Evaluation of mutation effects on UGT1A1 activity toward 17beta-estradiol using liquid chromatography-tandem mass spectrometry.
Mutations in the gene encoding UDP-glucuronosyltransferase 1A1 (UGT1A1) may reduce the glucuronidation of estradiol, bilirubin, etc. In the present study, we used a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method to assay the activities of recombinant mutated UGT1A1 toward 17beta-estradiol (E2), by determining its glucuronide (E2G) content. Direct evidence for glucuronide formation was provided by E2G-specific ion peaks. The UGT1A1 activities of G71R (exon 1), F83L (exon 1), I322V (exon 2) and G493R (exon 5) mutants were 24, 30, 18 and 0.6% of the normal UGT1A1 activity, respectively. In conclusion, our study showed that LC/MS/MS enabled accurate evaluation of the effects of mutations on recombinant UGT1A1 activity towards E2.
Wada
Keiko
K
Department of Public Health, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
Takeuchi
Atsuko
A
Saiki
Kayoko
K
Sutomo
Retno
R
Van Rostenberghe
Hans
H
Yusoff
Narazah Mohd
NM
Laosombat
Vichai
V
Sadewa
Ahmad Hamim
AH
Talib
Norlelawati Abdul
NA
Yusoff
Surini
S
Lee
Myeong Jin
MJ
Ayaki
Hitoshi
H
Nakamura
Hajime
H
Matsuo
Masafumi
M
Nishio
Hisahide
H
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004958:Estradiol / Q000737:chemistry / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D015394:Molecular Structure; D050505:Mutant Proteins / Q000378:metabolism; D020125:Mutation, Missense / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D000099148:UGT1A1 Enzyme
D004958:Estradiol; D014453:Glucuronosyltransferase; D050505:Mutant Proteins; D011994:Recombinant Proteins; D000099148:UGT1A1 Enzyme