\-. MAL.~SIiOVd Imtitutr
AND H. HOLEYSOVSKd
LDH isoenzymes in leukolysates prepared from isolated mixed population of leukocytes from normal persons were analyzed by agar electrophoresis and compared with leukolysates from patients with different blood diseases. In samples with high granulocytosis the pattern with high cathodic fractions was typical, in lymphocytosis the undifferentiated type with high intermediate fractions was found. In leukolysates of leukaemic blasts both types of LDH pattern were found, though no correlation was revealed with the morphological or cytochemical type of the cells examined. The authors suppose that the two patterns correspond to two different metabolic types of blast cells in acute leukaemia which are not necessarily followed by morphological changes or characteristics. In 5 examinations of 4 patients with paroxysmal nocturnal haemoglobinuria, an abnormal LDH pattern in granulocytes Possibly, this may be the expression of a metabolic abnormality olism in this red cell disease.
was found twice. of leukocyte metab-
Two different isoenzyme patterns of lactate dehydrogenase (LDH) activity were detected by several authors in leukolysates of human white blood cells1-6. In mature normal neutrophils and in neutrophils in granulocytoses of different origin all five isoenzyme fractions are present, cathodic fractions being predominant (Fig. I). In younger neutrophils of normal human bone marrow and in young cells in myeloid leukaemia this predominance of the cathodic fractions is less marked, as the middle and anodic fractions prevail in the immature cells 3y6. In the youngest, the blast cells, highest activity lies in the intermediate fractions. This intermediate type with low activity of the cathodic fractions is the second type of LDH isoenzyme pattern of human white blood cells, characteristic of both normal and leukaemic lymphocytes (Fig. 2) 7--9. The purpose of our study was to contribute to the differential diagnosis of acute leukaemias, as neither cytological, nor cytochemical or blood cell culture examinations can determine the type of malignant cells without possible error. We studied the LDH isoenzymes by agar electrophoresis on our own clinical material. Some of the * Presented at the 5th Meeting of the July 19, 1968. * Director Prof. Dr. J. Hoi-ejBi, Dr.%.
results obtained do not agree with other authors’ are presenting them in this paper.
and for this reasnn \vc
MATERIAL ANI) MISTHOI)5 I’or the isolation and lysis of human leukocytes from peripheral blood a metllod described by Dioguardi3 was used with a slight modification. The difference from tlic, original method lies in the use of the haemolytic effect of ammonium cllloride on plasma rich in leukocytes instead of on whole blood, collected by double sedimentation of heparinized whole blood with the addition of 116 vol. of pol~~in~ll~~~rrolidon. ‘IIICT concentration of leukocytes before lysis achieved by distilled water and threefold freezing and thawing only, was 150000~-Z~OOOOI.e/,~l. After final centrifugation at 3000 rev/min for IO min the supernatant was divided by agar electrophoresis according to Kamaryt’s modificationl” in veronal-Verona1 sodium buffer of pH 8.6 and ionic strengt Ii 0.5 ;I, voltage gradient 5.0 Y/cm. The LDH activit!. was detected by the tctrazolium evaluation was carried out spe(‘method according to \:an der HelmI’, quantitative using trophotometrically after extraction of the formed dyeI or densitometrically the apparatus ERI IO. In this part of the experiments the samples were not separated by more conplicated procedures to obtain a higher concentration of cells of a single line, but onI\, the samples leaving a large majority of one type of cell were evaluated. RESCLTS Table I shows the average activity
of individual LDH fractions
from 5 patients
blood of 16 blood donors, further of granulocytes
and from 4 patients
granulocytes with prevalence of ripe forms). In 9 samples from chronic lymphadenosis the lymphocytes of 90-98q/, purity and in IO samples from patients with acute leukaemia the blast cells of 62-9gq; purity were analyzed. The values are expressed in percentage of the total activity of the sample.
blood donors mvelosis M~elofibr&is Lytnphadenosis Acute leukaemias
5 4 9 1”
6.2 _I~ 2.0 3.0 : 2.3 3.2 _:_ 2.7 -I_ 9.1 8.5 -~ 7.5
‘9.4 ~~ 5.1 16.9 _,I 5.1 6.” 15.3
27.6 :I- 3.5
IX.7 -- 5.9 26.6 I 1.5 27.9 I- 3.”
~!I 6.5 f 10.3
23.1 : 5.3 23.4 3j.5 .i ::: 31.7 -1 7.3
17.5 ~7 9.5 20.9 1z 10.9
22.2 & j.0 32.” 7.8 12.3
5.9 : 9.-+ 6.1
U’ith the increasing number of neutrophils in the samples-as in chronic myelosis and myelofibrosis-the cathodic fractions, i.e. LDH, and LDH,, are higher than in normal mixed population of donors’ blood. In lymphadenosis the cathodic fractions are lower than normal, the intermediate fractions are higher, according to the absolute prevalence of lymphocytes. The average rate of activity in single LDH fractions in the group of acute leukaemias is almost the same as in lymphadenosis. However, from Table II where the isoenzyme patterns of individual patients are given, it is obvious that TABLE RATIO
4 5 0
I)1 s:\ x.0 lil< I:1 Tl< 1.0
7 t( 9 I”
.\veragc in 0; Deviation
7.4 i. 1 6. 7
25.3 3-t.9 21.3 26.8
3X.4 15.4 30.1
6.0 5.s 26.4
24.9 35.8 33.0 29.3 23.0
9.8 ‘9.7 17.0 2.6 27.3 1.3.” 36.2 22.”
