WO2005080993A1 - 血糖制御状態の検査方法 - Google Patents
血糖制御状態の検査方法 Download PDFInfo
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- WO2005080993A1 WO2005080993A1 PCT/JP2005/003438 JP2005003438W WO2005080993A1 WO 2005080993 A1 WO2005080993 A1 WO 2005080993A1 JP 2005003438 W JP2005003438 W JP 2005003438W WO 2005080993 A1 WO2005080993 A1 WO 2005080993A1
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- postprandial hyperglycemia
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- 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
- G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
Definitions
- the present invention relates to a method for testing a blood glucose control state, and a method for diagnosing a blood glucose control state. More specifically, the present invention relates to a method for examining and diagnosing a blood glucose control state using a marker reflecting postprandial hyperglycemia.
- BACKGROUND ART In recent years, it has become known that diabetic patients have a significantly higher risk of developing cardiovascular events due to atherosclerosis than non-diabetic patients. Thus, the ultimate goal of recent treatment for diabetes is not limited to lowering blood sugar, but has been focused on suppressing cardiovascular events due to atherosclerosis.
- ADA American Diabetes Association
- WHO World Health Organization
- JDS Japanese Diabetes Association
- Non-Patent Document 4 A prospective study of about 25,000 cases in Europe with a mean follow-up of about 7 years (DECODE study) showed that elevated blood glucose 2 hours after meals was strongly associated with an increased risk of death (Lancet 354: 617 (1999), Non-Patent Document 4). Epidemiological studies in Funagata-cho, Yamagata Prefecture, in Japan, have also reported that postprandial hyperglycemia has twice the risk of causing myocardial infarction and cerebral infarction compared to normal individuals (Diabetes Care 22: 920 (1999)). Non-Patent Document 5).
- advanced glycation end products also referred to as advanced glycation end products, hereinafter referred to as AGEs
- AGEs advanced glycation end products
- AGEs derived from glucose is referred to as AGE1
- AGE derived from dariceraldehyde is referred to as AGE2.
- Many studies have been made on AGEs. However, there is no report so far on what kind of glycemic fluctuation these AGEs actually reflect.
- Non-Patent Document 2 Diabet Med 15: 539 (1998)
- Non-Patent Document 4 Lancet 354: 617 (1999)
- Non-Patent Document 6 Clin Chem Lab Med 41: 1144 (2003)
- Non-Patent Document 7 Free Radic Res 33: 115 (2000)
- Non-Patent Document 8 J Neuropathol Exp Neurol 62: 486 (2003)
- the present invention provides a novel marker capable of monitoring a change in postprandial hyperglycemia, preferably a marker capable of monitoring a postprandial hyperglycemia control state,
- the task is to develop an inspection method.
- AGE 2 can be a marker for postprandial hyperglycemia.
- dariseraldehyde which is a source of AGE 2
- AGE 2 levels in blood may fluctuate due to very short-term changes in blood glucose, and as a result of diligent studies, we have found that blood levels of AGE 2 correlate with postprandial hyperglycemia.
- the present inventors administered insulin, which continuously lowers blood glucose, and nateglinide, which only suppresses postprandial hyperglycemia, to a diabetic model animal, compared and examined various blood markers, and Unlike HbAlc, which represents the average blood glucose level, we searched for a marker with a difference between the group that did not suppress postprandial hyperglycemia and the group that did.
- the present inventors have completed the present invention relating to a novel method for testing the control state of blood glucose based on such knowledge.
- the measurement of the AGE 2 concentration in the blood or body fluid of the patient is after administration of the drug that reduces postprandial hyperglycemia, and the value at another time in the same patient reduces the postprandial hyperglycemia.
- the test method according to [2] which is a value before administration of the drug, and is used for determining the effect of the drug for reducing postprandial hyperglycemia.
- the drug that reduces postprandial hyperglycemia is selected from the group consisting of a meglitinide drug, an ⁇ -glycosidase inhibitor, and a super-fast-acting insulin preparation;
- the AGE 2 concentration is measured by a measurement method selected from the group consisting of the Western plot method, the Enzymimnoassay method, the radioimmunoassay method, the liquid chromatography method, and the dot blot method.
- the method according to any one of the above [1] to [6], wherein the method is performed.
- a reagent for evaluating the control state of postprandial hyperglycemia which contains an anti-AGE2 antibody.
- FIG. 1 is a graph showing the relationship between blood HbAlc concentration and AGE concentration in streptozotocin (STZ) -induced diabetic rats.
- FIG. 1A is a graph showing the relationship between HMlc concentration and AGE 1 concentration
- FIG. 1B is a graph showing the relationship between HbAlc concentration and AGE 2 concentration.
- FIG. 2 is a graph showing blood glucose levels of Goto-Kakizaki (GK) rats.
- FIG. 3 is a graph showing the effects of nateglinide and insulin on postprandial blood glucose (blood glucose 1 hour after a meal) in Goto-Kakizaki (GK) rats.
- the “postprandial hyperglycemic state” in the present invention refers to a transient increase in blood glucose observed immediately after a meal in diabetic patients and healthy persons in the reserve diabetes group.
