JP7189537B2 - A measuring method comprising the step of measuring a reflected value of the amount of oxidized HDL in a blood sample - Google Patents

Description

TECHNICAL FIELD The present invention relates to a measurement method including a step of measuring a reflected value of the amount of oxidized HDL in a blood sample.

In recent years, there has been an increase in cases of "hidden diabetes," in which postprandial blood glucose levels are high despite normal fasting blood glucose levels. For example, since it is common to measure fasting blood glucose levels in health checkups, it is difficult to detect latent diabetes, and development of a method capable of detecting this disease is desired. In general, for the diagnosis of diabetes (measurement of glucose tolerance), a 75 g oral glucose tolerance test (OGTT) is performed, but OGTT requires fasting before the test, and the test time is Since it takes a long time, it has drawbacks such as requiring labor and time. Moreover, since the OGTT is a test in which the blood sugar level is intentionally increased, the burden on the test subject is heavy.

JP 2017-227618 A

Diabetes Treatment Guide 2018-2019, Japan Diabetes Society, 2018

An object of the present invention is to provide a method for simply measuring the risk of impaired glucose tolerance. An object of one embodiment is to provide a method for simply measuring hidden obesity risk.

As a result of intensive studies to solve the above problems, the present inventors found that the risk of impaired glucose tolerance or hidden obesity risk can be easily measured by using a specific indicator, and further improvements are made to the present invention. was completed.

The present invention encompasses subject matter described, for example, in the following sections.
Section 1.
A method for measuring the risk of impaired glucose tolerance in a subject from whom the blood sample was collected, comprising the step of measuring the reflected value of the amount of oxidized HDL in the blood sample.
Section 2.
3. The method of paragraph 1, wherein the blood sample is from a non-diabetic individual.
Item 3.
A method for measuring the latent obesity risk of a subject from whom the blood sample was collected, comprising the step of measuring the reflected value of the amount of oxidized HDL in the blood sample.
Section 4.
Item 4. The method of Item 3, wherein the blood sample is from a person with a BMI of less than 23.
Item 5.
Measurement of the reflected value of the amount of oxidized HDL is
(1) by a method comprising mixing a blood sample with a solution containing a transition metal compound and an acidic buffer;
Item 5. The method according to any one of Items 1 to 4.
Item 6.
Measurement of the reflected value of the amount of oxidized HDL is
moreover,
(2) by a method comprising the step of measuring the free radicals produced in step (1);
6. The method according to item 5.

By using a specific indicator, in a preferred embodiment, it is possible to provide a method for simply measuring the risk of impaired glucose tolerance. In one embodiment, a method for simply measuring hidden obesity risk can be provided.

Fig. 2 shows the results of analysis of the correlation between the reflected value of the oxidized HDL amount and the fasting blood sugar level. FIG. 10 shows the result of analysis of the correlation between the reflected value of the oxidized HDL amount and HbA1c. FIG. . When subjects were divided into two groups (low group/high group) by the median value obtained by dividing HbA1c by fasting blood glucose level, the reflected value of the amount of oxidized HDL in each group is shown. When the subjects were divided into two groups (Low group/High group) according to the median value reflected in the amount of oxidized HDL, the body fat percentage (%) of each group is shown.

The present invention will be described in more detail below.

The present invention includes a method for measuring the risk of impaired glucose tolerance of a subject from whom the blood sample was collected, including the step of measuring the reflected value of the amount of oxidized HDL in the blood sample.

The blood sample is not particularly limited as long as it is a sample derived from blood collected from a subject. For example, it may be whole blood, or blood isolates (eg serum, plasma, etc.). .

The value reflecting the amount of oxidized HDL means a value that reflects the amount of oxidized HDL contained in a blood sample, and examples thereof include the weight (mass) and concentration of oxidized HDL. Alternatively, for example, it may be a measurement value reflecting the amount of oxidized HDL contained in a blood sample, such as absorbance, fluorescence intensity, or the like. The measured value may be a value measured using a measurement auxiliary reagent. Examples of such measurement auxiliary reagents include fluorescent dyes, oxidized HDL-specific recognition molecules (eg, antibodies or fragments thereof), or combinations of two or more thereof. The measurement method is also not particularly limited, and a known measurement method can be appropriately selected and used.

