Method for identifying individual for vascular and cancer disease risk

FIELD: medicine, oncology, molecular pharmacology.

SUBSTANCE: invention relates to a method and set for identifying the individual subjected to risk for arising in it the vascular and cancer disease. Method involves stages for the quantitative determination of the analyte concentration, i. e. pepsinogen I (PGI), in serum sample taken in the personal individual; comparison of the analyte concentration determined by the proposed method with a method-specific boundary value for this analyte; determination of the homocysteine concentration in a serum sample taken in this individual. The set comprises the combination of separate components that are necessary for the quantitative determination of the PGI concentration. Method provides the early detection of the possibility for arising the vascular and cancer disease in the patient.

EFFECT: improved method for assay.

4 cl

 

The scope to which the invention relates.

The present invention relates to the combined diagnostic method for the identification of individuals with risk of coronary and vascular diseases, and cancer.

The method of the present invention is based on the combination of two tests carried out on samples of blood or serum to identify individuals who have a predisposition to form or manifestation of elevated homocysteine levels and are thus at risk of developing the diseases caused by these elevated levels, such as heart disease and cardiovascular disease, including atherosclerosis and ischemic stroke, and cancer.

Background of invention

Gastric cancer is preceded by a number of different gastric diseases or conditions, such as chronic atrophic gastritis, pernicious anaemia, stomach ulcers, polyps of the stomach and disease minetree (giant hypertrophic gastritis). Clearly identifiable changes in the mucosa are dysplasia and adenoma. It was found that almost all diseases risk arises due to the development of chronic atrophic gastritis.

Chronic gastritis is characterized by a prolonged inflammatory status shall eat of the gastric mucosa. This disease can be roughly divided into superficial and atrophic form. When superficial gastritis area of inflammatory cellular infiltrate is concentrated below the surface of the epithelium. When inflammation develops and spreads between specific gastric secretory glands, it can be attributed to chronic atrophic gastritis. In this case, normal glandular structure of the gastric mucosa, at least in part, undergo metaplastic changes.

Was estimated relative risk of gastric cancer in patients with atrophic gastritis in the body of the stomach, which was calculated based on statistics from the Finnish cancer, about 4-5 times the risk of cancer in persons with healthy mucosa of the stomach. In addition, the risk of cancer exists in patients with pernicious anemia due to deficiency of intrinsic factor and impaired absorption of vitamin B12. In severe atrophy in the area of privratnikovogo cavity risk increases even 18 times. If atrophic changes are observed both in the field of privratnikovogo cavity and in the body of the stomach (pangastrit), the risk is increased in 90 times.

In the publication WO 96/15456 described screening method for determining the risk of illness is Denmark gastric cancer, in accordance with which the atrophy of the mucosa of the body or privratnikovogo of the stomach cavity, or both is determined by measuring the levels of analytes, i.e. of pepsinogen I (PGI) and gastrin-17 (G-17), in the serum sample, and comparing the thus determined, the levels with method-specific boundary value for the corresponding analyte. Measured levels is also preferable compared to method-specific reference value for the respective analyte.

The value of PGI in the serum, which is below the specific limit values for PGI, indicates atrophic gastritis in the body of the stomach. If the concentration of G-17 in serum below its boundary values, the atrophy is localized in the area of privratnikovogo of the stomach cavity. When pangastrita, the concentration of serum PGI has a value below the cutoff, and the concentration of G-17 in serum is the lower limit of the reference value.

Methylenetetrahydrofolate-reductase (MTHFR) is an intracellular enzyme that is required for demetilirovania homocysteine to form methionine. Disruption of the functioning of this enzyme caused by defects in the structure of the gene MTHFR or deficiency of micronutrients such as folate, vitamin B6 and/or vitamin B12. Disruption of the functioning of the enzyme MTHFR leads to increased levels of homocysteine in Siwa ode/plasma (homocysteinemia) and homocysteinuria. Many studies have shown that elevated levels of homocysteine in serum/plasma are associated with increased risk of various coronary and vascular disease, and high homocysteine in serum/plasma, exceeding the reference value represents a major independent risk factor for coronary and vascular disease and ischemic stroke1-5.

It is assumed that the atherogenic effect of homocysteine based on the increased production of reactive oxygen molecules, which lead to perechislenie lipids. Ensuring a sufficient level of vitamin B12 is necessary for the metabolism of folate and normal production of blood cells, as well as for the functioning of nerve cells. Vitamin B12 forms a complex with the protein, the internal factor produced by the mucous membrane in the body of the stomach, where this complex is resorbed in the lower part of the ileum. This complex is the preferred form of resorbtive vitamin B12.

Deficiency of intrinsic factor, as a consequence of atrophic gastritis or gastric cancer, especially in the area of the body of the stomach, ultimately, leads to vitamin B12 deficiency, and hence to increased concentrations of homocysteine. Thus, it would be extremely important to identify the ex individuals, have a vitamin B12 deficiency or there is a high risk of vitamin B12 deficiency due to atrophic gastritis, and which therefore may have elevated levels of homocysteine in serum or plasma. Early introduction of supplementary vitamin B12 these individuals should favor the warning they have vascular disease. In addition, it would be extremely important to identify those individuals who have a risk of cancer due to overproduction intracellular oxygen radicals, and which may be favorable supplementation of vitamin B12 or other treatment.

