Classifications

• • 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
  • 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
  • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• • 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

Definitions

• the present invention is in the field of medicine and more particularly relates to a biomarker useful for the detection, characterization, treatment and prognosis of valvular heart diseases of rheumatic origin.
• Valvular heart disease is a major cardiac problem throughout the world and is responsible for about 20,000 deaths each year in the United States alone with an estimated 99, 000 inpatient valve procedures performed annually (Rosamond et al. 2007, Circulation, 115:el69-el71 ; Yoshioka et al. 2008, Circulation, 118: 1694-1696).
• Valvular heart diseases in United States population are mostly degenerative whereas rheumatic heart disease accounts for most valve pathology in developing nations. However rheumatic valve disease patients are expected to increase in US due to continuous immigration from the developing countries (Maganti et al. 2010, Mayo Clin Proc.85:483-500).
• Acute rheumatic fever is a common health problem in developing countries (Nordet, 1993, J. Int. Soc. Fed. Cardiol, 3: 4-5). It is estimated that around 12 million people, mainly children, suffer from the disease worldwide (Steer et al. 2002, J. Paediatr, Child Health, 38 : 229 -234; Report of a WHO Expert Consultation, Geneva, 29 October— 1 November 2001 WHO Technical Report Series 923). In India rheumatic fever related disease is responsible for 30 to 40% of cardiovascular disease related hospital admissions and a common indication for cardiac surgery (Kadir et. el. 2004, Annals of Thoracic surgery, 78:699-701).
• Rheumatic fever is an overwhelmingly common condition affecting heart valves among people with low socio-economic status and is related to overcrowding, poor nutrition and hygiene. In addition to the environmental factor, low socioeconomic status and poor access to health care have been associated with increase prevalence and incidence of rheumatic disease (World health organization. 2001 Report of a WHO Expert Consultation. Geneva, 2004:13-19). It is primarily a hypersensitive reaction to streptococcal proteins (acquired through streptococcal throat infections which may be clinically apparent or in apparent) and affects heart valves giving rise to an acute phase where there is fever, joint pain and features of frank heart failure like shortness of breath, palpitation and fatigue. The acute phase of the disease subsides giving rise to clinical sequelae in the form of valvular stenosis/regurgitation with haemodynamic and clinical consequences.
• Rheumatic heart disease a sequelae of rheumatic fever, is the most common acquired heart disease worldwide (Eisenberg et al. 1993, Eur. Heart J, 14 : 122 - 128). It is associated with fibrosis, calcification and fusion of commissures, leaflet thickening, and chordal fusion resulting in mitral stenosis (MS). MS may also be associated with mitral regurgitation (MR). In MR, the abnormality may be related to the valve or to the surrounding supporting structures. It is the second most common form of valve disease worldwide. Rheumatic disease is the leading cause of mitral regurgitation (MR) in the developing world. Rheumatic heart disease is also responsible for severe aortic regurgitation (AR). Abnormalities of the aortic leaf lets and their supporting structures usually lead to AR (Maganti et al 2010, Mayo Clin. Proc. 85:482-510).
• fibrosis is a hallmark of structural remodeling. In general, fibrosis is characterized by excessive deposition of extracellular matrix that can occur as a result of mechanical overload of the tissue combined with the action of other profibrotic factor or tissue damage.
• valvular heart disease accounts for a significant proportion of outpatient attendances in cardiology.
• Patient management usually involves monitoring for clinical symptoms or functional deterioration usually assessed by imaging studies, of which transthoracic echocardiography is the mainstay.
• Circulating biomarkers successfully incorporated into cardiology practice fall into the category of diagnostic biomarkers: troponin I and troponin T for myocardial infarction (Jaffe 2001, Cardiol Rev.9, 318-322) and brain natriuretic peptide (BNP) for heart failure (Cowie et al. 1997, Lancet. 8:1349-1353 Kazanegra 2001, J Card Fail. 1, 21-29).
• BNP brain natriuretic peptide
• Collagen I and collagen III are the major components of the myocardial matrix. Collagen 1 is synthesized in fibroblasts and released into the interstitial space which accounts for 80% of total cardiac collagen content. In mitral and tricuspid valves, the chordae tendineae is mostly composed of the collagenous tissue, 80% of which contains collagens and remaining 20% is composed of elastic fibers and layers of enodothelial cells on the basal lamina (Millington- Sanders et al, 1998, J Anat, 192:573-581; Yoshioka et al. 2008, Circulation, 118: 1694- 1696).
