RECOIL-FREE ABSORPTION IN THALASSEMIC RED BLOOD CELLS

(1)

HAL Id: jpa-00216752

https://hal.archives-ouvertes.fr/jpa-00216752

Submitted on 1 Jan 1976

HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

RECOIL-FREE ABSORPTION IN THALASSEMIC

RED BLOOD CELLS

S. Ofer, S. Cohen, E. Bauminger, E. Rachmilewitz

To cite this version:

S. Ofer, S. Cohen, E. Bauminger, E. Rachmilewitz. RECOIL-FREE ABSORPTION IN

(2)

JOURNAL DE PHYSIQUE Collogue C6, supplement au n° 12, Tome 37, Decembre 1976, page C6-199

RECOIL-FREE ABSORPTION IN THALASSEMIC RED BLOOD CELLS

S. OFER, S. G. COHEN and E. R. BAUMINGER The Racah Institute of Physics, The Hebrew University, Jerusalem, Israel

and

E. A. RACHMILEWITZ

Hadassah University Hospital, Hebrew University-Hadassah Medical School, Jerusalem, Israel

Résumé. — Le spectre de résonance sans recul des rayons gamma de 14,4 keV émis par une source de 57Co(Rh) a été observé dans des échantillons gelés de sang et de globules rouges prélevés

sur des malades atteints de différentes formes de thalassémie et sur des adultes normaux. Ces spectres sont composés de deux sous-spectres principaux, correspondant à l'oxi- et la déoxi-hémoglobine, et d'un troisième sous-spectre d'une intensité de 4 % dont l'origine est encore inconnue. Un qua-trième sous-spectre d'une intensité de 8 % a été détecté dans le sang thalassémien ; ses paramètres ressemblent à ceux trouvés dans les imidazole hémochromes artificiels. Une corrélation claire a été découverte entre l'intensité de la quatrième composante et les différents types de globules rouges thalassémiens examinés.

Abstract. — The recoilless absorption spectra of the 14.4 keV gamma ray, emitted from a

57Co(Rh) source, have been measured in frozen whole blood and red blood cells from patients

with different forms of thalassemia and from normal adults. All spectra were composed mainly of two subspectra, corresponding to oxy- and deoxy-haemoglobin and a third subspectrum of about 4 % total intensity of as yet unknown origin. In thalassemie blood a fourth subspectrum of about 8 % intensity was detected, with parameters similar to those found in artificial imidazole haemo-chromes. A clear correlation was found between the magnitude of the fourth component and the various types of thalassemie red blood cells that were examined.

1. Introduction. — The primary motivation of the present work was to investigate whether the technique of recoil-free absorption could be used in the study of human disease — in particular in diseases of the red cells. It was indeed found that in general the recoil-free absorption spectra in thalassemie red blood cells (RBCs) was different from that of normal RBCs and a clear correlation was found between the abnormal spectra and the different forms of thalassemia in the corresponding patients. This work represents, to our knowledge, the first application of the Mossbauer effect to human disease.

The thalassemia syndrome is a congenital haemoly-tic disease widely spread in the Mediterranean Basin, South-East Asia and Africa. The major deleterious effects are the result of a decreased synthesis of one of the a or /? globin chains which compose the major fraction of normal haemoglobin (HbA) in form of the Hb tetramer <x2 $%• The excess haemoglobin chains are unstable (both in vivo and in vitro) and eventually tend to precipitate as intercellular inclusions known also as Heinz bodies, with the outcome of severe haemolytic anaemia.

Beta-fhalassemia results from a decreased synthesis of /?-chains with respect to a-chains with a consequent

excess of a-chains. Alpha-thalassemia involves the converse situation and is associated with an excess of /5-chains. In heterozygotes (inheritance from one parent only) the ratio R between the amount of /? synthesis to a synthesis in peripheral blood reticulocytes is such that 0,5 < R < 1, and the disease is termed /^-thalassemia minor or ^-thalassemia trait.

In homozygotes (inheritance from both parents)

R < 0.5, the disease is severe, termed ^-thalassemia

major and is also known as Cooley's anaemia. A subclass of /^-thalassemia major is /^-thalassemia where R is zero. /S-thalassemia major with milder manifestations of the disease (requiring no blood transfusions) is called ^-thalassemia intermedia.

In interpreting the results of analysis of RBCs taken from patients suffering from severe forms of thalasse-mia, one must take into account the extent to which blood transfusions of extraneous normal blood might replace partially or wholly the patients own blood. 2. The technique of measurement and sample prepara-tions. — Since the Hb in the human blood samples contains only unenriched Fe5 7, the recoil-free effects

obtained in frozen samples were necessarily quite small ( ~ 1 %). In order to obtain reliable recoil-free spectra

(3)

C6-200 S. OFER, S. G. COHEN, E. R. BAUMINGER AND E. A. RACHMILEWITZ

in a finite time it was essential to maximalise counting TABLE I

statistics. To this end, a 100 mc Co57 in ~ h o d i u m source was used, and the y-rays were detected in a Harwell proportional counter generally operating at a total counting rate of about 105/s (I).

