LIPOPHILIC IMPURITY OF PHENOL RED IS A POTENT CATION-TRANSPORT MODULATOR

Previously, we described substantial alterations in the Na+ and K+ hom eostasis of human skin fibroblasts following removal of fetal bovine s erum (FBS). Herein, we report that FBS removal per se does not cause a ny cellular ionic changes unless a lipophilic impurity of commercial p henol red preparations is present. This substance accelerates Rb-86+ e fflux four to seven times, causes a four to eight time increase in cel lular Na+, and a 40-70% reduction in cellular K+ contents. FBS (10%) o r albumin (0.8%) appears to bind the impurity thus inhibiting its acti on. The increased cellular Na+ and decreased K+ contents do not return to baseline within 4 hours following the removal of the phenol red ex tract. However, albumin completely reverses the cellular cationic chan ges that develop during a 2 hour exposure of the cells to the free sub stance. The reversibility of its action by albumin suggests that the s ubstance exerts its effect on or within the cell membrane and not intr acellularly. Among seven different cell lines tested the Rb-86+ eff lu x from, and the Na+-K+ contents of, COS-7 and Hs68 cells also responde d to unpurified phenol red in a way similar to human fibroblasts. The amount of the phenol red contaminant is manufacturer dependent. As lit tle as 0.5 muM phenol red, from one vendor, was sufficient to elicit r esponse in the Rb-86+ efflux. Given that the impurity is unlikely to b e more than a small fraction of phenol red, it seems to be a potent io nic transport modulator. Based on these results, the presence of comme rcial phenol red in serum-free growth or test media, including the inc reasing variety of chemically defined culture media, should be conside red as a potential confounding factor in measurements that depend on i ntracellular Na+-K+ homeostasis. The findings of such earlier studies may need to be reconsidered if the cells were exposed to unbound pheno l red. We recommend that, until the manufacturers further refine their product, phenol red be purified by ether extraction before its use. T he evaluation of the potential physiologic or pharmacologic relevance of this potent cation transport modulator awaits its isolation. (C) 19 93 Wiley-Liss, Inc.