Clinica Chimica Acta, 203 (1991) 235-242

235

0 1991 Elsevier Science Publishers B.V. All rights reserved 0009-8981/91/%03.50

CCA 05141

In vitro stability of human atria1 natriuretic peptide (h-ANP) Erika Artner-Dworzak,

Herbert Lindner and Bernd Puschendorf

Institute of Medical Chemistry and Biochemistry, Unil~ersityof Innsbruck (Austria)

(Received 10 April 1991; revision received 29 August 1991; accepted 16 September 1991) Key words: h-ANP; In vitro stability; Aprotinin; Phosphoramidon

Introduction Human atria1 natriuretic peptide, h-ANP, is synthesized, stored and secreted by atria1 myocytes [l-3]. It acts by way of receptors in kidney, vascular system, adrenal gland and in central nervous system [4-61, producing natriuresis, diuresis, vasorelaxation and lowers renin and aldostirone levels [7-lo]. Concentrations of h-ANP are increased in plasma of patients with tachyarrhythmias [ll-131 and in those with impaired cardiac [14-161 and renal [17-191 function. The measurement of h-ANP by radioimmunoassay is associated with preanalytical (short half-life in vitro [20]) and analytical (immunoreactive cleavage products detected by antibodies used in radioimmunoassays [2,21]) difficulties, possibly caused by specific and unspecific proteolysis. Reliable results can be obtained only by immediate determination of h-ANP after blood collection with centrifugation at 4°C in chilled tubes or on plasma samples frozen at -20°C following centrifugation. The degradation of h-ANP (99-126) to ANP (99-105/ 106-126) is markedly caused by neutral membranemetallo-endopeptidase EC 3.4.24.11 (NEP, enkephalinase> activity an enzyme located in a number of tissues, preferentially in renal tissues [22] and is present in plasma [23,24]. In present study we report the effects of the protease inhibitors aprotinin 1251 and phosphoramidon [22,26] on the stabilisation of h-ANP in vitro. Materials and methods Peripheral venous blood was drawn into 1 mmol/l EDTA coated tubes either chilled or kept at room temperature from patients with presumptive evidence of

Correspondence to: Erika Artner-Dworzak, Pregl-Str. 3, A-6020 Innsbruck, Austria.

Institut Iiir Medizinische Chemie und Biochemie, Fritz-

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elevated h-ANP plasma levels (2 120 pg/ml) such as hypertonia or cardiac disorders (NYHA II-IV). The tubes contained aprotinin or phosphoramidon at various concentrations as indicated or were without additives except EDTA, and were centrifuged either at 4°C or 25°C without delay for 15 min at 2,000 Xg. The samples are kept for various time intervals exactly at 4°C or 25°C and 250 ~1 portions were frozen at -20°C until determination of h-ANP. Protease inhibitors: Aprotinin (Trasylol), an unspecific serine protease inhibitor from bovine lung in the form of a lyophijisate (Boehringer Mannheim, Germany), (approximately 1 mg protein/mg lyophilisate) was dissolved in Tris buffer, 0.1 mol/l, pH 8.0; 250, 500, 750 and 1,000 KIU/ml blood (1 kallikreininhibitor-unit (KII-0 = 0.17 pg of crystalline aprotinin) were used in experiments as indicated. Phosphoramidon, a specific inhibitor of neutral metalloendopeptidase (EC 3.4.24.11) and the metalloendopeptidase thermolysine (EC 3.4.24.4) was supplied by Boehringer Mannheim (Germany). Lyophilized ammonium salt dissolved in water at various concentrations in the range of lo-’ to lO_“’ mol/l blood was used in the stabilisation experiments. h-ANP (ANPI was supplied from Boehringer Mannheim (Germany) as a freeze-dried powder, sterile and pyrogen free. h-ANP radioimmunoassay: immunoreactive h-ANP was assayed with a ‘*‘J-labelled radioimmunoassay (HANP-Eiken, l-AR55), Eiken Chemical Co. Ltd. Tokyo, Japan; this is a direct double antibody assay based on the competitive method j27,28]. The interassay coefficient of variation of h-ANP concentration was 14%. Results Figure 1 demonstrates the in vitro kinetics of degradation of immunoreactive h-ANP in plasma of a patient suffering from hypertonia containing 1 mmol/l EDTA and stored at 4°C and 25°C. In accordance with the recommendations of the manufacturer of the used radioimmunoassay, HANP-Eiken, Japan, experiments were carried out with aprotinin, an unspecific serine protease inhibitor at different periods and concentrations. The stabilization effects of aprotinin were examined at the concentrations of 250, 500, 750 and 1,000 KIU/ml blood. By addition of 500 KIU/ml blood, the decay of h-ANP was delayed for 10 h at 4°C (Fig. 2); in the presence of 1,000 KIU/ml blood the delay is 48 h at 4°C. Storage of samples at 25°C led to early degradation or in some cases to elevation of h-ANP levels, limiting the reliable effectiveness of aprotinin on h-ANP stability. Since the enzyme metalloendopeptidase EC 3.4.24.11, known to degrade h-ANP in vivo, is present in a number of tissues as also in blood, we suspected that immunoreactive h-ANP may be cleaved by this enzyme in vitro as well. According to literature 1291which describes in vivo stabilization of h-ANP by phosphoramidon as a specific inhibitor of the atriopeptidase (metalloendopeptidase EC 3.4.24.111, we examined the influence of this substance on h-ANP stability in vitro (Fig. 3). 10s9 mol/l blood caused a stabilization for 50 h at 4°C and 25°C. Increasing the concentration to 5.10-’ mol/l blood results in an inhibition of cleavage of h-ANP for at least 100 h at 4°C and 25°C. Unspecific augmentation of h-ANP levels entailed by immunoreactive cleavage products in EDTA plasma