Subjects from which blood samples are collected are not particularly limited to humans, and may be mammals other than humans. Subjects include, for example, healthy subjects, non-diabetic subjects, patients with or suspected of having arteriosclerosis, patients with cardiovascular disease, people with or suspected of having high oxidized HDL or oxidized LDL levels, obese persons, and non-diabetics. An obese person with a BMI of less than 23 and the like can be mentioned. In addition, people who do not have diabetes are more preferable as subjects from whom blood samples are collected when the risk of impaired glucose tolerance is measured. In addition, a person with a BMI of less than 23 is more preferable as a subject from which a blood sample is collected when the hidden obesity risk is measured. In addition, although mammals are not limited, mammals raised as pets, domestic animals, laboratory animals, etc. are preferable. Examples of such mammals include dogs, cats, monkeys, cows, horses, sheep, goats, pigs, rabbits, mice, rats, camels, llamas, and the like.

The method for collecting a blood sample from a subject is not particularly limited, and it can be performed according to a conventional method using known instruments, devices, and the like. When serum or plasma is used as a blood sample, it is preferable to use a vacuum blood collection tube containing a serum or plasma separating agent from the viewpoint of ease of handling and prevention of infection. Moreover, the collected blood sample may be used as it is, or after being preserved by freeze-drying or the like, the freeze-dried product may be dissolved in an appropriate solvent described later and used. Further, the collected blood sample may be frozen and stored as it is, or may be dissolved in an appropriate solvent, frozen and stored, and then thawed before use. The method, conditions, and the like for storing the collected blood sample are not particularly limited, and can be performed according to a conventional method.

According to the present invention, by measuring the reflected value of the amount of oxidized HDL in a blood sample, in a group consisting of a plurality of subjects, the HbA1c value relative to the fasting blood glucose level of the subjects from whom the blood sample was collected is lower than the median value. can be determined to be high or low. For example, in a population of subjects, subjects with a (relatively) high oxidized HDL reflected value can be determined to have a higher than median HbA1c value relative to fasting blood glucose. In addition, in a group consisting of a plurality of subjects, subjects with a (relatively) low reflected value of the amount of oxidized HDL can be determined to have a lower HbA1c value relative to the fasting blood glucose level than the median value. The number of subjects that make up the population may be, for example, 10 or more, 30 or more, or 50 or more, in settings such as small-group clinical studies. The upper limit is not particularly limited, but in situations such as health checkups for a large number of people, for example, may be a person.

When the HbA1c value relative to the fasting blood sugar level is high, that is, when the HbA1c value is high despite the low fasting blood sugar level, it is expected that the postprandial blood sugar level is also high. be. Therefore, according to the present invention, by measuring the reflected value of the amount of oxidized HDL in a blood sample, in a population consisting of a plurality of subjects, the subject from whom the blood sample was collected has a (relatively) high postprandial blood glucose level. or low. For example, in a group of subjects, subjects with a (relatively) high reflected value of the amount of oxidized HDL can be determined to have a (relatively) high postprandial blood glucose level. In addition, in a group consisting of a plurality of subjects, subjects with a (relatively) low reflected value of the amount of oxidized HDL can be determined to have a (relatively) low postprandial blood glucose level.

Utilizing such a fact, according to the present invention, the glucose intolerance risk of a subject can be measured. In the present invention, the risk of impaired glucose tolerance means the risk of not being diagnosed with diabetes, but the function of suppressing an increase in blood sugar level is not working normally. A high reflected value of the oxidized HDL content indicates a high risk of impaired glucose tolerance, and a low reflected value of the oxidized HDL content indicates a low risk of impaired glucose tolerance. If the risk of impaired glucose tolerance is high, for example, a state that is judged to be borderline type that is not included in either the normal type or the diabetic type in the 75g oral glucose tolerance test, or the fasting blood sugar level is low but the postprandial blood sugar level is high. It is thought that it is a state seen in so-called "hidden diabetes", such as a high state.

