HK1160681B - In vitro-method for the diagnosis, prognosis, monitoring and therapy follow-up of disorders associated with the metabolic syndrome, a cardiovascular disease and/or insulin resistance - Google Patents

Description

In vitro method for the diagnosis, prognosis, monitoring and therapy follow-up of disorders associated with metabolic syndrome, cardiovascular diseases and/or insulin resistance

Subject of the present invention is an in vitro method for the diagnosis, prognosis, monitoring and therapy follow-up of disorders associated with metabolic syndrome, cardiovascular diseases and/or insulin resistance, comprising determining the relative level of one or more cardiovascular markers and uses thereof.

Hypertension is often accompanied by obesity, hypersecretion of insulin and insulin resistance. This type of hypertension is characterized by sodium retention, increased intravascular volume, and increased stroke volume and output (Messerli FH et al, 1981, "Obesity and primary hypertension: hemodynamics, intravascular volume, sodium excretion, and plasma renin activity" (Obesity and essential hypertension. Hemodynmics, intravascular volume, sodium evolution, and plasma reactivity), Arch Intern Med 141: 81-5; Stelfox HT et al, 2006 "Hemodynamic monitoring of body mass index effects on cardiac output and stroke volume in obese patients" (Hemodamic mon addressing in activities: the impact of the body mass of cardiac on cardiac output and stroke volume), Criti Care 34: 1243-6). In response to an increase in the stress of the blood vessel wall associated with heart failure or acute myocardial ischemia, Atrial Natriuretic Peptide (ANP) and Brain Natriuretic Peptide (BNP) are synthesized as prohormones in the Cardiac muscle cells, which are cleaved into ANP and BNP and N-terminal proANP and N-terminal proBNP (NT-proBNP), respectively (Ruskoaho H, 2003, "Cardiac hormones as diagnostic tools in heart failure" (cardio hormones as diagnostic in heart failure), Endocr Rev 24: 341-56; Potter LR et al, 2006, "Natriuretic peptides, their receptors, and cyclic guanylic acid-dependent signaling functions" (natural peptides, theirside monophosphators, and cyclic guanylic oxyphenon-dependent signaling functions), Endocr Rev 27: 47). High natriuretic peptide levels are a new promising Cardiovascular (CV) risk marker and have been associated with high Blood (BP), left ventricular hypertrophy, and proteinuria (Olsen MH et al, 2005, "N-terminal pro-natriuretic peptide is inversely related to metabolic cardiovascular risk factors and metabolic syndrome" (N-terminal pro-prandial biological peptides and metabolic syndrome), Hypertension 46: 660-6). Several large studies have in fact observed an independent association of mortality rates with natriuretic peptide levels (Wang TJ et al, 2004 "Plasma natriuretic peptide levels with cardiovascular events and risk of death" (Plasma urinary peptide levels and the risk of cardiovascular events and death), "N Engl J Med 350: 655-63; Bibbins-Domingo K et al, 2007" N-terminal fragment of brain-type natriuretic peptide prohormone (NT-proBNP), cardiovascular events and mortality rates in patients with stable coronary heart disease "(N-terminal fragment of the same, cardiovascular tissue-type urinary peptide), Jadiatascus large peptides (NT-proBNP), and mortalities in tissues with cardiovascular events and death probability" (N-terminal albumin), Jadiat protein responses: Ki-76; urine peptide levels in human patients with cardiovascular events and cardiovascular events 2005-brain death predicting protein responses and cardiovascular events "(N-terminal albumin, urine-peptide responses in human urinary protein responses 2005-cardiovascular events and death predicting urinary protein levels in patients with coronary heart disease: (N-cardiovascular events and death: Kirtin-urinary albumin) (N-peptide responses 2005-cardiovascular events and death predicting urinary protein responses in patients with cardiovascular events and death) (N-cardiovascular events 2005-pro-cardiovascular events and death in patients with stable coronary heart disease) natural peptides, C-reactive proteins, and urea album levels as predictors of mortalityand cardiovascular events in adapters), Jama 293: 1609-16; von haehling et al, 2007 "Comparison of predicted survival rates of Central atrial natriuretic peptide with N-terminal B-type natriuretic peptide in patients with chronic heart failure" (composite of central pro-atrial natriuretic peptide with N-terminal pro-B-type natriuretic peptide with compressing cardiac polypeptide), J Am Coll Cold card 50: 1973-80).

A recent Study described suppression of natriuretic peptide levels in obesity (Wang TJ et al, 2004 "Effect of obesity on plasma natriuretic peptide levels" (Impact of object on biological peptide levels), Circulation 109: 594-600; Das SR et al, 2005 "Effect of body weight and body composition on circulating levels of natriuretic peptide from Dallas Heart Study" (Impact of body mass and body composition on circulating peptides of biological peptides: results from the Dallas Heart Study), Circulation 112: 2163-8). Because obesity is associated with salt retention and increased cardiac output, it is expected that it will produce high natriuretic peptide levels. Obesity appears to have an opposite effect-a phenomenon that appears counterintuitive and is attributed to non-hemodynamic factors. Therefore, Wang and colleagues postulated that this inverse relationship may be due to increased natriuretic peptide clearance receptor (NPR-C) expression by adipose tissue, leading to increased clearance of natriuretic peptides in obese subjects (Wang TJ et al, 2004, "effect of obesity on plasma natriuretic peptide levels" (Circulation 109: 594-600). However, Das and colleagues determined lean and fat mass by DEXA in the Dallas Heart Study (Dallas Heart Study), and found a correlation between lower BNP levels and lean, but not fat mass (Das SR et al, 2005 "weight and body composition effects on circulating levels of natriuretic peptides:" results from Dallas Heart Study "(Impact of body mass and body composition on circulating levels of natural peptides: results from Dallas Heart Study), Circulation 112: 2163-8).

Several studies have observed associations of natriuretic peptides with other elements of the metabolic syndrome (Olsen MH et al, 2008 "prediction of Cardiovascular risk by age and sex through N-terminal pro-natriuretic peptide and high sensitivity C-reactive protein" (Cardiovascular risk by N-terminal pro-pulmonary promoter and high sensitivity protein after fermented by and sex), J Hypertens 26: 26-34; Wang TJ et al, 2007 "Association of plasma natriuretic peptide levels with metabolic risk factors in ambulatory individuals" (Association of plasma natriuretic peptides with metabolic risk factors), Circulation 115: 1345-53). In the Framingham cardiac study, large waist circumference, high triglycerides, low HD and high fasting glucose (Wang TJ et al, 2007 "Association of plasma natriuretic peptide levels with metabolic risk factors in ambulatory individuals" (Association of plasma natural peptides with metabolic risk factors in amyloid disorders), Circulation 115: 1345-53) are associated with lower plasma ANP levels and to a somewhat lower extent with lower BNP levels. In the Danish study, the association of BNP with BMI, insulin, glucose, triglycerides and Hypertension was observed (Olsen MH et al, 2005, "N-terminal pro-natriuretic peptide is inversely related to metabolic cardiovascular risk factors and metabolic syndrome" (N-tertiary pro-brain systemic peptide is involved in metabolic syndrome and the metabolic syndrome), Hypertension 46: 660-6). Although these studies demonstrate a close association of natriuretic peptides with several properties of the metabolic syndrome, the mechanisms behind these associations remain elusive.

Nutritional methods for lowering Postprandial insulin levels are very effective for lowering blood pressure (Appel LJ et al, 2005 "Effect of protein, monounsaturated fats and carbohydrate uptake on blood pressure and serum lipids:" results of OmniHeart random test "(Effects of protein, monounsaturated fatty, and carbohydrate intake on blood pressure and serum lipids: results of The dietary OmniHeart random trial, Jama 294: 2455-64), and it is believed that Postprandial status plays an important role in The early stages of metabolic Syndrome and in particular in The development of Atherosclerosis (Handfeld M et al, 1999" Postprandial plasma glucose is an independent risk factor for increased carotid-media thickness in non-diabetic individuals "(Postprandial plasma glucose is an independent risk factor for increased carotid-media thickness:" challenge of metabolic Syndrome: "manual-clinical Syndrome"; "metabolic Syndrome-metabolic Syndrome"; "see patent No. 35-;" metals-induced Syndrome ";" calcium-induced Syndrome ";" see patent Syndrome ";" calcium-induced Syndrome ";" calcium supplement and "see FIGS. 144), horm Metab Res 39: 625-6).

The object of the present invention is the diagnosis, prognosis and/or monitoring and/or therapy follow-up of disorders associated with the metabolic syndrome and/or diseases associated with the cardiovascular system and/or insulin resistance in a subject, wherein the relative change of one or more cardiovascular markers in said subject is determined.

It has been found that insulin induces cardiovascular markers, in particular MR-proANP_53-90And in insulin sensitivity with natriuretic peptides, in particular MR-proANP_53-90There is a significant positive correlation between the relative changes in. This correlation provides a direct link from insulin resistance to hypertension in the metabolic syndrome. The discovery of this correlation is further enhancedMethods for the diagnosis, prognosis and/or monitoring and/or therapy follow-up of disorders related to the metabolic syndrome and/or diseases related to the cardiovascular system and/or insulin resistance in a subject have been generated.