231

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Fig. 1. In vitro kinetics of degradation of immunoreactive h-ANP in EDTA plasma without addition of protease inhibitors. Blood was collected by peripheral venous punction from a 49-yr-old female patient suffering from untreated hypertension (180/110 mm Hg). Aliquots were frozen subsequently after centrifugation or kept at 4°C (+ - +l or 25°C (-•-@-l for different time intervals and frozen at -20°C until determination of h-ANP. The experiment was done in duplicate, the radioimmunoassay was carried out in duplicate too.

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10 21’5 45 70 93100 hours Fig. 2. Kinetics of h-ANP degradation in presence of 500 KIU aprotinin/l blood. Blood collection was carried out from the same patient as described in Fig. 1 into tubes containing 500 KIU of aprotinin/l blood and stored at 4°C or 25°C. Aliquots were taken at different time intervals at -20°C until determination. (+ - +l, chilled tubes, centrifugation at 4°C storage at 4°C until freezing of samples. (-*-a-), tubes kept at room temperature, centrifugation at 25”C, storage at 25°C until freezing of samples. As described in Fig. 1 all experiments were done in duplicate. 4

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Fig. 3. Stability of h-ANP in the presence of 5.10-” mol phosphoramidonper liter blood. Blood collection was conducted simultaneously from the same subject as for experiments described in Figs. 1 and 2. In this experiment tubes contained phosphoramidon in a final concentration of S.lO-’ mol per liter blood. Samples were kept at 4°C and 25°C for several time intervals and frozen at -20°C until measurement (+ - +I, chilled tubes, centrifugation and incubation at 4°C (@-e), tubes kept at room temperature, centrifugation and incubation at 25°C. All experiments were done in duplicate.

without addition of inhibitors at 4°C or in presence of aprotinin at 25°C was prevented by phosphoramidon at a concentration of 5.10-9 mol/l blood (Fig. 3). An increase of phosporamidon to lo-’ mol/l blood led to unspecific elevation of h-ANP levels probably due to alteration of the molecule structure. It appears from Table I that phosphoramidon has no .effect on exogenous h-ANP in aqueous solution. It disappears by hydrophobic linking to the tubes with TABLE I Stability of exogenous human atrial natriuretic peptide Concentration of h-ANP ” (pg/ml)

Preanalytical conditions

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h-ANP lyophilisate (Boehringer Mannheim) was dissolved in water to a final concentration of 500 pg/ml. 1, Aliquots were frozen immediately after dilution with water at - 20°C. 2, Aliquots of samples were kept at 25°C for 24 h and then frozen at -20°C. A, Exogenous h-ANP at a final concentration of 500 pg/ml aqueous solution in absence of any additive. B, A in the presence of 5.10-’ mol/l phosphoramidon. C, A dissolved in 0.1% bovine serum albumin.

