C. Manfredi

Circulating levels of cardiac natriuretic peptides (ANP and BNP) measured by highly sensitive and specific immunoradiometric assays in normal subjects and in patients with different degrees of heart failure

Plasma atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) levels increase in patients with heart failure with the progression of clinical symptoms and with the deterioration of hemodynamics; consequently, assay methods for these peptides may be useful in the follow-up of cardiac patients. Non-competitive immunoradiometric assay (IRMA) methods for ANP or BNP do not generally require preliminary extraction and/or purification of the plasma sample, and so may be more suitable than competitive immunoradiometric assay (RIA) methods for the routinary assay of plasma peptide concentrations. We evaluated the analytical characteristics and clinical usefulness of two IRMAs for plasma ANP and BNP, to verify whether these methods may be considered suitable for the follow-up of patients with heart failure. Both methods are based on the solid-phase sandwich IRMA system, which uses two monoclonal antibodies prepared against two sterically remote epitopes of peptide molecule; the first antibody was coated on the beads solid-phase and the second was radiolabeled with 125I. Blood samples were collected from a brachial vein in ice-chilled disposable polypropylene tubes containing aprotinin and EDTA after the patient had rested for at least 20 min in the recumbent position. Plasma samples were immediately separated by centrifugation and stored at −20 C until assay. The IRMA methods showed a better sensitivity and a wider working range sensitivity (about 2 ng/l) than those of RIA methods. Moreover, the normal range found with these methods (ANP= 16.1±8.6 ng/l, 5.2±2.8 pmol/l, BNP= 8.6±8.2 ng/l, 2.5±2.4 pmol/l) was similar to that generally reported using the most accurate methods, such as the other IRMAs or RIAs, using a preliminary extraction and purification of plasma samples with chromatographic procedures. Our results obtained in patients with different degrees of heart failure indicate that plasma ANP and BNP increase with the progression of clinical symptoms (NYHA class) (ANOVA p<0.0001). Indeed, circulating levels of ANP (R=− 0.701, no.=86) and BNP (R=−0.745, no.=55) were significantly (p<0.0001) and negatively correlated with the left ventricular ejection fraction values. Furthermore, a close curvilinear regression (R= 0.960, no.= 215) was found between ANP and BNP values, because plasma BNP progressively increases more than plasma ANP in patients with different stages of heart failure. In conclusion, IRMA methods are preferable for the measurement of plasma ANP and BNP for experimental studies and routine assay because they are more practicable, sensitive and accurate than RIA procedures. Finally, BNP assay appears to be better than ANP for discriminating between normal subjects and patients with different degrees of heart failure.

Preparation of mono-radioiodinated tracers for study of the in vivo metabolism of atrial natriuretic peptide in humans

In the present paper we evaluate the optimum chemical conditions for labelling atrial natriuretic peptide (ANP) and its metabolites and for preparing highly purified radiotracers which can be used for in vivo kinetic studies of ANP in humans. Synthetic a h1–28ANP and some hormone metabolites were iodinated with Na125I or Na131I by means of the lactoperoxidase (ANP) or the chloramine-T (ANP metabolites) technique. The biological activity of labelled ANP was tested by means of a binding study using mouse cardiac membranes. A high-performance liquid chromatography (HPLC) procedure was used to purify the labelled hormone and the principal labelled metabolites in venous plasma samples collected up to 50 min after the injection of125I-labelled ANP from nine healthy men. The main ANP kinetic parameters were derived from the disappearance curves of the [125I]ANP, which were satisfactorily fitted by a biexponential function in all subjects. The main advantages of this tracer technique are: (1) high accuracy, allowing the identification of the metabolites produced in vivo under steady-state conditions after injection of the precursor (labelled hormone); (2) high sensitivity, allowing the detection of minimal quantities of metabolites (that cannot be identified on the basis of the integrated areas from the ultraviolet-absorbing peaks on HPLC); (3) high specificity, allowing the detection of possible in vitro artefactual generation of cleavage products of ANP using an internal labelled standard. Utilizing this tracer method, it was possible to estimate the principal parameters of ANP kinetics and also to plot the appearance curves of the labelled metabolites produced in vivo after the injection of the labelled precursor.

