Profile study of some oxidant and antioxidant levels in leukemic patients

Objective: The aim of this study is to evaluate the oxidant and antioxidant level in cancer patients in Basrah during the period from the first of October 2002 till the end of April of the 2003. Material and methods:A total of 64 patients were investigated, who were admitted to the Basrah General Hospital, Teaching Hospital and Basrah Hospital for Maternity and Pediatric in Basrah City. 125 sex and agematched persons without malignancy served as controls. They were 64 persons as control for leukemic patients (32 male and 32 female). The parameters measured were glutathione (GSH) level, uric acid, malondialdehyde (MDA) and phagocytic functional activity. The leukemic patients were sub classified into two groups according to their age (30 patients ≤16 years and 16 patients >16 years). Results: The biochemical investigation showed a high significant depletion in GSH levels in leukemic patients (P<0.001). The phagocytic activity was high significantly decreased in all leukemic patients (P<0.001). There was no significant difference between male and female leukemic patients in phagocytic activity. The MDA level was highly significantly increased in 16 years old leukemic patients (P<0.01) and extremely high significant in >16 years old leukemic patients (P<0.001). The investigation of uric acid concentration shows extremely high significant increase in all leukemic patients (P<0.001). Conclusion: cancer patients suffer a high degree of ROS formation causing considerable oxidative stress which associated with decrease glutathione level and significant degree of lipid peroxidation. .(MJBU,30,2: 2012, Page 115-121) ىوتسم ةسارد ايميكوللا ىضرم يف دسكأتلل ةداضملاو ةدسكؤملا لماوعلا ضعب يبعلا ةياهن.د يد 1 ةمعنلا ىفطصم ءايمل.د.أ ، 2 يمشاهلا يلع.د.أ و 3 ( 3،1 ) و ةرصبلا ةعماج/بطلا ةيلك/ ةجلسفلا عرف 2 ةرصبلا ةعماج/بطلا ةيلك/ةيتايحلا ءايميكلا عرف فدهلا ىوتسم ميقتل ةساردلا هذه تيرجأ: و ةدسكؤملا لماوعلا نيرشت نم لولأا نم ةرتفلا للاخ ةرصبلا يف ناطرسلا ىضرم دنع دسكأتلل ةداضملا لولأا 2002 ناسين ةياهن ىلإ 2003 . :ةساردلا ةقيرط هعومجم ام ةعباتم تمت 89 ماعلا ةرصبلا ىفشتسم ىلإ اولخدا نيذلا ىضرملا نولثميو اضيرم لاو لفطلاو ةدلاولل ةرصبلا ىفشتسمو يميلعتلا ىفشتسم . ىضرملا ءلاؤه نم 64 باصم اضيرم ( مدلا ضاضيباب ا 26 اركذ 22 ىثنأ ) . ةفاضإ ىلإ 121 ىوتسم ةيتايحلا ةيئايميكلا تاصوحفل تلمش .سنجلاو رمعلا قفاوت ةاعارم عم ةطباض ةنيع مهفصوب مهرايتخا مت ناطرسلاب باصم ريغ اصخش ويثاتولغلا ( يئانثلا نولاملا دياهيدلاو كيرويلا ضماحو لزتخملا ن MDA ىلع اًدامتعا نيتعومجم ىلا مدلا ضاضيبا ىضرم مسق .ةيمعلبلا ةيلاعفلاو ) ( مهرامعأ 30 رمعب اًضيرم 14 و لقا وا ةنس 14 نم رثكا رمعب اًضيرم 14 .)ةنس جئاتنلا : ترهظأ لاع اًدتعم اًصقن ةيتايحلا ةيئايميكلا تارابتخلاا ( ضاضيبا ىضرم دنع لزتخملا نويثاتولغلا ىوتسم يف ايئاصحإ P<0.001 ) لاع دتعم لكشب ءاضيبلا مدلا ايلاخل ةيمعلبلا ةيلاعفلا تلق دقو . ( مدلا ضاضيبا ىضرم لك دنع ايئاصحإ P<0.001 صخي امب مدلا ضاضيبا ىضرم يف ثانلإاو روكذلا نيب ايئاصحإ دتعم قرف كانه نكي ملو.) بلا ةيلاعفلا ( يئانثلا نولاملا دياهيدلا ىوتسم نإ .ءاضيبلا مدلا ايلاخل ةيمعل MDA مدلا ضاضيبا ىضرم دنع ايئاصحإ لاع دتعم لكشب رثكأ ناك ) ةيرمعلا ةئفلل 14 ( لقاف ةنس P<0.01 مدلا ضاضيبا ىضرم لك يف ايئاصحإ اًدج لاع دادتعاب ةدايز كيرويلا ضمح رابتخا رهظأو .) ( P<0.001 .) :جاتنتسلاا إ ًادسكؤم اًدهج ببست يتلا ةطشنلا نيجسكولأا روذج نوكت نم ةيلاع ةجردب نوناعي ناطرسلا ىضرم نأ ترهظأ ةساردلا ن .نوهدلا ةدسكأ نم ةمهم ةجرد عمو نويثاتولغلا ىوتسم ةلق عم ًاطبترم ًامهم MJBU, VOL 30, No.2, 2012___________________________________________________________________________________________________ 116 INTRODUCTION ll living cells are prone to oxygen toxicity. This toxicity has been related to the intermediates of oxygen reduction referred to as reactive oxygen species (ROS) which include the super oxide anion radical (O2), hydrogen peroxide (H2O2), and hydroxyl radical (OH ). [1] These may lead to many human degenerative disease, such as atherosclerosis, certain types of cancer and cataract. [2-4] ROS are also formed during non enzymatic processes, for example, exposure to sunlight and ionizing radiation. [1] The MDA – modified proteins are potentially as deleterious as free MDA, and could be involved in aging as well as in degenerative complications of diseases with increased oxidative stress such as diabetes mellitus, atherosclerosis and cancer. [5] Serum MDA levels are increased in leukaemia and are higher in the active phase of disease as compared to those in remission, hence, serum MDA estimation in leukaemia can be of help in diagnosis and to predict the chances of relapse. [5,6] The present study aims to determine oxidant and antioxidant level of patients with leukemia. 1. Measurement of phagocytic activity by as oxidant by chemiluminesence (CL) in blood of leukemic patients to compare with those of their control. 2. Measurement of malondialdehyde as a biomarker of lipid peroxidation in blood of patients in comparism with control. 3. Measurement of uric acid and reduced glutathione as antioxidants in blood of patients in comparism with control. PATIENTS AND METHODS Patients Forty-six patients with leukemia, they were 22 females and 24 males; their age ranged from 1.5 to 50 years old. Control group for leukemic patients group; they were 64 persons include 32 females and 32 males; their age ranged from 2 to 45 years old having 32 with +ve and 32 with ve family history for leukemia. Data collections: From each subject full information were obtained using a questionnaire list that included name, age, sex, occupation, as well as the following questions about: 1. Medical history for any previous and recent illness and their type of treatment. 2. Family history for any malignant condition. 3. Social history for smoking and drinking habits. Physical and radiological examinations were done including U/S and X-ray.


