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European Journal of Heart Failure 2005 7(7):1145-1148; doi:10.1016/j.ejheart.2004.12.009
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© 2005 European Society of Cardiology

C-type natriuretic peptide plasma levels increase in patients with chronic heart failure as a function of clinical severity

Silvia Del Ry, Claudio Passino, Maristella Maltinti, Michele Emdin and Daniela Giannessi*

Institute of Clinical Physiology, National Research Council Pisa, Italy

* Corresponding author. CNR Institute of Clinical Physiology, Via Giuseppe Moruzzi 1, Pisa 56124, Italy. Tel.: +39 50 3152664; fax: +39 50 3152166. E-mail address: danielag{at}ifc.cnr.it


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Backgound: C-type natriuretic peptide (CNP), secreted by the endothelium and the heart, is structurally related to atrial and brain natriuretic peptides, but its clinical significance in chronic heart failure (CHF) is controversial.

Aim: To investigate the role of CNP in CHF, plasma CNP levels were determined in a prospective series of 133 patients with CHF (age 64±1 years, left ventricular ejection fraction (EF), 31.5±0.7%, mean±S.E.M.) and in 21 age-matched healthy subjects.

Methods and results: CNP was measured by a radioimmunoassay (sensitivity: 0.41±0.009 pg/tube) after a preliminary solid-phase extraction. Plasma level of CNP in healthy subjects was 2.7±0.2 pg/ml and significantly increased in CHF, as a function of clinical severity: 4.9±0.7 pg/ml in NYHA class I; 7.0±0.4 pg/ml in class II (p<0.001 vs. controls); 9.6±0.7 pg/ml in class III (p<0.001 vs. controls and class I and II), and 11.8±2.0 pg/ml in class IV (p<0.001 vs. controls, class I and II; Fisher's test after ANOVA). A significant relation was also found between CNP plasma levels and EF (R=0.40, p<0.001).

Conclusion: Plasma CNP elevation is related to clinical and functional disease severity. These findings suggest a pathophysiological role for this peptide that, for its vasorelaxing activity, could influence the endothelial vasomotor response in CHF.

Key Words: C-type natriuretic peptide (CNP) • Natriuretic peptides • Chronic heart failure (CHF)

Received September 16, 2004; Revised November 9, 2004; Accepted December 20, 2004


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
C-type natriuretic peptide (CNP), first isolated from porcine brain in 1990 [1], shares structural and physiological properties with the other natriuretic peptides, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), but it has a shorter circulatory half-life. It is produced by the vascular endothelium and by the heart, too, in patients with chronic heart failure (CHF) [2] and has vasodilator properties. In CHF patients, plasma levels of ANP and BNP are elevated in relation to clinical severity and have established diagnostic and prognostic relevance [3]. While a central pathophysiological role in CHF has been demonstrated for ANP and BNP [3], the significance of CNP is still controversial [4].

Some studies have investigated plasma CNP levels in stable CHF [5–7], but have failed to demonstrate any significant elevation. More recently, an elevation in plasma levels of NT-proCNP, the amino-terminal peptide derived from the cleavage of the prohormone proCNP, has been found in patients with CHF [8,9]. Moreover, increased urinary levels of CNP have been observed in CHF patients [10].

In order to evaluate the presence of an increase in peripheral levels of CNP in CHF according to clinical severity, we measured plasma levels of CNP in a population of CHF patients at different stages of the disease and in a group of age-matched healthy subjects.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Subjects
We enrolled a prospective cohort of 133 consecutive patients with a diagnosis of cardiomyopathy, referred to our institute for evaluation of CHF between September 2002 and March 2004. The cohort comprised 100 males and 33 females, mean age was 64±1 years, mean weight was 74.9±1.1 kg (range 54–112), mean height was 169±0.8 cm (range 149–192), and mean body surface area (BSA) was 1.86±0.2 (range 1.47–2.27). Serum creatinine levels were 1.17±0.03 mg/dl (mean±S.E.M.), and were >1.5 mg/dl in only 19 patients. Idiopathic dilated cardiomyopathy was diagnosed in 71 patients, 45 patients had ischemic cardiomyopathy, and 17 patients had valvular heart disease. The inclusion criterion was a significantly depressed (<50%) left ventricular ejection fraction (EF), measured by echocardiography [11]. Exclusion criteria were: acute myocardial infarction or unstable angina within the previous 6 months, significant primitive pulmonary disease, or severe renal failure (defined as a serum creatinine value above 2.5 mg/dl). Heart failure severity was evaluated clinically according to the New York Heart Association (NYHA) classification: 16 patients were in functional NYHA class I, 67 in class II, 41 in class III, and 9 in class IV.