• Type 1 collagen is the most common form of the collagens in the vertebrates. It comprises up to 90% of the skeletons of the mammals and is also widespread all over the body. The importance of type I collagen for medical research is that it is involved in many human and animal diseases, including fibrosis, osteoporosis, cancer, atherosclerosis etc. In spite of or because of the fact that it is widely distributed in the body the different parts (degradation products) of type I collagen molecule are frequently utilized to monitor physiological changes in tissues as well as being used as diagnostic tools in various pathological conditions.
• Collagen I is synthesized and secreted by fibroblasts as procollagen type I precursor which contains additional sequences of amino acids at the amino-terminal (N) and carboxy- terminals (C) During maturation process, the N terminal and C terminal propeptides are cleaved to yield the triple helical monomers (Prockop et al. 1995, Ann Rev Biochem, 64:403-34; Lopez et al. 2007, J Am Coll Cardiol, 50:859 -67). Importantly, the carboxyterminal propeptide of type I procollagen (PICP) is released into the bloodstream during the synthesis of collagen type I.
• PICP carboxyterminal propeptide of type I procollagen
• PICP carboxyterminal telopeptide
• the serum PICP concentration was shown to be increased in patients with hypertensive heart disease (HHD) when compared with normotensive subjects (Querejeta et al. 2000, CirculationJOl : 1729-1735 ; Lo pez et al.2001, Circulation, 104:286-291 ; Querejeta et al.2004, Circulation, 110:1263- 8). Furthermore, higher PICP level was also reported in the serum of heart failure patients (Lo'pez et al. 2004, J Am Coll Cardiol, 43: 2028-2035 ; Martos et al.2007 Circulation, 115:888-895; Gonzalez et al 2009 Cardiovasc Res. 15:509- 518).
• HHD hypertensive heart disease
• hypertrophic cardiomyopathy which is genetically defined according to mutation of relevant genes. The study involved 38 subjects with pathogenic sarcomere mutations and overt hypertrophic cardiomyopathy, 39 subjects with mutations but no left ventricular hypertrophy.
• valvular heart disease generally implies the degenerative valve disease, which are basically age related. However, till date there is no data or demonstration of PICP correlation with even degenerative valve disease.
• Rheumatic heart disease [RHD] a special subset of heart diseases with rheumatic etiology in which specifically mitral valve is affected. The inventors for the first time demonstrate the increase in PICP with extensive tissue fibrosis. There are no RHD patients in western population; hence there is no PICP data available for RHD. Therefore, simply mentioning (or remarks) in some literature about possibility of PICP as a marker for valvular disease should not conflict with use of PICP for RHD marker specifically.
• PICP as a marker for heart diseases which is very non specific.
• the present invention depicts that PICP is specific for RHD because removal of the defective valve reduces its level nearly to the normal.
• the aim of this study was to examine whether PICP is altered in the plasma of patients with rheumatic valvular disease which accounts for the significant cause of hospital admission with cardiac problems in developing countries.
• the present invention discloses a biomarker for valvular heart diseases of rheumatic origin useful for the detection, characterization and treatment thereof.
• the objective of the present invention is to provide a biomarker useful for detection of valvular heart diseases of rheumatic origin, characterization and treatment thereof.
• One more object of the invention is to develop a kit to detect valvular heart diseases of rheumatic origin.
• another object of the present invention is to develop a protocol to screen anti-fibrotic drug.
• the present invention relates to a biomarker and use thereof for diagnosis of valvular heart disease of rheumatic origin.
• the present invention provides a biomarker that is carboxyterminal propeptide of type I procollagen (PICP) for diagnosis of valvular heart disease useful for the detection, characterization and treatment thereof.
• PICP type I procollagen
• the present invention is directed towards characterizing the fibrosis and remodelling of the human heart valve in the patients suffering from valvular heart diseases.
• the present invention additionally provides methods of determining the risk of valvular disease progression in a subject, comprising analyzing biological sample of a subject in need thereof, estimating the cut off value of PICP in said sample , and determining that the subject is at increased risk of valvular disease progression if the PICP level exceeds the predetermined threshold value.
• the present invention provides a method for detecting the presence of PICP in blood, comprising providing blood from a subject and contacting reagents related for detection of PICP with blood sample under such conditions that the reagents detect the absence or presence of PICP in blood.
• the present invention provides a method for detecting PICP in urine comprising providing urine from a subject and contacting the reagent required for the detection of PICP with urine under such conditions that the reagents detect the absence or presence of PICP in urine.
• the present method is useful for the management of valvular disease in a subject by constant monitoring blood or urine sample of the subject.
• the presence of PICP during collagen synthesis is indicative of prognosis of valvular disease in said subject.
• the present invention describes the mechanism of increased PICP in valvular heart disease patients which is primarily due to altered remodelling of the valve tissue.
• the present invention characterizes the valvular tissue remodelling in patients suffering from heart valve defects.