239

0 Fig. 4. Stability of h-ANP the presence

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a half-life of 1 to 2 h (experiment A + B). No absorption or breakdown of h-ANP occurs during in vitro incubation of exogenous h-ANP in presence of 0.1% bovine serum albumin (experiment 0. The degradation of immunoreactive h-ANP could not be prevented in all cases by addition of inhibitors of proteolysis (Fig. 41, due to unspecific hydrolysis with pronounced individual variations. Discussion In present studies we note a progressive loss of immunoreactive h-ANP in human plasma in vitro with a half-life of about 60 min at 25°C. The rapid degradation of this peptide limits its usefulness in completing and even replacing invasive diagnostics in cardiac diseases. In order to establish this parameter for the clinical laboratory we conducted h-ANP stabilization experiments in vitro. Using aprotinin, an unspecific serine

240

protease inhibitor at concentrations of 500 to 1,000 KIU/ml blood, we found that the degradation of h-ANP was delayed only at 4°C for lo-48 h. Experiments with phosphoramidon, a specific inhibitor of metalloendopeptidase EC 3.4.24.11 at a concentration of 5.10-” mol/l blood showed in approximately 90% of cases a stabilization effect for 100 h at 4°C and 25°C. In approximately 10% of cases a loss of immunoreactive h-ANP could not be prevented by addition of these inhibitors to blood samples which is probably due to unspecific proteolysis with pronounced individual deviations. From our observations we conclude that h-ANP is degraded in vitro at 25°C almost exclusively by metalloendopeptidase EC 3.4.24.11 activity which is inhibited by phosphoramidon but not by aprotinin. At 4°C this enzyme is probably not active. In aqueous solution exogenous h-ANP disappears rapidly due to reversible hydrophobic interaction of the free peptide with the vial which might influence the structure or the assessibility of the epitope recognized by an antibody. This loss of immunoreactive ANP can be prevented by the addition of albumin. In addition to these results using phosphoramidon to inhibit cleavage on h-ANP in vitro we suggest that c-GMP should be measured routinely in cardiac diseases, since a close correlation between h-ANP and c-GMP release in human plasma could be evaluated in cardiac diseases and cGMP is stable for five days in blood samples containing EDTA [30,313. Furthermore, it must be examined how far other atriopeptides such as, for instance, human brain natriuretic peptide (h-BNP), which is supposed to have at least an equally important effect on cardiomyocytes as h-ANP 1321,can be stabilized in vitro by addition of phosphoramidon and other inhibitors of endopeptidases.

References 1 Atlas SA. Atrial natriuretic factor: a new hormone of cardiac origin. Ret Prog Hormone Res 1986;42:207-249. 2 Genest J, Cantin M. The atrial natriruetic factor: its physiology and biochemistry. Rev Physiol Biochem Pharmacol 1988;110:19-24. 3 Crozier IG, Nicholls MG, Ikram H, Espiner EA, Yandle TG, Jans S. Atrial natriuretic peptide in humans. Production and clearance by various tissues. Hypertension 1986;8:11-15. 4 Napier MA, Vandlen RL, Albers-Schonberg G, Nutt RT, Brady S, et al. Specific membrane receptors for atrial natriuretic factor in renal and vascular tissues. Proc Natl Acad Sci USA 1984;81:5946-5950. 5 Morel G, Chabot JG, Garcia-Caballero T, Gossard F, Dihl F, Belles-Isles M, Heisler S. Synthesis, internalization and localization of atrial natriuretic peptide in rat adrenal medulla. Endocrinology 1988;123:149-158. 6 Gardner DG, Vlasuk GP, Baxter JD, Fiddes JC, Lewicki JA. Identification of atrial natriuretic factor transcripts in the central nervous system of rats. Proc Natl Acad Sci USA 1987c;84:2175-2179. 7 Laragh JH, Cody RJ, Covit AB, Atlas SA. The renin system and atrial natriuretic hromone in congestive heart failure. Acta Med Stand (Suppl) 1986;707:45-53. 8 Lang RE, Unger T, Ganten D. Atrial natriuretic peptide:a new factor in blood pressure control. J Hypertension 1987;5:255-271. 9 Burnett JC Jr, Granger JP, Opgenorth TS. Effects of synethetic atrial natriuretic factor on renal function and renin release. Am J Physiol 1984;247:863-866.

