Quantification of phenol, phenyl glucuronide, and phenyl sulfate in blood of unanesthetized rainbow trout by online microdialysis sampling

Free concentrations of phenol (PH), phenyl glucuronide (PG), and phenyl sulfate (PS) were measured in the bloodstream of unanesthetized rainbow trout by online microdialysis (MD) sampling. Preliminary studies were conducted to optimize the MD system and evaluate three retrodialysis calibration standards: p-nitrophenyl glucuronide (PNPG), p-nitrophenyl sulfate (PNPS), and [14C]-phenol (14C-PH). PG and PNPG exhibited nearly identical dialyzing properties in vitro (saline and plasma) and in vivo (muscle tissue and dorsal aorta). A similar result was obtained for PS and PNPS. In vivo studies were therefore performed using PNPG, PNPS, and 14C-PH as retrodialysis calibrators for PG, PS, and PH, respectively. The utility of MD sampling for kinetic studies with fish was investigated by implanting MD probes into the dorsal aorta of spinally transected rainbow trout. Each fish was then exposed to PH in water in a respirometer-metabolism chamber. The free concentration of PH in blood reached a steady-state level within 12 h of initiating the exposure. A steady state for PS was generally established within 24 h, while free concentrations of PG tended to increase throughout the exposure. Terminal plasma samples were dialyzed using the same probe employed in each experiment. Analyte concentrations determined in this manner were in good agreement with calculated in vivo values. The methods described in this study can be used to collect kinetic data sets of high temporal resolution while eliminating artifacts often associated with conventional blood sampling methods.