Biochemical and Cytochemical Studies of Blood Esteratic Profile in Cancer Patients

Esterases are enzymes capable of hydrolyzing the simpler esters of N-free alcohols and organic acids. The majority of esterase enzymes are able to hydrolyze a synthetic ester substrate that is -naphthyl acetate; so they are called non-specific esterases. Nonspecific esterase activity was visualized from various types of cancer cells, where their activity showed to decrease in association with different tumor types. Aim; The aims of this study were to establish the normal esteratic profile in blood biochemically and in monocytes and lymphocytes cytochemically, study the esteratic pattern and the isoenzymatic state of esterases in patients with cancer, and characterization of esterase-substrate reaction using kinetic and thermodynamic parameters. Materials; All chemicals and standard solutions used in this work of the highest analytical grade, obtained from commercial sources and used without further purification. Hexazotized pararosaniline dye, phosphate buffer, -naphthyl acetate, polyacrylamide gel, and Biuret kit were used. Patients and Methods; Ninety seven patients with proven malignancy were investigated from Al- Kadhimiya Teaching Hospital / Department of Oncology, in Baghdad during the period from January 2004 till October 2005. Thirty six patients were diagnosed with adenocarcinoma of the breast, twenty nine patients with adenocarcinoma of the bronchus, twenty one patients with lymphoma - including Hodgkin's and
non-Hodgkin's lymphoma -, and eleven patients with adenocarcinoma of the colon), and compared with twenty normal male and female individuals. The samples were studied at the Departments of Physiological Chemistry and Human Anatomy, College of Medicine, Al-Nahrain University. The isoelectric focusing electrophoresis was used for studying the esteratic pattern in normal and cancer patients using slab gel and cylindrical gel techniques. The images generated by electrophoresis were processed using PhotoCaptMwt Software, and used in determination of enzyme units in standard carboxylesterase (E.C. 3.1.1.1) and all studied individuals. Membrane-associated human monocyte carboxylesterase was extracted from peripheral blood sample of breast cancer patient, by using monocyte isolation by adherence method. Spectrophotometric method was used to study the reaction of esterase enzyme with -naphthyl acetate substrate, and Michaelis-Menten constant (KM) and maximum velocity (Vmax) were determined. The thermodynamic parameters (DH, DS, and DG) of the reaction were determined. Blood smears were taken from both control and patients groups, and stained with azo-coupling dye (hexazotized pararosaniline) using -naphthyl acetate esterase as a substrate. Monocyte nonspecific esterase activity was quantitatively measured using microspectrophotometry technique. Results; The isoelectric point of the plasma fraction for the patient groups showed higher values (pH 7.2 – 7.5), while it was 6.3 in control group (male and
female). Lysate fraction for all patient groups showed band of pH ranges from 5.3 – 6.3, 8.2 – 9, and 9 – 9.8.
The calibration curve generated using this software gave a straight line equation with regression (R2) value 0.9952, and a relative standard deviation (RSD) 0.05 for peak height. Esterase enzyme units showed a significant lowering between control and patients groups (p 0.05) at a confidence limit 95 %. Isoelectric focusing electrophoresis of the extracted membrane-associated human monocyte carboxylesterase from peripheral blood sample of breast cancer patient gave two bands at pH 7.0 and 7.2, which may represent two isoenzymes. The specific activity of those two isoenzymes showed higher values compared with control group with a purification fold of 21 and 22 times for the two bands. Michaelis-Menten constant (KM) and maximum velocity (Vmax) were determined using Lineweaver-Burk plot at different temperatures. Esterase
enzymes in patient groups showed lower affinity Vmax towards substrate than the control group. Van’t-Hoff equation was used for determination of thermodynamic parameters. The results showed positive enthalpy that indicates an endothermic reaction, small entropy participation and spontaneous reaction in control and
patients groups. The mean of the measured optical density of the final reaction product of monocytes -naphthyl acetate esterase was; in control group (1787.4 ± 12.8), in adenocarcinoma of the breast (809.3 ± 8.9), in adenocarcinoma of the bronchus (906.3 ± 16.1), in adenocarcinoma of the colon (890.3 ± 12.7), and in lymphoma (1249.2± 54.2). Statistical analysis of variance (ANOVA) showed a significant difference between normal individuals and the disease group. This indicated that there is a significant decrease in the monocytes -naphthyl acetate esterase activity in neoplastic diseases than in normal control group.