In vivo determination of steric and electrostatic exclusion of albumin in rat skin and skeletal muscle

Abstract

In order to estimate the magnitude of electrostatic exclusion provided by the fixed negative charges of the skin and muscle interstitia of rat in vivo we measured the distribution volumes of two differently charged albumin probes within these tissues. An implanted osmotic pump was used to reach and maintain a steady-state extracellular concentration of a mixture containing two iodine-labelled probes: a charged-modified human serum albumin, cHSA (i.e. a positive probe, isoelectirc point (pI) = 7.6) and a native human serum albumin, HSA (i.e. a normally charged, negative probe, pI = 5.0). Steady-state tissue concentrations were achieved after intravenous infusion of probes for 5-7 days. At the end of this period the animals were nephrectomized and a bolus of 51Cr-EDTA was administered for estimating the extracellular volume. Plasma volumes were measured as 5-min distribution volume of 125I-HSA in separate experiments. The steady-state interstitial fluid concentrations of all probes were determined using nylon wicks implanted postmortem. Calculations of labelled probes were made for interstitial fluid volumes (Vi), extravascular albumin distribution volumes (Vav,a) and relative interstitial excluded volume fractions (Vex,a/Vi). We found that the positive probe is excluded from a significantly smaller fraction of the interstitium. Specifically, the average relative albumin exclusion fractions obtained were: 16% and 26% in skeletal muscle and 30% and 40% in skin, for cHSA and HSA, respectively. On average, the fixed negative charges of the interstitium are responsible for about 40% of the total albumin exclusion in skeletal muscle and 25% in the whole skin tissue and thus, contribute significantly to volume exclusion in these tissues.

Figure 1. Plasma concentration of tracer vs. duration of experiment

Relative plasma concentrations of cHSA (pI = 7.6, •) and HSA (pI = 5.0, ○). The duration of continuous infusion is t = 120 h (n = 4) and t = 168 h (n = 5). Values are given as means ±s.e.m.

Figure 2. Distribution volumes related to time of tissue sampling

Distribution volumes of HSA (pI = 5.0) and cHSA (pI = 7.6) in skin and muscle tissues at the time of sampling, i.e. t = 120 h (n = 4) and t = 168 h (n = 5). Values are given as means ±s.e.m.

Figure 3. Fractional excluded interstitial volumes

Fractional excluded interstitial volumes of cHSA and HSA. The number of rats is n = 9. Values are given as means ±s.e.m.*P < 0.05 and **P < 0.01 compared to the corresponding tissue involving uptake of albumin.

Figure 4. HPLC of final plasma after 168 h of tracer infusion

Radioactivity of cHSA (•) and HSA (□) in successive collections of excluded volume fractions V_e (ml) from a Superose 12 size exclusion column after applications of plasma final samples collected at t = 168 h of continuous infusion. The optimal separation range of the column is 1–300 kDa.

Figure 5. Fractional excluded interstitial volumes of differently charged HSA and IgG

Comparison between fractional exclusions of charged-modified cHSA (pI = 7.6), negative HSA (pI = 5.0) with positive IgG1 (pI = 8.7) and negative IgG4 (pI = 6.6). The number of rats is n = 9. Values are given as means ±s.e.m.