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OCR for page 172
THE ALKALI DENATURATION PROCEDURES* AMOZ I. CHERNOFF All current techniques for the determination of fetal hemoglobin based upon the resistance of the embryonic pigment to denaturation by highly alkaline solutions stem from the observations made almost one hundred years ago by von Korber,~ who demonstrated that hemoglobin solutions prepared from cord blood erythrocytes were resistant to the destructive effects of NaOH whereas hemoglobin preparations obtained from normal adults were rapidly destroyed under the same experimental conditions. Since the original observations of von Korber, relatively little has been learned con- cerning the kinetics of this reaction or about the general phenomenon of . . hemoglobin denaturation. Nevertheless, many procedures are available for the quantitative estimation of the fetal pigment based upon its resistance to alkali denaturation. In general, these techniques utilize two different ap- proaches. One method depends upon the change in light absorption at selected wave lengths as the hemoglobin is converted from the oxy form to the alka- line chromogen. If one follows these minute to minute changes in light ab- sorption, it is possible to determine not only the rate of reaction but also to calculate the percentage of Hb F in a mixture of hemoglobins. The second group of methods involves the precipitation of the denatured protein by 1/3 saturated (NH4~2 SO4 followed by the determination of the per cent of undenatured hemoglobin. If this procedure is carried out at varying intervals of time, the rate of reaction and per cent of alkali-resistant hemoglobin may be determined as in the first technique described. In practice, however, it becomes possible to select a convenient time period during which all alkali- sensitive hemoglobins are denatured whereas those which are alkali-resistant are only minimally affected. In the method described by Singer and Chernoff, . c> thlS time lIlterVa. . IS one mlnute.~ Although there is general agreement as to the usefulness of these two techniques in the quantitative determination of alkali-resistant hemoglobins, considerable disagreement is present concerning a number of features of the alkali denaturation procedures. These may be listed as follows: 1 ) Does the chemical state of the hemoglobin alter the results of alkali denaturation ? 2) What is the nature of the alkali-resistant fraction left after the re- action is complete when using the precipitation technique? 3) Is the alkali-resistant fraction always identical with fetal hemoglobin? 4) How do the two methods compare in their accuracy of Hb F deter- m~nat~on ~ Most investigators have used oxyhemoglobin as the starting material in the *; Some of the studies reported in this paper were supported by United States Public Health Service Grant No. A-1615. 172

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ALKALI DENATURATION PROCEDURES CHERNOFF 173 alkali denaturation procedures. Nevertheless, certain of the discrepancies which show up between the various techniques may be explained by the presence of hemoglobin forms other than oxyhemoglobin. Thus CO-hemo- globin is known to have a slower rate of denaturation than does oxybemo- globin.3 4 The results of the alkali denaturation procedures would therefore be elevated in the presence of significant amounts of this compound, as has been pointed out by [onxis and Huisman.5 The 'denaturation of methemo- globir~ precedes at the same rate, or somewhat more rapidly, than that of oxy- hemoglobin.3 Likewise, cyanhemoglobin demonstrates a reaction rate identical to that of oxyhemoglobin but is felt by Kunzer6 to constitute a better start- ing material because of the lower residual values after one minute of reac- tion an observation which wee will shortly refer to again. Finally, dif- ferences in the results of the alkali denaturation techniques may be caused by the method of preparation of the hemoglobin as, for example, when saponin is utilized as the hemolyzing agent. Baar and co-workers have shown that adult hemoglobin solutions prepared wild saponin often demonstrate two or more components on denaturation, whereas those hemolyzed with water be- have ire a manner suggestive of a monomolecular reaction.3 These observa- tions may explain the reports of a number of investigators that two or more hemoglobin components are present in normal adult hemoglobin solutions when studied by the denaturation technique's. Following what appears to tee ' complete denaturation of normal adult oxy- hemoglobin solutions, a residual spectrophotometric reading of up to 1.8 per cent of the original hemoglobin concentration is noted.