• the present invention provides methods for screening of drugs against fibrosis particularly by monitoring the level of PICP in a subject.
• the present invention provides methods for checking the efficiency of antifibrotic drugs not limited to valvular disease patients by monitoring the plasma PICP level in a subject.
• the present invention investigates whether marker of collagen biosynthesis is increased in valvular disease patients of rheumatic origin which could be used as biomarker.
• the present invention provides a method, wherein the level of PICP is used to predict the severity of valvular heart disease of rheumatic origin.
• the present invention provides a method wherein the diagnosis of valvular disease of rheumatic origin is performed using the said PICP level in blood or urine in said subject.
• the present invention provides a method, wherein the prognosis is performed using the said PICP in said blood or urine in said subject.
• the present invention provides a diagnostic kit for characterizing valvular disease in a subject, comprising diagnostic reagent comprising PICP as a probe.
• the present invention provides a method for diagnostic kit for characterizing valvular disease in a subject, comprising a diagnostic reagent comprising any component (s) during collagen biosynthesis as a probe.
• the present invention provides a method for screening antifibrotic drugs to valvular disease patients using PICP as a marker of fibrosis. In still another embodiment, the present invention provides a method wherein any component (s)/steps of collagen biosynthesis is utilized as a marker for screening antifibrotic drugs to valvular disease patients.
• the present invention provides a biomarker for valvular heart diseases of rheumatic origin useful for the detection, characterization, treatment and prognosis thereof in a subject, wherein the said biomarker is procollagen type 1 C peptide (PICP) represented by SEQ ID No. 1 and/or SEQ ID No. 2.
• PICP procollagen type 1 C peptide
• the invention also provides a diagnostic kit for characterizing valvular heart disease of rheumatic origin in a subject, comprising diagnostic reagents and the biomarker PICP as a probe.
• the invention further provides a method of detecting the presence of valvular heart disease of rheumatic origin in a subject comprising:
• PICPs are being selected from procollagen type 1 C peptide represented by SEQ ID No. 1 and/or SEQ ID No. 2, wherein presence of PICPs at a level more than 585.7 ng/ml is indicative of valvular heart disease of rheumatic origin.
• Procollagen type 1 C peptide represented by SEQ ID No. 1 and/or SEQ ID No. 2 useful in detection, characterization, treatment and prognosis of valvular heart diseases of rheumatic origin.
• the SEQ ID No. 1 comprises 246 amino acids representing amino acids 1219 to 1464 of the carboxy terminal propeptide of the Collagen alpha- 1(1) chain.
• the SEQ ID No. 2 comprises 264 amino acids representing amino acids 1103 to 1366 of the carboxy terminal propeptide of the Collagen alpha-2(I) chain.
• the Procollagen type 1 C peptide is useful in screening antifibrotic drugs.
• the present invention can be used to detect the presence of valvular heart disease of rheumatic origin in a subject, said method comprising the steps of:
• PICPs are being selected from procollagen type 1 C peptide represented by SEQ ID No. 1 and/or SEQ ID No. 2, wherein presence of PICPs at a level more than 585.7 ng/ml is indicative of valvular heart disease of rheumatic origin.
• the biological sample is collected from a group comprising tissue, blood and urine.
• the method is used to predict the severity of valvular heart disease of rheumatic origin.
• Figure 1A shows level of PICP in the plasma of normal human subjects (control) and valvular heart disease (VHD) patients before (VHD Pre Op) or after replacement of disease valve (VHD Post Op) with prosthetic device.
• Figure IB shows the receiver operating characteristic (ROC) curve of PICP in human subjects.
• Figure 1C shows level of PICP in the plasma of valvular heart disease patients with predominantly mitral stenotic lesions (MS), mitral regurgitant (MR) or mixed type of lesions (M).
• MS mitral stenotic lesions
• MR mitral regurgitant
• M mixed type of lesions
• Figure 2 shows detection of PICP in the blood sample of human subjects with valvular disease by high performance liquid chromatography (HPLC) .
• Figure 3 depicts a direct correlation of PICP level with severity of the valvular heart disease (reduced valve area) in mitral stenotic patients (MS).
• Figure 4 illustrates the level of PICP concentration in the urine of valvular disease patients
• Figure 5 illustrates histology showing valve architecture of human patients.
• Figure 5A and 5B depict the hematoxylene eosine stained sections of the mitral valve.
• Figure 5C shows the Masson's trichrome stained sections with excessive collagen deposition in the valve tissue.
• Figure 5D shows increase in collagen 1 fibrils in the disease valve of human subjects.
• the present invention demonstrates the increased level of procollagen type 1 C peptide (PICP) in the plasma of valvular heart disease patients of rheumatic origin.
