فصل انزيم اللايبوكسيجينيز من مصل الرجال الاصحاء ومعاملة خارجية للانزيم في مرضى الربو == Separation of Lipoxygenase from Sera of Healthy men and in Vitro Treatment of Enzyme in Asthmatic Patients
Author name:
احمد جياد عباس البرقعاوي
Supervisor name:
عودة مزعل ياسر الزاملي
General topic:
Chemistry
Specific topic:
Analytical Biochemistry
Degree:
Doctorate
University:
University of Babylon - College Of Science
Language:
English
University location:
Babylon
First pages:
25T1544 - p.pdf
Abstract:
This study is concerned with asthma. Sixty seven asthmatic male patients were examined and tested , their age ranged from 10 - 65 years and they previously diagnosed as asthmatic patient under the physician supervision in AL - Hilla center of Allergy and Asthma . They have the disease for more than five years. Forty patients were examined during the period between July2015 to October 2015, and twenty seven patients were examined during period between June to August 2016, also the control group consisted of sixty seven healthy males ranged from 10 - 65 years.In this study asthmatic patients were divided into three groups according to medication , the first group consisted of (17) asthmatic patients taking antihistamines such as loratadine , citrizine and allermine , while the second group included (24) asthmatic patients taking montelukast , and the third group included (26) asthmatic patients who were on different drugs other than antihistamines and montelukast such as steroids and beta agonists .Five milliliters of blood were obtained from each patient, serum collected absolutely free from hemolysis stored at - 20 °C until uses for measurement of the concentrations of Glutathione, Malondialdehyde, Catalase, Histamine, Serotonin, Lipoxygenase - 5, Leukotriene A4 Hydrolase, total Immunoglobulin E and specific immunoglobulin E.The results revealed that the mean of IgE level (measured by ELISA system)in serum of asthmatic group was (368.16 ±1.772 ng/ml) versus the control group (138.9±2.515 ng/ml) with a significant difference,)P < 0.001(. Asthmatic patients and patients treated with pharmacological drugs such as montelukast and antihistamines showed high sensitivity to some food, animals, plant, palm pollen, fungal and mite allergen and they showed different values between patients and control. The study showed that there is no association between the increased sensitization to food, animal, plant and fungal allergens with age of patients.The present study showed the relationship of count blood cells (WBC and RBC) in studied groups. In all studied groups, WBC values higher than control group while the RBC values showed little differences among all studied groups.According to levels of some variables (histamine, serotonin, LOX - 5 and LTA4H) in asthmatic patients, the results showed a higher mean value of serotonin in asthmatic patients (0.023± 0.004 ng /ml) compared with control group (0.002± 0.001 ng/ml, p˂0.001) In addition the value of histamine was observed high in asthmatic patients (8.511± 0.410 ng /ml)while the control group was (2.565 ± 0.140 ng/ ml) with a significant difference of the mean value of histamine between asthmatic patients and control group (p˂0.001). LOX - 5 level in serum of asthmatic patients was higher (11.080 ± 0. 190 ng/ml) than in control group (9.821 ± 0.203 ng/ml, p˂0.001.The data revealed significant high mean value of LTA4H in asthmatic patients (24.148 ± 0 .450 ng/ml) compared with control group (18.912 ± 0.459 ng/ml, p˂0.001).The result showed that histamine level was increased in the group (>40 years) with a mean value (9.67 ± 4.25 ng /ml ) . While LOX - 5 showed high mean value (11.532 ± 1.274) in the group 20 - 40 years . Also LTA4H enzyme showed high mean value (25.54 ± 3.504 ng/ml)in the group 20 - 40 years , while serotonin appeared with high mean value (0.027± 0.015 ng/ml) in the group (< 20 years) with a significant difference between levels of these variables in each age group (p ˂ 0.001)The mean concentration of an antioxidant, Glutathione(GSH), and Malondialdehyde (MDA) in serum of asthmatic patient and control groups. The mean concentration of MDA increased in asthmatic patients (11.317 ± 1.096 μmol/l) more than control group(8.733 ± 3.756 μmol/l)with a significant difference between these groups (p < 0.05). The mean concentration of GSH increased was in the control (5.106 ± 3.197 μmol/l) more than asthmatic patients(3.905 ± 1.638 μmol/l, p < 0.05).According to the results of the present study, the mean activity of catalase(CAT) was higher (1.555±0.982 U/l) in asthmatic patients when compared with control group(1.173 ± 0.502 U/l, p < 0.05).The current study also concerned with the extrection of lipoxygenase from serum of asthmatic patient and healthy men by taking 15 ml serum from ten asthmatic patients men and 15 ml from serum of ten healthy men. And these quantities of serum were precipitated by ammonium sulfate, The best ratio for precipitating serum and extraction of enzyme is 60%. The anion exchange chromatography was used for extraction of the enzyme. The molecular weight of the extracted enzyme was determined by SDS - PAGE . In this procedure one subunit of the extracted LOXs enzyme showed a molecular weight of 35KD for one chain while a recombinant pure human LOX - 5 showed a molecular weight of 38 KD. The current study used NATIVEN technique for purification of the extracted enzyme obtained from ion exchange chromatography.The results revealed the optimum pH and temperature for LOXs activity and they were 8 and 40°C, respectively.The velocity constant and maximum velocity of the extracted Lipoxygenase enzyme was detected by using PHYWE SYSTEME GMBH SYSTEM, this system had the ability to obtain the relationship between conductivity and concentration (an increase in concentration lead to an increase in conductivity).The extracted lipoxygenase enzyme Km was 2.2mM and Vmax= 140mM/min in asthmatic patients and Km was 2.7mM and Vmax= 135mM/min in controls (by applying Michaelis - Menten equation).While the enzyme Km= 5.5mM and Vmax= 238mM/min in asthmatic patients and Km= 5 mM and Vmax= 208 mM/min in controls (by applying Lineweaver - Burk equation).The extracted LOXs from asthmatic patients and controls was treated with montelukast. The Km and Vmax appeared to have low values Km 3.6mM and Vmax 153mM/min and Km 3.7mM and Vmax 147mM/min, respectively. Also the extracted LOXs from asthmatic patients and controls was treated with molsiodomine, showed low values Km 4.4mM and Vmax 182 mM/min and Km 4.3mM and Vmax 166 mM/min, respectively.
