JP5362487B2 - New use of proBNP-108 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new marker of a heart disease. <P>SOLUTION: A method for determining or detecting a load to a heart ventricle or a heart atrium, or a method for determining or detecting a progression of a level of a heat failure or a heart failure treatment includes the process for measuring (A) a pro-BNP108 and/or (B) a BNP-32 within a sample derived from a subject and overcomes the problem. The invention discriminates the heart failure, an atrial fibrillation, a mitral valve insufficiency; and a subvalvular aortic stenosis. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

  The present invention relates to cardiac medicine.

  Brain natriuretic peptide (BNP) is a cardiac hormone and a member of the natriuretic family (Non-patent Document 1). BNP is remarkably similar to atrial natriuretic peptide (ANP) with respect to amino acid sequence and pharmacological properties (Non-Patent Document 2). ANP is mainly produced and secreted in the atrium, while BNP is mainly produced and secreted in the ventricle (Non-patent Document 3). BNP gene expression in the ventricle is stimulated by ventricular wall stress and ischemia, and proBNP108 [1-108] (proBNP-108) is produced in the heart (Non-patent Documents 4 and 5). When proBNP-108 is secreted from ventricular myocardial cells (Non-patent Documents 4 and 5), proBNP-108 is an equimolar proBNP [77-108] by a prohormone converting enzyme Furin. ] (BNP-32) and N-terminal proBNP [1-76] (NT-proBNP). In particular, since both BNP-32 and NT-proBNP are useful as diagnostic markers for heart disease, antibodies and methods for measuring them have been developed (Patent Documents 1, 2, and 3).

  However, recent studies have found that not only BNP-32 and NT-proBNP but also proBNP-108 circulates in human plasma, and proBNP-108 concentration increases with heart failure (Non-Patent Document 6). Non-patent document 7, Non-patent document 8). In other studies, it was found that the current BNP-32 measurement kit shows high cross-reactivity between true BNP-32 and proBNP-108 (Non-patent document 8, Non-patent document 9).

  Also, the reason why proBNP-108 is secreted without specific conversion to BNP-32 and NT-proBNP remains unclear. Less has been studied about proBNP-108 in the plasma of heart failure patients. Furthermore, very recently, plasma proBNP-108 was reported to undergo O-glycosylation of the N-terminal peptide (Non-Patent Document 10, Non-Patent Document 11).

Japanese Patent No. 2665850 International Publication No. 02/083913 Pamphlet International Publication No. 99/013331 Pamphlet

Minamino N, Horio H, Nishikimi T. Chapter 165. Natriuretic peptides in the cardiovascular system, THE HANDBOOK OF BIOLOGICALLY ACTIVE PEPTIDES: Edited by Abba J. Kastin, Academic Press, pp, 1217-1225, 2006 Sudoh T, Kangawa K, Minamino N, Matsuo H. A new natriuretic peptide in porcine brain. Nature. 1988 Mar 3; 332: 78-81 Nishikimi T, Maeda N, Matsuoka H. The role of natriuretic peptides in cardioprotection. Cardiovasc Res. 2006; 69: 318-28 Daniels LB. Maisel AS. Natriuretic peptides. J Am Coil Cardiol. 2007; 50: 2357-68 Weber M, Hamm C. Role of B-type natriuretic peptide (BNP) and NT-proBNP in clinical routine. Heart. 2006; 92: 843-9 Lam CS, Burnett JC Jr, Costello-Boerrigter L, Rodeheffer RJ, Redfield MM. Alternate repeating pro-B-type natriuretic peptide and B-type natriuretic peptide forms in the general population. J Am Coil Cardiol. 2007; 49: 1193-202 Waldo SW, Beede J, Isakson S, Villard-Saussine S, Fareh J, Clopton P, Fitzgerald RL, Maisel AS. Pro-B-type natriuretic peptide levels in acute decompensated heart failure. J Am Coil Cardiol. 2008; 51: 1874-82 Seferian KR, Tamm NN, Semenov AG, Mukbaiyamova KS, Tolstaya AA, Koshkina EV, Kara AN, Krasnoselsky MI, Apple FS, Esakova TV, Filatov VL, Katrukha AG. The brain natriuretic peptide (BNP) precursor is the major immunoreactive form of BNP in patients with heart failure. Clin Chem. 2007; 53: 866-73 Shimizu H, Masuta K, Asada H, Sugita K, Sairenji T. Characterization of molecular forms of probrain natriuretic peptide in human plasma. Clin Chem Acta. 2003; 334: 233-9 Liang F, O'Rear J, Schellenberger U, Tai L, Lasecki M, Schreiner GF, Apple FS, Maisel AS, Pollitt NS, Protter AA. Evidence for functional heterogeneity of circulating B-type natriuretic peptide. J Am Coil Cardiol. 2007; 49: 1071-8 Schellenberger U, O'Rear J, Guzzetta A, Jue RA, Protter AA, Pollitt NS. The precursor to B-type natriuretic peptide is an O-linked glycoprotein. Arch Biochem Biophys. 2006; 451: 160-6

  An object of the present invention is to provide a new marker for heart disease.

  As a result of intensive research, proBNP-108 unexpectedly determined or detected the load on either the ventricle or the atrium (for example, aortic regurgitation, aortic stenosis / occlusive disease, It was solved by finding it effective for mitral regurgitation and atrial fibrillation.

  More specifically, the inventors considered that it is important to examine the ratio of proBNP-108 to total BNP in plasma and its relationship to the pathophysiological state of patients with heart failure, ProBNP-108 unexpectedly determines or detects the load on either the ventricle or the atrium (eg, aortic regurgitation, aortic stenosis / defatability, mitral regurgitation, atrial fibrillation, etc. ) Was found effective. That is, the present inventors have found and confirmed that proBNP-108 and BNP-32, which are high and low molecular weight forms of BNP, are present in the plasma of control subjects, patients with atrial fibrillation and patients with heart failure. .

Accordingly, the present invention provides the following.
(1) including a step of measuring pro-BNP108 in a sample derived from a subject,
A method of determining or detecting a load on either the ventricle or the atrium.
(2) measuring A) pro B NP - 108 and B) BNP-32 in a sample derived from a subject,
A method of determining or detecting a load on either the ventricle or the atrium.
Comprising measuring the 108 / total BNP, - (3) pro B NP in a sample from a subject
A method of determining or detecting a load on either the ventricle or the atrium.
(4) The determination or detection is directed to a symptom selected from the group consisting of grasping of aortic stenosis, aortic regurgitation, mitral regurgitation and atrial fibrillation pathology (progression). The method according to any one of items (1) to (3).
(5) including a step of measuring pro B NP - 108 in a sample derived from a subject,
A method of determining or detecting the progression of heart failure or the level of heart failure treatment.
(6) The determination or detection includes estimation of the stage and prognosis of heart failure patients, determination of therapeutic effects of β-blockers and ACE inhibitors in patients with heart failure, understanding of pathological conditions of dilated cardiomyopathy and determination of therapeutic effects, myocardial infarction And remodeling after infarction, classification of hypertrophic cardiomyopathy, index indicating decreased right ventricular function in right ventricular overload disease, and increase in right ventricular overload disease is a group consisting of indicators indicating capacity load 6. The method according to any one of items (1) to (5), which is directed to a selected symptom.
(7) The subject is a patient suspected of at least one disease selected from the group consisting of heart failure, atrial fibrillation, mitral regurgitation, aortic regurgitation and aortic stenosis ( The method according to any one of 1) to 6).
(8) as a marker for determining or detecting the load to either the ventricle or atrium, pro B NP - use of 108.
(9) as a marker for determining or detecting the load to either the ventricle or atrium, pro B NP - use of a combination of 108 and BNP-32.
(10) The determination or detection is directed to a symptom selected from the group consisting of grasping of aortic stenosis, aortic regurgitation, mitral regurgitation and atrial fibrillation pathology (progression). The use according to item (8) or (9).
Using 108 - (11) pro B NP as a marker for determining or detecting the level of progression or treatment of heart failure heart failure.
(12) The determination or detection includes estimation of stage and prognosis of heart failure patient, determination of therapeutic effect of β-blocker / ACE inhibitor of heart failure patient, understanding of pathological condition of dilated cardiomyopathy and determination of therapeutic effect, myocardial infarction And remodeling after infarction, classification of hypertrophic cardiomyopathy, index indicating decreased right ventricular function in right ventricular overload disease, and increase in right ventricular overload disease is a group consisting of indicators indicating capacity load The use according to any one of items (8) to (11), which is directed to the symptom selected.
(13) A diagnostic kit for determining or detecting a load on either the ventricle or the atrium, which contains a substance that specifically binds to pro B NP - 108.
(14) pro B NP - 108 in comprising a substance specifically binding to the BNP-32 substance that specifically binds to, diagnostic kit for determining or detecting the load to either the ventricle or atrium.
(15) A diagnostic kit for determining or detecting the progression of heart failure or the level of treatment for heart failure, comprising a substance that specifically binds to pro B NP - 108.
(16) The diagnostic kit according to any one of items (13) to (15), wherein the substance is an antibody.