Venous blood samples were examined throughout. The recoil-free absorption was measured in about 1 ml of whole blood in

a

container with an effective cross section of

-

1.25 cm2. In the first experiments, the blood was taken under paraffin in order to prevent contact with air. In following experiments this precau- tion was not found to be necessary, since the spectra were found to be almost independent of the contact with air for several hours.

Additional experiments showed that measurements on RBCs after removal of the plasma give recoil-free effects about 2.5 times greater than those obtained in corresponding whole blood samples, while the spectral shape remained identical. Most of the latter measurements were therefore carried out on RBCs.

The recoil-free absorption in all cases was measured in frozen samples at 82 K, after encapsulation in perspex containers. The RBC samples were frozen immediately after separation from the plasma and stored in liquid nitrogen before measurement. In general, counting statistics for a particular sample was accumulated during 24 hours. In most cases additional samples were left at 37 OC in closed testubes for periods varying from one day to two weeks before resonance absorption was remeasured, in order to detect any changes with time under these conditions. Computer analysis of the recoil-free spectrum was essential in view of its complex nature.

In the present work samples were examined from 3 normal adults and from 12 thalassemic patients, includ-

ing : 1 case with HbH disease (a-thalassemia), 7 cases

with P-thalassemia major (of which 4 were of the

Po

type), 2 cases with P-thalassemia intermedia and 2 with P-thalassemia minor. The diagnosis in each case was made on the basis of qualitative and quantitative Hb electrophoresis and determination of alkali resistant Hb. The distinction between

Po

and other types of P-thalassemia major (Pf) was based on measurements of Hb synthesis [I].

3. Results.

-

3 . 1 NORMAL RBC's.

-

All the spec-

tra were composed of two main sub-spectra, one corresponding to deoxy Hb (c a D) and the other to

oxy Hb ((( b >>) with isomer shifts and cluadrupole splittings as found in previous reports [2]. The para- meters are given in table 1. Figure 1 shows a typical spectrum of normal RBC's. The maximum recoil-free effect is about 1

%.

In all three cases examined, compu- ter analysis of the spectrum points to the existence of a minor quadrupole doublet in addition to the known

(1) We are greatly indebted to T. E. Cranshaw for recommend-

ing use of the Harwell proportional counter for their high counting rates.

Mossbauer parameters of subspectra of normal and thalassemic RBC's at 82 K Quadrupole splitting Component

-

(-1s) a >> (deoxy Hb) 2.30 (2) b >> (oxy Hb) 2.11 (2) << C >> 1.12 (4) << d >> 0.88 (4) Fe(I1) haemin, Imida-

zole (b) 0.95 Isomer (a) shift Linewidth (mmls) (-1s) 0.90 (1) 0.34 (2) 0.26 (1) 0.33 (2) 0.50 (3) 0.21 (2) 0.47 (3) 0.45 (5)

(a) Relative to metallic iron. (b) Ref. [41.

main components. This minor component is indicated by curve (( c )) in figure 1 and its parameters are given

in the table. The relative intensity of this component is about 4

%

of the total.

VELOCITY (mm/s)

FIG. 1.

-

Recoilles absorption spectrum of the 14.4 keV gamma-ray of 57Fe in normal RBC's at 82 k. The solid line is a least squares computer fit to the spectrum. The dashed << a >> subspectrum corresponds to deoxy Hb. The dashed << b D

subspectrum corresponds to oxy Hb. The dashed << c >> sub- spectrum corresponds to an unidentified component. The para-

meters of the subspectra are given in table I.

3 . 2 THALASSEMIC RBC's.

-

In nearly every case of

thalassemic RBC's a fourth component cr d )) was

identified in the recoil-free spectrum. The intensity of this component was most prominent in the spectrum obtained in the case of Hb H disease.

We will now consider the results pertaining to the various types of thalassemia.

3.2.1 Hb H disease (a-thalassemia). - The spec-

trum is displayed in figure 2. Computer analysis gives the following relative intensities (I) : I(a) = (64k 1)

%,

I(b)=(19+1)

%,

I(c)=(l+l)

%

andI(d)=(16+1)

%.

The parameters of the (< d )) component are given in

table I and are very different from those of oxy-Hb and deoxy-Hb.

