Application of HPLC to study the kinetics of a branched bi-enzyme system consisting of hypoxanthine-guanine phosphoribosyltransferase and xanthine oxidase--an important biochemical system to evaluate the efficiency of the anticancer drug 6-mercaptopurine in ALL cell line.
The thiopurine antimetabolite 6-mercaptopurine (6MP) is an important chemotherapeutic drug in the conventional treatment of childhood acute lymphoblastic leukemia (ALL). 6MP is mainly catabolized by both hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine oxidase (XOD) to form thioinosinic monophosphate (TIMP) (therapeutically active metabolite) and 6-thiouric acid (6TUA) (inactive metabolite), respectively. The activity of both the enzymes varies among ALL patients governing the active and the inactive metabolite profile within the immature lymphocytes. Therefore, an attempt was made to study the kinetic nature of the branched bi-enzyme system acting on 6MP and to quantitate TIMP and 6TUA formed when the two enzymes are present in equal and variable ratios. The quantification of the branched kinetics using spectrophotometric method presents problem due to the closely apposed lambda(max) of the substrates and products. Hence, employing an HPLC method, the quantification of the products was done with the progress of time. The limit of quantification (LOQ) of substrate was found to be 10nM and for products as 50 nM. The limit of detection (LOD) was found to be 1 nM for the substrate and the products. The method exhibited linearity in the range of 0.01-100 microM for 6MP and 0.05-100 microM for both 6TUA and TIMP. The amount of TIMP formed was higher than that of 6TUA in the bi-enzyme system when both the enzymes were present in equivalent enzymatic ratio. It was further found that enzymatic ratios play an important role in determining the amounts of TIMP and 6TUA. This method was further validated using actively growing T-ALL cell line (Jurkat) to study the branched kinetics, wherein it was observed that treatment of 50 microM 6MP led to the generation of 12 microM TIMP and 0.8 microM 6TUA in 6 h at 37 degrees C.
Kalra
Sukirti
S
Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector 67, Phase X, S.A.S. Nagar, Mohali, Punjab 160062, India. skalra11@yahoo.com
Paul
Manash K
MK
Balaram
Hemalatha
H
Mukhopadhyay
Anup Kumar
AK
D000293:Adolescent; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000378:metabolism*; D008297:Male; D015122:Mercaptopurine / Q000627:therapeutic use*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods; D014969:Xanthine Oxidase / Q000378:metabolism*
D000970:Antineoplastic Agents; D015122:Mercaptopurine; D014969:Xanthine Oxidase; D007041:Hypoxanthine Phosphoribosyltransferase