PMID- 11305778
OWN - NLM
STAT- MEDLINE
DCOM- 20010712
LR  - 20190906
IS  - 0340-5761 (Print)
IS  - 0340-5761 (Linking)
VI  - 74
IP  - 12
DP  - 2001 Feb
TI  - High-performance liquid chromatography/fluorescence detection of 
      S-methylglutathione formed by glutathione-S-transferase T1 in vitro.
PG  - 760-7
AB  - Glutathione-S-transferase T1 (GSTT1-1) is a major isoenzyme for the 
      biotransformation of halomethanes. The enzyme activity is located, among other 
      places, in human liver and erythrocytes and is subject to a genetic polymorphism. 
      Metabolism of the halomethanes via GSTT1-1 yields S-methylglutathione (MeSG). A 
      new HPLC assay for the enzymatic formation of MeSG was developed. The glutathione 
      conjugate was derivatized with 9-fluorenylmethyl chloroformate, followed by 
      reverse-phase HPLC with gradient elution and fluorescence detection. The limit of 
      detection was as low as about 39 pmol MeSG on-column. Including derivatization 
      and HPLC analysis, samples could be run at 42-min intervals, thus enabling a high 
      sample throughput. The entire method was validated for analyte recovery (78.2%) 
      and for variations in detector response with replicated injections (11.8%) and 
      with analyses on each of 11 consecutive days (15.2%) with erythrocyte lysate 
      incubations as the matrix. The time-, protein-, and substrate-dependences of the 
      enzymatic catalysis with the model substrates methyl bromide (MeBr) and methyl 
      chloride (MeCl) were studied. Due to its strong electrophilic character, MeBr 
      caused a high level of spontaneous MeSG formation from glutathione in a 
      protein-free medium and a substrate-trapping side reaction in the presence of 
      proteins. Therefore, enzymatic MeSG formation rates may only be determined with 
      MeBr concentrations of at least 3000 ppm in the presence of limited amounts of 
      protein (e.g. 100 microl erythrocyte lysate). In contrast, MeCl showed a lower 
      alkylating potential allowing enzymatic catalysis to be the dominant reaction in 
      incubations with 10,000 ppm MeCl and 2 ml erythrocyte lysate.
FAU - Muller, M
AU  - Muller M
AD  - Abteilung Arbeits- und Sozialmedizin, Georg-August-Universitat Gottingen, 
      Waldweg, Germany. mmuelle3@gwdg.de
FAU - Voss, M
AU  - Voss M
FAU - Heise, C
AU  - Heise C
FAU - Schulz, T
AU  - Schulz T
FAU - Bunger, J
AU  - Bunger J
FAU - Hallier, E
AU  - Hallier E
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Germany
TA  - Arch Toxicol
JT  - Archives of toxicology
JID - 0417615
RN  - 0 (Hydrocarbons, Brominated)
RN  - 9V42E1Z7B6 (methyl bromide)
RN  - A6R43525YO (Methyl Chloride)
RN  - EC 2.5.1.- (glutathione S-transferase T1)
RN  - EC 2.5.1.18 (Glutathione Transferase)
RN  - GAN16C9B8O (Glutathione)
RN  - OOW3025SR1 (S-methyl glutathione)
SB  - IM
MH  - Chromatography, High Pressure Liquid/*methods
MH  - Erythrocytes/metabolism
MH  - Glutathione/analogs & derivatives/*metabolism
MH  - Glutathione Transferase/*metabolism
MH  - Hemolysis
MH  - Humans
MH  - Hydrocarbons, Brominated/metabolism
MH  - In Vitro Techniques
MH  - Male
MH  - Methyl Chloride/metabolism
MH  - Spectrometry, Fluorescence/*methods
MH  - Substrate Specificity
EDAT- 2001/04/18 10:00
MHDA- 2001/07/13 10:01
CRDT- 2001/04/18 10:00
PHST- 2001/04/18 10:00 [pubmed]
PHST- 2001/07/13 10:01 [medline]
PHST- 2001/04/18 10:00 [entrez]
AID - 10.1007/s002040000201 [doi]
PST - ppublish
SO  - Arch Toxicol. 2001 Feb;74(12):760-7. doi: 10.1007/s002040000201.