Radioimmunoassay of vasopressin during pregnancy. Use and removal of cystylaminopeptidase inhibitors.

This paper describes a radioimmunoassay (RIA) for arginine-vasopressin in which o-phenanthroline effectively inhibits cystyl-amino-peptidase activity in whole blood and plasma from pregnant women but in which o-phenanthroline is removed during the extraction of vasopressin from plasma to prevent disturbance of the RIA. Cystyl-amino-peptidase causes immediate degradation of vasopressin unless cystyl-amino-peptidase enzyme inhibitors such as o-phenanthroline are applied. However, o-phenanthroline interferes with RIA. We report an extraction procedure over octadecasyl silica-packed Sep-Pak C18 columns, by which cystyl-amino-peptidase as well as most of the cystyl-amino-peptidase inhibitor is removed from plasma with chloroform. The average o-phenanthroline concentration (0.25 mmol/l) found in the assay medium after extraction appeared not to interfere with the RIA. Polyacrylamide gel isoelectric focusing of extracts of platelet-rich and platelet-poor plasma from pregnant women revealed a single vasopressin immunoreactive peak in the RIA. Recovery and between-assay coefficients of variation of 3.2 ng/l vasopressin from pregnancy whole blood were comparable with non-pregnant controls (57%/8% and 59%/13%, respectively). Results with this assay compare well with those of another assay using inhibitors in pregnant subjects and with results in non-pregnant subjects.

Preeclampsia is not associated with altered platelet vasopressin binding and cytosolic Ca++ concentration.

OBJECTIVES: Preeclampsia is an important cause of fetal and maternal morbidity and mortality. Recently it was described that platelet cytosolic Ca++ levels could be used to screen for preeclampsia. The current study investigated platelet arginine vasopressin receptor characteristics, platelet cytosolic Ca++ levels, plasma- and platelet-bound arginine vasopressin in white pregnant women. STUDY DESIGN: In a cross-sectional study nine third-trimester nulliparous pregnant women with gestational hypertension (seven with proteinuria, two with excessive weight gain without proteinuria) were compared with nine healthy nulliparous pregnant women matched for gestation length and age and 10 healthy age-matched nonpregnant women. Determined were (1) platelet arginine vasopressin receptor number and affinity, (2) platelet cytosolic Ca++ levels, both basal and on arginine vasopressin or thrombin stimulation, and (3) plasma- and platelet-bound arginine vasopressin levels. RESULTS: None of the measured parameters differed significantly among the three groups studied. Mean arginine vasopressin receptor number and affinity ranged from 108 to 143 receptors per platelet and 0.35 to 0.40 nmol/L, respectively. A single population of binding sites was found (Hill number 0.96). Basal Ca++ levels ranged from 113.4 to 133.3 nmol/L, on arginine vasopressin stimulation from 199 to 250 nmol/L. Median arginine vasopressin levels in platelet-poor plasma were between 1.2 and 2.4 pg/ml, with circulating platelets being estimated to possess two to five molecules of arginine vasopressin per platelet. A significant correlation was found between platelet cytosolic Ca++ levels before and after arginine vasopressin stimulation (r = 0.69, p < 0.001) and a weak correlation between platelet receptor density and arginine vasopressin-stimulated platelet cytosolic Ca++ levels (r = 0.38, p < 0.05). CONCLUSIONS: The studied parameters, platelet cytosolic Ca++ levels, whether basal or after stimulation with arginine vasopressin and vasopressin platelet receptor density and affinity, do not discriminate already hypertensive or preeclamptic white women from nondiseased subjects. A valuable test to screen for preeclampsia awaits further prospective studies.