ESR measurement of time-dependent and equilibrium volumes in red cells

Red cell water volumes were measured using ESR methods during transient osmotic perturbation, and under equilibrium conditions. Cell water contents were determined using the spin label Tempone (2,2,6,6-tetramethyl piperidine-N-oxyl) and the membrane impermeable quencher potassium chromium oxalate. With appropriate corrections for intracellular viscosity and changes in cavity sensitivity, equilibrium cell water measured both by electron spin resonance (ESR) and wet minus dry weight methods gave excellent agreement in solutions from 243-907 mOsm. Intracellular viscosities determined from the Tempone correlation times in the same cells gave values ranging from 9-47 centipoise at 21 degrees C. Osmotically induced transient volume changes were measured using Tempone and an ESR stopped-flow configuration. The Tempone response time was estimated at 17 msec compared to 250-350 msec for normal water relaxations. Nonlinear least square solutions to the Kedem-Katchalsky equations including a correction for the finite Tempone permeability gave 0.029 and 0.030 cm/sec for the osmotic permeability of RBCs in swell and shrink experiments, respectively. In stopped-flow experiments accurate water flux data are obtained very soon after challenging cells and do not require baseline subtractions. These results represent significant improvements over conventional light scattering techniques which necessitate corrections for long lasting optical artifacts (200-300 msec), and baseline drifts.