0021-972X/90/7004-ll03$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 70, No. 4 Printed in U.S.A.
Brain Natriuretic Peptide Administered to Man: Actions and Metabolism* ALASTAIR McGREGOR, MARK RICHARDSt, ERIC ESPINER, TIMOTHY YANDLE, and HAMID IKRAM Departments of Endocrinology and Cardiology, Princess Margaret Hospital, Christchurch 2, New Zealand
ABSTRACT. To investigate the effects and metabolism of brain natriuretic peptide (BNP) in man, eight normal subjects received 3-h infusions of synthetic porcine BNP (2 pmol/kgmin) in a placebo-controlled study. The MCR and plasma halflife of BNP were 2.69 L/min and 3.1 min, respectively. BNP clearly suppressed PRA to less than 50% of placebo values (P < 0.001). Plasma aldosterone concentrations were also significantly reduced by 30% (P < 0.05). Urinary sodium excretion tended to rise (P = 0.054), and urinary cGMP excretion was clearly enhanced (P < 0.01). Systemic and renal hemodynamics,
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hematocrit, plasma protein concentrations, plasma ACTH, arginine vasopressin, PRL, and catecholamines were unchanged. Porcine BNP has a similar range of effects and is similarly metabolized in man as human ANP. Further elucidation of the possible role of BNP as a circulating hormone in man awaits measurement of tissue and plasma concentrations of human BNP in health and disease and provision of fuller dose-response data for human as well as porcine BNP. (J Clin Endocrinol Metab 70: 1103-1107, 1990)
ECENTLY, a family of peptides with striking homology to, but clearly genetically distinct from, previously identified atrial natriuretic peptides (ANP) has been isolated from porcine brain and heart (1, 2). Whereas a considerable body of data is available to help define the role of ANP in volume and pressure homeostasis in mammalian species, information concerning the newly discovered brain natriuretic peptide (BNP) is scant. In particular, there are no published data concerning the effects of BNP administered to man. Porcine BNP is present in the brain as a 26-amino acid peptide containing a disulfide bond. The amino acid sequence contains eight substitutions and a single arginine residue C-terminal extension compared with human ANP-(103-126) (1). Molecular biologists, using genecloning techniques, have identified a unique 106-amino acid precursor (2). BNP and ANP differ in their distribution within the brain, suggesting differing or complementary roles within the nervous system (3). BNP is also found in cardiac atrial tissue, where it is present in higher concentrations than in the brain but at only 2% the level of ANP. This proportional difference between BNP and ANP immunoreactivity is also obReceived July 18, 1989. Address all correspondence and requests for reprints to: Dr. A. M. Richards, Department of Endocrinology, Princess Margaret Hospital, Christchurch 2, New Zealand. * This work was supported by the National Heart Foundation and Medical Research Council of New Zealand. t Senior Fellow of the National Heart Foundation of New Zealand.
served in vitro in the perfusate of isolated perfused porcine hearts and in vivo in porcine plasma (4). Both peptides appear to have equal affinity for the same receptors, and both induce similar dose-related generation and release of cGMP from an array of cultured tissues (5). The presence of BNP in the porcine heart and circulation raises the possibility that, like ANP, this newly discovered peptide may act as a circulating hormone. Although at the time this study was conducted BNP had not yet been identified in human tissue, in view of the striking conservation of the structure and bioactivity of ANP across mammalian species, it seemed reasonable to suspect that BNP-like peptides might also occur in human plasma and fulfill a role in circulatory regulation. Hence, we tested the effects and metabolism of BNP administered to man as a constant iv infusion. Subjects and Methods The experimental protocol was approved by the Hospital Ethical Committee. Eight normal men (aged 18-33 yr; mean, 26 yr; wt, 63-93 kg; mean, 74.5 kg) gave informed consent for participation in the study. The men were studied on two occasions (1-3 weeks apart) on the fourth day of identical constant sodium (150 mmol/day) and potassium (80 mmol/day) diets. After eating breakfast and drinking a water load (10 mL/kg distilled water) the men underwent venous cannulation of both arms for administration of infusions and withdrawal of blood samples. In three men the nondominant brachial artery was cannulated for measurement of blood pressure by the Oxford continuous recording technique (6). When review of blood
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pressure data from these first three subjects revealed no obvious acute or longer term trends, measurements for the remaining five men studied were obtained by an indirect blinded technique, using a semiautomated Rose box (Electronic Research and Development, Dunedin, New Zealand) to record blood pressure and heart rate at 10-min intervals throughout the study. The men remained seated except for standing to urinate every 30 min. Urinary indices measured included volume, sodium, potassium, calcium, magnesium, phosphate, creatinine, and cGMP (7). After each urine collection, the men drank 200 mL distilled water. A 1.5-h run-in period was followed by a 3h infusion of synthetic BNP (Peninsula, Belmont, CA; in haemaccel, at a dose of 2.0 pmol/kg- min in a volume of 15 mL/ h) or placebo (haemacel; 14 mL/h), administered in a single blind manner in random order. Haemaccel (Behring, Marburg, West Germany) is an iv solution commonly employed for plasma volume expansion consisting of 35 g degraded gelatine polypeptides cross-linked via urea