Thus, a subject of the present invention is an in vitro method for the diagnosis, prognosis and/or monitoring/therapy follow-up of metabolic and/or cardiovascular and/or insulin-resistant disorders in a subject, said method comprising:

-providing a sample from the subject,

-determining the relative level of one or more cardiovascular markers in said sample,

-using the relative level of said one or more cardiovascular peptides for diagnosis, prognosis and/or monitoring/therapy follow-up of metabolic and/or cardiovascular and/or insulin resistant disorders in said subject.

In a preferred embodiment of the invention, the postprandial relative level of one or more cardiovascular markers is determined.

According to the present invention, the "relative level" is defined as a relative concentration based on a base value, which can be mathematically expressed as follows:

wherein X is the change in the level of one or more cardiovascular markers expressed as a percentage, [ postprandial ] and [ basal ] are postprandial and basal levels, respectively.

It can also be specified as a percentage change in concentration from the basal value, which can be expressed mathematically as follows:

or

In a preferred embodiment of the method of the invention, the postprandial relative level of one or more cardiovascular markers is determined.

In the context of the present invention, the term "postprandial" refers to the period of time after food (nutrients) and/or beverages and/or drugs are ingested or administered to a subject, which may also be used in the context of diet and/or nutritional therapy.

In the case of glucose tolerance or glucose challenge tests, postprandial levels may also be determined. For example, an oral glucose tolerance test (oGTT) can be performed by oral administration (after an overnight fast) of 75g of glucose (usually provided as a glucose solution), which should be drunk within 5 minutes. Blood samples were drawn at baseline (before glucose was used) and at various time intervals, e.g., 15, 30, 60, 90, 120, or 180 minutes.

Glucose tolerance can also be determined using the intravenous glucose tolerance test (ivGTT). After an overnight fast, a glucose dose adjusted for body weight (e.g., 0.3g/kg up to about 25g) is administered via catheter, for example, into the forearm. Blood samples were drawn at baseline (before administration of glucose) and at various time intervals, e.g., 1, 2, 3, 5, 7, 10, 15, 20, 30, 45, 60, 90, 120, or 240 minutes.

In addition, postprandial levels can also be measured to investigate and quantify insulin resistance in the case of hyperinsulinemia, normal blood glucose clamp test. This test measures the amount of glucose required to compensate for increased insulin levels so as not to cause hypoglycemia. Through the peripheral vein at a rate of m²Insulin is infused at a rate of 10-120mU per minute. To compensate for insulin infusion, 20% glucose was infused to maintain blood glucose levels between 5 and 5.5 mmol/l. The glucose infusion rate is determined by examining blood glucose every 5 to 10 minutes. The rate of glucose infusion during the last 30 minutes of the experiment determines the pancreasIslanding sensitivity. If high levels (7.5mg/min or higher) are obtained, the patient is sensitive to insulin. Very low levels (4.0mg/min or less) indicate that the body is resistant to the action of insulin. Levels between 4.0 and 7.5mg/min are not critical and indicate "impaired glucose tolerance", an early sign of insulin resistance.

In the context of the present invention, a cardiovascular marker refers to a peptide and/or protein providing a diagnosis and/or prognosis and/or monitoring of a condition associated with metabolic syndrome (e.g. myocardial infarction, coronary artery disease, heart failure, type II diabetes, obesity, hypertension), said peptide and/or protein being selected from the group consisting of natriuretic peptides (e.g. atrial natriuretic peptide, brain natriuretic peptide), adrenomedullin, endothelin, vasopressin. The basal level of cardiovascular markers depends on a variety of factors, such as the age, body mass index, genetic susceptibility/family history to certain conditions, sex and ethnic background of the patient, and factors related to the overall health status of the subject. However, the present formula is based on the opposite finding that the relative change from the basal level of the cardiovascular marker to the postprandial level of the cardiovascular marker is substantially independent of these factors and strongly dependent on the food and/or beverage and/or nutritional therapy and/or medication administered to the subject.

The cardiovascular marker is preferably selected from ANP, BNP, ET-1, ADM, AVP and fragments thereof and prohormone and fragments of said prohormone.

In a particularly preferred embodiment of the method of the invention, the relative level of the one or more cardiovascular markers is determined using an assay with a lower detection limit of 1nmol/L or less, preferably 100pmol/L or less, more preferably 10pmol/L or less, more preferably 1pmol/L or less, most preferably 0.5pmol/L or less. Furthermore, the assay method preferably has an inter-assay accuracy of < 30%, more preferably < 20%, within the normal range. Furthermore, the determination method preferably has an in-determination accuracy of < 10%, more preferably < 5%, within the measurement range. As used herein, "intra-assay accuracy" refers to the deviation between measured values in a single lot of a particular assay method, and "inter-assay accuracy" refers to the deviation between measured values in multiple lots of a particular assay method, which may be performed at different locations, on different days, or by different operators. Thus, the terms mentioned above refer to a measure of the reproducibility of the results obtained using the assay method in question. "measurement range" refers to the upper and lower detection limits of the assay.

The assay is at least sensitive enough to detect changes and deterioration as increases and decreases. For healthy subjects, the normal range for a given biomarker corresponds to a gaussian distribution.

Furthermore, an embodiment of the invention is an in vitro method of the invention, further comprising:

a) determining a basal level of one or more cardiovascular markers in the subject,

b) determining postprandial levels of the one or more cardiovascular markers,

c) calculating the relative level of one or more cardiovascular markers from the values obtained in steps a and b.

Whereby the ingestion, ingestion or other form of administration of said food and/or beverage and/or diet and/or nutritional therapy and/or medication is correlated with its effect on the level of said one or more cardiovascular markers in said patient by a relative decrease in said level.

In the context of the present invention, the term "basal level" refers to the individual level of a certain compound, molecule or metabolite, such as a cardiovascular peptide, that a subject has in the absence of an influence of factors such as food, beverages, diet, nutritional therapy or medication. The basal levels were determined separately for each subject after approximately 12 hours of fasting. Fasting herein means that the subject does not eat or consume food, beverages or medicines for a period of time other than water and/or medication as necessary.

When referred to herein, an "assay method" or a "diagnostic assay method"It can be any type of assay used in the field of diagnostics. Such an assay method may be based on the binding of the analyte to be detected to one or more capture probes having a certain affinity. For the interaction between the capture molecule and the target molecule of interest, the affinity constant is preferably greater than 10⁸M^-1。

In this context, the term "fragment" when referred to in the context of a prohormone or other peptide refers to a smaller protein or peptide that can be derived from a larger protein or peptide and thus comprises a partial sequence of the larger protein or peptide. The fragments may be generated from larger proteins or peptides by saponification of one or more of their peptide bonds.

"fragments" of the cardiovascular markers pro ANP, proBNP, proET-1, proADM and pro-AVP preferably refer to fragments of at least 6 amino acids in length, more preferably at least 12 amino acid residues in length. Such fragments preferably can be detected using the immunoassay methods described herein.

In the context of the present invention, a "capture molecule" is a molecule that can be used to bind a target molecule of interest, i.e. an analyte (i.e. a cardiovascular peptide in the context of the present invention), from a sample. Thus, the capture molecules must be appropriately shaped in terms of spatial and surface characteristics such as surface charge, hydrophobicity, hydrophilicity, presence or absence of lewis donors and/or acceptors, etc., to specifically bind the target molecules of interest. Thus, binding may be mediated by, for example, ionic, van der Waals, π - π, hydrophobic or hydrogen bonding interactions or a combination of two or more of the above-mentioned interactions between the capture molecule and the target molecule of interest. In the context of the present invention, the capture molecule may for example be selected from a nucleic acid molecule, a carbohydrate molecule, an RNA molecule, a protein, an antibody, a peptide or a glycoprotein. Preferably, the capture molecule is an antibody, including fragments thereof having sufficient affinity to a target or molecule of interest, and including recombinant antibodies or recombinant antibody fragments, as well as chemically and/or biochemically modified derivatives of said antibodies or fragments thereof derived from a variant chain of at least 12 amino acids in length.

Preferred detection methods include immunoassays in various formats, such as Radioimmunoassays (RIA), chemiluminescent and fluorescent immunoassays, enzyme-linked immunoassays (ELISA), Luminex-based bead arrays, protein microarray assays, and rapid detection formats such as immunochromatographic strip tests.

The assays may be homogeneous or heterogeneous assays, competitive and non-competitive sandwich assays. In a particularly preferred embodiment, the assay is in the form of a sandwich assay, which is a non-competitive immunoassay, wherein the molecule to be detected and/or quantified is bound to a first antibody and to a second antibody. The first antibody may be bound to a solid phase such as a bead, well or other surface of a container, chip or strip, and the second antibody is an antibody labeled, for example, with a dye, radioisotope, or reactive or catalytically active moiety. The amount of labeled antibody bound to the analyte is then measured by a suitable method. The general compositions and procedures involved in a "sandwich assay" are well established and well known to those skilled in the art. (Handbook of immunoassays (the Immunoassay Handbook), eds. by David Wild, Elsevier LTD, Oxford; third edition (5.2005), ISBN-13: 978-.

In a particularly preferred embodiment, the assay method comprises two capture molecules, preferably antibodies, both present as a dispersed phase in a liquid reaction mixture, wherein a first label component is attached to the first capture molecule, wherein the first label component is part of a fluorescence or chemiluminescence quenching or amplification based labeling system, and a second label component of the labeling system is attached to the second capture molecule such that upon binding of the two capture molecules to the analyte a measurable signal is generated allowing detection of a sandwich complex formed in a solution comprising the sample.

