Determination of 5-fluorouracil and dihydrofluorouracil levels by using a liquid chromatography-tandem mass spectrometry method for evaluation of dihydropyrimidine dehydrogenase enzyme activity.
OBJECTIVE
5-Fluorouracil (5-FU), acting as a pyrimidine antagonist, is a major chemotherapy drug used for the treatment of tumors such as gastrointestinal, breast, ovary, and head and neck cancers. The key and rate-limiting enzyme in 5-FU catabolism is dihydropyrimidine dehydrogenase (DHPDH), whose partial or complete deficiency exposes to a severe 5-FU toxicity in patients. The determination of DHPDH activity in patients before the treatment and setting up a personalized therapy for each patient receiving the drug can help us to prevent the possible risk of toxicity.


METHODS
To isolate peripheral blood mononuclear cells (PBMCs), EDTA-anticoagulated blood samples were collected from randomly selected 47 patients and examined for 5-FU and its metabolite dihydrofluorouracil (FUH2) by using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) to observe DHPDH activity at different intervals (0 and 4th hour) indirectly.


RESULTS
Intra-assay and interassay CV % values of samples from the measurements of the modified methods are found 1.3-11.9, 2.3-9.4 for 5-FU and 3.1-14.4, 3.3-12.6 for FUH2, respectively. The reference values derived from 45 patients treated with 5-FU are 1.84 ± 0.34 ug/gr protein for 5-FU, 40.15 ± 11.43 ng/gr protein for FUH2, respectively. FUH2/5-FU ratio is 21.9 ± 3.72. In addition, the results determined from two patients, in which the lack of DHPDH is considered, were 3.24 and 4.16 ug/gr protein for 5-FU, 4.1 and 6.7 ng/gr protein for FUH2. FUH2/5-FU ratio is 1.26 and 1.61.


CONCLUSIONS
The measurements of 5-FU, FUH2, and especially their ratio (FUH2/5-FU) by the modified LC-MS/MS method could be used to determine DHPDH enzyme activity.
Department of Clinical Chemistry, Gulhane School of Medicine, Ankara, Turkey. maserdar@gata.edu.tr
Muhittin A
MA
Serdar
Erdim
E
Sertoğlu
Metin
M
Uyanık
Serkan
S
Tapan
Okhan
O
Akın
Murat
M
Cihan
D000964:Antimetabolites, Antineoplastic / Q000009:adverse effects / Q000097:blood* / Q000493:pharmacokinetics / Q000627:therapeutic use; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D054067:Dihydropyrimidine Dehydrogenase Deficiency / Q000150:complications / Q000175:diagnosis*; D042943:Dihydrouracil Dehydrogenase (NADP) / Q000097:blood*; D016903:Drug Monitoring / Q000379:methods; D057075:Enzyme Assays / Q000379:methods; D005472:Fluorouracil / Q000009:adverse effects / Q000031:analogs & derivatives* / Q000097:blood* / Q000493:pharmacokinetics / Q000627:therapeutic use; D006801:Humans; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000201:enzymology; D057230:Limit of Detection; D009369:Neoplasms / Q000097:blood* / Q000150:complications / Q000188:drug therapy; D057285:Precision Medicine / Q000379:methods; D015203:Reproducibility of Results; D012720:Severity of Illness Index; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*
D000964:Antimetabolites, Antineoplastic; C019389:5-fluorodihydrouracil; D042943:Dihydrouracil Dehydrogenase (NADP); D005472:Fluorouracil