bridges in 1000 mL water for injection plus physiological concentrations of sodium, potassium, calcium, chloride, phosphate, and sulfate. It was employed in this experiment as a peptide carrier to minimize loss of BNP from the infusate by adsorption onto the plastic of the infusion syringe and connecting tubing. Recordings were continued for 1.5 h after infusions were complete. The glomerular filtration rate and effective renal plasma flow were measured by standard inulin and para-aminohippuran clearance techniques. Serial venous blood samples were taken for measurement of plasma BNP concentrations at —60 and 0 min (relative to the start time of the BNP or placebo infusions) and then hourly until completion of the infusion. When the BNP infusions were halted, a series of rapid samples were taken 0, 2, 4, 6, 8, and 10 min after completion of the infusion to allow collection of data concerning the in vivo half-life of BNP immunoreactivity in human plasma. Final plasma samples for BNP assays were obtained 60 and 90 min after the infusions ended. At -60, 0, 60, 120, 180, 240, and 270 min, samples were obtained for determination of plasma ANP (8) in three cases and for measurement of PRA (9), aldosterone (10), cortisol (enzyme-linked immunosorbent assay), catecholamines (11), hematocrit, and serial automated multianalyzer plasma biochemistry profiles in all eight cases. Samples for assay of plasma ACTH (12), arginine vasopressin (AVP) (13), and PRL (14) were taken at 0, 180, and 270 min. For all hormone, para-aminohippuran, and inulin assays, all samples from an individual subject were assayed together. The intraassay coefficients of variations were 10% or less for all assays. The RIA for BNP was performed in a fashion similar to the previously described method for assay of ANP (8). BNP was extracted from plasma using Sep-Pak (C18) cartridges. The RIA incubation mixture contained 100 nh standard or sample, 100 fiL of a final 1:24,000 dilution of antiserum (Peninsula Laboratories, Belmont, CA), and 100 nL [125I]BNP (-15,000 cpm). Antiserum and sample/standard were incubated for 24 h at 4 °C, after which [125I]BNP was added, and incubation was continued for a further 24 h. Bound and free ligands were separated by the addition of 100 nL Sac-cell (IDS, Nottingham, England) and incubated for 30 min, followed by the addition of
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1 mL water. Centrifugation was followed by aspiration of unbound ligand. The precipitates were counted in a 7-counter. The minimal detectable quantity in the RIA was 0.5 fmol/tube synthetic BNP. The 50% binding intercept was 15.6 fmol/tube. Cross-reactivity with human ANP-(99-126) was less than 0.01%. Recovery of BNP added to buffer or human plasma (to achieve concentrations of 0-1,000 pmol/L; n = 20) was consistently greater than 70%. The data were analyzed by two-way analysis of variance, employing program P2V of the BMDP statistics package, with BNP or placebo and time as repeated measures. P < 0.05 was considered significant.
Results No subject incurred symptoms during the studies, and data collection was complete. Mean sodium excretion values for the 24 h before infusions were 143 ± 9 (± SE) and 145 ± 8 mmol/day before the BNP and placebo infusions, respectively (P = NS). The sodium excretion rates for the 90 min preceding the BNP and placebo infusions were 175 ± 17 and 157 ± 23 ^mol/min, respectively (P = NS). BNP infusions increased venous plasma BNP immunoreactivity to approximately 40 pmol/L above baseline and time-matched placebo values (Fig. 1; P < 0.001). BNP immunoreactivity measured in the study infusates yielded an average value of 74 ± 4% (mean ± SE) of that originally calculated, which, once corrected for extraction and assay recovery losses, suggested negligible loss of BNP onto syringe and/or storage vessel glass and plastic surfaces. The MCR of BNP immunoreactivity was calculated for each subject from MCR = infusion rate/ steady state — baseline plasma immunoreactivity. This yielded a value of 2.96 ± 0.81 L/min (mean ± SE). After infusions were halted, plasma BNP immunoreactivity returned rapidly toward baseline values. The initial decay in plasma BNP is illustrated in Fig. 1, and these data gave an initial plasma half-life of 3.1 min. Mean rates of urinary sodium excretion were greater for each urine collection period during BNP infusion than with placebo (Fig. 2), and sodium excretion was sustained, rather than exhibiting the diurnal fall in excretion rate observed with placebo. Subjects excreted an average of 8.6 ± 5 mmol (mean ± SE) more sodium during the 3-h infusion phase when receiving BNP than when placebo was given. Due to wide interindividual variation in natriuretic responses to infusions (one subject excreted slightly more sodium with placebo, and two showed only minimal increases in sodium excretion with infusion at BNP), this result was of only borderline statistical significance (P = 0.054). Urinary cGMP excretion was significantly enhanced by BNP (Fig. 2). Urinary volume and excretion of creatinine, potassium, calcium, magnesium, and phosphate were not al-
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EFFECTS OF BNP IN MAN
BNP
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FlG. 1. Plasma BNP immunoreactivity (BNP-IR) in eight normal male volunteers receiving BNP (A; 2 pmol/kgmin) or placebo (•; top panel). There was a decay in plasma BNP immunoreactivity (lower panel) in eight normal subjects after cessation of BNP infusions (0 min). The BNP immunoreactivity values given are the increment (A PLASMA BNP-IR) above baseline values plotted on a logarithmic scale, tua = 3.1 min. y = 1.6449 - 0.0957 x (r = 0.981; P < 0.001), where y = log BNP — IR, and x = time in minutes.
