Determination of endogenous norepinephrine levels in different chambers of the rat heart by capillary electrophoresis coupled with amperometric detection

Abstract

Capillary electrophoresis with end-column amperometric detection (CE-EC) was used to determine the regional distribution of norepinephrine (NE) in the hearts of sympathetically innervated (control) and chemically sympathectomized rats. Key features of the method are (i) the sample preparation and clean-up step that involved the application of off-line solid phase extraction (SPE) with a 95% NE recovery and (ii) the use of a diamond microelectrode for detection. NE was quantified in the left and right ventricle, the ventricular septum, and the left and right atrium. The NE concentration in the atria was three to five times higher than in the ventricles and ventricular septum of control rats. Basal NE levels in the left and right ventricle and the ventricular septum were reduced to below the detection limit (0.034 microg/g tissue) in tissues treated with the neurotoxin, 6-hydroxydopamine (6-OHDA), while only a moderate reduction was observed in the left and right atrium. Importantly, the diamond microelectrode provided low and stable background current and low peak-to-peak noise <or=0.65 pA at a detection potential of +0.86 V versus Ag/AgCl. A reproducible electrode response was observed for multiple injections of tissue homogenates with minimal response attenuation due to electrode fouling.

Figure 1

Electropherogram for a left atrium homogenate from a control rat recorded at a detection potential of 0.95 V (top curve) and 0.86 V vs Ag/AgCl (bottom curve). CE conditions: 29 μm i.d. × 76 cm fused silica capillary, 250 mM boric acid/KOH buffer, pH 8.8, applied voltage 24 kV, electrokinetic injection 18 kV/8s.

Figure 2

Comparison of electropherograms obtained for left ventricle (top) and ventricular septum (bottom) homogenates from a control rat. CE-EC conditions: 29 μm i.d. × 76 cm fused silica capillary, 250 mM boric acid/KOH buffer, pH 8.8, applied voltage 24kV, electrokinetic injection 18 kV/8s, detection potential +0.86 V vs Ag/AgCl.

Figure 3

Quantitation of norepinephrine by CE-EC in heart tissue compartments, left ventricle (LV), ventricular septum (VS), right ventricle (RV), left atrium (LA) and right atrium (RA). Columns represent values for rats 1, 2 and 3 of the control group (patterned columns) and rats 4 and 5 of the 6-OHDA treated group (solid columns). Values are expressed as μg of NE per g of frozen tissue (±RSD, vertical bar).

Figure 4

Comparison of electropherograms for right ventricle homogenates from a control (top curve) and a chemically sympathectomized (bottom curve) rat. The inset shows an enlarged view of 450–600 s time window. CE-EC conditions: 29 μm i.d. × 76 cm fused silica capillary, 250 mM boric acid/KOH buffer, pH 8.8, applied voltage 24kV, electrokinetic injection 18 kV/8s, detection potential +0.86 V vs Ag/AgCl.