests were performed,
the results of which are listed in Table 1.
The following results were obtained:
1. The concentration of total cholesterol was
low, with values not greatly different from those
observed in the blood serum of normal newborn
infants. The mean value was 87.3 mg. per 100
ml., with a range from 60 to 135 mg. per 100 ml.
2. The concentration of total bilirubin was
almost uniformly 0.3 mg. per 100 ml., with the
exception of 2 embiyos that had, respectively,
concentrations of 2.3 and S.O mg. per 100 ml.
3. The results of the cephalin reaction were
negative in the blood of all but 1 of the embryos.
4. The Weltmann coagulation band revealed,
in general, a slight shift to the left (6 tubes as an
average), as usually observed in the blood serums
of all of the newborn infants.
5. The results of the Takata Ara reaction were
negative in all instances.
G. The thymol turbidity test revealed low
values of 0.5 to 1.5 units, with 1 exception in
which there were 2.5 units in the blood of a
14-week-old embryo.
7. The results of the Lugol test were negative
in the blood of all but 1 of the embryos.
DISCUSSION AND CONCLUSION
The new embryonic hemoglobin that we discovered in a 10-week-old embryo, by means of
the electrophoretic method, was found in the
erythrocytes of 9 of the 15 embryos that ranged
in weight from 18 to 550 Gm. The existence of
this new embryonic hemoglobin, which is characteristic for the earliest stages of embryonic life,
was previously demonstrated by Drescher and
Kiinzer (in Germany), by means of a different
AL.
Vol. 29
method.2 Further investigations are being performed in our laboratory in order to separate
this embryonic hemoglobin, in its pure state,
from the other hemoglobins that normally coexist
with it, so that we may be able to establish the
physical and chemical characteristics that distinguish it from the other 2 normal hemoglobins
of human beings.
The new protein fraction that we detected in
the blood scrums of fetuses up to 5 months of
age, by means of the electrophoretic method, is
probably to be identified with the special fraction
of fetal protein discovered by Pedersen,4 in 1944,
in the blood serum of calves. Pedersen designated
the term fetuin for this fetal protein. Recently,
Deutsch 1 thought that he isolated this protein
fraction in its purest state, and he regarded it as
a mucoprotein with an isoelectric point at pH
4.4. Sandor 5 similarly admitted that this protein
fraction was a glucoprotein. Our investigations,
thus far, do not confirm this assumption. The
glucoproteinograms that we have obtained from
the blood serums of only 2 embryos (up to the
time of this writing) did not reveal any glucoprotein that was superposable on the specific
fraction present in the protcinogram of the same
embryo. We think that additional, detailed
studies are required in order to determine the
exact physical and chemical nature of this specific
fraction of protein, its physiologic significance,
and also its role in the first stages of embryonic
molecular maturation.
In contrast with this fraction of protein, characteristic for the earliest stages of the embryonic
development and normally disappearing from
the blood serum of the fetus after the fifth month
of the intrauterine life, the 7-globulin fraction is
found in the blood serum of the fetus much later.
On the other hand, no parallelism could be
established between the disappearance of the
specific embryonic protein and the appearance of
the 7-globulin fraction. In some instances, for
example, they were found coexisting in the blood
of the same fetus.
The results of the hepatic function tests with
the embryonic blood serums are especially
interesting, in that they arc closely similar to
those obtained with the blood of premature and
full-term, newborn infants. This fact warrants
the assumption that there is a relatively early
development of the functions of the embryonic
liver, but the full maturity of these functions is
April 195S
HEMOGLOBINS AND SERUM PROTEINS
Alb x <c, oc, /3
l
343
y
J
F I G . 4. Electrophoretic p a t t e r n of the fraction of serum protein in a 16-week-old fetus, with the specific fetal protein fraction (X) located between the albumin and a-i globulin fractions.
F i o . 5. Electrophoretic p a t t e r n of the serum proteins in a full-term, newborn infant. N o t e the absence of the specific fetal protein.
TABLE 1
R E S U L T S OF T H E H E P A T I C F U N C T I O N T E S T S ON THE BLOOD SERUM O F EMBRYOS AND IMMATURE F E T U S E S
( W E I G H T S , IS TO 550 G M . )
Proteins
Weight
Total
Gm.