tered by BNP. The glomerular filtration rate, effective renal plasma flow, and renal filtration fraction were also unchanged (data not shown). PRA and plasma aldosterone concentrations were suppressed by BNP (Fig. 3). Plasma cortisol concentrations during the two infusions were similar (Fig. 3). Plasma norepinephrine values tended to fall during BNP infusions and rise slightly with placebo (P = NS). Plasma concentrations of epinephrine, ACTH, AVP, and PRL were unchanged (data not shown). Plasma ANP concentrations (three subjects) were similar before (9 ± 2 us. 8 ± 1.5 pmol/L; P = NS), during (180 min values; 8 ± 1 us. 8 ± 2 pmol/L; P = NS), and after (270 min values; 7 ± 1 us. 8 ± 2 pmol/L; P = NS) BNP and placebo infusions, respectively. Hematocrit; plasma proteins; plasma concentrations of sodium, potassium, chloride, calcium, magnesium, phosphate, urea, creatinine, urate, glucose, total and conjugated bilirubin, and alkaline phosphatase; and plasma osmolality, were similar during both infusions.
TIME ( serial 30 nlnute periods )
FIG. 2. Urinary sodium (top panel) and cGMP (lower panel) excretion in serial 30-min urine collections from eight normal volunteers receiving BNP (2 pmol/kgmin; • ) or placebo TO. Enhancement of urinary sodium excretion by BNP was of borderline statistical significance (P = 0.054), whereas cGMP excretion was clearly significantly increased (P < 0.01).
Systolic and diastolic blood pressures, pulse pressure, and heart rate were not significantly affected by BNP (data not shown). Discussion The role, if any, of BNP in man is unknown. BNP immunoreactivity, using antisera raised to porcine BNP, has been detected in porcine and canine brain and cardiac tissue, but not monkey, rat, or human tissues (3). Synthetic porcine BNP does induce cGMP release in cultured tissues from other species (rat and bovine) (5). These findings plus previous knowledge of the interspecies conservation of ANP structure and function suggested that a human form of BNP was likely to exist. At the time of writing, Kojima et al. confirmed the presence of human BNP by molecular biology. The C-terminal bioactive form in man appears likely to differ from porcine BNP by at least six amino acid substitutions
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MCGREGOR ET AL.
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400 •
PLASMA CORTISOL
200 •
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-1
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2 TIME
(h)
FIG. 3. PRA and plasma aldosterone and cortisol concentrations in eight normal volunteers receiving BNP (2 pmol/kg-min; A) or placebo (•). BNP significantly suppressed PRA (P < 0.001) and plasma aldosterone (P < 0.05), whereas cortisol was unchanged (P = NS).
(15). In the current study a dose of peptide was chosen at which ANP is known to exert effects on renal and hormonal indices in association with a rise in plasma ANP concentrations barely beyond normal into the low pathophysiological range (16). If, indeed, BNP exists in the circulation at levels only about 2% those of ANP (4), such a dose seems likely to raise plasma BNP levels to or above the maximum that they are ever likely to attain in health or disease and should, therefore, offer an initial indication of the maximal short term effects possible for BNP to exert as a circulating hormone. We found baseline (BNP-like) immunoreactivity to be comparable to that of ANP, rather than the much lower level we might have expected from previous reports (4), but in the absence of high pressure liquid chromatography or other confirmatory data we cannot be certain of the degree to
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which nonspecific interference with the assay contributes to this baseline value. Recovery of BNP added to buffer or plasma was consistent (see Materials and Methods), and we are confident that the rise in plasma BNP immunoreactivity above baseline accurately reflected the change in plasma BNP concentrations with infusions. Hence, although we remain uncertain of the true proportional increment (i.e. fold-rise) in plasma BNP above baseline in the current study, our methods still permit accurate measurement of the MCR and plasma half-life of porcine BNP administered to man. The MCR and plasma half-life of BNP immunoreactivity were documented and found to be comparable to those for human ANP-(99-126) (17). BNP (2 pmol/kg-min) suppressed the renin-angiotensin-aldosterone system to a similar extent as ANP given to man at doses of 0.75 and 2.0 pmol/kg; min under similar conditions (16,18). BNP administered systematically had no effect on pituitary hormones, including ACTH, AVP, and PRL. Hence, it is unlikely that any effect BNP may in the future prove to have on these hormones will be via its role as a circulatory hormone, but it will rather act as a central nervous system neuropeptide. BNP also caused a trend toward enhanced natriuresis and an associated