Dilution of plasma with Tris buffer increases measured catecholamines in plasma.

We investigated the effects of dilution of plasma samples on the measured concentrations of catecholamines. Diluting samples of human plasma 10-, 50-, and 100-fold with Tris buffer (100 mol/L, pH 8.6) improved analytical recovery of internal standards, suggesting that it decreases the commonly observed inhibition of methylation in radioenzymatic assays of catecholamines in plasma. However, the dilution is not associated with a proportional decrease in counted radioactivity. This extra amount of radioactivity, which is unlikely to be nonspecific in origin, accounts for a significant increase in the calculated catecholamine concentration. Tentatively, we suggest that Tris buffer releases both catecholamines and conjugated catecholamines bound to some unidentified low-molecular-mass component of plasma.

Central adrenergic activity in the mediobasal hypothalamus is temporally related to surge outputs of gonadotrophins in oestrogen-stimulated infant female rats

ABSTRACT In the mediobasal hypothalamus (MBH) of prooestrous rats or acutely ovariectomized oestrogentreated adults a marked but short-lived increase in adrenergic activity occurs at 16.00 h, 2 h before the oestrogen-dependent surge of gonadotrophins at 18.00 h. In this study oestrogen-stimulated (noon on day 1) 22-day-old female rats were used which are known to produce surge levels of prolactin at 18.00 h on day 2 and surges of both prolactin and LH at 18.00 h on day 3; although similar treatment of 18-day-old animals or oil-treated 22-day-old rats failed to produce these effects. Radioenzymatic assays of adrenaline concentrations and of the activity of its synthesizing enzyme (phenylethanolamine-N-methyl transferase; PNMT, EC 2.1.1.28) in the MBH of oestrogen-treated 22-day-old rats showed significant (P< 0·05–0·01) increases in both parameters at 16.00 h (i.e. 2 h before surge levels of gonadotrophins) on days 2 and 3 when compared with other times of day. Such effects were not seen in oil-treated 22-day-old animals or in oestrogen-treated 16-day-old rats. Noradrenaline and dopamine concentrations in the MBH of oestrogen-treated 22-day-old rats remained at baseline levels on days 2 and 3 with the exception of noradrenaline at 17.00 h on day 3 when levels appeared higher (P<0·05) than at either 15.00 or 16.00 h. Subsequent measurements of PNMT activity in oestrogen-treated 22-day-old rats at 4-hourly intervals throughout days 2 and 3 showed the presence of a clear circadian rhythm with peak levels occurring at 16.00 h. In conclusion, a temporal relationship (not necessarily specific) exists between increased adrenergic activity in the MBH of oestrogen-treated 22-day-old rats and a surge of gonadotrophins (LH and/or prolactin) 2 h later. This relationship apparently depends on an oestrogen-stimulated circadian rhythm of PNMT activity. J. Endocr. (1986) 109, 45–51

Methods of plasma catecholamine measurement including radioimmunoassay

Fluorometric and radioenzymatic assays are presently the most widely used techniques for the estimation of plasma, urine, and tissue catecholamines. The fluorometric assay lacks specificity and sensitivity. The radioenzymatic assay is significantly more sensitive and specific but is technically very complex, time consuming, and expensive. A newer methodology has been developed by modification of a 125I radioimmunoassay for metanephrine. The assay utilizes an antibody that specifically binds metanephrine. Plasma and urinary epinephrine and norepinephrine are detected by conversion to metanephrine with the enzymes catechol-O-methyl-transferase (COMT) and phenylethanolamine-N-methyltransferase (PNMT). The major advantages of the radioimmunoassay are the savings in cost and time. The radioenzymatic assay utilizes an expensive tritium-labeled compound, S-adenosylmethionine, and requires multiple organic solvent-extraction steps, thin-layer chromatography, and liquid scintillation counting. The radioimmunoassay requires only one extraction with alumina to aid in specificity and to concentrate the catecholamines. Sample detection is by gamma counting. The radioenzymatic assay is presently the reference method for catecholamines and is best suited for small numbers of samples where sample volume is limited and exquisite sensitivity is required. The radioimmunoassay is rapid, has sufficient sensitivity, specificity, and precision for most applications and is best applied to the analysis of large numbers of samples.

Novel Double-Isotope Technique for Enzymatic Assay of Catecholamines, Permitting High Precision, Sensitivity and Plasma Sample Capacity

1. A novel use of a double-isotope method is described which allows radioenzymatic assays to combine precision and sensitivity. 2. In the catechol O-methyltransferase assay separate portions of each plasma sample are incubated with either S-[3H]- or S-[14C]-adenosyl-l-methionine. Standards of noradrenaline and adrenaline are added to the latter portions and are thus converted into standards of [14C]metadrenalines. These are added to the 3H-labelled portions after the incubation, where they function as tracers. 3. The final recovery of 14C radioactivity corrects for (a) the efficiency of methylation in the plasma sample concerned and (b) the recovery of metadrenalines during the extraction procedures. 4. The 3H/14C ratio is constant in each assay for a given catecholamine concentration and is determined for samples to which standards of noradrenaline and adrenaline are added to the 3H- (as well as the I4C-) labelled portions before the initial incubation. 5. The sensitivity of the assay is increased by using high specific radioactivity S-[3H]adenosyl-l-methionine (60-85 Ci/mmol), and low backgrounds are maintained by catecholamine depletion in vivo in the rats used for enzyme preparation. 6. Both catecholamines (1.5 pg/ml; 10 pmol/l) may be detected; the coefficients of variation are 3.0 and 3.2% for noradrenaline and adrenaline respectively (intra-assay) and 4.6 and 5.0% (inter-assay).