Glutathione conjugation with phosphoramide mustard and cyclophosphamide. A mechanistic study using tandem mass spectrometry.
The conjugations of cyclophosphamide and of phosphoramide mustard with glutathione are shown to be catalyzed by hepatic cytosolic glutathione-S-transferases. Cyclophosphamide conjugation is also catalyzed by microsomal glutathione-S-transferases, both in intact microsomes and after solubilization and immobilization. Deuterium isotope labels are used to test whether chloride is directly displaced by glutathione in the enzyme-catalyzed conjugations, or whether conjugation takes place via symmetrical cyclic aziridinium ions. Tandem mass spectrometry with high energy collisional activation is shown to provide reliable analysis of the isotope-labeling patterns in the conjugated products. This experiment leads to the conclusion that the aziridinium ion is opened in the conjugation of phosphoramide mustard in both the enzyme-catalyzed and the chemical reactions. Cyclophosphamide, on the other hand, is shown to be conjugated through direct displacement of chloride.
Yuan
Z M
ZM
Dept. of Chemistry and Biochemistry, University of Maryland Baltimore County, 21228.
Smith
P B
PB
Brundrett
R B
RB
Colvin
M
M
Fenselau
C
C
D000818:Animals; D001388:Aziridines / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003520:Cyclophosphamide / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D010752:Phosphoramide Mustards / Q000378:metabolism*; D011817:Rabbits
D001388:Aziridines; D010752:Phosphoramide Mustards; C030090:phosphoramide mustard; D003520:Cyclophosphamide; D005982:Glutathione Transferase; D005978:Glutathione