clear-cut rise in urinary cGMP. However, the urinary effects of BNP were considerably less than those previously observed for ANP given at only 0.75 pmol/kg-min (18). Extrapolations from studies of small numbers of subjects in which conditions were not entirely identical between groups do not allow definitive statements, but it seems possible that in man BNP has a somewhat diminished renal effect (on a molar basis) compared with ANP. Other variables measured, including other urine indices, systemic and renal hemodynamics, blood pressure, heart rate, hematocrit, and plasma proteins, were not significantly changed by BNP. These negative findings are in contrast to those found with ANP given at this dose, which resulted in significant increases in renal filtration fraction, hematocrit, and plasma protein concentrations (16). Differing background conditions, including sodium balance (the subjects receiving ANP were ingesting 200 mmol sodium/day, whereas those in the current study took only 150 mmol/day), may partly explain this discrepancy. In addition, it is possible that porcine BNP is sufficiently different from human BNP (15) that its affinity for ANP/BNP receptors is somewhat less than that of human ANP. In summary, BNP administered iv to man at 2 pmol/ kg-min clearly suppressed renin-angiotensin-aldosterone system activity in association with a borderline natriuretic effect and clear-cut enhancement of urinary cGMP excretion. The MCR and plasma half-life of BNP were comparable to those of ANP. Further elucidation
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EFFECTS OF BNP IN MAN
of the possible role of BNP as a circulating hormone in man must await provision of fuller dose-response data, identification of human BNP, and measurement of concentrations of this peptide in human tissue and plasma in health and disease.
Acknowledgments Assistance from the dietetic and laboratory technical staff is gratefully acknowledged. Secretarial assistance was provided by Mrs. P. Hollis.
References 1. Sudoh T, Kangawa K, Minamino N, Matsuo H. A new natriuretic peptide in porcine brain. Nature. 1988;332:78-80. 2. Minamino N, Kangawa K, Matsuo H. Isolation and identification of a high molecular weight brain natriuretic peptide in porcine cardiac atrium. Biochem Biophys Res Commun. 1988;157:402-9. 3. Itoh H, Nakao K, Saito Y, et al. Radioimmunoassay for brain natriuretic peptide (BNP): detection of BNP in canine brain. Biochem Biophys Res Commun. 1989;158:120-8. 4. Saito Y, Nakao K, Itoh H, et al. Brain natriuretic peptide is a novel cardiac hormone. Biochem Biophys Res Commun. 1989; 158:360-8. 5. Song D, Kohse KP, Murad F. Brain natriuretic factor augmentation of cellular cyclic GMP, activation of particulate guanylate cyclase and receptor binding. FEBS Lett. 1988;232:125-9. 6. Millar-Craig MW, Hawes D, Whittington J. New system for recording ambulatory blood pressure in man. Med Biol Engin Comput. 1978;16:727-31. 7. Steiner AL, Parker CW, Kipnis DM. Radioimmunoassay for cyclic
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nucleotides. J Biol Chem. 1972;247:1106-13. 8. Yandle TG, Espiner EA, Nicholls MG, Duff H. Radioimmunoassay and characterization of atrial natriuretic peptide in human plasma. J Clin Endocrinol Metab. 1986;63:72-8. 9. Dunn PJ, Espiner EA. Outpatient screening tests for primary aldosteronism. Aust NZ J Med. 1976;6:131-5. 10. Lun S, Espiner EA, Nicholls MG, Yandle TG. A direct radioimmunoassay for aldosterone in plasma. Clin Chem. 1983;29:26871. 11. Peuler JD, Johnson GA. Simultaneous single isotope radioenzymatic assays of plasma norepinephrine, epinephrine and dopamine. Life Sci. 1977;21:625-33. 12. Donald RA. Radioimmunoassay of corticotropin (ACTH). In: Abrahams G, ed, Handbook of endocrinology. New York, Basel: Marcel-Dekker; 1977;319-91. 13. Sadler WA, Lynskey CP, Gilchrist NL, Espiner EA, Nicholls MG. A sensitive radioimmunoassay for measuring plasma anti-diuretic hormone in man. NZ Med J. 1983;96:959-63. 14. Livesey JH, Donald RA. Prevention of adsorption losses during radioimmunoassay of polypeptide hormones: effectiveness of albumins, gelatin, caseins, Tween 20 and plasma. Clin Chim Acta. 1982;123:193-9. 15. Sudoh T, Maekawa K, Kojima M, Minamino M, Kangawa K, Matsuo H. Cloning and sequence analysis of cDNA encoding a precursor for human brain natriuretic peptide. Biochem Biophys Res Commun. 1989;159:1427-34. 16. Richards AM, Tonolo G, Montorsi P, et al. Low dose infusions of 26- and 28-amino acid human atrial natriuretic peptides in normal man. J Clin Endocrinol Metab. 1988;66:465-72. 17. Yandle TG, Richards AM, Nicholls MG, Cuneo R, Espiner EA, Livesey JH. Metabolic clearance rate and plasma half life of alphahuman atrial natriuretic peptide in man. Life Sci. 1986;38:182733. 18. Richards AM, McDonald D, Fitzpatrick MA, et al. Atrial natriuretic hormone has biological effects in man at physiological plasma concentrations. J Clin Endocrinol Metab. 1988;67:1134-9.
