Plasma levels of human atrial natriuretic peptide in

Plasma levels of human atrial natriuretic peptide in patients with hypertensive diseases' OSAMU IIMURA ,2 KAZUAKI SHIMAMOTO , TOSHIAKI ANDO,NOBUYUKI URA, HIROYUK~ ISHIDA, MOTOY A NAKAGAWA, TOYOHARU YOKOYAMA, SHUZABURO FUKUYAMA, YASUKAZU YAMAGUCHI, AND IZUMIYAMAJI

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Depository Services Program on 06/06/13 For personal use only.

The Second Department of Internal Medicitre, Sapporo Medical College, %-I W-16, Chuo-ku, Sapporo 860, Japan Received August 14, 1986 IIIMURA, 0..SHIMAMOTO, K., ANDB,T., ~ R AN.,, ISHIDB,H . , NAKAGAWA. M . , YOKOYAMA, T., FUKUYAMA, S . , YAMAGUCHI, Y., and YAMAJI,T. 1987. Plasma levels of human atrial natriuretic peptide in patients with hypertensive diseases. Can. 1. Physiol. Phamacol. 65: 1701- 1705. Three types of antihuman atrial natriuretic peptide antiserum were obtained. From the study of cross-reactivity to human atrial natriuretic peptide fragments, it was suggested that antisera-1 , -2, and -3 are mostly specific to 1-28, 5-25, and the ring structure, respectively. The estimated values of this horrnone were significantly lower in the order of antisera-1, -2, and -3. Moreover, high perfomance liquid chromatographic study showed that various types of fragments of atrial natriuretic peptide exist in human plasma. These findings suggested that the highly specific antiserum to 1-28 human atrial natriuretic peptide such as antiserum-1 should be used to estimate the 1-28 human atrial natriuretic peptide levels in human plasma. From the study by using antiserum- 1, it was concluded that the plasma human atrial natriuretic peptide increased in essential hypertensives, and in patients with primary aldosteronism, chronic renal failure, and inalignant hypertension. Regarding the pathophysiological significance of increased plasma atrial natriuretic peptide, it is unlikely that this plays an important role in the etiology of essential hypertension or other hypertensive diseases, because the plasma level of this hormone is elevated in these patients. The increase of plasma atrial natriuretic peptide level in these patients should be considered to be a secondary or compensatory reaction to high blood pressure. IIMUWA, O., SHIMAMOTO, K., ANDO.T.. URA,N., TSHIDA, H.,NAKAGAWA, M., YOKOYAMA, T., FUKUYAMA, S., YARZAGUCHI, Y.. et YAMAJI,I. 1987. Plasma levels of human atrial natriuretic peptide in patients with hypertensive diseases. Can. J . Physiol. Phm~acoB.65 : 1701- 19685. On a obtenu trois types d'antiskrum anti-peptide natriurktique auriculaire humain. Suite h l'ttude de rkaction croiske A des fragments de peptide natriurktique auriculaire hurnain, on a suggtre que les antiserums-1, -2 et -3 sont surtout specifiques au peptide 1-28, 5-25 et a la structure annulaire, respectivement. Les valeurs estimdes de cette hormone ont it6 significativement plus faibles, dam l'ordre des antiskmms- 1, -2 et -3. De plus, une etude par chromatographie liquide B haute performance a montrk qu'il existe divers types de fragments de peptide natriurttique auriculaire dans le plasma humain. Ges observations ont suggtrk que l'antisCrum trks spkifique au peptide natriurktique auriculaire humain 1-28, qu'est l'antis6rum- 1, devrait &re utilise pour tvaluer les taux de peptide natriurktique auriculaire humain 1-28 dans le plasma humain. Suite h l'ttude utilisant l'antistrum-1 , on a conclu que le peptide natriurktique auriculaire du plasma humain augmentait chez les hypertensifs essentiels, ainsi que chez les patients avec aldostCronisme primaire, insuffisance rdnale chronique et une hypertension maligne. Du point de vue pathophysiologique, l'augmentation du peptide natriurktique auriculaire plasmatique ne joue vraisemblablement pas un r61e important dans l'ktiologie de l'hypertension arterielle essentielle ou autres maladies hypertensives, 6tant donnt que 1e taux plasmatique de cette horrnone est Clcvk chez ces patients. L'augmentation du taux de peptide natriurktique auriculaire plasmatique chez ces patients devrait Ctre considtrt5e comrne uile riaction secondaire s u cornpensatrice a une hypertension artkrielle. [Traduit par la revue]

