title: Liquid chromatography-tandem mass spectrometry analysis of erythrocyte thiopurine nucleotides and effect of thiopurine methyltransferase gene variants on these metabolites in patients receiving azathioprine/6-mercaptopurine therapy.
abstract: BACKGROUND
  Polymorphic thiopurine S-methyltransferase (TPMT) is a major determinant of thiopurine toxicity.
  
  
  METHODS
  We extracted 6-thioguanine nucleotides (6-TGNs) and 6-methylmercaptopurine nucleotides (6-MMPNs) from erythrocytes with perchloric acid and converted them to 6-thioguanine (6-TG) and a 6-methylmercaptopurine (6-MMP) derivative during a 60-min acid hydrolysis step. The liquid chromatography system consisted of a C(18) column with an ammonium acetate-formic acid-acetonitrile buffer. 8-Bromoadenine was the internal standard. Analytes were measured with positive ionization and multiple reaction monitoring mode. With PCR-restriction fragment length polymorphism analysis and TaqMan allelic discrimination, common TPMT alleles (*1, *2, *3A, *3B, *3C) were determined in 31 792 individuals. We used perchloric acid extraction, acid hydrolysis, and HPLC with ultraviolet detection to measure erythrocyte 6-TG and 6-MMP nucleotide concentrations in 6189 patients with inflammatory bowel disease receiving azathioprine/6-mercaptopurine therapy.
  
  
  RESULTS
  Intra- and interday imprecision were <10% at low and high analyte concentrations. The conversion of 6-TG and 6-MMP nucleoside mono-, di-, and triphosphates was complete after hydrolysis. Allelic frequency for TPMT variant alleles ranged from 0.0063% (*3B) to 3.61% (*3A). Compared with wild types, TPMT heterozygotes had an 8.3-fold higher risk for 6-TGNs >450 pmol/8 x 10(8) erythrocytes (concentration associated with increased risk for leukopenia), but an 8.2-fold lower risk for 6-MMPNs >5700 pmol/8 x 10(8) erythrocytes (concentration associated with increased risk for hepatotoxicity).
  
  
  CONCLUSIONS
  The liquid chromatography-tandem mass spectrometry method can be applied to the routine monitoring of thiopurine therapy. The association between TPMT genotype and metabolite concentrations illustrates the utility of pharmacogenetics in the management of patients undergoing treatment with thiopurines.
authors.affiliation: Prometheus Laboratories, San Diego, CA 92121, USA. tdervieux@prometheuslabs.com
authors.forename: Thierry
authors.initials: T
authors.lastname: Dervieux
authors.forename: Gary
authors.initials: G
authors.lastname: Meyer
authors.forename: Robert
authors.initials: R
authors.lastname: Barham
authors.forename: Mariko
authors.initials: M
authors.lastname: Matsutani
authors.forename: Mary
authors.initials: M
authors.lastname: Barry
authors.forename: Roselyne
authors.initials: R
authors.lastname: Boulieu
authors.forename: Bruce
authors.initials: B
authors.lastname: Neri
authors.forename: Ernest
authors.initials: E
authors.lastname: Seidman
mesh_terms: D000328:Adult; D001379:Azathioprine / Q000097:blood / Q000627:therapeutic use*; D002853:Chromatography, Liquid; D015331:Cohort Studies; D004359:Drug Therapy, Combination; D004912:Erythrocytes / Q000737:chemistry*; D005260:Female; D014644:Genetic Variation; D006150:Guanine Nucleotides / Q000097:blood; D006801:Humans; D006868:Hydrolysis; D015212:Inflammatory Bowel Diseases / Q000097:blood / Q000188:drug therapy*; D008297:Male; D013058:Mass Spectrometry; D015122:Mercaptopurine / Q000031:analogs & derivatives / Q000097:blood / Q000627:therapeutic use*; D008780:Methyltransferases / Q000235:genetics*; D011685:Purine Nucleotides / Q000097:blood*; D013873:Thionucleotides / Q000097:blood
chemical_list: D006150:Guanine Nucleotides; D011685:Purine Nucleotides; D013873:Thionucleotides; C003964:6-thioguanylic acid; C010240:6-methylthiopurine; D015122:Mercaptopurine; D008780:Methyltransferases; C022745:thiopurine methyltransferase; D001379:Azathioprine