More preferably, the labeling system comprises a combination of a rare earth cryptate or a rare earth chelate with a fluorescent dye or a chemiluminescent dye, especially a dye of the cyanine type.

In the context of the present invention, fluorescence-based assay methods comprise the use of dyes which may, for example, be selected from FAM (5-or 6-carboxyfluorescein), VIC, NED, Fluorescein Isothiocyanate (FITC), IRD-700/800, cyanine dyes such as CY3, CY5, CY3.5, CY5.5, Cy7, xanthene, 6-carboxy-2 ', 4 ', 7 ', 4, 7-Hexachlorofluorescein (HEX), TET, 6-carboxy-4 ', 5 ' -dichloro-2 ', 7 ' -dimethoxyfluorescein (JOE), N, N, N ', N ' -tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-Rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (6), Rhodamine, rhodamine green, rhodamine red, rhodamine 110, BODIPY dyes such as BODIPY TMR, oregon green, coumarins such as umbelliferone, benzimidazoles such as Hoechst 33258; phenanthridines such as texas red, jagewasas yellow, Alexa Fluor, PET, ethidium bromide, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes and the like.

In the context of the present invention, the chemiluminescence-based assay method involves the use of a dye according to the physical principles of chemiluminescent materials, described in Kirk-Othmer Encyclopedia of chemical technology, fourth edition, J.I.Kroschwitz, executive eds, M.Howe-Grant, edited by John Wiley & Sons, 1993, vol.15, p.518-562, incorporated herein by reference, including the citation on page 551-562. The preferred chemiluminescent dye is an acridinium ester.

In another preferred embodiment, the basal level of the cardiovascular marker and the postprandial level of the cardiovascular marker in the patient are determined using an immunoassay.

As outlined above, the diagnostic assay may be any type of assay applied in the field of diagnostics, including but not limited to assays based on enzymatic reactions, chemiluminescence, fluorescence or radioactive chemicals. Preferred detection methods include strip assays, radioimmunoassays, chemiluminescent and fluorescent immunoassays, immunoblot assays, Enzyme Linked Immunoassays (ELISA), Luminex-based bead arrays, and protein microarray assays. The type of assay method may also be microplate-based, chip-based, bead-based, where the biomarker proteins may be attached to a surface or in solution. The assay methods may be homogeneous or heterogeneous assays, sandwich assays, competitive and non-competitive assays. (Handbook of immunoassays (The Immunoassay Handbook), eds David Wild, Elsevier LTD, Oxford; third edition (5.2005), ISBN-13: 978-.

In the most preferred embodiment of the invention, immunoassays are used as described in (Morgethaler NG et al, 2004Clin chem 50: 234-6).

In a particularly preferred embodiment of the in vitro method of the invention, one cardiovascular marker is proANP. More preferably, one marker is midstream proANP. Most preferred is a mid-range proANP_53-90。MR-proANP_53-90Refers to a mid-atrial natriuretic peptide precursor comprising amino acids 53 to 90 of atrial natriuretic peptide precursor (proANP).

In the context of the present invention, AVP refers to arginine vasopressin (vasopressin) or a fragment thereof or a precursor thereof or a fragment of said precursor. A preferred fragment of the AVP precursor is C-terminal proAVP (CT-proAVP or copeptin). In the context of the present invention, CT-proAVP_107-145(or CT-pre-proAVP_126-164) Are particularly preferred cardiovascular markers.

In the context of the present invention, ADM refers to adrenomedullin or a fragment thereof or a precursor thereof or a fragment of said precursor. A preferred fragment of ADM precursor is middle-stage proADM (MR-proADM). In the context of the present invention, MR-proADM_24-71(or MR-preproADM_45-92) Are particularly preferred cardiovascular markers.

In the context of the present invention, ET-1 refers to endothelin 1 or fragments thereof or precursors thereof or fragments of said precursors. ET-A preferred fragment of the precursor of 1 is C-terminal-proET 1(CT-proET 1). In the context of the present invention, CT-proET-1_151-195(or CT-preproeT-1_168-212) Are particularly preferred cardiovascular markers.

BNP in the context of the present invention refers to brain natriuretic peptide or a fragment thereof or a precursor thereof or a fragment of said precursor. A preferred fragment of the BNP precursor is N-terminal proBNP (NT-proBNP). NT-proBNP is a particularly preferred cardiovascular marker in the context of the present invention.

In a preferred embodiment of the invention, the one or more cardiovascular markers are selected from proANP or a fragment thereof (preferably MR-proANP, more preferably MR-proANP)_53-90) pro-BNP or a fragment thereof (preferably NT-proBNP), pro-ET-1 or a fragment thereof (preferably CT-proET1, more preferably CT-proET-1)_151-195) pro-AVP or a fragment thereof (preferably copeptin, more preferably CT-proAVP)_107-145) pro-ADM or a fragment thereof (preferably MR-proADM, more preferably MR-proADM)_24-71)。

According to the invention, preferably at least one marker is a peptide selected from the group consisting of: natriuretic peptides, endothelin-1, vasopressin, adrenomedullin, and pro-peptides thereof or fragments thereof of at least 3 amino acids, preferably 5 or more, more preferably 6 or more, more preferably 7 or more, more preferably 10 or more, more preferably 12 or more, more preferably 15 or more, most preferably 20 or more amino acids.

In a very preferred embodiment of the invention, the at least one cardiovascular marker is Atrial Natriuretic Peptide (ANP) or a pro-peptide and fragments thereof of at least 3 amino acids, preferably more than 5, more preferably more than 6, most preferably more than 7 amino acids.

In a particularly preferred embodiment, the at least one cardiovascular marker is MR-proANP_53-90Or a fragment thereof of at least 3 amino acids, preferably 5 or more, more preferably 6 or more, most preferably 7 or more amino acids.

The invention also relates to a particular embodiment of the in vitro method of the invention, wherein additionally the level of one or more other markers or clinical parameters having predictive value for the classification of the predisposition of said subject to a metabolic and/or cardiovascular disorder is determined, wherein a clinical parameter may be any parameter capable of influencing said predisposition, such as age, sex, past history of disease, in particular hypertension, obesity, in particular trunk obesity, body mass index, genetic predisposition/family history, ethnic background, patient's liking influencing said predisposition, such as smoking, drinking, diet, exercise or medication.

In another preferred embodiment of the in vitro method of the invention, the postprandial level of said one or more cardiovascular markers is determined within 4 hours, preferably within 2 hours, more preferably between 15 and 60 minutes after administration of said food and/or beverage and/or diet and/or nutritional therapy.

In another preferred embodiment of the in vitro method of the invention, the postprandial effect of the food and/or beverage and/or diet and/or nutritional therapy on the relative level of one or more cardiovascular peptides is monitored over an extended period of time, preferably over a period of one week, more preferably one month, more preferably two months, more preferably half a year, more preferably over the entire duration of the disease. For chronic diseases, monitoring may be performed for the lifetime of the patient.

The invention may further comprise comparing the relative level of the cardiovascular marker of the individual to a predetermined value. The predetermined value may take various forms. It may be a single cutoff value, e.g. the median or mean of the population or 75^th、90^th、95^thOr 99^thPercentile. It may be established from comparison groups, e.g. with twice the risk in one defined group as in the other. It may be a range, for example, grouping the test population equally (or unequally) such as a low risk group, a medium risk group, and a high risk group, or into quarters, the lowest quarter being the individual with the lowest risk and the highest quarter being the individual with the highest risk.

The predetermined value may vary with their preference, race, inheritance, etc. within a particular population selected. For example, an apparently healthy population of non-smokers (without detectable disease, in particular without diabetes) may have a different range of "normal" markers than a population in which the smoking population or a member thereof has a disease of the cardiovascular system and/or the metabolic system. Thus, the selected predetermined value may take into account the category to which the individual belongs. Those of ordinary skill in the art can select the appropriate range and class using no more than routine experimentation.

The threshold level may be obtained from, for example, a Kaplan-Meier analysis, in which the occurrence of disease is correlated with the quartile of cardiovascular markers in a population. According to this analysis, subjects with cardiovascular marker levels above the 75th percentile have a significantly increased risk of having the disease of the invention. This result is further supported by Cox regression analysis with well-tuned classical risk factors: the highest quarter is very clearly associated with an increased risk of suffering from the disease of the invention compared to all other subjects.

Other preferred cut-off values are, for example, 90 for the normal population^th、95^thOr 99^thPercentile. By using the ratio of 75^thHigher percentiles, one may reduce the number of false positive objects identified, but one may miss identifying objects with a moderate, although still increased risk. Thus, one can adjust the cutoff value based on whether it is considered more appropriate to identify the most at-risk subjects at the expense of also identifying "false positives", or to identify mainly high-risk subjects at the expense of missing several subjects with intermediate risk.

Other mathematical possibilities for calculating an individual's risk by using the individual's cardiovascular marker level values and other prognostic laboratory and clinical parameters are, for example, NRI (net reclassification index) or IDI (comprehensive differential index). The index can be calculated according to Pencina's literature (PencinaMJ et al: "assessing the increased predictive ability of a new marker from the area under the ROC curve to reclassification et al" (Evaluating the added predictive ability of a new marker: from area under the ROC curve to reclassification and beyond), Stat Med.2008; 27: 157 + 172).