According to the present invention, the risk of impaired glucose tolerance can be easily measured by measuring the fasting blood glucose level without performing OGTT for measuring glucose tolerance. In addition, according to the present invention, the risk of impaired glucose tolerance can be easily measured in non-diabetic individuals with HbA1c of less than 6.5% or fasting blood glucose level of less than 126 mg/dL, Hidden diabetes can be detected.

The present invention also includes a method of measuring the latent obesity risk of a subject from whom the blood sample was collected, comprising the step of measuring the reflected value of the amount of oxidized HDL in the blood sample.

According to the present invention, by measuring the reflected value of the amount of oxidized HDL in a blood sample, in a population consisting of a plurality of subjects, the body fat percentage of the subject from whom the blood sample was collected is (relatively) high or low. It is possible to determine whether For example, when a group consisting of a plurality of subjects is divided into two groups according to the median value of the reflected value of the oxidized HDL amount, subjects belonging to the group with a high reflected value of the oxidized HDL amount are It can be determined that the body fat percentage is higher than the subjects belonging to the group with low reflected values. Further, in a group consisting of a plurality of subjects, when the group is divided into two groups by the median value of the reflected value of the oxidized HDL amount, the subjects belonging to the group with the low reflected value of the oxidized HDL amount It can be determined that the body fat percentage is low compared to subjects belonging to a group with a high reflected value of . The number of subjects that make up the population may be, for example, 10 or more, 30 or more, or 50 or more, in settings such as small-group clinical studies. The upper limit is not particularly limited, but in situations such as health checkups for a large number of people, for example, may be a person.

In addition, according to the present invention, by measuring the reflected value of the amount of oxidized HDL in a blood sample, in a group consisting of a plurality of subjects, the amount of ectopic fat in the subject from whom the blood sample was collected (relatively ) can be determined. For example, when a group consisting of a plurality of subjects is divided into two groups according to the median value of the reflected value of the oxidized HDL amount, subjects belonging to the group with a high reflected value of the oxidized HDL amount are It can be determined that the amount of ectopic fat is greater compared to subjects belonging to the group with low reflected values. Further, in a group consisting of a plurality of subjects, when the group is divided into two groups by the median value of the reflected value of the oxidized HDL amount, the subjects belonging to the group with the low reflected value of the oxidized HDL amount It can be determined that the amount of ectopic fat is small compared to subjects belonging to a group with a high reflected value of .

Utilizing such a fact, according to the present invention, a subject's hidden risk of obesity can be measured. In the present invention, the hidden obesity risk means the risk of accumulation of visceral fat or ectopic fat, although obesity is not determined from the BMI value. It is important to measure this risk because it indicates a likely local fat accumulation. A high reflected value of the oxidized HDL amount indicates a high hidden risk of obesity, and a low reflected value of the oxidized HDL amount indicates a low hidden risk of obesity. When the risk of hidden obesity is high, for example, it is considered to be a state seen in so-called "hidden obesity", such as a state in which visceral fat or ectopic fat is accumulated.

According to the present invention, it is possible to easily measure hidden obesity risk in subjects with a BMI of less than 23 who are not judged to be obese.

The method for measuring the reflected value of the amount of oxidized HDL in a blood sample is not particularly limited, and can be carried out according to a conventional method. For example, a method of measuring oxidized HDL (malondialdehyde-modified HDL) using an ELISA method, a method of measuring nitration or chlorination of Apo-A1, a protein that constitutes HDL, by a mass spectrometer, d- ROMs method (method for measuring the concentration of hydroxyperoxide in a blood sample), a method comprising the step of mixing (1) a blood sample with a solution containing a transition metal compound and an acidic buffer, described in JP-A-2017-227618, and the like. is mentioned. Among them, (1) measurement is preferably performed by a method including a step of mixing a blood sample with a solution containing a transition metal compound and an acidic buffer. In this specification, this step may be referred to as "step (1)". Furthermore, it is more preferable to measure by a method comprising the step of (2) measuring the free radicals produced in step (1). In this specification, this step may be referred to as "step (2)".

A method for measuring the reflected value of the oxidized HDL amount in a blood sample may include a step of separating HDL from the blood sample. In this specification, this step may be referred to as "step (0)". HDL separated by step (0) can be used as a blood sample in step (1). A method for separating HDL from a blood sample is not particularly limited, and can be carried out according to a conventional method. For example, methods such as the dextran sulfate method using dextran sulfate and magnesium ions can be used.

