An improved radioenzymatic assay for plasma norepinephrine using purified phenylethanolamine N-methyltransferase.
Radioenzymatic assays have been developed for norepinephrine (NE) using either catechol O-methyltransferase (COMT) or phenylethanolamine N-methyltransferase (PNMT). Assays using PNMT are specific for NE but have been considered less sensitive than the more complex assay procedures employing COMT. An improved purification procedure for bovine PNMT has permitted development of a NE assay with substantially improved sensitivity (less than 0.5 pg), reproducibility, and decreased manipulative effort. PNMT was purified by sequential pH 5.0 treatment and dialysis and by column chromatographic procedures using DEAE-Sephacel, Sephacryl S-200 and Phenyl Boronate-agarose. Recovery of PNMT activity through the purification scheme was 50% while blank recovery was less than 0.001%. Norepinephrine can be directly quantified in 25 microliters of human plasma and a seventy-tube assay can be routinely completed within 4 h. The capillary to venous plasma NE gradient was examined in eight normotensive male subjects. Capillary plasma NE (211 +/- 21.7 pg/ml) was significantly lower than venous plasma NE (367 +/- 32.7 pg/ml) in all subjects (p less than 0.005). This difference suggests the concentration of NE in capillary blood may be a unique indicator of sympathetic nervous system activity in vivo.
D000313:Adrenal Medulla / Q000201:enzymology; D000818:Animals; D001794:Blood Pressure; D002417:Cattle; D002850:Chromatography, Gel; D006801:Humans; D007700:Kinetics; D009638:Norepinephrine / Q000097:blood*; D010625:Phenylethanolamine N-Methyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D013379:Substrate Specificity; D014316:Tritium
D014316:Tritium; D010625:Phenylethanolamine N-Methyltransferase; D009638:Norepinephrine