JP7585131B2 - Methods for measuring masticatory function - Google Patents

Description

This disclosure relates to a method for measuring masticatory function.

It is known that as people age, the number of people with 20 or more teeth decreases, and the percentage of people who can chew and eat anything also decreases. However, among the working generation, there are people who, even if they have a large number of teeth, respond that they have difficulty chewing and eating anything, and it is thought that factors other than the number of teeth are also related to chewing and eating.

When chewing function declines, in addition to direct problems such as inability to take in proper nutrition, reduced saliva secretion, difficulty eating hard foods, and a tendency to focus on soft foods, various indirect problems can also occur, such as reduced cerebral blood flow in the elderly due to reduced stimulation to the brain caused by chewing, and obesity and metabolic syndrome due to an unbalanced diet affected by chewing function in the working generation. Therefore, there is a need for the development of a method that can easily measure chewing function.

Patent No. 6779776

The objective of the present disclosure is to provide a method for measuring masticatory function. Another objective is to provide a method for measuring masticatory function without a dental checkup.

The inventors discovered that when it is difficult to chew food, the reflected value of oxidized HDL in blood samples is higher than when food can be chewed easily, and they have made further improvements.

The present disclosure includes, for example, the subject matter described in the following sections:
Item 1.
A method for measuring the masticatory function of a subject from whom a blood sample was taken, comprising measuring a value reflective of the amount of oxidized HDL in the blood sample, the method using the value reflective of the amount of oxidized HDL as an index.
Item 2.
The measurement of the reflection value of the amount of oxidized HDL
(1) The method is carried out by a method including a step of mixing a blood sample with a solution containing a transition metal compound and an acidic buffer solution,
Item 1. The method according to item 1.
Item 3.
The measurement of the reflection value of the amount of oxidized HDL
moreover,
(2) The method is carried out by a method including a step of measuring the free radicals produced in the step (1).
Item 3. The method according to item 2.
Item 4.
Item 4. The method according to any one of Items 1 to 3, wherein the reflection value of the amount of oxidized HDL is a change in absorbance per unit time.

A method for measuring masticatory function is provided. Also, a method for measuring masticatory function without a dental examination is provided.

FIG. 1 is a diagram showing tooth numbers and names. The results of plotting values reflecting the amount of oxidized HDL (oxHDL) in blood samples of each subject in a group that can chew anything and a group that sometimes has difficulty chewing are shown. The graph shows values reflecting the amount of oxidized HDL (oxHDL) in blood samples from a group that can chew anything and a group that has difficulty chewing, a group that currently has 24 or more teeth, and a group that currently has less than 24 teeth.

Each embodiment included in the present disclosure will be described in further detail below.
The present disclosure encompasses a method for measuring the masticatory function of a subject. In this specification, the method may be referred to as the "measurement method of the present disclosure."

The measurement method disclosed herein involves measuring a value reflecting the amount of oxidized HDL in a blood sample.

The subject may be a human or a non-human mammal.
The human may be, for example, a healthy individual, a human suffering from periodontal disease (a periodontal disease patient), or a human suspected of suffering from periodontal disease. Examples of the subject include a human with HbA1c of less than 5.6%, a human with fasting blood glucose of less than 110 mg/dL, a human with a BMI of less than 30, or a human with two or more of these characteristics. The subject may have 24 or more teeth, or less than 24 teeth.
The non-human mammal is not particularly limited, and examples thereof include mammals kept as pets, livestock, laboratory animals, etc. Examples thereof include dogs, cats, monkeys, cows, horses, sheep, goats, pigs, rabbits, mice, rats, camels, llamas, etc.

The blood sample is not particularly limited as long as it is a sample derived from blood collected from a subject, and may be, for example, whole blood or a blood separation product (e.g., serum, plasma, etc.). The method and conditions for storing the blood sample collected from the subject are not particularly limited, and may be carried out according to standard methods.

The method for collecting a blood sample from a subject is not particularly limited and may be performed according to a conventional method. 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 separator from the viewpoints of ease of handling, prevention of infection, etc.
The collected blood sample may be used as is, or may be freeze-dried or otherwise stored and then the freeze-dried product may be dissolved in a suitable solvent as described below and then used. The collected blood sample may be frozen and stored as is, or may be frozen and stored after being dissolved in a suitable solvent as described below and then thawed when ready for use.

The value reflecting the amount of oxidized HDL refers to a value reflecting the amount of oxidized HDL contained in the blood sample, such as the weight (mass) or concentration of oxidized HDL. Alternatively, it may be a measured value reflecting the amount of oxidized HDL contained in the blood sample, such as absorbance or fluorescence intensity. The measured value may be a value measured using a measurement auxiliary reagent. Examples of the measurement auxiliary reagent include a fluorescent dye, an oxidized HDL-specific recognition molecule (e.g., an antibody or a fragment thereof), or a combination of two or more of these.

