- Email: [email protected]
Comparison of vessel dilator and long-acting natriuretic peptide in the treatment of congestive heart failure
Comparison of vessel dilator and long-acting natriuretic peptide in the treatment of congestive heart failure David L. Vesely, MD, PhD, John R. Dietz, PhD, James R. Parks, RN, Ernest A. Antwi, MD, Rose M. Overton, BS, Michael T. McCormick, RPhMS, Guillermo Cintron, MD, and Douglas D. Schocken, MD Tampa, Fla
Background Long-acting natriuretic peptide (LANP; proANF 1-30) and vessel dilator (proANF 31-67) enhance sodium and water excretion in healthy human beings. The current investigation was designed to compare the beneficial effects of LANP and vessel dilator in persons with congestive heart failure (CHF). Methods and Results LANP and vessel dilator (100 ng/kg body weight/min, respectively) were given intravenously for 60 minutes to subjects with New York Heart Association class III CHF (n = 17) while their urine volume and sodium and potassium excretion were monitored. Vessel dilator increased urine flow more than 5-fold, which was still increased (P < .001) 3 hours after stopping its infusion. Vessel dilator enhanced sodium excretion 3-fold in subjects with CHF (P < .01), which was still significantly (P < .01) elevated 3 hours after infusion. The effects of LANP were diminished, with urine flow only increasing 2-fold (P < .05). The fractional excretion of sodium increased maximally 6-fold secondary to vessel dilator and 3-fold with LANP. The CHF control patients had no changes in the above parameters. Part of the diminished response to LANP was found to be caused by its rapid decrease in the circulation of individuals with CHF.
Conclusions These results indicate that vessel dilator has significant beneficial diuretic and natriuretic properties, which are not diminished, whereas the effects of LANP are diminished in human beings with CHF compared with healthy individuals. (Am Heart J 1999;138:625-32.)
See related Editorial on page 597. Vessel dilator and long-acting natriuretic peptide (LANP) consisting of amino acids 31-67 and 1-30, respectively, of the 126–amino acid atrial natriuretic factor prohormone (proANF) are hormones primarily synthesized in the heart that enhance sodium and water excretion in both human beings1 and animals.2 When administered to healthy human beings, vessel dilator (ie, proANF 31-67) and LANP (ie, proANF 1-30) enhance urine flow 4- to 12fold while increasing sodium excretion 3- to 8-fold.1 In subjects with congestive heart failure (CHF), the concentrations of vessel dilator and LANP increase in the circulation in direct proportion to the amount of sodium and water retention.3,4 Persons with more severe CHF have significantly (P < .001) higher circulating concentrations From the Departments of Medicine, Physiology, and Biophysics, James A. Haley Veterans Hospital, and the University of South Florida Health Sciences Center. Presented in part at the XIII World Congress of Cardiology, Rio de Janeiro, Brazil, April 26-30, 1998. Supported in part by a Merit Review Grant from the United States Department of Veteran Affairs (D.L.V.) and an American Heart Association grant-in-aid (D.L.V., D.D.S.). Submitted May 7, 1998; accepted July 24, 1998. Reprint requests: David L. Vesely, MD, Atrial Natriuretic Peptides Research Laboratories, J.A. Haley Veterans Hospital-151, 13000 Bruce B. Downs Blvd, Tampa, FL 33612. Copyright © 1999 by Mosby, Inc. 0002-8703/99/$8.00 + 0 4/1/96969
of these peptide hormones than persons with mild CHF.3,4 The increase in vessel dilator and LANP in the circulation is an apparent adaptive response that attempts to overcome the sodium and water retention that characterizes CHF.3-7 We have recently found that vessel dilator has beneficial natriuretic and diuretic effects in persons with CHF.8 The current investigation was designed to (1) determine whether LANP as well as vessel dilator has beneficial diuretic and natriuretic effects in human beings with CHF and (2) compare the beneficial effects of these two peptide hormones in the treatment of persons with CHF.
Methods Patients Seventeen men at the James A. Haley Veterans Hospital with clinically stable CHF (age 33 to 72 years; average 57 ± 6 years) were evaluated in this prospective study. These subjects had heart rates ranging from 68 to 102 beats/min, with respiration rates between 12 and 18 beats/min. These volunteers were divided into 3 similar groups, with age, weight, blood pressure, and heart rate of each shown in Table I. Subjectively, all patients had a history of heart failure, including 1 or more of the following symptoms: dyspnea on mild exertion, paroxysmal nocturnal dyspnea, ankle swelling, or effort-related fatigue. Objectively, chronic left ventricular systolic dysfunction and dilation were documented by cardiac catheterization, echocardiography, and/or radionuclide angiography. The left ventricular ejection fraction of each subject is listed in Table I. Patients
American Heart Journal October 1999
626 Vesely et al
Table I. Mean baseline blood pressure, heart rate, age, weight, sodium, potassium, and left ventricular ejection fraction of subjects with CHF receiving LANP or vessel dilator
CHF controls LANP Vessel dilator
Age (y)
Weight (kg)
BP (mm Hg)
Heart rate (beats/min)
Na+ (mmol/L)
K+ (mmol/L)
LVEF (%)
58 ± 6 54 ± 3 60 ± 6
88 ± 2 91 ± 5 85 ± 5
118/66 ± 6/6 126/75 ± 9/4 119/69 ± 8/6
86 ± 4 75 ± 5 81 ± 8
135 ± 2 136 ± 1.8 133 ± 3
4.41 ± 0.28 4.48 ± 0.20 4.05 ± 0.31
25 ± 3 23 ± 3 19 ± 6
BP, Blood pressure; LVEF, left ventricular ejection fraction. Each value is mean ± standard error of mean of 6 subjects with CHF each in the control and LANP groups and 5 subjects with CHF in the vessel dilator group. There was no significant difference in age, weight, blood pressure, heart rate, sodium, potassium, or medications between the 3 CHF groups when evaluated by 1-way analysis of variance.
Figure 1
Experimental protocol. After 60-minute baseline period, each volunteer received 100 ng/kg body weight/min venous infusion of LANP, vessel dilator, or a 0.9% saline control for 60 minutes followed by a 3-hour postinfusion period, with urine and plasma samples obtained at each of the times on the lower portion of the graph.
with a myocardial infarction within the preceding 6 months were excluded. All persons with kidney failure and/or cirrhosis with ascites were also excluded. The underlying cause of the CHF was ischemic for all the subjects except for control subject 4, in whom the cause was idiopathic. Each subject had class III New York Heart Association CHF. Each subject had CHF for at least 6 months (range 6 months to 3 years). All of the subjects in this study were in normal sinus rhythm with heart rates of ≤102 beats/min (Table I). None of the patients had primary valvular disease. Subjects with a creatinine level >1.5 mg/dL were excluded because vessel dilator and LANP increase in the circulation of human beings with kidney failure.9,10 LANP and vessel dilator increase in the circulation of persons with ascites,ll,12 therefore the subjects with ascites were excluded from the current study. None of the patients’ prescribed medications were taken the day of the study. All over-the-counter medications were stopped at least 24 hours before the study. Specifically, nonsteroidal antiinflammatory agents including aspirin were stopped 24 hours before the study because part of vessel dilator and LANP’S natriuretic effects work by increasing the synthesis of prostaglandin E2, which in turn inhibits Na+-K+-ATPase in the kidney.l3,14 Nonsteroidal agents block this effect in vitro13,14 and in vivo.8,15 Each of the subjects was receiving digoxin, an angiotensinconverting enzyme inhibitor, and a vasodilator, and 80% of each group was receiving a diuretic. Each of the subjects was at dry weight. Informed consent was obtained from each of the volunteers after the nature and possible consequences of the studies were fully explained. This study was approved by
the Institutional Review Board of the University of South Florida Health Sciences Center and the Research Committee of the James A. Haley Veterans Hospital. This study was also approved by the United States Food and Drug Administration (FDA IND No. 32,119). This investigation conforms with the principles outlined in the Declaration of Helsinki for investigations involving human subjects. For this comparative study, some of the subjects with CHF used previously8 in a study of a vessel dilator were used to compare the effects of vessel dilator with those of subjects with CHF given LANP for the first time.
