Liquid Chromatographic-Electrochemical Measurement of Dopamine in Hypophysial Stalk Blood of Rats*

Abstract

Evidence is accumulating that dopamine is the hypothalamic PRL[prolactin]-inhibiting factor, but the quantitative relationship between its secretion and that of PRL has not been established. A liquid chromatographic-electrochemical assay was developed for measurement of dopamine and other catecholamines in hypophysial stalk plasma. Dopamine in plasma was adsorbed onto alumina, eluted with 0.5 M HClO4 and injected into a cation exchange column. Elution of dopamine occurred in 17 min using a citrate-acetate buffer (pH 5.2) at a flow rate of 20-30 ml/h (500 psi). Detection resulted from oxidation of dopamine at the surface of a carbon plast electrode, with the resulting electrochemical current being linearly related to dopamine from 20 pg-10 ng. With a 50% extraction loss of dopamine, the limit of detection using 100 .mu.l plasma was 0.4-0.6 ng/ml. Specificity is provided by the limited number of compounds having oxidation potentials similar to dopamine, by alumina adsorbing only catechols and by separation of dopamine on the ion exchange column. Coefficients of variation for repeated determinations of dopamine concentrations were in the range of 3-7%. Dopamine concentrations in plasma of hypophysial stalk blood collected from rats under pentobarbital anesthesia at diestrus-2 and proestrus, ranged from 0.7-9.0 ng/ml. The dopamine ratios of hypophysial stalk vs. peripheral plasma of individual rats ranged from > 2-> 38. Dopamine levels in stalk plasma and PRL concentrations in peripheral plasma were not significantly correlated (r = 0.47; P > 0.05). Stalk plasma dopamine levels in diestrus-2 and proestrus animals (3.0 .+-. 1.5 vs. 1.2 .+-. 0.5 ng/ml, respectively) were not significantly different (P < 0.05). The results demonstrate the validity and usefulness of the liquid chromatographic-electrochemical assay for measurements of dopamine in hypophysial stalk plasma. This assay will permit quantitative studies on the role of dopamine as a regulator of PRL secretion.