US20150219669A1 - Diagnostic of Heart Failure - Google Patents

Diagnostic of Heart Failure Download PDF

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US20150219669A1
US20150219669A1 US14/419,566 US201314419566A US2015219669A1 US 20150219669 A1 US20150219669 A1 US 20150219669A1 US 201314419566 A US201314419566 A US 201314419566A US 2015219669 A1 US2015219669 A1 US 2015219669A1
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igfbp2
heart failure
concentration
patients
patient
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Philippe Rouet
Fatima Smih-Rouet
Franck Desmoulin
Michel Galinier
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Institut National de la Sante et de la Recherche Medicale INSERM
Centre Hospitalier Universitaire de Toulouse
Universite Toulouse III Paul Sabatier
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Institut National de la Sante et de la Recherche Medicale INSERM
Centre Hospitalier Universitaire de Toulouse
Universite Toulouse III Paul Sabatier
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Assigned to CENTRE HOSPITALIER UNIVERSITAIRE DE TOULOUSE, UNIVERSITÉ PAUL SABATIER TOULOUSE III, INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM) reassignment CENTRE HOSPITALIER UNIVERSITAIRE DE TOULOUSE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DESMOULIN, FRANCK, GALINIER, MICHEL, ROUET, PHILIPPE, SMIH-ROUET, Fatima
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4745—Insulin-like growth factor binding protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/4473—Arrangements for investigating the separated zones, e.g. localising zones by electric means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the invention relates to a method for classifying a patient at risk for heart failure, wherein said method comprises the steps of (i) measuring the concentration of IGFBP2 in a sample obtained from said patient and (ii) comparing the concentration of IGFBP2 measured in step (i) to a control value derived from the concentration of IGFBP2 in samples from patients who are at particular stages of heart failure and/or to a control value derived from the concentration of IGFBP2 in blood samples from healthy patients.
  • HF heart failure
  • BNP brain natriuretic peptides
  • Hassfeld S. et al 2007 discloses the use of IGFBP2 as a biomarker for the prognostic of patients with dilated cardiomyopathy who represents an etiological subset of HF patients and doesn't disclose the use of the IGFBP2 for the diagnostic of heart failure.
  • the inventors have launched a prospective monocentric case-control study and investigated for urinary polypeptides specific to acute (AHF) or chronic heart failure (CHF) with holistic analytical strategy by using the capillary electrophoresis-mass spectroscopy technique (CE-MS). They find that IGFBP2 concentration may be used as a biomarker of heart failure as a biomarker to classify patients with heart failure.
  • AHF acute
  • CHF chronic heart failure
  • CE-MS capillary electrophoresis-mass spectroscopy technique
  • the invention relates to a method for classifying a patient at risk for heart failure, wherein said method comprises the steps of (i) measuring the concentration of IGFBP2 in a sample obtained from said patient and (ii) comparing the concentration of IGFBP2 measured in step (i) to a control value derived from the concentration of IGFBP2 in samples from patients who are at particular stages of heart failure and/or to a control value derived from the concentration of IGFBP2 in blood samples from healthy patients.
  • the invention relates to a method for classifying a patient at risk for heart failure, wherein said method comprises measuring the concentration of IGFBP2 in a sample obtained from said patient.
  • said method further comprises the steps of:
  • step (ii) comparing the concentration of IGFBP2 measured in step (i) to a threshold value derived from the concentration of IGFBP2 in samples from patients who are at particular stages of heart failure and/or to a threshold value derived from the concentration of IGFBP2 in samples from healthy patients.
  • the invention also relates to a method for diagnosis of heart failure in a patient comprising the steps consisting of i) determining the concentration of IGFBP2 in a sample obtained from said patient; and ii) comparing said concentration to a control value.
  • the patient has significant comorbid conditions, including hypertension, coronary heart disease and diabetes for example diabetes mellitus.
  • the patient is on diuretics or antiplatelet agents.