Patients were treated with restriction of water–sodium intake (using a personalized, well-controlled diet with a sodium intake of 100–140 mmol/day) for 2 weeks prior to sampling and were on homogeneous optimal medical treatment (furosemide, ACE inhibitors, carvedilol, spironolactone for patients in class NYHA≥III). This treatment was continued during the study for ethical reasons.

A group of 21 age-matched healthy subjects was included in the study design, as a control group: mean age 61±2 years, 15 males and 6 females, mean weight 72.5±0.8 kg, and mean height 175±0.5 cm.

The investigation conforms with the principles outlined in the Declaration of Helsinki (Br Med J 1964; ii: 177). The study was approved by the local Ethical Committee and all patients provided signed informed consent.

2.2. Protocol
Blood samples were withdrawn on the study day at 8 a.m., from an antecubital vein, after a period of 20-min supine rest. Plasma samples were collected into ice-chilled disposable polypropylene tubes containing aprotinin, 500 KIU/ml, and EDTA, 1 mg/ml, to prevent the proteolytic breakdown of any peptide that might be present in plasma. Plasma samples were rapidly separated by centrifugation for 15 min at 4°C, and then stored in aliquots frozen at –20°C.

2.3. Extraction of CNP from plasma and immunometric assay
CNP was measured in plasma by a modified commercial radioimmunoassay (RIA) (C-type natriuretic peptide-22; Phoenix Pharmaceuticals, Belmount, CA, USA) after a preliminary extraction step: 2 ml of plasma was acidified with 2 ml of 1% trifluoracetic acid (TFA) and, after centrifugation at 1500xg for 10 min, loaded on activated Sep-Pak C18 cartridges (Waters Associated, Milford, MA, USA). The columns were washed three times with 3 ml of 1% TFA and CNP was eluted with 3 ml of a solution containing 60% acetonitrile in 1% TFA. The dry residue of this eluate was resuspended with 300–500 µl of the assay buffer and 100 µl used for the immunometric assay. Each sample was assayed in duplicate and the experiment was carried out in an ice bath. The values of the plasma CNP concentrations are corrected for a mean extraction yield of 78.3%, as determined by addition of both synthetic and radiolabelled CNP to plasma.

2.4. Statistical analysis
All sample values and other data for quality control of the RIA system were calculated using a previously described computer program [12]; the interpolation of the dose–response curves was computed using a four-parameter logistic function [12]. Because plasma CNP values are not normally distributed, natural logarithmic transformation of data was used for statistical analysis when needed. Differences among more than two independent groups were analyzed by Fisher's test after ANOVA. Relations among variables were assessed by linear regression. The results are expressed as mean±S.E.M. and a p value <0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
Plasma levels of CNP were significantly increased in CHF patients, as compared to healthy subjects (7.9±0.38 vs. 2.7±0.2 pg/ml, p<0.001). When patients were considered according to functional class, CNP concentration showed a significant increase with clinical severity: 4.9±0.7 pg/ml in NYHA class I, 7.0±0.4 pg/ml in class II (p<0.001 vs. controls), 9.6±0.7 pg/ml in class III (p<0.001 vs. controls and class I and class II), and 11.8±2.0 pg/ml in class IV (p<0.001 vs. controls, class I and II) (Fig. 1). A significant negative correlation was observed between CNP plasma values and EF in patients with CHF (R=0.40, p<0.001) (Fig. 2). Patients with a lower EF (<30%) presented higher CNP plasma levels, as compared to those with a higher EF (9.3±0.6 vs. 6.4±0.6 pg/ml, p<0.001). A slight, but significant, correlation was also observed between age and CNP plasma values in patients with CHF (R=0.25, p=0.004), although patients with more severe disease did not differ for age when compared with patients with milder disease (66.5±1.5 vs. 63.1±1.2 years, NS). Finally, a significant correlation between CNP plasma levels and serum creatinine was observed (R=0.29, p=0.0014).


