Fluorometric analysis of epinephrine and norepinephrine.

A modification of the classic trihydroxyindole method for the fluorometric estimation of epinephrine and norepinephrine is presented. Assay procedures for each catecholamine were separately optimized, significantly increasing the sensitivity and specificity of the procedures. Methodology has been simplified through the use of intersolution calibration, making this assay suitable for use in research or clinical laboratories. Performance of both epinephrine and norepinephrine assays are possible on 1 ml of deproteinated plasma or urine using these procedures.     

Cytochemistry of epinephrine and norepinephrine adrenomedullary cells

Summary Rat adrenomedullary tissue was fixed in 3% glutaraldehyde in a variety of buffers at different osmolarities. Incubations were performed in potassium dichromate (DC) at pH 4.1 to identify norepinephrine (NE) specifically and in osmium tetroxide to enhance contrast and to define membrane structures. Different buffer systems were also used for the DC and the osmium processes. It is evident from the results that both the osmolarity of the initial fixative and the incubation process itself are critical for adrenomedullary catecholamine (CAM) localization. Furthermore, the Schiff monobase formed by the amine and the aldehyde in the initial reaction was found to be reactive before and even after the tissue had been epoxy-embedded. Thus the technique of on-grid staining of tissue with DC has now been made possible and works sufficiently well to show a high degree of promise for future investigations. In vitro experiments were performed using purified CAMs. Reactions were conducted using the purified CAMs in solution with glutaraldehyde. The resulting reaction products then were incubated with DC and collected by centrifugation. The amount of chromium (Cr) present in the products was determined by the use of an electron probe as well as by chemical analyses by a commercial laboratory. In all cases it was evident that indeed Cr was in the reaction product, indicating this to be the electron-dense product seen microscopically in the tissue. In essence, as a result of this investigation, the specificity of the glutaraldehyde-DC pH 4.1 reaction has been made more sensitive, more utilizable cytochemically, and further substantiated by the chemical analyses of in vitro reaction products. Moreover, the on-grid staining technique leads to a new concept, not only in the electron microscopy of CAMs but in other heavy metal reactions as well.This investigation was supported by HEW Grant NS06980 and the Nathanael Henry Asten Fund for Research in Paralysis Agitans.     

Plasma norepinephrine and epinephrine levels in essential hypertension

Almost all comparative studies of plasma catecholamines in patients with essential hypertension (EH) and in normotensive controls have reported higher mean norepinephrine (NE) or epinephrine (E) levels in the hypertensive patients, but only about 40% of the studies have been positive, i.e., have reported statistically significant hypertensive-normotensive (H-N) differences. Virtually all studies of NE in young, consistently hypertensive patients were positive, as well as all studies of E in relatively tachycardic patients. Plasma NE increased with age in normotensive control but not EH groups. The likelihood that a study was positive with respect to NE was independent of the likelihood with respect to E. In 189 individuals with EH and 130 normotensive controls, NE increased with age in controls but not in patients with EH, so that the extent of the H-N difference in NE varied inversely with patient age. Among 41 other individuals with EH and 59 other normotensive controls, the distributions of NE and E values were shifted upward in EH. NE and E values were uncorrelated. Plasma NE levels are abnormally high in some patients with EH--especially those who are young and consistently hypertensive--and E levels are independent of age and NE. Increased sympathetic nervous system and/or sympathoadrenomedullary activity characterize a proportion of patients with EH.     

Urinary epinephrine and norepinephrine levels in women athletes during training and competition.

Training and competitive epinephrine and norepinephrine levels and proportions were compared in two groups of women athletes to determine whether changes in catecholamine excretion reflect the added mental stress of athletic competition on physical effort. An intercollegiate basketball team and a group of track and field athletes volunteered as subjects. Competitive epinephrine urinary levels were significantly (P less than 0.01) higher than training levels. A concomitant rise in the norepinephrine with an increase in physical effort was observed in both groups of athletes following training sessions as well as after athletic competition. Track and field athletes trying to qualify for an international team exhibited significantly ( less than 0.01) higher epinephrine levels than the team members; thus suggesting that anticipation of competition imposes a mental stress on an athlete. Constant changes in the catecholamine pattern as against a normal work load have yet to be established.     

ACTH stimulation of adrenal epinephrine and norepinephrine release

Epinephrine (E) and norepinephrine (NE) levels were measured simultaneously in the adrenal veins of 6 patients before and after stimulation with 0.25 mg beta 1-24 ACTH. In 1 patient with Cushing's syndrome, E and NE were also measured before and 30 min after dexamethasone. There was a significant increase in NE and E secretion (p less than 0.002) from both adrenal glands after ACTH stimulation. In the patient with Cushing's syndrome, there was also a slight increase in plasma E levels after dexamethasone. It is postulated that ACTH stimulated NE and E secretion by augmenting blood flow through the adrenals and by induction of tyrosine hydroxylase and dopamine beta-hydroxylase, although a direct effect of ACTH on NE and E secretion cannot be excluded. It is also possible that the increase in adrenal catecholamine secretion after ACTH may be due to ACTH augmentation of catecholamine secretion by endogenous opioids such as beta-endorphin.     

A radioimmunoassay for epinephrine and norepinephrine in tissues and plasma

An I¹²⁵ radioimmunoassay (RIA) has been developed for the measurement of plasma and tissue epinephrine (E) and norepinephrine (NE). The assay utilizes an antibody which specifically binds metanephrine. E and NE are detected by conversion to metanephrine with the enzymes catechol-0-methyltransferase and phenylethanol-amine-N-methyltransferase. The assay is very specific and will allow the measurement of E and NE in less than 500 μl of normal human plasma. E and NE concentrations were determined by both the RIA and a radioenzymatic assay in canine, human and rat biologic samples. The correlation coefficients between the two assays were .962 for E and .956 for NE. The RIA is sensitive, specific, precise and significantly less costly and time consuming than present radioenzymatic methods.     

A simplified radiometric assay for plasma norepinephrine and epinephrine

An assay for plasma norepinephrine and epinephrine levels has been developed by the modification of published procedures. The plasma norepinephrine and epinephrine assay, when compared to currently available methods, provides a substantial decrease in the assay time while providing a 10-fold increase in sensitivity which allows the analysis to be performed on 0.75 ml or less of plasma. Norepinephrine and epinephrine are converted to their O-methylated analogs in the presence of catechol-O-methyl transferase and S-adenosylmethionine-methyl-³H. Following purification of the labelled normetanephrine and metanephrine by solvent extraction and thinlayer chormatography, the amines are oxidized to vanillin, purified by solvent extraction and counted. The specificity, linearity and precision of the assay are discussed.