• Collagen I is synthesized from a precursor molecule known as pro collagen I.
• the amino and carboxy terminal propetides are cleaved to yield the triple helical monomers which are ultimately converted to matured collagen fibrils.
• the free propeptides are released onto the blood stream after cleaving off from the triple helical monomers.
• the release of carboxyterminal propeptide of procollagen I (PICP) in blood is increased as the synthesis of collagen I is increased (Prockop et al 1998. Matrix Biol, 16:399-408; Trackman 2005.
• PICP carboxyterminal propeptide of procollagen I
• 2 forms of PICP of which one is Part of Collagen alpha-l(l) chain represented by SEQ ID No. 1 [P02452 (COIAIJHUMAN) Reviewed, UniProtKB/ Swiss-Prot] and the other is Part of Collagen alpha-2(I) chain represented by SEQ ID No. 2 [P08123 (C01A2_HUMAN) Reviewed, UniProtKB/Swiss-Prot].
• the present invention demonstrates a reduced level of the present biomarker in the valvular heart disease patients of rheumatic origin after replacement of the defective heart valve with prosthetic one.
• the present invention further describes the method of prognosis of the valvular heart disease of rheumatic origin by quantitatively measuring the present biomarker PICP in blood and/or urine sample.
• the source of the biomarker is removed from the body and accordingly the level of the biomarker is reduced. This is primarily due to the defective valve which is the main source of the high level of the present biomarker in the blood of the valvular disease patients.
• the present invention also describes statistical methods for qualifying this biomarker for diagnosis of valvular disease.
• the statistical analysis such as ROC curve reveals the cut off value of this biomarker for predicting valvular disease in a subject.
• the cut off concentration of this biomarker is found to be above the normal limit of control subjects.
• the present invention demonstrates the correlation of the present biomarker PICP with the severity of valvular heart disease of rheumatic origin.
• the heart disease of the mitral valve is characterized by a decrease in the valve area. Therefore, the valvular area is decreased with increasing severity of the valvular heart disease.
• the plasma PICP concentration is increased with the reducing valvular area.
• the present invention describes the marker of collagen synthesis and or fibrosis as a biomarker for valvular defects in heart.
• the level of collagen deposition is increased in the heart valve leading to fibrosis. Accordingly, increased level of PICP is released into the circulation with the increased synthesis of collagen in the heart valve.
• the present invention also illustrates the occurrence of extensive fibrosis in mitral valve of valvular heart disease patients. It is demonstrated that collagen I deposition is increased significantly in the mitral valve of the valvular disease patients.
• the present invention also describes the method of detection of valvular heart disease of rheumatic origin using urine samples.
• the possibility of defects in heart valve can also be predicted based on the PICP level in the urine samples of the patients.
• the increased level of PICP in the urine sample was recorded in 10 human patients suffering from valvular heart defects confirmed by echocardiography.
• the present invention also describes the positive correlation of PICP in plasma with the urine samples of the patients suffering from heart valve defects. Though PICP is diluted to 500 fold in urine compared to plasma, however it was detectable by the assay method. The detection of the present biomarker in urine is useful for practical purpose.
• Biomarker may be defined as a specific physical trait used to measure or indicate the effects or progression of a disease or condition. From a clinical perspective, biomarkers have a variety of functions, which corresponds to different stages in the development of a disease. Biomarkers can assist in the care of patients who have no apparent disease (screening biomarkers), those who are suspected to have disease (diagnostic biomarkers) and those with overt disease (prognostic biomarkers (Gerszten & Wang, 2008, Nature 451,949-952).
• valve refers to the valve in the heart that normally allows blood to flow through it in only one direction. There are four in a heart and they determine the pathway of blood flow through the heart.
• valvular heart disease is any disease process involving one or more of the valves of the heart (the aortic and mitral valves on the left and the pulmonary and tricuspid valves on the right).
• valvular disease used herein refers to human subjects with valvular heart disease.
• “Rheumatic fever” is an inflammatory disease that occurs following a Group A streptococcal infection, (such as strep throat or scarlet fever). Being submitted to be caused by antibody cross-reactivity that can involve the heart, joints, skin, and brain.
• the term “subject” refers to humans which were included in this study or any human which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
• blood refers to the blood of human subjects used for this study.
• plasma refers to "blood plasma of human subjects" which is the yellow liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid. It is mostly water (90% by volume) and contains dissolved proteins, glucose, clotting factors, mineral ions, hormones and carbon dioxide (plasma being the main medium for excretory product transportation). Blood plasma is prepared by spinning a tube of fresh blood containing an anti-coagulant in a centrifuge until the blood cells fall to the bottom of the tube. The blood plasma is then poured or drawn off.
