BIOCHIMICA ET BIOPHYSICA ACTA 291

E R Y T H R O C Y T E 8 - A M I N O L E V U L I N I C ACID D E H Y D R A S E ACTIVITY

IN T H A L A S S E M I A M A J O R AND S I C K L E - C E L L A N E M I A

F E L I X FELDMAN AND H E R B E R T C. LICHTMAN

Department of Pediatrics, Maimonides Hospital and Department of Medicine, State University of New York, Downstate Medical Center, Brooklyn, N.Y. (U.S.A.)

(Received August I l th , 1961)

SUMMARY

An enzyme deficiency has been postulated to account for the defect in hemoglobin synthesis in certain hereditary anemias. The enzyme necessary for the conversion of $-aminolevulinic acid to porphobilinogen, an obligatory step in heme synthesis, was assayed. The values obtained from the circulating erythrocyte of patients with thalassemia and sickle-cell anemia were higher than normal.

INTRODUCTION

A variety of evidence a-~ suggests that there exists in patients with thalasscmia major and sickle-cell anemia, a quantitative defect in hemoglobin synthesis. That this defect could depend upon a qualitative or quantitative aberration in an enzymically con- trolled reaction necessary for heme production seemed worthy of investigation.

I t has been demonstrated that 3-aminolevulinic acid and porphobilinogen are effective precursors of porphyrin and heme 4, 5. The conversion of 3-aminolevulinic acid to porphobilinogen is catalyzed by an enzyme, 3-aminolevulinic acid dehydrase, which has been extracted from ox liver e, duck and human erythrocytes 7 and chicken erythrocytes s.

In the reports currently available very little has been published concerning $-aminolevulinic acid dehydrase activity in the erythrocytes of normal humans and patients with hereditary anemias. In the present study 3-aminolevulinic acid dehydrase activity was assayed in normal human erythrocytes and in the red blood cells of patients with thalassemia major and sickle-cell anemia.

Erythrocytes collected in heparin and kept at 0-4 ° were washed three times and resuspended in physiological saline solution. An aliquot of the incubation suspension was set aside for determination of hematocrit, hemoglobin, red blood cell count, white blood cell count, nucleated red blood cell count and reticulocyte count. The remainder of the suspension was hemolyzed by rapid freezing and thawing. It was then incubated anaerobically under nitrogen in a shaking water bath with added 3-aminolevulinic acid for I h at 37 °.

The porphobilinogen synthesized was determined by the method of MAUZERALL AND GRANICK ~. The 3-aminolevulinic acid dehydrase activity was expressed as/zmoles of porphobilinogen synthesized/ml of packed red blood cells/h of incubation.

Biochim. Biophys. Acta, 58 (1962) 291-293

2 9 2 F. FELDMAN, H. C. LICHTMAN

S-Aminolevulinic acid dehydrase activity was measurable in all specimens tested. Experiments in duplicate were performed on red blood cells of 16 normal individuals. The average enzyme activi ty was 18o. lO -3 ~moles of porphobilinogen/ml of packed red blood cells/h of incubation, with a range of 27.8- lO -3 to 327" lO -3. The average reticu- locyte count in the incubation mixtures of the normals was 0.5 % with a range of from o.I °/o to 1.2 %. Analysis of covariance 1° log. dehydrase activity on log. reticulocyte count of normal subjects revealed a poor coefficient of correlation between dehydrase act ivi ty and reticulocyte count (coefficient of correlation, r ----- o.164).

Experiments were performed in duplicate on erythrocytes from eleven patients with sickle-cell anemia. The average dehydrase activity was 421 .lO -3 /zmoles of porphobilinogen/ml of packed red blood cells/h of incubation with a range of from t71. lO -3 to 730" lO -3. The average reticulocyte count in the incubation mixtures of the sickle-cell anemics was 8.7 % with a range of from 2.4% to 25.4 %. In this group the correlation of dehydrase activity and reticulocyte count was quite good (r -~ 0.640 ). Fourteen experiments were performed in duplicate on erythrocytes of patients with thalassemia major. Blood was drawn from each patient just prior to his periodic transfusion at a t ime when the circulating blood cell population was most repre- sentative of the individual's own erythropoiesis. The average dehydrase activity was 366. lO -3/~moles of porphobilinogen/ml of packed red blood cells/h of incubation with a range of from lO8- IO -~ to 64 °. lO -3. The average reticulocyte count was 2.1% with a range of from o.I °/o to 7.8 o/o. In this group the correlation of dehydrase activity and reticulocyte count was good (r ---- 0.600).