Figure 1
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  		Fig. 1 Distribution of CNP plasma levels as a function of clinical severity: the 10th, 25th, 50th (median), 75th, and 90th percentiles of the variable are reported. All values above the 90th percentile and below the 10th percentile are plotted separately.

 


Figure 2
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  		Fig. 2 Correlation between CNP plasma values (logarithmic transformation) and EF.

 
The intra-assay variability was about 5%, inter-assay variability 7%, and sensitivity 0.41±0.015 pg/tube. The accuracy of the immunometric determination was evaluated by dilution and recovery tests: a linearity of response between 20 and 350 µl of extracted plasma and a recovery of about 80% were observed. As to the specific cross-reactions, human ANP or porcine and rat BNP do not interfere in the assay of CNP, as stated by the manufacturer of the assay. In addition, the mean values of CNP found in healthy subjects in this study closely agree with those of others [5,6,10], thus ruling out important interference effects.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The main finding of the present study is the evidence of an elevation of plasma CNP concentration in CHF patients, as compared to healthy subjects. Moreover, the increased secretion of this natriuretic peptide seems to be related to the severity of the disease, as indicated by the progressive rise in CNP plasma values with worsening of symptoms, and by the negative relation between CNP plasma values and left ventricular EF.

The knowledge of the importance of CNP for the regulation of the cardiovascular system has been delayed because initial studies on CNP failed to find this peptide in the heart [13], or the plasma of patients with CHF [5–7]. Subsequently, mRNA transcripts for NPR-B have been found within myocardial tissue [14]. The presence of CNP within the myocardium was confirmed by immunohistochemistry and radioimmunoassay techniques; its tissue concentrations were two to three times greater in CHF patients compared with controls [5].

More recently, it has been observed that CNP is produced by the failing heart [2] and an increase in plasma levels of NT-proCNP was observed in CHF patients [8,9]. These observations indicate a significant role for CNP in the pathophysiology of CHF [15]. The results of the present study, first evidencing an elevation of plasma CNP in CHF as a function of the severity of the disease, closely agree with these recent observations. As to the discrepancy between our findings and previous reports on CNP plasma values in CHF patients [5–7], the small numbers of patients evaluated in the previous studies and, mainly, differences in the assay methodology could account for this discrepancy (sensitivity level and accuracy in the evaluation of extraction yield).

In conclusion, CNP plasma levels are higher in patients with heart failure than in healthy subjects, and possibly contribute with other natriuretic peptides (ANP and BNP) to the neuro-hormonal activation, which is an hallmark of CHF, and counteracting the predominance of vasoconstrictor and sodium-retentive systems (sympathetic, renin–angiotensin–aldosterone, vasopressin, and endothelin systems). As for cardiac natriuretic hormone assay [3], CNP plasma level assessment might provide a complementary index of disease severity. Further studies are needed to clarify the pathophysiological properties of this peptide, to evaluate its relationship with neuro-hormonal activation, and its possible clinical importance.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Sudoh T., Minamino N., Kangawa K., Matsuo H. C-type natriuretic peptide (CNP): a new member of the natriuretic peptide family identified in porcine brain. Biochem. Biophys. Res. Commun. (1990) 168:863–870.[CrossRef][Web of Science][Medline]
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  4. Kalra P.R., Anker S.D., Struthers A.D., Coats A.J.S. The role of C-type natriuretic peptide in cardiovascular medicine. Eur. Heart J. (2001) 22:997–1007.[Free Full Text]
  5. Wei C.M., Heublein D.M., Perella M.A., Lerman A., Rodeheffer R.J., McGregor C.G., et al. Natriuretic peptide system in human heart failure. Circulation (1993) 88:1004–1009.[Abstract/Free Full Text]
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  12. Pilo A., Zucchelli G.C., Malvano R., Masini S. Main features of computer algorithms for RIA data reduction. Comparison of some different approaches for the interpolation of dose–response curve. J. Nucl. Med. Allied Sci. (1982) 26:235–248.[Web of Science][Medline]
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