Cardiovascular Research

Correction for Daggubati et al., Cardiovasc Res 44 (2) 452-453.
Cardiovascular Research 1997 36(2):246-255; doi:10.1016/S0008-6363(97)00164-8
© 1997 by European Society of Cardiology

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Copyright © 1997, European Society of Cardiology

Adrenomedullin, endothelin, neuropeptide Y, atrial, brain, and C-natriuretic prohormone peptides compared as early heart failure indicators¹

Sreedevi Daggubati^a,b,*, James R Parks^a,b, Rose M Overton^a,b, Guillermo Cintron^a,b, Douglas D Schocken^a,b and David L Vesely^a,b

^aDepartments of Medicine, Physiology and Biophysics, James A. Haley Veterans Hospital, 151, 13000 Bruce B. Downs Blvd., Tampa, FL 33612, USA
^bUniversity of South Florida Health Sciences Center, Tampa, FL, USA

^* Corresponding author. Tel.: +1 813 9727624; Fax: +1 813 9727623.

Received 18 February 1997; accepted 5 June 1997

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objectives: The present investigation was designed to determine the best endogenous plasma marker of early congestive heart failure (CHF). Methods: Forty volunteers with mild CHF (New York Heart Association Class I, n = 12), moderate (Class II, n = 8), or severe (Class III and Class IV, each=n of 5) and 10 age-matched healthy individuals had the simultaneous evaluation of their respective plasma samples by the following radioimmunoassays: atrial natriuretic peptide, ANP; three N-terminal ANP prohormone assays, i.e., proANPs 1–30, 31–67, and 79–98 with the numbers referring to their amino acid (a.a.) sequences in their 126 a.a. prohormone; brain (BNP) and C-natriuretic peptides; N-terminal BNP prohormone; adrenomedullin; neuropeptide Y and endothelin. Results: ProANPs 31–67, 1–30 and 79–98 had 100% (P = 0.01), 83% (P = 0.09) and 50% (P = 0.74) sensitivity in differentiating Class I CHF subjects from healthy subjects. The ANP, BNP, NT-proBNP, CNP, adrenomedullin, neuropeptide Y, and endothelin assays could not differentiate mild CHF subjects from healthy individuals. Logistic regression analysis revealed that only proANP 31–67 significantly (P = 0.0001) discriminated between early CHF (5226±377 pg/ml) and healthy individuals (1595±157 pg/ml). The positive and negative predictive values of proANP 31–67 were excellent (100% for each). The peptides measured in these assays were found to be independent markers of CHF with respect to left ventricular ejection fraction. Conclusions: ProANPs 31–67 is the most sensitive marker in discriminating NYHA Class I CHF subjects from healthy individuals. The ANP, BNP, NT-proBNP, CNP, adrenomedullin, neuropeptide Y and endothelin radioimmunoassays cannot discern mild CHF. These peptides are independent of left ventricular ejection fraction.

KEYWORDS Heart failure; Plasma; Endothelin; Neuropeptide-Y; Adrenomedullin; Humans

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The ability to detect early asymptomatic or minimally symptomatic congestive heart failure (CHF) by measuring a circulating marker of CHF would be a major advance in the diagnosis of CHF. The ability to diagnose a person with very early, i.e., Class I New York Heart Association (NYHA) CHF would permit early intervention and might diminish the morbidity and mortality associated with advanced CHF. In 1989, it was first demonstrated that a marker in plasma measured with an N-terminal atrial natriuretic peptide prohormone (proANP) radioimmunoassay could differentiate persons with NYHA Class I CHF from 54 normal subjects [1]. In this investigation, it was found that this assay, i.e., the proANP 31–67 (devised to amino acids 31–67 of the atrial natriuretic peptide prohormone) radioimmunoassay (RIA) could differentiate Class I CHF subjects from normal subjects while the atrial natriuretic peptide (ANP; amino acids 99–126 of the same 126 amino acid prohormone) RIA could not differentiate CHF subjects from normal persons until their CHF was advanced (i.e., Class III NYHA) [1]. In 1993 Lerman et al. confirmed that the ANP RIA does not detect increased ANP plasma levels until advanced CHF is present and that an N-terminal proANP assay could detect persons with early asymptomatic congestive heart failure [2].

