A sensitive radioimmunoassay of atrial natriuretic peptide in human plasma, using a tracer with an immobilized glycouril agent.

A highly specific and sensitive radioimmunoassay (RIA) for alpha-human atrial natriuretic peptide (hANP[1-28]) in plasma was developed. The assay used a [125I]monoiodotyrosyl-hANP[1-28] tracer, prepared with an immobilized glycouril agent (Protag) and purified by high pressure liquid chromatography (HPLC), and a highly specific antiserum raised against hANP[1-28], coupled to keyhole limpet haemocyanin, in sheep. Plasma was extracted using C-18 Seppak cartridges. A good parallelism was found after dilution prior to extraction of plasma of patients with congestive heart failure (CHF) or of plasma of healthy subjects. Recovery of hANP[1-28] added to plasma was 96%. The limit of detection was 0.8 pg/tube, intra- and inter-assay variation were 9 and 12%, respectively. Mean plasma ANP values in 25 normal persons with a normal salt intake was 26.0 +/- 15.5 (+/- SD) pg/ml. Plasma levels of 18 subjects (7 normals, 11 CHF) were measured using four different antisera after the extraction step. High correlations were found between the values obtained with these four antisera.

Evaluation of a direct assay for atrial natriuretic peptide.

A direct radioimmunoassay for the assessment of human atrial natriuretic peptide (ANP) in plasma, using a highly specific antibody and a well-defined monoiodotyrosyl-tracer was developed and evaluated by concurrent application of an extraction method. Sensitivity was 13.4 pg/ml; intra- and interassay variations were 3.1 and 5.5%, respectively; recovery averaged 99%; normal values ranged from 15-111 pg/ml (mean +/- SD = 59 +/- 25 pg/ml, n = 41). The results, including the effect of exercise, of the two methods correlated well. Pooling ANP values in normal subjects and patients with congestive heart failure gave a good correlation (p less than 0.01). However, due to processes in unextracted plasma, in the lower range the results from the direct method were erratic. Velocity and duration of centrifugation changed the number of platelets, but no effect on ANP levels, whether assessed by the direct or by the extraction method, was observed. Although the direct method is considerably less laborious than the extracted method its lack of reliability disqualifies it for most purposes.

Atrial natriuretic peptide--the influence of various physiological and sampling conditions.

A study of various physiological conditions, possibly influencing levels of atrial natriuretic peptide (ANP), is described. Atrial natriuretic peptide was determined by a radioimmunoassay, suitable for routine measurements, using 1 mL of human plasma. Atrial naturetic peptide was adsorbed onto Sep-pak C-18 cartridges, eluted with acidified ethanol and subsequently radioimmunoassayed. The detection limit was slightly less than 2 pg/tube (approximately 5 pg/mL plasma). Intra- and interassay coefficients of variation were 7 and 10%, respectively. Study of various sampling conditions revealed that blood sampling in EDTA-tubes kept on ice, and centrifuged within 1 h, gives the most reliable results. Reference values in 74 individuals ranged between 10 to 69 pg/mL (mean +/- SD = 30 +/- 11 pg/mL). We observed no difference in reference values when the blood sampling procedure was in the sitting (29 +/- 11 pg/mL, n = 43) or supine (31 +/- 12 pg/mL, n = 31) position. Venepuncture stress did not consistently change ANP-levels. No difference was observed in ANP between the follicular and the luteal phase of the menstrual cycle. ANP levels of 7 ambulant female subjects declined significantly during the study period from 7 a.m. to 3 p.m.

A sensitive radioimmunoassay of atrial natriuretic peptide in human plasma; some guidelines for clinical applications.

We report the development of an in-house RIA for human alpha-ANP. The method requires the extraction of EDTA-plasma on C18-cartridges followed by radioimmunoassay of the eluates with an antibody raised in sheep against human-alpha-ANP coupled to Keyhole Limped Hemocyanin, and a monoiodotyrosyl-alpha-ANP tracer purified with HPLC before use. The assay is precise and sensitive: intra- and interassay coefficients of variation were 8.6% and 11.6%, respectively, and the lower limit of detection was 0.8 pg/tube (4.0 pg/ml plasma). Normal values were 26.0 +/- 15.5 (mean +/- sd) pg/ml plasma (n = 25). Change from supine to sitting position significantly lowered the ANP immunoreactivity. No episodic variations were observed. Our method was applied in a study comparing plasma ANP values in samples of normal persons and CHF-patients. Assay of ANP after extraction using four different antibodies showed significant correlations. HPLC analysis of both plasma of CHF patients and plasma of normal persons revealed the existence of two major immunoreactive components besides alpha-ANP which were not detected in a radioreceptor assay.

Plasma immunoreactive endothelin-1 in pregnant women with and without pre-eclampsia.

In normotensive nonpregnant women (n = 23), the plasma concentrations of immunoreactive endothelin-1 (ir-ET-1) were not different from nonpregnant women with essential hypertension (n = 15): 3.6 (2.0-5.4) ng/L (mean, range) versus 3.8 (2.4-5.8) ng/L. In normotensive pregnant women (n = 25), the plasma level of ir-ET-1 was 2.1 (1.3-3.4) ng/L (p less than 0.01) lower than in normotensive nonpregnant women. Pre-eclamptic patients (n = 25) had elevated ir-ET-1 plasma levels of 5.0 (2.1-12.4) ng/L (p less than 0.001 for normotensive pregnant women, p less than 0.01 for nonpregnant women). The low level of ir-ET-1 in normotensive pregnant women may be explained by the increase in the distribution volume of ir-ET-1 during the course of pregnancy. Damage of vascular endothelium is a consistent morphological abnormality in pre-eclampsia. Elevated ir-ET-1 in plasma might be a biochemical marker of this abnormality and may contribute to the pathogenesis of pre-eclampsia.