  The present invention is intended to estimate the stage and prognosis of heart failure patients, determine the therapeutic effects of β-blockers and ACE inhibitors in heart failure patients, grasp the pathophysiology of dilated cardiomyopathy, determine the therapeutic effects, Understanding of modeling, classification of hypertrophic cardiomyopathy, increase in the ratio of proBNP-108 in right ventricular overload disease is an indicator of decreased right ventricular function, increase in the ratio of proBNP-108 in right ventricular overload disease There exists an effect that it can be used as a parameter | index to show.

FIG. 1 shows BNP molecular types in gel filtration HPLC extracts of venous plasma from normal subjects (A), atrial fibrillation patients (B), and heart failure patients (C). IR-proBNP-108 (high molecular weight immunoreactive (IR-) BNP including both proBNP-108 and glycosylated proBNP-108) and IR-BNP-32 (mainly corresponding to proBNP-108 and BNP-32, respectively) Two peaks of low molecular weight IR-BNP consisting of BNP-32) were observed. The arrows indicate 1: void volume, 2: glycosylated proBNP-108, 3: proBNP-108, 4: BNP-32. Sodium chloride (NaCl) was extracted in fraction 40 (not shown in this figure). FIG. 2 shows the proBNP-108 / total BNP ratio in normal subjects, atrial fibrillation patients (Af), and heart failure patients (HF). Data are shown as mean ± SD. FIG. 3 shows the correlation between plasma IR-proBNP-108 and IR-BNP-32. The unit is the horizontal axis and the vertical axis (pg / mL). FIG. 4 shows BNP molecular types in gel filtration HPLC extracts of venous plasma from patients with ventricular overload heart failure (A) and atrial overload heart failure (B). The arrows indicate 1: void volume, 2: glycosylated proBNP-108, 3: proBNP-108, 4: BNP-32. FIG. 5 shows the proBNP-108 / total BNP ratio in patients with atrial load heart failure and ventricular load heart failure. Data are shown as mean ± SD. FIG. 6 shows the BNP molecular types in gel filtration HPLC extracts of atrial tissue (A) and ventricular tissue (B) from heart failure patients undergoing cardiac surgery. The arrows indicate 1: void volume, 2: glycosylated proBNP-108, 3: proBNP-108, 4: BNP-32. FIG. 7 shows the proBNP-108 / total BNP ratio in atrial and ventricular tissue. Data are shown as mean ± SD. FIG. 8 shows BNP molecular types in gel filtration HPLC extracts of pericardial fluid and plasma obtained from patients with atrial stress heart failure (A, B) and ventricular stress heart failure patients (C, D) undergoing cardiac surgery. A and C show plasma extract data, and B and D show pericardial fluid data. The arrows indicate 1: void volume, 2: glycosylated proBNP-108, 3: proBNP-108, 4: BNP-32. FIG. 9 shows the proBNP-108 / total BNP ratio in the pericardial fluid and plasma of 8 patients. Data are shown as mean ± SD. FIG. 10 shows the relationship between total IR-BNP and proBNP-108 / total BNP ratio in heart failure patients when the heart failure status varies. A. Negative correlation between plasma proBNP-108 / total BNP ratio and total BNP concentration before treatment (black circle) and after treatment (open circle) of the same patient. B. Positive correlation between plasma proBNP-108 / total BNP ratio and total BNP concentration before worsening (black circle) and after worsening (white circle) in the same patient.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in the case of English) also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the above field unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition of terms)
Hereinafter, typical terms used in the present invention will be described together, and terms used for individual embodiments will also be described appropriately in individual sections.

  As used herein, the term “brain natriuretic peptide (BNP)” is a hormone that is a member of the natriuretic family secreted from the heart and is synthesized primarily in the ventricle. In humans, it is typically known to be produced as a prepro peptide having SEQ ID NO: 1, and is used herein as a term including all isoforms, splice variants, and the like. . The base sequences of the genes encoding the proteins are registered with GenBank as AAH25785 and NP002512, respectively. As used herein, the term immunoreactive (IR) may be used with reference, for example, the term “IR-BNP” refers to immunoreactive BNP. As used herein, “immunoreactivity” or “IR” means being reactive with an antibody for detection.

  As used herein, the term “proBNP-108” is a precursor of BNP, typically consisting of 108 amino acids (SEQ ID NO: 2), and ventricular by ventricular wall stress and ischemia. BNP gene expression is stimulated and produced in the heart. Also included is glycosylated proBNP-108. When proBNP-108 is secreted from ventricular cardiomyocytes, proBNP-108 is converted into equimolar amounts of proBNP [77-108] (BNP-32) and N-terminal proBNP [1-76] (NT-proBNP-76) by protease. It is also known that proBNP-108 circulates in human plasma (Non-Patent Documents 4 and 5), and that proBNP-108 concentration also increases in heart failure (Non-patent Document 4). References 6, 7 and 8).

  As used herein, the term “BNP-32” refers to a peptide having the sequence of amino acids 77-108 of proBNP-108 among BNP (SEQ ID NO: 3), which circulates in human plasma. It is known (Non-Patent Documents 6, 7, and 8).

As used herein, the term "total BNP" refers to the entire BNP-32 and its precursors and cleavage product, BNP-32 and pro B NP - 108, such as individually measuring each molecular form In addition to adding them together, it can be measured, for example, by using an antibody that can recognize these common regions (for example, the region 77-108). Such antibodies are commercially available and can be easily obtained (for example, Shionoria BNP kit, manufactured by Shionogi & Co.).

In the present specification, “pro B NP - 108 / total BNP” refers to the ratio of the pro B NP - 108 amount to the total BNP amount.

As used herein, “subject-derived sample” refers to a part of a body part from a subject that is expected to contain pro B NP - 108, BNP-32, etc., for example, pericardial fluid, blood, or Although what processed blood (for example, plasma, serum, etc.) can be mentioned, it is not limited to this. In order to make a more accurate diagnosis, various samples such as tissues, cells, body fluids (for example, brain fluid, lymph fluid) obtained from a living body of a subject can be used.

  As used herein, “subject” or “subject” refers to an organism to which the diagnosis of the present invention is applied, and is also referred to as a “patient”. The patient or subject may preferably be a human.

In the present specification, the “ peripheral blood-derived sample” refers to a sample derived from the peripheral blood of a subject and its components, and examples thereof include plasma and platelet fraction in peripheral blood and peripheral blood. The peripheral blood-derived sample can be collected from the subject according to, for example, DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, the 4th Ed.).

  As used herein, the term “pericardial fluid”, also referred to as “pericardial fluid”, refers to the fluid that accumulates between the pericardium, a bag that surrounds the heart, and the heart.

  As used herein, “load” on either the ventricle or the atria refers to any load on the subject (here, the ventricle or the atrium), typically pressure and volume (blood Increase). Representative examples of the cause of such a load include not only the disorder, injury, and disease of the subject, but also an increase in blood pressure throughout the body (an increase in peripheral resistance).

The term "heart failure" as used herein, is used in the broadest sense used in the art, refers to a syndrome that causes the failure of the heart function to push a sufficient amount of blood flow, decreased venous pressure associated therewith heart stroke volume And the resulting various clinical symptoms are included. For example, the causes of heart failure targeted by the present invention include valvular heart disease, ischemic heart disease, congenital heart disease, dilated cardiomyopathy, hypertrophic cardiomyopathy, atrial septal defect, ventricular septal defect And symptomatic heart disease. More specific valvular heart diseases that are the subject of the present invention include aortic regurgitation, aortic stenosis, mitral regurgitation, and mitral stenosis. Heart failure, unlike atrial fibrillation, reduced cardiac stroke volume is the cause. That is, for example, isolated atrial fibrillation is exemplified in atrial fibrillation, but blood circulation is impossible because the heart is partially and frequently contracted frequently without sufficient coordination. It is different from the stroke volume.

  As used herein, a “marker” refers to a characteristic biological factor that indicates the genetic or phenotypic nature of an organism, typically a nucleic acid, or RNA or protein that is a gene. It can be a gene product. Such markers can be measured by any suitable biological assay known in the art. The presence of the marker can be used as an indicator of a certain disease state or condition.

  As used herein, “immunoassay” refers to any assay using an immunological technique such as an antigen-antibody reaction, such as dot blot, Western blot, enzyme immunoassay ( EIA), in particular solid-phase enzyme immunoassay (ELISA), radioimmunoassay (RIA), electrochemiluminescence immunoassay (ECLIA), chemiluminescence immunoassay (CLIA), chemiluminescence enzyme immunoassay (CLEIA), Examples include competitive protein binding analysis.

In this specification, “measurement” such as pro B NP - 108 can be performed by any method known in the art. That is, in this specification, it can be performed by a known protein assay method, for example, Western blotting using a specific polyclonal antibody or monoclonal antibody against a target protein (for example, pro B NP - 108 etc.), The measurement can be performed using an immunological technique such as EIA method, RIA method, FIA, chemiluminescence immunoassay, or ECLIA method. For assays using antibodies, reference can be made to Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; JP-A-10-160735.

