Porous graphitic carbon shows promise for the rapid screening partial DPD deficiency in lymphocyte dihydropyrimidine dehydrogenase in Chinese, Indian and Malay in Singapore by using semi-automated HPLC-radioassay.
OBJECTIVE
Dihydropyrimidine dehydrogenase (DPD) catalyzes the degradation of thymine, uracil, and the chemotherapeutic drug 5-Fluorouracil. In general reverse-phase high pressure liquid chromatography is the standard method for separating 5-[2-(14)C]Fluorouracil and 5-[2-(14)C]Fluoro-5,6-dihydrouracil. However, the use of 100% aqueous solution (as HPLC mobile phase) may collapse the C-18 bonded phase and result in a retention time shift. The aim of this study is to develop a rapid, reproducible, sensitive method for screening partial DPD deficiency in healthy volunteers.


METHODS
The activity of DPD was measured using 5-[2- (14)C]Fluorouracil (5-[2-(14)C]FUra) followed by separation of substrate and product 5-[2-(14)C]FUraH(2) with a 15 x 4.6 mm I.D., 5 microm particle size (d(p)) porous graphitic carbon (PGC) column (Hypercarb(R)) and HPLC with online detection of the radioactivity. This was standardized using the protein concentration of the cytosol (NanoOrange(R) Protein Quantitation).


RESULTS
Complete baseline separation of 5-[2-(14)C]Fluorouracil (5-[2-(14)C]FUra) and 5-[2-(14)C]Fluoro-5,6-dihydrouracil (5-[2-(14)C]FUraH(2)) was achieved using a porous graphitic carbon (PGC) column. The detection limit for 5-[2-(14)C]FUraH(2) was 0.4 pmol.


CONCLUSIONS
By using linear gradient separation (0.1% Trifluoroacetic acid [TFA] in water to 100% Methanol) protocols in concert with PGC columns (Hypercarb(R)), we have demonstrated that a PGC column has a distinct advantage over C-18 reverse phase columns in terms of column stability (pH 1-14). This method provides an improvement on the specific assay for DPD enzyme activity. It is rapid, reproducible and sensitive and can be used for routine screening for healthy and cancer patients for partial and profound DPD deficiency before treatment with 5- FUra.
Liem
Lie Kuo
LK
Donner Laboratory, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. lkliem@lbl.gov
Choong
Lina Hui Lin
LH
Woo
Keng Thye
KT
D000328:Adult; D000368:Aged; D044466:Asian People; D001331:Automation / Q000379:methods; D002681:China; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003955:Diagnostic Tests, Routine / Q000379:methods*; D042943:Dihydrouracil Dehydrogenase (NADP); D005472:Fluorouracil / Q000378:metabolism; D006108:Graphite / Q000737:chemistry*; D006801:Humans; D007194:India; D008214:Lymphocytes / Q000201:enzymology*; D008296:Malaysia; D008403:Mass Screening / Q000379:methods; D008875:Middle Aged; D010088:Oxidoreductases / Q000097:blood* / Q000172:deficiency*; D011869:Radioligand Assay / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018709:Statistics, Nonparametric
D006108:Graphite; D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP); D005472:Fluorouracil