allosteric effect by UDPGA on substrate binding (as proposed by Ethell and Wrighton

2004) is not known. Does the low-affinity component of the biphasic glucuronidation

exist in vivo, or is it an in vitro artifact of the excess UDPGA concentrations?

 In summary, the implication here is that the UGT activity obtained using saturating

concentrations of UDPGA that are in excess of physiological concentrations in a certain

tissue belonging to a specific species can render extrapolating in vitro data to in vivo

situations potentially useless. One can overestimate the efficacy of glucuronidation of a

xenobiotic because the maximal rate determined by conventional kinetic experiments

may be greater than the maximal rate in vivo. In addition, compounds which are

glucuronidated at rates ranging from the low nmol/min per mg to <1 pmol/min per mg

may all be regarded as substrates in the presence of high concentrations of UDPGA

(Miners et al., 2006).

 Objective

 To develop a reproducible method for the determination of UDPGA concentrations

in channel catfish liver and intestine.

 Method Development

 Previous attempts to separate UDPGA by HPLC utilizing a C18 or a C4 column

gave rise to results which were not reproducible. It was decided that, since UDPGA is

acidic at physiological pH, an ion-exchange column would be used in order to separate

this compound from other components of biological tissue that absorb at 260 nm (mainly

nucleotides and nucleotide sugars).