In this specification, pro B NP - 108 and BNP-32 are identified using, for example, an elution position in gel filtration high performance liquid chromatography (HPLC) (for example, TSK gel G2000SWXL column (TOSOH), etc.) as an index. Can be implemented. Using such a gel filtration technique, pro B NP - 108 and BNP-32 can be distinguished. Such identification is subjected the sample to gel filtration, pro B NP - by comparison with the elution position of the standard, such as 108, it can be achieved. Glycosylated pro B NP by setting condition strictly - 108 and non-glycosylated (e.g., recombinant) pro B NP of - can be carried out identification of such 108. However, for the purposes of the present invention, glycosylated pro B NP - 108 and non-glycosylated (e.g., recombinant) pro B NP of - to distinguish 108 is not necessarily required.

In the diagnostic method of the present invention, preferably, when pro B NP - 108 is measured in the above assay, it recognizes and binds to a specific amino acid sequence of pro B NP - 108, but does not bind to BNP-32. Antibodies (eg, available from BIO-RAD, Phoenix, Hytest, etc.) are used. As long as the antibody used by this invention is an antibody which can recognize the said protein, any of a polyclonal antibody and a monoclonal antibody may be sufficient. The antibody of the present invention can be produced according to a known antibody or antiserum production method using a target protein as an antigen.

  In order to produce a monoclonal antibody-producing cell, an antibody can be produced by administering a protein or the like as a subject of the present invention to a site capable of producing an antibody by administration to an animal itself or with a carrier and a diluent. . In order to enhance the antibody producing ability upon administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be administered. The administration is usually performed once every 2 to 6 weeks, 2 to 10 times in total. Examples of animals to be used include mammals such as monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, etc., and mice and rats are preferably used. The antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with antiserum and then measuring the activity of the labeling agent bound to the antibody.

  An individual having an antibody titer is selected from an animal immunized with an antigen, such as a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization, and the antibody-producing cells contained therein are myeloma cells (for example, NS-1, P3U1, SP2 / 0, etc.) can be fused to prepare a monoclonal antibody-producing hybridoma. The fusion operation can be performed according to known methods, for example, the method of Koehler and Milstein (Nature 256: 495 (1975)). Examples of the fusion promoter include polyethylene glycol (PEG), Sendai virus and the like, and preferably PEG is used.

Polyclonal antibodies can be produced according to known methods or similar methods. For example, a complex of an immunizing antigen ( proBNP-108 antigen) and a carrier is prepared, a mammal is immunized in the same manner as the above-described monoclonal antibody production method, and an antibody-containing product against proBNP-108 is prepared from the immunized animal. It can be produced by collecting and purifying the antibody.

(Judgment, detection and diagnostic methods)
In one aspect, the present invention provides a method for determining or detecting a load on either the ventricle or the atrium. This method comprises measuring A) pro B NP - 108 (SEQ ID NO: 2) and / or B) BNP-32 (SEQ ID NO: 3) in a sample from a subject. By determining the load on either the ventricle or the atrium, the state of the heart can be determined, and symptoms such as heart failure can be determined more precisely. As these applications, for example, it is possible to grasp the pathology (progression) of aortic stenosis, aortic regurgitation, mitral regurgitation, and atrial fibrillation.

In one embodiment, these measurements are performed in a pro B NP - 108 / 108 sample from a subject.
The total BNP may be measured. The ratio of pro B NP - 108 / total BNP was determined by measuring the molecular forms of BNP in vivo (for example, pre-pro form, pro-form pro B NP - 108, mature form BNP-32). Thus, pro B NP - 108 can be divided by this sum, or a measurement using a monoclonal antibody that can recognize these common regions (for example, region 77-108) can be substituted for total BNP. These can be used for measurement.

In one embodiment, the method of the present invention, pro B NP in a sample from a subject - comprises measuring 108. In the present invention, pro B NP in a sample from a subject - from the 108 value of itself, revealed that reflects the status of load on the ventricle and atrium, the use of this number alone Can do.

In one aspect, the present invention provides a method for determining or detecting the progression of heart failure or the level of heart failure treatment. This method, pro B NP in a sample from a subject - comprises measuring 108. Alternatively, pro B NP - may measure the ratio of 108 / total BNP.

  The present invention can be used to distinguish between atrial fibrillation and heart failure, as well as hypertrophic cardiomyopathy, aortic stenosis, aortic regurgitation, mitral stenosis, mitral regurgitation, etc. Can be identified.

As used herein, “progression of heart failure” refers to the state, stage, or transition of the stage of heart failure. The progression of heart failure can be classified according to the following criteria: symptoms such as fatigue and shortness of breath (NYHA heart function classification), heart expansion on chest X-ray and pulmonary congestion, left ventricle on echocardiography Expansion of the cavity, cardiac function, inferior vena cava diameter, Doppler index, etc. The progression of heart failure and pro B NP - 108 are found to have the following correlation. If heart failure progresses, pro B NP - 108 increases, and at the same time the ratio of pro B NP - 108 / total BNP Also rises.

  Symptoms such as fatigue and shortness of breath (NYHA heart function classification), heart expansion on chest X-ray and lung congestion, left ventricular enlargement by echocardiography, cardiac function, inferior vena cava diameter, Doppler The proBNP-108 increases when the index or the like deteriorates, and the ratio of proBNP-108 / total BNP also increases. Therefore, if the pro-BNP level is known, the progression of heart failure can be determined in the following manner. Increased proBNP-108 level triggered exacerbation of symptoms, heart enlargement on chest X-ray and worsening of pulmonary congestion, enlargement of left ventricular cavity by echocardiography, cardiac function, inferior vena cava diameter Since it becomes possible to estimate the deterioration of the Doppler index or the like, the diagnostic ability is improved as compared with the diagnosis performed only with the current measured value of BNP.

  As used herein, “level of heart failure treatment” refers to the level of how much heart failure treatment has improved heart failure and is used in the evaluation of the treatment used or in determining subsequent treatment. be able to. The level of heart failure therapy can be determined by the progression or level of heart failure, for example, stopping or improving progression. Further, it is determined based on symptoms (NYHA heart function classification), heart expansion on chest X-ray and degree of lung congestion, enlargement of left ventricular cavity by echocardiography, cardiac function, inferior vena cava diameter, Doppler index, and the like. Since proBNP-108 levels are biochemical markers that change prior to the level of these heart failure treatments, it is conceivable that diagnostic performance will be improved over current measurements of BNP alone. Utilizing the present invention, estimation of stage and prognosis of heart failure patients, determination of therapeutic effects of β-blockers and ACE inhibitors of heart failure patients, understanding of pathological conditions of dilated cardiomyopathy and determination of therapeutic effects, myocardial infarction and infarction An increase in proBNP-108 in later remodeling, hypertrophic cardiomyopathy type classification, and right ventricular overload disease can be used as an index indicating a decrease in right ventricular function. As a right ventricular load disease, an increase in the ratio of proBNP-108 and proBNP-108 / total BNP can be used as an index indicating pressure load.

  Examples of treatments that can be determined in the present specification include treatment by surgery in addition to treatment by drugs. Examples of drug treatment include ACE treatment. Examples of treatment by surgery include aortic valve replacement (AVR), mitral valve replacement (MVR), two-valve replacement (DVR), coronary artery bypass grafting (CABG), maze, Dor.

  As used herein, “atrial load” means that a load is applied to the atrium.

  As used herein, “ventricular load” means that a load is applied to the ventricle.

  It is known that heart failure develops under stress in one or both of the atria and ventricles, and it is known that the policy of subsequent treatment should be changed depending on the type.

  As used herein, “atrial load heart failure” refers to a type of heart failure in which the atrium is mainly loaded. Examples of atrial stress heart failure include mitral valve stenosis, mitral regurgitation, and atrial septal defect. In the case of atrial load heart failure, it is preferable to administer a drug that reduces atrial load, and examples of such a drug include diuretics, vasodilators, hANP (carperitide), and the like.

  As used herein, “ventricular load heart failure” refers to a type of heart failure in which the ventricle is mainly loaded. Examples of ventricular load heart failure include aortic regurgitation and aortic stenosis. In the case of ventricular overload heart failure, it is preferable to administer a drug that mainly acts on the ventricle or to perform a treatment, such as an ACE inhibitor, an angiotensin receptor antagonist, a beta blocker, an aldosterone antagonist Drugs, etc., and can also be used to determine the time of treatment for aortic valve replacement.

  The ratio of proBNP-108 / total BNP is generally smaller for an atrial load than for a ventricular load.

  For example, the ratio of proBNP-108 / total BNP is in the range of about 0.3 to 0.9 for ventricular load, as determined from fractions by gel filtration HPLC using plasma from heart failure patients, preferably about It is between 0.4 and 0.8. In the case of an atrial load, it can be between about 0.1 and 0.5, preferably between about 0.2 and 0.4. These values may tend to disperse with specific symptom changes. For example, in a patient with mitral stenosis, a value such as about 0.15 ± 0.03 is taken.

  Moreover, when measured using pericardial fluid, the value tends to be larger than that of plasma. For example, it takes a value of 0.7 to 0.9.

  The determination can also be performed using the amount or concentration of proBNP-108 as an index. When measured by concentration, in the case of ventricular overload heart failure, the proBNP-108 level can be 0 to 2000 pg / mL, preferably 50 to 100 pg / mL. In the case of atrial stress heart failure, it can be 0-400 pg / mL.