• Fibrosis is the formation or development of excess fibrous connective tissue in an organ or tissue as a reparative or reactive process, as opposed to the formation of fibrous tissue as a normal constituent of an organ or tissue. Fibrosis used herein refers to the formation of fibrous tissue within the heart valve of human subjects.
• Collagen is a group of naturally occurring proteins. In nature, it is found exclusively in animals, especially in the flesh and connective tissues of mammals. It is the main component of connective tissue, and is the most abundant protein in mammals, making up about 25% to 35% of the whole-body protein content. Collagen, in the form of elongated fibrils, is mostly found in fibrous tissues such as tendon, ligament and skin, and is also abundant in cornea, cartilage, bone, blood vessels, the gut, and intervertebral disc. Echocardiography refers to electrocardiogram creating two dimensional picture of the cardiovascular system. "Echocardiography” used herein refers to imaging the heart of human subjects by cardiac ultrasound.
• TPR true positive rate
• FPR false positive rate
• ROC curve herein refers to the graphical plot of the sensitivity and specificity of the present biomarker between the control and valvular heart disease subjects.
• SBP refers to systolic blood pressure.
• DBP refers to diastolic blood pressure. The highest pressure within the bloodstream, occurring during each heart beat, because of the systole.
• Systolic pressure is peak pressure in the arteries, which occurs near the end of the cardiac cycle when the ventricles are contracting.
• Diastolic pressure is minimum pressure in the arteries, which occurs near the beginning of the cardiac cycle when the ventricles are filled with blood.
• An example of normal measured values for a resting, healthy adult human is 120 mmHg systolic and 80 mmHg diastolic (written as 120/80 mmHg).
• Systolic and diastolic arterial BPs are not static but undergo natural variations from one heart beat to another and throughout the day (in a circadian rhythm). They also change in response to stress, nutritional factors, drugs, disease, exercise, and momentarily from standing up. Sometimes the variations are large.
• LA refers to left atrium of the heart.
• the left atrium is one of the four chambers in the human heart. It receives oxygenated blood from the pulmonary veins, and pumps it into the left ventricle, via the atrioventricular valve.
• Atrial fibrillation AF or A-fib
• AF or A-fib is the most common cardiac arrhythmia (abnormal heart rhythm) and involves the two upper chambers (atria) of the heart. Its name comes from the fibrillating (i.e., quivering) of the heart muscles of the atria, instead of a coordinated contraction. It can often be identified by taking a pulse and observing that the heartbeats don't occur at regular intervals.
• ECG electrocardiogram
• AF electrocardiogram
• disorganized electrical impulses that originate in the atria and pulmonary veins leading to conduction of irregular impulses to the ventricles that generate the heartbeat.
• the result is an irregular heartbeat, which may occur in episodes lasting from minutes to weeks, or it could occur all the time for years.
• the natural tendency of AF is to become a chronic condition.
• LVIDd refers to left ventricular internal diastolic diameter. It is an echocardiographic parameter for monitoring dilation of the ventricle. LVIDd is useful for assessing left ventricular hypertrophy.
• LVIDs refers to left ventricular internal diameter at systole. It is an echocardiographic parameter for monitoring left ventricular hypertrophy.
• LVPW refers to left ventricular posterior wall .
• the left ventricle is cone shaped. Although the limits are imprecise it can be divided, except at the apex, into four walls, named classically septal, anterior, lateral, and inferoposterior. The basal part of the inferoposterior wall often branches upward and then becomes really posterior and for that reason it was named the posterior wall.
• Blood supply of posterior wall Coronary arteries- Circumflex (Cx), Obtuse Marginal (OM), Posterolateral (PL).
• the ST segment depression is "reciprocal” or “reflected” ST elevation from the back of the heart. T waves will become upright. In true posterior infarction, no abnormality is seen in the limb leads. However, posterior infarction is usually accompanied by infarction of another area, such as the inferior wall
• IVSD refers to interventricular septal thickness in diastole. It is an echocardiographic parameter for assessing left ventricular hypertrophy.
• EF refers the fraction of blood pumped out of ventricles with each heart beat.
• the term ejection fraction applies to both the right and left ventricles; one can speak equally of the left ventricular ejection fraction (LVEF) and the right ventricular ejection fraction (RVEF).
• RVEF and LVEF may vary widely from one another incumbent upon physiologic state.
• the volume of blood within a ventricle immediately before a contraction is known as the end-diastolic volume.
• the volume of blood left in a ventricle at the end of contraction is end-systolic volume.
• the difference between end-diastolic and end-systolic volumes is the stroke volume, the volume of blood ejected with each beat.