Experimental protocol The experimental protocol is outlined in Figure 1. After written informed consent was obtained, an Insyte-w (Becton Dickinson Infusion Therapy Systems Inc, Sandy, Utah), 20-gauge, 11⁄2inch catheter was placed into one forearm of each subject for infusion and an identical catheter was placed in the opposite forearm of each subject for blood sampling. A 60-minute baseline period preceded any infusion. A total volume of 20 mL of normal saline (0.9% sodium chloride, with or without vessel dilator or LANP) was infused by a constant-rate infusion pump over a 60-minute time period. Blood and urine samples were obtained every 20 minutes during the infusion and at 30-minute time intervals during the 1-hour baseline and 3-hour postinfusion time periods. Urine volume was precisely measured by use of graduated cylinders. One hundred nanograms per kilogram of body weight per minute was chosen for the infusion dosage of LANP and vessel dilator because the rate of release of LANP and vessel dilator from the atrium of the heart with physiologic stimuli are 139 and 138 ng/kg body weight/min, respectively.16 This is also the concentration that has been shown to cause a marked natriuresis and diuresis in healthy human beings.1 Molar equivalents of the 100 ng/kg body weight dose of LANP and vessel dilator are 29 and 28 pmol/kg body weight, respectively. Each of the subjects ingested their usual diet until the evening before the study. All subjects were studied in the morning after an overnight fast, beginning their baseline period at 8 AM. Each volunteer was studied in the seated position. After completion of the 60-minute baseline period, to maintain a similar plasma volume throughout the study, water was given orally in milliliters for each milliliter of urine output at the above time periods. Each volunteer received only 1 vehicle, LANP or vessel dilator infusion.
Purity of LANP and vessel dilator The human forms of LANP and vessel dilator were synthesized by Peninsula Laboratories, Belmont, Calif. Before their use in
American Heart Journal Volume 138, Number 4, Part 1
Vesely et al 627
Figure 2
Vessel dilator (open circles) and LANP (solid circles) increase fractional excretion of sodium (FENa) in persons with CHF. *Time points when filtration fraction of sodium was significantly (P < .05) increased secondary to vessel dilator and LANP infusion at their 100 ng/kg body weight/min concentration for 60 minutes when evaluated by repeated-measures analysis of variance. The increase in FENa secondary to vessel dilator was significantly greater (P < .05) than that secondary to LANP, from 100 to 300 minutes in this investigation when evaluated by Student t test (n = 5 for vessel dilator and n = 6 for LANP group).
these studies, samples of these commercially synthesized peptides were subjected to high-pressure liquid chromatography (HPLC) to determine purity by use of a Novapak C18 (5 µm) cartridge column. The flow rate for the HPLC study was 1 mL/min with 0.1% trifluoroacetate (TFA) solvent in pump A and 60% acetonitrile in 0.1% TFA in pump B, with a gradient of 0% to 60% acetonitrile achieved in 40 minutes. This evaluation verified purity and authenticity when compared with the known HPLC elution profile of these peptides. After determination that the respective peptides were pure, the peptides were dissolved in 0.9% saline solution in the hospital pharmacy, where pyrogen and sterility testing was performed before dispensing the 100 ng/kg body weight concentrations of each peptide into two 10mL syringes. The contents of each 10-mL syringe were infused over 30 minutes. After completing the infusion, each of the syringes and the infusion catheter was examined by the radioimmunoassay described below to determine the amount of residual peptide that may have remained within the syringes or tubing. Approximately 5% of LANP and vessel dilator remained on the walls of the syringes and tubing after completion of the infusion.
Measurement of LANP and vessel dilator Each of the blood samples and flushings of the syringes and tubing with 4 mL of 0.9% sodium chloride was collected into chilled 5-mL ethylenediaminetetraacetic acid (EDTA) tubes to prevent proteolytic breakdown of any peptides that might be present. Each sample was extracted with 100% ethanol (1:2
dilution).1,3 Vessel dilator was measured by a radioimmunoassay devised to amino acids 31-67 of its 126-amino acid prohormone, whereas LANP was measured with a radioimmunoassay devised to amino acids 1-30 of this same prohormone as described in detail previously by our laboratory.l,3,17 The intraassay coefficient(s) of variation for the LANP and vessel dilator radioimmunoassays were 4.8% and 5.3%; their interassay coefficient of variation was 8%. Serial dilution of pooled plasma has revealed excellent parallelism of standards and unknowns in these assays.18
Measurement of sodium, potassium, creatinine, and osmolality Sodium and potassium concentrations in the study were measured by flame photometry (Instrumentation Laboratory 943, Lexington, Mass). Osmolality was measured by a microosmeter (Microosmett 5004, Precision Systems, Inc, Sudbury, Mass). Serum and urine creatinine were measured with the use of a colorimetric diagnostic kit (Sigma Chemical Co, St Louis, Mo) monitored at 500 nm. Creatinine clearance was calculated by multiplying the urine creatinine times the urine flow rate and dividing by the plasma creatinine. The creatinine clearance in this model system is a reflection of the glomerular filtration rate.
Statistical analysis The data obtained in this investigation are illustrated as mean ± standard error. Differences in measurements between sub-
American Heart Journal October 1999
628 Vesely et al
Table II. Vessel dilator enhances urine volume and urine flow rate in patients with CHF Infusion time (min)
CHF control patients Mean ± SE V LANP Mean ± SE V Vessel dilator Mean ± SE V
0
30
60
80
100
120
112 ± 43 —
75 ± 34 2.3 ± 0.24
62 ± 20 2.0 ± 0.33
38 ± 10 1.91 ± 0.27
33 ± 7 1.65 ± 0.31
35 ± 6 1.75 ± 0.42
97 ± 13 —
68 ± 18 2.27 ± 0.60
43 ± 10 1.43 ± 0.33
36 ± 10 1.80 ± 0.50
25 ± 13 1.25 ± 0.65
55 ± 13 2.75 ± 0.65
107 ± 37 —
60 ± 12 2.0 ± 0.40
51 ± 9 1.70 ± 0.30
83 ± 13* 4.15 ± 0.65
75 ± 8* 3.75 ± 0.40
179 ± 56* 8.95 ± 2.8
Values for urine volume are in milliliters; values for urine flow rate (V) are in milliliters of urine per minute. Zero-time urine reflects amount in the bladder after overnight fasting. This value varied markedly from individual to individual, in part because some individuals voided at home before arriving for the study. The 1-hour baseline was thus used to ensure that each subject was at their baseline. The 60-minute time period is thus the control time period for comparison of any increase in urine volume. Enhancement of urine volume by vessel dilator was significant (*) at P < .01; enhancement of urine flow rate at each time point rate was significant at P < .05; LANP enhanced urine flow rate only between 120 to 210 minutes at P < .05 when evaluated by 1-way analysis of variance (n = 6 for LANP and control groups, n = 5 for vessel dilator group).
Table III. Decrease in urine osmolality secondary to vessel dilator in human beings with CHF Infusion time (min) 0 CHF control patients Vessel dilator LANP
30
60
308 ± 6 300 ± 8 550 ± 93
305 ± 7 298 ± 9 560 ± 96
80 300 ± 9 158 ± 11* 498 ± 78
100 303 ± 7 152 ± 14* 474 ± 95
120 305 ± 10 127 ± 17* 555 ± 101
Values are in milliosmoles per liter. Urine osmolality decreased significantly (*) (P < .01) during and for 3 hours after infusion of 100 ng/kg body weight/min concentration of vessel dilator for 60 minutes, whereas LANP did not significantly affect urine osmolality when evaluated by repeated-measures analysis of variance. Values are mean ± standard error of 6 subjects with CHF in each group.
Table IV. Vessel dilator enhances sodium excretion in patients with congestive heart failure Infusion time (min) 0 CHF control patients LANP Vessel dilator
30 193 ± 77 104 ± 46 132 ± 20
60
80
204 ± 102 87 ± 31 109 ± 11
143 ± 52 68 ± 18 229 ± 33*
100 95 ± 25 39 ± 15 175 ± 28*
120 95 ± 25 84 ± 22 286 ± 74*
Values are microequivalents of Na+ excreted per minute. There is no “zero” time value because this time period was after an overnight fast; some subjects had to void at home before arriving for the study, making the “zero” different for each individual. True baseline for comparison of sodium excretion is the 60-minute time period (ie, immediately before respective infusions). CHF control subjects received 20 mL of 0.9% saline (vehicle) infused during a 60-minute time period. Enhancement of sodium by vessel dilator from 60 to 300 minutes was significant (*) at P < .01; for LANP as group, sodium excretion was not significantly enhanced when evaluated by 1-way analysis of variance (n = 6 for LANP and control subjects; n = 5 for vessel dilator group).
jects or groups of subjects were evaluated by 1-way analysis of variance. Measurements obtained in the same subject over time were evaluated by repeated-measures analysis of variance. Maximum changes in urine volume and sodium excretion within groups were determined by a paired Student’s t test. To be considered statistically significant, we required a probability value to be < .05 (95% confidence limits).