The method for measuring the reflected value of the amount of oxidized HDL in a blood sample is not particularly limited, and can be performed according to a conventional method. For example, a method for measuring oxidized HDL (malondialdehyde-modified HDL) using an ELISA method, a method for measuring the nitration or chlorination of Apo-A1, a protein that constitutes HDL, using a mass spectrometer, and a method including the step of (1) mixing a blood sample with a solution containing a transition metal compound and an acidic buffer solution, as described in Japanese Patent Publication No. 6779776 (Patent Document 1), are included. Among these, it is preferable to perform the measurement by a method including the step of (1) mixing a blood sample with a solution containing a transition metal compound and an acidic buffer solution. In this specification, this step may be referred to as "step (1)". Furthermore, it is more preferable to perform the measurement by a method including the step of (2) measuring the free radicals produced by the step (1). In this specification, this step may be referred to as "step (2)".

The method for measuring a value reflecting the amount of oxidized HDL 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)". The HDL separated in step (0) may be used as the blood sample in step (1). The method for separating HDL from a blood sample is not particularly limited, and may be performed according to a conventional method. For example, a method such as the dextran sulfate method using dextran sulfate and magnesium ions may be used.

The transition metal compound is not particularly limited as long as it can be ionized in the mixed solution to become a metal ion and can react with hydroxyperoxide (R-OOH) to produce a free radical, and examples thereof include copper (II) compounds, iron (II) compounds (divalent iron compounds), and iron (III) compounds (trivalent iron compounds). 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), advantageous effects such as 1) the presence of a sufficient amount of transition metal ions that react with hydroxyperoxide makes it possible to measure oxidized HDL with a small amount of blood sample and 2) the measurement of oxidized HDL is possible without being affected by the amount of iron contained in the blood are achieved.

The reflected value of oxidized HDL is greater in a solution containing a divalent iron compound than in a solution containing a trivalent iron compound. In other words, a measurement method using a divalent iron compound can measure oxidized HDL with higher sensitivity than a trivalent iron compound. Therefore, among the iron compounds mentioned above, a divalent iron compound is 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.

There are no particular limitations on the acidic buffer solution, so long as it has an acidic pH and a buffering effect, and any known buffer solution can be used. For example, an acetate buffer solution can be used.

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 even more preferably 4.5 to 6. When a color development method is used as a method for measuring free radicals in step (2) described below, 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, but is, for example, 0.005 to 0.5 M, preferably 0.01 to 0.2 M.

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 free radicals such as peroxy radicals (R-OO ^. ) and alkoxy radicals (R-O ^. ). By measuring the free radicals produced in step (1), oxidized HDL can be measured.

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

The method for measuring free radicals is not particularly limited, and any known method can be used. Examples include the color development method, the chemiluminescence method, and the electron spin resonance method (ESR method).

The color-developing method uses a substance that reacts with free radicals to produce color (a color-developing substance), and measures the absorbance of the colored substance using a spectrophotometer or similar device.

The color-forming substance is not particularly limited as long as it is a compound that reacts with a free radical or an alkoxy radical to produce a color, and any known compound can be used. For example, a compound represented by the following general formula (1) or a salt thereof can be used.

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

Preferred compounds represented by general formula (1) are N,N-dimethyl-p-phenylenediamine (DMPD) and N,N-diethyl-p-phenylenediamine (DPD).

Examples of salts of the compound represented by general formula (1) include sulfates, oxalates, and diacetates.

The compound represented by general formula (1) or a salt thereof used in the reaction step may be used alone or in combination of two or more.

The compound represented by general formula (1) can also be dissolved in a solvent as necessary. Examples of the solvent include, but are not limited to, pure water, a buffer solution, and dimethyl sulfoxide (DMSO). The concentration of the solution is not particularly limited, and can be, for example, about 0.1 to 50 mM, and preferably about 1 to 20 mM.

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

When measuring absorbance, it is preferable to measure the time course of absorbance between the start and end of the measurement in order to measure quantitatively. For example, the rate of increase in absorbance (change in absorbance per unit time) may be measured starting from 3 minutes after the start of the reaction between the free radicals and the color-forming substance and ending from 6 minutes after the start of the reaction.

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

The luminescent 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 include luminol, dansyl-TEMPO, lucigenin, 2-methyl-6-p-methoxyphenylethylimidazopyrazine (MPEC), hydroxyphenyl fluorescein (HPF), aminophenyl fluorescein (APF), and Cypridina luciferin derivative (MCLA).