- diabetes occurs when the blood glucose level exceeds 200 mg / dl 2 hours after a meal
- impaired glucose tolerance occurs when the blood glucose level is 140-200 mg / dl (i immediately ai red glucose tolerance, (Hereinafter referred to as IGT).
- IGT immediately ai red glucose tolerance
- Control of postprandial hyperglycemia refers to treatment aimed at lowering the blood glucose level for 2 hours after a meal, mainly based on the above criteria. This includes reducing blood glucose and the area under the blood glucose curve during meals or trans-glucose tolerance tests, including dietary restrictions such as postprandial blood glucose reduction and calorie restriction with drugs that reduce postprandial hyperglycemia. Exercise therapy and the like are included.
- AGE 2 as measured in the present invention is a final saccharification product formed by binding dariceraldehyde to a protein, peptide or amino acid.
- Candidate proteins specifically glycated include albumin, globulin, lipoproteins, various intracellular proteins, basement membrane proteins, and the like, but are not limited thereto, and proteins that function in vivo. The whole can be a candidate for a protein to be converted into AGE2.
- the subject of the test method of the present invention is not particularly limited, but is mainly a healthy subject or a diabetic patient suspected of having diabetes.
- the patient referred to here is not limited to a human, but is a mammal other than a human (for example, a monkey, a rat, a mouse, a hamus, a guinea pig, a dog, a cat, a cat, a heron, a bush). , Higgins, goats, horses, horses, etc.) are also covered by the present invention.
- the gender, age, weight, etc. of the patient are not particularly limited.
- the diagnostic criteria for diabetes according to AM, WHO, and JDS are: fasting blood glucose ⁇ 126 mg / dl; Diagnosis is diagnosed if any of the 2-hour blood glucose levels ⁇ 200 mg / dl at (0GTT) is confirmed, but the subject of the test method of the present invention includes diabetic patients satisfying these values, Patients judged to be close to the numerical value of are included.
- the AGE2 concentration in the patient's blood or body fluid obtain a sample of the patient's blood or body fluid (eg, blood, plasma, serum, lymph, urine, etc.) and perform appropriate pretreatment if necessary. After the measurement.
- Means for measuring the AGE 2 concentration include the Western blot method, the enzymimnoassy method (EIA method), the radioimnoatsay method (RIA method), the liquid chromatography method, and the dot blot method. And other known methods.
- the Western plot method means that a protein separated according to molecular weight by gel electrophoresis is transferred to a transfer membrane, and an antibody against a specific protein is used to perform a specific reaction on the transfer membrane by an antigen-antibody reaction.
- Many known methods such as coloring a protein with a fluorescent reagent and coloring it using an enzymatic reaction can be applied.
- the EIA method includes the ELISA method, the IEMA method (Immuno-enzymometallic assay), the EMIT method (Enzyme multiplied immunoassay techniaue), and the like. After reacting by adding an enzyme-labeled antibody to the complex obtained by the reaction, a substrate for the enzyme is added, the color is developed, and colorimetric determination is performed based on the absorbance. Various protocols are well known.
- the dot plot method is a method in which a test sample is spotted on a hydrophobic membrane and a specific protein is quantified in the same manner as in the EIA method.
- Preferred methods for the present invention include the ELISA method, the RIA method, and the dot blot method using an anti-AGE2 antibody such as an anti-AGE2 polyclonal antibody or an anti-AGE2 monoclonal antibody. Particularly preferred is the ELISA method.
- the anti-AGE 2 polyclonal antibody is prepared by mixing ⁇ 2, which is a mixture of ⁇ egan serum albumin (hereinafter referred to as RSA) and daliceraldehyde, in ⁇ heron to first produce an antiserum, which is then used as an antigen. It can be prepared by a method such as affinity purification using an affinity column (AGE2 / Sepharose 4B column) in which the obtained AGE 2 is immobilized as a carrier.
- ⁇ 2 is a mixture of ⁇ egan serum albumin (hereinafter referred to as RSA) and daliceraldehyde
- RSA ⁇ egan serum albumin
- daliceraldehyde daliceraldehyde
- an anti-AGE2 monoclonal antibody can be prepared according to a conventional method using AGE2 prepared by mixing RSA and dariceraldehyde.
- the measurement by the ELISA method using the above-mentioned anti-AGE 2 polyclonal antibody or monoclonal antibody is performed by the following procedure. First, a test sample and an anti-AGE2 antibody solution are added to a 96-well plate on which AGE2 has been immobilized in advance, and reacted for a certain period of time. AGE 2 in the test sample and immobilized AGE 2 Anti-AGE2 antibody, which reacts competitively with the immobilized AGE2 to form a complex with the immobilized AGE2, is further reacted with an enzyme-labeled antibody, a substrate for the enzyme is added, color is developed, and colorimetric determination is performed by absorbance. .
- the AGE 2 concentration of the same patient or the healthy subject The post-hyperglycemic state is controlled by comparing the AGE 2 concentration with the AGE 2 concentration of each sample after a predetermined period (for example, about 1 to 4 weeks, preferably about 1 week) after the start of the administration. It can be easily determined whether or not it has been performed.