  The present invention can also be used as an index for treatment of heart failure. For example, in decompensated heart failure, both BNP-32 and proBNP-108 were found to increase. It was also found that these values and proBNP-108 / total BNP increase as heart failure worsens. On the other hand, it was also found that patients who improved with treatment had decreased values of both BNP-32 and proBNP-108 and also decreased proBNP-108 / total BNP. These values were similarly reduced in patients with spontaneous healing. Thus, both BNP-32 and proBNP-108, and proBNP-108 / total BNP can be utilized to determine the level of heart failure, level of treatment or level of cure.

  In one embodiment, in the present invention, as a sample, pericardial fluid, blood, or a processed product thereof (for example, plasma) can be used, but is not limited thereto. It will be understood that the criteria will vary depending on the sample selected. Such criteria can be appropriately selected and determined by those skilled in the art based on the contents described in this specification.

  Such a sample may be from a patient suspected of at least one disease selected from the group consisting of heart failure, atrial fibrillation, mitral regurgitation and aortic stenosis.

  In all embodiments of the method according to the invention, other markers used in the art can also be used. The combination of the marker of the present invention and other markers is advantageous in that the types of diseases to be identified in one diagnostic step are wide.

In another aspect, the present invention, pro B NP in a sample from a subject - comprising measuring the 108, to provide a method for diagnosing heart failure. As described above, such a diagnosis can diagnose a disease state in consideration of medical knowledge by determining the load on the atrium or the ventricle. For such medical knowledge, reference can be made to: Braunwald's Heart Disease, A Textbook of Cardiovascular Medicine, Saunders; 7 edition 2004.

  As used herein, “diagnosis” refers to identifying various parameters related to a disease, disorder, or condition in a subject and determining the current state or future of such a disease, disorder, or condition. By using the method, apparatus, and system of the present invention, the state in the body can be examined, and such information is used to formulate a disease, disorder, condition, treatment to be administered or prevention in a subject. Alternatively, various parameters such as methods can be selected. In this specification, “diagnosis” in a narrow sense refers to diagnosis of the current state, but includes “pre-diagnosis” in a broad sense.

  The diagnostic method of the present invention is industrially useful because, in principle, the diagnostic method can be used from the body and can be carried out away from the hands of medical personnel such as doctors. In this specification, in order to make it clear that it can be performed away from the hands of medical personnel such as doctors, it is sometimes referred to as “assisting diagnosis”.

  As used herein, “treatment” refers to preventing a disease or disorder from deteriorating, preferably maintaining the status quo, more preferably reducing, when a certain disease or disorder becomes such a condition. Preferably, it means elimination.

  In the diagnostic method of the present invention, the concentration of at least one of the target proteins of the present invention is measured as a marker. And the target disease etc. are diagnosed by comparing the measured value with a healthy value. Here, “diagnosis” not only determines whether or not the target disease or the like is affected, but also determines whether or not the target disease or the like may be affected for the purpose of preventing the target disease or the like. And monitoring the improvement status and recurrence of the target disease.

  In a preferred embodiment of the disease diagnosis method of the present invention, a carrier on which a substance having affinity for a marker is immobilized is used. Then, a body fluid or a body fluid component is brought into contact with the carrier, and a marker contained in the body fluid or body fluid component is captured on the carrier via a substance having affinity for the marker, and the body fluid is based on the amount of the captured marker. Calculate the concentration of the marker inside. According to the disease diagnosis method of the present invention, since the marker captured on the carrier is the measurement target, the influence of contaminants contained in the measurement sample can be reduced, and the marker is more sensitive and accurate. Concentration can be measured. Examples of the body fluid component include serum or plasma when the body fluid is blood.

  In a preferred embodiment of the disease diagnosis method of the present invention, a carrier having a planar portion is used, and a substance having affinity for the marker is immobilized on a part of the planar portion. With this configuration, a substance having affinity for the marker can be spot-fixed at a plurality of locations on the carrier. As a result, it is possible to simultaneously process a plurality of measurement samples with one carrier, and simultaneously measure the concentrations of a plurality of markers with one carrier, and work efficiency is improved. Furthermore, by reducing the area of each spot, the marker concentration can be measured even from a very small amount of measurement sample. An example of the carrier having a planar portion is a substrate such as a chip.

  In a preferred embodiment of the disease diagnosis method of the present invention, an ion exchanger, a metal chelate or an antibody is used as a substance having affinity for a marker, and the sample in the measurement sample is passed through the ion exchanger, the metal chelate or the antibody. The marker is captured on the carrier. When the substance is an ion exchanger or a metal chelate, various substances are easily available, and a carrier for capturing a marker can be easily prepared. Further, when the substance is an antibody, the marker can be captured more specifically. Examples of the method for measuring the amount of the captured marker include mass spectrometry and immunoassay (in the case of an antibody).

  Moreover, the differential diagnosis can be performed by the method according to the present invention. The purpose of differential diagnosis is to distinguish and identify one or more specific diseases within a group of diseases that show similar or possibly identical symptoms. Surprisingly, the method according to the present invention makes it possible to distinguish or identify aortic stenosis and insufficiency, mitral insufficiency, atrial fibrillation, etc., which often show very similar symptoms.

(Use as a marker)
In one aspect, the present invention is, as a marker for detecting the load to either the ventricle or atrium, pro B NP - provides for the use of 108. Conventionally, BNP-32 has been known to be used as a marker for heart failure or the like, but pro B NP - 108 itself has not been measured and can be used as such a marker for the first time in the present invention. It has been shown. More detailed embodiments of the use as a marker can be carried out in consideration of the matters described in the section of (Method of determination, detection and diagnosis). For example, measurement of total BNP can be actually performed using an antibody that recognizes BNP-32. Since the BNP-32 region is shared by all molecules including the pro-form, it can be measured by using an antibody that can recognize this portion.

In another aspect, the present invention is, as a marker for detecting the load to either the ventricle or atrium, pro B NP - provides the use of a combination of 108 and BNP-32. By using such a combination, it is possible to detect the load on either the ventricle or the atrium, grasp the pathology (progression) of aortic stenosis / insufficiency, the condition of mitral regurgitation ( Progression) and the pathology (progression) of atrial fibrillation can be understood. Further, a more detailed embodiment of the use as a marker can be carried out in consideration of the matters described in the section of (Method of determination, detection and diagnosis).

In another aspect, the present invention, pro B NP as a marker for detecting levels of progression or treatment of heart failure heart failure - provides for the use of 108. In this case, similarly, the purpose of pro B NP - 108 is to estimate the stage and prognosis of patients with heart failure, to determine the therapeutic effects of β-blockers and ACE inhibitors in patients with heart failure, and to understand the pathology of dilated cardiomyopathy determination and the therapeutic effect, grasp of myocardial infarction and remodeling after infarction, it is possible to perform the typing of hypertrophic cardiomyopathy, pro B NP in right ventricular load diseases - increased 108 shows the reduction in right ventricular function An increase in pro B NP - 108 in the index, right ventricular load disease, can be used as an index indicating the capacity load. Further, a more detailed embodiment of the use as a marker can be carried out in consideration of the matters described in the section of (Method of determination, detection and diagnosis).

(Diagnostics, treatment kit, treatment system)
In one aspect, the present invention, pro B NP - containing a substance that specifically binds to the 108, to provide a diagnostic kit for detecting the load to either the ventricle or atrium.

In another aspect, the present invention relates to a diagnostic for detecting a load on either the ventricle or the atrium comprising a substance that specifically binds to pro B NP - 108 and a substance that specifically binds to BNP-32. Provide kit. More detailed embodiments of the diagnostic kit can be carried out in consideration of the matters described in the section of (determination, detection and diagnosis method).

In another aspect, the present invention, pro B NP - containing a substance that specifically binds to the 108, to provide a diagnostic kit for detecting the level of progression or treatment of heart failure heart failure. More detailed embodiments of this diagnostic kit can be carried out in consideration of the matters described in the section of (determination, detection and diagnosis method).

As used herein, "pro B NP - a substance that specifically binds to 108" is, pro B NP - as long as it can measure 108 may be any material. For example, pro B NP - can be exemplified such as an antibody that specifically binds to 108 are not limited thereto. More detailed embodiments of antibodies that specifically bind can be carried out in consideration of the matters described in the section of (Determination, detection and diagnostic methods). As long as it is a specific substance, it may be any substance or other element as long as the intended purpose can be achieved. Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.)) , These complex molecules are included, but not limited thereto.

  In the present specification, the “substance that specifically binds to BNP-32” may be any substance that can measure BNP-32. Examples thereof include, but are not limited to, an antibody that specifically binds to BNP-32. A more detailed embodiment of the antibody that specifically binds can be carried out in consideration of the matters described in the section of (Method of determination, detection and diagnosis).

  As used herein, “specific” typically means that the affinity for a particular biological agent is greater than the affinity for other unrelated (especially less than 30% identity) polypeptides. Equivalent or higher or preferably significantly (eg, statistically significantly) higher. Such affinity can be measured, for example, by a binding assay.

  The term “binding” as used herein means a physical or chemical interaction between two proteins or compounds or related proteins or compounds, or a combination thereof. Bonds include ionic bonds, non-ionic bonds, hydrogen bonds, van der Waals bonds, hydrophobic interactions, and the like. A physical interaction (binding) can be direct or indirect, where indirect is through or due to the effect of another protein or compound. Direct binding refers to an interaction that does not occur through or due to the effects of another protein or compound and does not involve other substantial chemical intermediates.