The method for measuring the light emitted by the luminescent substance is not particularly limited and can be appropriately determined depending on the type of luminescent substance used, etc.

The electron spin resonance method (ESR method) is a spectroscopic analysis that observes the transition between levels that occurs when an unpaired electron is placed in a magnetic field. The electron spin resonance method is not particularly limited and can be performed using a known method and device, and may be either a direct method in which free radicals are directly measured or an indirect method in which a spin trapping agent is reacted with a free radical. When an indirect method is adopted, the spin trapping agent is not particularly limited and a known spin trapping agent can be used. Examples of the spin trapping agent include nitrone-based spin trapping agents such as 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 2,5,5-triethyl-1-pyrroline-N-oxide (M ₃ PO), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (TMPO), and N-tert-α-phenylnitrone (PBN); and nitroso-based spin trapping agents such as 2-methyl-2-nitrosopropane (MNP) and nitrosodurene (ND).

As will be shown in the examples below, when the value reflected by the amount of oxidized HDL in a blood sample is high, it is possible that the person has difficulty chewing food, and their chewing function is considered to be low. Therefore, according to the measurement method disclosed herein, by measuring the value reflected by the amount of oxidized HDL in a blood sample, the chewing function of the subject from whom the blood sample was taken can be measured using the value reflected by the amount of oxidized HDL as an index.

As will be shown in the examples below, when the value reflected by the amount of oxidized HDL in a blood sample is high, there is a possibility that the state of "difficulty chewing" when chewing food, regardless of the number of teeth. Therefore, according to the measurement method disclosed herein, by measuring the value reflected by the amount of oxidized HDL in a blood sample, the masticatory function of the subject from whom the blood sample was taken can be measured using the value reflected by the amount of oxidized HDL as an index, regardless of the number of teeth.

In this specification, chewing function means the ability to crush food.

According to the measurement method disclosed herein, for example, when the reflected value of the amount of oxidized HDL in a blood sample taken from a subject is equal to or greater than a reference value, it is possible to predict (determine) that the chewing function is low, compared to when the reflected value is less than the reference value.

The reference value to be compared with the value reflecting the amount of oxidized HDL in the blood sample collected from the subject can be, for example, 32.0 mOD/min, which is the rate of increase in absorbance (change in absorbance per unit time), with the value reflecting the amount of oxidized HDL in the blood sample starting from 3 minutes after the start of the reaction between the free radicals and the color-forming substance and ending at 6 minutes after the start of the reaction. This reference value is highly likely to be able to predict chewing ability with high sensitivity, and can predict positively for more than 70% (10 out of 14 subjects) of the subjects classified into the group "sometimes having difficulty chewing" in the test example of the present disclosure.

The reference value to be compared with the value reflecting the amount of oxidized HDL in a blood sample taken from a subject can be, for example, a value reflecting the amount of oxidized HDL in a blood sample taken from a subject with normal chewing function, a subject with normal values in a chewing function evaluation using gummy jelly, or a subject who can chew anything when eating food. The value may be standardized from data obtained in the past.

The reference value to be compared with the value reflecting the amount of oxidized HDL in a blood sample taken from a subject can be, for example, the average value, median value, 25th percentile value, 75th percentile value, or mode value of the values reflecting the amount of oxidized HDL in blood samples from a group of multiple subjects.

Furthermore, according to the measurement method disclosed herein, it is also possible to measure the change in the chewing function of the same subject over time. For example, if the value reflecting the amount of oxidized HDL in a blood sample taken from the subject increases over time, it is possible to predict (determine) that the chewing function may have decreased. For example, if the value reflecting the amount of oxidized HDL in a blood sample taken from the subject decreases over time, it is possible to predict (determine) that the chewing function may have improved.

According to the measurement method disclosed herein, by measuring the value reflecting the amount of oxidized HDL in a blood sample, even when a dental professional is not present or a dental checkup has not been conducted, it is possible to measure chewing function based on the results of a blood test, for example, during a health checkup.

The present disclosure also encompasses a method for assisting in determining a subject's chewing function. In this specification, the method may be referred to as the "assisting method of the present disclosure."

The auxiliary method disclosed herein includes measuring a value reflective of the amount of oxidized HDL in a blood sample.

The above-mentioned description of the "measurement method of the present disclosure" can be used to determine the reflected value of the amount of oxidized HDL in the blood sample.

The assistance method disclosed herein can assist in assessing a subject's chewing function.

In addition, in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure includes all of the constituent elements described in this specification in any combination.

Furthermore, the various characteristics (properties, structures, functions, etc.) described for each embodiment of the present disclosure above may be combined in any way to identify the subject matter encompassed by the present disclosure. In other words, the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described in this specification.

