Inter- and intralaboratory variation of in vitro diffusion cell measurements: an international multicenter study using quasi-standardized methods and materials.

In vitro measurements of skin absorption are an increasingly important aspect of regulatory studies, product support claims, and formulation screening. However, such measurements are significantly affected by skin variability. The purpose of this study was to determine inter- and intralaboratory variation in diffusion cell measurements caused by factors other than skin. This was attained through the use of an artificial (silicone rubber) rate-limiting membrane and the provision of materials including a standard penetrant, methyl paraben (MP), and a minimally prescriptive protocol to each of the 18 participating laboratories. "Standardized" calculations of MP flux were determined from the data submitted by each laboratory by applying a predefined mathematical model. This was deemed necessary to eliminate any interlaboratory variation caused by different methods of flux calculations. Average fluxes of MP calculated and reported by each laboratory (60 +/- 27 microg cm(-2) h(-1), n = 25, range 27-101) were in agreement with the standardized calculations of MP flux (60 +/- 21 microg cm(-2) h(-1), range 19-120). The coefficient of variation between laboratories was approximately 35% and was manifest as a fourfold difference between the lowest and highest average flux values and a sixfold difference between the lowest and highest individual flux values. Intralaboratory variation was lower, averaging 10% for five individuals using the same equipment within a single laboratory. Further studies should be performed to clarify the exact components responsible for nonskin-related variability in diffusion cell measurements. It is clear that further developments of in vitro methodologies for measuring skin absorption are required.

Ammonium assimilation by Candida albicans and other yeasts: a 13N isotope study.

Nuclear magnetic resonance spectra of cultures of Candida albicans incubated in the presence of 15N-labelled ammonium demonstrated that glutamine and glutamate were the only initial products of ammonium assimilation. The nature of the route of assimilation in the yeasts Candida albicans, Saccharomyces cerevisiae, and Candida tropicalis was further examined by the use of the short-lived isotope 13N. [13N]ammonium was generated in the reaction 16O(p,alpha)13N, induced by proton bombardment of water in tandem accelerator. High-pressure liquid chromatography was used to separate and identify the products of assimilation, and radioactivity was detected and corrected for decay, using a computer-linked NaI scintillation detector. In the three yeasts studied, the labelled ammonium was assimilated into the acid-extractable fraction of cell suspensions within 1 min, and over 75% was converted to glutamine and glutamate. Subsequent to exhaustion of the labelled ammonium, the stoichiometry of the distribution of radiolabel was consistent with a net transfer of radiolabel from glutamine to glutamate, confirming the operation of glutamate synthase (EC 1.4.1.14) in these yeasts. Initial assimilation of label was mostly into glutamine (at a maximal rate within 10 s in C. albicans), whereas accumulation in glutamate did not occur at maximal rate until more than 70% of the labelled ammonium had been assimilated (between 30 and 60 s in C. albicans). We conclude that the glutamine synthetase-glutamate synthase pathway is the major route of ammonium assimilation in C. albicans and also in nitrogen-starved cultures of S. cerevisiae and C. tropicalis.