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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.

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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.

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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

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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 techniqueswhether 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-

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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.