In a particular embodiment of the invention, a postprandial relative decrease of more than 5%, preferably more than 10%, more preferably more than 15%, more preferably more than 20% of the level of said one or more cardiovascular markers in said subject is associated with a diagnosis and an adverse prognosis of a metabolic system and/or cardiovascular system and/or insulin resistant disorder and an increased risk of the subject to develop a metabolic system and/or cardiovascular system and/or insulin resistant disorder.

In another particular embodiment of the invention, a postprandial relative increase of more than 5%, preferably more than 10%, more preferably more than 15%, more preferably more than 20% of the level of said one or more cardiovascular markers in said subject is associated with a favorable prognosis for the metabolic system and/or cardiovascular system and/or insulin resistant disorder and an increased risk for the subject to develop a metabolic system and/or cardiovascular system and/or insulin resistant disorder.

Furthermore, the subject of the present invention is the use of an assay, preferably having a sensitivity of 1nmol/L or less, preferably 100pmol/L or less, more preferably 10pmol/L or less, more preferably 1pmol/L or less, most preferably 0.5pmol/L or less, for determining a change in the level of one or more cardiovascular markers of a subject relative to a basal level of said markers of said subject, wherein the assay is capable of determining a decrease.

It is important to note that the ability of the assay method used in the present invention to measure a decrease in the level of said one or more cardiovascular markers, wherein the decrease results in a very low level of said one or more cardiovascular markers, is crucial. Thus, the assay method used herein is preferably ultrasensitive so as to be able to locate at a basal level 97.5 of the level of the one or more cardiovascular markers in a healthy population^thMeasuring the one or more cardiovascular diseases in a subject within a percentileA decrease in marker levels.

Another subject of the present invention is the use of the assay described above for determining the postprandial change in the level of one or more cardiovascular markers of a subject relative to the basal level of said marker of said subject.

Preferably, the assay method is an immunoassay.

Another subject of the present invention is the use of the assay method outlined above, wherein said change in the level of one or more cardiovascular markers is used for diagnosing, prognosing and/or monitoring a subject having a condition associated with metabolic syndrome.

In another embodiment of the invention, the subject is a human having a medical condition associated with metabolic syndrome.

In a particular embodiment, said change in the level of one or more cardiovascular markers is used for the diagnosis/prognosis and/or monitoring of diabetes, in particular type 2 diabetes.

As used herein, the term "metabolic syndrome" refers to the collection of several risk factors for cardiovascular disease and type II diabetes, as defined by the American Heart Association (AHA) and the National Heart, Lung and Blood Institute (NHLBI) (Grundy et al, 2005.Circulation 112: 2735-3752), which are incorporated herein by reference. Important elements of metabolic syndrome include glucose intolerance and dyslipidemia, hypertension, and trunk obesity, among others. Metabolic syndrome can be diagnosed if 3 of the following 5 criteria are met: coarse waist circumference (> 102cm in men and > 88cm in women), high triglycerides (> 150mg/dL/1.7mmol/L respectively, or for medication of high triglycerides), low HDL cholesterol (< 40mg/dL/1.03mmol/L respectively in men and < 50mg/dL/1.3mmol/L respectively in women, or for medication of low HDL cholesterol), hypertension (systolic pressure > 130mmHg or diastolic pressure > 85mmHg, or for antihypertensive medication in patients with a history of hypertension), and high fasting glucose (> 100mg/dL or for medication of hyperglycemia).

More recently, the definition with some modifications is given by the International Diabetes Federation (International Diabetes Federation) ((http://www.idf.org/webdata/docs/IDF_Meta_def_final.pdf)。

In another preferred embodiment of the invention, the condition associated with metabolic syndrome is selected from Myocardial Infarction (MI), coronary syndrome, Congestive Heart Failure (CHF), coronary artery disease (atherosclerosis), stroke, Transient Ischemic Attacks (TIA), peripheral arterial disease, cardiomyopathy, type II diabetes, renal failure and/or patients with one or more symptoms of the above mentioned diseases such as obesity, hypertension, headache, chest pain and dyspnea.

Insulin Resistance (IR) is a state in which a given concentration of insulin produces less than the expected biological effect. Insulin resistance has also been arbitrarily defined as requiring more than 200 units of insulin per day to achieve glycemic control and prevent ketosis. High plasma insulin and glucose levels, caused by insulin resistance, often lead to metabolic syndrome and type 2 diabetes, including complications thereof. Symptoms of IR may include fatigue, brain blurriness, inattention, hypoglycemia, intestinal distension, lethargy, weight gain, fat storage, difficulty losing weight, high blood triglyceride levels, hypertension and depression.

Cardiovascular disease is characterized by dysfunction of the myocardium or vascular system that supplies blood to the heart, brain, and other critical organs. The term "cardiovascular disease" covers a wide range of conditions including atherosclerosis, coronary artery disease, valvular heart disease, arrhythmia, heart failure, hypertension, orthostatic hypotension, shock, endocarditis, diseases of the aorta and its branches, conditions of the peripheral vascular system, congenital heart disease, stroke.

Another subject of the present invention is a method for determining a change in the level of one or more cardiovascular markers of a subject relative to a basal level of said markers of said subject, wherein the determination method is capable of detecting a decrease in the level of said one or more cardiovascular markers and is capable of detecting an increase in the level of said one or more cardiovascular markers.

In a preferred embodiment of the assay method, the change is an increase or a decrease, and wherein the assay method has a sensitivity of 1nmol/L or less.

Another embodiment of the invention is the use of an assay to determine postprandial changes in the level of one or more cardiovascular markers in a subject relative to a basal level of said markers in said subject.

The assay methods are useful for the diagnosis, prognosis and/or monitoring/therapy follow-up of metabolic and/or cardiovascular and/or insulin resistance disorders in a subject.

Another object of the invention is the use of a cardiovascular peptide for the diagnosis, prognosis and/or therapy follow-up of a disorder of the metabolic system and/or of the cardiovascular system of a patient.

As used herein, the term "subject" refers to a living human or non-human organism. In this context, the subject is preferably a human subject.

As used herein, the term "sample" refers to a sample of bodily fluid obtained for the purpose of diagnosis, prognosis or assessment of a subject of interest, such as a patient. Preferred test samples include blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusion. Furthermore, those skilled in the art will recognize that certain test samples may be more easily analyzed after fractionation or purification steps, such as separating whole blood into serum or plasma components.

Thus, in a preferred embodiment of the invention, the sample is selected from the group consisting of a blood sample, a serum sample, a plasma sample, a cerebrospinal fluid sample, a saliva sample and a urine sample, or an extract of any of the above mentioned samples. Preferably, the sample is a blood sample, most preferably a serum sample or a plasma sample.

The level of the marker obtained by the method of the present invention or by using the assay method of the present invention can be analyzed in various ways well known to those skilled in the art. For example, each assay result obtained may be compared to a "normal" value or a value indicative of a particular disease or outcome. The particular diagnosis/prognosis may depend on the comparison of each assay result to such a value, which may be referred to as a diagnostic or prognostic "threshold".

The sensitivity and specificity of a diagnostic and/or prognostic test depends not only on the analytical "quality" of the test, but also on the definition of what constitutes an abnormal result. In practice, a subject operating characteristic curve (ROC curve) is typically calculated by plotting the variable values of the "normal" (i.e. apparently healthy) and "diseased" populations (i.e. patients with diabetes, insulin resistance and/or metabolic syndrome) against their relative frequencies. For any particular marker, the marker level distributions of subjects with or without disease may overlap. Under these conditions, the test cannot absolutely distinguish normal from disease with 100% accuracy, and the overlapping region represents where the test cannot distinguish normal from disease. A threshold is chosen above which (or below which, depending on how the marker changes with the disease) the test is considered abnormal and below which the test is considered normal. The area under the ROC curve is a measure of the probability that a perceived measurement will allow a condition to be correctly identified. The ROC curve can be used even if the test results do not necessarily give an exact number. One can generate an ROC curve as long as one can sort the results. For example, the results of tests performed on "diseased" samples may be ranked by degree (e.g., 1-low, 2-normal, 3-high). This ranking can be correlated with the results in the "normal" population and generate a ROC curve. Such methods are well known in the art. See, e.g., Hanley et al, Radiology 143: 29-36(1982). Preferably, the threshold value is selected to provide a ROC curve area greater than about 0.5, more preferably greater than about 0.7, more preferably greater than about 0.8, more preferably greater than about 0.85, and most preferably greater than about 0.9. In this context, the term "about" refers to +/-5% of a given measurement.

The horizontal axis of the ROC curve represents (1-specificity), which increases with the false positive rate. The vertical axis of the curve represents sensitivity, which increases with true positive rate. Thus, for a particular cut-off value chosen, a value of (1-specificity) can be determined and a corresponding sensitivity can be obtained. The area under the ROC curve is a measure of the probability that the measured marker level will allow the correct identification of a disease or disorder. Thus, the area under the ROC curve can be used to determine the effectiveness of the test.