Results Basal urine volume of the subjects with CHF before infusion of vehicle, LANP, or vessel dilator was 2-fold
higher (Table II) than the basal urine volume (25 ± 5 mL per 30 min) of healthy individuals examined previously under an identical protocol. Likewise, the basal urine flow rate of the subjects with CHF immediately before infusion (Table II) was also 2-fold higher than that of healthy individuals (0.83 ± 0.17 mL/min).1 LANP did not enhance mean urine flow rate more than 2-fold in persons with CHF either during its infusion or after stopping its infusion (Table II). Vessel dilator, on the other hand, markedly increased urine volume and the urinary flow rate
American Heart Journal Volume 138, Number 4, Part 1
Vesely et al 629
150
180
270
300
35 ± 7 1.75 ± 0.36
42 ± 10 1.46 ± 0.29
40 ± 10 1.1 ± 0.39
40 ± 10 1.1 ± 0.26
37 ± 14 1.2 ± 0.59
47 ± 20 1.5 ± 0.64
57 ± 9 1.90 ± 0.30
55 ± 7.0 1.83 ± 0.23
68 ± 23 2.27 ± 0.77
33 ± 6 1.10 ± 0.20
47 ± 17 1.57 ± 0.57
44 ± 23 1.47 ± 0.77
209 ± 42* 6.90 ± 1.43
163 ± 36* 5.43 ± 1.4
201 ± 54* 6.70 ± 1.8
217 ± 36* 7.23 ± 1.2
178 ± 19* 5.93 ± 0.63
237 ± 23* 7.90 ± 0.77
210
240
270
310 ± 6 117 ± 15* 548 ± 119
315 ± 11 107 ± 12* 454 ± 91
325 ± 13 106 ± 7* 565 ± 102
338 ± 14 111 ± 8* 540 ± 77
349 ± 12 108 ± 13* 519 ± 98
150
180
210
240
270
300
96 ± 31 115 ± 35 248 ± 38*
66 ± 23 109 ± 19 227 ± 40*
107 ± 49 84 ± 7 241 ± 21*
69 ± 29 63 ± 13 224 ± 42*
154 ± 106 68 ± 26 189 ± 35*
139 ± 90 38 ± 12 246 ± 51*
150 310 ± 8 119 ± 11* 555 ± 118
180
210
(Table II) (P < .001) of persons with CHF. At the end of the 60-minute infusion of vessel dilator, mean urinary flow had increased to 4.8-fold and was 4.3-fold higher than the baseline 3 hours after stopping its infusion (Table II). Urinary flow did not increase significantly in the CHF control subjects during the combined 5-hour baseline and experimental periods (Table II). The serum osmolality increased slightly but not significantly secondary to both LANP and vessel dilator, whereas urine osmolality decreased significantly (P <.01)
240
300
secondary to the infusion of vessel dilator but did not decrease significantly in the CHF group receiving LANP or vehicle only (Table III). In this and the results that follow, each individual also serves as his or her own control. The 60-minute time period referred to in tables and figures (which is the time period immediately before beginning one of the respective infusions) serves as the control (baseline) value in the individual subjects with which one can compare any effects observed at later time points in this investigation. The diuresis and the results of enhancing sodium excretion are the amount of increase in
American Heart Journal October 1999
630 Vesely et al
Figure 3
Increase in LANP (solid circles) and vessel dilator (open circles) in circulation with infusion of 100 ng/kg body weight/min for 60 minutes of vessel dilator or LANP in persons with CHF. Vessel dilator increased 5.7-fold (P < .01) during infusion and for 30 minutes after cessation of infusion and then began to decrease, becoming not significantly different from baseline 21⁄2 hours after cessation of infusion when evaluated by analysis of variance. LANP increased 3-fold (P < .01) during infusion but within 60 minutes of cessation of infusion, its plasma concentration was not significantly different from its preinfusion values when evaluated by analysis of variance. Infusion of vehicle only (ie, 20 mL normal saline) did not cause circulating concentration of vessel dilator or LANP to increase (n = 6 for LANP and n = 5 for vessel dilator group).
sodium and water excretion compared with the respective preinfusion measurement (that is, 60-minute time period) in each of the subjects. The control group consisting of individuals with CHF who received 20 mL of 0.9% saline (vehicle) has been added to demonstrate that sodium and water excretion do not change by chance alone during the time period used in this investigation. LANP did not significantly enhance sodium excretion in the subjects with CHF when considered as a group (Table IV). LANP did not enhance sodium excretion at all in 2 of the subjects with CHF (ie, subjects 2 and 4). In 2 of the other subjects, that is, subjects 1 and 3, however, LANP enhanced (ie, 2-fold, P < .05) sodium excretion at the end of its infusion (subject 1) and 30 minutes after completion of the infusion (subject 3). CHF subject 5 had 2-fold enhanced sodium excretion for 2 hours after cessation of the LANP infusion; CHF subject 6 had 2-fold enhanced sodium excretion for 3 hours after the LANP infusion was stopped. Vessel dilator significantly (P < .01) increased sodium excretion, with a doubling in sodium excretion occurring within 20 minutes (Table IV). Three hours after the vessel dilator infusion was stopped, sodium excretion was 3-fold greater than baseline sodium
excretion (Table IV). The control subjects who received 0.9% saline did not have a significant increase in sodium excretion (Table IV). Fractional excretion of sodium (FENa) increased a maximum of 6-fold (P < .001) secondary to vessel dilator in the subjects with CHF (Figure 2). LANP increased the fractional excretion of sodium 2-fold during its infusion and was 3-fold increased at 60 minutes after infusion, returning to preinfusion values 2 1⁄2 hours after its infusion (Figure 2). Neither LANP nor vessel dilator significantly increased creatinine clearance or glomerular filtration rate while increasing the filtration fraction of sodium. Potassium excretion and the fractional excretion of potassium (FEK+) did not significantly increase in the subjects with CHF secondary to LANP or vessel dilator. Serum sodium and potassium did not significantly change during the 5 hours of this investigation. Serum sodium and potassium never varied by >3 mEq/L or 0.4 mEq/L, respectively, from their baseline values (Table I) in the subjects with CHF who received LANP, vessel dilator, or vehicle. Hematocrit increased 3% ± 1% with vessel dilator infusion compared with no increase in the subjects with CHF who received LANP or vehicle only. The measured basal circulating concentration of LANP in persons with CHF was increased >2-fold (P < .05)
American Heart Journal Volume 138, Number 4, Part 1
compared with that of 54 healthy individuals3 (Figure 3). Infusion of LANP augmented the circulating concentration of LANP 4-fold (P < .01), with its concentration becoming maximal at 40 and 60 minutes of infusion (Figure 3). With cessation of the infusion, LANP rapidly decreased within the circulation, approaching its preinfusion values within 30 minutes of stopping infusion (Figure 3). LANP concentration did not return to preinfusion values for 2 hours after cessation of a similar infusion in healthy individuals.19 We therefore next examined whether LANP was being rapidly metabolized by the kidney, which would be reflected by an increased amount of LANP and its breakdown products in the urine. LANP excretion rate into the urine of subjects with CHF during and after its infusion was markedly decreased compared with healthy individuals rather than increased, with only one fifth of LANP being excreted in individuals with CHF compared with healthy individuals at each time point. The measured basal circulating concentration of vessel dilator was increased 3-fold (P < .01) in the subjects with CHF compared with 54 healthy adults3 (Figure 3). Infusion of vessel dilator increased the circulating concentration of vessel dilator another 3-fold (P < .01) during its infusion (Figure 3). The postinfusion decrease in vessel dilator in the circulation of the subjects with CHF (Figure 3) mirrored that of healthy individuals.19 There were no side effects with the use of LANP or vessel dilator in subjects with CHF.