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Vol. 70, No. 4 Printed in U.S.A.
Brain Natriuretic Peptide Administered to Man: Actions and Metabolism* ALASTAIR McGREGOR, MARK RICHARDSt, ERIC ESPINER, TIMOTHY YANDLE, and HAMID IKRAM Departments of Endocrinology and Cardiology, Princess Margaret Hospital, Christchurch 2, New Zealand
ABSTRACT. To investigate the effects and metabolism of brain natriuretic peptide (BNP) in man, eight normal subjects received 3-h infusions of synthetic porcine BNP (2 pmol/kgmin) in a placebo-controlled study. The MCR and plasma halflife of BNP were 2.69 L/min and 3.1 min, respectively. BNP clearly suppressed PRA to less than 50% of placebo values (P < 0.001). Plasma aldosterone concentrations were also significantly reduced by 30% (P < 0.05). Urinary sodium excretion tended to rise (P = 0.054), and urinary cGMP excretion was clearly enhanced (P < 0.01). Systemic and renal hemodynamics,
R
hematocrit, plasma protein concentrations, plasma ACTH, arginine vasopressin, PRL, and catecholamines were unchanged. Porcine BNP has a similar range of effects and is similarly metabolized in man as human ANP. Further elucidation of the possible role of BNP as a circulating hormone in man awaits measurement of tissue and plasma concentrations of human BNP in health and disease and provision of fuller dose-response data for human as well as porcine BNP. (J Clin Endocrinol Metab 70: 1103-1107, 1990)
ECENTLY, a family of peptides with striking homology to, but clearly genetically distinct from, previously identified atrial natriuretic peptides (ANP) has been isolated from porcine brain and heart (1, 2). Whereas a considerable body of data is available to help define the role of ANP in volume and pressure homeostasis in mammalian species, information concerning the newly discovered brain natriuretic peptide (BNP) is scant. In particular, there are no published data concerning the effects of BNP administered to man. Porcine BNP is present in the brain as a 26-amino acid peptide containing a disulfide bond. The amino acid sequence contains eight substitutions and a single arginine residue C-terminal extension compared with human ANP-(103-126) (1). Molecular biologists, using genecloning techniques, have identified a unique 106-amino acid precursor (2). BNP and ANP differ in their distribution within the brain, suggesting differing or complementary roles within the nervous system (3). BNP is also found in cardiac atrial tissue, where it is present in higher concentrations than in the brain but at only 2% the level of ANP. This proportional difference between BNP and ANP immunoreactivity is also obReceived July 18, 1989. Address all correspondence and requests for reprints to: Dr. A. M. Richards, Department of Endocrinology, Princess Margaret Hospital, Christchurch 2, New Zealand. * This work was supported by the National Heart Foundation and Medical Research Council of New Zealand. t Senior Fellow of the National Heart Foundation of New Zealand.
served in vitro in the perfusate of isolated perfused porcine hearts and in vivo in porcine plasma (4). Both peptides appear to have equal affinity for the same receptors, and both induce similar dose-related generation and release of cGMP from an array of cultured tissues (5). The presence of BNP in the porcine heart and circulation raises the possibility that, like ANP, this newly discovered peptide may act as a circulating hormone. Although at the time this study was conducted BNP had not yet been identified in human tissue, in view of the striking conservation of the structure and bioactivity of ANP across mammalian species, it seemed reasonable to suspect that BNP-like peptides might also occur in human plasma and fulfill a role in circulatory regulation. Hence, we tested the effects and metabolism of BNP administered to man as a constant iv infusion. Subjects and Methods The experimental protocol was approved by the Hospital Ethical Committee. Eight normal men (aged 18-33 yr; mean, 26 yr; wt, 63-93 kg; mean, 74.5 kg) gave informed consent for participation in the study. The men were studied on two occasions (1-3 weeks apart) on the fourth day of identical constant sodium (150 mmol/day) and potassium (80 mmol/day) diets. After eating breakfast and drinking a water load (10 mL/kg distilled water) the men underwent venous cannulation of both arms for administration of infusions and withdrawal of blood samples. In three men the nondominant brachial artery was cannulated for measurement of blood pressure by the Oxford continuous recording technique (6). When review of blood
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MCGREGOR ET AL.
