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Circulating immunoreactive proANP (1-30) and proANP (31-67) in sedentary subjects and athletes.

Atrial natriuretic peptide (ANP)[4] was first described by De Bold et al. (1). Since then, a family of natriuretic peptides has been characterized, and the structures of their active forms have been identified. In atrial myocytes, ANP is stored in secretory granules as a prohormone, proANP(1-126). When released, this peptide is cleaved into its active COON terminus ([alpha]-ANP) and a second larger fragment, the N-terminal proANP(1-98), in equimolar amounts. The N-terminal fragment is further proteolytically cleaved in plasma to form the peptides proANP(1-30), proANP(31-67), and proANP(79-98) (2,3). Winters et al. (4) suggested that both the [NH.sub.2] and COON termini of the proANP are released simultaneously with exercise; in addition, Macauluay et al. (5) agreed that the peptides are cosecreted from the heart. However, the metabolism of these circulating fragments is unknown. The main stimulus for the secretion of natriuretic peptides is atrial stress in response to increased intracardiac volume (6-8), and their effects are natriuresis and vasodilatation (9).

The release of ANP increases in response to physical exercise (10, 11), reflecting increased venous return to the heart and perhaps the higher heart rate. Freund et al. (10) observed that, during marathon runs, the release of ANP increased in a dose- and time-dependent manner and returned to values nearer basal values by the end of the exercise.

Measurement of the N-terminal proANP requires an immunoradiometric assay. Numata et al. (12) established a two-step assay that uses monoclonal antibodies and synthetic N-terminal proANP(1-67). During physical exercise, increases in the plasma [alpha]-ANP [proANP(99-126)] concentration also suggests that atrial proANP(1-126) synthesis increases, leading to increased formation of all fragments. The N-terminal proANP fragments have longer half-lives in the circulation than [alpha]-ANP, with corresponding higher plasma concentrations. Little is known about the metabolism of proANP(1-98), and the structures of the circulating forms are unclear. Nevertheless, the proteolytic cleavage could be in equimolar quantities, and the different circulating concentrations of the proANP fragments reflect their degradation and clearance rates (13). Furthermore, the responses of proANP peptides to various stimuli seem slow both when ANP increases or decreases (14,15). The aim of this study was to develop enzymes immunoassays (EIAs) for plasma immunoreactive (ir)-proANP(1-30) and ir-proANP(31-67) without extraction. These new detailed EIAs were also devised to determine whether a difference in the circulating concentrations of these peptides exists between trained athletes and sedentary healthy subjects.

Materials and Methods

PEPTIDES AND REAGENTS

Human N-terminal proANP(1-30), middle proANP(3167), C-terminal biotinylated proANP(1-30), and C-terminal biotinylated proANP(31-67) were purchased from Pichem. For cross-reactivity tests, proANP(79-98), [alpha]-ANP(99-126), and proANP(1-98) were purchased from the Institute of Mikrobiologie and Genetik der Universitat Wien. A solution of biotinylated peptides with assay buffer was prepared before each assay. Peroxidase-streptavidin was purchased from Southern Biotechnology Associates. 3,3',5,5'-Tetramethylbenzidine solution was purchased from Sigma. The stopping solution was 3 mol/L sulfuric acid in distilled water.

BUFFERS

Buffer A (for coating) was 0.05 mol/L sodium borate buffer, pH 9.6. Buffer B (for washing) was sodium phosphate-buffered saline (0.06 mol/L sodium phosphate), pH 7.4, containing 0.1 mL/L Triton X-100. Buffer C (for blocking) was sodium phosphate-buffered saline (0.06 mol/L sodium phosphate), pH 7.5, containing 1 g/L nonfat dried milk (Skim Milk Powder; Fluka), 20 g/L fetal calf serum (JRH), 20 g/L peptone (Merck), and 20 g/L Karion F (Merck). Buffer D (assay buffer) was sodium phosphate-buffered saline (0.05 mol/L sodium phosphate), pH 7.4, containing 5 g/L bovine serum albumin.