241 10 Maack T, Marion DN, Camargo MJF, Kleinert HD, Laragh JH, Vaughan ED Jr, Atlas SA. Effects of auriculin (atrial natriuretic factor) on blood pressure, renal function and the renin aldosterone system in dogs. Am J Med 1984;77:1069-1075. 11 Yamaji T, Ishibashi M, Nakaoka M, Imataka K, Amano M, Fujii J. A possible role of ANF in polyuria associated with paroxysmal atrial arrhythmias. Lancet 1985;i:1211. 12 Crozier I, Ikram H, Nicholls MG, Espiner EA, Yandle TG. Atrial natriuretic peptide in spontaneous tachycardias. Br Heart J 1987;58:96-100. 13 Espiner EA, Crozier IG, Nicholls MG, Cuneo R, Yandle TG, Ikram H. Caridac secretion of ANP. Lancet 1985;i:398-399. 14 Anderson JU, Gibbs JSR, Woodruff PWR, Greo C, Rowland E, Bloom SR. The plasma atrial natriuretic response to treatment of acute cardiac failure, spontaneous supraventricular tachycardia and induced rentrant tachycardia in man. Hypertension 1986;4:137-141. 15 Hirata Y, Ishii M, Matsuo H, et al. Plasma concentrations of alpha-human ANP and cyclic GMP in patients with heart disease. Am Heart J 1987;113:1463-1469. 16 Burnet JC, Kao PC, Hu DC, et al. Atrial natriuretic peptide elevation in congestive heart failure in human. Science 1986;231:1145-1147. 17 Rascher W, Tulassay T, Lang RE. ANP in plasma of volume overloaded children with chronical renal failure. Lancet 1985;i:303-305. 18 Hasegawa K, Matsushita Y, Inove T, Morii H, Ishibashi M, Yamaji T. Plasma levels of atrial natriuretic peptide in patients with chronic renal failure. J Clin Endocrinol Metab 1986;63:819-822. 19 Suda S, Weidmann P, Saxenhofer H, Cottier C, Shawand SG, Ferrier C. ANF in mild to moderate chronic renal failure. Hypertension 1988;11:483-490. 20 Hartter E, Kargl R, Woloszczuk W. Methodical problems in radioimmunological measurement of ANP. Z Kardiol 1988;Suppl 2:11-19. 21 Schwartz D, Geller DM, Manning PT, Siegel NR, Fok FK, Smith CE, Needleman P. Ser-Leu-ArgArg-Atriopeptin III: the major circulating form of atrial peptide. Science 1985;229:397-400. 22 Kenny AJ, Stephenson SL. Role of endopeptidase 24.11. in the inactivation of atrial natriuretic peptide. FEBS Lett 1988;232:1-8. 23 Yandle TG, Brennan SO, Espiner EA, Nicholls MG, Richards AM. Endopeptidase 24.11 in human plasma degrades atrial natriuretic factor (ANF) to ANF (99-105/106-126). Peptides 1989;10:891894. 24 Ura N, Carretero OA, Erdds EG. Role of renal endopeptidase 24.11 in kinin metabolism in vitro and in vivo. Kidney Intern 1987;32:507-513. 25 Yandle TG, Espiner EA, Nicholls MG, Duff H. Radioimmunoassay and characterization of atrial natriuretic peptide in human plasma. J Clin Endocrinol Metab 1986;63:72-79. 26 Fulcher IS, Matsas R, Turner AJ, Kenny AJ. Kidney neutral endopeptidase and the hydrolysis of enkephalin by synaptic membranes show similar sensitivity to inhibitors. Biochem J 1982;203:519522. 27 Wencker M, Hauptlorenz S, Moll W, Puschendorf B. Influence of blood pressure, heart rate, age and sex on concentrations of ANF and cGMP in 124 volunteers. Clin Chem 1989;35:1519-1523. 28 Fridrich L, Szekeres TH, Hartter E, Schweighofer F, Gassner A, Laczkovic A. Correlation between heart disorders and concentrations of directly measured atrial natriuretic peptide in plasma. Clin Chem 1989;35:435-439. 29 Ura N, Carretero OA, Erdijs EG. Role of endopeptidase 24.11. in kininmetabolism in vitro and in vivo. Kidney Intern 1987;32:507-513. 30 Vordetwinkler KP, Artner-Dworzak E, Jakob G, Mair J, Dienstl F, Pichler M, Puschendorf B. Release of cyclic guanosine monophosphate evaluated as a diagnostic tool in cardiac diseases. Clin Chem 1991;37:186-190. 31 Weil J, Lang RE, Suttmann H, Rampf U, Bidlingmaier F, Gerzer F. Concomitant increase in plasma ANP and cyclic GMP during volume loading. Klin Wochensch 1985;63:1265-1268. 32 Mukoyama M, Nakao K, Saito Y, Ogawa J, Hosoda K, Suga S, Shirakami G, Jougasaki M, Imura H. Human brain natriuretic peptide, a novel cardiac hormone. Lancet 1990;335:801.

In vitro stability of human atrial natriuretic peptide (h-ANP).

Clinica Chimica Acta, 203 (1991) 235-242 235 0 1991 Elsevier Science Publishers B.V. All rights reserved 0009-8981/91/%03.50 CCA 05141 In vitro st...
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