  • the patient is more than 50 years old. In another particular embodiment, the patient is more than 60 years old.
  • the heart failure may be an asymptomatic heart failure, a chronic heart failure or an acute heart failure.
  • the sample according to the invention may be a blood, plasma, serum, lymph, urine sample, cardiac tissues like atria or ventricle or liver.
  • said sample is plasma or urine.
  • IGFBP2 Insulin-like Growth Factor-Binding Protein 2
  • IGF I Insulin-like growth factor 1
  • IGF II Insulin-like growth factor 2
  • fragments of IGFBP2 denotes shorter peptides becoming from chemical or biochemical hydrolysis of IGFBP2.
  • the invention relates a method for classifying a patient at risk for heart failure or to a method for diagnosis of heart failure in a patient according to the patient by determining the concentration of fragments of IGFBP2.
  • heart failure denotes inability of the heart to supply sufficient blood flow to meet the body's needs and this pathology is well-described in medicine practice. This term encompasses chronic heart failure, acute heart failure, myocardial infarction, unstable angina, diastolic dysfunction, systolic dysfunction and diabetic cardiomyopathy.
  • chronic heart failure denotes a long term situation, usually with stable treated symptomatology.
  • acute heart failure denotes to sudden onset heart failure, as well as acute “exacerbated” or “decompensated” heart failure, referring to episodes in which a patient with known chronic heart failure or devoid of chronic heart failure abruptly develops worsening symptoms and requires hospitalization.
  • Common symptoms of complications due to acute heart failure include, but are not limited to, dyspnea due to pulmonary congestion or cardiogenic shock due to low cardiac output, easy fatigueability (exercise intolerance), peripheral edema, anasarca (pronounced generalized edema), nocturia (frequent nighttime urination), bradycardia, heart block, hypotension, dizziness, syncope, diabetes, oliguria or anuria, hypokalemia, bronchospasm, cold sweat, and asthma.
  • a patient with a heart failure is classified according to an international gradation namely the New York Heart Association (NYHA) functional classification.
  • Functional classification of heart failure is generally done by the New York Heart Association Functional Classification (Criteria Committee, New York Heart Association. Diseases of the heart and blood vessels). Nomenclature and criteria for diagnosis, 6th ed. Boston: Little, Brown and co, 1964; 114). This classification stages the severity of heart failure into 4 classes (I-IV).
  • a patient with cardiac disease but resulting in no limitation of physical activity is classified as a NYHA class I.
  • Ordinary physical activity does not cause undue fatigue, palpitation, dyspnea or anginal pain.
  • a asymptomatic patient is classified as a NYHA class I.
  • a patient with cardiac disease resulting in slight limitation of physical activity is classified as a NYHA class II.
  • Ordinary physical activity results in fatigue, palpitation, dyspnea or anginal pain. They are comfortable at rest.
  • a patient with cardiac disease resulting in marked limitation of physical activity is classified as a NYHA class III. Less than ordinary activity causes fatigue, palpitation, dyspnea or anginal pain. They are comfortable at rest.
  • a patient with cardiac disease resulting in inability to carry on any physical activity without discomfort is classified as a NYHA class IV. Symptoms of cardiac insufficiency or of the anginal syndrome may be present even at rest. If any physical activity is undertaken, discomfort is increased.
  • a patient with a high concentration of IGFBP2, for example more than 1300 ng/ml in plasma, will be classified as having an heart failure of class IV.
  • IGFBP2 concentrations may be measured for example by capillary electrophoresis-mass spectroscopy technique (CE-MS) or ELISA performed on the sample.
  • CE-MS capillary electrophoresis-mass spectroscopy technique
  • the invention relates to a method for diagnosis of heart failure in a patient comprising a step a) consisting of measuring IGFBP2 concentration in a sample obtained from said patient.
  • the method of the invention further comprises a step of comparing the concentration of IGFBP2 obtained in step a) to a threshold level.
