Collagen and glycosaminoglycans (GAGs) constituting the extracellular matrix may limit the space available and thus exclude macromolecules from a fraction of the interstitial fluid phase. This exclusion phenomenon is of importance for transcapillary fluid and solute exchange. The purpose of the study was to examine the range of interstitial exclusion in rat skin by using probes within a span of molecular weights and electrical charge, and also to test if a change in interstitial composition occurring as a consequence of aging affected exclusion. To this end we used a novel approach, involving the exact determination of albumin concentration and mass in interstitial fluid and tissue eluate by high performance liquid chromatography and thereafter expressing the corresponding numbers relative to albumin for a set of probe proteins assessed by quantitative proteomics. Albumin was excluded from 55 ± 4% (n=8) of the extracellular fluid phase. There was a highly significant positive correlation between probe Stokes-Einstein (SE) radius and fractional excluded volume (VEF) described by VEF = 0.078*SE radius + 0.269 (p<0.001), and oppositely, a negative correlation between probe isoelectric point (pI) and exclusion for proteins with comparable size, VEF = -0.036 * pI +0.719 (p=0.04). Aging resulted in a significant reduction in skin hydration and sulphated GAGs and a moderate increase in hyaluronan, and a corresponding reduced VEF for albumin and the other macromolecular probes. Our findings suggest that the changes in the extracellular matrix in aged skin may result in delayed adjustments of fluid perturbations and reduced ability for salt storage.
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