Abstract
Red cell glucose-6-phosphate dehydrogenase (G6PD) levels in 8 patients of chronic myeloid leukaemia (CML), who underwent remission, showed a decrease in G6PD levels from 283.7 ± 47.63 mU/109 RBCs to 221.71 ± 31.71 mU/109 RBCs after 6 weeks of chemotherapy (p < 0.001). 12 patients who didn’t undergo remission showed a decline from 296.33 ± 48.34 mU/109 RBC’s to 273.63 ± 65.39 mU/109 RBC’s after chemotherapy (p < 0.05) as compared to levels in controls of 125.95 ± 8.41 mU/109 RBCs. Therefore, red cell G6PD levels can act as prognostic indicator in patients of CML and help in predicting the response to chemotherapy.

Introduction
Haematological malignancies are the neoplastic proliferation of blood cells. Leukaemic cells rapidly accumulate in the bone marrow, ultimately replacing most of the normal haematopoietic cells, thus resulting in the signs and symptoms of the disease. Chronic myeloid leukaemia (CML) is a chronic myeloproliferative disorder of pluripotent haematopoietic progenitor cells characterized by excessive proliferation and accumulation of granulocytes and occasionally red blood cells and platelets.¹

Various acquired enzymopathies have been described in leukaemias. These biochemical markers may be useful for making an early diagnosis and monitoring of the disease activity. Enzymes like Glucose-6-phosphate dehydrogenase, Pyruvate kinase, 5’-Nucleotidase, Adenosine deaminase and Lactate dehydrogenase have been studied in various haematological malignancies.^2-4

Glucose-6-phosphate dehydrogenase (G6PD) levels have been studied in patients of haematological malignancies. Both red blood cell (RBC) and white blood cell (WBC) G6PD values have been found to correlate with disease activity and have prognostic significance.⁵ Glucose-6-phosphate dehy-drogenase (EC 1.1.1.49; D-glucose-6-phosphate: NADP oxidoreductase, G6PD) is the first and rate limiting enzyme of hexose monophosphate pathway (HMP).⁶

This study was planned to correlate red cell G6PD levels before and after chemotherapy and also to find out any prognostic significance of red cell G6PD activity in patients of CML.

Material and Methods
The study was conducted in 20 patients of CML admitted in the medical ward and 20 age and sex matched healthy controls after getting their informed consent and approval from local ethical committee. Only newly diagnosed and proven cases of chronic myeloid leukaemia in which diagnosis was made by history, clinical examination, complete haemogram and bone marrow examination and receiving no medication for at least last one month were selected for the study. The staging of CML patients was done as per the criteria given by Wetzler et al and Kantarjian et al.^7,8

In addition to haematological parameters and routine investigations, red cell G6PD was estimated at diagnosis and then 6 weeks after start of standard chemotherapy (consisting of hydroxyurea, 6-mercaptopurine and prednisolone) or at remission, whichever is earlier. It was also estimated in 20 healthy controls. Venous blood was taken by aseptic technique in heparinised autoclaved syringe. Red cell G6PD estimation was done after preparing the haemolysate using the principle that G6PD oxidizes glucose-6-phosphate to 6-phosphogluconate reducing NADP to NADPH causing an increase in absorbance at 340 nm. This increase is directly proportional to G6PD activity. Production of a second molar equivalent of NADPH was prevented by using maleimide, an inhibitor of red cell 6-phosphogluconate dehydrogenase. This activity was expressed mU/109 red blood cells after noting change in absorbance for 5 minutes and taking into account the temperature correction factor and red cell count.⁹

Results
The mean age of presentation in patients was 46.15 years (range 45-75 years) and there were 10 males and 10 females. The controls consisted of 10 males and 10 females and their mean age was 42.9 years (range 24-77 years).

The patient presented with a variety of complaints including generalized weakness, easy fatigability, mass abdomen, weight loss, fever, easy bruisability and bleeding tendencies. On examination, all the patients had pallor and moderate to massive splenomegaly. 75% of patients had mild to moderate hepatomegaly. Purpuric spots were seen in 3 cases and no patients showed lymphadenopathy.