  • the “control” may be a healthy subject, i.e. a subject who does not suffer from any heart failure.
  • the control may also be a subject suffering from heart failure.
  • said control is a healthy subject.
  • Detection of IGFBP2 concentration in the sample may also be performed by measuring the level of IGFBP2 protein.
  • the “level of IGFBP2 protein” means the quantity or concentration of said IGFBP2 protein.
  • the “level of IGFBP2” means the level of IGFBP2 fragments.
  • Such methods comprise contacting a sample with a binding partner capable of selectively interacting with IGFBP2 protein peptides present in the sample.
  • the binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.
  • the presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays.
  • immunoassays such as competition, direct reaction, or sandwich type assays.
  • assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, capillary electrophoresis-mass spectroscopy technique (CE-MS).
  • the reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
  • the aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound.
  • Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
  • an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.
  • Methods of the invention may comprise a step consisting of comparing IGFBP2 protein and fragments concentration in circulating cells with a control value.
  • concentration of IGFBP2 refers to an amount or a concentration of a transcription product, for instance the protein IGFBP2.
  • a level of a protein can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example.
  • relative units can be employed to describe a concentration.
  • concentration of IGFBP2 may refer to fragments of IGBP2.
  • fragments of IGFBP2 may also be measured.
  • the invention relates to a method for classifying a patient at risk for heart failure, wherein said method comprises measuring the concentration of IGFBP2 in a sample obtained from said patient.
  • concentration of IGFBP2 in a sample obtained from said patient.
  • the inventors have established threshold values which are able to classifying patient with heart failure.
  • a patient with a concentration of IGFBP2 with less than 600 ng/ml, preferably less than 500 ng/ml, even preferably less than 400 ng/ml most preferably less than 300 ng/ml is indicative of a heart failure of stage I according to the NYHA heart failure classification.
  • a patient with a concentration of IGFBP2 comprised between about 600 ng/ml and about 1100 ng/ml, preferably between about 800 ng/ml and about 1050 ng/ml, preferably between about 900 ng/ml and about 1000 ng/ml, most preferably between about 925 ng/ml and about 975 ng/ml is indicative of a heart failure of stage II according to the NYHA heart failure classification.
  • a patient with a concentration of IGFBP2 comprised between about 1100 ng/ml and about 1300 ng/ml, preferably between about 1150 ng/ml and about 1250 ng/ml, most preferably between about 1175 ng/ml and about 1225 ng/ml is indicative of a heart failure of stage III according to the NYHA heart failure classification.
  • a patient with a concentration of IGFBP2 more than 1300 ng/ml, preferably more than 1350 ng/ml, even preferably more than 1400 ng/ml, most preferably more than 1450 ng/ml is indicative of a heart failure of stage IV according to the NYHA heart failure classification.
  • the invention in another embodiment, relates to a method for diagnosis heart failure in a patient comprising determining the concentration of IGFBP2 in a sample obtained from said patient and comparing said concentration to a threshold value.
  • the level of IGFBP2 in a patient suffering of heart failure is increased by at least 50%, preferably by at least 70%, preferably by at least 100%; preferably by at least 150%, preferably by at least 200%, preferably by at least 250%, more preferably by at least 300%, even more at least 400% compared to a control reference.
  • the quantity of IGFBP2 protein in a patient suffering of heart failure is increased by at least 50%, preferably by at least 70%, preferably by at least 100%; preferably by at least 150%, preferably by at least 200%, preferably by at least 250%, more preferably by at least 300%, even more at least 400% compared to a control reference.
  • Concentration of IGFBP2 in plasma has been measured by Elisa technique.
  • the inventors have established a threshold value for concentration of IGFBP2 to easily diagnose heart failure.
  • this threshold value is more than 150 ng/ml, preferably more than 200 ng/ml, even most preferable more than 250 ng/ml, most preferably said threshold value is more than 300 ng/ml.