A023 The circolatory model in metabolic studies of rapidly renewed hormones: Application to ANP kinetics

Pilo, A., G. Iervasi, A. Clerico, F. Vitek, S. Berti, C. Palmieri, A. Biagini, and L. Donato. Circulatory model in metabolic studies of rapidly renewed hormones: application to ANP kinetics. Am. J. Physiol. 274 (Endocrinol. Metab. 37): E560–E572, 1998.—In an attempt to identify and quantify the sites of atrial natriuretic peptide (ANP) degradation, a new tracer experiment has been developed. 125I-ANP was injected as a bolus just upstream from the right atrium, and blood was sampled from two different sites (pulmonary artery and aorta) in eight cardiac patients. Data were analyzed using a physiologically based circulatory model consisting of three blocks in series (right heart, lungs and left heart, and periphery) supplied by the same flow (cardiac output, measured by thermodilution); the extraction coefficients of the three blocks and of the whole body could be determined from the areas under tracer concentration curves in plasma (AUCs). The values for AUCs (means 6 SD) were 64.8 6 9.4 and 65.5 6 10.7% dose·l21 ·min21 for pulmonary artery and aorta curves, respectively; the area under the pulmonary artery curve could be subdivided into the area under the first-pass curve (30.6 6 4.7% dose· l21 ·min21) and the area under the recirculating curve (34.0 6 7.7% dose· l21 ·min21). The metabolic clearance rate of 125IANP, computed as dose divided by the area under the recirculating curve, was 3.1 6 0.7 l/min, and the whole body extraction was 47.6 6 6.6%. In our patients with myocardial dysfunction, neither right heart block nor lungs and left heart block significantly extracted ANP, and periphery block accounted for almost all removal of the hormone from the blood.

In vivo measurement of ANP overall turnover and identification of its main metabolic pathways under steady state conditions in humans

Using a tracer method, we evaluated, in vivo, the main turnover parameters and the main metabolic pathways of ANP in 10 normal subjects. HPLC was used to purify the labeled hormone and the principal labeled metabolites present in venous plasma samples collected at determined times after tracer injection. The main ANP kinetic parameters were derived from the disappearance curves of [125I] ANP, which were satisfactorily fitted by a biexponential function in all subjects. Newly produced ANP initially distributes in a large, plasma equivalent space (10.9±3.6 l/m2 body surface); the hormone rapidly leaves this space due to both degradation and to distribution in peripheral spaces. The mean residence time in the body (19.4±19.8 min) and the plasma equivalent total distribution volume (28.2±11.5 l/m2) indicate that ANP is also widely distributed outside the initial space in humans (circulating ANP is no more than 1/15 of the body pool). Metabolic clearance rate values were distributed across a wide range (from 740 ml/min/m2 to 2581 ml/min/m2, mean 1849 ml/min/m2), and were shown to strongly correlate (R=0.962) with the daily urinary excretion of sodium. A complete separation of labeled ANP from its labeled metabolites was achieved by the HPLC technique; at least 3 different peaks due to labeled metabolites in vivo produced from the injected [125I]ANP1–28 were found. The first Chromatographic peak eluted showed an identical elution time to monoiodotyrosine. At least two other peaks due to in vivo generated labeled metabolites were well identified in the chromatograms: one peak (coeluting with labeled COOH-terminal tripeptide, H-Phe-Arg-Tyr-OH) was eluted ahead and one (coeluting with labeled peptide fragments ANP7–28, ANP13–28, and ANP18–28) behind the elution peak of the labeled ANP. The peak of labeled tyrosine appearing in the plasma ranged between 3 and 5 min after tracer injection; the other two peaks of radioiodinated metabolites showed their highest activity in the first sample (1.5 min), suggesting an earlier occurrence of their peaks. These labeled metabolites seem to be intermediate peptides, between the intact circulating form of the hormone and the final labeled metabolite (tyrosine), which is the last amino acid of the peptide hormone, produced in vivo after injection of the tracer. In conclusion, our kinetic data indicate that: 1) newly produced ANP is rapidly distributed and degraded; 2) the body pool of the hormone can be considered a combination of two exchanging spaces, circulating ANP representing no more than 1/15 of the body pool; 3) MCR of ANP is closely related to sodium intake; 4) labeled tyrosine is the main endogenous metabolite of the hormone in humans; 5) both receptor-mediated and enzymatic degradation play an important role in the turnover of ANP in humans.

Acute effects of intravenous amiodarone on sulphate metabolites of thyroid hormones in arrhythmic patients

Factors that contribute to the remarkably rapid decrease in serum T3 and increase in reverse T3 (rT3) levels during illness, fasting, or treatment with some drugs (e.g. amiodarone) are not clear. In order to understand better the effect of acute amiodarone administration on T3 metabolism, especially the sulphation pathway, we performed a prospective study in 8 arrhythmic in‐patients treated with a loading dose of amiodarone.