At approximately the same time that one of the N-terminal ANP prohormone assays was found to be an indicator of early CHF [1], another peptide was discovered in porcine brain that was named brain natriuretic peptide (BNP) [3]. This peptide contains 32 a.a. and is formed by a different gene from the above atrial natriuretic peptides [3–5]. BNP has structural homology to ANP and is also synthesized in the heart with more BNP actually being found in the heart than in the brain [4, 5]. BNP has been found to be elevated in the plasma of some CHF subjects [6–8]. The N-terminus of the BNP prohormone has also been reported to be increased in CHF but the severity (i.e., NYHA classification) was not reported for the CHF subjects [9]. C-type natriuretic peptide (CNP) is a 28 a.a. peptide also first isolated from porcine brain and which consists of the C-terminal end of a prohormone distinct from the ANP and BNP prohormones but CNP has structural homology to ANP and BNP [10]. This peptide has been reported not to increase in CHF [11].

Adrenomedullin, a 52 a.a. peptide originally isolated from pheochromocytoma and also found in the human adrenal [12], has been reported to be elevated in circulation of patients with Class III and IV CHF, but not to be significantly elevated in Class I and II CHF [13]. Neuropeptide Y, a 36 a.a. peptide released from sympathetic nerve endings, has been reported to be increased in the plasma of 6 of 17 individuals with CHF [14], while other investigators have found that there is no difference in the circulating levels of neuropeptide Y in CHF subjects compared to healthy individuals [15]. Finally, endothelin, a 21 a.a. vasoconstrictive peptide [16, 17](as opposed to all of the above peptides having vasodilating properties), has been reported to be increased in CHF [18, 19]possibly secondary to the known ability of atrial natriuretic peptides to increase endothelin in the circulation [20].

The present investigation was designed to compare in individuals with mild, moderate, and severe CHF which is the best plasma marker of early CHF by simultaneously measuring all of the above markers and comparing their circulating concentrations with age matched healthy individuals. Thus, ANP, BNP, three N-terminal proANP assays (proANP 1–30, proANP 31–67, proANP 79–98), an N-terminal proBNP assay, CNP, adrenomedullin, neuropeptide Y and endothelin RIAs were each utilized to evaluate the same plasma samples from CHF subjects and age-matched healthy volunteers as to which of these peptides is the best indicator of early CHF. There has not been a direct comparison of each of these peptide markers of congestive heart failure as to which is the best to diagnose mild CHF and/or to clarify which of these endogenous peptides increase in the circulation early in heart failure as opposed to increasing when heart failure progresses to a more severe form.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Healthy volunteers and CHF subjects
Forty men (65±1.6 [mean±SEM] years old) were studied. Thirty subjects with congestive heart failure (CHF) were subdivided according to functional symptoms (New York Heart Association Classification [21]) into Class I (n = 12), Class II (n = 8); Class III (n = 5) and Class IV (n = 5). More CHF subjects with early CHF (Class I and II, n = 20) rather than severe CHF (Class III and IV, n = 10) were incorporated into this investigation since this study was designed to determine which of the peptides measured simultaneously by the 10 radioimmunoassays (RIAs) can reliably differentiate early CHF patients (63.7±2.4 years) from healthy age matched (63.2±3.6 years, n = 10) human volunteers. Subjectively, each of the volunteers had symptoms of heart failure [22–24], including one or more of the following symptoms: dyspnea on exertion, paroxysmol nocturnal dyspnea, or effort related fatigue. Objectively, left ventricular systolic function was documented by radionuclide angiography. The functional class of CHF (NYHA I to IV) [21]was determined independently by a cardiologist and a cardiovascular nurse based upon the individual's symptoms. The classification of the severity of CHF was done immediately before drawing the blood sample for the respective assays. Thus, the persons grading the severity of CHF had no knowledge of what respective peptide levels were going to be (i.e., blinded to the results) as they had not been assayed at the time of classifying the severity of symptoms of CHF. The cardiologist and cardiovascular nurse, likewise, did not know the left ventricular ejection fractions (LVEF) of the CHF subjects at the time of their classification of the severity of CHF. The left ventricular ejection fraction was calculated by ‘area-counts technique’ utilizing multiple-gated acquisition (MUGA) radionuclide angiography. All of the control and CHF subjects were normotensive with blood pressures less than 138/88 mm Hg. These 40 subjects had heart rates ranging from 60 to 92 with respiration rates between 12 and 16 min^–1.