After incubating the sample at 37 OC for 3 days the intensity of the ct d >) component increased to about

(4)

RECOIL-FREE ABSORPTION I N THALASSEMIC RED BLOOD CELLS C6-201

FIG. 2. - Recoilles absorption spectrum of the 14.4 keV gamma-ray of 57Fe in RBC's a t 82 K obtained from a patient with Hb-H disease. The solid line is a least squares computer fit to the experimental spectrum. The dashed ee a >> subspectrum corresponds t o deoxy Hb. The dashed (( b )> subspectrum cor-

responds to oxy Hb. The dashed <c d n subspectrum is charac- teristic of thalassemia and is believed to correspond to a haemochrome. The parameters of the subspectra are given in

table I.

3 .2.2 j?-thalasseinia major.

-

In 3 cases, subspectra corresponding to the << d >> component with relative intensities of about 8

%,

where observed. No signifi- cant change in the spectra were recorded after incubation for 6 days at 37 OC. A typical spectrum is shown in figure 3. Analysis of this spectrum yields for the

,

-2.0 -1.0 0.0 1.0 2 . 0 3.0

VELOCITY (m/s)

FIG. 3. - Recoilles absorption spectrum of the 14.4 keV gamma-ray of 57Fe in RBC's at 82 K obtained from a patient with fithalassemia major. The RBC's were kept for 6 days at 37 "C in a closed test-tube before freezing. The solid line is a least squares computer fit to the experimental spectrum. The (c a )>, cc b >>, ce c >> and cc d >> curves correspond to subspectra

with parameters given in table I and intensities given in the text.

various intensities : I(a) = (80

k

1)

%,

I(b) = (2

k

1)

%,

I(c) = (5

+

1)

%

and I(d) = (13

+

2)

%.

In 4 cases of Po-thalassemia, only very small intensities of the

<< d )> component ((0-2)

%)

were observed. Again the

spectra showed no appreciable change after incubation for 6 days at 37 OC.

3 .2.3 P-thalassemia intermedia. - The first case initially showed the << d )) component with a relative

intensity of 7.5

%

increasing to 12

%

after 24 hours at 37 0C (Fig. 4). The second case showed an initial intensity of 2

%,

increasing to about 7

%

after 6 days. 3.2.4 fithalassemia minor.

-

In the 2 cases inves- tigated, the initial intensity of the << d >> component was

(0 f 1)

%.

In one of the cases the intensity increased

I I I I

I

-2.0 -1.0 0.0

-

1.0 2.0 3.0

VELOCITY (mm/s)

FIG. 4. - Recoilless absorption spectra of the 14.4 keV gamma- rays of 57Fe in RBC's a t 82 K, obtained from a patient with Bthalassemia intermedia. Spectrum (1) was obtained with a sample frozen immediately after the RBC's were separated. Spectrum (2) was obtained with a sample frozen after incubation for 24 hours at 37 "C in a closed test-tube. The <c a w, cc b >>,

cc c >) and cc d >> curves correspond to subspectra with para-

meters given in table I.

to about 5

%

after one week and to 9

%

after 2 weeks incubation at 37 OC. The effect of incubation on the second case was not examined.

4. Discussion.

-

4.1 The above results demonstrate

the presence of the (( d )) component in thalassemic

RBC's. At first sight, the relatively small amounts of the

<( d )) component found in the severe forms of

Po-

thalassemia would seem to argue against this. However, in these cases the peripheral blood which was examined is mainly transfused blood from normal subjects, since the patients cannot synthesize their own haemoglobin A (a, P,). The only components of their own blood is haemoglobin A, (a, 6,) and fetal haemoglobin-F (a, y,) which compose a small fraction of the blood examined. In addition, the excess a-chains are unstable and tend to precipitate already in young erythroid cells in the bonemarrow [3] and can be found only in trace amounts in a soluble form. In patients with

Pf

-thalassemia major some haemoglobin A is synthesized in addition to haemoglobin A, and F and the (( d ))

component can therefore be identified. On the other hand, excess j3 chains present in Hb H disease can form their own tetramer and are relatively stable in the peripheral blood. Therefore patients with haemoglobin H disease and also P-thalassemia intermedia usually do not require blood transfusions and the measurements are actually performed on their genuine blood. The same applies to the cases with P-thalassemia minor. 4.2 NATURE OF THE << d )) COMPONENT. - We have noticed that the parameters of the <( d )) component as

measured in our present work are close to those of synthesized imidazole haemochromes [4]. The parameters are compared in table I. This similarity strongly suggests that the (( d >> component consists of protein

(5)

C6-202 S. OF=, S. G. COHEN, E. R. BAUMINGER ABD E. A. RACHMILEWITZ

following the spontaneous oxidation of oxy-Hb subunits in vitro [5] and were also identified in precipitated a and