  • Concentration of IGFBP2 in urine has been measured by Elisa technique in urine.
  • the inventors have established a threshold value for concentration of IGFBP2 to easily diagnose heart failure.
  • this threshold value is more than 2.5 ng/ml, most preferably said threshold value is more than 3 ng/ml.
  • a “threshold value”, “threshold level” or “cut-off value” can be determined experimentally, empirically, or theoretically.
  • a threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art.
  • the person skilled in the art may compare the concentration of IGFBP2 obtained according to the method of the invention with a defined threshold value.
  • said threshold value is the mean concentration of IGFBP2 of a population of healthy individuals.
  • the term “healthy individual” denotes a human which is known to be healthy, i.e. which does not suffer from heart failure, has never been subjected to such chronic heart failure, and does not need any medical care.
  • said threshold value is the mean concentration of IGFBP2 of a population of sick individuals.
  • sick individual denotes a human which is known to be sick, i.e. which suffers from heart failure at any stage of heart failure as according to the NYHA heart failure classification.
  • the skilled person in the art may determine the concentration of IGFBP2 in a biological sample, preferably plasma or urine, of 100 individuals known to be healthy or sick.
  • the mean value of the obtained concentrations is then determined, according to well known statistical analysis, so as to obtain the mean concentration of IGFBP2.
  • Said value is then considered as being normal and thus constitute a threshold value.
  • the physician is then able to diagnose heart failure or classifying patients. Indeed, by comparing the concentrations of IGFBP2 obtained in a biological sample, preferably plasma or urine, of a given subject to a threshold value, one can easily determine whether said subject suffers from heart failure or not or can easily determine the stage of the heart failure according to the NYHA heart failure classification.
  • the physician would be able to adapt and optimize appropriate medical care of a subject in a critical and life-threatening condition suffering from heart failure.
  • the determination of said prognosis is highly appropriate for follow-up care and clinical decision making.
  • the invention is drawn to a method for diagnosis of heart failure in a patient or for classifying a patient at risk for heart failure comprising the following steps:
  • methods of the invention comprise measuring the concentration of at least one further biomarker.
  • biomarker refers generally to a molecule, the expression of which in a sample from a patient can be detected by standard methods in the art (as well as those disclosed herein), and is predictive or denotes a condition of the subject from which it was obtained.
  • the other biomarker may be selected from the group of heart failure biomarkers consisting of brain natriuretic peptide (BNP), amino-terminal pro-brain natriuretic peptide (NT-pro BNP), norepinephrine, troponin, heart-type fatty acid binding protein, myosin light chain-1, matrix metalloproteinase, tissue inhibitor of matrix metalloproteinase, C-reactive protein (CRP), TNFalpha, soluble tumor necrosis factor receptor 1 (sTNFR1), soluble TNFR2 receptor, soluble IL-2 receptor, CD40-CD154, CCAM-I, P-selectin, tissue factor and von Willebrand factor, urocortin, myeloperoxidase, and uric acid.
  • BNP brain natriuretic peptide
  • NT-pro BNP amino-terminal pro-brain natriuretic peptide
  • norepinephrine troponin
  • the further biomarker of heart failure is BNP or NT-pro BNP.
  • kits for performing a method of the invention comprising means for measuring the concentration of IGFBP2 in a sample obtained from a patient.
  • the kit may include an antibody, or a set of antibodies as above described. In a particular embodiment, the antibody or set of antibodies are labelled as above described.
  • the kit may also contain other suitably packaged reagents and materials needed for the particular detection protocol, including solid-phase matrices, if applicable, and standards.
  • the kit may also contain one or more means for the detection of a further biomarker.