pressure data from these first three subjects revealed no obvious acute or longer term trends, measurements for the remaining five men studied were obtained by an indirect blinded technique, using a semiautomated Rose box (Electronic Research and Development, Dunedin, New Zealand) to record blood pressure and heart rate at 10-min intervals throughout the study. The men remained seated except for standing to urinate every 30 min. Urinary indices measured included volume, sodium, potassium, calcium, magnesium, phosphate, creatinine, and cGMP (7). After each urine collection, the men drank 200 mL distilled water. A 1.5-h run-in period was followed by a 3h infusion of synthetic BNP (Peninsula, Belmont, CA; in haemaccel, at a dose of 2.0 pmol/kg- min in a volume of 15 mL/ h) or placebo (haemacel; 14 mL/h), administered in a single blind manner in random order. Haemaccel (Behring, Marburg, West Germany) is an iv solution commonly employed for plasma volume expansion consisting of 35 g degraded gelatine polypeptides cross-linked via urea bridges in 1000 mL water for injection plus physiological concentrations of sodium, potassium, calcium, chloride, phosphate, and sulfate. It was employed in this experiment as a peptide carrier to minimize loss of BNP from the infusate by adsorption onto the plastic of the infusion syringe and connecting tubing. Recordings were continued for 1.5 h after infusions were complete. The glomerular filtration rate and effective renal plasma flow were measured by standard inulin and para-aminohippuran clearance techniques. Serial venous blood samples were taken for measurement of plasma BNP concentrations at —60 and 0 min (relative to the start time of the BNP or placebo infusions) and then hourly until completion of the infusion. When the BNP infusions were halted, a series of rapid samples were taken 0, 2, 4, 6, 8, and 10 min after completion of the infusion to allow collection of data concerning the in vivo half-life of BNP immunoreactivity in human plasma. Final plasma samples for BNP assays were obtained 60 and 90 min after the infusions ended. At -60, 0, 60, 120, 180, 240, and 270 min, samples were obtained for determination of plasma ANP (8) in three cases and for measurement of PRA (9), aldosterone (10), cortisol (enzyme-linked immunosorbent assay), catecholamines (11), hematocrit, and serial automated multianalyzer plasma biochemistry profiles in all eight cases. Samples for assay of plasma ACTH (12), arginine vasopressin (AVP) (13), and PRL (14) were taken at 0, 180, and 270 min. For all hormone, para-aminohippuran, and inulin assays, all samples from an individual subject were assayed together. The intraassay coefficients of variations were 10% or less for all assays. The RIA for BNP was performed in a fashion similar to the previously described method for assay of ANP (8). BNP was extracted from plasma using Sep-Pak (C18) cartridges. The RIA incubation mixture contained 100 nh standard or sample, 100 fiL of a final 1:24,000 dilution of antiserum (Peninsula Laboratories, Belmont, CA), and 100 nL [125I]BNP (-15,000 cpm). Antiserum and sample/standard were incubated for 24 h at 4 °C, after which [125I]BNP was added, and incubation was continued for a further 24 h. Bound and free ligands were separated by the addition of 100 nL Sac-cell (IDS, Nottingham, England) and incubated for 30 min, followed by the addition of
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1 mL water. Centrifugation was followed by aspiration of unbound ligand. The precipitates were counted in a 7-counter. The minimal detectable quantity in the RIA was 0.5 fmol/tube synthetic BNP. The 50% binding intercept was 15.6 fmol/tube. Cross-reactivity with human ANP-(99-126) was less than 0.01%. Recovery of BNP added to buffer or human plasma (to achieve concentrations of 0-1,000 pmol/L; n = 20) was consistently greater than 70%. The data were analyzed by two-way analysis of variance, employing program P2V of the BMDP statistics package, with BNP or placebo and time as repeated measures. P < 0.05 was considered significant.
Results No subject incurred symptoms during the studies, and data collection was complete. Mean sodium excretion values for the 24 h before infusions were 143 ± 9 (± SE) and 145 ± 8 mmol/day before the BNP and placebo infusions, respectively (P = NS). The sodium excretion rates for the 90 min preceding the BNP and placebo infusions were 175 ± 17 and 157 ± 23 ^mol/min, respectively (P = NS). BNP infusions increased venous plasma BNP immunoreactivity to approximately 40 pmol/L above baseline and time-matched placebo values (Fig. 1; P < 0.001). BNP immunoreactivity measured in the study infusates yielded an average value of 74 ± 4% (mean ± SE) of that originally calculated, which, once corrected for extraction and assay recovery losses, suggested negligible loss of BNP onto syringe and/or storage vessel glass and plastic surfaces. The MCR of BNP immunoreactivity was calculated for each subject from MCR = infusion rate/ steady state — baseline plasma immunoreactivity. This yielded a value of 2.96 ± 0.81 L/min (mean ± SE). After infusions were halted, plasma BNP immunoreactivity returned rapidly toward baseline values. The initial decay in plasma BNP is illustrated in Fig. 1, and these data gave an initial plasma half-life of 3.1 min. Mean rates of urinary sodium excretion were greater for each urine collection period during BNP infusion than with placebo (Fig. 2), and sodium excretion was sustained, rather than exhibiting the diurnal fall in excretion rate observed with placebo. Subjects excreted an average of 8.6 ± 5 mmol (mean ± SE) more sodium during the 3-h infusion phase when receiving BNP than when placebo was given. Due to wide interindividual variation in natriuretic responses to infusions (one subject excreted slightly more sodium with placebo, and two showed only minimal increases in sodium excretion with infusion at BNP), this result was of only borderline statistical significance (P = 0.054). Urinary cGMP excretion was significantly enhanced by BNP (Fig. 2). Urinary volume and excretion of creatinine, potassium, calcium, magnesium, and phosphate were not al-
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EFFECTS OF BNP IN MAN
BNP
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URINE SODIUM ( imol )
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END BNP INFUSION
i 6 4 TIME (min)
10
FlG. 1. Plasma BNP immunoreactivity (BNP-IR) in eight normal male volunteers receiving BNP (A; 2 pmol/kgmin) or placebo (•; top panel). There was a decay in plasma BNP immunoreactivity (lower panel) in eight normal subjects after cessation of BNP infusions (0 min). The BNP immunoreactivity values given are the increment (A PLASMA BNP-IR) above baseline values plotted on a logarithmic scale, tua = 3.1 min. y = 1.6449 - 0.0957 x (r = 0.981; P < 0.001), where y = log BNP — IR, and x = time in minutes.