~ Although the ma- terial is benzidine positive, the evidence is inconclusive as to whether the com- pour~d causing this residual reading represents fetal hemoglobin, is a particu- larly al'~ali-resistant non-fetal hemoglobin fraction, is a denatured hemo- chromogen which is not precipitated by saturated (NH4~2SO4 or is, indeed, a heme pigment at all. Prolonged reaction times of up to 24 hours reduce the amount of, but do not eliminate, this substance. That part of this material is truly fetal hemoglobin is suggested by a number of studies indicating the presence of small quantities of fetal hemoglobin in the normal adult. These studies have been carried out by immunologic techniques,7 by amino acid determinations on the residual protein,S and by solubility determinations such as described by Roche and Derrien.9 The remaining portion of the residual material has been ascribed to one of two causes. Betke believes that the de- natured hemochromogen unites with albumin carried along with incompletely washed erythrocytes in the preparation of the hemoglobin solution.~ Others have postulated that some of the products of denaturation are not precipi- tated by (NH4~2SO4 and hence enter the filtrate to contribute to the resid- ual reading. Although direct proof of the latter postulate is lacking, the idea seems to provide a logical explanation for the presence of the residual ma- terial noted above.

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174 PART III. ABNORMAL HEMOGLOBINS The third problem for consideration is that of the possible identity of the alkali-resistant pigment with fetal hemoglobin. There is considerable evi- dence that small quantities of fetal hemoglobin are present in normal adults, up to approximately 0.5 per cent of the total pigment being of the fetal variety. Tl~e studies suggesting this identity have been alluded to above and involve three entirely different approaches immunologic, amino-acid com- position and solubility studies. In pathologic states, however, there is some disagreement as to the identity of the alkali-resistant compound and fetal hemoglobin. Thus, Larsen and co-workers believe tl-~at the alkali-resistant material in untreated pernicious anemia is probably related to a stromal factor found in the macrocytes of this disease.'' The alkali-resistant pigment in sickle cell anemia is claimed by van der Schaaf and Huisman to be different from fetal hemoglobin on the basis of careful amino-acid analyses of the pro- te~n when separated by ion exchange chromatography.13 On the other hand, there is considerable evidence that the alkali-resistant fraction in sickle cell anemia, as well as in all other hereditary hemoglobin diseases, is identical with the fetal compound. To cite only a few of the techniques used in arriving at this conclusion, one may mention studies of the electrophoretic, spectrophctometric, immunologic, amino-acid composition and solubility char- acteristics of the resistant material. It should seem, therefore, that, in the present stage of our knowledge, the a.lkali-resistant pigment is probab~v identical with fetal hemoglobin. That startle alkali-resistant f factions may however not be Hb F cannot be definitely excluded. Of the various methods for determining Hb r, else alkali denaturation technique is by far the most sensitive practical method. Ultraviolet spectro- photometry cannot be used with less than 10-20 per cent of the fetal com- pound. Electrophoretic techniques cannot determine less than ten per cent of this pigment when in combination with adult hemoglobin. Chromato- graphic procedures are not feasible with less than 5-10 per cent of this com- pound, when Hb A is present. Amino acid determinations, as