In another aspect, the present invention comprises means for distinguishing pro B NP - 108 from BNP-32, for detecting a load on either the ventricle or the atrium, or for the progression of heart failure or level of heart failure treatment Provide a diagnostic system for detection of A more detailed embodiment of this diagnostic system can be carried out in consideration of the matters described in the section (determination, detection and diagnosis method).

In one embodiment, “means for identifying pro B NP - 108 from BNP-32” is any means for identifying pro B NP - 108 from BNP-32. May be. Examples of such means include antibodies or combinations thereof, or gel filtration HPLC. As for gel filtration, a more detailed embodiment can be carried out in consideration of the matters described in the section of (Method of determination, detection and diagnosis).

  In another embodiment, the diagnostic kit can optionally include SDS-polyacrylamide gel electrophoresis reagents, Western blotting PVDF membranes, proteins, and the like. When the diagnostic kit is used for a technique such as EIA, the antibody can be bound to a solid phase carrier in advance. Generally, a reaction vessel, beads (preferably magnetic beads) and the like are used for the solid phase.

  In this specification, “system” refers to an arbitrary system for diagnosis, and generally includes one or a plurality of components, and when there are a plurality of components, these components act and relate to each other. It means a system that satisfies the three conditions of exhibiting harmonious behavior and function as a whole. The system can be in any form, such as a device, composition, diagnostic agent. Therefore, the system is, for example, a large-scale system including a measuring device, a system including a chromatography, a kit using an immune reaction, a composition including an antibody (that is, a diagnosis that is an in vitro medicine including a monoclonal antibody as a marker substance) (Medicine) and the like.

In this specification, the “label” refers to a presence (for example, a substance, energy, electromagnetic wave, etc.) for distinguishing a target molecule or substance from others. Examples of such a labeling method include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method and the like. When both the nucleic acid fragment and the complementary oligonucleotide are labeled by the fluorescence method, the labeling is performed with fluorescent substances having different fluorescence emission maximum wavelengths. The difference in the maximum fluorescence emission wavelength is preferably 10 nm or more. As the fluorescent substance, any substance can be used as long as it can bind to the base portion of the nucleic acid, but cyanine dyes (for example, CyDye ^™ series Cy3, Cy5 etc.), rhodamine 6G reagent, N-acetoxy-N 2 -acetyl. Aminofluorene (AAF), AAIF (iodine derivative of AAF) or the like is preferably used. Examples of the fluorescent substance having a difference in fluorescence emission maximum wavelength of 10 nm or more include a combination of Cy5 and rhodamine 6G reagent, a combination of Cy3 and fluorescein, a combination of rhodamine 6G reagent and fluorescein, and the like. In the present invention, by using such a label, the target object can be modified so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can appropriately carry out such methods depending on the label and the target object.

  In a preferred embodiment of the present invention, the means used are mass spectrometer, nuclear magnetic resonance analyzer, X-ray analyzer, SPR, chromatography (eg, HPLC, thin layer chromatography, gas chromatography), immunology (E.g., Western blotting, ELISA, RIA), biochemical means (e.g., pI electrophoresis, Southern blotting, two-dimensional electrophoresis), electrophoresis instrument, chemical analysis instrument, fluorescence two-dimensional differential electrophoresis ( 2DE-DIGE), isotope labeling method (ICAT), tandem affinity purification method (TAP method), physical means, laser microdissection, and combinations thereof.

  In a preferred embodiment of the invention, the system of the invention further comprises a marker standard. Such a standard may be used to confirm whether a marker detection means (such as a factor that specifically interacts with the marker or a means for selectively recognizing the marker) is functioning normally. preferable.

  In a preferred embodiment, the present invention may further comprise means for purifying the sample of interest. Examples of such purification means include chromatography. Since purification can increase the accuracy of the diagnosis, it can be used in preferred embodiments, but this is not essential.

  In one embodiment, the means used in the present invention have the ability to quantify the markers of the present invention. Such quantification may be a means or factor that can draw a calibration curve properly when a standard curve is drawn. Preferable examples include antibodies, mass spectrometry, and chromatographic analysis. Therefore, in one embodiment, the system of the present invention further comprises a quantification means for quantifying the marker substance.

  In one embodiment, the quantification unit includes a determination unit that compares the standard curve with the measurement result to determine whether the marker is within the normal value range. Such determination means can be realized using a computer.

  In one embodiment, the system of the present invention is a composition comprising a marker or a substance that specifically interacts with the marker.

  The formulation procedure of the preparation of the present invention is known in the art, and is described in, for example, the Japanese Pharmacopeia, the US Pharmacopeia, the pharmacopoeia of other countries, and the like. Thus, one skilled in the art can determine the amount to be administered without undue experimentation as described herein.

(General technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the art, and are described in, for example, Sambrook J. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method”, Yodosha, 1997, etc., and related parts (which may be all) are incorporated herein by reference.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  The present invention has been described with reference to the preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration, not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is limited only by the scope of the claims.

  Hereinafter, although an example explains the composition of the present invention in detail, the present invention is not limited to this. The reagents used in the following were commercially available unless otherwise specified. For human subjects, informed consent was obtained, and the experiments were conducted after satisfying all internationally recognized ethical standards.

(Target patient)
A total of 132 Japanese patients with heart failure (65 men, 67 women, age 25-90 years, average age 67 ± 11 years) participated in this study. The main causes of heart failure are valvular heart disease (55 cases), ischemic heart disease (49 cases), congenital heart disease (13 cases), dilated cardiomyopathy (8 cases), hypertrophic cardiomyopathy (8 cases) And others (9 cases). Patients with symptomatic heart failure were administered medications such as angiotensin converting enzyme inhibitor / angiotensin receptor antagonist (67%), digitalis preparation (35%), and diuretic (72%). The classification of cardiac function according to the New York Heart Association (NYHA) was as follows: Class I, 31 cases (12 men, 19 women), mean age 65 ± 10 years; Class II, 69 cases (38 men, 31 women), average age 68 ± 12 years; class III, 24 cases (12 men, 12 women), average age 71 ± 8 years; class IV, 8 cases (3 men, 5 women), average Age 63 ± 11 years.

  Informed consent was obtained from each patient, the protocol was approved by our institutional ethics committee and / or the study was conducted according to the recommendations of the Dokkyo Medical University Ethics Committee.

Example 1: Comparison of immunoreactive BNP-32 and proBNP-108 concentrations in plasma of normal control subjects, patients with atrial fibrillation and patients with heart failure
Plasma BNP-32 and proBNP-108 concentrations in normal control subjects, patients with atrial fibrillation and heart failure were measured. Atrial fibrillation patients were isolated atrial fibrillation patients and other cardiovascular diseases were excluded by physical examination, clinical examination, chest radiograph, electrocardiogram and echocardiography. Normal control subjects were 10 (4 males, 6 females, 35-77 years old, mean age 65 ± 12 years), normal findings by physical examination, clinical examination, chest radiograph, electrocardiogram, echocardiography Met. The main causes of heart failure were assessed based on past history, physical examination, chest radiograph, electrocardiogram, echocardiography, and / or cardiac catheterization. Patient characteristics are shown in Table 1 below.

A. Blood sampling A blood sample (3 mL) was collected from the anterior cubital vein of all subjects. The blood was immediately placed in a refrigerated glass tube containing EDTA.2 sodium (1 mg / mL) and aprotinin (500 U / mL). The blood was immediately centrifuged at 4 ° C. and the plasma was frozen and stored at −80 ° C. until measurement.

B. Measurement of BNP-32 and proBNP-108 in plasma Plasma stored at −80 ° C. was extracted with a Sep-Pak C18 cartridge (Waters, Milford, Mass., USA). As previously reported (Tateyama H, Hino J, Minamino N, Kangawa K, Minamino T, Sakai K, Ogihara T, Matsuo H. Concentrations and molecular forms of human brain natniuretic peptide in plasma. Biochem Biophys Res Commun. 1992; 185: 760-7) Prewashed sequentially with 5 mL each of chloroform, methanol, 50% acetonitrile containing 0.1% trifluoroacetic acid (TFA), 0.1% TFA, and saline. 3 mL of plasma was acidified with 3 mL of physiological saline containing 28 μL of 1 mol / L HCl, diluted with 3 mL of physiological saline, and loaded onto a Sep-Pak C18 cartridge. After washing with 5 mL of physiological saline, 0.1% TFA and 20% acetonitrile containing 0.1% TFA, the adsorbate was extracted with 4 mL of 50% acetonitrile containing 0.1% TFA. The extract was lyophilized. Thereafter, the lyophilized product was dissolved in 30% acetonitrile containing 0.1% TFA, and gel filtration high performance liquid chromatography using a TSK gel G2000SWXL column (7.8 × 300 mm, Tosoh, Tokyo, Japan) with the same buffer ( HPLC) and separated at a rate of 0.2 mL / min. Twenty minutes after injection, the column eluate was fractionated every minute into polypropylene tubes containing bovine serum albumin (50 μg) and Triton X-100 (25 μg). Each fraction was freeze-dried, dissolved in a radioimmunoassay buffer and centrifuged, and the clarified lysate was subjected to BNP-32 fluorescent enzyme immunoassay (Tosoh). Details of the BNP-32 fluorescent enzyme immunoassay were reported previously, and BNP-32 monoclonal antibody from Shionogi & Co. (Osaka, Japan) was used in this assay (Emi Ota et al., E-test “TOSOH” II ( Examination of brain natriuretic peptide measurement using BNP), medical and test equipment / reagents Vol. 28 No. 3 (2005) 255-261).