tered by BNP. The glomerular filtration rate, effective renal plasma flow, and renal filtration fraction were also unchanged (data not shown). PRA and plasma aldosterone concentrations were suppressed by BNP (Fig. 3). Plasma cortisol concentrations during the two infusions were similar (Fig. 3). Plasma norepinephrine values tended to fall during BNP infusions and rise slightly with placebo (P = NS). Plasma concentrations of epinephrine, ACTH, AVP, and PRL were unchanged (data not shown). Plasma ANP concentrations (three subjects) were similar before (9 ± 2 us. 8 ± 1.5 pmol/L; P = NS), during (180 min values; 8 ± 1 us. 8 ± 2 pmol/L; P = NS), and after (270 min values; 7 ± 1 us. 8 ± 2 pmol/L; P = NS) BNP and placebo infusions, respectively. Hematocrit; plasma proteins; plasma concentrations of sodium, potassium, chloride, calcium, magnesium, phosphate, urea, creatinine, urate, glucose, total and conjugated bilirubin, and alkaline phosphatase; and plasma osmolality, were similar during both infusions.
TIME ( serial 30 nlnute periods )
FIG. 2. Urinary sodium (top panel) and cGMP (lower panel) excretion in serial 30-min urine collections from eight normal volunteers receiving BNP (2 pmol/kgmin; • ) or placebo TO. Enhancement of urinary sodium excretion by BNP was of borderline statistical significance (P = 0.054), whereas cGMP excretion was clearly significantly increased (P < 0.01).
Systolic and diastolic blood pressures, pulse pressure, and heart rate were not significantly affected by BNP (data not shown). Discussion The role, if any, of BNP in man is unknown. BNP immunoreactivity, using antisera raised to porcine BNP, has been detected in porcine and canine brain and cardiac tissue, but not monkey, rat, or human tissues (3). Synthetic porcine BNP does induce cGMP release in cultured tissues from other species (rat and bovine) (5). These findings plus previous knowledge of the interspecies conservation of ANP structure and function suggested that a human form of BNP was likely to exist. At the time of writing, Kojima et al. confirmed the presence of human BNP by molecular biology. The C-terminal bioactive form in man appears likely to differ from porcine BNP by at least six amino acid substitutions
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MCGREGOR ET AL.
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BNP
A/
PLACEBO
1.0 -
PRA ( rmol/l/h )
0.5 -
PLASMA AIDOSTERONE
1QQ
( pnol/1 )
400 •
PLASMA CORTISOL
200 •
( nml/1 )
-1
1
2 TIME
(h)
FIG. 3. PRA and plasma aldosterone and cortisol concentrations in eight normal volunteers receiving BNP (2 pmol/kg-min; A) or placebo (•). BNP significantly suppressed PRA (P < 0.001) and plasma aldosterone (P < 0.05), whereas cortisol was unchanged (P = NS).
