A Practical Non-Extraction Direct Liquid Chromatography Method for Determination of Thiopurine S-Methyltransferase Activity in Inflammatory Bowel Disease
Thiopurine drugs remain pivotal therapies for the wide varieties of diseases such as inflammatory bowel disease (IBD). Here, thiopurine S-methyltransferase (TPMT) phenotype, the main metabolizing enzyme of thiopurine-drugs, was studied. This is for the first time that TPMT activity is measured in Iranian IBD patients. We used an improved direct liquid chromatography assay without need for solvent extraction and minimize excess labor handling making it ideal for use in routine referral medical centers. TPMT activity in whole blood was determined by a non-extraction HPLC method. We evaluated 427 individuals including 215 IBD patients and 212 unrelated healthy individuals as control group from Iran’s western population. TPMT phenotyping of this study demonstrated no frequency for deficient, 2.8 % for low and 97.2% for normal activity, which is different with results of other studies. There was a significant negative correlation between TPMT activities as calculated based on nmol/grHb/h and the Hb-levels in IBD and control groups (r= -0.54, P<0.001 and r= -0.27, P<0.001), respectively. Interestingly a significant positive correlation between Hb levels and TPMT-activities were seen when the activity calculated in mU/L in IBD patients and control subjects (r=0.14, P=0.05 and r=0.43, P<0.001), respectively. We strongly suggest the use of international unit (mU/L) is more appropriate than nmol6MTG/grHb/h for expressing TPMT-activity in IBD patients. In addition, in comparison with other providers of TPMT test activity and centers around the world the risk of toxicity is much lower after utilizing thiopurine drugs for IBD patients in this region.
Al Hadithy A, De Boer N, Derijks L, Escher J, Mulder C,Brouwers J. Thiopurines in inflammatory bowel disease:pharmacogenetics, therapeutic drug monitoring andclinical recommendations. Digestive and liver disease.2005;37:282-97.
Lennard L. The clinical pharmacology of 6-mercaptopurine. Eur J Clin Pharmacol 1992;43:329-39.
Lowry P, Franklin C, Weaver A, Pike MG, Mays D,Tremaine W, et al. Measurement of thiopurine methyltransferase activity and azathioprine metabolites inpatients with inflammatory bowel disease. Gut2001;49:665-70.
Sandborn WJ, Tremaine WJ, Wolf DC, Targan SR,Sninsky CA, Sutherland LR, et al. Lack of effect of intravenous administration on time to respond toazathioprine for steroid-treated Crohn's disease.Gastroenterology 1999;117:527-35.
Graham V. Thiopurine methyltransferase phenotypingand genotyping in clinical practice preferred accessarrangement: University of Birmingham; 2010.
Darakhshan F, Khojeini EV, Balaii H, Naderi N, FirouziF, Farnood A, et al. Epidemiology of nflammatoryBowel Disease in Iran: A review of 803 cases.Gastroenterol Hepatol Bed Bench 2009;1:19-24.
McLeod HL, Siva C. The thiopurine S-methyltransferasegene locus-implications for clinical pharmacogenomics.Pharmacogenomics 2002;3:89-98.
Holme S, Duley J, Sanderson J, Routledge PA, AnsteyAV. Erythrocyte thiopurine methyl transferase assessmentprior to azathioprine use in the UK. QJM 2002;95:439-44.
Evans WE, Horner M, Chu YQ, Kalwinsky D, RobertsWM. Altered mercaptopurine metabolism, toxic effects,and dosage requirement in a thiopurine metyltransferasedeficientchild with acute lymphocytic leukemia. J Pediatr1991;119:985-9.
O'Kane DJ, Weinshilboum RM, Moyer TP.Pharmacogenomics and reducing the frequency ofadverse drug vents.Pharmacogenomics 2003;4:1-4.
Ossaily S, Zgheib NK. The pharmacogenetics of drugmetabolizing enzymes in the Lebanese population. DrugMetab Drug Interact 2014;29:81-90.
Szumlanski CL, Honchel R, Scott MC, WeinshilboumRM. Human liver thiopurine methyltransferasepharmacogenetics: biochemical properties, livererythrocytecorrelation and presence of isozymes.Pharmacogenet Genomics 1992;2:148-59.
Ford LT, Berg JD. Determination of thiopurine Smethyltransferaseactivity in erythrocytes using 6-thioguanine as substrate and a non-extraction liquidchromatographic technique. J Chromatogr B2003;798:111-5.
Kröplin T, Weyer N, Gutsche S, Iven H. Thiopurine methyltransferaseactivity in human erythrocytes: a newHPLC method using 6-thioguanine as substrate. Eur JClin Pharmacol 1998;54:265-71.
Kröplin T, Fischer C, Iven H. Inhibition of thiopurine Smethyltransferaseactivity by impurities in commerciallyavailable substrates: a factor for differing results ofTPMT measurements. Eur J Clin Pharmacol1999;55:285-91.
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