The contents of this disclosure will be specifically explained using the following test examples. However, this disclosure is in no way limited to these. In the following, unless otherwise specified, the experiments were performed under atmospheric pressure and room temperature conditions.

The study participants were 219 men (mean age 53.0 years, standard deviation 6.8), excluding those with a BMI of 30 or more.
In addition, the current number of teeth was confirmed by counting the number of teeth currently held out of 32 teeth, including wisdom teeth.

Test Example 1 Calculation of the value reflecting the amount of oxidized HDL (oxHDL) in blood samples
(1) Preparation of blood samples : Blood was collected from the vein of the subject using a vacuum blood collection tube, and then allowed to stand at room temperature for 1 hour. Then, the blood was centrifuged under general serum separation conditions to obtain the supernatant (serum) as a blood sample, which was then stored at −80° C.

(2) Measurement of the reflected value of oxidized HDL (oxHDL) amount A mixture 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. Next, N,N-diethyl-p-phenylenediamine sulfate solution (solvent: DMSO) was added to each well, and then the serum obtained in (1) above was added. The absorbance at a wavelength of 505 nm was measured using a microplate reader (manufactured by Bio-Rad) set at 37° C. The absorbance was measured by setting it to Kinetics, measuring a total of 48 times (for a total of 8 minutes) every 10 seconds, and calculating the absorbance change per unit time (unit: mOD/min) from each value 3 minutes to 6 minutes after the start of the measurement.

The subjects' oxHDL average was 30.7 mOD/min with a standard deviation of 4.9.

Test Example 2 Condition when chewing food The subjects were interviewed to confirm their condition when chewing food, whether they "can chew anything,""there are parts of the mouth that bother me and it is sometimes difficult to chew," or "can hardly chew at all."

The subjects were classified into a group of "can chew anything" and a group of "sometimes has difficulty chewing", and the results of plotting oxHDL in each group are shown in Figure 2. Note that "there are areas of concern in the oral cavity that make it difficult to chew" and "can barely chew at all" were collectively grouped into the "sometimes has difficulty chewing" group. In Figure 2, the tips of the whiskers extending upward from the columns indicate the maximum value or the interquartile range multiplied by 1.5 and added to the 75th percentile value, the top of the columns indicate the 75th percentile value, the horizontal line in the columns indicates the 50th percentile value (median), the bottom of the columns indicates the 25th percentile value, and the tips of the whiskers extending downward from the columns indicate the minimum value or the interquartile range multiplied by 1.5 and subtracted from the 25th percentile value.

As shown in Figure 2, it was confirmed that the oxHDL of the group who sometimes have difficulty chewing was significantly higher than that of the group who can chew anything. This suggests that when oxHDL is high, it is possible that the state of mind when chewing food is "sometimes difficult." In other words, it is suggested that masticatory function can be measured using oxHDL. Note that this data was the result of an analysis conducted on male subjects only, but similar results were obtained when the analysis was conducted including female subjects.

The subjects were classified into a group with a current number of teeth of "24 or more" or "fewer than 24," and the oxHDL levels in each group were plotted, as shown in Figure 3. In Figure 3, the tips of the whiskers extending upward from the columns indicate the maximum value or the value obtained by multiplying the interquartile range by 1.5 and adding it to the 75th percentile value, the top of the columns indicate the 75th percentile value, the horizontal line in the columns indicates the 50th percentile value (median), the bottom of the columns indicates the 25th percentile value, and the tips of the whiskers extending downward from the columns indicate the minimum value or the value obtained by multiplying the interquartile range by 1.5 and subtracting it from the 25th percentile value.

As shown in Figure 3, it was confirmed that regardless of the number of teeth (whether they had a large number of teeth (24 or more) or a small number of teeth (fewer than 24)), the oxHDL of the group who sometimes had difficulty chewing was higher than the oxHDL of the group who could chew anything. This suggests that when oxHDL is high, regardless of the number of teeth, there is a possibility that the condition when chewing food may be "sometimes difficult to chew." Note that this data was the result of an analysis conducted on men only, but similar results were obtained when the analysis was conducted including women.

Claims (3)

A method for measuring the chewing function of a subject from whom a blood sample was taken, comprising measuring a value reflecting the amount of oxidized HDL in the blood sample, and using the value reflecting the amount of oxidized HDL as an index. The measurement of the reflection value of the amount of oxidized HDL
(1) The method is carried out by a method including a step of mixing a blood sample with a solution containing a transition metal compound and an acidic buffer solution,
The method of claim 1. The measurement of the reflection value of the amount of oxidized HDL
moreover,
(2) The method is carried out by a method including a step of measuring the free radicals produced in the step (1).
The method of claim 2.

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