Blood pressure, heart rate, weight, height, and age of each subject are shown in Table 1. None of the healthy volunteers had any known disease including any salt or water metabolism abnormalities. The cause of congestive heart failure included ischemic cardiomyopathy (n = 22), idiopathic dilated cardiomyopathy (n = 3), and valvular heart disease (n = 5). All patients had a complete physical and laboratory evaluation. Exclusion critera included all patients with a creatinine greater than 1.5 mg/dl and any person with hypertension, cirrhosis and/or ascities. All patients with symptomatic CHF were on treatment, which included digitalis, diuretics, ACE inhibitors, beta blockers and/or vasodilators. Venipuncture for measurement of the respective peptides was performed in the sitting position in all subjects with each of these ambulatory subjects being in the sitting position 15 min prior to a single venipuncture sample from the antecubital vein. 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.

View this table: [in this window] [in a new window]   Table 1 Blood pressure, heart rate, age, weight, height, and ejection fractions of controls and congestive heart failure subjects

 
Informed written 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 investigation conforms with the principles outlined in the Declaration of Helsinki for investigations involving human subjects.

2.2 Extraction of the respective peptides from plasma
To determine if one or more of the respective plasma peptides measured with the ten radioimmunoassays described below are indicators of early CHF, plasma samples from healthy subjects and patients with congestive heart failure were collected in cold 5 ml tubes containing EDTA (7.5 mg, Vacutainer®, Becton Dickinson, Rutherford, NJ) to prevent the proteolytic breakdown of any peptides that might be present in plasma. These samples were then transported on ice, and immediately centrifuged at 3000 g for 15 min. After centrifugation, the plasma was extracted with 100% ethanol (1:1 dilution), vortexed, and allowed to stand at 4°C for 30 min as described previously [1, 25, 26]. After 30 min at 4°C, the samples were centrifuged at 3000 g for 15 min and the supernatants taken to dryness via controlled nitrogen flow. We have found that at this point these samples can be frozen at –80°C with no loss in the concentration of the various peptides measured [27].

2.3 Measurement of atrial natriuretic peptides
Radioimmunoassays to measure peptides from the N-terminus of the ANP prohormone were devised to amino acids 1–30 (3506 M_r), 31–67 (3876 M_r), and 79–98 (2182 M_r) of the 126-amino acid prohormone; the C-terminal ANP prohormone assay measures amino acids 99–126 of the prohormone, i.e., ANP (3078 M_r), as described in detail previously by our laboratory [1, 25, 26]. Serial dilution of pooled plasma has revealed excellent parallelism of standards and unknowns in these assays [1, 28]. None of these assays or the other assays described below have any cross-reactivity with each other.

2.4 Measurement of brain and c-natriuretic peptides
Brain natriuretic peptide (i.e., BNP-32, the final 32 a.a. of the 108 a.a. BNP prohormone, proBNP 77–108; 3462 M_r), an N-terminal proBNP assay (devised to a.a. 22 to 46 of the N-terminus of the BNP prohormone; or a.a. 22–46=2859 M_r) and C-natriuretic peptide (CNP, the C-terminal 28 a.a. end of its 126 a.a. prohormone, 2196 M_r) were each measured ulitizing radioimmunoassay (RIAs) kits from Phoenix Pharmaceuticals Inc., Belmont, CA.