The transition metal compound is not particularly limited as long as it can ionize into metal ions in the mixed solution and can react with hydroxyl peroxide (R-OOH) to produce free radicals. , copper (II) compounds, iron (II) compounds, (divalent iron compounds), iron (III) compounds (trivalent iron compounds), and the like. Among these, iron compounds such as iron (II) compounds and iron (III) compounds are preferred. Examples of iron compounds include ammonium iron (II) sulfate hexahydrate and iron (III) chloride hexahydrate. By using a transition metal compound in step (1), 1) the presence of a sufficient amount of transition metal ions that react with hydroxyl peroxide enables the measurement of oxidized HDL in a small blood sample, and 2) it is contained in blood. Advantageous effects such as enabling the measurement of oxidized HDL without being affected by the amount of iron are exhibited.

It should be noted that the reflected value of the amount of oxidized HDL is greater in a solution containing a divalent iron compound than in a trivalent iron compound. In other words, a measurement method using a divalent iron compound rather than a trivalent iron compound can measure oxidized HDL with higher sensitivity. Therefore, among the above iron compounds, divalent iron compounds are particularly preferred.

The concentration of the transition metal compound in the mixed solution is not particularly limited, and can be, for example, about 0.1 to 150 μM, preferably 1 to 100 μM.

The acidic buffer is not particularly limited as long as it has an acidic pH and a buffering action, and known buffers can be used. Examples thereof include acetate buffers.

The pH of the acidic buffer solution is not particularly limited as long as it is acidic, and is preferably about 2 to 6.9, more preferably about 3 to 6.5, and 4.5 to 6. is more preferred. In step (2), which will be described later, when a coloring method is employed as a method for measuring free radicals, it is particularly preferable that the pH of the acidic buffer solution is about 3 to 6.9.

The concentration of the acidic buffer is not particularly limited, and is, for example, 0.005-0.5M, preferably 0.01-0.2M.

The reaction temperature in step (1) is not particularly limited, and can be, for example, about 20 to 40°C.

As described above, in step (1), hydroperoxides (R—OOH) in oxidized HDL contained in the blood sample react with transition metal ions to produce peroxy radicals (R ^— OO) and alkoxy radicals (R—O^・) and other free radicals are produced. Oxidized HDL can be measured by measuring the free radicals produced in step (1).

Therefore, it is preferable that the method for measuring the reflected value of the oxidized HDL amount further includes the step of (2) measuring the free radicals produced in step (1) above.

The method for measuring free radicals is not particularly limited, and known methods can be employed. For example, a chromogenic method, a chemiluminescence method, an electron spin resonance method (ESR method) and the like can be mentioned.

The chromogenic method is a method in which a substance (chromogenic substance) that reacts with free radicals to develop color is used, and the absorbance of the colored substance is measured using a spectrophotometer or the like.

The color-developing substance is not particularly limited as long as it is a compound that reacts with free radicals or alkoxy radicals to develop color, and known compounds can be used. Examples thereof include compounds represented by the following general formula (1) and salts thereof.

In general formula (1) above, each R is the same or different and represents a hydrogen atom, a halogen atom, methyl or ethyl. In particular, at least two of each R are preferably methyl or ethyl, and more preferably both Rs substituting the same nitrogen atom are methyl or ethyl.

As the compound represented by general formula (1), N,N-dimethyl-p-phenylenediamine (DMPD) and N,N-diethyl-p-phenylenediamine (DPD) are preferable.

Salts of the compound represented by formula (1) include, for example, sulfates, oxalates, diacetates and the like.

The compounds represented by formula (1) or salts thereof used in the reaction step may be used singly or in combination of two or more.

Moreover, the compound represented by the general formula (1) can be used by dissolving it in a solvent, if necessary. The solvent is not particularly limited, and examples thereof include pure water, buffer solution, dimethylsulfoxide (DMSO) and the like. The concentration in the solution is not particularly limited, and can be, for example, about 0.1 to 50 mM, preferably about 1 to 20 mM.