ANTIBODIES

Donkey anti-sheep IgG Fc-specific antiserum was purchased from Guilday. The sheep antibody against proANP(1-30) IgG and the sheep antibody against proANP(31-67) IgG were purified by immunoaffinity chromatography on HiTrap minicolumns (Pharmacia) according to the protocol of the manufacturer. In brief, 0.5 mg of each peptide was bound to the column. The immunogen was column-coupled using 1 mL of the ligand solution (0.5 g/L immunogen in 0.2 mol/L sodium bicarbonate and 0.5 mol/L sodium chloride, pH 8.3). After the column was washed and non-coupled active groups were deactivated, 10 mL of the proANP-specific antiserum (diluted 1:2 in 50 mmol/L sodium borate buffer, pH 7.0) was loaded (0.5 mL/min) onto the column at room temperature. After suitable column washing, the proANP-specific antibody was eluted with sodium citrate buffer (100 mmol/L pH 1.7). Fractions (0.5 mL) were collected in tubes containing 0.5 mL of 500 mmol/L sodium borate buffer (pH 10.0) for immediate neutralization. The protein concentrations of the eluted IgG-containing fractions were determined with a commercial protein assay (Pierce).

MICROTITER PLATES

Microwells (6.5 mm diameter; Nunc Maxisorp High Binding) were coated with 200 [micro]L of diluted donkey antisheep IgG Fc-specific antiserum (200 ng/well) in buffer A overnight at 4 [degrees]C. After the wells were washed, they were blocked using buffer C (350 [micro]L). The microplates were then washed with buffer B (350 [micro]L), dried, and stored at 4 [degrees]C before use. Before the assay, the microwells were coated with the appropriate proANP antibody (200 [micro]L; 0.5 ng/well in buffer D) by incubation for 5 h at room temperature.

SUBJECTS AND PLASMA SAMPLE PREPARATION

Blood samples were drawn from an antecubital vein after the subjects had been supine for at least 15 min. Vacutainer Tubes containing heparin were used. The blood was placed on ice and centrifuged (800-10008) for 15-20 min; the plasma samples were stored at -80 [degrees]C.

Blood samples were obtained from internationally competitive male rowers (14 males) and from age-matched sedentary control subjects (22 males). Table 1 shows the mean ([+ or -] SE) ages, weights, height, blood pressure, and heart rates of the athletes and control subjects.

All subjects fasted overnight, and the blood samples were collected in the morning (0900-1100). A repeat blood collection was obtained from seven randomly chosen athletes on a second day under the same conditions.

The Ethics Committee of Copenhagen approved the experimental procedures.

EIA ASSAYS

Calibrators for proANP(1-30) (range, 0-600 pmol/L) and proANP(31-67) (range, 0-1250 pmol/L) were prepared in assay buffer. The coated wells were incubated with the sheep antibody directed against proANP for 5 h at room temperature as described. These antibody solutions were then discarded, the wells were washed, and the proANP calibrators, diluted samples (1:20 in assay buffer), or controls (200 [micro]L) were added to each well. Tracer solution [50 [micro]L of 360 ng/L biotinylated proANP(1-30) and 3.6 [micro]g/L biotinylated proANP(31-67)] was pipetted into the mixture and incubated overnight at room temperature. On the following day, the contents of the wells were discarded. After the wells were washed three times (350 [micro]L each time), the peroxidase-conjugated streptavidin (200 [micro]L) was added and incubated for 1 h with shaking at 37 [degrees]C. After the wells were washed four times (350 [micro]L each time), 200 [micro]L of 3,3',5,5'-tetramethylbenzidine solution was added. The reaction was stopped after 20-30 min by the addition of 50 [micro]L of the sulfuric acid solution. The absorbance was read at 450 nm. Calibration curves were prepared using a logit/log curve (absorbance vs concentration).

STATISTICAL ANALYSIS

The equation for the interpolation of the dose-response curve was computed with a four-parameter logistic function. Concentrations were calculated with commercial software. Detection limits were measured as the mean values at the 95% confidence limit of the absorbance of the [St.sub.0] samples. ANOVA was used for the statistical analysis. P [less than or equal to]0.05 was considered statistically significant.

Results

ANALYTICAL EVALUATION

Calibration curves. For each proANP peptide, a calibration curve was prepared with synthetic peptides (Figs. 1 and 2). The concentration of peptide at 95% B/[B.sub.0] was 2.4 pmol/L for proANP(1-30) and 10 pmol/L for proANP(31-67).