2.5 Adrenomedullin and neuropeptide y radioimmunoassays
Adrenomedullin (6029 M_r) was measured with a human 1–52 adrenomedullin radioimmunoassay kit while neuropeptide Y (4272 M_r) was measured with a human neuropeptide Y radioimmunoassay (RIA) kit (both from Phoenix Pharmaceuticals, Inc., Belmont, CA) as previously described from our laboratory [29].

2.6 Endothelin radioimmunoassay
Endothelin (2490 M_r) was measured in plasma after extraction as above with an ET-12 (high sensitivity) -assay kit with Amerlex-M® magnetic separation (Amersham, Arlington Heights, IL) as previously described from our laboratory [20].

2.7 High-performance gel permeation chromatography
The molecular forms of the immunoreactive peptides in CHF plasma were determined by high-performance gel permeation chromatography (HP-GPC). 50 ml of plasma pooled from 5 congestive heart failure subjects was extracted with 100 ml of 100% ethanol (1:2 dilution) and then dried with low-flow nitrogen. This plasma extract was then resuspended in 100 µl of HP-GPC column mobile phase (10 mmol/l trifluoroacetic acid containing 0.3 mol/l sodium chloride and 30% acetonitrile) for HP-GPC assay as described previously from our laboratory [25, 26, 30, 31].

2.8 Statistical analysis
The data obtained in this investigation are illustrated as mean±SEM. Differences in measurements between subjects or groups of subjects were evaluated by analysis of variance (ANOVA) followed by Tukey's analysis of significant differences (ASD). In deriving a model for predicting which peptide assay best distinguished between Class I patients and healthy subjects, both forward and backward stepwise discriminant analyses were performed. To be considered statistically significant, we required a probability value to be <0.05 (95% confidence limits).

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The age, height and mean blood pressures of NYHA Class I to IV subjects were not significantly different from healthy individuals although the mean arterial pressure of the CHF subjects tended to be lower (Table 1). The heart rates of CHF subjects also tended to be lower than the healthy controls but this did not reach a level of statistical significance (Table 1). The mean ejection fractions of the Class III and Class IV CHF groups were significantly (P<0.05) lower than the Class I and II CHF subjects (Table 1).

Two of the N-terminal proANP assays, i.e., proANP 1–30 and proANP 31–67 RIAs, were able to differentiate Class I NYHA congestive heart failure (CHF) patients from healthy individuals (Table 2). The proANP 31–67 RIA differentiated each of the 12 Class I CHF subjects from the 10 healthy individuals (i.e., 100% sensitivity, P = 0.014). While the proANP 1–30 values were higher in each of the Class CHF I subjects than healthy subjects it is important to note that two of the Class I CHF subjects (i.e., subjects 10 and 11) were not significantly higher than the values found in the healthy individuals (Table 2) (P = 0.087 for group). None of the other eight peptide assays could differentiate Class I CHF subjects from healthy humans (Table 2). The proANP 79–98 concentrations in plasma were higher in 50% of the subjects in the Class I CHF group versus the healthy volunteers (Table 2) (P = 0.744). The ANP, BNP, NT-proBNP, CNP, adrenomedullin, neuropeptide Y, and endothelin assays could not differentiate early CHF subjects from healthy individuals (Table 2).

View this table: [in this window] [in a new window]   Table 2 Comparison of the circulating concentration of ANP, BNP, CNP, ProANPs 1–30, 31–67, 79–98, N-terminal ProBNP, adrenomedullin, neuropeptide Y and endothelin in subjects with mild, moderate and severe congestive heart failure with healthy aged matched human volunteers

 
In the evaluation of Class I CHF versus controls, the proANP 31–67 RIA was found to be a very sensitive (100%) assay in determination of which persons have CHF and a very specific (100%) assay in determining which persons do not have CHF (Table 2). The proANP 1–30 RIA had 83% sensitivity and 100% specificity while the proANP 79–98 RIA had a 50% sensitivity and 100% specificity (Table 2). All of the other RIAs evaluated had 0% sensitivity, i.e., they could not determine which persons had mild CHF (Table 2).

