A microelectrode for continuous recording of volume fluxes in isolated perfused tubule segments

Manufacture, properties and use of a micro enzyme electrode for continuous monitoring of volume fluxes in the isolated tubule preparation is described. The specific electrode is a galactose-oxidase enzyme electrode, which can be used to detect changes in raffinose concentrations. The electrode's response to raffinose is almost linear over concentrations from 0-12 mmol/l. The electrode equally responds to galactose as to raffinose but is insensitive to other sugars, to pH changes (from 6.0-8.0), CO2 (from 1-10%) and electrolytes tested. Reducing O2 from 100 to 10% and to 1%, leads to a reduction of the reading by 10% and 30%, respectively. The reading is almost doubled when the temperature is increased from 20-40 degrees C. Furthermore, reducing agents such as uric acid and ascorbic acid interfere with the reading. If these substances and raffinose are omitted from the perfusate for isolated perfused proximal mouse tubules, the reading is identical in perfusate and collected fluid, indicating that the tubular epithelium does not produce substances in sufficient amounts to interfere with the electrode reading. After addition of 6 mmol/l raffinose to the perfusate the raffinose concentration in the collected fluid of 0.76 +/- 0.05 mm segments of straight proximal mouse tubules (perfusion rate = 3.4 +/- 0.45 nl/min) is 10.2 +/- 0.3 mmol/l, indicating a volume reabsorption of 1.5 +/- 0.3 nl/min. Peritubular application of acetazolamide reduces the volume reabsorption by 42 +/- 4%.