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

Abstract

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]ANP_1–28 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 ANP_7–28, ANP_13–28, and ANP_18–28) 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.