Discussion The effects of LANP were markedly diminished in persons with CHF compared with that in healthy individuals.1 In healthy subjects, with the use of an identical protocol, LANP enhances urine flow 4-to 5-fold,l whereas in the current investigation LANP did not enhance mean urine flow more than 2-fold either during or after stopping its infusion. In healthy subjects, LANP enhances sodium excretion 3- to 8-fold,1 but LANP did not significantly enhance total sodium excretion in the subjects with CHF when considered as a group, although it did increase the fractional excretion of sodium 2- to 3-fold. In addition to the attenuation of natriuretic and diuretic effects of LANP, these effects also did not generally last as long in the subjects with CHF as they do in healthy subjects.1 Part of the reason for their shortened period of activity appears to be that in the subjects with CHF, LANP returned to preinfusion concentrations in the circulation within 30 minutes, unlike healthy individuals in whom LANP concentration does not return to baseline for 2 hours after cessation of infusion.19 The current investigation revealed that this shortened halflife was not a result of the kidney causing an increased metabolic breakdown of LANP because there was no increased excretion of LANP and its breakdown products into the urine. There was rather a decreased excretion rate of LANP by the kidneys of individuals with CHF
Vesely et al 631
that may contribute to the increased circulating concentration of LANP found in the basal state of CHF. When LANP is incubated in vitro with blood of healthy individuals, 25% of it is metabolically degraded within 2 hours, with no further degradation at 8 and 24 hours.20 The data of the current investigation would suggest that the metabolic processing of LANP within the circulation or tissues other than the kidney of individuals with CHF is progressing at a much more rapid rate because its concentration had returned to preinfusion values within 30 minutes of stopping its infusion. Because vessel dilator is not metabolically degraded in human plasma,20 this may be the reason that the effects of vessel dilator are not blunted whereas those of LANP are; that is, LANP is being degraded at a rapid rate in CHF plasma with loss of biological effects, whereas vessel dilator, which is not degraded by protease(s) in plasma, is therefore present to produce its biological effects (ie, diuresis and natriuresis). Vessel dilator caused a significant diuresis in persons with CHF that was not diminished compared with healthy individuals.1 The 2- to 13-fold increase in urine volume in the subjects with CHF is nearly identical to the amount of diuresis (4- to 12-fold) found in healthy human beings given vessel dilator.1 Vessel dilator also stimulated a natriuresis (3- to 4-fold) in the human subjects with CHF that was similar to the amount of natriuresis previously observed in healthy human subjects secondary to vessel dilator.1 The ability of long-acting natriuretic peptide and vessel dilator to increase the excretion of sodium and the filtration of sodium without enhancing creatinine clearance or glomerular filtration rate suggests that they are inhibiting the reabsorption of sodium in the renal tubules of persons with congestive heart failure. Vessel dilator is known to inhibit sodium reabsorption in the inner medullary collecting duct and renal tubules by inhibiting their Na+-K+-ATPases secondary to its ability to enhance the synthesis of prostaglandin E2, which appears to be the final mediator of the inhibition of renal Na+-K+-ATPase.13,14 Long-acting natriuretic peptide, likewise, inhibits renal Na+-K+-ATPase through its ability to enhance the synthesis of prostaglandin E2.14 Finally, it should be noted that the basal urine flow in the patients with CHF was 1.5 to 2 times higher than that of healthy individuals studied under identical conditions.1 The water retention of CHF is therefore not caused by a decrease in the excretion of water because these patients actually excrete more water than healthy individuals.1 This enhanced excretion of water in individuals with CHF is most likely caused, at least in part, by the endogenous increase of long-acting natriuretic peptide and vessel dilator.3,4 The finding that water excretion is increased in patients with CHF suggests that the endogenous increased synthesis21-23 and release3-4 of vessel dilator and long-acting natriuretic
632 Vesely et al
peptide (in some patients) increased water and sodium excretion of patients with CHF over and above that produced by endogenous atrial peptides suggests that part of the problem in CHF is that the heart does not produce enough of vessel dilator and long-acting natriuretic peptide for the amount of sodium and water retention that is present. This phenomenon may play a role in the development and/or maintenance of CHF. Further, the dramatic increase of water and sodium excretion when exogenous vessel dilator is administered likewise suggests that the kidney is able to respond appropriately if enough vessel dilator reaches it, suggesting that the sodium and water retention of CHF may be ameliorated if one can either (l) enhance atrial natriuretic peptide gene expression to increase synthesis of the atrial peptides including vessel dilator to a sufficient extent or (2) add vessel dilator on a chronic (ie, daily) basis to eliminate the sodium and water retention. We thank Charlene Pennington for excellent secretarial assistance.
References 1. Vesely DL, Douglass MA, Dietz JR, et al. Three peptides from the atrial natriuretic factor prohormone amino terminus lower blood pressure and produce a diuresis, natriuresis and/or kaliuresis in humans. Circulation 1994;90:1129-40. 2. Martin DR, Pevahouse JB, Trigg DJ, et al. Three peptides from the ANF prohormone NH2-terminus are natriuretic and/or kaliuretic. Am J Physiol 1990;258:F1401-8. 3. Winters CJ, Sallman AL, Baker BJ, et al. The N-terminus and a 4000 molecular weight 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. Daggubati S, Parks JR, Overton RM, et al. Adrenomedullin, endothelin, neuropeptide Y, atrial, brain, and C-natriuretic prohormone peptides compared as early heart failure indicators. Cardiovasc Res 1997;36:246-55. 5. Vesely DL. Atrial natriuretic hormones. Englewood Cliffs, NJ: Prentice-Hall; 1992. p. 256. 6. Vesely DL. Atrial natriuretic hormones originating from the N-terminus of the atrial natriuretic factor prohormone. Clin Exp Pharmacol Physiol 1995;22:108-14. 7. Vesely DL. Vessel dilator, long acting natriuretic peptide, and kaliuretic peptide: new peptide hormones originating from the atrial natriuretic factor prohormone. In: Vesely DL, editor. Atrial natriuretic peptides. Trivandrum, India: Research Signpost; 1997. p. 87-110.
American Heart Journal October 1999
8. Vesely DL, Dietz JR, Parks JR, et al. Vessel dilator enhances sodium and water excretion and has beneficial hemodynamic effects in persons with congestive heart failure. Circulation 1998;98:323-9. 9. Winters CJ, Sallman AL, Meadows J, et al. Two new hormones: Prohormone atrial natriuretic peptides 1-30 and 31-67 circulate in man. Biochem Biophys Res Commun 1988;150:231-6. 10. Winters CJ, Vesely DL. Change in plasma immunoreactive N-terminus, C-terminus, and 4000 Dalton mid portion of atrial natriuretic factor prohormone with hemodialysis. Nephron 1991;58:17-22. 11. Vesely DL, Winters CJ. Presence of the N-terminal and C-terminal portions of the prohormone of atrial natriuretic factor in the ascitic fluid of cirrhotic patients. J Med 1990;21:265-75. 12. Vesely DL, Preston R, Winters CJ, et al. Increased release of the N-terminal and C-terminal portions of the prohormone of atrial natriuretic factor during immersion-induced central hypervolemia in cirrhotic humans. Am J Nephrol 1991;11:207-16. 13. Gunning ME, Brady HR, Otuechere G, et al. Atrial natriuretic peptide (31-67) inhibits Na+ transport in rabbit inner medullary collecting duct cells: role of prostaglandin E-2. J Clin Invest 1992;89:1411-7. 14. Chiou S, Vesely DL. Kaliuretic peptide: the most potent inhibitor of Na+-K+-ATPase of the atrial natriuretic peptides. Endocrinology 1995;136:2033-9. 15. Habibullah AA, Villarreal D, Freeman RH, et al. Effects of cyclooxygenase inhibition on the renal actions of atrial natriuretic prohormone fragment 31-67 in normal dogs [abstract]. FASEB J 1995; 9:A296. 16. Ackerman BH, Wyeth RP, Vesely DL, et al. Pharmacokinetic characterization of the postdistribution phase of prohormone atrial natriuretic peptides amino acids 1-98, 31-67 and atrial natriuretic factor during and after rapid ventricular pacing in dogs. J Clin Pharmacol 1992;32:415-21. 17. Vesely DL, Douglass MA, Dietz JR, et al. Negative feedback of atrial natriuretic peptides. J Clin Endocrinol Metab 1994;78:1128-34. 18. Vesely DL, Norsk P, Winters CJ, et al. Increased release of the N-terminal and C-terminal portions of the prohormone of atrial natriuretic factor during immersion-induced central hypervolemia in normal humans. Proc Soc Exp Biol Med 1989;192:230-5. 19. Ackerman BH, Overton RM, McCormick MT, et al. Disposition of vessel dilator and long-acting natriuretic peptide in healthy humans after a 1-hour infusion. J Pharmacol Exp Ther 1997;282:603-8. 20. Overton RM, Vesely DL. Processing of long-acting natriuretic peptide and vessel dilator in human plasma and serum. Peptides 1996; 17:1155-62. 21. Tsuchimochi H, Yazaki Y, Ohno H, et al. Ventricular expression of atrial natriuretic peptide. Lancet 1987;2:336-7. 22. Edwards BS, Ackermann DM, Lee ME, et al. Identification of atrial natriuretic factor within ventricular tissue in hamsters and humans with congestive heart failure. J Clin Invest 1988;81:82-6. 23. Poulos JE, Gower WR Jr, Sullebarger JT, et al. Congestive heart failure: increased cardiac and extracardiac atrial natriuretic peptide gene expression. Cardiovasc Res 1996;32:909-19.
