EP1210452A1 - Method for identifying an individual at risk for vascular and cancer disease - Google Patents
Method for identifying an individual at risk for vascular and cancer diseaseInfo
- Publication number
- EP1210452A1 EP1210452A1 EP00956556A EP00956556A EP1210452A1 EP 1210452 A1 EP1210452 A1 EP 1210452A1 EP 00956556 A EP00956556 A EP 00956556A EP 00956556 A EP00956556 A EP 00956556A EP 1210452 A1 EP1210452 A1 EP 1210452A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- concentration
- homocysteine
- serum
- individual
- determining
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6806—Determination of free amino acids
- G01N33/6812—Assays for specific amino acids
- G01N33/6815—Assays for specific amino acids containing sulfur, e.g. cysteine, cystine, methionine, homocysteine
Definitions
- the present invention relates to a combination diagnostic method for identifying individuals who are at risk for coronary and vascular, as well as cancer diseases.
- the method according to the invention is based on the combination of two tests carried out on a blood or serum sample in order to identify individuals prone to develop or exhibiting elevated levels of homocysteine, which individuals are thus at risk of developing afflictions resulting from such elevated levels, such as car- dio- and cerebrovascular disease, including atherosclerosis and ischaemic stroke, as well as cancer diseases.
- gastric diseases or conditions such as chronic atrophic gastritis, pernicious anaemia, ventricular ulcer, gastric polyposis and the Menetrier disease (giant hypertrophic gastritis) precede gastric cancer.
- Menetrier disease giant hypertrophic gastritis
- mucosa dysplasia and adenoma. It has been established that in almost all diseases the risk is mediated over chronic atrophic gastritis.
- Chronic gastritis means a prolonged inflammatory condition of the gastric mucosa.
- the disease can coarsely be divided into the superficial and the atrophic form.
- superficial gastritis the inflammatory cell infiltration is concentrated below the surface epithelium.
- chronic atrophic gastritis the normal glandular structures of the gastric mucosa are at least partly substituted by metaplastic changes.
- the relative risk of gastric cancer in patients suffering from atrophic gastritis in the corpus area of the stomach has been estimated, as calculated from the Finnish cancer statistics, to be about 4- to 5-fold as compared to persons having a healthy mucosa.
- the publication WO 96/15456 discloses a method for screening for the risk of gastric cancer according to which atrophy in the mucosa either of the corpus or the antrum area, or of both, is determined by determining the pepsinogen I (PGI) and gastrin-17 (G-17) analyte levels in a serum sample, and comparing the levels so determined to a method-specific cut-off value for respective analyte.
- the levels determined are preferably also compared to a method-specific reference value for respective analyte.
- a serum PGI value below the specific cut-off value for PGI indicates atrophic gastritis in the corpus area of the stomach. If the serum G-17 concentration is below its cut-off value, the atrophy is located in the antrum area of the stomach. In pangastritis, the serum PGI is below the cut-off value and the serum G-17 value is at the lower limit of its reference value.
- MTHFR Methylenetetrahydrofolate reductase
- Impaired function of this enzyme is caused by defects in the structure of the MTHFR gene, or by nutritional deficiencies of i.a. folate, vitamin B6 and/ or vitamin B12. Impaired function of the MTHFR enzyme results in an increase in the serum/plasma level of homocysteine (homocysteinemia) and in homocysteinuria.
- Increased serum/plasma levels of homocysteine has in many studies shown to be associated with an increased risk for various coronary and vascular diseases, a high serum/plasma level of homocysteine above the reference value being a serious independent risk factor for coronary and vascular disease and ischaemic stroke. 5
- the atherogenic influence of homocysteine is thought to be based on an increased production of reactive oxygen species which enables lipid peroxidation.
- Vitamin B12 forms a complex with a protein, the intrinsic factor, produced by the mucosa of the corpus area of the stomach, which complex is resorbed in the lower part of the ileum. This is the preferred resorption form of vitamin B12.
- a deficiency of intrinsic factor as a result of atrophic gastritis or stomach cancer, especially in the corpus area of the stomach, will ultimately lead to a deficiency in B12 vitamin in the body and hence to an increase in the homocysteine concentration. It would thus be highly valuable to identify those subjects who have or are at a high risk for vitamin B12 deficiency due to atrophic gastritis, and who therefore might have an elevated serum or plasma level of homocysteine. In those persons an early initiation of vitamin B12 supplementation would be beneficial in the prevention of vascular diseases. It would also be valuable to be able to identify those individuals which would be at risk of cancer due to overproduction of intra- cellular oxygen radicals, and in whom a B12 vitamin supplementation or other treatment could be beneficial.
- the present invention is directed to a method for combining an assay of determining, in a serum sample, a marker for atrophic gastritis in combination with an assay for homocysteine, in order to assist in the diagnosis of, or in the determination of the risk of vascular, as well as cancer diseases in an individual, the term vascular being understood broadly to include any coronary or vascular disease which can result from the atherogenic influence of homocysteine.