Brief description of the invention

The present invention relates to a method of combining analysis to determine marker for atrophic gastritis in a sample of serum, with analysis of homocysteine to facilitate diagnosis or to identify the risk of cardiovascular and cancer in an individual, where the term "vascular" has a wide meaning and applies to any coronary or vascular disease that may develop as a result of atherogenic effect of homocysteine.

The method of the present invention is a method of identification of the individual with the risk of vascular disease and cancer, DG is the method involves the following stages:

- quantitative determination of the analyte concentration, pepsinogen I (PGI), in a sample of serum taken from a particular individual,

selection method-specific boundary values for the specified analyte,

- comparison of certain thus the concentration of the analyte with the method-specific boundary value for a given analyte and

- determine the concentration of homocysteine in the serum sample taken from the individual, and its comparison with method-specific reference value for homocysteine.

The present invention also allows the identification of the individual with the concentration of pepsinogen I in serum following method-specific boundary values for serum pepsinogen I and the concentration of homocysteine in serum above the reference values for homocysteine as an individual with an increased risk of vascular and/or cancer, or prone to these diseases.

The identification of concentrations of PGI and homocysteine in serum can be carried out in any order in order to simultaneously receive data on the levels of both PGI and homocysteine in serum to facilitate diagnosis of cardiovascular diseases or cancer, or to assess the risk of these diseases. However, in accordance with one variant of the invention, the first about Radelet the concentration of serum PGI and compare it with a specific or selected method-specific boundary value and for the subsequent diagnosis of selected individual from whom the PGI concentration in serum following method-specific boundary values. In this embodiment of the invention on homocysteine examine only those individuals in the serum which there are low levels of PGI, indicating atrophy of the mucosa of the gastric body.

In accordance with one embodiments of the invention determine the concentration of B12 in the serum of a specified individual, and comparing it with the method-specific reference boundary value for vitamin B12.

The present invention includes a step of comparing the measured concentrations of the analyte with the method-specific boundary value or a reference value for the specified analytes. The choice of these values is well known in the art and depends on the specificity and sensitivity selected for the test method used for determination of the concentration of the analyte, see for example, William J. Marshall, Clinical Chemistry, Third Edition, 1995, Mosby.

In accordance with the preferred embodiment, the present invention mainly aims to identify those individuals who still do not have vitamin B12 deficiency, that is, which have mostly normal levels of vitamin B12, but which, due to the low values of PGI, was diagnosed with atrophy in the area of the tee body of the stomach, and which also have high levels of homocysteine in the serum. Such identification enables early to resort to precautionary measures, for example, to the introduction of additional B12 to combat disease, associirowannyh with elevated homocysteine levels. Detailed description of the invention

1. Determination of pepsinogen I (PGI)

The method of determining the PGI in the serum sample can be carried out, as described in the publication WO 96/15456, which is incorporated into this description by reference.

This method preferably includes the stage of use of poly - or monoclonal antibodies against pepsinogen I in immunological method for the determination of pepsinogen I. Usually the reaction is carried out in a suitable medium such as plastic, glass or cellulose media, such as a microplate. The immunological methods can be carried out in a known manner, for example by the method of optical absorption, luminescence method or fluorescence method for measuring the concentration of a specified pepsinogen I in the sample.

If the concentration of pepsinogen I in serum below the cutoff value, which depends on the specificity and sensitivity of agreed upon for the method in question, and is 20-30 μg/l, corresponding to approximately 450-mol/l, it means that atrophy is confined in the body region of the stomach. Normal or reference value for PGI is in the range from 25 to 120 mg/L.

2. Determination of homocysteine

Homocysteine levels in serum can be determined by any known per se methods, which are used for these purposes, and which are also commercially available, for example, in the form set. Known method for the quantitative determination of total homocysteine in plasma or serum is liquid chromatography high resolution with radioactive, fluorescent or electrochemical detection. In addition, a method was developed enzyme-linked immunosorbent assay (ELISA) (Bio-Rad Laboratories; Axis-Shield A/S), and method of fluorescent polarization immunoassay (FPIA)(FPIA; Abbott Laboratories), where the specified immunoassay involves the pre-processing of samples dithiothreitol and adenosine followed by the enzymatic stage of obtaining S-adenosyl-L-homocysteine, and where total homocysteine was determined using monoclonal antibodies against S-adenosyl-L-homocysteine, see, for example, U.S. patent 5631127.

Reference values for homocysteine are, to some extent, method-specific, but generally, they range from approximately 5 to 15 µmol/l homocysteine in serum greater than the second method-specific reference levels for homocysteine, is considered as a component of the risk factor, as explained above. In most cases, we can assume that homocysteine levels above 15 µmol/l represents such a high level, which indicates a clear risk factor.