These data suggest the following: (a) ~-Aminolevulinic acid dehydrase activity persists in the circulating erythrocytes of normal individuals and patients with sickle- cell anemia and thalassemia major. (b) The enzyme activity and the reticulocyte counts are statistically significantly higher in both anemic groups than in the normal. There is not a statistically significant difference in the ~-aminolevulinic acid dehydrase act ivi ty between the two anemic groups. The reticulocyte counts, however, tend to be distinctly higher among the sickle-cell patients when compared with the patients with thalassemia. (c) In the normal there is poor correlation between 8-amino- levulinic acid dehydrase activity and reticulocyte count. (d) In both anemic groups the correlation between S-aminolevulinic acid dehydrase activity and reticulocyte count is very good.

N o r r n o l Io 9 x = - 0 . 2 2 8 log y = 2.18.5

'3.0 R e g r e s s C o e f f = - 0 . 2 2 3

>~

.> 2 .5

~ 2.o

-x t~.

T h o l l a s s e m i o I ~ = + 0 .0531

Io-- '~ = 2 . 5 0 9

Regress Coef f = ÷ 0 .286

0 1 Log Retlcylocyte Count

Sickle Cell Anemio log x = + 0 . B 3 6

I '-~-'~ = 2,..578

0 I 2

Fig. i . Log. 6 -amino levu l in i c acid a c t i v i t y p lo t t e d as a func t ion of log. r e t i cu locy te count .

Biochim. Biophys. Acta, 58 (1962) 291-293

~-AMINOLEVULINIC ACID DEHYDRASE IN HUMAN RED CELLS 293

The reticulocyte counts seem to affect dehydrase activity in the same manner in both anemic groups (Fig. I). There is no statistical proof, but there is a suggestion that the 8-aminolevulinic acid dehydrase act ivi ty in sickle-cell anemia and thalassemia major are actually different by amounts not accounted for by reticulocyte differences. This might be referable to the presence of circulating nucleated red blood cells in some of the patients with thalassemia. Nucleated red blood cell counts varied in those patients from 0-2.5 %. They were noted in nine of the fourteen patients studied with thalassemia. No nucleated red blood cells were observed in any of the normal or sickle-cell patients.

I t has been well established that heme is not synthesized in the mature erythrocyte We have made the assumption that the level of enzyme activity which persists in the mature erythrocyte is a reflection of the level of activity which occurs in the nucleated red cell, i.e., the site of heme synthesis in the bone marrow. There appears to be no evidence that there is a deficiency of 8-aminolevulinic acid dehydrase act ivi ty in either thalassemia maj or or sickle-cell anemia. The observed increase in enzyme activity may be due to the increased numbers of immature erythrocytes found in these conditions.

ACKNOWLEDGEMENTS

We are grateful to Mr. P. NEMENYI, Depar tment of Environmental Medicine, for statistical analysis of the data.

This s tudy was supported in part by United States Public Health Service Grant

No. H-3877. This s tudy was completed with the technical assistance of Mrs. R. HERZ and

Mrs. S. STEINSCHREIBER.

R E F E R E N C E S

1 p. STURGEON AND C. A. FINCH, Blood, I2 (1957) 64. M. GRINSTEIN, R. M. BANNERMAN, J. D. VAVRA AND C. V. MOORE, Am. J. Med., 29 (196o) 18.

3 R. M. BANNERMAN, M. GRINSTEIN, AND C. V. MOOP,~, Brit. J. Hematol., 5 (1959) lO2. 4 D. SHEMIN AND C. S. RUSSELL, J., Am. Chem. Soc. 75 (1953) 4873 • s j . E. FALK, E. I. B. DRESEL AND C. RIMINGTON, Nature, 172 (1953) 292.

K. D. GIBSON, A. NEUBERGER AND J. J. SCOTT, Biochem. f . , 61 (1955) 618. R. SCHilD AND D. SHEMIN, J. Am. Chem. Soe., 77/(1955) 5o6.

s S. GRANICK, Science, 12o (1954) 11o5. g D. MAUZERALL AND S. GRANICK, J . Biol. Chem., 219 (1956) 435.

10 G. W. SNEDECOR, Iowa State College Press 5th Ed., 1957.

Biochim. Biophys. Acta, 58 (1962)291-293