  • the kit may also contain means for the detection of one or more heart failure biomarker selected from the group consisting of brain natriuretic peptide (BNP), amino-terminal pro-brain natriuretic peptide (NT-pro BNP), norepinephrine, troponin, heart-type fatty acid binding protein, myosin light chain-1, matrix metalloproteinase, tissue inhibitor of matrix metalloproteinase, C-reactive protein (CRP), TNFalpha, soluble tumor necrosis factor receptor 1 (sTNFR1), soluble T2 receptor, soluble IL-2 receptor, CD40-CD154, CCAM-I, P-selectin, tissue factor and von Willebrand factor, urocortin, myeloperoxidase, galectin-3 and uric acid.
  • BNP brain natriuretic peptide
  • NT-pro BNP amino-terminal pro-brain natriuretic peptide
  • kit of the invention comprises means for measuring the concentration of IGFBP2 and means for measuring the concentration of BNP or NT-pro BNP.
  • a further object of the invention relates to the use of IGFBP2 as a biomarker for heart failure.
  • FIG. 2 Plasma concentration of IGFBP2 (ng/ml) in Control; Chronic Heart failure (CHF), and Acute heart failure (AHF) patients. Multiple comparison was performed with Anova and Bonferroni post hoc test (*** p ⁇ 0.001).
  • FIG. 4 Plasma concentration of IGFBP2 according to the NYHA classification of heart failure stage. Multiple comparison was performed with Anova and Bonferroni post hoc test (* p ⁇ 0.01).
  • FIG. 5 Scatter plot of IGFBP2 versus BNP concentration in plasma. Estimated regression line is plotted including its 95% Confident interval (dashed lines).
  • FIG. 6 Plasma concentration of IGFBP2 and the left ventricular ejection fractions are correlated. IGFBP2 Plasma concentration was measured by ELISA and left ventricular ejection fraction (LVEF) by transthoracic echocardiography.
  • FIG. 7 IGFBP2 and BNP levels in plasma from the discovery-test set. Biomarkers measurements were performed with plasma from control patient with cardiovascular risk factors (CRF) or heart failure (HF) patients (acute and chronic, see FIG. 1 ). * Significant difference; p ⁇ 0.05.
  • CRF cardiovascular risk factors
  • HF heart failure
  • FIG. 8 IGFBP2 and BNP levels in plasma from the validation set.
  • IGFBP2 and BNP levels were assessed in cardiovascular risk factor patients (CRF), non cardiac dyspnea (NCD), chronic heart failure (CHF) and acute heart failure (AHF) patients. * Significant difference; p ⁇ 0.05. Horizontal dashed line corresponds to optimal cut-point at 556 ng/ml.
  • FIG. 9 IGFBP2 and BNP levels in plasma from NCD and AHF patients from the validation set.
  • FIG. 10 Analysis of a rat model for ischemic heart failure.
  • CHF Chronic Heart Failure Patients
  • AHF Acute Heart Failure
  • Control patients were included through the artherosclerosis prevention department of Rangueil University Hospital during day hospitalization.
  • anthropometric data weight, height, gender
  • electrocardiographic and biological data plasmatic sodium, creatinine, hepatic status, prothrombine, CRP, hematocrite and hemoglobin
  • BNP levels data were collected for CHF and AHF patients. All these examinations were requested during the treatment of the by the physician in charge of the patients and were performed to monitor the heart failure stage but also the kidney and liver function, hydratation and inflammatory level. All medications were recorded.
  • TTE Transthoracic echocardiography
  • TTE allowed for systematic volumes and diameters measurements, left ventricle systolic and diastolic function. In addition, right ventricle function was analyzed as well as aortic, mitral or tricuspid valvulopathies. TTE was considered as ⁇ gold standard>> for heart failure diagnosis that was completed with its etiology according to the European and American current recommendations.
  • Urine were collected in standard polypropylene tubes and immediately frozen and maintained at ⁇ 80° C. Plasma were collected on EDTA tubes, centrifuged, aliquoted on ice and immediately frozen at ⁇ 80° C.