The predictive value of the proANP 31–67 assay in early CHF was excellent. The positive predictive value of the proANP 31–67 was 100% with this assay being able to determine the probability of CHF in a person in every case with a positive result (i.e., significantly elevated plasma concentration). The negative predictive value of the proANP 31–67 was also 100% as it was able to determine in every individual if early CHF was not present. The positive and negative predictive values of the proANP 1–30 RIAs were 100% and 83%, respectively. The other RIAs did not have any predictive value when Class I CHF subjects were evaluated in comparison to persons without disease. Data from all the peptide assays were examined for entry into a multivariate discriminate analysis model to predict which peptide or group of peptides could best distinguish between control subjects and Class I CHF patients. Whether by forward or backward stepwise analysis only the single variable proANP 31–67 could discriminate between these two groups at a statistically significant level (P = 0.0001 for proANP 31–67).

With increasing severity of CHF the proANP 1–30 and proANP 31–67 assays could reliably differentiate Class II, III and IV CHF from Class I CHF subjects and from each other (Table 2). In Class II CHF individuals the proANP 79–98 assay could now definitely differentiate these individuals from healthy subjects with a measured plasma sample (Table 2). In contrast, the ANP, BNP, NT-proBNP, CNP, adrenomedullin and endothelin assays were unable to differentiate Class II subjects from healthy subjects (Table 2).

The ANP, BNP, NT-proBNP, and endothelin assays could discern subjects with severe, i.e., Class III and especially in Class IV CHF with a single plasma sample from that of healthy subjects (Table 2). The adrenomedullin and CNP assays could not reliably differentiate persons with even severe CHF from healthy individuals although one subject (CHF IV subject #4) did have an elevated CNP concentration compared to healthy individuals (Table 2).

To help determine which peptide(s) the respective assays were immunologically recognizing in the plasma of subjects with CHF, plasma from CHF subjects (n = 5) was subjected to high-performance gel permeation chromatography (HP-GPC) followed by each of the respective assays. This evaluation revealed that the proANP 1–30 assay is immunologically recognizing (1) proANP 1–30, and (2) proANP 1–98 (i.e., N-terminus of ANP prohormone without the C-terminus, that is ANP, attached to it) in the plasma of CHF subjects (Fig. 1A). HP-GPC evaluation of plasma of persons with CHF followed by proANF 31–67 assay revealed that this assay is only recognizing a single peptide with a molecular weight consistent with proANP 31–67 (Fig. 1B). As one observes in Fig. 1B, the proANP 31–67 assay does not recognize proANP 1–98 (i.e., the N-terminus of the ANP prohormone) which would elute at 9 800 to 10 000 molecular weight (M_r) or the whole ANP prohormone (which would elute at approximately 13 000 M_r) in the plasma of CHF individuals.

View larger version (27K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 High-performance gel permeation chromatography evidence that (A) atrial natriuretic peptide prohormone (proANP) 1–30, (B) atrial natriuretic peptide prohormone (proANP) 31–67, (C) atrial natriuretic peptide (ANP), and (D) atrial natriuretic peptide prohormone 79–98 exist as separate entities in human congestive heart failure (CHF) plasma. (A) In evaluation of CHF plasma, a sharp peak consistent with proANP 1–98 was observed at 9 800 Mr (9.8 kD) together with second peak in fractions 69–72 with proANP 1–30 assay. The pure human synthetic form of proANP 1–30 elutes in fractions 69–70 (arrow). In (B) the elution of proANP 31–67 from CHF plasma was identical with the elution of the pure human synthetic form of proANP 31–67 (arrow). As observed in (B), neither proANP 1–98 nor the ANP prohormone (proANP 1–126) was immunologically recognized in CHF plasma by the proANP 31–67 assay. (C) A distinct elution peak in CHF plasma consisting of 0.3-ml fractions 74–79 with the peak in fraction 75 was found by HP-GPC evaluation followed by ANP radioimmunoassay. (D) Pure synthetic proANP 79–98 (open circles) eluted in 0.3-ml fractions 76–81, with the peak in fraction 78. -labeled proANP 79–98 was similar, with its peak in fraction 79. The elution of proANP 79–98 from CHF plasma was in a peak where the human synthetic form of proANP 79–98 elutes. In addition, two other peaks are observed consistent with (1) proANP 1–98 and (2) proANP 68–98. Elution profiles of molecular weight markers are denoted by arrows on the top of the graph. Vt= total volume collected; Vo= void volume. Each fraction equals 0.3 ml.