• Ejection fraction is the fraction of the end-diastolic volume that is ejected with each beat; that is, it is stroke volume (SV) divided by end-diastolic volume (EDV). Ejection fraction is commonly measured by echocardiography, in which the volumes of the heart's chambers are measured during the cardiac cycle. Ejection fraction can then be obtained by dividing stroke volume by end-diastolic volume as described above. Healthy individuals typically have ejection fractions between 50% and 65%.
• PASP refers to pulmonary artery systolic pressure. It is a measure of the blood pressure found in the pulmonary artery. It is an echocardiographic parameter.
• ACE inhibitors refer to angiotensin converting enzyme. ACE inhibitors block the conversion of angiotensin I to angiotensin II. With ACE inhibitor use, the effects of angiotensin II are prevented, leading to decreased blood pressure. They therefore lower arteriolar resistance and increase venous capacity; increase cardiac output and cardiac index, stroke work and volume, lower renovascular resistance, and lead to increased natriuresis (excretion of sodium in the urine). ACE inhibitors have been shown to be effective for indications other than hypertension even in patients with normal blood pressure.
• Beta blockers (sometimes written as ⁇ -blocker) is a class of drugs used for various indications, but particularly for the management of cardiac arrhythmias, cardioprotection after myocardial infarction (heart attack), and hypertension. As beta adrenergic receptor antagonists, they diminish the effects of epinephrine (adrenaline) and other stress hormones. Invented by Sir James W. Black in the late 1950s, Propranolol was the first clinically useful beta blocker; it revolutionized the medical management of angina pectoris and is considered to be one of the most important contributions to clinical medicine and pharmacology of the 20th century (Pritchett and Redfield, 2002. Mayo Clin. Proc. 77: 839-845).
• Beta blockers may also be referred to as beta-adrenergic blocking agents, beta-adrenergic antagonists, or beta antagonists.
• ACE inhibitors herein refers to angiotensin-converting enzyme inhibitors, that are used primarily in treatment of hypertension and congestive heart failure.
• Digoxin also known as digitalis, is a purified cardiac glycoside extracted from the foxglove plant, Digitalis lanata (Hollman 1985 Br. Heart J, 54: 258-261) Its corresponding aglycone is digoxigenin, and its acetyl derivative is acetyldigoxin. Digoxin is widely used in the treatment of various heart conditions, namely atrial fibrillation, atrial flutter and sometimes heart failure that cannot be controlled by other medication.
• the main pharmacological effects of digoxin are on the heart. Extracardiac effects are responsible for many of the adverse effects. It has mechanical effects as it increases myocardial contractility; however, the duration of the contractile response is just slightly increased. Overall, the heart rate is decreased, while blood pressure increases as the stroke volume is increased, leading to increased tissue perfusion. Myocardial efficiency improves due to improved hemodynamics, and the ventricular function curve is improved.
• Diuretic refers to any drug that elevates the rate of urination and thus provides a means of forced diuresis.
• diuretics There are several categories of diuretics. All diuretics increase the excretion of water from bodies, although each class does so in a distinct way.
• High ceiling diuretics are diuretics that may cause a substantial diuresis - up to 20% [1] of the filtered load of NaCl and water. This is huge when compared to normal renal sodium reabsorption which leaves only -0.4% of filtered sodium in the urine. Loop diuretics have this ability, and are therefore often synonymous with high ceiling diuretics.
• Loop diuretics such as furosemide, inhibit the body's ability to reabsorb sodium at the ascending loop in the kidney which leads to a retention of water in the urine as water normally follows sodium back into the extracellular fluid (ECF).
• ECF extracellular fluid
• Other examples of high ceiling loop diuretics include ethacrynic acid, torsemide and bumetanide.
• diuretics are used to treat heart failure, liver cirrhosis, hypertension and certain kidney diseases. Diuretics herein used to treat heart failure in VHD patients.
• NYHA class refers to the New York Heart Association (NYHA) Functional
• Classification It provides a simple way of classifying the extent of heart failure. It places patients in one of four categories based on how much they are limited during physical activity; the limitations/symptoms are in regards to normal breathing and varying degrees in shortness of breath and or angina pain:
• the blood samples and heart valve specimen were obtained from patients attending clinic of cardiology and cardio-thoracic surgery department of Institute of Postgraduate Medical Education and Research (IPGME&R), Seth Sukhlal Kanoria Memorial Hospital (SSKM), Rico, India between January to August 2010 with Institutional Review Board approval.
• Written informed consent was obtained from all subjects before participation in the study.
• Heart valve tissue samples were derived from patients undergoing replacement of diseased heart valves with prosthetic device after receiving donor's consent.