In certain embodiments, a particular threshold value for one or more markers in a panel is not relied upon to determine whether a marker level profile obtained from a subject is indicative of a particular diagnosis/prognosis. In contrast, the present invention can utilize the "distribution" of the marker panel as an integral whole for evaluation. The particular "fingerprint" combination of changes in this marker panel can be used effectively as a particular diagnostic or prognostic indicator. As discussed herein, a combination of changes can be obtained from the then-current change (or response value of a group) of a single sample or one or more members of a group. Here, a group refers to a group of markers.

As described later herein, the response values of the panel are preferably determined by plotting the sensitivity of a particular marker panel at various cut-off values against the 1- (specificity) curve of the panel. In these methods, the distribution of marker measurements from a subject taken together is considered to provide a global probability (expressed as a numerical score or percentage risk) of diagnosis or prognosis. In such embodiments, an increase in certain subsets of markers may be sufficient to indicate a particular diagnosis/prognosis in one patient, while an increase in different subsets of markers may be sufficient to indicate the same or different diagnosis/prognosis in another patient. A weighting factor may also be applied to one or more markers in a panel, for example, when a marker is particularly highly effective in identifying a particular diagnosis/prognosis, it may be weighted such that at a given level it alone is sufficient to convey a signal of positive result. Likewise, a weighting factor may also be provided such that any given level of a particular marker is insufficient to deliver a signal of a positive result, but only if another marker also contributes to the analysis.

In certain embodiments, the marker and/or set of markers is selected to exhibit a sensitivity of at least about 70%, more preferably a sensitivity of at least about 80%, more preferably a sensitivity of at least about 85%, more preferably a sensitivity of at least about 90% and most preferably a sensitivity of at least about 95%, in combination with a specificity of at least about 70%, more preferably a specificity of at least about 80%, more preferably a specificity of at least about 85%, more preferably a specificity of at least about 90% and most preferably a specificity of at least about 95%. In particularly preferred embodiments, both sensitivity and specificity are at least about 75%, more preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95%. In this case, the term "about" refers to +/-5% of a given measurement.

In other embodiments, a positive likelihood ratio, a negative likelihood ratio, an opportunity ratio, or a risk ratio is used as a measure of the test's ability to predict risk or diagnose a disease. In the case of a positive likelihood ratio, a value of 1 indicates that the likelihood of a positive result is equal in subjects of both the "diseased" and "control" groups; a value greater than 1 indicates a greater likelihood of a positive result in the diseased group; a value less than 1 indicates a greater likelihood of a positive result in the control group. In the case of negative likelihood ratios, a value of 1 indicates equal likelihood of negative results in subjects in both the "diseased" and "control" groups; a value greater than 1 indicates a greater likelihood of a negative result in the test group; a value less than 1 indicates a greater likelihood of a negative result in the control group. In certain preferred embodiments, the markers and/or marker panels are preferably selected to exhibit a positive or negative likelihood ratio of at least about 1.5 or more or about 0.67 or less, more preferably at least about 2 or more or about 0.5 or less, more preferably at least about 5 or more or about 0.2 or less, more preferably at least about 10 or more or about 0.1 or less, and most preferably at least about 20 or more or about 0.05 or less. In this case, the term "about" refers to +/-5% of a given measurement.

In the case of the odds ratio, a value of 1 indicates equal likelihood of positive outcome in subjects in both the "diseased" and "control" groups; a value greater than 1 indicates a greater likelihood of a positive result in the diseased group. A value less than 1 indicates a greater likelihood of a positive result in the control group. In certain preferred embodiments, the marker and/or set of markers are preferably selected to exhibit a odds ratio of at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, more preferably at least about 4 or more or about 0.25 or less, more preferably at least about 5 or more or about 0.2 or less and most preferably at least about 10 or more or about 0.1 or less. In this case, the term "about" refers to +/-5% of a given measurement.

In the case of the risk ratio, a value of 1 indicates that the relative risk of endpoint (e.g., death) is equal in both the "diseased" and "control" groups; a value greater than 1 indicates a higher risk in the diseased group; a value less than 1 indicates a higher risk in the control group. In certain preferred embodiments, the marker and/or marker panel is preferably selected to exhibit a risk ratio of at least about 1.1 or more or about 0.91 or less, more preferably at least about 1.25 or more or about 0.8 or less, more preferably at least about 1.5 or more or about 0.67 or less, more preferably at least about 2 or more or about 0.5 or less and most preferably at least about 2.5 or more or about 0.4 or less. In this case, the term "about" refers to +/-5% of a given measurement.

The skilled artisan will appreciate that correlating a diagnostic or prognostic indicator with a diagnosis or with a prognostic risk of a future clinical outcome is a statistical analysis. For example, a marker level greater than X may signal that the patient is more likely to suffer adverse consequences than a patient whose level is less than or equal to X, as determined by a level of statistical significance. Statistical significance is typically determined by comparing two or more populations and determining a confidence interval and/or p-value. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983. Preferred confidence intervals for the present invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001 and 0.0001.

Sequence of

The amino acid sequence of the precursor peptide of adrenomedullin (pro-adrenomedullin) is provided in SEQ ID NO: 1 in (c). Adrenomedullin refers to amino acid residues 22 to 185 of the pro-adrenomedullin sequence. The amino acid sequence of pro-adrenomedullin (pro-ADM) is provided in SEQ ID NO. 2. The pro-ADM N-terminal 20 peptide (PAMP) refers to amino acid residues 22-41 of pre-proADM. The amino acid sequence of PAMPs is provided in SEQ ID NO: 3 in (b). MR-pro-adrenomedullin (MR-pro-ADM) refers to amino acid residues 45-92 of pre-pro-ADM. The amino acid sequence of MR-pro-ADM is provided in SEQ ID NO: 4 in (b). The amino acid sequence of mature Adrenomedullin (ADM) is provided in SEQ ID NO: 5 in (c).

The amino acid sequence of ANP is provided in SEQ ID NO: 8 in (c). The sequence of pre-pro-ANP of 153 amino acids is shown in SEQ ID NO: 6 in (A). After cleavage of the N-terminal signal peptide (25 amino acids) and of the two C-terminal amino acids (127/128), proANP is released (SEQ ID NO: 7). ANP comprises residues 99-126 from the C-terminus of prohormone pro-ANP. This prohormone is cleaved into the mature 28 amino acid peptide ANP, also known as ANP (1-28) or α -ANP, and the amino-terminal fragment ANP (1-98) (NT-proANP, SEQ ID NO: 9). A middle-segment proANP (MR-proANP) is defined as NT-proANP or any fragment thereof comprising at least amino acid residues 53-90 of proANP (SEQ ID NO: 10).

The sequence of the 164 amino acid vasopressin precursor peptide (pro-vasopressin) is provided in SEQ ID NO: 11 in (b). Pro-vasopressin refers to amino acid residues 29 to 164 of the pro-vasopressin sequence. The amino acid sequence of pro-vasopressin is provided in SEQ ID NO: 12 in the above step (1). The pro-vasopressin is cleaved into mature vasopressin, the posterior leaflet hormone transporter II and the C-terminal pro-vasopressin (CT-proAVP or copeptin). Vasopressin refers to the amino acid residues 20 to 28 of pro-vasopressin. The amino acid sequence of vasopressin is shown in SEQ ID NO: 13 (c). And propeptide refers to amino acid residues 126 to 164 of prepro-vasopressin. And the amino acid sequence of copeptin is provided in SEQ ID NO: 14 (c). The posterior leaflet hormone transporter II comprises amino acid residues 32 to 124 of pro-vasopressin, the sequence of which is shown in SEQ ID NO: 15, in (b).

The sequence of the precursor peptide of endothelin-1 of 212 amino acids (proendothelin-1) is provided in SEQ ID NO: 16, respectively. Pro-ET-1 refers to amino acid residues 18 to 212 of the pre-Pro-ET-1 sequence. The amino acid sequence of pro-ET-1 is provided in SEQ ID NO: 17 (c). Pro-ET-1 is cleaved into mature ET-1, big-ET-1 and C-terminal proET-1 (CT-proET-1). ET-1 refers to amino acid residues 53 to 73 of pre-pro-ET-1. The amino acid sequence of ET-1 is shown in SEQ ID NO: 18, respectively. CT-proET-1 refers to amino acid residues 168 to 212 of pre-pro-ET-1. The amino acid sequence of CT-proET-1 is provided in SEQ ID NO: 19 in (b). Big-ET-1 comprises amino acid residues 53 to 90 of pre-pro-ET-1, the sequence of which is shown in SEQ ID NO: 20 (c).

The sequence of the precursor peptide of the 134 amino acid brain natriuretic peptide (pre-pro-BNP) is provided in SEQ ID NO: 21, respectively. Pro-BNP means amino acid residues 27 to 134 of pre-Pro-BNP. The sequence of pro-BNP is shown in SEQ ID NO: 22, respectively. Pro-BNP is cleaved into N-terminal Pro-BNP (NT-Pro-BNP) and mature BNP. NT-pro-BNP comprises amino acid residues 27 to 102, the sequence of which is shown in SEQ ID NO: 23, respectively. SEQ ID NO: 24 shows the sequence of BNP comprising amino acid residues 103 to 134 of the pre-pro-BNP peptide.

