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COMPARTMENTAL DIFFUSION 98

Chapter 5

Yeast Experiments

The goal of these experiments was to characterize the diffusion behavior of water in atwo-compartment system. Yeast cell suspensions have been well characterized by othersand are fairly uncomplicated model systems to understand. The following work containsmultiple sets of experiments using yeast cells as a model system to characterize thediffusion behavior of water.

5.1. Deconvolution of Restriction Effects onCompartmental Diffusion Using CombinedRelaxometric and Diffusimetric NMR

5.1.1. Abstract

Diffusion signal attenuation curves in yeast-cell suspensions show non-monoexponentialsignal decay which is assumed to arise from separate compartmental contributions to theoverall signal. However, restricted diffusion effects also give rise to non-monoexponential signal decay and are difficult to separate from compartmental signalcontributions. Combined relaxometry and diffusion measurements allows differentiation

COMPARTMENTAL DIFFUSION 99

between compartmental diffusion constants by first separating the compartmentalcontributions on the basis of differences in their respective relaxation times. Diffusion-weighted inversion-recovery spin-echo experiments were carried out at different

-values. Intra- and extracellular compartments in yeast-cell suspensions were separated onthe basis of

relaxation by adding an MR contrast agent to the extracellular space.Once the compartmental signals were distinguished on the basis of

, the relative signalattenuation for each compartment was used to calculate the separate compartmentalADCs. With this method, even compartmental diffusion coefficients with similar valuescan be distinguished.

5.1.2. Introduction

The cell membrane plays an important role in cell physiology by acting as a protectivebarrier between the extra- and intracellular compartments. Since the cellular membraneis a lipid bilayer structure with a highly hydrophobic core (Fox, 1993), it is unclear howwater is transported between the intra- and extracellular spaces through a seeminglyimpermeable membrane. It has been speculated that transmembrane transport of wateroccurs

via

osmo-regulated water channels (Jap and Li, 1995; Skach

et al

., 1994) or inconjunction with water-soluble ion transport.Movement of water between the extra- and intracellular spaces, as well as within thecells, is of particular importance in biomedicine. Studies have shown that changes in the

COMPARTMENTAL DIFFUSION 100

apparent diffusion coefficient (ADC) of water in biological systems are a function of membrane permeability and changes in the extracellular volume fraction (Latour

et al

.,1993b, 1994). Since the water NMR signal is measured simultaneously from the intra-and extracellular space, it is difficult to ascertain whether the changes in the ADC arecaused by drastic changes in one or both of the compartments, or by permeabilitychanges in the cell membrane.Non-monoexponential behavior in the diffusion-signal attenuation curves has beenobserved in a number of biological systems and has been attributed to compartmentaleffects (Niendorf

et al

., 1996; Andrasko, 1976b; Vétek

et al

., 1994; Szafer

et al

., 1995).Although it has been reported that changes in the extracellular volume fraction mirror thechanges observed in the rapidly decaying component of the signal decay curve (Trouard

et al

., 1997; Niendorf

et al

., 1994), the volume fraction calculated for the slowlydiffusing component in these experiments differ significantly from the true intracellularvolume fraction (Niendorf

et al

., 1994). While multi-compartment systems in the slow-exchange regime are expected to exhibit non-monoexponential signal decay curves,Helmer

et al

. (1995) have shown that non-monoexponential diffusion-signal attenuationcurves can be obtained in polystyrene bead packs (a single compartment system) due torestriction effects alone. Consequently, since the compartmental contributions do notdefinitively represent the cellular compartments, it is difficult to ascertain whether thenon-monoexponential behavior in a highly restricted two-compartment system is due tocompartmental contributions, restrictions, or both of these effects.