Identification of glucoside and carboxyl-linked glucuronide conjugates of mycophenolic acid in plasma of transplant recipients treated with mycophenolate mofetil.
1. Mycophenolic acid (MPA), is primarily metabolized in the liver to 7-O-MPA-beta-glucuronide (MPAG). Using RP-h.p.l.c. we observed three further MPA metabolites, M-1, M-2, M-3, in plasma of transplant recipients on MMF therapy. To obtain information on the structure and source of these metabolites: (A) h.p.l.c. fractions containing either metabolite or MPA were collected and analysed by tandem mass spectrometry; (B) the metabolism of MPA was studied in human liver microsomes in the presence of UDP-glucuronic acid, UDP-glucose or NADPH; (C) hydrolysis of metabolites was investigated using beta-glucosidase, beta-glucuronidase or NaOH; (D) cross-reactivity of each metabolite was tested in an immunoassay for MPA (EMIT). 2. Mass spectrometry of M-1, M-2, MPA and MPAG in the negative ion mode revealed molecular ions of m/z 481, m/z 495, m/z 319 and m/z 495 respectively. 3. Incubation of microsomes with MPA and UDP-glucose produced M-1, with MPA and UDP-glucuronic acid MPAG and M-2 were formed, while with MPA and NADPH, M-3 was observed. 4. Beta-Glucosidase hydrolysed M-1 completely. Beta-Glucuronidase treatment led to a complete disappearance of MPAG whereas the amount of M-2 was reduced by approximately 30%. Only M-2 was labile to alkaline treatment. 5. M-2 and MPA but not M-1 and MPAG cross-reacted in the EMIT assay. 6. These results suggest that: (i) M-1 is the 7-OH glucose conjugate of MPA; (ii) M-2 is the acyl glucuronide conjugate of MPA; (iii) M-3 is derived from the hepatic CYP450 system.
Shipkova
M
M
Abteilung Klinische Chemie, George-August-Universität Göttingen, Germany. ewieland@med.uni-goettingen.de
Armstrong
V W
VW
Wieland
E
E
Niedmann
P D
PD
Schütz
E
E
Brenner-Weiss
G
G
Voihsel
M
M
Braun
F
F
Oellerich
M
M
D002851:Chromatography, High Pressure Liquid; D005960:Glucosides / Q000302:isolation & purification; D005965:Glucuronates / Q000302:isolation & purification; D020719:Glucuronides; D006801:Humans; D006868:Hydrolysis; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000378:metabolism; D009173:Mycophenolic Acid / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D016377:Organ Transplantation
D005960:Glucosides; D005965:Glucuronates; D020719:Glucuronides; D007166:Immunosuppressive Agents; C113145:mycophenolic acid glucuronide; D009173:Mycophenolic Acid