Precision. The CVs for the proANP(31-67) assay, assessed in 10 intraassay replicates of two peptide concentrations (545 and 1843 pmol/L) and in 6 different assays at 1885 pmol/L proANP(1-30), were 6%, 4%, and 9%, respectively. For 12 replicates of two proANP(31-67) concentrations (900 and 3400 pmol/L) and for six different assays at a peptide concentration of 3350 pmol/L, the CVs were 6%, 5%, and 9%, respectively.

[FIGURE 1 OMITTED]

Detection limit. The detection limits of the proANP(1-30) and proANP(31-67) assays were 4.7 [+ or -] 0.8 pmol/L (n = 8) and 14.2 [+ or -] 2.1 pmol/L (n = 8), respectively.

Dilution and recovery tests. Dilution curves of plasma samples gave linearity in the EIA measurements of proANP(1-30) and proANP(31-67). The equations for the two curves were: y = 0.9945x - 0.7291 and y = 1.0001x 3.428 (Fig. 3). The recoveries of added proANP(1-30) and proANP(31-67) at different concentrations were 102-112% for 30 and 150 pmol/L proANP(1-30) and 106-102% for 25 and 750 pmol/L proANP(31-67).

Interference and cross-reactivity. Cross-reactivities between the two peptides and with proANP(79-98) and [alpha]-ANP(99-126) were <1%. The two EIAs, Nt-ANP and and-ANP, recognized proANP(1-98) with 68% and 108% cross-reactivity, respectively.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

CONCENTRATIONS IN PLASMA SAMPLES

In the control subjects (n = 22), the ir-proANP(1-30) plasma concentration was 318 [+ or -] 38 pmol/L, whereas in athletes (n = 14), the concentration was 312 [+ or -] 25 pmol/L; there was no significant difference between these two groups. For ir-proANP(31-67), the plasma concentration was lower (P <0.005) in the control group (387 [+ or -] 71 pmol/L) than in the athletes (713 [+ or -] 81 pmol/L).

The relationships between the concentrations of the two peptides in plasma obtained from control volunteers and athletes were assessed by the calculation of a correlation coefficient by linear regression (Fig. 4).

Discussion

This study demonstrated that the two EIAs had good performance for measurement of ir-proANP(1-30) and ir-proANP(31-67) in human plasma samples without prior extraction. Both assays can overcome the analytical problems involved with the determination of ANP(99-126) related to its short half-life. The cross-reactivity results indicate that the antibodies used react specifically with their epitopes. In fact, the anti-proANP(1-30) antiserum did not demonstrate cross-reactivity with [alpha]-ANP(99-126) hormone. As expected, proANP(1-98) demonstrated a cross-reactivity with the antiserum anti-proANP(31-67) and with the antiserum anti-proANP(1-30).

[FIGURE 4 OMITTED]

With regard to what these assays were actually measuring, it should be taken into account that the two utilized antibodies were immunoaffinity purified; the measured immunoreactivities recognized epitopes in the proANP(1-30) and proANP(31-67) peptides, but other circulating fragments, particularly proANP(1-98), were also measured. Furthermore, other peptides such as proANP(1-67) and proANP(31-98) might be carefully investigated.

The plasma proANP fragment immunoreactivity determination by specific antibodies suggests that the irproANP(1-30) is present at a lower plasma concentration than the ir-proANP(31-67). These results are in agreement with the findings of Numata et al. (12) and Winters et al. (3). In fact, the molar ratio of ir-proANP(31-67) to ir-proANP(1-30) was 2.3 [+ or -] 0.2 in the athletes and 1.2 [+ or -] 0.2 in the sedentary subjects. Thus, the ratio for the athletes was higher (P <0.001) than that of the sedentary subjects. In any case, the good positive correlation (Fig. 4) between plasma concentrations of ir-proANP(1-30) and ir-proANP(31-67) and their ratio may suggest cosecretion but different half-lives of the fragments in the circulation, and renal clearance and protease activity might be involved (3). ProANP(31-67) has been shown to have a role in fluid and electrolyte homeostasis (13). Synthetic proANP(31-67) and [alpha]-ANP(99-126) are diuretic and natriuretic (14,15). Thus, in agreement with the hypothesis of Winters et al. (3), it could be suggested that the same ANF prohormone, similarly to pro-opiomelanocortin, contains several hormones within its amino acid sequence.