B

haemoglobin subunits in the form of inclusion bodies within RBC ghosts .in patients with haemoglobin H disease and P-thalassemia major on the basis of microspectrophotometry of single inclusions and electron paramagnetic resonance. It has been therefore suggested that the precipitation of excess Hb subunits in thalassemic RBC's occurs as a result of haemichrome formation. One of the most common and reversible haemichromes is thought to result from bonding of the distal histidine at helical position E7 to the sixth coordination position of the haeme ion, while the norlpal bond between histidine F8 and the fifth position remains intact. Haemochromes retain the basic struc- ture of haemichromes except for a change of ferric to ferrous iron:

We have not found any other known recoil-free spectrum of a haeme derivative which resembles the spectrum of component << d >>. Methaemoglobin is always found in small amounts in RBC's as a reaction product between oxy-Hb and water and in larger amounts in the RBC's of patients with abnormal and unstable Hb's, including thalassemia. However, the recoil-free spectrum of methaemoglobin is widely different from that of the d )) component [2].

The present work indicates the presence of haemochrome proteins in thalassemic RBC's, whereas previous reports referred to above demonstrated haemichrome formation in both the spontaneous oxidation of separated Hb subunits and also in their precipitated form (inclusion bodies) within thalassemic RBC's ghosts. There is not necessarily a contradiction between these different observations for the following reasons :

(a) The present measurements were performed on fresh samples of intact RBC's. (b). The EPR technique is not sensitive to haemochromes. (c) An exact quantitative analysis of the amount of haemichromes by EPR is not possible. It is possible that small amounts of haemichromes are present in the samples examined in the present study, but there is no doubt that their amount is much less than that of the predominant haemochrome. This observation raises the question as to whether the haemochromes are perhaps formed from the excess Hb subunits via a pathway not involving formation of

Refer

[I] CIVIDALLI, G., KEREM, H. and RACHMILEWITZ, E. A., sub-

mitted for publication.

[2] LANG, G. and MARSHALL, W., Proc. Phys. Soc. 87 (1966) 3. [3 1 RACHMILEWITZ, E. A. and THORELL, B., Blood 39 (1972) 794. [4] EPSTEIN, L. M., STRAUB, D. K. and MARICONDI, C., Inorg.

Chem. 6 (1967) 1720.

[5] RACHMILEWITZ, E. A., PEISACH, J. and BLUMBERG, W. E., J.

Biol. Chem. 246 (1971) 3356.

ferri Hb (methaemoglobin) as has previously been supposed [ 6 ] .

B. H. Huynh et al. [7] in a recent study have compared the recoil-free spectra in deoxy-HbA and its isolated a and

p

units. Within experimental error no difference was found between the Mossbauer parameters charac- terizing the main components of these three proteins. However, the authors discovered in the spectra of the isolated a and /? subunits only, a weak component (20

%

of the main component) consistent with a low spin ferrous six-coordinated compound, which they ascribe to haemochrome formation as the result of an inherent instability of the isolated subunits and their mode of preparation. Within the experimental errors there is agreement between the parameters o f this component found in the latter work and that of component << d >> in the present work. We regard this as supporting evidence that the (( d >) subspectrum corres-

ponds to a haemochrome produced as a consequence of instability of the excess Hb subunits within thalassemic RBC's.

In some of the samples the intensity of the (( d )>

component increased with time when the sample was incubated at 37 OC for a few days. This is further evi- dence for the instability of the Hb in thalassemic RBC's. However, there are instances where the intensity of the

<< d )> component does not change with time. A possible

explanation is that in these cases of P-thalassemia major all the isolated chains have been already trans- formed to haemochromes.

The present work shows that recoil-free absorption can be used for the quantitative detection of haemochromes in thalassemic RBC's.

4.3 COMPONENT c D. - At the present time we do not know the origin of this component present in healthy RBC's and in most of the patients' samples. It is possible that this component is associated with the presence of a smdl amount of anhydrous Hb which was perhaps produced during the freezing of the RBC's samples. Spectra similar to that of the <( c >> component

were observed by Papaefthymiou et al. in anhydrous Hb and its isolated subunits [8].

We intend to pursue further studies of thalassemia and other blood diseases using Mossbauer spectros- COPY-

[6] RACHMILEWITZ, E. A., PEISACH, J., BRADLEY, T. B. and BLUMBERG, W. E., Nature 222 (1969) 248.

[7j HUYNH, B. H.,. PAPAEFTHYMIOU, G. C., YEN, C. S., GROVES,

J. L. and Wu, C. S., Biochem. and Biophys. Res. Comm. 60 (1974) 1295.

[8] PAPAEFTHYMIOU, G. C., HUYNH, B. H., YEN, C. S., GROVES,