A new bioartificial pancreas, in which rat islets within a Millipore chamber are perifused with blood ultrafiltrate (polyacrylonitrile membrane), has been developed and its function evaluated in vitro and in vivo. Use of ultrafiltration (instead of diffusion) permitted rapid transmission of changes in blood glucose to the islet compartment (less than 10 min). To determine whether ultrafiltrate of Krebs-Ringer bicarbonate buffer (KRBB) and blood could support normal islet Bcell function, insulin secretion of freshly isolated islets in response to a 20-mM glucose challenge was examined during perifusion with KRBB and then with ultrafiltrate produced either from KRBB itself or from blood of normal and streptozotocin-induced diabetic rats. In the latter case, perifusate was reinfused to the animals, whose plasma glucose and insulin were then measured.
Insulin responses to glucose were virtually identical when the same islets were perifused with KRBB and then with ultrafiltrate of KRBB (47 ± 12 versus 41 ± 9 ng/100 islets/24 min), or when islets were perifused with KRBB and then with ultrafiltrate produced from blood of normal rats (69 ± 6 versus 73 ± 11 ng/100 islets/24 min). When five diabetic rats were connected to the device containing 600 islets, their plasma glucose decreased from 19.8 ± 1.2 to 6.1 ± 0.5 mM (P < 0.001) within 120 min without overshoot hypoglycemia, and remained normal for up to 6 h. Plasma insulin increased from 2.0 ± 0.1 to 11.4 ± 1.4 ng/ml (P < 0.02) at 30 min and then decreased to 2.9 ± 0.6 ng/ml. After intravenous glucose administration (0.5 g/kg, N = 4), plasma insulin increased twofold within 15 min, and glucose disappearance rates were identical to those obtained in four normal rats (K = 2.13 ± 0.18 versus 2.23 ± 0.09, NS).
These results indicate that ultrafiltrate of KRBB and blood can acutely support normal islet B-cell function, and that ultrafiltration can provide satisfactory glucose-insulin kinetics for an implantable closed-loop insulin delivery system, in which nonsyngeneic islets may be protected against immune rejection.
Among the middle molecule fractions obtained by high performance gel chromatography on Sephadex G-15 combined with gradient ion exchange chromatography on DEAE Sephadex A-25 from plasma ultrafiltrates of six polyneuropathic patients, only peak b4-2 was at a significantly higher concentration than that obtained from uremic patients with neuropathy. Purification of the b4-2 solute allows its quantitative determination in biological fluids. The b4-2 plasma concentrations are 1 mg/L in healthy subjects (n=30), 4.6 +/- 0.2 mg/L in uremic patients (n=67) and 13-19 mg/L in six polyneuropathic patients. The 24-hr urinary excretion is 13 +/- 1 mg. The weekly removal rates in hemofiltration or in hemodialysis, using high permeability membrane three times a week, are 38-40 mg. In an in vitro sural nerve test for the evalution of middle molecule neurotoxicity, the b4-2 solute exhibits a neurotoxic effect at concentrations similar to those found in plasma of neuropathic patients. Preliminary results of an attempt to identify the neurotoxin indicate that it is an acid-polyol derivative.
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