A.C.I.T.L. Tan

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.

Atrial natriuretic peptide after myocardial infarction

Plasma concentrations of atrial natriuretic peptide (ANP) after acute myocardial infarction were measured at fixed times during 48 hours in 38 patients admitted to the hospital within 4.4 hours after the onset of symptoms. Three hours after admission, the mean concentration of ANP was significantly lower than that at the time of admission. Thereafter it rose steadily until 15 hours after admission. ANP concentrations measured in each patient at the time of admission and the individual mean ANP concentrations during the first 48 hours after admission correlated weakly but significantly with the size of the infarct and the left ventricular function. Neither the site of the infarct, the occurrence of reperfusion, nor the number of coronary vessels affected influenced the ANP concentration. In 24 patients in whom cardiac catheterization was performed, no relationship between ANP concentrations and left ventricular pressures was observed. Determination of ANP concentrations seems to be of little value in assessing cardiac function after acute myocardial infarction.

Atrial Natriuretic Peptide

SummaryThe atrial natriuretic peptide (ANP) is part of a new family of cardiac hormones regulating water and salt homeostasis. Besides acting as a blood pressure-lowering agent, it also exerts potent natriuretic and diuretic effects. ANP can be considered an endogenous antagonist of the renin-angiotensin-aldosterone system and the antidiuretic hormone. One of the roles of ANP is to protect the body against fluid overload: it decreases intravascular fluid volume, which in turn diminishes cardiac secretion of ANP. The pharmacokinetic parameters of ANP reported in the literature vary widely. In general, ANP rapidly disappears from plasma with a high total body clearance. This is in agreement with the short-lived effects of the hormone. The actions of ANP have led to efforts to use this peptide hormone in the treatment of various cardiovascular dis-orders such as hypertension and congestive heart failure. Intravenous ANP administration indeed resulted in beneficial effects in these disorders. However, the peptide nature of ANP and its rapid elimination from the circulation limit its suitability as a drug. More promising is the development of long-acting ANP analogues and inhibitors of ANP degradation. Proper understanding of ANP pharmacokinetics is essential for the clinical use of these pharmacological agents.

The effect of volume expansion on atrial natriuretic peptide and cyclic guanosine monophosphate levels in young and aged subjects.

The relationship between atrial natriuretic peptide (ANP) and age was studied. In elderly subjects (65 to 84 years) higher basal ANP levels were found as compared with those found in young subjects (21 to 23 years). Volume loading (750 mL 0.9% NaCl in 60 minutes) increased ANP levels only in the aged group, and plasma renin activity decreased only in the young subjects. Plasma aldosterone levels did not change. Basal cyclic guanosine monophosphate values did not differ in either group, and rose to a similar extent during volume loading. High pressure liquid chromatography analysis did not suggest different molecular forms of immunoreactive ANP in elderly subjects. The main circulating component both basal and after stimulation corresponds to the (99–126)hANP form.

Kinetics of atrial natriuretic peptide in young and elderly subjects

SummaryTo study the influence of age on the kinetics of atrial natriuretic peptide (ANP) in man, human (99–126) ANP 2.0 μg·min−1 was infused IV for 60 min in 8 healthy young (18 to 25 y) and 9 healthy elderly (71 to 84 y) subjects.Both baseline ANP values and the levels at the end of infusion were higher in the elderly subjects. The mean residence time of ANP in the two age groups was not significantly different, whereas total body clearance (CL) was markedly diminished in the elderly as compared to the young subjects (mean±SD 3.1±1.0 l·min−1 and 6.2±4.1 l·min−1, respectively). The apparent volume of distribution at steady state was lower in the elderly than in the young, but the difference was not significant (mean±SD 44±19 and 103±111, respectively. The decrease in CL largely explained the higher ANP levels found in the elderly subjects. The MRT and the plasma half-life of the terminal phase did not differ between the two groups.In the elderly but not in the young subjects the calculated endogenous creatinine clearance was closely correlated with the CL (r=0.90, P<0.001), thereby emphasizing the importance of the kidney in the metabolic clearance of ANP in the elderly.

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.