Our findings demonstrate that the ir-proANP(31-67) plasma concentration is higher in trained athletes than in sedentary control subjects. This proANP fragment might change more slowly than [alpha]-ANP (16,17). The ir-proANP(31-67) might be maintained at a high concentration as a consequence of the continuous daily stimuli of endurance training, but blood pressure might also be involved. In fact, a strong positive correlation with systolic and diastolic blood pressure has been observed in exercising healthy individuals (18). Stimulation of atrial ANP(1-126) synthesis and the release of [alpha]-ANP(99-126) and proANP-related fragments could be caused by atrial stretch and distension associated with physical activity. Proveda et al. (19) recently demonstrated that diastolic blood pressure at rest was lower in a group of trained athletes than in control subjects. This finding is in agreement with the present data (P <0.005; Table 1), which were generated not to study this application, but to suggest one of the uses of these analytical methods.

Among elderly women, differences in the response of the N[H.sub.2] terminus of proANP were found that are suggested to reflect differences in postsecretory mechanisms (20). Other factors may include renal function and other hormones, as well as other biochemical variables of hemodynamic involvement.

In conclusion, this study reinforces the idea that proANP fragments with a longer half-life, such as proANP(1-30) and proANP(31-67), can be useful biochemical indices in a study of physical exercise. The measurements carried out on plasma samples obtained from healthy sedentary subjects and from athletes demonstrated no statistical difference for the ir-proANP(1-30), but plasma ir-proANP(31-67) was higher in the trained athletes than in the sedentary subjects. This finding suggests a use of these assays for evaluation of fluid homeostasis with physical exercise.

We thank Dr. Gianni Cavatton for cooperation and assistance with the control group subjects, and Federica Lancerin and Dr. Albert Missbichler for excellent technical assistance and analytical suggestions in the development of these methods. We thank also Julia Stevens for excellent and patient help in the English grammar.

Received July 8, 1999; accepted February 15, 2000.

References

(1.) De Bold AJ, Borenstein HB, Veress AT, Sonnenberg H. A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sci 1980;28:89-94.

(2.) Winters CJ, Sallman AL, Meadows J, Rico DM, Vesely DL. Two new hormones: prohormone atrial natriuretic peptides 1-30 and 31-67 circulate in man. Biochem Biophys Res Commun 1988;150:231-6.

(3.) Winters CJ, Sallman AL, Baker BJ, Meadows J, Rico DM, Vesely DL. The N-terminus and a 4,000-MW peptide from the midportion of the N-terminus of the atrial natriuretic factor prohormone each circulate in humans and increase in congestive heart failure. Circulation 1989;80:438-49.

(4.) Winters CJ, Baker BJ, Dinh HA, Sallman AL, Rico DM, Vesely DL. Prohormone atrial natriuretic peptide 1-98 and 31-67 increase with exercise in congestive heart failure patients. Am J Med Sci 1989;298:377-82.

(5.) Macauluay Hunter EF, Kelly PA, Prowse C, Woods RJ, Lowry PJ. Analysis of peptides derived from pro atrial natriuretic peptide that circulate in man and increase in heart disease. Scand J Clin Lab Investig 1998;58:205-16.

(6.) Maack T. Role of atrial natriuretic factor in volume control. Kidney Int 1996;49:1732-7.

(7.) Melin B, Jimenez C, Savourey G, Bittel J, Cottet-Emard JM, Pequignot JM, et al. Effect of hydration state on hormonal and renal responses during moderate exercise in the heat. Eur J Appl Physiol 1997;76:320-7.

(8.) Grant SM, Green HJ, Phillips SM, Enns DL, Sutton JR. Fluid and electrolyte hormonal responses to exercise and acute plasma volume expansion. J Appl Physiol 1996;81:2386-92.

(9.) Martin DR, Prevahouse J B, Trigg DJ, Vesely DL, Buerkert JE. Three peptides from the ANF prohormone NHZterminus are natriuretic and/or kaliuretic. Am J Physiol 1990;258:11401-8.

(10.) Freund BJ, Wade CE, Claybaugh JR. Effects of exercise on atrial natriuretic factor: release mechanisms and implications for fluid homeostasis. Sports Med 1988;6:364-76.

(11.) Hosoda K, Nakao K, Mukoyama M, Saito Y, Jougasaki M, Shirakami G, et al. Expression of brain natriuretic peptide gene in human heart. Production in the ventricle. Hypertension 1991;17: 1152-6.

