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OCR for page 215
ELECTROPHORETIC FINDINGS IN THALASSEMIA GERALD 215
l l | O Normal
(by ( I I 1- [A ~ I I ~ Hgb H
~ b b ~ ~ ~ Microcytosis
FIG. 4. Family pedigree of an hereditary microcytosis associated with Hb H disease.
the father's side we have found three members with microcytosis and a mini-
mal degree of anemia. Excepting for the propositus, the electrophoretic pat-
terns are qualitatively and quantitatively normal. The hereditary microcytosis
of: this family accordingly differs from thalassemia trait in lacking an elevation
of the A2 content.
We have thus encountered three electrophoretically distinguishable varieties
of hereditary microcytosis. That usually present in thalassemia major families
of Mediterranean extraction and characterized by an increased A2 content
we have called thalassemia trait. That form without increased A, content
and so far observed in a single fIb H family perhaps warrants the designation
"pseudo-thalassemia." Finally, that form associated with an S-like hemo-
globin will be named after the abnormal hemoglobin if it proves distinct
from hemoglobins S and D.
REFERENCES
1. Kunl~el, H. G., and Wallenius, G.: New hemoglobin in normal adult blood, Sci-
ence 122: 288, 1955.
2. Motulsky, A. G.: Genetic and haematological significance of haemoglobin H. Na-
ture 173: 1055, 1956.
3. We are indebted to Dr. F. H. Gardner for permission to report the hematologic
findings in this family.
DISCUSSION
J9r. A. Josephson: I just want to make a point about the A2. We have
studied approximately 35 people with thalassemia, proven, and although many
fall into a range comparable to the severity of the disease, there are a number
of exceptions. Some of our thalassemia minimas have had elevation of the A2
level. One complicating feature is that we have studied the A2 in other hema-
tologic disorders, primarily red cell disease, and found that in three out of six
patients with pernicious anemia the A2 has been elevated.
Dr. /1. G.~ll/lo~zllsky: I would first like to confirm Dr. Gerald's results.
We have studied six patients with Hb H-thalassemia disease from three fami-
lies. A diminished amount of the As component was found.
I would like to turn now to another component we found in normal hemo-
lysates. These studies were fully presented at the meeting of the American
Federation for Clinical Research in Atlantic City.
OCR for page 216
216 PART III. ABNORMAL HEMOGLOBINS
Using paper electrophoresis at acid pH (pH 5.8-6.0) and staining with
bromphenol blue, we found a distinct minor component with diminished mo-
bility in the hemolysates of normal subjects and in patients with a variety of
disorders (fig. 1~. This component was markedly diminished in cord blood.
Artifacts as source of the component could be ruled out. We first thought we
were dealing with a heterogeneous hemoglobin.
FIG. I. Paper electropho-
resis at acid pH of hemoly-
sates of normal subjects and
patients. Location of minor
component discussed in text
is indicated by arrows on the
normal column. ( Note that
line of origin and relative
size are not same for left
column as for other two.)
Initially this hypothesis appeared substantiated when the component was
found to have identical electrophoretic mobility to Hb H at acid pH (fig 19.
However, when electrophoretic runs were done at multiple pH's, it was found
that the component under study had a different electrophoretic behavior from
Hb H near the neutral and alkaline range. The component could be concen-
trated by salting out at 72.5~ ammonium sulfate. No precipitate was ob-
tained from cord broad at this ammonium sulfate concentration (fig. 2~.
PIG. 2.—Paper electrophoresis of
( NfI4) .,SO4 precipitates of hemo-
lysates.
OCR for page 217
DISCUSSION
217
Searching for a non-hemoglobin red cell constituent to explain the fraction,
we were fortunate in that Dr. Huex~nekens (Department of Biochemistry,
University of Washington J recently had isolated a yellow protein from human
red cells which had TPNH-activated methemoglobin reductase activity. At
all pH's studied, this yellow protein had electrophoretic mobility identical to
that of the fraction under study (fig. 3~. We concluded therefore that the
component represented methemoglobin reductase.
—2
—1
my
1
o
+2
` \ HbA
Hb H\ \
b.U b.3 t.U f . ~ b.u b.0 Y.u
pH
-
~ _
O-
~ +
-
—
~ O-
~ +
.~ Component
I I I I I I ~ 1 1 ~
6.0 7.0 8.0 9.0
pH
Methemog robin
__- Reductase
I ~ 1 ~ 1 1 1 · 1 1 1 1
6.0 7.0 8.0 9.0
pH
FIG. 3. Relative electrophoretic mobilities of the subject component, hemoglobins
A arid H and methemoglobin reductase.