The following formula based on the sum of high molecular weight immune response (proBNP-108) and low molecular weight immune response (BNP-32):
proBNP-108 / total BNP ratio = proBNP-108 / (proBNP-108 + BNP-32)
The proBNP-108 / total BNP ratio was calculated according to

  The knowledge that glycosylated proBNP having a molecular weight (MW) of about 35K circulates in plasma has been clarified by recent studies (Non-patent Document 10 and Non-patent Document 11), so that recombinant proBNP-108 And glycosylated proBNP-108 (HyTest, Finland) and synthetic BNP-32 (Peptide Institute, Japan) were purchased, and the elution positions of these three peptides during gel filtration were examined. In order to evaluate the cross-reactivity in the enzyme immunoassay of proBNP-108 and glycosylated proBNP-108, each peptide was desalted with Monotip C18 cartridge (GL Science, Tokyo, Japan) according to the manufacturer's protocol. In order to estimate the content of each peptide, a certain amount of desalted peptide was subjected to enzyme immunoassay, and another amount was subjected to amino acid analysis after acid hydrolysis at 110 ° C. for 22 hours (L-8500 analyzer, Hitachi, Tokyo, Japan).

C. Statistical analysis All measured values (variables) were expressed as mean ± SD. Statistically significant differences between the two groups were assessed with Fisher's exact test or unpaired Student's t test, as appropriate. Logarithmic transformation was used when distribution normalization was appropriate for plasma peptide concentrations. Classification variables were compared using chi-square test. For comparison among the three groups, Bonferroni's multiple comparison test was performed after one-way analysis of variance. The correlation coefficient was calculated by linear regression analysis. A P value of less than 0.05 was regarded as a statistically significant difference.

D. Results 1) Measurement results of immunoreactive BNP-32 and proBNP-108 concentrations In order to know the characteristics of the molecular type of BNP in human plasma, the peptide fraction obtained from reverse phase C-18 column extraction was analyzed using a TSK gel G2000SWXL column. As shown in FIG. 1, IR-proBNP-108 (high molecular weight immunoreactive (IR-) BNP containing both proBNP-108 and glycosylated proBNP-108) and IR- Two IR-BNP peaks of BNP-32 (low molecular weight IR-BNP consisting mainly of BNP-32) were always observed in normal control subjects, patients with atrial fibrillation and patients with heart failure. The first peak (IR-proBNP-108) is found in fractions # 9-16 with a molecular weight greater than 13K, and the second peak (IR-BNP32) has a molecular weight of 3.5K corresponding to BNP-32. It was seen in fractions # 18-21. In this gel filtration HPLC, glycosylated proBNP-108 and recombinant proBNP-108 eluted mainly in fraction # 14 and fraction # 15, respectively, and could not be separated from each other.

  The cross-reactivity of proBNP-108 and glycosylated proBNP-108 in the BNP-32 fluorescent enzyme immunoassay was 52.8% and 72.0% as estimated by amino acid analysis based on net weight. Both proBNP-108 and glycosylated proBNP-108 share the C-terminal structure of BNP-32, and the fluorescent enzyme immunoassay for BNP-32 strictly recognizes the structure of BNP-32. The N-terminal sugar chain of proBNP-76 could interfere with antibody recognition of glycosylated proBNP-108, but this possibility was denied by the high cross-reactivity seen with glycosylated proBNP-108. Rather, since these two proBNP-108 are produced by different recombinant techniques (from E. coli and HEK293 cells), this difference in cross-reactivity is related to BNP-32 of unglycosylated proBNP-108 and glycosylated proBNP-108. This is presumed to be due to the difference in the structural suitability of the parts. Thus, the complete molecule of glycosylated proBNP-108 and non-glycosylated proBNP-108 is expected to show greater than 70% cross-reactivity, and we correct IR-proBNP108 concentration based on cross-reactivity. I didn't. On the other hand, synthetic BNP-32 was always measured in the range of 100 to 110% in this measurement.

2) Plasma IR-BNP-32 and IR-proBNP-108 concentrations in normal control subjects, atrial fibrillation patients, and heart failure patients The proBNP-108 / total BNP ratio is shown in FIG. Since recombinant proBNP-108 and glycosylated proBNP-108 could not be separated from each other, the ratio proBNP-108 / total BNP was calculated by the formula: proBNP-108 / total BNP ratio = proBNP-108 / (proBNP-108 + BNP-32 ). The distribution of proBNP-108 / total BNP ratio in normal control subjects and patients with atrial fibrillation was narrow, but was broadly distributed in heart failure patients. As a result, the average proBNP-108 / total BNP ratio in heart failure patients was significantly lower than in normal control subjects and atrial fibrillation patients. The average IR-BNP-32 concentration and the average IR-proBNP-108 concentration are shown in Table 1. In patients with heart failure, not only IR-BNP-32 but also IR-proBNP-108 was significantly higher than the other two groups. The IR-BNP-32 concentration measured by this method and the IR-proBNP-108 concentration were sufficiently correlated (FIG. 3).

(Example 2: Plasma proBNP-108 / total BNP ratio in patients with heart failure; comparison of atrial stress heart failure and ventricular stress heart failure)
62 cases out of 132 heart failure patients of Example 1 were divided into two groups, atrial stress heart failure and ventricular stress heart failure. The breakdown of atrial stress heart failure (32 cases) was mitral stenosis, mitral regurgitation, and atrial septal defect. The breakdown of ventricular overload heart failure (30 cases) was aortic regurgitation and aortic stenosis. The ratio of proBNP-108 / (BNP-32 + proBNP-108) for atrial stress heart failure was compared to that for ventricular stress heart failure. Patient characteristics are shown in Table 2.

A. Blood sampling A blood sample (3 mL) was collected from the anterior cubital vein of all subjects. The blood was immediately placed in a refrigerated glass tube containing EDTA.2 sodium (1 mg / mL) and aprotinin (500 U / mL). The blood was immediately centrifuged at 4 ° C. and the plasma was frozen and stored at −80 ° C. until measurement.

B. Measurement of plasma BNP-32 and proBNP-108 Plasma BNP-32 and proBNP-108 were measured in the same manner as in Example 1, and the proBNP-108 / total BNP ratio was calculated.

C. Statistical analysis All measured values (variables) were expressed as mean ± SD. Statistically significant differences between the two groups were assessed with Fisher's exact test or unpaired Student's t test, as appropriate. Logarithmic transformation was used when distribution normalization was appropriate for plasma peptide concentrations. Classification variables were compared using chi-square test. For comparison between the three groups, Bonferroni's multiple comparison test was performed after one-way analysis of variance. The correlation coefficient was calculated by linear regression analysis. A P value of less than 0.05 was regarded as a statistically significant difference.

D. Results In order to examine the reason why the distribution of proBNP-108 / total BNP ratio is wide, the proBNP-108 / total BNP ratio was measured in patients with atrial stress heart failure and patients with ventricular stress heart failure. As shown in FIG. These two peaks were observed in both groups. Interestingly, in atrial stress heart failure, the IR-BNP-32 peak was superior to the IR-proBNP-108 peak (FIG. 4B). In contrast, in ventricular overload heart failure, IR-proBNP-108 and IR-BNP-32 peaks were nearly equivalent (FIG. 4A). As a result, the mean proBNP-108 / total BNP ratio for ventricular overload heart failure was higher than for atrial overload heart failure (FIG. 5).

E. Discussion We measured plasma IR-BNP-32 and IR-proBNP-108 concentrations in patients with atrial stress and ventricular stress and compared the proBNP-108 / total BNP ratio in these two conditions did. The proBNP-108 / total BNP ratio of patients with ventricular stress heart failure was higher than that of patients with atrial stress heart failure, but the plasma concentrations of both peptides were correlated with each other.

(Example 3: Comparison of proBNP-108 concentration and BNP-32 concentration in atrial tissue and ventricular tissue)
To elucidate the mechanism by which the proBNP-108 / total BNP ratio of atrial stress heart failure was lower than ventricular stress heart failure, the BNP molecular types in the atrial and ventricular tissues of other patients undergoing cardiac surgery (11 cases) analyzed. Atrial tissue was obtained from mitral valve disease (6 cases) and autopsy (1 case), and ventricular tissue was obtained from cardiac surgery (5 cases) and autopsy (1 case). The characteristics of patients who provided specimens of atrial and ventricular tissue are shown in Table 3.

