Effect of charge on interstitial distribution of albumin in rat dermis in vitro



At physiological pH, negatively charged glycosaminoglycans in the extracellular matrix may influence distribution volume of a probe. We hypothesized that by varying the probe charge we would be able to observe a graded response of available volume fraction. Human serum albumin (HSA) (isoelectric point (pI) 5.0) was made more positive by cationization. Using reaction times of 10, 45 and 60 min, cationized HSA (cHSA) with respective pIs of 6.5, 7.3 and 8.0 were made. After eight days of equilibration in a buffer containing labelled native HSA and cHSA, the distribution volumes were calculated relative to that of 51Cr-EDTA, an extracellular tracer. The available volume in fully swollen dermis for native albumin relative to that of the extracellular tracer averaged 0.485 ± 0.008 (n= 49), with corresponding volumes for cHSA-10 min, cHSA-45 min and cHSA-60 min of 0.554 ± 0.012 (n= 17), 0.647 ± 0.026 (n= 17) and 0.718 ± 0.021 (n= 12), respectively. Increasing the ionic strength of the bathing solution to 1 M NaCl, thereby screening the fixed charges of tissue elements and probes alike, resulted in similar available and thereby excluded volumes of native HSA and neutral cHSA-45 min. These experiments suggest that fixed negative charges, most likely glycosaminoglycans, contribute significantly to interstitial exclusion of charged macromolecules, a phenomenon of importance for hydration of the interstitial fluid phase and therefore for body fluid balance. Moreover, the data indicate that previous findings of similar excluded volumes for the two differently sized major plasma proteins albumin (molecular mass 66 kDa) and IgG (molecular mass 160 kDa) may be explained by a more pronounced electrostatic repulsion of the former by the extracellular matrix.