Introduction Recently, atrial natriuretic peptide (ANP) has been thought to be involved in the water and sodium metabolism as weal as blood pressure regulation (Kangawa and Matsuo 1984; Palluk et al. 1985; Yamaji et a1. 1985). Numerous repc~rts(Schiffrin et al. 1985;Tikkanenetal.1985;Sagnellaetal.l985;Kuribayashiet al. 1985; Larose et al. 1985; Nakagawa et al . 1985;Sugawara et al. 1985) have been published on the measurement of ~ l a s m a levels of human ANP, and increased plasma ANP leveis were reported in patients with paroxysmal atrial tachycardia (Schiffrin et al. 1985; Yamaji et al. 1985). heart failure (Hartter et al. 1985; TiEekanen et al. 1985; Shenker et al. 1985), and renal failure (Nakagawa et al. 1985; Iimura et al. 1986). From these results, it has been suggested that blood volume change is an important regulating factor for the ANP release from the atria. However, it should be noted that various values from 6.4 to 200 pg/rnL have been reported in normal subjects in those studies

(Sagnella et al. 1985; Kuribayashi et al. 1985; Earose et aH. 1985;Nakagawa et al. 1985; ~ a k a o k et a al. 1985; Shenker et al. 1985; Iimura et al. 1986; Yamaji et a]. 1985). Therefore, to further investigate the patho~h~siological role of ANP, the establishment of a refined procedure for the determination of ANPinplasmaseemst0beessentialRegarding the plasma ANP levels in hypertensive diseases, it was repofled from Sagnella et al- (898619 Sugawara et a10 (1985), and Arendt et al. (1986) that its levels were significantly higher in essential hypertensives than in norrnotensives. In the present study, the cause of the large variation of plasma ANP values reported from different laboratories was investigated by using three types s f antisera with a different specificity and fractionating the plasma extract by high perfomance liquid chromatography. Thus, the pathophysiologica8 significance of a-human ANP (hANP) was evaluated in patients with essential hypertension and other hypertensive diseases.

his paper was presermted at the Hypertension Symposium (Whistler, B.C., July 19-21, 1986). a Satellite Symposium of the 30th IUPS Congress, and has undergone the Journal's usual peer review. 2 ~ u t h oto r whom all ccrarcspondence should be addressed.

~adioi~nrnunoassay ofcx-WP En the preparation of antiserum, synthetic a-hANP (28 amino acid, Peptide Institute, Inc., Osaka, Japan) was conjugated to bovine serum

Materials and methods


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CAN. J. PHYSICBL. PHARMACOL. VOL. 65, 198'7