Hemodynamic effects of atrial natriuretic factor in young and elderly subjects

Because levels of plasma atrial natriuretic factor (ANF) increase with advancing age, a diminished hemodynamic responsiveness to ANF in the elderly has been hypothesized in the literature. Therefore hemodynamic effects after two infusion rates (0.25 and 2.0 μg/min) of atrial natriuretic factor (99‐126) were investigated in young (n = 8) and elderly (n = 9) volunteers in a double‐blind, randomized, and placebo‐controlled protocol. After low‐rate infusion, ANF concentrations increased to the upper normal range, and only minor effects were observed. In contrast, high‐rate infusion resulted in a decrease in blood pressure and forearm vascular resistance, whereas an increase in heart rate was observed in both groups. Between young and elderly subjects, a significant difference was observed in the ANF‐induced decrease in systolic blood pressure (mean ± SD, − 4% ± 4% versus − 12% ± 7%, p < 0.05) and mean arterial pressure (− 6% ± 5% versus −11% ± 4%, p < 0.05) during the high rate infusion. When compared with the concentrations of the young subjects, the ANF concentrations reached at both ANF dosages were higher in the elderly subjects; this was the result of a diminished ANF clearance in the elderly subjects. After correction of the changes of systolic blood pressure and mean arterial pressure for the higher ANF levels reached within this elderly group, no difference between young and elderly subjects remained. We conclude that a diminished cardiovascular responsiveness to ANF with advancing age could not be demonstrated. In contrast, the high‐rate infusion of ANF induced an increased hemodynamic response in the elderly subjects, but this seems to be the result of the higher ANF levels reached within this group.

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 (∼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.

Atrial Natriuretic Peptide (ANP) in Normal Pregnancy; A Longitudinal Study

Plasma levels of atrial natriuretic peptide (ANP) were studied in 23 nulliparous women longitudinally during normal pregnancy in relation to various cardiovascular and urinary parameters. Concentrations of ANP were significantly increased at the 20th gestational week as compared with values 6 weeks post partum. During the second half of pregnancy ANP levels showed a significant decrease towards levels not much different from those 6 weeks after delivery. Gestational ANP levels were significantly inversely correlated with weight, systemic vascular resistance and systolic blood pressure. ANP was neither correlated with atrial or ventricular dimensions, natriuresis nor diuresis. The results might suggest that in the course of pregnancy the regulation of ANP secretion takes place around a new volume ‘set-point’. The finding of gestational plasma levels of ANP within the non-pregnant range does, however, not exclude some enhanced cardiac production of ANP, in view of the increased volume of distribution and pr...

Attenuated forearm vasodilator response to atrial natriuretic factor in the elderly.

The vasodilator potency of human atrial natriuretic factor-(99–126) was investigated in the forearm vascular bed of 10 young and 10 elderly normotensive volunteers with venous occlusion strain gauge plethysmography. Atrial natriuretic factor was infused at six increasing dose steps into the brachial artery from 0.001 up to 03 μg/min/100 ml of forearm volume. This induced a mean±SEM increase in blood flow from 1.4 ±0.2 up to 6.0 ±1.0 ml/min/100 ml in the young and from 1.4±0.2 up to 3.9±0.6 ml/min/100 ml in the elderly. The dose-response curves of forearm blood flow and of forearm vascular resistance after increasing infusion rates of atrial natriuretic factor were shifted to the right in the elderly when compared with the young subjects. The mean percent decrease in forearm vascular resistance, induced by atrial natriuretic factor, during this dose-response curve averaged -31 ±3% in the elderly versus -56±3% in the young subjects (p=0.0002). The calculated forearm spillover of the second messenger of atrial natriuretic factor, cyclic guanosine monophosphate, significantly increased from baseline values of 1.2±1.1 and 0.7±0.5 pmol/min/100 ml in young and elderly subjects, respectively, up to 23.2±5.0 and 30.5±7.0 pmol/min/100 ml during the highest dose of atrial natriuretic factor (bothp<0.01 versus baseline). There were no significant differences in the increments of the forearm spillover of this second messenger between both age groups. Further, both age groups demonstrated an equal vasodilator response to increasing infusion rates of the control vasodilator sodium nitroprusside, with a maximal decrease in forearm vascular resistance of 65±3% and 61±2% in the young and elderly group, respectively. This study demonstrates that advancing age in humans is accompanied by a specific reduction in vascular responsiveness to atrial natriuretic factor, without evidence for a diminished response of its second messenger. Therefore, we hypothesize that changes in postreceptor processes are involved in the age-related reduction in vascular sensitivity for atrial natriuretic factor.