Lactate dehydrogenase isoenzyme patterns of human lymphatic and myeloid leukemic leukocytes

CLINICA

LACTATE

DEHYDROGENASE

LYMPHATIC

V.

349

CHIMICA ACTA

AND MYELOID

ISOENZYME LEUKEMIC

PATTERNS

OF HUMAN

LEUKOCYTES

HIJLE

Department of Clinical Biockemist?y, (Revised manuscript

University of Brno (Czechoslovakia)

received April 17th, 1967)

SUMMARY

The isoenzymes of lactate dehydrogenase (LDH) of human leukocytes were studied in 20 patients with chronic lymphatic leukemia and IO patients with chronic myeloid leukemia. The separation of isoenzymes was performed with agar gel electrophoresis. Isoenzymes were visualized with nitroblue tetrazolium salt and the activities estimated by direct photometric assay. The activity of isoenzyme V was low in lymphatic leukemia and high in myeloid leukemia. It seems that there is a biochemical difference between the LDH isoenzyme pattern of chronic lymphatic and myeloid leukemia, especially in the activity of LDHv.

INTRODUCTION

Dioguardi et al.192demonstrated by DEAE cellulose column chromatography, electrophoresis on starch gel and on cellulose acetate foils five fractions of lactate dehydrogenase (LDH) in normal human leukocytes and in cells of myeloid leukemia. Their studies indicated that immature granulocytes showed a very marked reduction of LDHv (30.7’;/0), especially the myeloblasts (5.6%) as compared to normal granulocytes (46.5%). After completing our studies on LDH isoenzymes in leukemias, Bottomley et aL3 presented a paper about LDH isoenzyme pattern in the leukocytes of IO patients with chronic lymphatic leukemia. As our results differ from the observations cited by these authors, we shall report our findings in the following. MATERIALS

AND METHODS

Peripheral white cells were obtained from 20 patients with chronic lymphatic leukemia in various stages of the disease, and from IO patients with chronic myeloid leukemia. Leukocytes were separated by slow centrifugation of plasma. The bottom layer is practically free from erythrocytes and contains only few thrombocytes. In every case, the isoenzymogram of thrombocytes and erythrocytes is different from the isoClin.

Chim. Ada,

17 (1967) 349-352

HIXE

350

enzymogram of leukocyte+. The leukocytes resuspended in distilled water disrupt. We no longer use freezing and thawing, and ultrasonic disruption is too drastic in our opinion. The separation of isoenzymes was performed by agar gel electrophoresis. In the presence of barbital buffer, ionic strength 0.05, pH 8.6, a current of 800 V was applied to 6 samples for 45 min, at 12~. Isoenzymes were visualized by nitroblue tetrazolium salt and phenazine methosulphate as described in a previous paper4. Activity was estimated by direct photometric assay, using a MCI instrument for paper electrophoresis. RESULTS

The activity of LDH isoenzymes in per cent of the total activity in human leukocytes after planimetric estimation is shown in Tables I and II. Fig. I shows the striking difference between isoenzyme activities of leukocytes in lymphatic and myeloid leukemia. The activity of the fifth isoenzyme, LDHv (the slowest moving isoenzyme) of leukocytes in chronic leukemia has a mean value of 7.3 f 4.1:/o. One patient with

Fig. I. The LDH isoenzymes patterns of leukocytes in chronic lymphatic (138 U) and myeloid (74 T) leukemia. From bottom to top: LDGv (the slowest moving isoenzyme)-LDHI. Fig. z. LDH isocnzymograms of leukocytes in chronic lymphatic leukemias and in terminai myeloblastic stage of chronic myeloid leukemia (1025J), From bottom to top : LI3I-I~ (the slowest moving isoenzyme)-LDHI.

ACTIVITY LEUKEMIA!5 --..

OF THE FIVE

Mean

0, /O

LDHI

LDH

ISOENZYMES

EXPRESSED IN 4’0 OF THE _. .-____ value

ISOLATED

TOTAL

?%7

0: /O

FROM

ACTIVITY -~~ S

(Standard error)

!b

8.42 26.52

-t 3.34 ii 5.31

LDHIV

39.02 "8.77

x4.35 * 6.39

T ~Z 1.22 _::I.00 i 1.46

LDHv

7.27

= 4.06

1 0.93

LDHII J=%II

Cliw. Chim. Acta, 17 (1967) 349-352

LYMPHOCYTES

._____~_~

l- 0.77

IN _

20 CHRONIC

LYMPHATIC

LDH ISOENZYMESIN LEUKOCYTES TABLE

351

II

ACTIVITY LEUKEMIAS _.._.. -__I

OF THE

LDH

FIVE

EXPRESSED illem

IN

vaEue

% _.__ _.“._______.____

LDHr LDHrr LDHm

LDHI\F LDHv --i..

