Metabolism of an Anionic Fluorescent Dye, 1-Anilino-8-naphthalene Sulfonate (ANS) by Rat Liver Microsomes

The present study was designed to examine the metabolism of 1-anilino-8-naphthalene sulfonate (ANS), an anionic compound which is transported into liver via "multispecific organ ic anion transporter", with rat hepatic microsomes. TLC analysis indicated that the fluorescent metabolites were not produced to a measurable extent, which made it possible to assess the ANS metabolism by measuring the fluorescence disappearance. The metabolism of ANS was remarkably inhibited by the presence of SKF-525A as well as by the substitution of 02 by CO gas. ANS metabolism by microsomes also required NADPH as a cofactor. These results indicated that the microsomal monooxygenase system might be mainly responsible for the ANS metabolism. The maximum velocity ($V_{max}$) and Michaelis constant ($K_m$) were calculated to be $4.3{\pm}0.2$ nmol/min/mg protein and $42.1{\pm}2.0\;{\mu}M$, respectively. Assuming that 1g of liver contains 32mg of microsomal protein, the $V_{max}$ value was extrapolated to that per g of liver ($V_{max}^I$). The intrinsic metabolic clearance ($CL_{int}$) under linear conditions calculated from this in vitro metabolic study was 3.3ml/min/g liver, being comparable with that (3.0ml/min/g liver) calculated by analyzing the in vivo plasma disappearance curve in a previous study. Furthermore, the effects of other organic anions on the metabolism of ANS were examined. Bromophenolblue (BPB) and rose bengal (RB) competitively inhibited the metabolism of ANS, while BSP inhibited it only slightly. The inhibition constant ($K_i$) of BPB ($6\;{\mu}M$) was much smaller than that of RB ($200\;{\mu}M$). In conclusion, the microsomal monooxygenase system plays a major role in the metabolism of ANS, and other unmetabolizable organic anions (BPB and RB) compete for this metabolism.