• the study population consisted of 80 patients of various ages (15-65 years) referred for cardio-thoracic assessment from the cardiology service.
• the patients with clinical and echocardiographic evidence of valvular heart disease were selected for the study.
• the data was recorded in a structured proforma and clinical examination was done by trained physicians and the information entered into a database.
• Echocardiography Two dimensional echocardiography with targeted M-mode and Doppler ultrasound measurements were performed in the patients by experienced cardiologists at the SSKM hospital, Rico, India. All measurements were made by blinded observers. Left atrial dimension and left ventricular dimension both at systole and diastole were recorded. The mitral valve area (MVA) were measured by planimetry as well as pressure half time (PHT) method and the mean of these two readings were taken in patients with mitral valve disease. Pulmonary artery systolic pressure (PASP) was also measured in patients. Left ventricular ejection fraction (EF) was calculated by the Teichholz method. None of the patients studied left ventricular systolic dysfunction as assessed by an ejection fraction ⁇ 50%. Doppler ultrasound measurements were studied to record pressure gradients across valves and to assess the haemodynamic severity of lesions. Valve lesions were graded as mild, moderate or severe at final impression.
• the baseline characteristics of the patients are demonstrated in Table 1.
• Out of 80 valvular disease patients, stenotic (mitral stenosis), regurgitant (mitral regurgitant) and mixed lesions were present in 32, 23 and 25 subjects, respectively.
• About 51 % of patients (41 out of 80 patients) were female.
• 52.5 % patients had definite history of rheumatic fever as documented in their medical record or as suggested by history of large joint arthritis, carditis, fever and history of documented penicillin prophylaxis.
• the major clinical and demographic variables were comparable between the three subgroup of patients as assessed by one-way ANOVA.
• 61 % patients were NYHA class I symptomatic.
• Peripheral venous blood samples were drawn during clinical assessment and immediately subjected to plasma isolation. Each sample was centrifuged for 10 minutes at 4°C. The plasma was then separated into aliquots and stored at -80°C before analysis. Duplicate measurements were performed and averaged.
• the plasma PICP concentration was determined by enzyme immunoassay (EIA) with a monoclonal anti-PICP antibody using commercial assay system (Takara Bio Inc).
• EIA enzyme immunoassay
• the intra assay and inter-assay variations for determining PICP concentrations were 6 % and 5% respectively.
• Plasma was isolated from blood and diluted to 10 times with the diluent solution supplied with the PICP assay kit. Duplicated determinations of all samples and standards were performed.
• Immunological reaction 100 ⁇ of antibody-peroxidase labelled conjugate solution was transferred into each well, and subsequently 20 ⁇ sample (plasma) or standard PICP protein solution was added. The microtiter plate was sealed and mixed well and then kept undisturbed for 3 hours at 37°C.
• Data are expressed as mean ⁇ SEM for continuous variables, while frequencies summarize categorical variables. Normal distribution of all continuous variables was tested using the 1- sample Kolmogorov-Smirnov test. One way Analysis of Variance (ANOVA) was used to evaluate differences in continuous variables between the three groups of valve disease patients. Otherwise a non parametric test (Kruskal Wallis test) was used. Comparisons between categorical variables were done by x 2 Fisher's exact test when necessary. Differences within the group of patients were tested by a Student's t test for paired data once normality was demonstrated. Differences between the control and patient groups were tested by a Student's t test for unpaired data once normality was demonstrated. The correlation between continuously distributed variables was tested using Pearson's correlation coefficient r.
• Partial correlation coefficients were calculated to assess the relationship between echocardiographic parameters and biochemical markers. ROC curves were plotted to assess the usefulness of changes in PICP levels in predicting valvular heart disease. A probability value of ⁇ 0.05 was considered statistically significant.
• Figure 1A demonstrated that the level of plasma PICP was increased in valvular heart disease (VHP Pre Op patients of rheumatic origin (1404 ⁇ 81 ng/ml) compared to control (352 ⁇ 19). Student's T Test analysis reveals / 0.0001. The level of PICP was significantly reduced one month after the defective valve was replaced (VHD Post Op) with prosthetic one (580 ⁇ 118 ng/ml). To test the specificity and sensitivity of the biomarker for predicting valvular disease, ROC curve analysis was performed.
• ROC analysis is a useful tool for evaluating the performance of diagnostic tests and more generally for evaluating the accuracy of a statistical model that classifies subjects into 1 of 2 categories, diseased or non- diseased (Lloyd 1998. J Am Stat Assoc. 93: 1356-1364; Pepe 2003. The Statistical Evaluation of Medical Tests for Classification and Prediction. Oxford, UK: Oxford University Press; Zou 2007, Circulation 6:654-657).
• Example 2 HPLC determination of PICP presence in human blood.