Acute enoximone effect on systemic and renal hemodynamics in patients with heart failure

SummaryPatients with heart failure generally show improvement in their clinical condition after enoximone infusion over the period of treatment; this effect cannot be ascribed only to the known hemodynamic action of this drug. Thirty-six patients (age range 44–82 years) with heart failure (NYHA class II–IV) underwent 48-hour enoximone infusion to study whether this prolonged improvement might depend on changes in systemic or renal hemodynamics or in neurohormonal balance. All patients underwent Swan-Ganz hemodynamic monitoring; renal plasma flow, glomerular filtration rate, plasma atrial natriuretic factor (ANF), and plasma renin activity (PRA) were all measured at baseline, at the peak of the enoximone action, and 48 hours after drug discontinuation. The main hemodynamic parameters were significantly improved during enoximone infusion and after drug discontinuation. The cardiac index basal value of 2.2±0.1 l/min/m2 increased to 3.1±0.1 l/min/m2 after 24-hour therapy (p<0.01); similarly, pulmonary wedge pressure, mean pulmonary arterial pressure, and right atrial pressure decreased markedly (p<0.01). Beneficial effects were also observed in renal hemodynamics; indeed, renal plasma flow (basal value 485±39 ml/min) increased significantly after 24-hour enoximone infusion (575±35 ml/min; p<0.01), and this tendecy was also observed 48 hours after drug discontinuation. No significant modifications were observed in plasma hormone data; however, the PRA plasma level had a tendency to decrease. We conclude that in patients with heart failure, enoximone infusion has a less marked effect on renal hemodynamics, but this is more lasting than systemic hemodynamic effects. The tendency of PRA to decrease (although not statistically significant), still detectable 2 days after treatment in the presence of steady high plasma ANF concentrations, may also contribute to the paradoxical longlasting benefit despite the short-lived improvement in systemic hemodynamles after brief cycles of enoximone infusion.

Peripheral Resistance to Atrial Natriuretic Peptide in Patients with Idiopathic Dilated Cardiomyopathy

Atrial natriuretic peptide (ANP) has been suggested to play an important role in asymptomatic left ventricular dysfunction, preserving cardiorenal homeostasis through the maintenance of the sodium balance and the inhibition of the detrimental effects of the neurohormonal vasoconstrictor system. The current study was designed to investigate whether differences in the renewal and distribution of ANP may play a role in the pathogenesis and evolution of heart failure in idiopathic dilated cardiomyopathy (IDC). A tracer method was used to study ANP kinetics in the steady-state condition in 10 normal subjects and in 13 patients with IDC with different degrees of hemodynamic dysfunction at variable sodium intakes. [125I]-labeled ANP was bolus injected and high-pressure liquid chromatography (HPLC) was used to purify the labeled hormone in venous plasma samples collected up to 50 min after injection. The main ANP kinetic parameters were then derived from the disappearance curve of the labeled hormone. Patients with IDC showed a gradual reduction in the total distribution volume (on average from 20.5 ± 4.51/m2 to 12.2 ± 7.21/m2, p < 0.0279) with the progression of disease, mainly due to a contraction of the peripheral distribution spaces in the early phases of the disease and to a reduction in both the initial distribution volume and the peripheral spaces in the late phases of the disease. Moreover, the ANP production rate, which was in the normal range (120.0 ± 104.7 ng/ min/m2) in the early stages (99.8 ± 52.3ng/min/m2), greatly (more then three times, p < 0.0055) increased in patients with more severe myocardial involvement (378.9 ± 189.1 ng/min/m2). Different relationships between the metabolic clearance rate (MCR) values and daily sodium excretion were observed in patients (r = 0.837, p < 0.0001) and controls (r = 0.962, p < 0.0001). The significantly (p < 0.02) different linear regression coefficients (slopes) indicate that, on average, for each millimole rise in sodium excretion, the ANP MCR increased by 17ml/min/m2 in the patients, that is, there was an increase of about twofold with respect to the controls. Our study demonstrates a markedly altered peripheral distribution and degradation of ANP in patients with IDC, even those in the early phase of the disease (NYHA class I and II), who have plasma levels in the normal range. This alteration of ANP metabolism indicates the presence of a peripheral resistance to hormone effects and also suggests disturbed renal handling of sodium in patients with IDC and asymptomatic left ventricular dysfunction.