The method for measuring the absorbance of the colored substance is not particularly limited, and a conventional method can be used. For example, radical cations exhibiting a reddish purple color are generated by the reaction between free radicals and a chromogenic substance, so radical cations can be measured by measuring absorbance using a known device. The wavelength for measuring the absorbance can be, for example, 460 to 570 nm, preferably 500 to 560 nm.

Moreover, when measuring the absorbance, it is preferable to measure the absorbance over time between the measurement start time and the measurement end time in order to measure it quantitatively. For example, it is preferable to measure the rate of increase in absorbance with a starting point of 2 minutes after the start of the reaction between the free radical and the chromogenic substance and an end point of 5 minutes after the start of the reaction.

The chemiluminescence method uses a substance (luminescent substance) that reacts with free radicals to emit light, and measures the light emitted when the excited substance returns to the ground state using a luminometer or the like. be.

The light-emitting substance is not particularly limited as long as it is a compound that reacts with free radicals to emit light, and known compounds can be used. Examples thereof include luminol, Dansyl-TEMPO, lucigeninle, 2-methyl-6-p-methylphenylethylimidazopyrazineone (MPEC), Hydroxyphenyl Fluorescein (HPF), Aminophenyl Fluorescein (APF), Cypridina luciferin (MCLA) derivatives and the like.

The method for measuring the light emitted from the substance is not particularly limited, and can be determined as appropriate according to the type of the light-emitting substance used.

The electron spin resonance method (ESR method) is a spectroscopic analysis that observes transitions between levels that occur when unpaired electrons are placed in a magnetic field. The electron spin resonance method is not particularly limited, and can be performed using known methods and equipment. A direct method of directly measuring free radicals, an indirect method of reacting a spin trapping agent and free radicals. Either can be used. When the indirect method is employed, the spin trapping agent is not particularly limited, and known spin trapping agents can be used. Spin trapping agents include, for example, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 2,5,5-triethyl-1-pyrroline-N-oxide (M ₃ PO), 3,3, Nitrone-based spin trap agents such as 5,5-tetramethyl-1-pyrroline-N-oxide (TMPO), N-tert-α-phenylnitrone (PBN); 2-methyl-2-nitrosopropane (MNP), nitroso Nitroso-based spin trapping agents such as duren (ND) and the like can be mentioned.

In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present invention encompasses all arbitrary combinations of the constituent elements described herein.

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. In this example, the change in absorbance (unit: mOD/min) was used as a value reflecting the amount of oxidized HDL.

Test Example 1 (Measurement of Reflected Value of Oxidized HDL Amount)
(1) Preparation of blood sample Blood is collected from the vein of the subject using a vacuum blood collection tube, left at room temperature for 1 hour, centrifuged at 4 ° C., 3500 rpm (1100 g) for 10 minutes, and the supernatant is obtained. (serum) was obtained as a blood sample and stored at -80°C. Specifically, the blood of a complete medical checkup recipient was used.

(2) Purification of HDL To 45 μl of the serum obtained in (1) above, 5 μl of a mixture of 1% dextran sulfate and 0.5 M magnesium chloride (pH 7.3) was added and mixed at room temperature. After allowing to stand at room temperature for 10 minutes, centrifugation was performed at 4°C and 1500g for 30 minutes, and the resulting supernatant was obtained as an HDL fraction and stored at -80°C.

(3) Measurement of reflected value of oxidized HDL A mixed solution of 0.1 M acetate buffer (pH 4.8) and 100 μM ammonium iron (II) sulfate hexahydrate was added to each well of the microplate and kept at 37°C. did. Next, after adding an N,N-diethyl-p-phenylenediamine sulfate solution (solvent: DMSO) to each well, the HDL obtained in (2) above was added. Absorbance at a wavelength of 505 nm was measured using a microplate reader (manufactured by Bio-Rad) set at 37°C. The absorbance measurement is set to Kinetics, measured 30 times every 10 seconds (total 5 minutes), and absorbance change per unit time from each value 2 minutes to 5 minutes after the start of measurement (unit: mOD /min).

