Simultaneous quantification of IMPDH activity and purine bases in lymphocytes using LC-MS/MS: assessment of biomarker responses to mycophenolic acid.
BACKGROUND
The development of biomarkers describing the individual responses to the immunosuppressant mycophenolic acid (MPA) has focused on the target enzyme activity [inosine 5'-monophosphate dehydrogenase (IMPDH)]. An extended strategy is to quantify the metabolic consequences of IMPDH inhibition. The aim of this study was to develop an assay for quantification of IMPDH activity and related purine bases and to provide preliminary data on the behavior of these biomarkers during clinical exposure to MPA.


METHODS
Liquid chromatography-mass spectrometry was used to determine xanthine (IMPDH activity in incubated cell lysate), hypoxanthine, guanine, and adenine derived from free nucleotides in lymphocytes. Analytical performance was assessed, and the biomarkers were examined in CD4⁺ cells from 2 groups: Healthy individuals in a single-dose MPA study (n = 5) and liver transplant recipients on MPA therapy (n = 15).


RESULTS
Coefficients of variation between series were below 10% and 15% for measurement of the purines and IMPDH activity, respectively. Although IMPDH was inhibited, the purine levels increased in response to MPA in 3 of the 5 healthy individuals, and this positive response seemed to be associated with IMPDH1 c.579 + 119 G/G and c.580 - 106 G/G. In the liver transplant study, guanine was not reduced in response to the transient drop in IMPDH activity after MPA dosing. However, there were trends toward decrease in guanine and elevation of hypoxanthine during prolonged MPA therapy. The guanine/hypoxanthine ratio (median) was 37% lower and the adenine level was 21% lower at day 17 compared with day 4 after transplantation.


CONCLUSIONS
The assay allows precise quantification of IMPDH activity, hypoxanthine, guanine, and adenine in lymphocytes. Some individuals may possess a counteracting purine response to the MPA-mediated inhibition of IMPDH. Reduction of the guanine/hypoxanthine ratio may be related to prolonged inhibition of IMPDH and seems as an intriguing pharmacodynamic biomarker for MPA.
*Department of Medical Biochemistry, Oslo University Hospital; †School of Pharmacy, University of Oslo; and Departments of ‡Anaesthesiology, §Pharmacology, and ¶Transplantation, Oslo University Hospital, Oslo, Norway.
Nils Tore
NT
Vethe
Ali Mohamad
AM
Ali
Pål Aarstrand
PA
Reine
Anders M
AM
Andersen
Sara
S
Bremer
Pål-Dag
PD
Line
Helge
H
Rootwelt
Stein
S
Bergan
D000328:Adult; D000368:Aged; D015415:Biomarkers / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D018592:Cross-Over Studies; D005260:Female; D006801:Humans; D007168:IMP Dehydrogenase / Q000378:metabolism*; D007166:Immunosuppressive Agents / Q000008:administration & dosage / Q000494:pharmacology*; D016031:Liver Transplantation; D008214:Lymphocytes / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009173:Mycophenolic Acid / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology; D011687:Purines / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods; D013997:Time Factors; D055815:Young Adult
D015415:Biomarkers; D007166:Immunosuppressive Agents; D011687:Purines; D007168:IMP Dehydrogenase; D009173:Mycophenolic Acid