Abstract
Human glutathione transferase T2-2 (GSTT2-2) is one of the enzymes considered to play a role in inactivation of toxicants and carcinogens. The expression level of this enzyme is determined by genetic and environmental factors, which may lead to differences in susceptibility. As a specific assay for GSTT2-2 so far a spectroscopical assay based on GSH-conjugation of menaphthyl sulfate (MSu) was used. This spectrophotometric assay, however, appeared too insensitive to accurately quantify the GSTT2-2 activities in a panel of 20 human liver samples. More recently, expression levels of GSTT2-2 in biological samples are quantified by measuring mRNA levels. Since mRNA-levels do not always correlate well with enzyme activity, a specific and sensitive assay is required. In the present study a highly sensitive high-performance liquid chromatography (HPLC)-based method was developed. By applying the new method, firstly, the specificity of GSTT2-2 among 15 recombinant human GST isoforms in catalyzing GSH-conjugation of MSu was confirmed. In addition, a 65-fold inter-individual variation of GSTT2-2 activity was found from the individual liver fractions. By applying the method to individual liver fractions, a 65-fold inter-individual variation of GSTT2-2 activity was found. As a second application, the role of GSTT2-2 in GSH-conjugation of the environmental carcinogen 1-methylpyrene sulfate (MPS) was studied by correlation analysis with GSTT2-2-catalyzed MSu conjugation. The relatively poor correlation suggested that other GSTs also contribute to MPS-conjugation, as confirmed by incubations with recombinant GSTs.
| Original language | English |
|---|---|
| Pages (from-to) | 181-188 |
| Number of pages | 8 |
| Journal | Journal of Pharmaceutical and Biomedical Analysis |
| Volume | 156 |
| Early online date | 23 Apr 2018 |
| DOIs | |
| Publication status | Published - 15 Jul 2018 |
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Keywords
- 1-Menaphthyl sulfate
- 1-Methylpyrene sulfate
- Conjugation
- Glutathione
- GSTT2-2
- Human liver cytosol
Cite this
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High-performance liquid chromatography-based assay for glutathione transferase theta 2 activity : Application to characterize interindividual variability in human liver fractions. / Zhang, Yongjie; Wijaya, Lukas; Dekker, S.J.; Vermeulen, N.P.E.; Commandeur, J.N.M.
In: Journal of Pharmaceutical and Biomedical Analysis, Vol. 156, 15.07.2018, p. 181-188.Research output: Contribution to Journal › Article › Academic › peer-review
TY - JOUR
T1 - High-performance liquid chromatography-based assay for glutathione transferase theta 2 activity
T2 - Application to characterize interindividual variability in human liver fractions
AU - Zhang, Yongjie
AU - Wijaya, Lukas
AU - Dekker, S.J.
AU - Vermeulen, N.P.E.
AU - Commandeur, J.N.M.
PY - 2018/7/15
Y1 - 2018/7/15
N2 - Human glutathione transferase T2-2 (GSTT2-2) is one of the enzymes considered to play a role in inactivation of toxicants and carcinogens. The expression level of this enzyme is determined by genetic and environmental factors, which may lead to differences in susceptibility. As a specific assay for GSTT2-2 so far a spectroscopical assay based on GSH-conjugation of menaphthyl sulfate (MSu) was used. This spectrophotometric assay, however, appeared too insensitive to accurately quantify the GSTT2-2 activities in a panel of 20 human liver samples. More recently, expression levels of GSTT2-2 in biological samples are quantified by measuring mRNA levels. Since mRNA-levels do not always correlate well with enzyme activity, a specific and sensitive assay is required. In the present study a highly sensitive high-performance liquid chromatography (HPLC)-based method was developed. By applying the new method, firstly, the specificity of GSTT2-2 among 15 recombinant human GST isoforms in catalyzing GSH-conjugation of MSu was confirmed. In addition, a 65-fold inter-individual variation of GSTT2-2 activity was found from the individual liver fractions. By applying the method to individual liver fractions, a 65-fold inter-individual variation of GSTT2-2 activity was found. As a second application, the role of GSTT2-2 in GSH-conjugation of the environmental carcinogen 1-methylpyrene sulfate (MPS) was studied by correlation analysis with GSTT2-2-catalyzed MSu conjugation. The relatively poor correlation suggested that other GSTs also contribute to MPS-conjugation, as confirmed by incubations with recombinant GSTs.
AB - Human glutathione transferase T2-2 (GSTT2-2) is one of the enzymes considered to play a role in inactivation of toxicants and carcinogens. The expression level of this enzyme is determined by genetic and environmental factors, which may lead to differences in susceptibility. As a specific assay for GSTT2-2 so far a spectroscopical assay based on GSH-conjugation of menaphthyl sulfate (MSu) was used. This spectrophotometric assay, however, appeared too insensitive to accurately quantify the GSTT2-2 activities in a panel of 20 human liver samples. More recently, expression levels of GSTT2-2 in biological samples are quantified by measuring mRNA levels. Since mRNA-levels do not always correlate well with enzyme activity, a specific and sensitive assay is required. In the present study a highly sensitive high-performance liquid chromatography (HPLC)-based method was developed. By applying the new method, firstly, the specificity of GSTT2-2 among 15 recombinant human GST isoforms in catalyzing GSH-conjugation of MSu was confirmed. In addition, a 65-fold inter-individual variation of GSTT2-2 activity was found from the individual liver fractions. By applying the method to individual liver fractions, a 65-fold inter-individual variation of GSTT2-2 activity was found. As a second application, the role of GSTT2-2 in GSH-conjugation of the environmental carcinogen 1-methylpyrene sulfate (MPS) was studied by correlation analysis with GSTT2-2-catalyzed MSu conjugation. The relatively poor correlation suggested that other GSTs also contribute to MPS-conjugation, as confirmed by incubations with recombinant GSTs.
KW - 1-Menaphthyl sulfate
KW - 1-Methylpyrene sulfate
KW - Conjugation
KW - Glutathione
KW - GSTT2-2
KW - Human liver cytosol
UR - http://www.scopus.com/inward/record.url?scp=85046152345&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85046152345&partnerID=8YFLogxK
U2 - 10.1016/j.jpba.2018.04.037
DO - 10.1016/j.jpba.2018.04.037
M3 - Article
VL - 156
SP - 181
EP - 188
JO - Journal of Pharmaceutical and Biomedical Analysis
JF - Journal of Pharmaceutical and Biomedical Analysis
SN - 0731-7085
ER -