Test Example 2 (Measurement of fasting blood glucose level (FPG) and hemoglobin A1c (HbA1c))
(1) Measurement method of fasting blood glucose level (FPG) Using the blood of the complete medical checkup recipient obtained in Test Example 1, the fasting blood glucose level was measured by an enzymatic method.

(2) Method for measuring hemoglobin A1c (HbA1c) Using the blood of the medical checkup recipient obtained in Test Example 1, HbA1c was measured by HPLC (high performance liquid chromatography) method or immunoassay method.

Test Example 3 (Analysis of Correlation between Reflected Value of Oxidized HDL and Fasting Blood Glucose Level or HbA1c)
The correlation between the reflected value of the oxidized HDL amount and the fasting blood glucose level or HbA1c was analyzed. FIG. 1 shows the results of 185 subjects whose oxidized HDL reflected levels and fasting blood glucose levels were measured, and FIG. 2 shows the results of 163 subjects whose oxidized HDL reflected levels and HbA1c were measured.

As shown in FIGS. 1 and 2, both the blood sugar level and HbA1c were found to have a positive correlation with the reflected value of the oxidized HDL amount. On the other hand, no significant correlation was observed between the reflected value of oxidized HDL content and total cholesterol, triglycerides, HDL cholesterol, and LDL cholesterol.

Test Example 4 (measurement of impaired glucose tolerance risk)
HbA1c was divided by the fasting blood glucose level in 156 examinees who had HbA1c levels of 6.5% or higher and fasting blood sugar levels of 126 mg/dL or higher, which were diagnosed as diabetes, were excluded from the complete checkup examinees whose HbA1c was measured. The subjects were divided into two groups (Low group/High group) according to the median value (HbA1c/FPG) obtained, and the reflected values of the oxidized HDL amount in each group were compared. The results are shown in FIG.

As shown in FIG. 3, it was found that the reflected value of the amount of oxidized HDL was significantly higher in the High group than in the Low group. Generally, HbA1c is an index that reflects glycemic control over the past 1-2 months. It is also becoming known that there is a positive correlation between HbA1c and postprandial blood glucose levels, and it is thought that postprandial blood glucose levels are high when HbA1c is high. When the HbA1c/FPG is high, even if the fasting blood sugar level is low and apparently normal, the postprandial blood sugar level is considered to be high because the HbA1c is high. The results of FIG. 3 indicate that the reflected value of the oxidized HDL level is high when the HbA1c/FPG is high, and thus it was found that the reflected value of the oxidized HDL level can measure the risk of impaired glucose tolerance.

Test Example 5 (measurement of hidden obesity risk)
BMI (kg/m ² ) was calculated by dividing the body weight (kg) by the square of the height (m) for the subjects whose reflective value of the oxidized HDL amount was measured. The 62 subjects under 23 were divided into two groups (Low group/High group) according to the median reflected value of the amount of oxidized HDL, and the body fat percentage (%) of each group was compared. The results are shown in FIG.

As shown in FIG. 4, it was found that the body fat percentage was significantly higher in the High group than in the Low group. In addition, no significant difference was observed when the BMI of each group was compared. Based on this result, it is expected that people with high oxidized HDL reflected values have a large amount of visceral fat and ectopic fat, which cannot be judged from BMI or appearance, and that the oxidized HDL reflected value can measure hidden obesity risk. I found out.

Claims (6)

A method for measuring the risk of impaired glucose tolerance in a subject from whom the blood sample was collected, comprising the step of measuring the reflected value of the amount of oxidized HDL in the blood sample. 2. The method of claim 1, wherein said blood sample is from a non-diabetic individual. A method for measuring the latent obesity risk of a subject from whom the blood sample was collected, comprising the step of measuring the reflected value of the amount of oxidized HDL in the blood sample. 4. The method of claim 3, wherein said blood sample is from a person with a BMI less than 23. Measurement of the reflected value of the amount of oxidized HDL is
(1) by a method comprising mixing a blood sample with a solution containing a transition metal compound and an acidic buffer;
The method according to any one of claims 1-4. Measurement of the reflected value of the amount of oxidized HDL is
moreover,
(2) by a method comprising the step of measuring the free radicals produced by the step (1);
6. The method of claim 5.

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