title: LC-MS/MS Analysis of Erythrocyte Thiopurine Nucleotides and Their Association With Genetic Variants in Patients With Neuromyelitis Optica Spectrum Disorders Taking Azathioprine.
abstract: Azathioprine is a first-line drug in treating neuromyelitis optica spectrum disorders (NMOSD). To exhibit its bioactivity, azathioprine needs to be converted to thiopurine nucleotides (TPNs) including 6-thioguanine nucleotides (6-TGNs) and 6-methylmercaptopurine nucleotides (6-MMPNs) that are affected by genetic polymorphisms. This study aims to develop an LC-MS/MS method for the analysis of erythrocyte concentrations of TPNs and to evaluate their associations with variants of various genes (MTHFR, TPMT, HLA, SLC29A1, SLC28A2, SLC28A3, ABCB1, and ABCC4) in patients with NMOSD.
  
  
  
  Erythrocyte 6-TGNs and 6-MMPNs were converted to their free bases 6-thioguanine and 6-methylmercaptopurine derivative by 1-hour acid hydrolysis at 95°C. An LC-MS/MS method was developed, validated, and used to study 32 patients with NMOSD to determine these free bases. Genetic variants were identified by MassARRAY (Sequenom) and multiple SNaPshot techniques. The associations between genetic variants and the concentrations of TPNs or the 6-MMPNs:6-TGNs ratio were evaluated by PLINK software using linear regression.
  
  
  
  Methanol and water were used for separation with a total run time of 6.5 minutes. The lowest limit of quantification was 0.1 μmol/L with an injection volume of 10 μL. rs10868138 (SLC28A3) was associated with a higher erythrocyte concentration of 6-TGNs (P = 0.031), whereas rs12378361 (SLC28A3) was associated with a lower erythrocyte concentration of 6-TGNs (P = 0.0067). rs507964 (SLC29A1) was significantly associated with a lower erythrocyte concentration of 6-MMPNs (P = 0.024) and a lower 6-MMPNs:6-TGNs ratio (P = 0.029).
  
  
  
  An LC-MS/MS method for the analysis of erythrocyte TPNs was developed, validated, and used to study 32 patients with NMOSD. SLC29A1 and SLC28A3 were associated with the erythrocyte concentrations of TPNs and 6-MMPNs:6-TGNs ratio. Further studies are needed to confirm these results.
authors.affiliation: *Department of Pharmacy, Beijing Tiantan Hospital, Capital Medical University; †Department of Clinical Pharmacology, College of Pharmaceutical Sciences, Capital Medical University; and ‡Neuroinfection and Neuroimmunology Center, Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China.
authors.forename: Shenghui
authors.initials: S
authors.lastname: Mei
authors.forename: Xindi
authors.initials: X
authors.lastname: Li
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Gong
authors.forename: Xingang
authors.initials: X
authors.lastname: Li
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.forename: Heng
authors.initials: H
authors.lastname: Zhou
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Liu
authors.forename: Anna
authors.initials: A
authors.lastname: Zhou
authors.forename: Leting
authors.initials: L
authors.lastname: Zhu
authors.forename: Xinghu
authors.initials: X
authors.lastname: Zhang
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Zhao
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001379:Azathioprine / Q000008:administration & dosage / Q000493:pharmacokinetics*; D002648:Child; D002853:Chromatography, Liquid / Q000379:methods*; D004912:Erythrocytes / Q000378:metabolism; D005260:Female; D014644:Genetic Variation; D006150:Guanine Nucleotides / Q000032:analysis; D006801:Humans; D007166:Immunosuppressive Agents / Q000008:administration & dosage / Q000493:pharmacokinetics; D008297:Male; D008875:Middle Aged; D009471:Neuromyelitis Optica / Q000188:drug therapy* / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism; D011110:Polymorphism, Genetic; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013873:Thionucleotides / Q000032:analysis; D055815:Young Adult
chemical_list: D006150:Guanine Nucleotides; D007166:Immunosuppressive Agents; D009711:Nucleotides; D013873:Thionucleotides; C003964:6-thioguanylic acid; D001379:Azathioprine