FI107808B - A method for identifying an individual at risk for vascular and cancerous disease - Google Patents

Description

107808

A method for identifying an individual at risk for vascular and cancer disease

The invention relates to a combination diagnostic method for identifying individuals susceptible to coronary and vascular diseases as well as cancers.

5

The method of the invention is based on a combination of two assays performed on a blood or serum sample to identify individuals who have a tendency to develop or are shown to have elevated levels of homocysteine and thus are susceptible to developing diseases such as cardiovascular or cerebrovascular disease. atherosclerosis and ischemic stroke; and cancer.

Many different gastric diseases or conditions, such as chronic atrophic gastritis, pernicious anemia, stomach ulcer, gastric polyposis, and Mdndtrier disease 15 (giant hypertrophic gastritis) precede gastric cancer. Clearly identifiable changes in the mucosa are dysplasia and adenoma. It has been speculated that in almost all diseases, susceptibility is mediated through chronic atrophic gastritis.

Chronic gastritis is a prolonged inflammatory condition of the stomach lining. Illness. 20 can be roughly divided into superphyseal and atrophic forms. In superphysical ** · *: in the gastric tract, inflammatory cell infiltration is concentrated under the surface epithelium. When · ·: V: Inflammation progresses and diffuses between specific gastric secretory glands, it is ·: ··: chronic atrophic gastritis. In this case, metaplastic changes at least partially replace the normal glandular mucosal structures.

· «» • · · o r ·. · · 25

Patients suffering from atrophic gastritis in the gastrointestinal tract have an estimated * * * risk of gastric cancer, calculated from Finnish cancer statistics, at approximately 4- to 5-fold compared to individuals with a healthy mucosa. .

• · ·, * ·· In addition, there is a risk of developing anemia due to intrinsic-deficiency and a malabsorption of vitamin B12. In severe Antrum •: · ·: The risk of atrophy is up to 18-fold. If atrophic changes occur in both the antrum and corpus region (pangastritis), the risk can increase up to 90-fold.

2 107808

WO 96/15456 discloses a method for screening for the risk of gastric carcinoma, which involves detecting atrophy in either the corpus or antrum region, or both, by analyzing serum concentrations of pepsinogen I (PGI) and gastrin-17 (G-17) in a serum sample and comparing method-specific cut-off value for the analyte corresponding to the specified concentrations. Preferably, the concentrations determined are also compared to the method-specific reference value of the corresponding analyte.

A serum PGI value below the specific cut-off value of PGI is indicative of 10 atrophic gastritis in the corpus cavernosum. If serum G-17 concentration is below its cut-off value, atrophy is located in the antrum region of the stomach. In Pangastritis, the PGI of the serum is below the cut-off value and the G-17 of the serum is below the lower limit of its reference value.

Methylene tetrahydrofolate reductase (MTHFR) is an intracellular enzyme required for the re-methylation of homocysteine to methionine. The impaired function of this enzyme is due to deficiencies in the structure of the MTHFR gene or. mm. nutritional deficiencies of folate, vitamin B6 and / or vitamin B12. MTHFR- • ♦ • ♦ ♦ Dysfunction of the enzyme results in serum / plasma levels of homocysteine • ♦ · *. I 20 for elevation (homo-cysticemia) and homocystinuria. Elevated serum levels of homocysteine • · ·] have been shown in many studies to be associated with different levels of • · # ··· (increased risk of cardiovascular disease, high serum levels of homocysteine • 1mL / plasma above the reference value, cardiovascular disease). and a severe independent risk factor for ischemic stroke.1'5 25 • ♦ ** '1; The atherogenic effect of homocysteine is thought to be due to increased production of reactive oxygen species, allowing lipid peroxidation.

* 1 ·. ···, Adequate supply of vitamin B12 is essential for folate metabolism and normal blood production, as well as for nerve function. Vitamin B12 forms a complex • ·. 3 0 sin protein, intrinsic factor, the gastric mucosa of the corpus area • · · formed and that the complex resporboituu the lower part of the ileum. This 3,107,808 is the most preferred form of vitamin B12 resorption.

Deficiency of the intrinsic factor due to atrophic gastritis or gastric cancer, particularly in the corpus cavernosum, eventually results in a deficiency of vitamin B12 in the body 5 and thus an increase in homocysteine concentration. Thus, it would be particularly valuable to identify individuals who are, or are at high risk of, vitamin B12 deficiency due to atrophic gastritis and who, for this reason, may have elevated serum or plasma levels of homocysteine. Early initiation of an additional vitamin B12 dose in these individuals would be beneficial in preventing vascular disease.

