Brain Natriuretic Peptide as a Cardiac Hormone in Essential Hypertension MASAKAZUKOHNO, M.D., TAKESHIHORIO, M.D., KOJIYOKOKAWA,M.D., KOH-ICHIMURAKAWA,M.D., KENICHIYASUNARI,M.D., KANAMEAKIOKA,M.D., AKIRATAHARA,M.D., IKUTODA, M.D., KAZUHIDETAKEUCHI,M.D., NAOTSUGUKURIHARA,M.D., TADANAOTAKEDA,M.D., Osaka, Japan
PURPOSE: A natriuretic peptide, brain natriuretic peptide (BNP), has been isolated from porcine hearts. We performed this study to determine if BNP is secreted from the heart and to identify changes, if any, in the plasma BNP concentration in essential hypertension. PATIENTSANDMETHODS: Wemeasuredtheimmunoreactive (ir) BNP concentration at intracardiac sites including the coronary sinus of five patients with heart disease during cardiac catheterization. We examined plasma ir-BNP in 48 hypertensive patients, 15 borderline hypertensive patients, and 25 normotensive subjects. RESULTS: Plasma ir-BNP in the coronary sinus was greater than at other cardiac sites. The concentration was significantly higher in hypertensive subjects than in borderline hypertensive or normotensive subjects. Hypertensive patients with left ventricular hypertrophy (LVH) established by echocardiography had higher plasma ir-BNP levels than those without LVH. In the hypertensive group, plasma ir-BNP was closely correlated with the LV mass index. In these patients, BNP levels were correlated with mean arterial pressure and inversely correlated with the LV ejection fraction, although these correlations were weak. Reverse-phase high-pressure liquid chromatography showed that the major component of circulating ir-BNP in the hypertensive and normotensive subjects corresponded to authentic human BNP-32. CONCLUSIONS: Human BNP-32 was secreted through the coronary siuus from the heart and may act as a cardiac hormone. Plasma BNP was increased in many of the hypertensive subjects with LVlXTheiucreaseiuBNPseemedtoberelatedto LXH or the cardiac overload associated with LVH. From the First Department of Internal Medicine, Osaka City University Medical School, Osaka, Japan. This work was supported by a Grant-in-Aid for Scientific Research (6148210) from the Ministry of Education, Science, and Culture, Japan. Requests for reprints should be addressed to Masakazu Kohno, M.D., First Department of Internal Medicine, Osaka City University Medical School, l-5-7 Asahi-machi, Abeno-ku. Osaka 545, Japan. Manuscript submitted February 4, 1991, and accepted in revised form September 1. 1991.
rain natriuretic peptide (BNP) was first identified in the porcine brain [l] and later isolated from porcine heart . Porcine BNP consists of 26 amino-acid residues that share considerable homology with the sequence of atria1 natriuretic peptide (ANP) [l]. BNP elicits a spectrum of diuretic, natriuretic, and hypotensive effects similar to that of ANP [l]. BNP interacts with the same receptors as ANP in vascular smooth muscle cells [3,4]. Recently, a low-molecular-weight form of human BNP, human BNP-32, which corresponds to the C-terminal sequence (77-108) of the human BNP precursor deduced from the cDNA sequence, was found in the human atrium 151. Subsequently, immunoreactive (ir) BNP was found in human plasma; its concentration is high in plasma from patients with congestive heart failure . Here, we measured ir-BNP concentrations in plasma samples obtained from various intracardiac sites including the coronary sinus of patients with ischemic or valvular heart disease during cardiac catheterization. The results suggested that human BNP-32 was secreted through the coronary sinus from the heart and that this peptide may act as a cardiac hormone (Protocol 1). Then we measured plasma ir-BNP concentrations in patients with essential hypertension with or without left ventricular hypertrophy (LVH) as defined by echocardiography. The results were compared with those in normotensive and borderline hypertensive subjects. We also examined the circulating form by reverse-phase high-pressure liquid chromatography (HPLC) coupled with a radioimmunoassay (Protocol 2).
PATIENTSAND METHODS Protocol I We studied four patients with ischemic heart disease and one with valvular heart disease (three men and two women; mean age, 66 years; range, 56 to 77 years) who were undergoing diagnostic right and left cardiac catheterization. Right and left heart studies were performed with standard techniques through the femoral artery and venous entry sites. Venous samples were taken from the inferior vena January
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cava, right atrium, coronary sinus, and pulmonary artery. Arterial samples were taken from the femoral artery.