4.88 20.65 26.72 23.60

24.09

ISOENZYMES

ISOLATED

o/o OF THE TOTAL

S.D. “/

.-.-- - * 1.69 :/ 4.28 i 5.47 i

4.71

1x il.16

--.

--

FROM

ACTIVITY

LEUKOCYTES

IN

IO

CHRONIC

MYELOIU

.--

S (Standard error) oi /*

i 9.56 :I: I.42 f I.82 i 5

._

I.57 2.05

__~_.

abnorl~ally high value presented an atypical clinical picture. The spleen was not enlarged, lymphadenopathy was not present. Only absolute lymphocytosis persisted. In contrast to chronic lymphatic leukemia, the leukocytes of chronic myeloid leukemia showed a high activity of isoenzyme V. The mean value was 24.1 f 6.2% of the whole activity. One patient with abnormally low isoenzyme V activity (11.8%) had at the time of examination 23% of myeloblasts in circulating blood. We followed up 2 patients to the final myeloblastic stage and observed a marked reduction of LDHv from 29.6% activity to 5.3%, and from 30.0% to 5% of the total activity. Fig. z presents the isoenzymograms of 4 caseS of chronic lymphatic leukemia and of patient No. 1025 J in the final myeloblastic stage. Fig. 3 shows 3 isoenzymograms of chronic myeloid leukemias.

LDH isoenzymograms of leukocytes in chronic my&id LDHv (the slowest moving isoenzymej-LDHI.

Fig. 3.

leukemia.

From bottom

to top:

DISCUSSION

Bottomley et al.3 studied the isoenzyme pattern of lactate dehydrogenase in the leukocytes of IO patients with chronic lymphatic leukemia. The isoenzymes were not quantitated. From Figs. I and 2 in their study it can, however, be seen that 7 samples showed no activity of isoenzyme IV and V, in one sample the activity of isoenzyme I was lost. The data described in this paper indicate that in all isoenzyme patterns of leukocytes in zo cases of chronic lymphatic leukemia the 5 isoenzymes of LDH were C&n. Chim. Acta, 17 (1967) 34g-3.p

HULE

3.52

present. The activity of isoenzyme V was 7.3 i 4.1% and according to Bottomley et aL3, there is some loss of activity of isoenzyme V when stored at -20’. The mean value of the activity of LDHrv in our experiments was 18.7 * 6.4%, which completely disagrees with no LDHrv activity in 7 samples from IO studied by Bottomley et aL3. In good agreement is the finding that LDHirr activity was highest. From 30 observations of LDH isoenzyme patterns of human chronic lymphatic and myeloid leukemia leukocytes it can be concluded that in lymphatic and myeloid leukemias a biochemical difference exists in the LDH isoenzyme pattern, especially in the activity of LDHv. During the course of the disease the pattern changes. In chronic myeloid leukemia the high LDHv activity diminishes to very low in the final myeloblastic stages. This agrees with the observation of Dioguardi et al.’ who, for their 8 acute leukemias, gave the mean value to be 5.6 & 1.3 (S)u/,. We observed LDHv activity to diminish from 29.6% to 5.3% and from 30.0% to 5.0% of the total activity. Further experiments about the possible significance of inhibitors are in progress. ACKNOWLEDGEMENTS The author wishes to thank Dot. Dr. B. Vec’erek (I. Inst. of Medical Chemistry, Prague) for synthesis of nitroblue tetrazolium, Vaiikova for valuable technical assistance.

and to Mrs. M. Teclova

and Mrs. L.

REFERENCES I N. DIOGUARDI, A. AGOSTONI, G. FIORELLI AND B. LAMENTO, J. Lab. Clin. Med., L N. DIOGUARDI AND A. AGOSTONI, Enzymol. Biol. Clin., 2 (1962/63) 116. 3 R. H. BOTTOMLEY, S. J, LOCKE AND H. C. INGRAM, Blood, 27 (1966) 85. 4 V. HULE, Clin. Chinz. Acta, 13 (1966) 431. Clin.

Chim.

Acta.

17 (1967) 349-352

61 (1963)

713.