  • CE-MS was performed using standard procedure [Mischak, H. et al., 2010]. Briefly, peptides were electrophoretically separated on 90 cm long and 50 ⁇ m diameter silice capilar (Beckmann-Coulter, Fullerton, Calif., USA) coupled to a ESI-TOF mass spectrometer (electrospray ionisation—time of flight) (MicroTOF, Brucker-Daltonic, Bremen, Germany). CE-MS buffer was 20% (v/v) acetonitrile and 250 mM formic acid in HPLC water. Electrophoretic separation is performed during 60 min under an electric field (+35 to ⁇ 40 kV) leading to a 13 ⁇ A intensity. Capillary temperature is maintained to +35° C. during runs.
  • Enzyme-linked immunosorbent assay (ELISA) IGFBP2 quantization was performed using R&D SYSTEMS EUROPE LTD reagents according to the manufacturer ELISA reagent protocol.
  • IGFBP2 as a putative biomarker was performed by ELISA using both plasma and urine concentration measurements in 200 patients.
  • ROC curve analysis provided an AUC value of 0.988, p ⁇ 0.0001 ( FIG. 1 ).
  • Clearly plasma ( FIG. 2 ) and urinary ( FIG. 3 ) concentration of IGFBP2 are strongly enhanced in CHF and AHF patients.
  • the elevation of IGFBP2 concentration in plasma was dependant on the severity of heart failure as indicated by the NYHA classification ( FIG. 4 ).
  • IGFBP2 and BNP levels were weakly correlated giving more importance to this new biomarker as an almost independent indicator ( FIG. 5 ).
  • IGFBP2 plasma level and the left ventricular ejection fraction were negatively correlated, indicating a physiological link between bloodstream IGFBP2 level and the heart function and bringing IGFBP2 levels as possible estimate of the heart pump status ( FIG. 6 ). Therefore, we propose that elevated IGFBP2 concentration could be used as a biomarker of heart failure.
  • the case group of the discovery-validation cohort was constituted of patients suffering from chronic (CHF) or acute (AHF) heart failure.
  • CHF patients had a known stable HF with >3 months without any decompensation episodes, whatever the stage of clinical severity (stage I to IV of NYHA classification) and, regardless of etiology.
  • Diagnosis of heart failure had been formally established from clinical observations, heart disease follow-up, transthoracic echocardiography (TTE) and BNP monitoring. These patients were included during their regular scheduled visit at the hospital.
  • AHF patients were recruited whatever the clinical presentation (left, right, mixed, low flow-cardiogenic shock.
  • the control group of the discovery-validation cohort was constituted of patients without HF but with cardiovascular risk factors (CRF) or CRF and non cardiac dyspnea (NCD) for the discovery step and the validation step, respectively.
  • CRF patients were recruited during their scheduled visit at the atherosclerosis prevention center of the Rangueil University Hospital. Inclusion in this group required the exclusion of all patients with a history, clinical signs, biological, or echocardiographic evidence of heart failure (systolic or diastolic dysfunction).
  • External validation cohort was constituted at the Lariboisière University Hospital (Paris, France) with COPD patients (with BNP ⁇ 20 pg/ml to test “pure” COPD without any right or left cardiac stress) as control patient and AHF patients as cases patients.
  • TTE was performed for all subjects included by a single cardiologist. Echocardiography (Konton Imagic, Kontron, Saint German en Laye, France) allowed for systematic volumes and diameter measurements, left ventricle systolic and diastolic function and ejection fraction measurements. Patients with renal dialysis or transplant (stage 5D and 5T) were excluded.
  • the research protocol was registered in a clinical database (ClinicalTrials.gov NCT01024049) conforms to the ethical guidelines of the 1975 Declaration of Helsinki.
  • the protocol was approved by the institution's human research (COSSEC) and regional ethics committee (Comotti de Protection des Personnes (CPP) # DC 2008-452). Written informed consent was obtained from all participants and/or their legally authorized representatives.