SEQ ID NO: 1 (amino acid sequence of pre-pro-ADM):

1 MKLVSVALMY LGSLAFLGAD TARLDVASEF RKKWNKWALS RGKRELRMSS

51 SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMN

101 NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR

151 RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL

SEQ ID NO: 2 (amino acid sequence of pro-ADM):

1 ARLDVASEFR KKWNKWALSR GKRELRMSSS YPTGLADVKA GPAQTLIRPQ

51 DMKGASRSPE DSSPDAARIR VKRYRQSMNN FQGLRSFGCR FGTCTVQKLA

101 HQIYQFTDKD KDNVAPRSKI SPQGYGRRRR RSLPEAGPGR TLVSSKPQAH

151 GAPAPPSGSA PHFL

SEQ ID NO: 3 (amino acid sequence of pro-ADM N20):

1 ARLDVASEFR KKWNKWALSR

SEQ ID NO: 4 (amino acid sequence of MR-pro-ADM):

1 ELRMSSSYPT GLADVKAGPA QTLIRPQDMK GASRSPEDSS

SEQ ID NO: 5 (amino acid sequence of ADM):

1 YRQSMNNFQG LRSFGCRFGT CTVQKLAHQI YQFTDKDKDN VAPRSKISPQ

51 GY

SEQ ID NO: 6 (amino acid sequence of pre-pro-ANP):

1 MSSFSTTTVS FLLLLAFQLL GQTRANPMYN AVSNADLMDF KNLLDHLEEK

51 MPLEDEVVPP QVLSEPNEEA GAALSPLPEV PPWTGEVSPA QRDGGALGRG

101 PWDSSDRSAL LKSKLRALLT APRSLRRSSC FGGRMDRIGA QSGLGCNSFR

151 YRR

SEQ ID NO: 7 (amino acid sequence of pro-ANP):

1 NPMYNAVSNA DLMDFKNLLD HLEEKMPLED EVVPPQVLSE PNEEAGAALS

51 PLPEVPPWTG EVSPAQRDGG ALGRGPWDSS DRSALLKSKL RALLTAPRSL

101 RRSSCFGGRM DRIGAQSGLG CNSFRY

SEQ ID NO: 8 (amino acid sequence of ANP):

1 SLRRSSCFGG RMDRIGAQSG LGCNSFRY

SEQ ID NO: 9 (amino acid sequence of NT-proANP):

1 NPMYNAVSNA DLMDFKNLLD HLEEKMPLED EVVPPQVLSE PNEEAGAALS

51 PLPEVPPWTG EVS PAQRDGG ALGRGPWDSS DRSALLKSKL RALLTAPR

SEQ ID NO: 10 (amino acid sequence of amino acids 53 to 90 of proANP):

1 PEVPPWTGEV SPAORDGGAL GRGPWDSSDR SALLKSKL

SEQ ID NO: 11 (amino acid sequence of pre-pro-AVP):

1 MPDTMLPACF LGLLAFSSAC YFQNCPRGGK RAMSDLELRQ CLPCGPGGKG

51 RCFGPSICCA DELGCFVGTA EALRCQEENY LPSPCQSGQK ACGSGGRCAA

101 FGVCCNDESC VTEPECREGF HRRARASDRS NATQLDGPAG ALLLRLVQLA

151 GAPEPFEPAQ PDAY

SEQ ID NO: 12 (amino acid sequence of pro-AVP):

1 CYFQNCPRGG KRAMSDLELR QCLPCGPGGK GRCFGPSICC ADELGCFVGT

51 AEALRCQEEN YLPSPCQSGQ KACGSGGRCA AFGVCCNDES CVTEPECREG

101 FHRRARASDR SNATQLDGPA GALLLRLVQL AGAPEPFEPA QPDAY

SEQ ID NO: 13 (amino acid sequence of AVP):

1 CYFQNCPRG

SEQ ID NO: 14 (amino acid sequence of CT-pre-proAVP or copeptin):

1 ASDRSNATQL DGPAGALLLR LVQLAGAPEP FEPAQPDAY

SEQ ID NO: 15 (amino acid sequence of the posterior leaflet hormone transporter II):

1 AMSDLELRQC LPCGPGGKGR CFGPSICCAD ELGCFVGTAE ALRCQEENYL

51 PSPCQSGQKA CGSGGRCAAF GVCCNDESCV TEPECREGFH RRA

SEQ ID NO: 16 (amino acid sequence of pre-pro-ET-1):

SEQ ID NO: 17 (amino acid sequence of pro-ET-1):

1 APETAVLGAE LSAVGENGGE KPTPSPPWRL RRSKRCSCSS LMDKECVYFC

51 HLDIIWVNTP EHVVPYGLGS PRSKRALENL LPTKATDREN RCQCASQKDK

101 KCWNFCQAGK ELRAEDIMEK DWNNHKKGKD CSKLGKKCIY QQLVRGRKIR

151 RSSEEHLRQT RSETMRNSVK SSFHDPKLKG KPSRERYVTH NRAHW

SEQ ID NO: 18 (amino acid sequence of ET-1):

1 CSCSSLMDKE CVYFCHLDII W

SEQ ID NO: 19 (amino acid sequence of CT-pro-ET-1):

1 RSSEEHLRQT RSETMRNSVK SSFHDPKLKG KPSRERYVTH NRAHW

SEQ ID NO: 20 (amino acid sequence of Big-ET-1):

1 CSCSSLMDKE CVYFCHLDIIWVNTPEHVVP YGLGSPRS

SEQ ID NO: 21 (amino acid sequence of pre-pro-BNP):

1 MDPQTAPSRA LLLLLFLHLA FLGGRSHPLG SPGSASDLET SGLQEQRNHL

51 QGKLSELQVE QTSLEPLQES PRPTGVWKSR EVATEGIRGH RKMVLYTLRA

101 PRSPKMVQGS GCFGRKMDRI SSSSGLGCKV LRRH

SEQ ID NO: 22 (amino acid sequence of pro-BNP):

1 HPLGSPGSAS DLETSGLQEQ RNHLQGKLSE LQVEQTSLEP LQESPRPTGV

51 WKSREVATEG IRGHRKMVLY TLRAPRSPKM VQGSGCFGRK MDRISSSSGL

101 GCKVLRRH

SEQ ID NO: 23 (amino acid sequence of NT-pro-BNP):

1 HPLGSPGSAS DLETSGLQEQ RNHLQGKLSE LQVEQTSLEP LQESPRPTGV

51 WKSREVATEG IRGHRKMVLY TLRAPR

SEQ ID NO: 24 (amino acid sequence of BNP):

1 SPKMVQGSGC FGRKMDRISS SSGLGCKVLR RH

description of the drawings

FIG. 1 sequence of pre-pro-ADM

FIG. 2 sequence of pro-ADM

FIG. 3 sequence of pro-ADM N20

FIG. 4 sequence of MR-pro-ADM

FIG. 5 sequence of ADMs

FIG. 6 sequence of pre-pro-ANP

FIG. 7 sequence of pro-ANP

FIG. 8 sequence of ANP

FIG. 9 sequence of NT-pro ANP

FIG. 10, amino acid sequence 53-90 of pro ANP

FIG. 11 sequence of pre-pro-AVP

FIG. 12 sequence of pro-AVP

FIG. 13, sequence of AVPs

FIG. 14, the sequence of CT-pre-pro AVP or copeptin

FIG. 15 sequence of the posterior leaflet hormone transporter II

FIG. 16 sequence of pre-pro-ET-1

FIG. 17 sequence of pro-ET-1

FIG. 18, ET-1 sequence

FIG. 19, CT-pro-ET-1 sequence

FIG. 20 shows the sequence of Big-ET-1

FIG. 21 sequence of pre-pro-BNP

FIG. 22 sequence of pro-BNP

FIG. 23 sequence of NT-pro-BNP

FIG. 24, sequence of BNP

FIG. 25, plasma MR-proANP during oral glucose tolerance test in non-obese normotensive subjects (black diamonds), normotensive subjects with trunk obesity (white diamonds) and hypertensive subjects (black triangles)_53-90(A) And serum insulin (B):^*non-obese normotensive subjects with obesity p < 0.05;obese normotensive blood pressure p < 0.001 for hypertensive subjects;hypertension p < 0.01 for nonobese normotensive subjects.

(C) In hyperinsulinemia and normal blood sugar clampSerous MR-proANP_53-90Horizontal inhibition. (D) Delta MR-proANP in subjects with low and high insulin sensitivity_53-90(0-120min), respectively, determined to be less than 25 in the steady state of the clip^thAnd higher than 75^thPercentile glucose perfusion rate (GIR) values. Data are shown in box and whisker plots. Box 25^thExtend to 75^thPercentile, line at median indicate 50^thPercentile. Must represent a range extending from the lowest to the highest value.

FIG. 26 post-oGTT NT-proBNP and MR-proANP in n-10 subjects_53-90Comparison of relative concentrations.

FIG. 27, (Metabolic syndrome) -correlation of insulin sensitivity with changes in relative MR-proANP at 120min [ insulin sensitivity index (ISI or SI) ]: for healthy people ISI > 4.5 when t 0 [ fasting; ISI ≧ 6 at 30, 60, 90, and 120 minutes; for insulin-resistant people, ISI < 6 at t-30, 60, 90 and 120 minutes.

Examples

Research design and method

Study protocol 1(OGTT and jaw test)

The study protocol was approved by the ethical committee of the university of borstem and the Charite medical school of berlin, germany. Prior to the study, formal written informed consent was obtained from all participants.

