Phenotypic Approaches

DPD is a ubiquitous enzyme. Several reports have shown that evaluating DPD activity in easily available surrogate tissues such as lymphocytes or fibroblasts can be used as a marker for the actual liver function, despite mixed correlations between activities (91,92). Admittedly, PBMC DPD levels below 150pmol/min/mg protein are associated with a partial deficiency syndrome, and patients displaying DPD <50 pmol/min/mg protein are considered as totally deficient (17,93,94,95,96). Precise measurement of ex vivo DPD activity from blood mononuclear cells was the first historical method proposed in the late 1980s to evaluate overall DPD functionality (97). Enzymatic activity is mainly assessed using a radio-HPLC method that measures the conversion of radio-labelled 5-FU, uracil or thymine into dehydrogenated metabolites (32, 91, 93, 98, 99,100,101). More recently, a HPLC method that does not require radio-labelled substrates has been proposed, thus rendering this approach easier to be used in most laboratories (102,103).

Besides the actual and direct measurement of DPD activity, an estimation of the global DPD functionality can be achieved by monitoring physiological substrates used as probes. Because DPD converts uracil (U) to dihydrouracil (UH2), expressing levels of both compounds as a ratio was first proposed in the mid-1990s as a surrogate marker for overall DPD activity (104). Numerous methods have been published since then to monitor U and UH2, sometimes along with thymine and dihydrothymine (105), in either urine (106,107) or plasma (108,109,110,111,112).

Such determinations can be achieved using simple UV-HPLC (109,112,113,114,115), GC-MS or LC-MS/MS systems (105,106,113). Study of the correlations between U, UH2-U levels, and DPD activities or 5-FU PK parameters have yield conflicting data (16,22,116). However, a clear relationship has been demonstrated between U-UH2 ratio values and the occurrence of severe/lethal toxicities with 5-FU or capecitabine as a clinical endpoint (20,27,28).

More recently, two novel methods based upon the ingestion of exogenous uracil as a substrate for DPD prior to in vivo metabolism monitoring have been proposed. The Uracil Breath Test is based on the ingestion of a stable isotope of uracil (13C-uracil) and monitoring of expired 13C-CO2 using an infrared spectrometer breath analyzer (117,118). Following the same principle of exogenous uracil intake, the Uracil Challenge Test is an alternative, time- and cost-effective method which requires un-labelled uracil followed by standard UV-HPLC analysis, and so far has shown promising preliminary results (119,120).

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