albumin by a modification of the method reported by Goodfriend et al. (1964). Conjugated a-hANP was injected intradermally into five rabbits after emulsification with an equal volume of complete Freund's adjuvant. Each injected solution contained 0.5-0.6 mg of conjugated a-hANP. Blood was drawn 3 months after the first immunization and was tested for antibodies. Synthetic a-hANP was Iabeled with 12" by a modification of the chloramine-T method described by Greenwood et al. (1963). The reaction mixture was applied to high performance liquid chromatography (HPEC), and the Iabeled a-hANP was purified by this column system. Phosphate-buffered saline (PBS; pH 7.0) containing 0.1 % egg albumin, 30 mk! EDTA, and 3 nil4 1,lO-phenanthroline was used as the assay buffer. The assay buffer was added to the standard a-hANP (1- 125 pg) or sample to yield 200 pL. Then, 100 pL of antiserum and an equal volume of nonimmune rabbit semm (1 :100) were added. The tubes were incubated at 4'C for 24 h. Afer incubation. radioiodinated a-hANP (4000-50QO cpm) in 100 ~ J . L assay buffer was added to the tubes. Forty-eight hours after the addition of the labeled a-hANP, a sufficient amount of antirabbit y-globulin antiserum was added. After the tubes were centrifuged at 30W rpm for 20 min at 4'C, the supernatant was carefully decanted, and the radioactivity in the precipitates was counted with an Aloka autogamina spectrometer. The tubes containing the assay buffer and normal rabbit semm were assigned as the control tubes. The counts of the precipitate were expressed as the percent of the control tubes. The cross-reactivities to the rat ANP (28 amino acid, Peptide Institute, h c . ) , the hANP fragments 9-28, 1828, and 5-28 (Beptide Institute, Inc.), and 5-25 rat ANP (Peninsula Lab., Inc.) were determined in three hANP antisera. Plasma hANB levels were determined in 10 normal subjects by hANP radioimunoassays (RIA) using three different antisera. A plasma sample from a patient with heart failure was extracted by Sep-Pak C18 cartridge (described later). The extract was then applied to HPEC and ANP concentration in each fraction (0.5 n1L each) was measured by the t h e e different radioirnmunoassays. Plasma samples were collected into plastic syringes in the early morning after overnight fasting, and mixed with EDTA (1 m g l r d ) and aprotinin (500 kallikein inhibitor unitslml) immediately after the sampling. Plasma was quickly separated by centrifugation at 4°C and subjected to extraction. Extraction was performed by a Sep-P& C18 cartridge (Waters Associates). The adsorbed peptide was eluted with acetonitrile and 0.5% ammonium acetate solution. The elutions were evaporated under a room airstream, dissolved in the assay buffer, and subjected to radioimmunoassay.

FIG. 1. Comparison of plasma atrial natriuretic peptide (p-ANP) levels measured with three different radioirnmunc~assaysin 10 normal subjects. Ab, antiserum. Statistical unalg~sis Statistical analysis was performed employing Student's t-test with a Canon Bx-1, for paired or unpaired data. Correlation was calculated using linear regression analysis.

Wesuits Rc~dioimmtaraoasscayof a-WP The high titered antisera were obtained from three immunized rabbits (antisera-l, -2, and -3) and were used at a find dilution of 1:50000. In radioiodination, three peaks were obtained after the HPEC procedure. The first peak represents 90% binding to excess anti-hANP antiserum. but the other two peaks showed a lower binding than the first. The first peak was used as '"51labeled hANP in this radioimmunoassay system. The specific Puehophysis~ogicadrole c$h4NP in hyper~ensivediseases activity of this tracer was 2180 Ci/lnnaol(1 Ci = 37 GBq). Plasma hANP levels were determined by antiserum-1 radioimmuTypical standard curves s f antisera- 1, -2, and -3 radioimmunoassay in 12 nomotensive subjects (aged 47.2 k 1.8 years, mean 5 noassays were obtained in the range of H .O- B 25.0 pg of hANP/ SE), I B patients with essential hypertension (aged 48.1 -+ 2.9 years), 4 patients with primary aldosteronism (aged 34.3 + 4.9 years), 23 tube. Sensitivity was 2.0 pgttube with a 95% confidence limit. patients with renal failure (aged 5 1.$ -+ 2.8 years), and 4 patients with The 50% intercept was at 28.0 pgttube. In five deteminaticms, malignant hypertension (aged 42.0 2 4.7 years). All of these subjects the 7-28, 88-28, 5-25, and 5-28 ANP showed no crossexcept the patients with renal failure were hospitalized and had a reactivity in the antiserum-] radioimmumoassay. In the anticonstant diet containing 120 mequiv./day of sodium and 75 mequiv.1 serum-%radioirnmunoassay, the 7-28, 5-25, and 5-28 ANP day of potassium. On the 2nd day the measurements were performed in showed22.9 2 2.8,130 5 9.6, and'SB.4 6.4% to 1-28 ANP, patients with malignant hypertension and on the 14th day of hospitalrespectively. In the antiserum-3 radioimnnunoassay, the 7-28? ization in other subjects who had not received any antihypertensive 5-25,s-28, and 18-28 ANP showed 121 -t- 12.0. 109 5.4, drugs for at least 2 weeks prior to the measurements. 1 16 i 3.5, and 0% to 1-28 AMP, respectively. 1-28 Rat ANP Afterwards, sc)dium intake was restricted (35 mequiv.lday) in four showed a 100% cross-reactivity to 1-28 hANP in the three essential hypertensives for 7 days. In these patients, the IoIoHowing variables were measured before and after the sodium restriction. radioinsmunoassay s . Plasma lrenin activity (PRA), plasma angiotensin I1 (PANG-11), and Figure I shows the results of the simultaneous determination plasma aldosterone concentration (PAC) were measured by the method of plasma hANP by three different antisera in nom~alsubjects. of Haber et a]. (1969), direct radioi~nmunoassay(Shimamoto et al. Plasma ANP in normal subjects were 10.8 P 8 . 5 , 21.9 -b 0.9, B984), and radioi~nmunoassay(Iinuma et al. 19777, respectively . Plasand 1 B 8 C 0.9 pg/mL in antisera- 1 , -2, and -3 RIAs, respectivema vc)lume (PV) was determined by the ""--radioiodide human scrum ly. When correlations were investigated among the plasma ANP albumin (WISA) dilution method and was expressed as the percent of s , a highly the mean value per height of mormal men and women (~nL/cm-% values measured with three rddio~mmunoassay significant correlation was observed only between antisera- l normal) (Tarazi et al. 1968). Blood pressure was measurcd by an and -2 radioimmuncsassay, but not between antisera- Z and -3, auscultatory method three times a day. and the rnean arterial pressure -2 and -3 rddioirnmaanoassays (Fig. 2). was calculated.