  • a second validation cohort was constituted with 40 patients from Paris Lariboisière University Hospital. This external validation cohort included 10 COPD and 30 AHF patients (Table 5).
  • CE-MS analysis was performed as described using a P/ACE MDQ capillary electrophoresis system (Beckman Coulter, Villepinte, France) on-line coupled to a MicroQTOF MS (Bruker Daltonics, Bremen, Germany).
  • the ESI sprayer (Agilent Technologies, Palo Alto, Calif., USA) was grounded, and the ion spray interface potential was set between ⁇ 4.0 and ⁇ 4.5 kV.
  • Data acquisition and MS acquisition methods were automatically controlled by the CE via contact-close-relays. Spectra were accumulated every 3 s, over a range of m/z 350 to 3000. Details on accuracy, precision, selectivity, sensitivity, reproducibility, and stability of the CE-MS method have been provided previously.
  • Mass spectra were processed using MosaiquesVisu software (Mosaiques, Hannover, Germany), including peak picking, deconvolution, and de-isotoping. Migration time and peak intensity were normalized using internal polypeptide standards. These fragments are believed to be the result of normal biological processes and appear to be unaffected by any disease state studied to date on the basis of 20,000 samples in our database. The resulting peak list characterizes each polypeptide by its molecular mass, normalized capillary electrophoresis migration time, and normalized signal intensity. All detected polypeptides were deposited, matched, and annotated in a Microsoft SQL database, allowing further analysis and comparison of multiple patient groups.
  • the urine samples were analysed on a Dionex Ultimate 3000 RSLS nano flow system (Dionex, Camberly UK), essentially as described (Carty et al., 2011; Metzger et al., 2012).
  • the samples (5 ⁇ l) were loaded onto a Dionex 100 ⁇ m ⁇ 2 cm 5 ⁇ m C18 nano trap column at a flowrate of 5 ⁇ l/min by a Ultimate 3000 RS autosampler (Dionex, Camberley UK)
  • the composition of the loading solution was 0.1% formic acid and acetonitrile (98:2).
  • the eluent from the column was directed to a Proxeon nano spray ESI source (Thermo Fisher Hemel UK) operating in positive ion mode then into an Orbitrap Velos FTMS.
  • the ionisation voltage was 2.5 kV and the capillary temperature was 200° C.
  • the mass spectrometer was operated in MS/MS mode scanning from 380 to 2000 amu.
  • the fragmentation method was HCD at 35% collision energy.
  • the ions were selected for MS2 using a data dependant method with a repeat count of 1 and repeat and exclusion time of 15 s. Precursor ions with a charge state of 1 were rejected.
  • the resolution of ions in MS1 was 60,000 and 7,500 for HCD MS2.
  • IPI:IPI00297284.1 insulin like growth factor binding protein 2
  • IGFBP2 in plasma showed a significant increase in heart failure patients vs control individuals with 1350 ⁇ 635 (p ⁇ 0.001) and 214 ⁇ 136 ng/ml, respectively ( FIG. 7 ).
  • BNP levels in these patients displayed with a higher concentration in HF than in control patients; 806 ⁇ 693 and 39 ⁇ 28 (p ⁇ 0.001), respectively ( FIG. 7 ).
  • CRF patients have cardiovascular risk factors without any symptoms or objective parameters of HF.
  • AHF patients were the oldest group.
  • AHF Patients had significant comorbid conditions, including hypertension (81%) coronary heart disease (52%) and diabetes mellitus (24%). Most patients were on diuretics (89%) and antiplatelet agents (60%).
  • Age distribution of patients of the CHF group is nestled in the control group between CRF and NCD groups.
  • the CHF patients were 62 ⁇ 13 years old with 68% male and the LVEF was ⁇ 45% in 42 patients. Patients had significant comorbid conditions, including hypertension (37%) coronary heart disease (51%) and diabetes mellitus (30%).
  • IGFBP2 concentrations were significantly increased in CHF and AHF vs CRF or NCD patients. This increase of IGFBP2 levels followed the similar pattern as BNP levels in this set of patients ( FIG. 8 ).
