Polarographic measurement of ascorbate washout in isolated perfused rabbit hearts

Abstract

To study the myocardial washout of ascorbate, the applicability of polarographic detection of ascorbate ions by a platinum electrode (sensitive area 0.03 mm2) was investigated, in both a calibration setup (sampling flow along the electrode: 100 microliter X s-1) and isolated, retrogradely perfused rabbit hearts. In the calibration setup at pH 7.4, the sensitivity of the electrode was 70 microA/mol. This sensitivity increased moderately with increasing pH (13%/unit pH) and increasing sampling flow rate (14% at an increase from 100 to 150 microliter X s-1). In the isolated hearts, ascorbate infused into the aorta was detected in a right ventricular drain by the electrode as well as by the use of 14C-labeled ascorbate. Both recorded time courses were similar except for a scaling factor dependent on flow velocity. During continuous infusion the arteriovenous difference of ascorbate was 2 +/- 2% (SD), indicating a relatively low consumption of ascorbate by the isolated heart. We conclude that polarographic measurement of ascorbate in the coronary effluent of an isolated rabbit heart can be performed on-line and relatively easily.

FIG. 1

Design of the electrodes. Upper panel: platinum electrode. A, platinum wire; B, heat shrink insulation; C, wire connection. Lower panel: Ag-AgCl reference electrode. D, cotton wick; E, 0.1 N KCl solution; F, chlorided silver wire; G, wire connection; H, tubing filled with KCl solution; J, stopcock.

FIG. 2

Current amplifier circuit for electrodes. Pt, platinum electrode; Ag-AgCl, silver chloride reference electrode; FB, auxiliary current feedback electrode.

FIG. 3

Schematic representation of the experimental setup for obtaining ascorbate outflow dilution curves via platinum electrode detection and sequential sampling for measurement of tracer concentration-time curves. Indicator is injected or infused into cannulated aorta of isolated, perfused rabbit hearts. Sample collection tubes are translated by a rotating collector.

FIG. 4

Calibration of electrode system for ascorbic acid in calibration setup. Electrode current as a function of ascorbic acid concentration.

FIG. 5

Calibration of electrode system for ascorbic acid in calibration setup. Sensitivity of electrode current to changes in sampling flow velocity.

FIG. 6

Ascorbatae outflow dilution curves from an isolated perfused rabbit heart following aortic injection at 3 different flows. Continuous lines, polarographic detection. Symbols: [¹⁴C]ascorbate at flows of 39 (○), 103 (●), and 147 (□) μl·g^–1·s^–1. Scale factors for multiplying each electrode curve to superimpose [¹⁴C]ascorbate curves were 5.3, 5.4, and 3.7 counts/pA, respectively. Perfusion pressures at various flow levels were 35, 85, and 125 mmHg. Lower apparent concentration by Pt electrode in tail portion of curve obtained at lowest flow suggests a small degree of chemical transformation.