On laboratory investigations, all of these patients were found to be anaemic with mean haemoglobin being 6.87 g% (range 4.3-10.5 g%). Mean RBC count was found to be 4.41 x 106/µl (range 3.2-5.2 x 106/µl). Total leucocyte count (TLC) was 93.25 x 103/µl (range 23-210 x 103/µl) with platelet and reticulocyte counts being within the normal limits.

The mean red cell G6PD level in healthy controls was 125.95 mU/109 RBC’s (range 113-6-140.3) while that in CML patients at presentation was 291.28 mU/109 RBC’s (range 210.6-375.3). This difference is highly significant with a p value < 0.001. After chemotherapy, G6PD levels showed a decrease with a mean of 252.86 mU/109 RBC’s (range 182.4-365.3) and this change is statistically highly significant (p < 0.001).

Out of 20 patients, 8 showed haematological remission during the study period. Their G6PD levels have been depicted in Table 1.

Discussion
In our study, G6PD level is found to be significantly higher in CML patients than the control cases. Red cell G6PD levels, after chemotherapy, decreased from 283.7 ± 47.63 mU/109 RBC’s to 221.71 ± 31.71 mU/109 RBC’s in eight patients who achieved haematological remission and change is highly significant (p < 0.001). Twelve patients who didn’t achieve haematological remission also showed a decrease in red cell G6PD levels from 296.33 ± 48.34 mU/109 RBC’s to 273.63 ± 65.39 mU/109 RBC’s after chemotherapy which is significant statistically (p < 0.05) though not as in remission group. The levels of G6PD after chemotherapy are significantly low in remission group (p < 0.001) as compared to

Table 1 : G6PD levels (mU/109 RBC’s) in CML patients and in controls
		Mean ± SD	Range	P value
Controls		125.95 ± 8.41	(113.6 - 140.3)	< 0.001*
Group 1 (with remission, n=8)	Before Chemotherapy	283.7 ± 47.63	(210.6 - 360.1)	< 0.001
	After Chemotherapy	221.7 ± 31.71	(182.4 - 268.6)
Group II (without remission, n=12)	Before Chemotherapy	296.33 ± 48.34	(233.7 - 375.3)
	After Chemotherapy	273.63 ± 65.39	(184.9 - 365.3)
*As compared to Group I and Group II

patient without remission. In some studies, red cell G6PD levels were found to be consistently elevated in CML patients as compared to controls^2,5,10 but levels of G6PD have been shown to be decreased in acute leukaemias by some authors.^2,11

Many factors may be contributing towards increased levels of red cell G6PD in CML patients - Increased red cell G6PD activity is thought to be the result of various chromosomal aberrations present in CML patients.^12-14 Valentile et al have proposed that multiple perturbations in genetic material lead to disturbance in the production of several enzymes which may affect the metabolic pathways.¹⁵ It has also been found that post translational modifications are increased with aging as well as various haematological malignancies leading to change in kinetic properties or change in enzyme levels.¹⁶ Rochant et al have proposed that young cells have increased levels of various enzymes including G6PD and these are abundant in CML.¹⁷ Increased oxidative stress with tumour load also explains increased activity of HMP pathway and thus of red cell G6PD.¹⁸ HMP pathway may also be activated by intercellular stress of Leukaemic blasts on red cells leading to temporary inhibition of red cells glycolysis.¹⁹

Thus, estimation of G6PD levels implies a prognostic significance. No studies are available for comparison of results after chemotherapy. So we suggest more research and large sample number to unravel the significance of these findings.

List of Abbreviations
G6PD   Glucose-6-Phosphate Dehydrogenase
RBC      Red Blood Cell
CML      Chronic Myeloid Leukaemia
HMP      Hexose MonoPhosphate
NADP   Nicotinamide Adenine Dinucleotide Phosphate

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