Design of research

The subjects were part of an ongoing case-control association study of metabolic syndrome and etiology of type 2 diabetes (metabolic syndrome berlin-borstem study, MESY-BEPO). Volunteers were recruited from the general population in borstem and berlin, germany. Baseline exams included human measurements, blood sampling, 75g oral glucose tolerance test (oGTT), and personal interviews regarding lifestyle, hobbies, and medical history. A subset of the population (n ═ 31) underwent hyperinsulinemic, euglycemic clamp tests, which were performed another day after oGTT.

Object

108 non-hypertensive subjects (55 non-obese and 53 subjects with trunk obesity) and 54 patients with essential hypertension were studied. Hypertension is defined as systolic pressure ≥ 140mmHg, diastolic pressure ≥ 90mm Hg, or treatment with antihypertensive. All drug-treated hypertensive subjects had stable drug treatment within the last 6 months prior to the study. Subjects with elevated liver enzymes above two times the corresponding upper normal limit, or with high plasma creatinine concentrations (> 1.3mg/dl) or with severe conditions including systemic inflammation, heart failure or advanced malignant disease were excluded from the study. All subjects were instructed to maintain their normal physical activity and consume a normal diet containing 200g of sugars within 3 days prior to oGTT and clamp trials. Subjects with diabetes-resistant treatment or newly diagnosed type 2 diabetes were excluded from the examination. The definition of glucose metabolism disorder is based on the criteria of glucose values obtained after an overnight fast and a 2-hour 75g oGTT by the American Diabetes Association in 1997 (2000, "Report of the expert Committee on the Diagnosis and Classification of Diabetes mellitus" (Report of the expert Committee on the Diagnosis and Classification of Diabetes mellitus), Diabetes Care 23 Suppl 1: S4-19).

Experimental procedure

All tests were performed in the morning after a 12 hour overnight fast. BP by trained research Nurse Using OmronThe HEM705CP sphygmomanometer (Omron, Germany) measures when a patient assumes a sitting position. Three measurements were taken at 2 minute intervals and the mean values were used to determine the clinical systolic and diastolic blood pressure. For oGTT, venous blood samples were taken at 0, 30, 60, 90, 120 and 180 minutes relative to oral glucose infusion.

Euglycemic, hyperinsulinemic blood clamp:

the clip is used for treating hyperinsulinemia and normal blood sugar²Variable perfusion of 100mU human insulin (Actrapid; Novo Nordisk, Bagsvaerd, Denmark) and 20% Glucose (Serag Wiesser, Naila, Germany) on the body surface was carried out for 120 minutes (Deonzo RA et al, 1979 "Glucose clamping technique: a method for quantifying insulin secretion and resistance" (Glucose clamp technique: a method for quantifying insulin secretion and resistance), Am J Physiol 237: E214-23). Under steady state conditions in the jaws, capillary blood glucose was adjusted to 5.5mmol/l for at least 60 minutes. A deviation of > 10% of the single capillary glucose concentration during the assumed steady state conditions is defined as non-steady state. Capillary blood glucose concentration was monitored every 5 minutes throughout the clamp experiment and used to regulate plasma glucose by adjusting the variable perfusion of glucose.

Analytical procedure

All venous blood samples were immediately centrifuged and frozen at-70 ℃ until analysis. The capillary blood Glucose concentration was determined using the Glucose oxidase method on a Dr.Muller G-L Glucose Analyzer (Dr.Muller Glucose analyzer, Fritital, Germany). Serum triglycerides, total cholesterol and HDL-cholesterol were determined by standard enzymatic assays and LDL-cholesterol was calculated from these data (certified laboratory for clinical chemistry analysis). HbA1c was determined using the Hi-AutoA1C HA-8140 system (Menarini Diagnostics, Germany). Serum insulin was measured using a commercial enzyme-linked immunosorbent assay (insulin ELISA, Mercodia AB, Uppsala, Sweden). Steady-State model assessment Insulin Resistance (HOMA-IR) was calculated as fasting Insulin (IU/L) x fasting glucose (mmol/L)/22.5(Matthews DR et al, 1985 "Steadstate model assessment: assessment of Insulin Resistance and beta-cell function from human fasting plasma glucose and Insulin concentrations" (Insulin Homeostasis model: Insulin Resistance and Insulin Resistance center), Diabetologia 28: 412-9).

Human plasma MR-proANP53-90 was determined as described previously (MorgethalerrG et al, 2004 "Immunolutionmetric assay for mid-atrial natriuretic peptides in human plasma" (Clin Chem 50: 234-6).

NT-proBNP was determined by electrochemiluminescence immunoassay (ELICIA, Roche diagnostics, Basel, Switzerland).

Statistical analysis

We divided the study population into three groups: patients with essential hypertension (n-54), non-hypertensive subjects with trunk obesity (n-53), and non-hypertensive subjects without trunk obesity (n-55). Trunk obesity is diagnosed according to metabolic syndrome criteria defined by ATP III (National Cholesterol emission Program (NCEP) adult high blood Cholesterol Detection, assessment and Treatment Expert Committee (National Cholesterol emission Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of high blood Cholesterol in Adults) (adult Treatment Committee III), National Cholesterol emission Committee (NCEP) Expert Committee for the Third report on Detection, assessment, and Treatment of adult high blood Cholesterol (adult Treatment Committee III) Final report (Third report of the National Cholesterol Emission Program (NCEP) Expert Panel on Detection, assessment, and Treatment, Evaluation, and Treatment of high blood Cholesterol in Adapts (NCEP) examination, and Treatment of high blood Cholesterol in Adapts (added Panel III) patent report 3443), health report of high blood Cholesterol in Adapts (3443: 31).

General characteristics are provided as mean ± SD. All other data are shown as mean ± SEM. All data were logarithmically transformed prior to analysis. Calculating Δ MR-proANP from 0 to 180min in oGTT_53-90. Group comparisons were performed by ANOVA, followed by Sidak test as post hoc group comparisons. Comparison of MR-proANP during oGTT between three groups using repeated measures ANOVA analysis_53-90Time course. Relevance analysis was performed by Pearson's relevance analysis. In thatIn the clamp test, insulin sensitivity was determined as the glucose perfusion rate (GIR; mg per kg body weight min) in the steady state (at least 30 minutes) of the clamp test^-1) Divided by circulating insulin concentration in steady state (pmol/l). Data from baseline and steady state were compared using the nonparametric Wilcoxon's signed rank test for paired samples. In all analyses, P values < 0.05 were considered significant. All statistical analyses were performed using SPSS version 14 for Windows (SPSS inc., Chicago, Illinois).

Results

The characteristics of the study population are summarized in table 1. Obese normotensive subjects have higher insulin resistance compared to non-obese normotensive subjects: they have higher fasting insulin and blood glucose concentrations, lower HDL-cholesterol concentrations and triglyceride levels, and higher SPB and DBP. Except for higher age, HbA1c levels, and SBP, obese subjects with hypertension were comparable to obese normotensive subjects in terms of BMI, waist circumference, and insulin resistance.

Among obese normotensive subjects, the lowest MR-proANP observed was compared to non-obese normotensive subjects and obese subjects with hypertension_53-90Fasting concentrations (53.9. + -. 28.0pmol/l vs 64.1. + -. 25.6pmol/l and 77.5. + -. 30.8 pmol/l). The difference was still significant after adjustment for age and BMI. Fasting MR-proANP in normotensive subjects_53-90Levels are clearly positively correlated with age (r ═ 0.429, p < 0.0001), HDL-cholesterol (r ═ 0.270, p ═ 0.006), and clearly negatively correlated with BMI (r ═ 0.313, p ═ 0.001), DBP (r ═ 0.251, p ═ 0.009), fasting insulin (r ═ 0.276, p ═ 0.004) and HOMA-IR index (r ═ 0.268, p ═ 0.005). In contrast, in obese subjects with hypertension, MR-proANP_53-90The positive correlation of (b) is limited to age (r ═ 0.546, p < 0.0001).

MR-proANP in all subjects_53-90Levels rapidly decreased at 30 minutes following oral glucose challenge and remained inhibited throughout the test (p < 0.0001; basal levels vs. oGTT 30, 60, 90,Levels at 120 and 180 min). MR-proANP from 90 to 180 minutes post challenge in obese normotensive subjects compared to non-obese normotensive subjects and obese hypertensive subjects_53-90The concentration was significantly lower (fig. 25A). However, at 180 minutes MR-proANP_53-90The relative inhibition was similar (20.0 + -13.4% in non-obese normotensive subjects, 21.4 + -19.5% in obese normotensive subjects, 21.2 + -13.4% in hypertensive subjects, NS). Insulin levels were significantly lower in obese normotensive subjects after fasting and glucose challenge (FIG. 25B), and compared to MR-proANP from 60 to 180 minutes after fasting and challenge in normotensive subjects_53-90Horizontal negative correlations (r ═ 0.198- -0.358; p < 0.0001-0.05), and these correlations were much weaker in hypertensive subjects. Blood glucose levels and MR-proANP after fasting and challenge during oGTT in normotensive and hypertensive subjects_53-90There was no correlation between the levels (data not shown).

Although BNP appears to play a secondary physiological role in healthy subjects, it is often regulated in a similar manner to ANP. Therefore, we tested whether NT-proBNP levels also decreased in response to oral glucochallenge. NT-proBNP levels were assessed in 10 subjects and compared to MR-proANP in this subgroup_53-90A comparison is made. In fact, the relative concentration of NT-proBNP also decreased following glucose challenge, but compared to MR-proANP_53-90Shorter duration and smaller than response (fig. 26).