1
IS
2
IS
3
SO
4 220
5 220
G 220
7 240
S 240
9 240
10 240
11 310
12 350
13 350
14 350
15 550
Albumin
Globulin
Bilirubin
A/G
ratio
Cm./
Gm./
Gm./
100 ml. 100 ml. 100 ml.
2.9
3.7
4.4
4.2
4.4
4.3
4.3
4.5
4.4
4.4
4.1
4.4
4.4
4.0
4.4
2.5
3.3
3.4
3.1
3.2
3.3
3.4
3.4
3.3
3.9
3.1
3.0
3.0
2.9
3.5
0.4
0.4
3.0
1.1
1.2
1.0
0.9
1.1
1.1
0.5
1.0
1.4
1.4
1.1
0.9
Cholesterol
Cephalin
Total
Indirect
Direct
Welt- Takata
man
Thymol
Ara
(Tubes)
100 ml. 100 ml. 100 ml. 100 ml.
G.25
9.25
3.40
2.SI
2.66
3.30
3.77
3.09
3.00
7.SO
3.10
2.10
2.10
2.60
3.60
7S
110
75
97
7S
75
65
IIS
75
90
133
60
60
0.3
0.3
0.3
S.O
0.3
0.3
2.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
7.3
0.3
0.3
1.9
0.3
0.3
0.3
0.3
0.3
0.3
0
0
0
0.3
0
0
0.4
0
0
0
0
0
0
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
++++
Neg.
Neg.
Neg.
Neg.
Neg.
6
6
6
6
5 to 6
7 toS
6
7
6
5
5
6
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
2.5
0.5
1.0
1.5
0.5
0.5
1.5
0.5
1.5
1.0
1.5
1.0
1.5
Neg.
Neg.
Neg.
Neg.
Pos.
Neg.
Neg.
Neg.
Nog.
Neg.
Neg.
Neg.
Neg.
344
Vol. 29
H A L B E E C H T ET AL.
never reached at birth, even in the full-term,
newborn infant.
SUMMARY
1. A specific embryonic hemoglobin that is
characterized by its slow electrophoretic mobility
on filter paper was found in the blood of 11 of 15
embryos that ranged in .weight from 18 to 550
Gm. Until now, this hemoglobin had not been
observed in the blood of older fetuses.
2. By means of the electrophoretic method, a
new protein fraction was discovered in the blood
serums of fetuses up to 5 months of age. This
new fraction, located on the proteinogram between the albumin and a-\ globulin fractions, was
hitherto known to be present only in the blood
serums of calves. Its exact physical and chemical
characteristics still remain to be determined by
means of other methods.
SUMMAKIO IN INTEHLINGUA
1. Un specific hemoglobina embryonic, charactcrisate per su lente mobilitate in papiro-filtro,
esseva trovate in le sanguine de 11 ex 15 embryones con pesos de inter IS c 550 g. Usque
nunc, isto hemoglobina ha non essite obscrvate
in le sanguine de fetos plus matur.
2. Per medio del mcthodo electrophoretic, un
nove fraction proteinic esseva discoperite in le
seros sanguinee de fetos de etatcs de usque a 5
menses. Istc fraction, locate in le proteinogamma
inter le fraction albuminic c le fraction globulinic a - 1 , esseva previemente cognoscite solmente como occurrente in seros de sanguine
vitellin. Su exacte characteristicas physic e
chimic debe ancora esser determinate per medio
de altere methodos.
REFERENCES
1. DEUTSCH, H. F.: Fctuin; the mucoprotein of
fetal calf serum. J. Biol. Chem., 208: 669-678,
1954.
2. DRESCHEU, H., AND KUNZER, W.: Der Blut-
farbstoff des menschlichen
Wchnschr.,32:92, 1954.
Feten.
3. HALBRECHT, I., AND KLIBANSKI,
Klin.
C.: Identi-
fication of a new normal hemoglobin. Nature, 178: 794, 1956.
4. PEDERSEN, K. O.: Fetuin; a new globulin
isolated from serum. Nature, 164:575, 1945.
5. SANDOR, G.: Quelques considerations concernant la pathogdnie de 1'augmentation du taux
des glucoprotdides et des alpha-globulines
du se>um sanguin. Sang, 27: 597-602, 1956.
6. SINGER, K., CHERNOFF, A. I., AND SINGER, L.:
Studies on abnormal hemoglobins; their
demonstration in sickle cell anemia and other
hematologic disorders by means of alkali
denaturation. Blood, 6: 413-428, 1950.