A. Sampling of left atrial tissue and left ventricular tissue Left atrial resection tissue sample of 7 patients (mitral valve replacement (MVR) + maze operation 5 cases, 2 valve replacement operation (DVR) + maze operation 1 case, autopsy 1 case)) Frozen with nitrogen and stored at -80 ° C. Left ventricular resected tissue specimens obtained from 6 patients (5 coronary artery bypass grafting + 5 Dor procedures, 1 autopsy) were frozen in liquid nitrogen and stored at -80 ° C. Our goal was to obtain biochemical evidence for the BNP molecular types of atrial and ventricular tissue.

B. BNP-32 and proBNP-108 measurements of heart tissue Atrial and ventricular tissues stored at -80 ° C were weighed and boiled in 10 volumes of 1 mol / L acetic acid as previously reported (Yoshihara F, Nishikimi T, Sasako Y, Hino J, Kobayashi J, Minatoya K, Bando K, Kosakai Y, Horio T, Suga S, Kawano Y, Matsuoka H, Yutani C, Matsuo H, Kitamura S, Ohe T, Kangawa K. Plasma atrial natniuretic peptide concentration inversely correlates with left atrial collagen volume fraction in patients with atrial fibrillation: plasma ANP as a possible biochemical marker to predict the outcome of the maze procedure. J Am Coll Cardiol. 2002; 39: 288-94). The tissue was then homogenized (homogenized) with a Polytron mixer. The homogenate was centrifuged at 3000 × g and the supernatant was again centrifuged at 15000 × g for 15 minutes. B. of Example 1 The supernatant was extracted a second time using a Sep-Pak C18 cartridge as described above. The extract was lyophilized and subjected to gel filtration HPLC using a TSK gel G2000SWXL column. A certain amount of each fraction was freeze-dried, and the IR-BNP concentration was measured in the same manner as in Example 1.

C. Statistical analysis All measured values (variables) were expressed as mean ± SD. Statistically significant differences between the two groups were assessed with Fisher's exact test or unpaired Student's t test where appropriate. Logarithmic transformation was used when distribution normalization was appropriate for plasma peptide concentrations. Classification variables were compared using chi-square test. For comparison between the three groups, Bonferroni's multiple comparison test was performed after one-way analysis of variance. The correlation coefficient was calculated by linear regression analysis. A P value of less than 0.05 was regarded as a statistically significant difference.

D. Results Similar to plasma samples, two peaks corresponding to BNP-32 and proBNP-108 were observed in atrial and ventricular tissues. Interestingly, in atrial tissue, the low molecular weight IR-BNP peak corresponding to BNP-32 was superior to the high molecular weight IR-BNP peak (7 cases, 77 ± 5%) (FIG. 6A). In contrast, in ventricular tissue, the high molecular weight IR-BNP peak corresponding to proBNP-108 was superior to the low molecular weight IR-BNP peak (6 cases, 66 ± 4%, P <0.0001) ( FIG. 6B). As a result, the mean proBNP-108 / total BNP ratio of ventricular tissue was higher than that of atrial tissue (FIG. 7).

E. Discussion To elucidate why the proBNP-108 / total BNP ratio in patients with ventricular overload heart failure was higher than in patients with atrial overload heart failure, IR-BNP-32 concentrations and IR-proBNP-108 in atrial and ventricular tissues Concentration was measured. Interestingly, more than 70% IR-BNP was present as proBNP-108 in ventricular tissue, whereas more than 75% IR-BNP was present as BNP-32 in atrial tissue. Two IR-BNP molecular forms of molecular weight 4K and molecular weight 13-15K are present in human atrial extracts and they are separated using anti-BNP IgG immunoaffinity chromatography and reverse phase HPLC, direct N of each peptide It was shown by Hino et al. That it was identified as BNP-32 and proBNP-108 by terminal sequencing (Hino J, Tateyama H, Minamino N, Kangawa K, Matsuo H. Isolation and identification of human brain natniuretic peptides in cardiac atrium. Biochem Biophys Res Commun. 1990; 167: 693-700). Hino et al. Also reported that BNP-32 is the major molecular type in human atrial tissue, a finding consistent with our results. However, as far as investigated so far, there has been no previous study examining the BNP molecular type in human ventricular tissue. Goetze et al. (Goetze JP, Friis-Hansen L, Rehfeld JF, Nilsson B, Svendsen JH. Atnial secretion of B-type natniunetic peptide. Eur Heart J. 2006; 27: 1648-50) is important for atrial-derived BNP in heart failure Suggested sex, but not much attention. In view of the above, it has been obtained that proBNP-108 is the main molecular form of BNP rather than BNP-32 in ventricular tissue, and most of proBNP-108 is secreted from the ventricle without being processed. Suggested by the evidence. A recent report showed that plasma concentrations of proBNP-108 as well as BNP-32 increased in patients with heart failure, and plasma proBNP-108 concentration was strongly correlated with plasma BNP-32 concentration ( Non-patent document 6, Non-patent document 7, Non-patent document 8, Non-patent document 9). These results are consistent with the results of this example.

Example 4: Comparison of IR-BNP-32 and IR-proBNP-108 concentrations in the pericardial fluid and plasma of heart failure patients undergoing cardiac surgery
In order to confirm the BNP molecular type produced and secreted from ventricular tissue, proBNP− in pericardial effusion and plasma was analyzed in 8 heart failure patients who underwent cardiac surgery (4 cases of aortic valve replacement, 4 cases of mitral valve replacement). The concentration of 108 and BNP-32 was measured. Table 4 shows the characteristics of patients who provided pericardial fluid.

A. Sample collection of plasma and pericardial fluid Immediately after pericardiotomy, as previously reported (Nishikimi T, Shibasaki I, Iida H, Asakawa H, Matsushita Y, Mori H, Mochizuki Y, Okamura Y, Horinaka S, Kangawa K, Shimada K, Matsuoka H. Molecular forms of adrenomedullin in pericardial fluid and plasma in patients with ischaemic heart disease.Clin Sci (London) .2002; 102: 669-77, Nishikimi T, Asakawa H, Iida H, Matsushita Y, Shibasaki I, Tadokoro K, Mori Y, Mori H, Mochizuki Y, Okamura Y, Miyoshi S, Kangawa K, Matsuoka H. Different secretion patterns of two molecular forms of cardiac adrenomedullin in pressure- and volume-overloaded human heart failure. 10: 321-7) An undiluted pericardial fluid sample was collected. At the same time, a blood sample was collected from the brachial artery inserted into the catheter. Samples were stored at −80 ° C. as described above.

B. Measurement of BNP-32 and proBNP-108 in plasma and pericardial fluid Plasma or pericardial fluid specimens stored at −80 ° C. were processed according to the method described in Example 1 to measure BNP-32 and proBNP-108, and The proBNP-108 / total BNP ratio was calculated.

C. Statistical analysis All measured values (variables) were expressed as mean ± SD. Statistically significant differences between the two groups were assessed with Fisher's exact test or unpaired Student's t test where appropriate. Logarithmic transformation was used when distribution normalization was appropriate for plasma peptide concentrations. Classification variables were compared using chi-square test. For comparison between the three groups, Bonferroni's multiple comparison test was performed after one-way analysis of variance. The correlation coefficient was calculated by linear regression analysis. A P value of less than 0.05 was regarded as a statistically significant difference.

D. Results IR-BNP-32 and IR-proBNP-108 were measured in the pericardial fluid and plasma of 8 patients undergoing cardiac surgery to confirm the BNP molecular type produced and secreted from ventricular tissue. FIG. 8 shows two IR-BNP peaks corresponding to BNP-32 and proBNP-108 in plasma and pericardial fluid of patients with mitral regurgitation (FIGS. 8A, B) and aortic stenosis (FIGS. 8C, D). The presence of In plasma, IR-BNP-32 was the major molecular type of patients with mitral regurgitation, while IR-proBNP-108 was the major molecular type of patients with aortic stenosis. However, in the pericardial fluid of both groups of patients, IR-proBNP-108 was the predominant major molecular type. As a result, the average proBNP-108 / total BNP ratio in pericardial fluid was higher than that of plasma (FIG. 9).

E. Discussion It is known that pericardial fluid is rich in various physiologically active substances produced in the heart (Non-patent Document 10, Non-patent Document 11), and its composition is similar to ventricular interstitial fluid. (Fujita M, Ikemoto M, Kishishita M, Otani H, Noliara R, Tanaka T, Tamaki S, Yamazato A, Sasayama S. Elevated basic fibroblast growth factor in pericardial fluid of patients with unstable angina. Circulation. 1996 94: 610-3). Furthermore, it has been reported that the concentration of physiologically active substances such as adrenomedullin, BNP, ANP, basic fibroblast growth factor, and vascular endothelial growth factor is higher in pericardial fluid than in plasma (Nishikimi T, Shibasaki I, Iida H, Asakawa H, Matsushita Y, Mori H, Mochizuki Y, Okamura Y, Horinaka S, Kangawa K, Shimada K, Matsuoka H. Molecular forms of adrenomedullin in pericardial fluid and plasma in patients with ischaemic heart disease.Clin Sci (Lond 2002; 102: 669-77, Nishikimi T, Asakawa H, Iida H, Matsushita Y, Shibasaki I, Tadokoro K, Mori Y, Mori H, Mochizuki Y, Okamura Y, Miyoshi S, Kangawa K, Matsuoka H. Different. secretion patterns of two molecular forms of cardiac adrenomedullin in pressure- and volume-overloaded human heart failure.J Card Fail.2004; 10: 321-7, Fujita M, Ikemoto M, Kishishita M, Otani H, Noliara R, Tanaka T, Tamaki S, Yamazato A, Sasayama S. Elevated basic fibroblast growt h factor in pericardial fluid of patients with unstable angina.Circulation.1996; 94: 610-3, Tambara K, Fujita M, Nagaya N, Miyamoto S, Iwakura A, Doi K, Sakaguchi G, Nishimura K, Kangawa K, Komeda M ．Increased pericardial fluid concentrations of the mature form of adrenomedullin in patients with cardiac remodelling. Heart. 2002; 87: 242-6). Interestingly, in our results, most IR-BNP in the pericardial fluid was present as IR-proBNP-108 regardless of the type of heart failure. These results are consistent with the hypothesis that proBNP-108 is the ventricular BNP major molecular form and most of proBNP-108 is secreted from the ventricle without undergoing proteolytic processing.