Background Long-acting natriuretic peptide (LANP; proANF 1-30) and vessel dilator (proANF 31-67) enhance sodium and water excretion in healthy human beings. The current investigation was designed to compare the beneficial effects of LANP and vessel dilator in persons with congestive heart failure (CHF). Methods and Results LANP and vessel dilator (100 ng/kg body weight/min, respectively) were given intravenously for 60 minutes to subjects with New York Heart Association class III CHF (n = 17) while their urine volume and sodium and potassium excretion were monitored. Vessel dilator increased urine flow more than 5-fold, which was still increased (P < .001) 3 hours after stopping its infusion. Vessel dilator enhanced sodium excretion 3-fold in subjects with CHF (P < .01), which was still significantly (P < .01) elevated 3 hours after infusion. The effects of LANP were diminished, with urine flow only increasing 2-fold (P < .05). The fractional excretion of sodium increased maximally 6-fold secondary to vessel dilator and 3-fold with LANP. The CHF control patients had no changes in the above parameters. Part of the diminished response to LANP was found to be caused by its rapid decrease in the circulation of individuals with CHF.
Conclusions These results indicate that vessel dilator has significant beneficial diuretic and natriuretic properties, which are not diminished, whereas the effects of LANP are diminished in human beings with CHF compared with healthy individuals. (Am Heart J 1999;138:625-32.)
See related Editorial on page 597. Vessel dilator and long-acting natriuretic peptide (LANP) consisting of amino acids 31-67 and 1-30, respectively, of the 126–amino acid atrial natriuretic factor prohormone (proANF) are hormones primarily synthesized in the heart that enhance sodium and water excretion in both human beings1 and animals.2 When administered to healthy human beings, vessel dilator (ie, proANF 31-67) and LANP (ie, proANF 1-30) enhance urine flow 4- to 12fold while increasing sodium excretion 3- to 8-fold.1 In subjects with congestive heart failure (CHF), the concentrations of vessel dilator and LANP increase in the circulation in direct proportion to the amount of sodium and water retention.3,4 Persons with more severe CHF have significantly (P < .001) higher circulating concentrations From the Departments of Medicine, Physiology, and Biophysics, James A. Haley Veterans Hospital, and the University of South Florida Health Sciences Center. Presented in part at the XIII World Congress of Cardiology, Rio de Janeiro, Brazil, April 26-30, 1998. Supported in part by a Merit Review Grant from the United States Department of Veteran Affairs (D.L.V.) and an American Heart Association grant-in-aid (D.L.V., D.D.S.). Submitted May 7, 1998; accepted July 24, 1998. Reprint requests: David L. Vesely, MD, Atrial Natriuretic Peptides Research Laboratories, J.A. Haley Veterans Hospital-151, 13000 Bruce B. Downs Blvd, Tampa, FL 33612. Copyright © 1999 by Mosby, Inc. 0002-8703/99/$8.00 + 0 4/1/96969
of these peptide hormones than persons with mild CHF.3,4 The increase in vessel dilator and LANP in the circulation is an apparent adaptive response that attempts to overcome the sodium and water retention that characterizes CHF.3-7 We have recently found that vessel dilator has beneficial natriuretic and diuretic effects in persons with CHF.8 The current investigation was designed to (1) determine whether LANP as well as vessel dilator has beneficial diuretic and natriuretic effects in human beings with CHF and (2) compare the beneficial effects of these two peptide hormones in the treatment of persons with CHF.
Methods Patients Seventeen men at the James A. Haley Veterans Hospital with clinically stable CHF (age 33 to 72 years; average 57 ± 6 years) were evaluated in this prospective study. These subjects had heart rates ranging from 68 to 102 beats/min, with respiration rates between 12 and 18 beats/min. These volunteers were divided into 3 similar groups, with age, weight, blood pressure, and heart rate of each shown in Table I. Subjectively, all patients had a history of heart failure, including 1 or more of the following symptoms: dyspnea on mild exertion, paroxysmal nocturnal dyspnea, ankle swelling, or effort-related fatigue. Objectively, chronic left ventricular systolic dysfunction and dilation were documented by cardiac catheterization, echocardiography, and/or radionuclide angiography. The left ventricular ejection fraction of each subject is listed in Table I. Patients
American Heart Journal October 1999
626 Vesely et al
Table I. Mean baseline blood pressure, heart rate, age, weight, sodium, potassium, and left ventricular ejection fraction of subjects with CHF receiving LANP or vessel dilator
CHF controls LANP Vessel dilator
Age (y)
Weight (kg)
BP (mm Hg)
Heart rate (beats/min)
Na+ (mmol/L)
K+ (mmol/L)
LVEF (%)
58 ± 6 54 ± 3 60 ± 6
88 ± 2 91 ± 5 85 ± 5
118/66 ± 6/6 126/75 ± 9/4 119/69 ± 8/6
86 ± 4 75 ± 5 81 ± 8
135 ± 2 136 ± 1.8 133 ± 3
4.41 ± 0.28 4.48 ± 0.20 4.05 ± 0.31
25 ± 3 23 ± 3 19 ± 6
BP, Blood pressure; LVEF, left ventricular ejection fraction. Each value is mean ± standard error of mean of 6 subjects with CHF each in the control and LANP groups and 5 subjects with CHF in the vessel dilator group. There was no significant difference in age, weight, blood pressure, heart rate, sodium, potassium, or medications between the 3 CHF groups when evaluated by 1-way analysis of variance.
Figure 1
Experimental protocol. After 60-minute baseline period, each volunteer received 100 ng/kg body weight/min venous infusion of LANP, vessel dilator, or a 0.9% saline control for 60 minutes followed by a 3-hour postinfusion period, with urine and plasma samples obtained at each of the times on the lower portion of the graph.
with a myocardial infarction within the preceding 6 months were excluded. All persons with kidney failure and/or cirrhosis with ascites were also excluded. The underlying cause of the CHF was ischemic for all the subjects except for control subject 4, in whom the cause was idiopathic. Each subject had class III New York Heart Association CHF. Each subject had CHF for at least 6 months (range 6 months to 3 years). All of the subjects in this study were in normal sinus rhythm with heart rates of ≤102 beats/min (Table I). None of the patients had primary valvular disease. Subjects with a creatinine level >1.5 mg/dL were excluded because vessel dilator and LANP increase in the circulation of human beings with kidney failure.9,10 LANP and vessel dilator increase in the circulation of persons with ascites,ll,12 therefore the subjects with ascites were excluded from the current study. None of the patients’ prescribed medications were taken the day of the study. All over-the-counter medications were stopped at least 24 hours before the study. Specifically, nonsteroidal antiinflammatory agents including aspirin were stopped 24 hours before the study because part of vessel dilator and LANP’S natriuretic effects work by increasing the synthesis of prostaglandin E2, which in turn inhibits Na+-K+-ATPase in the kidney.l3,14 Nonsteroidal agents block this effect in vitro13,14 and in vivo.8,15 Each of the subjects was receiving digoxin, an angiotensinconverting enzyme inhibitor, and a vasodilator, and 80% of each group was receiving a diuretic. Each of the subjects was at dry weight. Informed consent was obtained from each of the volunteers after the nature and possible consequences of the studies were fully explained. This study was approved by
the Institutional Review Board of the University of South Florida Health Sciences Center and the Research Committee of the James A. Haley Veterans Hospital. This study was also approved by the United States Food and Drug Administration (FDA IND No. 32,119). This investigation conforms with the principles outlined in the Declaration of Helsinki for investigations involving human subjects. For this comparative study, some of the subjects with CHF used previously8 in a study of a vessel dilator were used to compare the effects of vessel dilator with those of subjects with CHF given LANP for the first time.