31 obese subjects (17 normotensive subjects and 14 hypertensive subjects) underwent a normoglycemic-hyperinsulinemic clamp test. Normal and hypertensive subjects were matched with respect to age, BMI, waist circumference and insulin sensitivity (mean SE; 30.6 + -3.5 kg/m, respectively²For 32.0 +/-3.4 kg/m²P is 0.341; 101.0 ± 7.3cm to 103.6 ± 8.8cm, p is 0.526; 6.1 + -1.6 mg/kg body weight x min^-1For 5.5 plus or minus 2.0mg/kg body weight x min^-1And p is 0.388). Fasting blood glucose, insulin and MR-proANP between normotensive and hypertensive subjects_53-90The levels were not different (mean. + -. SE; 5.2. + -. 0.5mmol/l, respectively)P is 0.147 for 5.4 plus or minus 0.6 mmol/l; 66.0 ± 40.2pmol/l vs 76.2 ± 39.0pmol/l, p ═ 0.470; 59.5 ± 18.3pmol/l vs 61.0 ± 31.5pmol/l, p ═ 0.874). In the euglycemic clamp trial, circulating insulin levels increased to 1223 (range 708-. In both groups, MR-proANP at 120min bracketing_53-90The levels were significantly reduced compared to the basal values, but there was no difference between the two groups (Δ MR-proANP)_53-900-120min is 11.1(-1.3-25.1) pmol/l in normal blood pressure subjects and 8.5(-35.2-34.6) in hypertension subjects; p is 0.297) (fig. 25C). In subjects with low and high insulin sensitivity, no MR-proANP was observed_53-90Inhibition (determined as less than 25 each)^thAnd higher than 75^thPercentile values of glucose perfusion rate) (fig. 25D).

In healthy individuals (n-264), the mean MR-ProADM concentration was 0.33nmol/L (standard deviation 0.07nmol/L, range 0.1-0.64nmol/L, 99^thPercentile of 0.52nmol/L, 97.5^thPercentile of 0.49nmol/L, 2.5^thPercentile of 0.17nmol/L, 1^stThe percentile is 0.14 nmol/L. The lower limit of detection for the assay is 0.08nmol/L (Morgethaler et al, 2005.Clin Chem 51 (10): 1823-1829).

In healthy individuals (n 325), the median MR-ProANP concentration was 45pmol/L, ranging from 9.6 to 313pmol/L, 99^thThe percentile is 197.5pmol/L, 97.5^thPercentile 163.9pmol/L, 2.5^thPercentile 18.4pmol/L, 1^stThe percentile was 13.6 pmol/L. The lower limit of detection of the assay is 6.0pmol/L (Morgethaler et al, 2004.ClinChem 50 (1): 234-.

In healthy individuals (n 359), the median CT-ProAVP concentration was 4.2pmol/L, ranging from 1-13.8pmol/L, 99 pmol/L^thPercentile 13.5pmol/L, 97.5^thPercentile of 11.25pmol/L, 2.5^thThe percentile is 1.7 pmol/L. The lower limit of detection of the determination method is 1.7pmol @L (Morgethaler et al, 2006, Clin Chem 52 (1): 112-. 9 of 359 individuals had CT-proAVP values below the lower limit of detection and were defined as 1.0 pmol/L.

In healthy individuals (n 326), the mean CT-ProET1 concentration was 44.3pmol/L (standard deviation 10.6pmol/L), ranging from 10.5-77.4pmol/L, 99^thPercentile 72.8pmol/L, 97.5^thPercentile 66.6pmol/L, 2.5^thPercentile of 24.8pmol/L, 1^stThe percentile is 17.4 pmol/L. The lower limit of detection by the assay method was 0.4pmol/L (Papassotriou et al, 2006.Clin Chem 52 (6): 1144-.

In healthy subjects (n ═ 2264), the mean NT-proBNP concentration was 5.94pmol/l (standard deviation 7.36pmol/l), the median was 3.25pmol/l, 97.5^thPercentile 19.94pmol/l, 95^thThe percentile was 17.58 pmol/l. The lower limit of detection by the measurement method was 0.59 pmol/l.

TABLE 1 clinical and Biochemical characteristics of the study subjects

Unless otherwise indicated, values are mean ± SD. All values were unadjusted.^ap＜0.0001；^bp＜0.01；^cp < 0.05, relative to non-obese non-hypertensive subjects, and^dp＜0.0001；^ep < 0.05, relative to obese hypertensive subjects. Obesity is defined as "trunk obesity" according to the criteria used by ATIII for metabolic syndrome: waist circumference > 88cm for women and > 102cm for men.

The study protocol was approved by the ethical committee of the university of borstem and the Charite medical school of berlin, germany. Prior to the study, formal written informed consent was obtained from all participants.

Analysis step (bite ü berpriifen und ü berarbeiten)

All venous blood samples were immediately centrifuged and frozen at-70 ℃ until analysis. The capillary blood Glucose concentration was determined using the Glucose oxidase method on a Dr.Muller G-L Glucose Analyzer (Dr.Muller Glucose analyzer, Fritital, Germany). HbA1c was determined using the Hi-AutoA1C HA-8140 system (Menarini Diagnostics, Germany). Serum insulin was measured using a commercial enzyme-linked immunosorbent assay (insulin ELISA, Mercodia AB, Uppsala, Sweden). Steady-State model assessment Insulin Resistance (HOMA-IR) was calculated as fasting Insulin (IU/L) x fasting glucose (mmol/L)/22.5(Matthews DR et al, 1985 "Steadstate model assessment: assessment of Insulin Resistance and beta-cell function from human fasting plasma glucose and Insulin concentrations" (Insulin Resistance and Insulin Resistance), Diabetologia 28: 412-9)

Human plasma MR-proANP53-90 was determined as described previously (MorgethalerrG et al, 2004 "Immunolutionmetric assay for mid-atrial natriuretic peptides in human plasma" (Clin Chem 50: 234-6).

NT-proBNP was determined by electrochemiluminescence immunoassay (ELICIA, Roche diagnostics, Basel, Switzerland).

Claims (11)

1. Use of one or more capture molecules in the preparation of a reagent for use in an in vitro method for the diagnosis, prognosis and/or monitoring/therapy follow-up of a disorder of the metabolic system and/or cardiovascular system and/or insulin resistance in a subject, said method comprising:

a) providing a sample from the subject and,

b) determining the postprandial relative level of one or more cardiovascular markers in the sample,

c) using the postprandial relative levels of the one or more cardiovascular markers for diagnosis, prognosis and/or monitoring/therapy follow-up of a disorder of the metabolic system and/or cardiovascular system and/or insulin resistance in the subject;

wherein the one or more capture molecules are capable of binding to the one or more cardiovascular markers; wherein the postprandial relative level of the one or more cardiovascular markers is the relative change from the basal level of the cardiovascular marker to the postprandial level of the cardiovascular marker; and wherein the term "postprandial" refers to a period of time within 4 hours after administration of food and/or nutrients and/or beverages and/or diet and/or nutritional therapy to the subject.

2. Use according to claim 1, wherein the cardiovascular marker is selected from the group consisting of ANP, proANP, NT-proANP, MR-proANP, BNP, pro-BNP, NT-pro-BNP, ET-1, pro-ET-1, CT-proET-1, ADM, pro-ADM, MR-proADM, AVP, pro-AVP, metafolacin II and copeptin.

3. Use according to claim 1, wherein the relative level of one or more cardiovascular markers is determined using an assay with a sensitivity of 1nmol/L or less.

4. Use according to claim 3, wherein the relative level of one or more cardiovascular markers is determined using an assay having a sensitivity of 0.5pmol/L or less.

5. Use according to claim 1, wherein the determination of the relative level comprises the steps of:

a. determining a basal level of one or more cardiovascular markers in the subject,

b. determining postprandial levels of the one or more cardiovascular markers,

c. calculating the relative level of one or more cardiovascular markers from the values obtained in steps a and b.

6. The use of claim 1, wherein the level of said cardiovascular marker in said subject is determined using an immunoassay.

7. Use according to claim 1, wherein the term "postprandial" refers to a period of time within 2 hours after administration of food and/or nutrients and/or beverages and/or diet and/or nutritional therapy to the subject.

8. Use according to claim 1, wherein the term "postprandial" refers to a period of time between 15 and 60 minutes after administration of food and/or nutrients and/or beverages and/or diet and/or nutritional therapy to the subject.

9. Use according to any one of the preceding claims, wherein additionally the level of one or more other markers or clinical parameters is determined, said one or more other markers or clinical parameters having predictive value for the classification of the predisposition of said subject to a disorder of the metabolic system and/or the cardiovascular system, wherein a clinical parameter is any parameter capable of affecting said predisposition, said parameters comprising: age, sex, past history of disease, body mass index, genetic susceptibility/family history, ethnic background, and patient preference for affecting the predisposition.

10. The use of claim 9, wherein the past history of the disease is selected from the past history of hypertension and the past history of obesity, and the preference is selected from the group consisting of smoking, drinking, eating, exercise, and medication.

11. The use of claim 9 wherein the past history of disease is a past history of trunk obesity.