• Eluant A Water containing 0.5% TFA.
• Eluant B Acetonitrile containing 0.5% TFA Injection Volume: 5 ⁇ 1. Run Time: 55 min. Detection: 280nm.
• the HPLC profile of PICP is presented in Figure 2.
• the peak of standard PICP was detected at run time 22.91 min and similarly a peak was also detected in valvular heart disease plasma at run time 22.65 min.
• Example 3 Monitoring the effect of medications on plasma PICP in valvular heart disease patients of rheumatic origin.
• the severity of the valvular disease in MS patients was examined by measuring the valve area by echocardiography as described above.
• the mitral valve area is expressed in sq cm. Because of the extensive stenosis and fibrosis the mitral valve area is reduced in the valvular heart disease patients with stenotic lesions (MS). Therefore, it is considered as the marker of severity of valvular disease in MS patients (Maganti et al 2010, Mayo Clin Proc. 85:482- 510).
• the plasma PICP is increased in MS patients.
• r 0.4196
• Urine was collected from the patients admitted to the cardiothoracic department of SSKM hospital, Marie, India for valve replacement. 100 ⁇ urine sample was used for the estimation of PICP. The level of PICP was estimated as described above. The level of PICP in patient with valvular disease was higher than the control. Student's T test for unpaired data reveals p ⁇ 0.05. The results are presented in Figure 4.
• Example 6 Histochemical examination of collagen deposition (fibrosis) in human heart valve
• Mitral tissue samples from valvular heart disease patients undergoing valve replacement with prosthetic one were collected arid fixed in 10% formalin.
• Mitral valve from control (dead) subject during post mortem analysis in SSKM hospital, India was also collected and fixed in 10 % formalin.
• the samples were then embedded in paraffin and 5 ⁇ thick sections were prepared according to the standard procedure. The sections were either stained with hematoxylin - eosin (sigma chemical Co., St Louis, MO, USA) as described earlier (Ghose Roy et al 2007, Matrix Biol.26:269-279) or subjected to Masson Trichrome staining to examine fibrosis.
• the sections were deparaffinised by dimethyl benzene and soaked into a series of gradient concentration from 100 to 75% of alcohol. The sections were then put into haematine solution, dyed for 5 min, and washed with acid water twice for 30 s. After being washed in distilled water for 1 h, they were dewatered in 70 and 90% alcohol for 10 min. The sections were subjected to eosine staining for 2-3 min, dewatered, and mounted with neutral gum. The representative images of valvular histology are shown in Figure 5A. The gross examination of mitral valve showed thickening of valve leaflet with irregular valve margin.
• Picrosirius red staining of the valvular tissue was performed to quantitate collagen content and alignment and inner type of collagen fibres (Elizabeth et al 2008, Circulation, 1 18:S243- S249). When picrosirius stained sections are viewed under polarized light, the collagen I appears red and collagen III appears green (Elizabeth et al 2008, Circulation, 118:S243-S249; Whittaker et al. 1994, Basic Res. Cardiol. 89: 397-410). Mitral tissue samples from valvular heart disease patients undergoing valve replacement were sectioned and processed as described above in example 6 and the sections were stained with picrosirius red solution (0.1 % sirius red F3B solution in picric acid). The picrosirius stained sections were imaged by confocal microscope and the results are presented in Figure 5D showing the increase in red lines of collagen I fibers in disease valve compared to the normal valve.
• the main advantage of the present invention is to provide a biomarker for valvular heart disease patients useful for the diagnosis, characterization and treatment of valvular heart disease.
• Another advantage of the present invention is to provide an easy and fast biochemical method for diagnosis, detection and characterization of valvular heart disease using blood or urine sample of the subject.
• the current and only available method of diagnosing valvular heart disease is the imaging of defective heart valve by echocardiography which requires sophisticated instrument and specialized trained personnel.
• the present biomarker is detectable by easy and simple biochemical procedure and therefore accessible to mass population.
• Still another advantage of the present invention is the demonstration of a cut off value of the said biomarker which is useful for predicting the valvular heart disease with maximum precision (with about 99 % specificity and approximately 99% sensitivity).
• Yet another advantage of the present invention is to demonstrate the cause effect relationship between the increased biomarker and the defective valve in valvular heart disease patients.
• it describes the method of histological alteration of the heart valve as the main cause of increased biomarker in the plasma/urine of valvular heart disease patients.
• Another advantage of the present invention is prediction of the severity of the valvular heart disease useful for the decision making process for surgical intervention for correction / replacement of the defective valve.
• Yet another advantage of the present invention is to provide a method for screening of therapeutics for the treatment of defective heart valve in valvular heart disease patients.

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