3. Determination of vitamin B12

In accordance with the present invention, the diagnostic method includes, but is not necessarily, the concentration of vitamin B12 in the serum sample. The concentration of vitamin B12 (cobalamin) can be determined using any of the methods known per se and used for this purpose. Such known methods include conducting microbiological analysis to determine the B12 in serum using micro-organisms such as Euglena gracilis or Lactobaclllus leichmanii that its growth requires the presence of cobalamin. To determine B12 were also used analysis method of cultivation radioisotopes, such analytical methods are well described in the literature, for example, Lau et al. "Measurement of serum B12 levels using Radioisotope Dilution and Coated Charcoal, Blood, 26 (1965), 202. Methods of breeding radioisotopes are faster and give results comparable with the results of the analysis, for example, using Euglena, provided that the binding protein is specific for bioactive cobalamin. A standardized preparation of pure or purified internally what about the factor is most suitable as a binding protein, because it specifically binds to the true cobalamin, and not with analogues of cobalamin.

Analysis on B12 method of cultivation radioisotopes typically involves the step of separating endogenous B12 from its natural binding protein, for example, by boiling at the desired pH and then adding a measured quantity of the radioisotope57Co-B12 and the limited number of binding protein. All binding protein will bind with a specific form of B12, because the number of added radioisotope-labeled B12 is sufficient for binding of a small amount of protein. Because both natural and radioactive B12 compete for binding to the binding protein, the degree to which inhibited the amount of radioactivity associated with the protein B12, indicates the amount of B12 in the sample. This method was modified Lau, see above, by separating unbound B12 from vitamin B12-related protein, using coated with protein coal dust, and counting the radioactivity of the supernatant liquid containing the mixture associated radioactive B12 and associated non-radioactive vitamin B12. Then based on this number, calculate the concentration of B12 in serum, often by comparison to a standard curve. To implement this method, there are commercially available kit for radioisotope the analysis.

Deficiency of vitamin B12 can also be determined using, for example, chemoluminescent receptor assays (Wentworth S. et al., Clin. Chem., vol.40, 537-540), radioimmunoassays (Endres, D.B., et al. Clin. Chem., Vol.24, 460-465), as well as tests for non-isotropic binding, CEDIA, i.e. immunofermentnyh assays using cloned donor enzyme (van der Weide, J. et al. Clin. Chem., Vol. 38, 766-768).

Reference value for B12 varies from 200 to 900 ng/l, corresponding to approximately 170-700 pmol/L.

In recent unpublished work, it was found that 50% of individuals suffering from atrophy of the gastric body (SPGI < 25 µg/l)the concentration of vitamin B12 in serum was below the lower limit of the reference values (<170 pmol/l)had a significantly increased concentration of homocysteine in serum (mean 33.3 mmol/l, 16-157 µmol/l). In addition, 22% of these individuals, in which the concentration of vitamin B12 in serum was 180-230 pmol/l (reference values 170-700 pmol/l)also had elevated concentrations of homocysteine (an average of 18.9 µmol/l, 16-25 µmol/l).

The present invention also relates to a kit for use in the method of the present invention, where a specified set includes:

- means for determining the concentration of PGI in the sample serum,

- means for determining the concentration of gomati theine in a sample of serum.

The kit of the present invention may contain a combination of single components necessary for quantitative determination of the concentration of pepsinogen I and homocysteine in a sample of blood serum. For these purposes specified set may contain a separate vials or containers for required components, such as antibodies and substrates that can be used for exploration of the analyte.

Links

1. C.J. Boushey, S.A.A. Beresford, G.S. Omenn, A.G. Motulsky, JAMA 1995; 274:1049-57.

2. Graham I.M., Daly LE, Rafsum H.M., et. al. The European Concerted Action Project, JAMA 1997; 277:1775-81.

3. Jacobsen OW, Clin Chem 1998; 44:1833-43.

4. Mogadashian M.H., B.M. McManus, Frohlich J.J., Arch Intern Med 1997; 157:2299-2308.

5. Rafsum H., Ueland P.M.,O, Vollset S.E., Rev Medicine 1998; 49:31-62.

1. The method of determining the risk of an individual cardiovascular or cancer, which includes stages of quantitative determination of the analyte concentration, pepsinogen I (PGI) in a sample of serum taken from a specified individual, the choice of method-specific boundary values for the specified analyte, the specific comparison thus the concentration of the analyte with the method-specific boundary value for a given analyte and determining the concentration of homocysteine in a sample of serum taken from a specified individual, and its comparison with method-specific reference value for homocysteine, and the concentration of the situation of serum pepsinogen I, having a value below the method-specific boundary values, and the concentration of homocysteine in serum in excess of its method-specific reference value, indicates that this individual has an increased risk of vascular disease and cancer.

2. The method according to claim 1, comprising determining the concentration GI in serum and the selection of the individual from whom serum PGI below its boundary values, to further determine the concentration of homocysteine.

3. The method according to claim 1 or 2, providing for additional stages determine the concentration of vitamin B12 in the sample and its comparison with method-specific reference value.

4. A kit for carrying out the method according to claim 1, including means for determining the concentration of PGI in the serum sample, a means for determining the concentration of homocysteine in a sample of serum.

5. The kit according to claim 4, where said funds to determine the concentration of pepsinogen I and homocysteine include a tool for immunological analysis.



 

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