Lipophilic impurity of phenol red is a potent cation transport modulator.
Previously, we described substantial alterations in the Na+ and K+ homeostasis of human skin fibroblasts following removal of fetal bovine serum (FBS). Herein, we report that FBS removal per se does not cause any cellular ionic changes unless a lipophilic impurity of commercial phenol red preparations is present. This substance accelerates 86Rb+ efflux four to seven times, causes a four to eight time increase in cellular Na+, and a 40-70% reduction in cellular K+ contents. FBS (10%) or albumin (0.8%) appears to bind the impurity thus inhibiting its action. The increased cellular Na+ and decreased K+ contents do not return to baseline within 4 hours following the removal of the phenol red extract. However, albumin completely reverses the cellular cationic changes that develop during a 2 hour exposure of the cells to the free substance. The reversibility of its action by albumin suggests that the substance exerts its effect on or within the cell membrane and not intracellularly. Among seven different cell lines tested the 86Rb+ efflux from, and the Na(+)-K+ contents of, COS-7 and Hs68 cells also responded to unpurified phenol red in a way similar to human fibroblasts. The amount of the phenol red contaminant is manufacturer dependent. As little as 0.5 microM phenol red, from one vendor, was sufficient to elicit response in the 86Rb+ efflux. Given that the impurity is unlikely to be more than a small fraction of phenol red, it seems to be a potent ionic transport modulator. Based on these results, the presence of commercial phenol red in serum-free growth or test media, including the increasing variety of chemically defined culture media, should be considered as a potential confounding factor in measurements that depend on intracellular Na(+)-K+ homeostasis. The findings of such earlier studies may need to be reconsidered if the cells were exposed to unbound phenol red. We recommend that, until the manufacturers further refine their product, phenol red be purified by ether extraction before its use. The evaluation of the potential physiologic or pharmacologic relevance of this potent cation transport modulator awaits its isolation.
Hopp
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Children's Hospital of Pittsburgh, Division of Nephrology, Pennsylvania.
Bunker
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D000418:Albumins / Q000378:metabolism; D000818:Animals; D001798:Blood Proteins / Q000378:metabolism; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004958:Estradiol / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D008055:Lipids / Q000494:pharmacology*; D010637:Phenolsulfonphthalein / Q000737:chemistry / Q000494:pharmacology*; D011188:Potassium / Q000378:metabolism*; D012413:Rubidium / Q000378:metabolism; D012867:Skin / Q000166:cytology; D012964:Sodium / Q000378:metabolism*
D000418:Albumins; D001798:Blood Proteins; D016895:Culture Media, Serum-Free; D008055:Lipids; D004958:Estradiol; D012964:Sodium; D010637:Phenolsulfonphthalein; D012413:Rubidium; D011188:Potassium