- the method according to the invention is a method for identifying an individual at risk for vascular and cancer disease, the method comprising the steps of
- PKI pepsinogen I
- the present invention thus allows for the identification of an individual having a serum pepsinogen I concentration below the method-specific cut-off value for serum pepsinogen I and a serum homocysteine concentration above the reference value for homocysteine, as being an individual with an increased risk of, or having a predisposition for vascular and/or cancer disease.
- the determination of the serum PGI and homocysteine concentrations can take place in any order, in order to simultaneously obtain knowledge of both the serum PGI and homocysteine levels for the purpose of assisting in the diagnosis of, or assessing the risk of vascular disease or cancer.
- the serum PGI concentration is determined and compared to its determined or selected method-specific cut-off value, and for further diagnosis an individual is selected who has a serum PGI concentration value below its method-specific cut-off value. In this embodiment, only the individuals which exhibit low serum PGI values indicative of corpus atrophy, are screened for homocysteine.
- the B 12 concentration is determined in the serum of the said individual, and compared to a method-specific reference value for vitamin B12.
- the invention includes a step of comparing the measured analyte concentrations to method- specific cut-off or reference values for said analytes. The selection of such values is well known to a person skilled in the art, and depends on the specificity and sensitivity chosen for the test method used for the determination of the analyte concentrations, see e.g. William J Marshall, Clinical Chemistry, Third
- the present invention is especially aimed at screening such individuals, which still do not exhibit a B12 vitamin defi- ciency, that is which exhibit substantially normal B12 vitamin levels, but which due to low PGI values are diagnosed for atrophy in the corpus area of the stomach and which also have high homocysteine serum levels.
- a B12 vitamin defi- ciency that is which exhibit substantially normal B12 vitamin levels, but which due to low PGI values are diagnosed for atrophy in the corpus area of the stomach and which also have high homocysteine serum levels.
- the method of determining PGI in a serum sample can be carried out as is described in the publication WO 96/15456, which publication is included herein for reference.
- the said method preferably includes the steps of using poly- or monoclonal antibodies to pepsinogen I in an immunological method for the determination of pepsinogen I.
- the reaction is carried out on a suitable support, such as a plastic, glass or cellulose support, for example on a microplate.
- the immunological methods can be carried out in a known manner, using e.g. absorbance, lu- minescence or fluoresence techniques for measuring the said pepsinogen I concentration in the sample. If the serum pepsinogen I concentration is below the cut-off value, which depending on the specificity and sensitivity agreed upon for the method in question, is 20-30 ⁇ g/1, which corresponds to appr. 450 - 690 pmol/1, there is atrophy in the corpus area of the stomach.
- the normal or reference value for PGI is in the range of 25 - 120 ⁇ g/1.
- Homocysteine levels in serum can be determined according to any of the methods known per se for this purpose and which are also commercially available, e.g. in kit form.
- the established method for quantifying total homocysteine in plasma or serum is high performance liquid chromatography with radioactive, fluorescent or electrochemical detection.
- EIA enzyme immunoassay
- FPIA fluorescence pola- risation immunoassay
- the immunoassay including pretreating the specimens with dithiothreitol and adenosine, followed by an enzymatic step to form S-adenosyl-L-homocysteine, and total homocysteine is measured e.g. using monoclonal anti-S-adenosyl-L-homocysteine antibodies, see e.g. US 5,631,127.
- homocysteine is to some degree method-specific, but generally varies between appr. 5 to 15 ⁇ mol/L.
- a serum homocysteine level which is above the method-specific reference level for homocysteine is taken as constituting a risk factor, as explained above.
- a homocysteine level of above 15 ⁇ mol/L can in most cases be considered to constitute such an elevated level constituting a clear risk factor.
- the method of diagnosis includes an optional method of determining the B 12 vitamin concentration in the serum sample.
- the B 12- vitamin (cobalamin) concentration can be determined according to any of the methods per se known for this purpose.
- Such known methods include microbiological assay of serum B12 employing an organism, such as Euglena gracilis or Lactoba- cillus leichmannii which requires cobalamin for growth.
- radioisotope dilution assays for B 12 vitamin have been utilized and such assay techniques are well documented in the literature, e.g. Lau et al , "Measurement of serum B12 levels using Radioisotope Dilution and Coated Charcoal", Blood, 26 (1965), 202.
- Radioisotope dilution methods are more rapid and give results comparable with those of e.g. the Euglena assay, provided the binding protein is specific for biologically active cobalamin.
- a standardized pure or purified intrinsic factor preparation is most satisfactory as the binding protein as it binds specifically to true cobalamin rather than cobalamin analogues.
- the radioisotope dilution assay of B12 generally includes the step of freeing endogenous B12 from its natural binding protein e.g by boiling at a selected pH and then adding a measured amount of the radioisotope 57Co-B12, and a limited amount of binding protein. All of the binding protein will be bound by some form of B12 since the amount of radioisotope B12 added is sufficient to bind the small amount of protein. As both the natural and the radioactive B12 compete to bind with the binding protein, the degree to which the radioactive count of the protein bound B12 was inhibited is indicative of the amount of B12 in the sample.