p-AMP with Ab- 1 $PQ/~L

p-ANP with Ab-2

p-AMP with Ab-1 ( P O / ~1 L

( P U ~B L

FIG. 2. Correlations among the values of plasma human atrial natriuretic peptide (p-ANPb determined with antisera (Ab)-1, -2, and -3 in 10 normal subjects. 9 2 pe0.06d +l p&0.005 +3 pe0.01 vs. NT

40

A

E 30 a

-

€3

c2.

p

20

Q

I0

0

FIG.4. Plasma human atrial natriuretic peptide (ANP) Ievels of 12 nsnnotensives (NIT), i i patients with essential hypertension (EHT'), 23 patients with renal failure (RF) , 4 patients with primary aldos tercanism (PA), and 3 patients with malignant hypertension (MH). 1 Oil

80 60 40

20

0 F r a c t i o n no. FBG.3. Human atrial natriairetic peptide (hANP) soncentr;ition in each of the fractions of high performance liquid chromatography (HPLC), measured with antisera (Ab)-1 , -2, and -3.

The results of the HPLC study were shown in Fig. 3 . One big peak of 2 -28 ANP was found by antiserum- l radioimmunoassay in addition to the small peaks of 5-27, 4-28, and 7-28 ANP. In antiserum-2 radioimmunoassay, not only 1-28, but also 5-27,4-28, and small peaks of 7-28 ANP and unknown fragments were observed. In antiserum-3 radioimmunoassay,

obvious peaks of 4-28, 5-27, and 5-28, and unknown peaks were found in addition to many small peaks. In antiserum- l radioimmunoassay , plasma ANP level in the normal control was 9.7 + 1.3 pgimL. Intra-assay and interassay coefficients of variation were 4.4 and 5.796, respectively. P%Clthcdphysiologic*ul role ofANP in various hypertensive diseuses Plasma ANP levels were significantly higher in essential hypertensives (23.6 + 4.5 pgimL), patients with primary aldosteronism 422.3 + 4.4 pgiml), patients with renal failure (3 B .8 t 3.8 pg/mL), and patients with malignant hypertensives (30.0 + 2.9 p g i d ) than those of normal subjects (9 -7 + 1.3 pgimL) (Fig. 4). Figure 5 shows the effects of sodium restriction on BRA, PANG-11, PAC, PV, blAP, and plasma hANP in essential hypertensives. Following the sodium restriction, PV and plasma hANP decreased and PAC increased significantly. On tbe other hand, BRA and gANG-II showed a tendency to increase, and MAP a tendency to decrease by the sodium restriction, but these were not significant.

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CAN. J. PHYSHOE. PHARMACOL. TO&. 65, 1987


PWA

(mk8cm% normal

120

DANG-II

PAC

PV MAP kANP 057 (rnrnHg1 'pg8mL' rPXO.05,

yP