 
Evaluation of human CHF plasma by HP-GPC followed by ANP RIA also revealed only one peak which eluted identically with the pure synthetic form of ANP (Fig. 1C). HP-GPC evaluation of human CHF plasma followed by proANP 79–98 assay revealed that this assay recognizes three peptides whose molecular weights are consistent with this assay recognizing proANPs 1–98, 68–98, and 79–98 in CHF plasma (Fig. 1D). The elution pattern immunologically recognized in CHF subjects with these assays are essentially identical to what has been previously found in the plasma of healthy human subjects [25, 26]. Evaluation of CHF plasma by HP-GPC followed by CNP, NT-proBNP, adrenomedullin, neuropeptide Y and endothelin RIAs revealed a single eluted peak consistent with their known molecular weights (data not shown). HP-GPC evaluation of CHF plasma followed by BNP assay, on the other hand, revealed two eluted peaks, one consistent with BNP-32 and the other consistent with a high molecular peptide of approximately 10 to 11 000 MW (Fig. 2). It is important to note in Fig. 2 that the BNP assay is actually measuring more of the high molecular weight peptide than BNP itself in CHF plasma.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 High-performance gel permeation chromatography evaluation of congestive heart failure plasma followed by brain natriuretic peptide (BNP) radioimmunoassay of each 0.3-ml fraction obtained from human congestive heart failure plasma revealed two elution peaks. The smaller peak eluted where the pure synthetic form of BNP elutes (arrow). The larger peak eluted at 10 000 to 11 000 Mr (10 to 11 kD) which is consistent with this peak being a large BNP precursor peptide. Molecular weight markers are on the top of the graph. Vt= total volume collected; Vo= void volume.

 
None of the peptides measured with the 10 radioimmunoassays correlated with the left ventricular ejection fraction (LVEF) of the respective subjects with CHF. Thus, when the ejection fractions of the subjects in the various NYHA classes of CHF in Table 1 were compared with the circulating concentrations recognized in the respective peptide assays (Table 2) there was no correlation between LVEF and any of the peptides measured. The r-values for LVEF versus each of the peptides when all of the CHF subjects were considered as a group are as follows: ANP (r = –0.257), BNP (r = –0.345), CNP (r = –0.165), adrenomedullin (r = –0.053), endothelin (r = –0.322), neuropeptide Y (r = –0.196), Nt–proBNP (r = –0.366), proANP 1–30 (r = –0.327), proANP 31–67 (r = –0.344), and proANP 79–98 (r = –0.311). It should also be noted that left ventricular ejection fraction alone did not discriminate between the various NYHA classes (Table 1).

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The present investigation demonstrates that a simple and inexpensive measurement of a peptide marker in a plasma sample can differentiate persons with early (NYHA Class I) congestive heart failure from healthy humans. ProANP 1–30 and proANP 31–67 radioimmunoassays were found to be far superior to ANP, BNP, CNP, N-terminal proBNP, adrenomedullin, neuropeptide Y, and endothelin in the ability to differentiate NYHA Class I CHF persons from healthy individuals. The ANP, BNP, N-terminal proBNP, CNP, adrenomedullin, neuropeptide Y and endothelin assays could not distinguish between persons with mild CHF and healthy individuals. Yoshimura et al. have pointed out another deficiency in utilizing BNP as a screen for CHF [8]. In patients with valvular lesions such as mitral stenosis in whom the atria as opposed to the ventricles are overloaded, BNP does not increase in the circulation as one progresses from NYHA Class I to Class III and IV CHF [8]making it an unreliable assay for quantifying the progression of CHF [8]as well as being a poor screen for early CHF.