INTRODUCTION
ll living cells are prone to oxygen toxicity. This toxicity has been related to the intermediates of oxygen reduction referred to as reactive oxygen species (ROS) which include the super oxide anion radical (O 2 ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (OH -). [1] These may lead to many human degenerative disease, such as atherosclerosis, certain types of cancer and cataract. [2][3][4] ROS are also formed during non enzymatic processes, for example, exposure to sunlight and ionizing radiation. [1] The MDAmodified proteins are potentially as deleterious as free MDA, and could be involved in aging as well as in degenerative complications of diseases with increased oxidative stress such as diabetes mellitus, atherosclerosis and cancer. [5] Serum MDA levels are increased in leukaemia and are higher in the active phase of disease as compared to those in remission, hence, serum MDA estimation in leukaemia can be of help in diagnosis and to predict the chances of relapse. [5,6] The present study aims to determine oxidant and antioxidant level of patients with leukemia. 1. Measurement of phagocytic activity by as oxidant by chemiluminesence (CL) in blood of leukemic patients to compare with those of their control. 2. Measurement of malondialdehyde as a biomarker of lipid peroxidation in blood of patients in comparism with control. 3. Measurement of uric acid and reduced glutathione as antioxidants in blood of patients in comparism with control.

Patients
Forty-six patients with leukemia, they were 22 females and 24 males; their age ranged from 1.5 to 50 years old. Control group for leukemic patients group; they were 64 persons include 32 females and 32 males; their age ranged from 2 to 45 years old having 32 with +ve and 32 withve family history for leukemia.

Data collections:
From each subject full information were obtained using a questionnaire list that included name, age, sex, occupation, as well as the following questions about: 1. Medical history for any previous and recent illness and their type of treatment. 2. Family history for any malignant condition. 3. Social history for smoking and drinking habits. Physical and radiological examinations were done including U/S and X-ray.

Blood samples:
Blood samples (5ml) were collected by venopuncture using a sterile disposable syringe in a heprinized tubes, (4ml) of this blood was centrifuged; the plasma was transferred to sample tube and stored at (-4c) until analyzed. While the rest fresh (1ml) of whole blood used for determination of the glutathion concentration and phagocytic activity.