  • Logistic regression of IGFBP2+BNP raised the AUC to 0.942 but did not significantly increased the performance of IGFBP2 (data not shown).
  • IGFBP2 Levels and Correlations with Clinical Parameters
  • Plasma BNP or NT-proBNP levels are valuable tools to diagnose patients with HF 2. Therefore, we compared both IGFBP2 diagnostic value to the one of BNP and the putative added value of IGFBP2 to BNP.
  • the diagnostic performance of IGFBP2 measurement led us to discriminate HF cases from control cases without heart disease (CRF and NCD patients) with an increased AUC compared to the one of BNP.
  • Logistic combination of IGFBP2 and BNP led to an increased AUC compared to BNP alone when testing the use of IGFBP2 and BNP in the discrimination between NCD and AHF patients.
  • the added value of IGFBP2 diagnostic performance was even more evident in patients with moderate BNP elevation i.e. in the 100-600 pg/ml concentration range.
  • IGFBP2 up-regulation point out a potential role for Insulin-like growth factor (IGF)-binding proteins (IGFBPs) in HF and more largely involves the somatotrope axis that encompass hypothalamic hormones such as hypothalamic Growth-hormone-releasing-hormone (GHRH), hypohyse Growth-hormone and IGF 1 and 2.
  • IGFBPs confer regulation to IGF bioactivity but can also have a direct role.
  • IGF1's availability is regulated by Insulin Growth Factor Binding Protein IGFBPs. Only one to 5% of IGF1 is free in the blood stream, the remaining IGF1 is tightly bound to IGFBPs with an affinity that is greater or equal to the one for its receptor. Thus, IGFBPs modulate IGF1 and IGF1 receptor interaction.
  • IGFBPs were recently found to have their own activity even in the absence of IGF, through a direct interaction with transcription factors and modulation of gene expression.
  • IGFBP2 is the second most abundant IGFBP in human plasma. IGFBP2 is mostly an IGF1 inhibitor. Our correlation data confirmed that BNP and IGFBP2 plasma levels are not dependant on sex but are positively increased with age and negatively with the body mass index.
  • IGF1 was recognized to be involved in growth and myocardial development 28. IGF1 and it receptor are expressed in heart since the foetal stage and induce mycocardial hypertrophy through MAP-Kinases and PI-3 kinase pathways. IGF1 also stimulates myocardial protein production, including some with contractile functions such as troponine and actin. Therefore, IGF1 relates to cardiac contractile function and IGF1 levels are correlated to the left ventricular ejection fraction (LVEF). IGF1 is also involved in cardiomyocytes survival and apoptosis.
  • LVEF left ventricular ejection fraction
  • Murine models and also small clinical trials involving GH or IGF1 injection have revealed a reduction of heart early remodeling, an improvement of the systolic and diastolic function, and a contractility improvement. Recently, the addition of the neutralising IGFBP2 antibody upon cell differentiation, led to an important myoblasts hypertrophy.
  • IGFBP2 is mainly produced by the liver and the heart therefore is likely to be indicative of liver or heart function. These points were evaluated in the ischemic rat model of HF which also revealed a stronger IGFBP2 mRNA level in atria vs ventricle or liver. Above all, IGFBP2 mRNA levels were significantly increased in the atria from HF animals. This observation raises the question of the putative role of IGFBP2 in atria. One could speculate that IGFP2 increased synthesis could be involved in a protective mechanism against excessive remodeling in heart because reduction in vitro antibodies based neutralization of IGFBP2 upon myoblasts differentiation led to hypertrophy 34. In addition, overexpression of IGFBP2 led to a reduction in muscle mass in the mouse 36. However, despite solid backgrounds, this hypothesis will have to be further tested in animals' models of HF.
  • IGFBP2 is a reliable biomarker that could reveal the heart functional status.

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