(12.) Numata Y, Dohi K, Furukawa A, Kikuoka S, Asada H, Fukunaga T, et al. Immunoradiometric assay for the N-terminal fragment of proatrial natriuretic peptide in human plasma. Clin Chem 1998; 44:1008-13.

(13.) Vesely DL, Douglass MA, Dietz JR, Gower WR, McCormick MT, Rodriguez-Paz G, Schocken DD. Three peptides from the atrial natriuretic factor prohormone amino terminus lower blood pres sure and produce diuresis, natriuresis, and/or kaliuresis in humans. Circulation 1994;90:1129-40.

(14.) Habibullah AA, Villareal D, Freeman RH, Dietz JR, Vesley DL, Simmons JC. Atrial natriuretic peptide fragments in dogs with experimental heart failure. Clin Exp Pharmacol Physiol 1995;22:130-5.

(15.) Benjamin BA. Effects of ANF prohormone peptides in conscious primates. Clin Exp Pharmacol Physiol 1995;22:125-9.

(16.) Steele IC, McDowell G, Moore A, Campbell NPS, Shaw C, Buchanan KD, Nicholls DP. Responses of atrial natriuretic peptide and brain natriuretic peptide to exercise in patients with chronic heart failure and normal control subjects. Eur J Clin Investig 1997;27:270-6.

(17.) Friedl W, Mair J, Thomas S, Pichler M, Puschendorf B. Relationship between natriuretic peptides and hemodynamics in patients with heart failure at rest and after ergometric exercise. Clin Chim Acta 1999;281:121-6.

(18.) Baker BJ, Wu WCL, Winters CJ, Dinh H, Wyeth R, Sallman AL, Vesely DL. Exercise increases the circulating concentration of the N-terminus of the atrial natriuretic factor prohormone in normal individuals. Am Heart J 1991;122:1395-402.

(19.) Poveda JJ, Riestra A, Salas E, Cagica ML, Lopez-Sumoza C, Amado JA, Berrazueta JR. Contribution of nitric oxide to exercise induced changes in healthy volunteers: effects of acute exercise and long-term physical training. Eur J Clin Investig 1997;27:967-71.

(20.) Kallinen M, Suominen H, Vuolteenaho O, Alen M. Effort tolerance in elderly women with different physical activity backgrounds. Med Sci Sports Exerc 1998;30:170-6.

[4] Nonstandard abbreviations: ANP, atrial natriuretic peptide; EIA, enzyme immunoassay; and ir, immunoreactive.

ELIO F. DE PALO, [1] * WOLFGANG WOLOSZCZUK, [2] MARTINA MENEGHETTI, [1] CARLO B. DE PALO, [1] HENNING B. NIELSEN, [3] and NIELS H. SECHER [3]

[1] Section of Clinical Biochemistry, Department of Medical Diagnostic Sciences and Special Therapy, University of Padua, 35100 Padua, Italy.

[2] Ludwig Boltzmann Institut for Experimentelle Endokrinologie, A-1100 Wien, Austria.

[3] Department of Anesthesia, University of Copenhagen, DK-2100 Copenhagen, Denmark.

* Author for correspondence. Fax 039-49-657391; e-mail depalo@ux1.unipd.it.
Table 1. Anthropologic and physiologic variables (mean [+ or -] SE)
in athletes (n = 14) and control subjects (n = 22).

Group studied Age, years Height, cm Weight, kg

Athletes 25 [+ or -] 1 182 [+ or -] 2 74.4 [+ or -] 0.5
Controls 24 [+ or -] 1 179 [+ or -] 2 79.9 [+ or -] 2.9

 Blood pressure, mmHg

Group studied Systolic Diastolic Heart rate,
 beats/min

Athletes 127 [+ or -] 7 64 [+ or -] 1 (a) 52.6 [+ or -] 0.6
Controls 132 [+ or -] 3 82 [+ or -] 2

(a) P <0.005.
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Title Annotation:Endocrinology and Metabolism
Author:De Palo, Elio F.; Woloszczuk, Wolfgang; Meneghetti, Martina; De Palo, Carlo B.; Nielsen, Henning B.;
Publication:Clinical Chemistry
Date:Jun 1, 2000
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