Biochemical parameters: Determination of glutathione (GSH):
The GSH determined according to method [7] The GSH concentration was obtained by using the following equation:

Estimation of malondialdehyde (MDA):
MDA, formed from the breakdown of polyunsaturated fatty acids, serves as a convenient index for determining the extent of the peroxidation reaction. The thiobarbituric acid assay of Buege and Aust (1978) was used to measure the (MDA). [8] Principle: MDA has been identified as the product of lipid peroxidation by its reaction with thiobarbituric acid to give a red species absorbing at 535nm. The absorbance of the sample was determined at 535nm against a A
Calculation: MDA = (ΔA /1.56) X 10 mol/L For accuracy and precision, the intra-coefficient of variation were estimated to be 1.6% and 6.0% respectively.

Chemiluminescence (CL):
CL was taken to indicate that phagocytes were generating singlet oxygen during the respiratory burst. [9] So the CL was taken to determine meyloperoxidase activities an indicator for leucocytes phagocytic activity.
Principle: [10] The luminol-amplified chemiluminescence activity can be simplified by the following formula Peroxidase Luminol + reactive oxygen species aminophtalate + N2 + light (O2 and H2O2) catalyst Chemiluminescence inducer and measurement: [11][12][13] Calculations The area under CL kinetic curve represented the granulocytes functional activity. The peak high of CL kinetic curve represent the functional activity. The granulocytes functional activity yield were estimated using the formula below:

Peak heights Functional activity yield =------------------------------------Number of granulocytes
Enzymatic determination of uric acid: [14,15] Calculation: The results were expressed as meanSD. The comparisons between groups were performed with analysis of variance (ANOVA). While correlation was assessed by the Pearson correlation coefficient (r) using computerized SPSS program (Statistical Program for Social Sciences). P<0.05 was considered to be the lowest limit of significance. Glutathione: GSH levels in different age groups and each sex in leukemic and their control group are shown in (Table-2). The GSH concentration in blood of leukemic patients was lower than their control group with significant difference (P<0.001).

Table 2. GSH levels in different age groups and sex in leukemic and control subjects.
Values expressed as XSD E.S: Extremely significant (P<0.001)

Malondialdehyde:
The MDA levels in leukemic and their control group with respect to age group and sex are shown in (Table-3). MDA levels in leukemic patients are higher than those of control subjects with highly significant difference in age group 16 (P<0.01) and with extremely significant difference in age group >16 (P<0.001). The MDA levels in females patients are significantly higher than their controls (P<0.05), but significantly higher in males leukemics than their controls (P<0.01).

DISCUSSION
The study was designed to measure the oxidant and antioxidant levels in leukemic patients. The study showed depletion of reduced glutathione concentration (the major endogenous antioxidant) in leukemic patients as shows in (Table-2), these results were in agreement with other studies. [16,17] There can be two reasons for GSH depletion in cancer. Firstly; elevated glutathione peroxidase will use more GSH in an attempt to cope with the excessive production of oxyradicals as revealed by elevated lipid peroxidation. Secondly, if little replenishment of GSH occurred, the level of GSH would become lower. [18,16,19,20] Uric acid with powerful antioxidant properties was proposed to play a key role in the antioxidant protection in humans; it is a very effective scavenger such that it is as effective as ascorbic acid. [21] The present study shows an increase in plasma urate concentration of leukemic patients. This results were in agreement with other studies, [22][23][24][25] but in disagreement with observation of others. [26] High plasma urate concentration may be caused by increased turnover of nucleic acids due to rapidly growing malignant tissue, especially in leukemia's, or may caused by increased tissue breakdown by the treatments of cancer. [27] This study revealed a significant elevation of MDA among all groups of patients. This implies that cancer patients are exposed to a considerable degree of lipid peroxidation. This finding in consistent with observations of others. [5,6,22,[28][29][30] The cause of elevated MDA level in cancer patients may be the free radicals (which increased in cancer) are associated with an impaired antioxidant defense enzyme activity resulting in increased lipid peroxidation. [31] The principle phagocytes are neutrophils and monocytes because the melonperoxidase (MPO) is more abundante in these cells. [32] In this study, phagocytic activity was reduced significantly in leukemic patients. This result in agreement with other studies [33] and in disagreement with others. [34] This may be due to neutropenia, which is the most important deficiency phenomenon in acute leukemia. [35] Cancer chemotherapy may also produce defects in neutrophil function, and combination of some drugs can produce a significant decrease in the phagocytic killing capability. [36] Other study suggested that the H 2 O 2 inhibits the ability of different antineoplastic chemotherapeutic drugs to induce the phagocytosis. [37] In conclusions, leukemic patients suffer from a significant degree of free radical formation as indicated by the significantly higher MDA level among those patients. As a result of continuous production of ROS, the level of GSH is depleted. Elevated urate concentration in cancer patients caused by increase of nucleic acid turnover. The WBC phagocytic activity was reduced in leukemic patients due to neutropenia. No significant difference in the level of GSH, uric acid, MDA and phagocytosis activity in patients with leukemia regarding to age and sex of those patients.