Experimental protocol The experimental protocol is outlined in Figure 1. After written informed consent was obtained, an Insyte-w (Becton Dickinson Infusion Therapy Systems Inc, Sandy, Utah), 20-gauge, 11⁄2inch catheter was placed into one forearm of each subject for infusion and an identical catheter was placed in the opposite forearm of each subject for blood sampling. A 60-minute baseline period preceded any infusion. A total volume of 20 mL of normal saline (0.9% sodium chloride, with or without vessel dilator or LANP) was infused by a constant-rate infusion pump over a 60-minute time period. Blood and urine samples were obtained every 20 minutes during the infusion and at 30-minute time intervals during the 1-hour baseline and 3-hour postinfusion time periods. Urine volume was precisely measured by use of graduated cylinders. One hundred nanograms per kilogram of body weight per minute was chosen for the infusion dosage of LANP and vessel dilator because the rate of release of LANP and vessel dilator from the atrium of the heart with physiologic stimuli are 139 and 138 ng/kg body weight/min, respectively.16 This is also the concentration that has been shown to cause a marked natriuresis and diuresis in healthy human beings.1 Molar equivalents of the 100 ng/kg body weight dose of LANP and vessel dilator are 29 and 28 pmol/kg body weight, respectively. Each of the subjects ingested their usual diet until the evening before the study. All subjects were studied in the morning after an overnight fast, beginning their baseline period at 8 AM. Each volunteer was studied in the seated position. After completion of the 60-minute baseline period, to maintain a similar plasma volume throughout the study, water was given orally in milliliters for each milliliter of urine output at the above time periods. Each volunteer received only 1 vehicle, LANP or vessel dilator infusion.
Purity of LANP and vessel dilator The human forms of LANP and vessel dilator were synthesized by Peninsula Laboratories, Belmont, Calif. Before their use in
American Heart Journal Volume 138, Number 4, Part 1
Vesely et al 627
Figure 2
Vessel dilator (open circles) and LANP (solid circles) increase fractional excretion of sodium (FENa) in persons with CHF. *Time points when filtration fraction of sodium was significantly (P < .05) increased secondary to vessel dilator and LANP infusion at their 100 ng/kg body weight/min concentration for 60 minutes when evaluated by repeated-measures analysis of variance. The increase in FENa secondary to vessel dilator was significantly greater (P < .05) than that secondary to LANP, from 100 to 300 minutes in this investigation when evaluated by Student t test (n = 5 for vessel dilator and n = 6 for LANP group).
these studies, samples of these commercially synthesized peptides were subjected to high-pressure liquid chromatography (HPLC) to determine purity by use of a Novapak C18 (5 µm) cartridge column. The flow rate for the HPLC study was 1 mL/min with 0.1% trifluoroacetate (TFA) solvent in pump A and 60% acetonitrile in 0.1% TFA in pump B, with a gradient of 0% to 60% acetonitrile achieved in 40 minutes. This evaluation verified purity and authenticity when compared with the known HPLC elution profile of these peptides. After determination that the respective peptides were pure, the peptides were dissolved in 0.9% saline solution in the hospital pharmacy, where pyrogen and sterility testing was performed before dispensing the 100 ng/kg body weight concentrations of each peptide into two 10mL syringes. The contents of each 10-mL syringe were infused over 30 minutes. After completing the infusion, each of the syringes and the infusion catheter was examined by the radioimmunoassay described below to determine the amount of residual peptide that may have remained within the syringes or tubing. Approximately 5% of LANP and vessel dilator remained on the walls of the syringes and tubing after completion of the infusion.
Measurement of LANP and vessel dilator Each of the blood samples and flushings of the syringes and tubing with 4 mL of 0.9% sodium chloride was collected into chilled 5-mL ethylenediaminetetraacetic acid (EDTA) tubes to prevent proteolytic breakdown of any peptides that might be present. Each sample was extracted with 100% ethanol (1:2
dilution).1,3 Vessel dilator was measured by a radioimmunoassay devised to amino acids 31-67 of its 126-amino acid prohormone, whereas LANP was measured with a radioimmunoassay devised to amino acids 1-30 of this same prohormone as described in detail previously by our laboratory.l,3,17 The intraassay coefficient(s) of variation for the LANP and vessel dilator radioimmunoassays were 4.8% and 5.3%; their interassay coefficient of variation was 8%. Serial dilution of pooled plasma has revealed excellent parallelism of standards and unknowns in these assays.18
Measurement of sodium, potassium, creatinine, and osmolality Sodium and potassium concentrations in the study were measured by flame photometry (Instrumentation Laboratory 943, Lexington, Mass). Osmolality was measured by a microosmeter (Microosmett 5004, Precision Systems, Inc, Sudbury, Mass). Serum and urine creatinine were measured with the use of a colorimetric diagnostic kit (Sigma Chemical Co, St Louis, Mo) monitored at 500 nm. Creatinine clearance was calculated by multiplying the urine creatinine times the urine flow rate and dividing by the plasma creatinine. The creatinine clearance in this model system is a reflection of the glomerular filtration rate.
Statistical analysis The data obtained in this investigation are illustrated as mean ± standard error. Differences in measurements between sub-
American Heart Journal October 1999
628 Vesely et al
Table II. Vessel dilator enhances urine volume and urine flow rate in patients with CHF Infusion time (min)
CHF control patients Mean ± SE V LANP Mean ± SE V Vessel dilator Mean ± SE V
0
30
60
80
100
120
112 ± 43 —
75 ± 34 2.3 ± 0.24
62 ± 20 2.0 ± 0.33
38 ± 10 1.91 ± 0.27
33 ± 7 1.65 ± 0.31
35 ± 6 1.75 ± 0.42
97 ± 13 —
68 ± 18 2.27 ± 0.60
43 ± 10 1.43 ± 0.33
36 ± 10 1.80 ± 0.50
25 ± 13 1.25 ± 0.65
55 ± 13 2.75 ± 0.65
107 ± 37 —
60 ± 12 2.0 ± 0.40
51 ± 9 1.70 ± 0.30
83 ± 13* 4.15 ± 0.65
75 ± 8* 3.75 ± 0.40
179 ± 56* 8.95 ± 2.8
Values for urine volume are in milliliters; values for urine flow rate (V) are in milliliters of urine per minute. Zero-time urine reflects amount in the bladder after overnight fasting. This value varied markedly from individual to individual, in part because some individuals voided at home before arriving for the study. The 1-hour baseline was thus used to ensure that each subject was at their baseline. The 60-minute time period is thus the control time period for comparison of any increase in urine volume. Enhancement of urine volume by vessel dilator was significant (*) at P < .01; enhancement of urine flow rate at each time point rate was significant at P < .05; LANP enhanced urine flow rate only between 120 to 210 minutes at P < .05 when evaluated by 1-way analysis of variance (n = 6 for LANP and control groups, n = 5 for vessel dilator group).
Table III. Decrease in urine osmolality secondary to vessel dilator in human beings with CHF Infusion time (min) 0 CHF control patients Vessel dilator LANP
30
60
308 ± 6 300 ± 8 550 ± 93
305 ± 7 298 ± 9 560 ± 96
80 300 ± 9 158 ± 11* 498 ± 78
100 303 ± 7 152 ± 14* 474 ± 95
120 305 ± 10 127 ± 17* 555 ± 101
Values are in milliosmoles per liter. Urine osmolality decreased significantly (*) (P < .01) during and for 3 hours after infusion of 100 ng/kg body weight/min concentration of vessel dilator for 60 minutes, whereas LANP did not significantly affect urine osmolality when evaluated by repeated-measures analysis of variance. Values are mean ± standard error of 6 subjects with CHF in each group.
Table IV. Vessel dilator enhances sodium excretion in patients with congestive heart failure Infusion time (min) 0 CHF control patients LANP Vessel dilator
30 193 ± 77 104 ± 46 132 ± 20
60
80
204 ± 102 87 ± 31 109 ± 11
143 ± 52 68 ± 18 229 ± 33*
100 95 ± 25 39 ± 15 175 ± 28*
120 95 ± 25 84 ± 22 286 ± 74*
Values are microequivalents of Na+ excreted per minute. There is no “zero” time value because this time period was after an overnight fast; some subjects had to void at home before arriving for the study, making the “zero” different for each individual. True baseline for comparison of sodium excretion is the 60-minute time period (ie, immediately before respective infusions). CHF control subjects received 20 mL of 0.9% saline (vehicle) infused during a 60-minute time period. Enhancement of sodium by vessel dilator from 60 to 300 minutes was significant (*) at P < .01; for LANP as group, sodium excretion was not significantly enhanced when evaluated by 1-way analysis of variance (n = 6 for LANP and control subjects; n = 5 for vessel dilator group).
jects or groups of subjects were evaluated by 1-way analysis of variance. Measurements obtained in the same subject over time were evaluated by repeated-measures analysis of variance. Maximum changes in urine volume and sodium excretion within groups were determined by a paired Student’s t test. To be considered statistically significant, we required a probability value to be < .05 (95% confidence limits).