- This method has been modified by Lau, supra, by separating unbound B12 from protein bound B12 by protein coated charcoal and the radioactivity of the supernatant liquid containing the mixture of bound radioactive B12 and bound non-radioactive B12 is counted for radioactivity. The serum B12 concentration is then calculated from the count, often by comparison with a standard chart. Radioassay kits are commercially available for carrying out the method.
- B12 vitamin deficiency has also been determined using e.g. chemiluminiscence receptor assays (Wentworth, S. et al , Clin. Chem., vol 40 537-540), radioim- munoassays (Endres, D.B., et. al. Clin. Chem. , Vol. 24, 460-465) as well as no- nisotopic binding assays, CEDIA, cloned enzyme donor immunoassays (van der Weide, J. et al. Clin. Chem. , Vol. 38, 766-768).
- the reference value for B12 varies between 200-900 ng/1, corresponding to appr. 170 to 700 pmol/1.
- the present invention also relates to a kit for use in the method according to the invention the kit comprising
- the kit according to the invention can comprise a combination of the individual components needed to quantitatively determine the serum pepsinogen I and the homocysteine concentration in a blood serum sample.
- the kit can comprise separate vials or containers for the necessary components, such as antibodies and substrates to be used for the determination of the analyte.
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FI991836A FI107808B (fi) | 1999-08-31 | 1999-08-31 | Menetelmä verisuoni- sekä syöpäsairaudelle riskialttiin yksilön identifioimiseksi |
| FI991836 | 1999-08-31 | ||
| PCT/FI2000/000733 WO2001016356A1 (en) | 1999-08-31 | 2000-08-30 | Method for identifying an individual at risk for vascular and cancer disease |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP1210452A1 true EP1210452A1 (en) | 2002-06-05 |
Family
ID=8555217
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP00956556A Ceased EP1210452A1 (en) | 1999-08-31 | 2000-08-30 | Method for identifying an individual at risk for vascular and cancer disease |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP1210452A1 (enExample) |
| JP (1) | JP2003508059A (enExample) |
| CN (1) | CN1158389C (enExample) |
| AU (1) | AU6845600A (enExample) |
| FI (1) | FI107808B (enExample) |
| RU (1) | RU2247389C2 (enExample) |
| WO (1) | WO2001016356A1 (enExample) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2312356C2 (ru) * | 2005-12-28 | 2007-12-10 | Федеральное государственное лечебно-профилактическое учреждение "Научно-клинический центр охраны здоровья шахтеров" Федерального агентства по энергетике РФ | Способ оценки степени тяжести ишемического инсульта у лиц в возрасте до 50 лет |
| RU2413940C2 (ru) * | 2009-02-02 | 2011-03-10 | Государственное образовательное учреждение высшего профессионального образования Воронежская государственная технологическая академия | Способ определения витамина b12 в водных растворах |
| RU2453001C1 (ru) * | 2010-10-18 | 2012-06-10 | Государственное образовательное учреждение высшего профессионального образования "Курский государственный медицинский университет Федерального агентства по здравоохранению и социальному развитию" | Способ коррекции гипергомоцистеин индуцированного дефицита оксида азота l-норвалином в эксперименте |
| CN114924016B (zh) * | 2022-04-28 | 2024-03-08 | 中国医学科学院北京协和医院 | 同时检测硫酸化及未硫酸化胃泌素g17的方法及其检测试剂盒 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FI97304C (fi) * | 1994-11-16 | 1996-11-25 | Locus Genex Oy | Menetelmä mahasyövän riskin seulonnaksi |
-
1999
- 1999-08-31 FI FI991836A patent/FI107808B/fi active
-
2000
- 2000-08-30 JP JP2001520901A patent/JP2003508059A/ja active Pending
- 2000-08-30 WO PCT/FI2000/000733 patent/WO2001016356A1/en not_active Ceased
- 2000-08-30 EP EP00956556A patent/EP1210452A1/en not_active Ceased
- 2000-08-30 RU RU2002107982/15A patent/RU2247389C2/ru active
- 2000-08-30 AU AU68456/00A patent/AU6845600A/en not_active Abandoned
- 2000-08-30 CN CNB008122261A patent/CN1158389C/zh not_active Expired - Lifetime
Non-Patent Citations (1)
| Title |
|---|
| See references of WO0116356A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| RU2247389C2 (ru) | 2005-02-27 |
| CN1371427A (zh) | 2002-09-25 |
| WO2001016356A1 (en) | 2001-03-08 |
| FI19991836L (fi) | 2001-02-28 |
| CN1158389C (zh) | 2004-07-21 |
| FI107808B (fi) | 2001-10-15 |
| AU6845600A (en) | 2001-03-26 |
| JP2003508059A (ja) | 2003-03-04 |
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