Dr. J. L. Cook: In his remarks made earlier concerning our column chrom-
atographic procedure, Or. Morrison mentioned our results with regard to
thalassemia. I would like now to show two typical ion-exchange chromato-
grams of patients from the Hematology Clinic at the University of Rochester.
Figure 1 shows thalassemia minor. The forerunnir~g component has its peak
in the position characteristic of the feta1 peak from cord hemoglobin. The
second peak is in the region of the principal component of normal adult
hemoglobin. The last peak is in the area of the final peak of normal adult
hemoglobirl, but differs in being significarltly larger than in the normal.
Attention is invited to the peak at tube 40, which we find characteristic of
thalassemia minor, though it is absent in thalassemia major and in normal
hemoglobin.
Figure 2 shows typical thalassemia maj or. Note the sharp peak which
moves in the fetal region. This component represents about 30 per cent of
OCR for page 218
218
800
700
-
o 600
-
~S
500
he
z 400
o
Z 300
o
~ 200
o
LL
I 100
PART III. ABNORMAL HEMOGLOBINS
FIG. 1
_
_ THALASSE M IA M I NOR
ALKALINE RESISTANT HO 3 %
0:
by
my
G
V
I
1
-
1
1
~ _ = REGION OF NORMAL
/ l ADULT PEAK
-
o J I I, I , I , \
1 0 20 30 40 50 60
TUBE NUMBER
900
800
700
600
500
400
300
200
THALASSEMIA MAJOR
ALKALINE RESISTANT HS 24 %
AREA I 30 %
—- REGION OF NORMAL ADULT PEAK
1 1
1 11
FIG. 2
ol J ~
10 20 30 40 50 60
TUBE NUM 3ER
(Figure 1 appears in Federation Proceedings 16: 765, 1957, and is reproduced by
permission of the publishers.)
the hemoglobin and, when isolated, is alkaline-resistant. In thalassemia
major, as well as thalassemia minor, our technique demonstrates an increase
in the final component in the tube 54 region.
Dr. 1. M. London: One of the questions that has arisen, and has in fact
been asked by some of the people here in the audience, is concerned with the
question of heterogeneity, particularly in normal human hemoglobin. Do
the various groups that are using different techniques—whether these be
zone electrophoresis, column chromatography, or paper electrophoresis
feel that the minor components that they are observing are the same? Would
any of you care to comment on the possible identity of the minor components
that you note by these various techniques?
Dr. H. G. Kinked: This is not a question that can readily be answered.
The minor components that we have studied are observed with a wide variety
of electrophoretic procedures. Free solution electrophoresis demonstrates the
A2 component particularly in patients with thalassemia, as Drs. Josephson
and Singer have shown. Paper electrophoresis also shows the minor components
when special conditions are employed. There also is a correspondence between
these and the subfractions obtained by Drs. Morrison and Cook. We are at
present exchanging samples to resolve some quantitative differences, particu-
larly in one of the components. The heterogeneity of hemoglobin noted in low
ionic strength cacodylic acid does not correspond well with these other studies.
Here the quantities involved are far greater than the minor subfractions re-
ported.
Dr. Martin Morrison: I think it is clear that the rapidly-moving com-
OCR for page 219
DISCUSSION
219
ponent on our column and Dr. Kunkel's rapidly-moving electrophoretic com-
ponent at pH 8.6 are very close to being the same, both in percentages and in
electrophoretic movement. I do not think there is any question about this.
With regard to the slow-moving component, we did get Dr. Kunkel's sample
and it does move very much like ours. It is exactly like ours in position. The
percentages may be off by virtue of the partial oxidation of Hb A, let us say
one iron atom per molecule of normal A, which might give us a small in-
crease. That is, if the major component A is partially oxidized, it would move
in the same region as the third component. As much as 10 per cent of our
third component could be composed of such molecules and we may not have
detected that.
Dr. Alfred Charting: One obtains three distinct components when a red
cell extract of a freshly-drawn sample of blood is analyzed by free electro-
phoresis in a cacodylic acid buffer at pH 6.5 and 0.06 M concentration. The
fastest-moving component is colorless and has been designated as F. The two
remaining components designated as A and B comprise about 60 and 30 per
cent, respectively, of the total area of the pattern. These two components
appear to be hemoglobins. It has been observed that the slow-moving com-
ponent (B) gradually disappears when ACD blood is stored at 4°. It can be
demonstrated that this component may be restored to its original concentra-
tion by the addition of inosine. We believe that the evidence obtained in our
laboratory supports the concept of heterogeneity of hemoglobin in the red
cell of a normal individual.
REFERENCE
1. Berry, E. R., and Chanutin, A.: Electrophoretic studies of red cell extracts of
stored blood, J. Clin. Invest. 36 225, 1957.
Representative terms from entire chapter:
red cell