Results Basal urine volume of the subjects with CHF before infusion of vehicle, LANP, or vessel dilator was 2-fold
higher (Table II) than the basal urine volume (25 ± 5 mL per 30 min) of healthy individuals examined previously under an identical protocol. Likewise, the basal urine flow rate of the subjects with CHF immediately before infusion (Table II) was also 2-fold higher than that of healthy individuals (0.83 ± 0.17 mL/min).1 LANP did not enhance mean urine flow rate more than 2-fold in persons with CHF either during its infusion or after stopping its infusion (Table II). Vessel dilator, on the other hand, markedly increased urine volume and the urinary flow rate
American Heart Journal Volume 138, Number 4, Part 1
Vesely et al 629
150
180
270
300
35 ± 7 1.75 ± 0.36
42 ± 10 1.46 ± 0.29
40 ± 10 1.1 ± 0.39
40 ± 10 1.1 ± 0.26
37 ± 14 1.2 ± 0.59
47 ± 20 1.5 ± 0.64
57 ± 9 1.90 ± 0.30
55 ± 7.0 1.83 ± 0.23
68 ± 23 2.27 ± 0.77
33 ± 6 1.10 ± 0.20
47 ± 17 1.57 ± 0.57
44 ± 23 1.47 ± 0.77
209 ± 42* 6.90 ± 1.43
163 ± 36* 5.43 ± 1.4
201 ± 54* 6.70 ± 1.8
217 ± 36* 7.23 ± 1.2
178 ± 19* 5.93 ± 0.63
237 ± 23* 7.90 ± 0.77
210
240
270
310 ± 6 117 ± 15* 548 ± 119
315 ± 11 107 ± 12* 454 ± 91
325 ± 13 106 ± 7* 565 ± 102
338 ± 14 111 ± 8* 540 ± 77
349 ± 12 108 ± 13* 519 ± 98
150
180
210
240
270
300
96 ± 31 115 ± 35 248 ± 38*
66 ± 23 109 ± 19 227 ± 40*
107 ± 49 84 ± 7 241 ± 21*
69 ± 29 63 ± 13 224 ± 42*
154 ± 106 68 ± 26 189 ± 35*
139 ± 90 38 ± 12 246 ± 51*
150 310 ± 8 119 ± 11* 555 ± 118
180
210
(Table II) (P < .001) of persons with CHF. At the end of the 60-minute infusion of vessel dilator, mean urinary flow had increased to 4.8-fold and was 4.3-fold higher than the baseline 3 hours after stopping its infusion (Table II). Urinary flow did not increase significantly in the CHF control subjects during the combined 5-hour baseline and experimental periods (Table II). The serum osmolality increased slightly but not significantly secondary to both LANP and vessel dilator, whereas urine osmolality decreased significantly (P <.01)
240
300
secondary to the infusion of vessel dilator but did not decrease significantly in the CHF group receiving LANP or vehicle only (Table III). In this and the results that follow, each individual also serves as his or her own control. The 60-minute time period referred to in tables and figures (which is the time period immediately before beginning one of the respective infusions) serves as the control (baseline) value in the individual subjects with which one can compare any effects observed at later time points in this investigation. The diuresis and the results of enhancing sodium excretion are the amount of increase in
American Heart Journal October 1999
630 Vesely et al
Figure 3
Increase in LANP (solid circles) and vessel dilator (open circles) in circulation with infusion of 100 ng/kg body weight/min for 60 minutes of vessel dilator or LANP in persons with CHF. Vessel dilator increased 5.7-fold (P < .01) during infusion and for 30 minutes after cessation of infusion and then began to decrease, becoming not significantly different from baseline 21⁄2 hours after cessation of infusion when evaluated by analysis of variance. LANP increased 3-fold (P < .01) during infusion but within 60 minutes of cessation of infusion, its plasma concentration was not significantly different from its preinfusion values when evaluated by analysis of variance. Infusion of vehicle only (ie, 20 mL normal saline) did not cause circulating concentration of vessel dilator or LANP to increase (n = 6 for LANP and n = 5 for vessel dilator group).
sodium and water excretion compared with the respective preinfusion measurement (that is, 60-minute time period) in each of the subjects. The control group consisting of individuals with CHF who received 20 mL of 0.9% saline (vehicle) has been added to demonstrate that sodium and water excretion do not change by chance alone during the time period used in this investigation. LANP did not significantly enhance sodium excretion in the subjects with CHF when considered as a group (Table IV). LANP did not enhance sodium excretion at all in 2 of the subjects with CHF (ie, subjects 2 and 4). In 2 of the other subjects, that is, subjects 1 and 3, however, LANP enhanced (ie, 2-fold, P < .05) sodium excretion at the end of its infusion (subject 1) and 30 minutes after completion of the infusion (subject 3). CHF subject 5 had 2-fold enhanced sodium excretion for 2 hours after cessation of the LANP infusion; CHF subject 6 had 2-fold enhanced sodium excretion for 3 hours after the LANP infusion was stopped. Vessel dilator significantly (P < .01) increased sodium excretion, with a doubling in sodium excretion occurring within 20 minutes (Table IV). Three hours after the vessel dilator infusion was stopped, sodium excretion was 3-fold greater than baseline sodium
excretion (Table IV). The control subjects who received 0.9% saline did not have a significant increase in sodium excretion (Table IV). Fractional excretion of sodium (FENa) increased a maximum of 6-fold (P < .001) secondary to vessel dilator in the subjects with CHF (Figure 2). LANP increased the fractional excretion of sodium 2-fold during its infusion and was 3-fold increased at 60 minutes after infusion, returning to preinfusion values 2 1⁄2 hours after its infusion (Figure 2). Neither LANP nor vessel dilator significantly increased creatinine clearance or glomerular filtration rate while increasing the filtration fraction of sodium. Potassium excretion and the fractional excretion of potassium (FEK+) did not significantly increase in the subjects with CHF secondary to LANP or vessel dilator. Serum sodium and potassium did not significantly change during the 5 hours of this investigation. Serum sodium and potassium never varied by >3 mEq/L or 0.4 mEq/L, respectively, from their baseline values (Table I) in the subjects with CHF who received LANP, vessel dilator, or vehicle. Hematocrit increased 3% ± 1% with vessel dilator infusion compared with no increase in the subjects with CHF who received LANP or vehicle only. The measured basal circulating concentration of LANP in persons with CHF was increased >2-fold (P < .05)
American Heart Journal Volume 138, Number 4, Part 1
compared with that of 54 healthy individuals3 (Figure 3). Infusion of LANP augmented the circulating concentration of LANP 4-fold (P < .01), with its concentration becoming maximal at 40 and 60 minutes of infusion (Figure 3). With cessation of the infusion, LANP rapidly decreased within the circulation, approaching its preinfusion values within 30 minutes of stopping infusion (Figure 3). LANP concentration did not return to preinfusion values for 2 hours after cessation of a similar infusion in healthy individuals.19 We therefore next examined whether LANP was being rapidly metabolized by the kidney, which would be reflected by an increased amount of LANP and its breakdown products in the urine. LANP excretion rate into the urine of subjects with CHF during and after its infusion was markedly decreased compared with healthy individuals rather than increased, with only one fifth of LANP being excreted in individuals with CHF compared with healthy individuals at each time point. The measured basal circulating concentration of vessel dilator was increased 3-fold (P < .01) in the subjects with CHF compared with 54 healthy adults3 (Figure 3). Infusion of vessel dilator increased the circulating concentration of vessel dilator another 3-fold (P < .01) during its infusion (Figure 3). The postinfusion decrease in vessel dilator in the circulation of the subjects with CHF (Figure 3) mirrored that of healthy individuals.19 There were no side effects with the use of LANP or vessel dilator in subjects with CHF.