(15). In the current study a dose of peptide was chosen at which ANP is known to exert effects on renal and hormonal indices in association with a rise in plasma ANP concentrations barely beyond normal into the low pathophysiological range (16). If, indeed, BNP exists in the circulation at levels only about 2% those of ANP (4), such a dose seems likely to raise plasma BNP levels to or above the maximum that they are ever likely to attain in health or disease and should, therefore, offer an initial indication of the maximal short term effects possible for BNP to exert as a circulating hormone. We found baseline (BNP-like) immunoreactivity to be comparable to that of ANP, rather than the much lower level we might have expected from previous reports (4), but in the absence of high pressure liquid chromatography or other confirmatory data we cannot be certain of the degree to
JCE & M • 1990 Vol70«No4
which nonspecific interference with the assay contributes to this baseline value. Recovery of BNP added to buffer or plasma was consistent (see Materials and Methods), and we are confident that the rise in plasma BNP immunoreactivity above baseline accurately reflected the change in plasma BNP concentrations with infusions. Hence, although we remain uncertain of the true proportional increment (i.e. fold-rise) in plasma BNP above baseline in the current study, our methods still permit accurate measurement of the MCR and plasma half-life of porcine BNP administered to man. The MCR and plasma half-life of BNP immunoreactivity were documented and found to be comparable to those for human ANP-(99-126) (17). BNP (2 pmol/kg-min) suppressed the renin-angiotensin-aldosterone system to a similar extent as ANP given to man at doses of 0.75 and 2.0 pmol/kg; min under similar conditions (16,18). BNP administered systematically had no effect on pituitary hormones, including ACTH, AVP, and PRL. Hence, it is unlikely that any effect BNP may in the future prove to have on these hormones will be via its role as a circulatory hormone, but it will rather act as a central nervous system neuropeptide. BNP also caused a trend toward enhanced natriuresis and an associated clear-cut rise in urinary cGMP. However, the urinary effects of BNP were considerably less than those previously observed for ANP given at only 0.75 pmol/kg-min (18). Extrapolations from studies of small numbers of subjects in which conditions were not entirely identical between groups do not allow definitive statements, but it seems possible that in man BNP has a somewhat diminished renal effect (on a molar basis) compared with ANP. Other variables measured, including other urine indices, systemic and renal hemodynamics, blood pressure, heart rate, hematocrit, and plasma proteins, were not significantly changed by BNP. These negative findings are in contrast to those found with ANP given at this dose, which resulted in significant increases in renal filtration fraction, hematocrit, and plasma protein concentrations (16). Differing background conditions, including sodium balance (the subjects receiving ANP were ingesting 200 mmol sodium/day, whereas those in the current study took only 150 mmol/day), may partly explain this discrepancy. In addition, it is possible that porcine BNP is sufficiently different from human BNP (15) that its affinity for ANP/BNP receptors is somewhat less than that of human ANP. In summary, BNP administered iv to man at 2 pmol/ kg-min clearly suppressed renin-angiotensin-aldosterone system activity in association with a borderline natriuretic effect and clear-cut enhancement of urinary cGMP excretion. The MCR and plasma half-life of BNP were comparable to those of ANP. Further elucidation
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EFFECTS OF BNP IN MAN
of the possible role of BNP as a circulating hormone in man must await provision of fuller dose-response data, identification of human BNP, and measurement of concentrations of this peptide in human tissue and plasma in health and disease.
Acknowledgments Assistance from the dietetic and laboratory technical staff is gratefully acknowledged. Secretarial assistance was provided by Mrs. P. Hollis.
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nucleotides. J Biol Chem. 1972;247:1106-13. 8. Yandle TG, Espiner EA, Nicholls MG, Duff H. Radioimmunoassay and characterization of atrial natriuretic peptide in human plasma. J Clin Endocrinol Metab. 1986;63:72-8. 9. Dunn PJ, Espiner EA. Outpatient screening tests for primary aldosteronism. Aust NZ J Med. 1976;6:131-5. 10. Lun S, Espiner EA, Nicholls MG, Yandle TG. A direct radioimmunoassay for aldosterone in plasma. Clin Chem. 1983;29:26871. 11. Peuler JD, Johnson GA. Simultaneous single isotope radioenzymatic assays of plasma norepinephrine, epinephrine and dopamine. Life Sci. 1977;21:625-33. 12. Donald RA. Radioimmunoassay of corticotropin (ACTH). In: Abrahams G, ed, Handbook of endocrinology. New York, Basel: Marcel-Dekker; 1977;319-91. 13. Sadler WA, Lynskey CP, Gilchrist NL, Espiner EA, Nicholls MG. A sensitive radioimmunoassay for measuring plasma anti-diuretic hormone in man. NZ Med J. 1983;96:959-63. 14. Livesey JH, Donald RA. Prevention of adsorption losses during radioimmunoassay of polypeptide hormones: effectiveness of albumins, gelatin, caseins, Tween 20 and plasma. Clin Chim Acta. 1982;123:193-9. 15. Sudoh T, Maekawa K, Kojima M, Minamino M, Kangawa K, Matsuo H. Cloning and sequence analysis of cDNA encoding a precursor for human brain natriuretic peptide. Biochem Biophys Res Commun. 1989;159:1427-34. 16. Richards AM, Tonolo G, Montorsi P, et al. Low dose infusions of 26- and 28-amino acid human atrial natriuretic peptides in normal man. J Clin Endocrinol Metab. 1988;66:465-72. 17. Yandle TG, Richards AM, Nicholls MG, Cuneo R, Espiner EA, Livesey JH. Metabolic clearance rate and plasma half life of alphahuman atrial natriuretic peptide in man. Life Sci. 1986;38:182733. 18. Richards AM, McDonald D, Fitzpatrick MA, et al. Atrial natriuretic hormone has biological effects in man at physiological plasma concentrations. J Clin Endocrinol Metab. 1988;67:1134-9.
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