What is the role of these assays in diagnosing congestive heart failure? Since proANP 1–30 and proANP 31–67 are very stable in frozen plasma (concentrations being almost identical in the same plasma samples evaluated seven years apart [27]), the utilization of one of these two assays can provide an objective measure of early CHF. These radioimmunoassays are simple to perform [32, 33]and could easily be performed in any hospital's clinical laboratory. The ability to detect early CHF by an inexpensive, non-invasive screening method described herein has become of greater importance with clinical studies suggesting that patients with mild or asymptomatic heart failure may benefit from early pharmacologic intervention [34, 35]. The prevalence of asymptomatic left ventricular dysfunction in a population based echocardiographic study has been reported to be 3.4% and symptomatic left ventricular dysfunction was found in 8% of the general population [36], indicating that CHF is a public health problem of major importance.

In choosing which assay should be used to screen for CHF, one should consider what is actually being measured in CHF plasma. The high-performance gel permeation chromatography (HP-GPC) evaluation of CHF plasma in the present investigation revealed that proANP 31–67 measured only proANP 31–67 while the proANP 1–30 and BNP assays (see Figs. 1 and 2) immunologically recognized and measured high molecular weight peptides as well as proANP 1–30 and BNP. Interpretation of the values measured in plasma is, thus, easier with the proANP 31–67 since one is measuring only one peptide. This point has previously been stressed by Yandle et al. [7]with respect to the BNP assay. These authors demonstrated with high pressure liquid chromatography that the BNP assay is not primarily measuring BNP but rather measuring a high molecular peptide which increases in greater proportion than BNP itself when CHF plasma is compared to plasma of healthy subjects [7]. The present investigation confirms the study of Yandle et al. [7]that the BNP assay in CHF subjects is primarily not measuring BNP but rather more of what the assay is measuring is a high molecular weight peptide.

Importantly, it should be noted that the peptides measured in proANP 1–30 and proANP 31–67 RIAs did not correlate with the left ventricular ejection fraction (LVEF). This information indicates that the proANP 1–30 and proANP 31–67 measured plasma concentrations are independent indicators of CHF that can be utilized independent of ejection fraction to help determine the severity of CHF. Thus, the evaluation of a single plasma sample with either the proANP 1–30 or proANP 31–67 RIAs gives diagnostic information over and above traditional criteria such as ejection fraction. As one observes in Table 2, the measured plasma concentration of either proANP 1–30 or proANP 31–67 actually correlates much better with increasing severity of CHF than does the ejection fraction. Left ventricular ejection fraction is an indirect measurement of systolic or contractile performance of the left ventricle. Although left ventricular ejection fraction is a powerful prognostic predictor in patients with severe heart failure, correlation between ejection fraction and symptomatic functional class (NYHA) has previously been shown to be poor [37]. Atrial peptides, which are secreted in response to elevation in atrial pressure and stretch, appear to be more sensitive indicators of the early circulatory abnormalities in heart failure. The early changes in atrial pressure may actually precede the structural changes which result in ventricular remodeling and depression of left ventricular systolic function and ejection fraction which are the hallmarks of advanced CHF.

Time for primary review 30 days

	Acknowledgements

 
This study was supported in part by a Merit Review Grant from the United States Department of Veterans Affairs (DLV). The authors gratefully acknowledge the excellent secretarial assistance of Charlene Pennington in the preparation of the manuscript.

	Notes

 
¹ Presented in part at the American Society of Clinical Investigation Meeting, Washington, DC, April 25–27, 1997.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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