Discussion The effects of LANP were markedly diminished in persons with CHF compared with that in healthy individuals.1 In healthy subjects, with the use of an identical protocol, LANP enhances urine flow 4-to 5-fold,l whereas in the current investigation LANP did not enhance mean urine flow more than 2-fold either during or after stopping its infusion. In healthy subjects, LANP enhances sodium excretion 3- to 8-fold,1 but LANP did not significantly enhance total sodium excretion in the subjects with CHF when considered as a group, although it did increase the fractional excretion of sodium 2- to 3-fold. In addition to the attenuation of natriuretic and diuretic effects of LANP, these effects also did not generally last as long in the subjects with CHF as they do in healthy subjects.1 Part of the reason for their shortened period of activity appears to be that in the subjects with CHF, LANP returned to preinfusion concentrations in the circulation within 30 minutes, unlike healthy individuals in whom LANP concentration does not return to baseline for 2 hours after cessation of infusion.19 The current investigation revealed that this shortened halflife was not a result of the kidney causing an increased metabolic breakdown of LANP because there was no increased excretion of LANP and its breakdown products into the urine. There was rather a decreased excretion rate of LANP by the kidneys of individuals with CHF
Vesely et al 631
that may contribute to the increased circulating concentration of LANP found in the basal state of CHF. When LANP is incubated in vitro with blood of healthy individuals, 25% of it is metabolically degraded within 2 hours, with no further degradation at 8 and 24 hours.20 The data of the current investigation would suggest that the metabolic processing of LANP within the circulation or tissues other than the kidney of individuals with CHF is progressing at a much more rapid rate because its concentration had returned to preinfusion values within 30 minutes of stopping its infusion. Because vessel dilator is not metabolically degraded in human plasma,20 this may be the reason that the effects of vessel dilator are not blunted whereas those of LANP are; that is, LANP is being degraded at a rapid rate in CHF plasma with loss of biological effects, whereas vessel dilator, which is not degraded by protease(s) in plasma, is therefore present to produce its biological effects (ie, diuresis and natriuresis). Vessel dilator caused a significant diuresis in persons with CHF that was not diminished compared with healthy individuals.1 The 2- to 13-fold increase in urine volume in the subjects with CHF is nearly identical to the amount of diuresis (4- to 12-fold) found in healthy human beings given vessel dilator.1 Vessel dilator also stimulated a natriuresis (3- to 4-fold) in the human subjects with CHF that was similar to the amount of natriuresis previously observed in healthy human subjects secondary to vessel dilator.1 The ability of long-acting natriuretic peptide and vessel dilator to increase the excretion of sodium and the filtration of sodium without enhancing creatinine clearance or glomerular filtration rate suggests that they are inhibiting the reabsorption of sodium in the renal tubules of persons with congestive heart failure. Vessel dilator is known to inhibit sodium reabsorption in the inner medullary collecting duct and renal tubules by inhibiting their Na+-K+-ATPases secondary to its ability to enhance the synthesis of prostaglandin E2, which appears to be the final mediator of the inhibition of renal Na+-K+-ATPase.13,14 Long-acting natriuretic peptide, likewise, inhibits renal Na+-K+-ATPase through its ability to enhance the synthesis of prostaglandin E2.14 Finally, it should be noted that the basal urine flow in the patients with CHF was 1.5 to 2 times higher than that of healthy individuals studied under identical conditions.1 The water retention of CHF is therefore not caused by a decrease in the excretion of water because these patients actually excrete more water than healthy individuals.1 This enhanced excretion of water in individuals with CHF is most likely caused, at least in part, by the endogenous increase of long-acting natriuretic peptide and vessel dilator.3,4 The finding that water excretion is increased in patients with CHF suggests that the endogenous increased synthesis21-23 and release3-4 of vessel dilator and long-acting natriuretic
632 Vesely et al
peptide (in some patients) increased water and sodium excretion of patients with CHF over and above that produced by endogenous atrial peptides suggests that part of the problem in CHF is that the heart does not produce enough of vessel dilator and long-acting natriuretic peptide for the amount of sodium and water retention that is present. This phenomenon may play a role in the development and/or maintenance of CHF. Further, the dramatic increase of water and sodium excretion when exogenous vessel dilator is administered likewise suggests that the kidney is able to respond appropriately if enough vessel dilator reaches it, suggesting that the sodium and water retention of CHF may be ameliorated if one can either (l) enhance atrial natriuretic peptide gene expression to increase synthesis of the atrial peptides including vessel dilator to a sufficient extent or (2) add vessel dilator on a chronic (ie, daily) basis to eliminate the sodium and water retention. We thank Charlene Pennington for excellent secretarial assistance.
References 1. Vesely DL, Douglass MA, Dietz JR, et al. Three peptides from the atrial natriuretic factor prohormone amino terminus lower blood pressure and produce a diuresis, natriuresis and/or kaliuresis in humans. Circulation 1994;90:1129-40. 2. Martin DR, Pevahouse JB, Trigg DJ, et al. Three peptides from the ANF prohormone NH2-terminus are natriuretic and/or kaliuretic. Am J Physiol 1990;258:F1401-8. 3. Winters CJ, Sallman AL, Baker BJ, et al. The N-terminus and a 4000 molecular weight 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. Daggubati S, Parks JR, Overton RM, et al. Adrenomedullin, endothelin, neuropeptide Y, atrial, brain, and C-natriuretic prohormone peptides compared as early heart failure indicators. Cardiovasc Res 1997;36:246-55. 5. Vesely DL. Atrial natriuretic hormones. Englewood Cliffs, NJ: Prentice-Hall; 1992. p. 256. 6. Vesely DL. Atrial natriuretic hormones originating from the N-terminus of the atrial natriuretic factor prohormone. Clin Exp Pharmacol Physiol 1995;22:108-14. 7. Vesely DL. Vessel dilator, long acting natriuretic peptide, and kaliuretic peptide: new peptide hormones originating from the atrial natriuretic factor prohormone. In: Vesely DL, editor. Atrial natriuretic peptides. Trivandrum, India: Research Signpost; 1997. p. 87-110.
American Heart Journal October 1999
8. Vesely DL, Dietz JR, Parks JR, et al. Vessel dilator enhances sodium and water excretion and has beneficial hemodynamic effects in persons with congestive heart failure. Circulation 1998;98:323-9. 9. Winters CJ, Sallman AL, Meadows J, et al. Two new hormones: Prohormone atrial natriuretic peptides 1-30 and 31-67 circulate in man. Biochem Biophys Res Commun 1988;150:231-6. 10. Winters CJ, Vesely DL. Change in plasma immunoreactive N-terminus, C-terminus, and 4000 Dalton mid portion of atrial natriuretic factor prohormone with hemodialysis. Nephron 1991;58:17-22. 11. Vesely DL, Winters CJ. Presence of the N-terminal and C-terminal portions of the prohormone of atrial natriuretic factor in the ascitic fluid of cirrhotic patients. J Med 1990;21:265-75. 12. Vesely DL, Preston R, Winters CJ, et al. Increased release of the N-terminal and C-terminal portions of the prohormone of atrial natriuretic factor during immersion-induced central hypervolemia in cirrhotic humans. Am J Nephrol 1991;11:207-16. 13. Gunning ME, Brady HR, Otuechere G, et al. Atrial natriuretic peptide (31-67) inhibits Na+ transport in rabbit inner medullary collecting duct cells: role of prostaglandin E-2. J Clin Invest 1992;89:1411-7. 14. Chiou S, Vesely DL. Kaliuretic peptide: the most potent inhibitor of Na+-K+-ATPase of the atrial natriuretic peptides. Endocrinology 1995;136:2033-9. 15. Habibullah AA, Villarreal D, Freeman RH, et al. Effects of cyclooxygenase inhibition on the renal actions of atrial natriuretic prohormone fragment 31-67 in normal dogs [abstract]. FASEB J 1995; 9:A296. 16. Ackerman BH, Wyeth RP, Vesely DL, et al. Pharmacokinetic characterization of the postdistribution phase of prohormone atrial natriuretic peptides amino acids 1-98, 31-67 and atrial natriuretic factor during and after rapid ventricular pacing in dogs. J Clin Pharmacol 1992;32:415-21. 17. Vesely DL, Douglass MA, Dietz JR, et al. Negative feedback of atrial natriuretic peptides. J Clin Endocrinol Metab 1994;78:1128-34. 18. Vesely DL, Norsk P, Winters CJ, et al. Increased release of the N-terminal and C-terminal portions of the prohormone of atrial natriuretic factor during immersion-induced central hypervolemia in normal humans. Proc Soc Exp Biol Med 1989;192:230-5. 19. Ackerman BH, Overton RM, McCormick MT, et al. Disposition of vessel dilator and long-acting natriuretic peptide in healthy humans after a 1-hour infusion. J Pharmacol Exp Ther 1997;282:603-8. 20. Overton RM, Vesely DL. Processing of long-acting natriuretic peptide and vessel dilator in human plasma and serum. Peptides 1996; 17:1155-62. 21. Tsuchimochi H, Yazaki Y, Ohno H, et al. Ventricular expression of atrial natriuretic peptide. Lancet 1987;2:336-7. 22. Edwards BS, Ackermann DM, Lee ME, et al. Identification of atrial natriuretic factor within ventricular tissue in hamsters and humans with congestive heart failure. J Clin Invest 1988;81:82-6. 23. Poulos JE, Gower WR Jr, Sullebarger JT, et al. Congestive heart failure: increased cardiac and extracardiac atrial natriuretic peptide gene expression. Cardiovasc Res 1996;32:909-19.