Milford, MA). After the cartridge was washed with distilled water, the adsorbed peptides were eluted with 86% ethanol in 4% acetic acid. After evaporation of the eluent with a centrifugal evaporator (Model RD-31, Yamato Scientific Co., Tokyo, Japan), the dry residue was dissolved in the assay buffer described below. The recovery rate was calculated by the addition of three amounts of cold human BNP-32 (5,20, and 100 pg/mL) to hormonefree plasma prepared by being passed through a Sep-Pak Cls cartridge. The recovery rate of 5, 20, and 100 pg/mL by our extraction method was 67.3%, 67.5%, and 68.5%, respectively. The concentration of plasma ir-BNP was measured with antibody against synthetic human BNP-32 and lz51labeled human BNP-32 (Peninsula Laboratories, Inc., Belmont, CA). This antibody reacted 100% with human BNP-32 and cross-reacted 0.05% with rat BNP-32. It did not have any cross-reactivity with a-human ANP(l-28) or ANP(5-28), a-rat ANP(l-28), porcine BNP-26, rat BNP-45, ,&endorphin, angiotensin II, vasopressin, or endothelin-1. The radioimmunoassay was done in an assay buffer of 0.01 M sodium phosphate, pH 7.4, containing 0.05 M sodium chloride, 0.1% bovine serum albumin, 0.1% Nonidet P-40, and 0.01% NaNs as reported elsewhere for ANP . In brief, 100 PL of antiserum (final dilution: 1:300,000) and 100 PL of the sample or 100 PL of standard human BNP-32 were dissolved in the assay buffer and then incubated for 24 hours at 4’C. Approximately 15,000 counts/minute of 1251-labeled human BNP-32 was added to each reaction, and the reaction mixture was incubated for an additional 24 hours. After the second 24-hour incubation, 100 PL of diluted normal rabbit serum and 100 PL of goat anti-rabbit immunoglobulin G serum, also diluted, were added, and the mixture was again incubated for 24 hours. After the third incubation, the precipitate was collected by centrifugation at 1,700g for 30 minutes. The supernatant was removed by aspiration, and the pellet was counted for 1251 with a gamma counter. The effective range of the standard curve was between 0.5 and 100 pg of human BNP-32 (Figure 1). To calculate the coefficient of variation, we assayed 10 human plasma samples 4 times each for the interassay variation and assayed 20 human plasma samples for the intra-assay variation. The inter-assay variation was 11.7%, and the intra-assay variation was 7.0%. Reverse-phase HPLC was performed with an octadecylsilica column (4.6 X 250 mm, Tosoh Corp., Tokyo, Japan) eluted with a linear gradient of acetonitrile from 15% to 60% in 0.09% trifluoroacetic acid with a flow rate of 1 ml/minute; 1-mL frac-
Protocol 2 Between June 1990 and February 1991, we recruited 88 patients for this study from a population of some 350 patients with various diseases seen in our department. Routine laboratory studies of all patients included assays of serum electrolytes, serum creatinine, blood urea nitrogen, and the fasting blood glucose level, liver function tests, urinalysis, a chest roentgenogram, and an electrocardiogram. Based on the results of laboratory tests and the World Health Organization Classification , subjects were placed into one of the following three diagnostic categories: normal, hypertensive, or borderline. Normal blood pressure was defined as a systolic pressure of 140 mm Hg or less and a diastolic pressure of 90 mm Hg or less. Hypertension was defined as a systolic pressure of 160 mm Hg or more or a diastolic pressure of 95 mm Hg or more, or both. The term “borderline hypertension” was used to denote blood pressure values between these normal and hypertensive ranges. Secondary hypertension was excluded by the taking of a clinical history, physical examination, routine laboratory tests including measurements of plasma renin activity, aldosterone, catecholamines, and cortisol, and an excretory urogram or renal arteriogram. None of these patients had signs or symptoms of cardiac, renal, or hepatic failure or of diabetes, and none had clinical evidence of pulmonary disease, angina pectoris, or myocardial infarction. Only hypertensive patients who had not been treated earlier with antihypertensive drugs or whose antihypertensive drug therapy had been discontinued for at least the preceding 2 weeks were included in the study. Arterial blood pressure was measured with a mercury sphygmomanometer after the patient had rested sitting in a quiet, warm room for 30 minutes. The mean of three blood pressure measurements obtained at two different times was used to classify the subjects. A blood sample (6 mL) was drawn directly into siliconized disposable glass tubes containing aprotinin (5 X lo5 kallikrein inactivator units/L) and EDTA (1 g/L) chilled on ice. Plasma was separated by centrifugation for 10 minutes at 4°C and immediately frozen and stored at -80°C for several days. BNP was extracted essentially as reported for ANP . Briefly, 2 mL of plasma was diluted with 5 mL of 4% acetic acid. After centrifugation, the solution was pumped at the rate of 1 ml/minute through a Sep-Pak Cls cartridge (Water Associates,
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TABLE I Regional Plasma Concentrationsof ir.BNP ir-BNP Concentration (pg/mLI Mean f SD Range Coronary sinus Right atrium Pulmonary artery Inferior vena cava Femoral artery
12:6-42:3 6.1-16.1 9.6-25.2
15.7 ; 6:8
Significance* p p p p
zo5* < 0.05*