- screening of an unknown drug for reducing postprandial hyperglycemia can be performed in the same manner as in the performance evaluation of the known drug for reducing postprandial hyperglycemia described above.
- test method of the present invention is useful not only for performance evaluation and screening of these drugs for reducing postprandial hyperglycemia, but also for confirming compliance of patients treated with these drugs.
- the present invention also provides a reagent for evaluating the control state of a postprandial hyperglycemia state, comprising an anti-AGE2 antibody.
- a reagent for evaluating the control state of a postprandial hyperglycemia state comprising an anti-AGE2 antibody.
- the anti-AGE2 antibody in the reagent include the above-mentioned anti-AGE2 monoclonal antibody ⁇ anti-AGE2 polyclonal antibody.
- a kit in the reagent of the present invention, it is also preferable to package a kit together with necessary reagents and the like in accordance with each analysis method using the reagent, to form a kit.
- a kit can be prepared by combining a support (such as a gel plate), a secondary antibody (such as an enzyme-labeled antibody), a coloring reagent, and a buffer.
- a support such as a gel plate
- a secondary antibody such as an enzyme-labeled antibody
- a coloring reagent such as an enzyme-labeled antibody
- a buffer such as an enzyme-labeled antibody
- Rats in the insulin treatment group were implanted with implantable insulin (LINPLANT, LINCHIN CANADA Inc.) subcutaneously on the back at the start of the study.
- a diet containing 0.3% pyridoxamine was provided to the pyridoxamine-treated group from the start of the study.
- Food and water were given by ingestion, and blood was collected after 8 weeks to measure the concentrations of HbAlc, AGE derived from daricheraldehyde (AGE2), and AGE derived from glucose (AGE1).
- the concentration of AGE1 or AGE2 was measured using an anti-AGE1 or anti-AGE2 polyclonal antibody prepared from Pergum (An anti-AGE1 antibody was prepared by immunizing Pergum with AGE1 prepared by mixing BSA and glucose. Make serum, and then
- the anti-AGE2 antibody was prepared by immunizing rabbits with AGE2, which was prepared by mixing BSA and dalyseraldehyde, to first produce an antiserum, and then performing affinity purification using an AGE2 / Sepharose 4B column.)
- the ELISA was performed according to an ordinary method.
- the HbAlc value in the disease control group was increased about three times compared to the normal rats, and the increase was significantly suppressed in the insulin-treated group.
- the AGE 1 concentration was about 5 times higher in the disease control group than in the normal group, but was significantly suppressed in the insulin and pyridoxamine groups.
- the AGE 2 concentration was about twice as high in the disease control group as in the normal group, but no significant decrease was observed in either the insulin treatment group or the pyridoxamine group.
- AGE 1 showed a significant correlation with HbAlc
- AGE 2 showed no significant correlation with HbAlc (FIG. 1).
- Goto-Kakizaki (GK) rats a spontaneous type 1 diabetes model, were acclimated to a daily restricted-feeding regimen of 1 hour twice a day according to the prior literature and used in experiments.
- the concentration of AGE1 or AGE2 was measured by ELISA using an anti-AGE1 or anti-AGE2 polyclonal antibody (see Example 1) prepared from a perch, according to a conventional method.
- the FBG concentration was measured from the tail vein immediately before feeding the first diet on the measurement day (17 hours after the end of HI feeding twice on the previous day).
- the blood glucose level of the whole blood sample was measured.
- the present invention can provide a novel marker for diagnosing a glycemic control state and a method for diagnosing and testing a glycemic control state, and thus can be used in the fields of medical treatment and diagnostic agents.
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JP2019041668A (ja) * | 2017-09-01 | 2019-03-22 | Bloom Technology 株式会社 | 終末糖化産物に対する抗体およびその使用 |
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JP2003230382A (ja) * | 2002-02-08 | 2003-08-19 | Univ Kanazawa | Age−rage拮抗剤 |
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JP2003230382A (ja) * | 2002-02-08 | 2003-08-19 | Univ Kanazawa | Age−rage拮抗剤 |
Non-Patent Citations (2)
Title |
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TAKEUCHI ET AL: "Tonyobyo Kekkan Gappeisho no Hassho. Sinten ni Okeru Glyceraldehyde Yurai Shumatsu Toka Sanbutsu (AGE-2) no Igi.", SEIKAGAKU., vol. 74, no. 8, 2002, pages 819 - 2P-687 * |
TAKEUCHI M. ET AL: "Neurotoxicity of advanced glycation end-products for cultured cortical neurons.", J.NEUROPATHOL.EXP.NEUROL., vol. 59, no. 12, December 2000 (2000-12-01), pages 1094 - 1105, XP008084489 * |
Cited By (2)
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JP2019041668A (ja) * | 2017-09-01 | 2019-03-22 | Bloom Technology 株式会社 | 終末糖化産物に対する抗体およびその使用 |
JP7149509B2 (ja) | 2017-09-01 | 2022-10-07 | Bloom Technology 株式会社 | 終末糖化産物に対する抗体およびその使用 |
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