It would also be valuable to be able to identify individuals at risk of cancer due to overproduction of intracellular oxygen radicals and who would benefit from additional vitamin B12 or other treatment.

The invention relates to a method of combining a test for the determination of an atrophic gastritis marker in a serum sample with a homocysteine assay to aid in the diagnosis or assessment of the risk of individual vascular and cancerous diseases, the term vascular being broadly understood to include any cardiovascular disease. for that effect.

The method of the invention is a method for identifying an individual at risk for vascular and cancer disease, the method comprising the steps of: · · ·: - quantifying the analyte concentrations of pepsinogen I (PGI) • · ·: Determining a method specific cut-off value for said analyte λ * * in a serum sample of said individual, - comparing the thus determined analyte concentration with the specific cut-ofF value of the analyte, and · · - determining the homocysteine concentration in an individual's serum sample and comparing it with a method specific reference for homocysteine.

,:. = 30 • ·: · j Serum PGI and homocysteine concentrations can be determined in any order of 4,107,808 to provide simultaneous information on both serum PGI and homocysteine levels, which are intended to assist in the diagnosis or risk assessment of vascular disease or cancer. However, according to one embodiment of the invention, a serum PGI concentration of 5 is determined and compared to its determined or selected method-specific cut-ofF and homocysteine concentration is determined when the serum PGI concentration is less than its method-specific cut-ofF. In this embodiment, homocysteine assay is performed on individuals with low serum PGI values as evidence of corpus atrophy.

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According to one embodiment of the invention, the serum concentration of B12 in said individual is also determined and compared to a method specific reference value of vitamin B12.

Thus, according to a preferred embodiment, the object of the invention is in particular to screen individuals who are not yet deficient in vitamin B12, i.e., who have essentially normal levels of vitamin B12 but who, due to low PGI values, have been diagnosed with gastric corpus region atrophy and also has high serum homocysteine levels. Such identification is possible. 20 early on, for preventive measures, such as an additional dose of vitamin B12, elevated homocysteine levels. l to prevent the development of related damage.

1. Pepsinogen I (PGI) Assay • · · · · 25

A method for determining PGI in a serum sample can be carried out as described in WO 96/15456, which is incorporated herein by reference.

Preferably, said method involves the use of poly or monoidonal antibodies to pepsinogen I in an immunological method for the determination of pepsinogen I. sex. The reaction is preferably carried out on a suitable carrier, such as a plastic, glass or cellulose carrier such as a microplate. Immunological methods may be performed in a known manner using, for example, absorbance, luminescence or fluorescence techniques to measure the concentration of said pepsinogen I in a sample.

5 Serum pepsinogen I concentration below the cut-off value, which, depending on the specificity and sensitivity of the method in question, is 20-30 lg / l, corresponding to approximately 450-690 pmol / l, is atrophy of the corpus luteum. . Normal or reference value for PGI is in the range of 25-120 µg / L.

10 2. Assay for homocysteine

Homocysteine serum levels can be determined by any of the methods known for this purpose, which are also commercially available, e.g. in the form of a test kit. A known method for the quantitative determination of total homocysteine in plasma or serum is high performance liquid chromatography with radioactive, fluorescent, or electrochemical detection. An enzyme-linked immunoassay (EIA) method has also been developed (Bio-Rad Laboratories; Axis-Shield A / S) in the same way. ·: Resistance Polarization Immunoassay (FPIA; Abbot Laboratories) which Immunoassay • »·: V 20 contains sample pretreatment with dithiothreitol and adenosine followed by • · an enzymatic step to form S-adenosyl-L-homocysteine, and total * * homocysteine is measured e.g. using monoldonal anti-S-adenosyl-L- * * * * '' homocysteine antibodies, see, e.g., US 5,631,127.

i · · # 25 Homocysteine reference values are somewhat method specific, but generally * ... range from about 5-15 pnol / l. Serum homocysteine levels above the method specific reference level of homocysteine present a risk, as described above. A concentration of homocysteine greater than 15 pnol / l may be

• · '·; · * in which cases it is considered to be so high that it constitutes a clear risk factor 1.

6 107808 3. Determination of Vitamin B12

According to the invention, the diagnostic method comprises an alternative method of determining the vitamin B12 concentration in a serum sample. The concentration of vitamin B12 (cobalamin) 5 can be determined according to any method known in the art. Such known methods include a microbiological assay of serum B12 using an organism such as Euglena gracilista. or Lactobacillus leichmanni, which requires cobalamin to grow. Also, radiolabelled dilution assays of vitamin B12 have been used and such assay techniques are well described in 10 kits, e.g., "Measurement of Serum B12 Levels Using Lau et al.