  Yandle et al. (Yandle TG, Richards AM, Gilbert A, Fisher S, Holmes S, Espiner EA. Assay of brain natriuretic peptide (BNP) in human plasma: evidence for high molecular weight BNP as a major plasma component in heart failure. J Clin Endocrinol Metab. 1993; 76: 832-8) BNP molecular types in plasma of coronary sinus and peripheral veins of patients with heart failure have been analyzed previously, BNP-32 is the main molecular type of coronary sinus and proBNP-108 is intravenous It was shown to be the major molecular type of plasma. The present inventors have previously reported that proBNP-108, rather than BNP-32, is the BNP major molecular form of normal subjects (Tateyama H, Hino J, Minamino N, Kangawa K, Minamino T, Sakai K, Ogihara). T, Matsuo H. Concentrations and molecular forms of human brain natniuretic peptide in plasma. Biochem Biophys Res Commun. 1992; 185: 760-7). These results suggest the possibility that proBNP-108 has a longer half-life compared to BNP-32, probably because its affinity for the receptor is different. In fact, BNP-32 production ability of cGMP in vascular smooth muscle cells and endothelial cells is 10 to 20 times higher than proBNP-108 production ability (Non-patent Document 10), and receptor-dependent metabolism of BNP-32 is proBNP-. It is suggested that it is earlier than 108. In this example, plasma BNP-32 and proBNP-108 in the coronary sinus were not measured. From these results, the metabolism of the two molecular forms of BNP also determines the proBNP-108 / total BNP ratio in the peripheral circulation. It is suggested that this is an important factor.

(Example 5: Comparison of plasma BNP-32 concentration and proBNP-108 concentration after treatment or natural course in patients with heart failure)
In order to examine the influence of the pathophysiological state of heart failure on the plasma BNP molecular type, proBNP-108 concentration and BNP-32 concentration were measured before and after the heart failure state changed. Plasma proBNP-108 and BNP-32 concentrations were measured in 5 patients before and after improvement of symptoms by treatment. In addition, the plasma proBNP-108 concentration and BNP-32 concentration of four patients were measured before and after deterioration of symptoms due to natural course. Patient characteristics are shown in Table 5.

A. Blood sampling A blood sample (3 mL) was collected from the anterior cubital vein of all subjects. The blood was immediately placed in a refrigerated glass tube containing EDTA.2 sodium (1 mg / mL) and aprotinin (500 U / mL). The blood was immediately centrifuged at 4 ° C. and the plasma was frozen and stored at −80 ° C. until measurement.

B. Measurement of BNP-32 and proBNP-108 in plasma Plasma BNP-32 and proBNP-108 were measured in the same manner as in Example 1, and the proBNP-108 / total BNP ratio was calculated.

C. Statistical analysis All measured values (variables) were expressed as mean ± SD. Statistically significant differences between the two groups were assessed with Fisher's exact test or unpaired Student's t test where appropriate. Logarithmic transformation was used when distribution normalization was appropriate for plasma peptide concentrations. Classification variables were compared using chi-square test. For comparison between the three groups, Bonferroni's multiple comparison test was performed after one-way analysis of variance. The correlation coefficient was calculated by linear regression analysis. A P value of less than 0.05 was regarded as a statistically significant difference.

D. Results IR-BNP-32 and IR-proBNP-108 concentrations were measured before and after treatment of heart failure patients to investigate whether the pathophysiological state of heart failure affects the plasma BNP molecular type. As shown in FIG. 10A, the elevated plasma IR-BNP concentration decreased after treatment and the proBNP-108 / total BNP ratio also decreased. In cases where central failure worsened during the observation period, the IR-BNP concentration increased simultaneously with the increase in the proBNP-108 / total BNP ratio (FIG. 10B).

E. Discussion In decompensated heart failure, both IR-BNP-32 and IR-proBNP-108 were increased. In patients with improved medical treatment, plasma IR-BNP-32 and IR-proBNP-108 were decreased, with a decrease in the proBNP-108 / total BNP ratio. In contrast, as heart failure worsened, both plasma IR-BNP-32 and IR-proBNP-108 concentrations increased, with an increase in the proBNP-108 / total BNP ratio. Thus, it is estimated that the proBNP-108 / total BNP ratio is dependent on the pathophysiological state of heart failure. The increase in the proBNP-108 / total BNP ratio in severe heart failure can be explained in part by an increase in proBNP-108 production and secretion from the ventricles. Another possibility is that mRNA cleavage of the proteolytic processing enzyme does not increase in parallel with increased mRNA expression of the BNP precursor in high degree of heart failure. As a result, the conversion of proBNP-108 to BNP-32 is thought to decrease when proBNP-108 is secreted. Very recent studies have shown that proBNP-108 processing by proteases is inhibited by O-glycosylation at sites near the degradation site (Semenov AG, Postnikov AB, Tamm NN, Seperian KR, Karpova NS, Blochchitsyna). MN, Koshkina EV, Krasnoselsky MI, Serebrianaya DV, Katrukha AG. Processing of pro-Brain natriuretic peptide is suppressed by O-glycosylation in the region close to the cleavage site. Clin Chem. 2009 Jan 23. [Epub ahead of print]) . Although the regulatory mechanism in O-glycosylation of proBNP-108 is currently unknown, it is thought that O-glycosylase in dysfunctional myocardium is associated with increased proBNP-108 plasma concentration in heart failure.

  Based on the results of this example, atrial load increased the production and secretion of low molecular weight IR-BNP mainly composed of BNP-32, and ventricular load increased the production and secretion of high molecular weight IR-BNP corresponding to proBNP-108. increase. As a result, in either case, even if the plasma IR-BNP concentration increases, the proBNP-108 / total BNP ratio decreases at atrial load and increases at ventricular load.

  In summary, we analyzed the molecular form of plasma BNP in heart failure by gel filtration HPLC, and not only IR-BNP-32 but also IR-proBNP-108 in the plasma of control subjects, atrial fibrillation patients, heart failure patients. It was shown to exist. The proBNP-108 / total BNP ratio varies depending on the pathophysiological state of heart failure primarily related to atrial or ventricular load. In the measurement system currently used in the clinical field, the BNP-32 kit shows a high cross-reactivity with proBNP-108 (Non-patent document 8, Non-patent document 9, Hammerer-Lercher A, Halfinger B, Sarg B, Mair J , Puschendorf B, Griesmacher A, Guzman NA, Lindner HH. Analysis of circulating forms of proBNP and NT-proBNP in patients with severe heart failure. Clin Chem. 2008; Matsuoka H. Do commercially available assay kits for B-type natriuretic peptide measure Pro-BNP-108, as well as BNPI-32? Hypertension. 2007; 50: e163), this is the ambiguity of BNP in the diagnosis of heart failure Causes variability. By separately measuring BNP-32 and proBNP-108 molecules, it is possible to provide cardiologists and general clinicians treating heart failure patients with more useful information based on the cause mechanism.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  According to the present invention, a simple and accurate method for diagnosing fine classification of heart failure can be provided. According to the present invention, a simple diagnostic kit for determining treatment for heart failure can also be provided.

  SEQ ID NO: 1 is the amino acid sequence of PrePro-BNP (full length).

SEQ ID NO: 2, pro B NP - is the amino acid sequence of 108.

  SEQ ID NO: 3 is the amino acid sequence of BNP-32.

Claims (7)

Measuring proBNP-108 in a sample from a subject,
How heart failure detect whether the load to such type or atrial load on the ventricle is any such type of. A) proBNP-108 and B) BNP-32 in samples from subjects
Comprising measuring the method of claim 1. Measuring proBNP-108 / total BNP in a sample from a subject,
The method of claim 1. The discovery is aortic stenosis, aortic regurgitation, mitral regurgitation, are directed to a condition selected from the group consisting of mitral valve stenosis and atrial septal defect, claim The method in any one of 1-3. Heart failure as a marker to detect which one of the types load is applied to the type or atrial load on the ventricle, the use of proBNP-108. Heart failure as a marker to detect which one of the types load is applied to the type or atrial load on the ventricle, use of a combination of proBNP-108 and BNP-32. The discovery is aortic stenosis, aortic regurgitation, mitral regurgitation, are directed to a condition selected from the group consisting of mitral valve stenosis and atrial septal defect, claim 6. Use according to 6.

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