Determination of adenosine in fetal perfusates of human placental cotyledons using fluorescence derivatization and reversed-phase high-performance liquid chromatography

Abstract

A simple, sensitive HPLC method using fluorescence detection was developed for determination of adenosine in fetal venous perfusates of dual-perfused cotyledons from human term placentas. Maternal and fetal circuits of in vitro placental cotyledons were perfused with physiological salt solution containing dextrose and dextran (Earle's medium). Conditions were established for optimal formation of fluorescent 1,N 6-ethenoadenosine from adenosine and chloroacetaldehyde in Earle's medium and for optimal resolution of 1,N 6-ethenoadenosine by reversed-phase HPLC of the reaction mixture. The yield of 1,N 6-ethenoadenosine was enhanced by dilution and acidification of the sample matrix. Perfusate samples in autosampler vials were diluted 40% with water and reacted with chloroacetaldehyde for 40 min at 100°C; replicate 100-μl injections were made automatically from each reaction mixture for HPLC analysis with fluorescence detection on a column packed with 3 μm octadecylsilica (Hypersil). Calibration curves were prepared similarly from 4–100 n m adenosine in Earle's medium. Alternatively, perfusate samples were diluted twofold with dilute phosphoric acid to give a final pH of 5.4 before reaction with chloroacetaldehyde, and replicate 50-μl injections were made automatically for HPLC; calibration curves were prepared from 2–400 n m adenosine in Earle's medium. 1,N 6-Ethenoadenosine was well resolved from Earle's-derived artifactual peaks on chromatography with either a linear or a concave gradient of methanol in ammonium phosphate buffer. Total run times were 15 and 19 min, respectively. Sensitivity of measurement of adenosine was 2–4 n m. Derivatization of adenosine using the acidified reaction mixture gave a limit of detection of 100 fmol of adenosine per injection. Application of the method to analysis of adenosine in fetal venous perfusates of eight dual-perfused cotyledons, each from a different placenta, gave a range of 3.5–52 n m adenosine. Ischemia, imposed by cessation of maternal perfusion, caused a two- to sixfold increase in fetal venous perfusate adenosine concomitant with an increase in fetoplacental perfusion pressure; perfusion pressure and perfusate adenosine returned to baseline levels on reperfusion of the maternal circuit. This facile method of determination of perfusate adenosine should allow investigation of the role of placental adenosine release in regulation of fetoplacental vascular resistance and should be applicable to study of adenosine release by other isolated perfused organs.