Radioisotope Dilution and Coated Charcoal ", Blood, 26 (1965), 202. Radioisotope spoilage methods are faster and yield results comparable to those obtained, e.g., with the Euglena assay, provided that the binding protein is specific for biologically active cobalamin or pure. The -15 intrinsic factor preparation is the most satisfactory as a binding protein because it binds specifically to real cobalamin rather than to cobalamin analogs.

: *; The B12 radioisotope dilution assay generally includes the step where the endogenous B12 • · ·; * «*; 20 is released from its native binding protein, e.g., by boiling at a selected pH, «·; V: followed by addition of a measured amount of 57C0-B12 radioisotope and a limited amount of • i *: **: binding protein. All binding protein must be bound to some form of B12 because the amount of radioisotope B12 added is sufficient to bind a small amount of protein.

Because both natural and radioactive B12 compete for binding to binding protein, the degree to which the radioactive amount of protein-bound B12 is inhibited is an * · ** · indication of the amount of B12 in the sample. This method has been modified by Lau, supra, by separating the unbound B12 from the B12 bound to protein with protein-coated activated carbon and the supernatant liquid containing a mixture of bound radioactive B12 and bound non-radioactive B12. calculated.

·; ··; * 30 The B12 concentration of serum is then calculated from the result obtained, often by comparing it to a standard table. Radio test kits are commercially available • · 7 107808 to perform the method.

Vitamin B12 deficiency has also been determined using, for example, chemiluminescent receptor assays (Wentworth, S. et al., Clin. Chem., Vol. 40, 537-549), radioimmunoassays (Enders, DB, et al., Clin. Chem., Vol. 24, 460-465) as well as non-isotopic binding assays, CEDIA, immunoassays of a cloned enzyme donor (van der Weide, J. et al., Clin. Chem., Vol. 38, 766-768).

The reference value for B12 ranges from 200-900 ng / l, corresponding to about 170-700 pmol / l.

10 In our recent, unpublished work, we found that 50% of patients suffering from corpus atrophy (S-PGI <25 l & V and having a serum vitamin B12 concentration below the lower limit of reference (<170 pmoles / L) had significantly elevated serum homocysteine levels. (mean 33.3 15 pnol / l, 16-157 pnol / l) In addition, 22% of patients with serum vitamin B12 concentrations between 180 and 230 pmol / l (reference values 170-700 pmol / l) homocysteine concentration (mean 18.9 pnol / l, 16-25 pnol / l).

The invention also relates to a test kit for use in a method according to the invention, wherein the kit contains - means for determining PGI concentration in serum. in a sample, · · · means for determining the concentration of homocysteine in a serum sample.

• · · * · «. The test kit of the invention may include a combination of the individual components required to quantify the concentration of serum pepsinogen I and homocysteine in a blood serum sample. For this purpose, the test kit may contain separate ampoules or containers of required components, such as antibodies and substrates, for use in assaying the analyte.

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References 1. Boushey, C.J., Beresford, S.A.A., Omenn, G.S., Motulsky, A.G., JAMA 1995; 274: 1049-57-5 2. Graham, I.M., Daly, L.E., Rafsum, H.M. you do not. al., The European Concerted Action Project, JAMA 1997; 277: 1775-81 3. Jacobsen, O.W., Clin Chem 1998; 44: 1833-43 10 4. Mogadashian, M.H., McManus, B.M., Frohlich, J.J., Arch Intern Med 1997; 157: 2299-2308 5. Rafsum, H., Ueland, P.M., Nygärd, O., Vollset, S.E., Rev Medicine 1998; 15 49: 31-62.

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Claims (5)

9 107808 A method for identifying an individual at risk for cardiovascular disease, characterized in that the method comprises the steps of: 5. quantitatively determining the concentration of analyte pepsinogen I (PGI) in a serum sample of said individual, - determining a method specific cut-off value for said analyte, comparing the analyte concentration so determined with the method-specific cut-off value of the analyte; and 10. determining the concentration of homocysteine in an individual serum sample and comparing it with the method-specific reference value of homocysteine. Method according to claim 1, characterized in that the PGI concentration of the individual serum sample is determined and then the homocysteine concentration when the PGI concentration of the serum is below its cut-of ¥. Method according to claim 1 or 2, characterized in that it also comprises the step of determining the vitamin B12 concentration of the sample and comparing it with a method specific reference value. »· * ·» A test kit for carrying out the method of claim 1, comprising: means for determining the concentration of PGI in a serum sample, means for measuring the concentration of homocysteine in a serum sample. The test kit according to claim 4, wherein the means for determining the concentration of pepsinogen I and • · 1 »··· 'homocysteine are immunological means. V-bo ·· ♦ m 4 m · • ♦ · • • • • • 10 107808