well as immun- ologic determinations, both of which are sensitive to considerably less than O.3 per cent of Hb ~ in combination with fIb A, are tedious and can only be done in special laboratories. Thus the alkali denaturation technique remains as the single most practical method for Hb F determination. The technique employing the change in light absorption at selected wave lengths is claimed by [onxis and HuismanS to be a more accurate method than the precipitation technique of Singer and Chernoff.'' Nevertheless, the former procedure cannot be utilized adequately with less than ten per cent Hb F and the technique is somewhat more cumbersome. The one minute denaturation pro- cedure is sensitive to at least 2 per cent and as modified by Kunzer to approximately one per cent Hb in any mixture of hemoglobins, is easily performed in clinical laboratories and does not require elaborate apparatus. The difficulties of falsely high values in the lower concentrations of Hb F

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ALKALI DENATURATION PROCEDURES CHERNOFF 175 and falsely low values in the higher ranges, as suggested by [onxis and Huisman, are not of sufficient magnitude to detract from the clinical useful- ness of the procedure. Using the one minute alkali denaturation test, we have now examined many thousand blood specimens. Tn~ren~A amount nT Wh F malt he found in almost all the hereditary hemolytic syndromes, particulary in thalassem~a and the hereditary hemoglobin diseases, as well as in a number of acquired hematologic diseases in which the marrow Is infiltrated by malignant or foreign cells. The use of the alkali denaturation procedure may serve, there- fore, as an important adjunct to the investigation of a number of obscure hematologic diseases. REFEREN CES 1. Von Korber, E.: Uber Diderenzen des Blutfarbstoffes, Inaugural dissertation. Dorpat, 1866. Cited by Bischoff, H., Ztschr. f. d. yes. expert Med. 48: 472~89, 1926. 2. Singer, K., Chernoff, A. I., and Singer, L.: Studies on abnormal hemoglobins. I. Their demonstration in sickle cell anemia and other hematologic disorders by means of alkali denaturation, Blood 6: 413~28, 1951. 3. Baar, H. S., and Hickmans, B. M.: Alkali denaturation of oxyhemoglobin, hemo- globin, carbonmonoxybemoglobin, methemoglobin and cyanmethemoglobin, l. Physiol. 100: 3P, 1941. 4. Singer, K., and Fisher, B.: Studies on abnormal hemoglobins. VII. Composition of non-S hemoglobin fraction in sickle-cell anemia bloods: comparative quan- titative study by methods of electrophoresis and alkali denaturation, J. Lab. & Clin. Med. 42: 193 - 204, 195 3. Jonxis, l. H. P., and Huisman, T. H. J.: The detection and estimation of fetal hemoglobin by means of the alkali denaturation test, Blood 11: 1009-1018, 1956. 6. Kunzer, W.: Untersuchungen uber das Vorkommen fetalen Hamoglobins bei Blutl;rankheiten. Zeitschrift fur Kinderheilkunde 76: 58-72. 1955. 7. 8. Chernoff, A. I.: Immunologic studies of hemoglobins. I. Production of antihemo globin sera and their immunologic characteristics, Blood S: 399~12, 1953. Huisman, T. H. J., Jonxis, l. H. P., and Dozy, A.: Is foetal haemoglobin present in the blood of normal human adults ? Biochim. Biophys. Acta 18: 576 - 577, 1955. 9. Roche, J., Derrien, Y., Reynaud, J., Laurent, G., and Roques, M.: Sur l'heteroge- neite des hemo~lobines. I. Technique d'etablissement des courbes de solu- bilite et premiers essais de fractionnement, Bull. Soc. chim. biol. 36: 51 - 63, 19 j4. 10. Betide, K., Greinacher, I., and Leber, E.: fiber die 13indung von Hamatin an Plasmaeiweiss. Zugleich ein Beitrag our Methodik der Alkalidenaturierung von Blutiarbstoff, Biochemische Zeitschrift 326: 1-8, 1954. 11. Chernoff, A. I.: Immunologic studies of hemoglobins. II. Quantitative precipitin test using anti-fetal hemoglobin sera, Blood S: 413~21, 1953. 12. Iversen, O. H., and Larsen, G.: EIemoglobin in pernicious and allied anemias. The significance of an abnormal alkali denaturation curve, Scand. l. of Clin. and Lab. Invest. 8: 159 - 167, 1956. 13. van der Schaaf, P. C., and Huisman, T. H. l.: The estimation of some different kinds of human hemoglobin, Recueil des Travaux Chimiques des Pays-gas 74: 563-570, 1955.