27.4 3.9 2.9 12.7 24.6 7.2 10.3 0.1 17.”
Dediff, Dediff. Dcdiff. 1Iono + para L~mpho -+ ~-‘ar;, Monomt para l’ara ml_ atyp.n1yc1o I’ara I’arapdediff. Dediff. + mono
97 94 73 85 87 75 62
though in most of the samples analyzed (i.e., in 6) the intermediate, lymphocytic type is found (Fig. 3), the other type, the cathodic or granulocytic pattern is also present in two of them (Fig. 4), besides a normal pattern in two other patients. This finding being rather surprising we attempted to find an explanation for it by correlation with the morphological type of the blast cells examined. In the last column of Table II we can see that no such correlation exists, every type of blast can be connected with Cli~z.Chum.
Acta, 2-1 (1969)
either type of LDH isoenzyme pattern. Kor could any other cytochemical correlation, not shown in this table, be found. The most unusual result was obtained in patient No. 5. The activities of LDH isoenzymes in the Table were obtained in a period of peripheral “lymphocytosis” of 85: i,, which was explained as a lymphocytosis in remission after cytostatics. As the isozyme pattern had a very distinct granulocytic character, we re-examined the blood and bone marrow smears but could only confirm the original morphological diagnosis. This cathodic type of isozyme pattern remained unchanged even in a second analysis two months later, wllcn there were c]o”;, of typical mycloblasts in this patient’s blood and marrow (results not included in the Table). This irregular and unusual finding of a granulocytic type of LDH isocnzymes in lymphocytes was not the only one we found. In leukocytes of two patients with chronic lymphadenosis the 5th fraction was also high in three examinations. Als it was revealed at the beginning of our study we attributed this irregularity to inflammator!. complications and did not include the results in the Table. On the contrai->-, we found the lymphocytic type of LDH pattern even in granulocytes of scvcral cases of panof granulocvtcs from four myelopathy, pancytopenia and in z of 5 examinations patients with paroxysmal nocturnal haemoglobinuria (a corpuscular red cell disease) (Table III). As these exceptional cases were not so frequent and as WCwere not able to follow these patients individually in different periods of their illness, WC 11a\e not yet been able to draw definite conclusions.
of white blood cells, tlrc
LDH ISOENZYMES OF LEUKOCYTES TXHLI<
25.8 26.4 26.1
4 3 9
19.5 25.0 0.0 20.1
J ;\ so
27.0 9.25 20.5
intermediate type of isoenzyme pattern in lymphocytes and the cathodic type in ripe granulocytes are in good agreement with references in literature1-9. The findings of the granulocytic type of LDH isoenzymes in lymphocytes and in some cases of acute leukaemia are mentioned only exceptionally ,13,i4, the finding of the lymphatic type in ripe granulocytes in some cases of paroxysmal nocturnal haemoglobinuria, panmyelopathy and other diseases has not yet been described. Only Rabinowitz and Dietzlb and Hellung-Larsen and Anderseni6 observed in vitro the change of lymphatic type of LDH isoenzymes to a granulocytic one, i.e. the rise in the proportion of lM subunits in cultured lymphocytes stimulated by phytohaemagglutinin (PHA). The stimulation by PHA is followed by an increase in proliferative activity of the cultured cells and increase in glycolysis. As it is generally accepted that the 5th fraction of LDH is connected with aerobic metabolism and the anodic fractions with anaerobic turnover of the cells, one might be tempted to look here for an explanation for the two different patterns of LDH isoenzymes. In the literature, there seems to be no agreement either in experimental results or in theories concerning the type of metabolism of lymphocytes and of granulocytes. It seems most probable that both kinds of cells reveal both types of metabolism, suppressed
and anaerobic, the type
but in normal
of glvcolysis _ ”
one of them
does not seem to be simpl!
correlated with the type of LDH isoenzyme pattern, as in other tissues. The studies on the isoenzymes in cultured cells mentioned above show that WRC cultures enable us to study metabolic regulations from new angles. The studies of Rabinowitz and Dietz’”
and of Hellung-Larsen and 4ndersen16 completed the \vide biochemic studies of Seitzl”yl” and opened some new aspects. Sow it seems most probable that not onl\ changing maturity or dedifferentiation of the cells, but also the type of glycolysis or proteosynthesis, and the metabolism of nucleic acids in stimulated cultured cells may be closely connected with the ratio of M and H subunits of LDH15~20~21.The cause of it may be a direct effect of partial pressure of oxygen, not mediated through some other metabolic cliangei6. i\ssumingtliat some similar changes mayoccur in living organisni in haematologic disorders, be it abnormal metabolism due to unclarified primary causes or abnormalintcrmediates (nucleoproteins, proteosvnthesis, minerals), we would have an explanation for the “atypical” LDH isoenzyme pattern both in ripe lymphocytes and granulocytes, as well as for the existence of two types of blast cells in acute leukaemia; the “1yn~pl~ocytic” and the “granulocytic” ones. The latter arc also in good agreement with similar metabolic types of blasts observed by broader biochemical parameters by Seitz et al.‘*. The clinical, therapeutical or prognostic evaluation of these findings is a matter for future studies.
8 9 IO II I2 I3 '4 I5 16 I7 I8 '9 20 2I Clin. Chiw.