Bioassay Method for Polyene Antibiotics Based on the Measurement of Rubidium Efflux from Rubidium-Loaded Yeast Cells

Abstract

A bioassay method for the polyene antibiotics nystatin and amphotericin B is proposed based on the measurement of the efflux of rubidium ions from a rubidium-loaded yeast culture challenged with the antibiotics. For this purpose a major proportion of the intracellular K+ ions in a Saccharomyces cerevisiae culture has been substituted by Rb+ ions. The rubidium leakage is measured by atomic absorption spectrophotometry, and a straight-line, dose-response correlation has been obtained for both antibiotics.It has been recognized since the early 1960s that one of the principal effects of polyene antibiotic action on susceptible microorganisms is the leakage of vital cytoplasmic constituents from the cells (8-11). The work of Sutton et al. (11) and Marini et al. (10) highlighted the leakage of K+ ions and NH4+ ions particularly. Since these early papers, many authors have examined induced potassium efflux from yeast cells by polyenes (4,5,7,(12)(13)(14).From some of the literature referred to above a correlation between polyene concentration and potassium efflux can be identified, and the prognosis for a possible rapid bioassay of this class of antibiotics, by measuring ion release from cells, is good.Smithler To overcome this problem, an alternative ion, which could be introduced into the yeast cells without affecting their viability or membrane function but which had a very low level of natural occurrence, was sought.To reproduce as closely as possible the characteristics of K+ ion leakage across the nystatin-impaired yeast cell membrane, it is necessary to find an ion of like charge and ionic radius. K+ ions exist in solution in a hydrated form, and the hydrated ionic radii of K+ (0.232 nm) and Rb+ (0.228 nm) are very similar, as are the ionic mobilities of these two ions.Rubidium ions are known to be well tolerated by erythrocytes, and an examination of the literature revealed a number of papers reporting the uptake of Rb+ ion by the yeast S. cerevisiae (1-3). Rubidium can also be determined at low levels with precision by atomic absorption spectrophotometry. It was therefore decided to examine the feasibility of using Rb+ uptake and subsequent release from yeast cells by the action of the polyene antibiotics nystatin and amphotericin as a bioassay system. The work reported here clearly shows such a system to be feasible in that a linear dose-response relationship can be obtained. MATERIALS AND METHODSYeast culture. S. cerevisiae SQ.1600 (from the Squibb Culture Collection) was used throughout.

A Rapid Microbiological Assay for Nystatin Using Rt+ Enriched Yeast Cells

“…The cost of reagents and media for the assay is negligible when compared to conventional methods. The Rb+ assay has been shown to be applicable with amphotericin B (Cosgrove & Fairbrother 1977) and preliminary results indicate its possible use clinically.…”

Clinical Pharmacokinetics of Systemic Antifungal Drugs

“…A number of microbiological methods for amphotericin B estimation in serum, CSF and other body fluids are available (Bannatyne et aI., 1977;Bindschadler and Bennett, 1969;Cosgrove and Fairbrother, 1977;Gale, 1974;Green et aI., 1965;Shadomy et aI., 1969). These are time consuming and interference from other drugs is a problem.…”

Chemotherapy of the Systemic Mycoses

“…Within 5 minutes of exposure to amphotericin B, fungal cells begin to "leak" out their cytoplasmic potassium. 10 The selectivity for ergosterol is relative, and at high concentrations amphotericin B binds to cholesterol in mammalian cell membranes, causing similar disruption of cell integrity. 11 "Leaky membranes" probably account for some of the nephrotoxicity of amphotericin B and also may account for the arrhythmias and acute hyperkalemia occasionally seen in patients with renal failure.…”