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.
Dept. of Chemistry and Biochemistry, University of Maryland Baltimore County, 21228.
Z M
ZM
Yuan
P B
PB
Smith
R B
RB
Brundrett
M
M
Colvin
C
C
Fenselau
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