Determination of metanephrines in plasma by liquid chromatography with electrochemical detection

Metanephrines are O-methylated metabolites of catecholamines. We report the use of liquid chromatography with electrochemical detection to determine plasma concentrations of normetanephrine (NMN) and metanephrine (MN). Plasma NMN and MN in 32 normal volunteers and inpatients were compared with concentrations in 23 patients with pheochromocytoma. Metanephrines were adsorbed from plasma onto a cation-exchange column and eluted with ammoniacal methanol. The dried residue was dissolved in mobile phase and injected onto a reversed-phase column. Recoveries of NMN and MN from 1 mL of plasma averaged 50-70%, and results varied linearly with quantity injected over a range of 0.13-55 pmol. The detection limit was 25 fmol for NMN and 50 fmol for MN. Intra-assay CVs were < 5%. In normal volunteers and inpatients, plasma concentrations of NMN ranged between 0.12 and 0.73 nmol/L (mean 0.38 nmol/L), and MN between 0.06 and 0.63 nmol/L (mean 0.19 nmol/L). Plasma NMN concentrations were increased in all 23 patients with pheochromocytoma (range 1-172 nmol/L), whereas MN concentrations (range 0.10-382 nmol/L) were increased in only 9 patients. The assay method is reliable and sensitive and offers an approach to examine the extraneuronal metabolism of catecholamines. The method may also be useful in the diagnosis of pheochromocytoma.

Uptake and metabolism of norepinephrine by endothelium of dog pulmonary artery

The contribution of endothelium of dog pulmonary artery to the extraneuronal metabolism of norepinephrine was determined. Pulmonary artery was cut into helical strips; the endothelium was removed from half of the strips by gently stroking them with a wooden applicator stick. All strips were immersed in l-[3H]norepinephrine (2 X 10(-7) M) and mounted for superfusion. Superfusate was collected continuously before, during, and after electrical stimulation (10 V, 2 ms, 2 Hz). Column chromatography was used to separate [3H]norepinephrine and its radiolabeled metabolites in superfusate. Quantitation was by liquid scintillation spectrometry. Previous studies have established that 3,4-dihydroxyphenylglycol is of neuronal origin and that O-methylated metabolites are of extraneuronal origin. Since cocaine prevented neuronal uptake of norepinephrine, the reduction in metabolites of norepinephrine of extraneuronal origin in arteries with endothelium removed represented the contribution of endothelium to extraneuronal metabolism. O-Methylated metabolites were decreased from 3.50 to 2.17 X 10(3) dpm/2 ml of superfusate during basal conditions preceding electrical stimulation and from 11.16 to 6.94 X 10(3) dpm/2 ml of superfusate during electrical stimulation when endothelium had been removed. Decreases in extraneuronal metabolite production continued throughout the basal periods following stimulation. These studies suggest that in small pulmonary artery a substantial amount of the total norepinephrine that is released at the neuroeffector junction may be metabolized following uptake into endothelium.

Metabolism of norepinephrine in vitro by dog pulmonary arterial endothelium

The importance of endothelial cells in the removal of norepinephrine from synaptic clefts in dog pulmonary artery was studied. Strips of artery cut helically were denuded of endothelium by gently stroking the intimal surface with a wooden applicator stick or were studied with endothelium intact. All strips were prelabeled in L-[3H]norepinephrine (2 X 10(-7) M) and mounted for superfusion. Superfusate was collected continuously before, during, and after electrical stimulation (10 V, 2 ms, 2 Hz). Measurements were made of the amounts of [3H]norepinephrine and its metabolites in superfusate and in tissue. These studies have established that 3,4-dihydroxyphenylglycol is of neuronal origin and O-methylated metabolites are of extraneuronal origin. The formation of extraneuronal metabolites in smooth muscle and endothelium was examined by either blocking uptake of norepinephrine into each, in turn, or by combining blockade of uptakes with the elimination of access of norepinephrine to endothelial tissue by completely removing it. Electrical stimulation elicited the overflow of large amounts of norepinephrine, 3,4-dihydroxyphenylglycol, and O-methylated metabolites into superfusate in strips with intact endothelium; in strips denuded of endothelium there were striking decreases in the amounts of O-methylated metabolites produced. These studies show that pulmonary arterial endothelium participates in the extraneuronal metabolism of norepinephrine released by sympathetic nerve stimulation.