Methods for measuring blood glucose levels

Abstract

The present disclosure aims to provide a method for accurately measuring blood glucose levels without causing pain to a subject. The method for measuring the blood glucose concentration of a subject according to the present disclosure includes the steps of collecting hair from the subject and analyzing the collected hair by infrared spectroscopy to obtain an infrared absorption spectrum, and -1 The blood glucose concentration is measured based on the absorption area within the range or the absorbance or transmittance of the peak within the range.

Description

[Technical Field]

[0001] The present disclosure relates to a method for accurately measuring blood glucose levels without causing pain to a subject. [Background technology]

[0002] In recent years, lifestyle-related diseases such as diabetes have become a problem in Japan due to factors such as changes in diet and lack of exercise. Diabetes can lead to blindness, kidney failure, leg amputation, and nerve damage, so early treatment is necessary. Therefore, monitoring blood glucose levels is important.

[0003] Blood glucose concentration is generally measured using an enzymatic reaction in a collected blood sample. For example, glucose oxidase or glucose dehydrogenase is used to oxidize glucose, and the amount of hydrogen peroxide and protons produced is measured as a change in current. Alternatively, the produced hydrogen peroxide is further reacted with peroxidase to produce a coloring agent, and the absorbance at a specific wavelength is measured.

[0004] However, taking a blood sample is painful for the subject. Therefore, non-invasive methods for measuring blood glucose concentration have been developed. For example, Non-Patent Document 1 describes a method for analyzing a hair sample by FTIR-ATR to measure the 1015cm related to glycogen in hair. -1 It is also described that, although it is not a non-invasive method, the wavelength of 1020 to 1040 cm is used to predict diabetes. -1 The present invention discloses a method for measuring the glucose concentration in blood based on the integral of the absorption intensity in a wavelength range of 1000 nm to 1000 nm. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2006 / 011487 Pamphlet [Non-patent literature]

[0006] [Non-Patent Document 1] Sundaramoorthi Kamatchi et al., Journal of Pharmaceutical and Biomedical Analysis, Volume 136, 20 March 2017, Pages 10-13 Summary of the Invention [Problem to be solved by the invention]

[0007] As mentioned above, methods have been developed to measure blood glucose levels using hair samples and infrared spectroscopy. However, for example, in permanent waving, the disulfide bonds between the keratin that makes up the hair are reduced, waving, and then reoxidized, so thiol groups (-SH) may remain in the hair. Furthermore, when dyeing hair, the dye that has penetrated the hair is oxidized to produce color, and the thiol groups may be oxidized to sulfinyl groups [-S(=O)-]. As a result, symmetrical SO and S=O bonds are formed at 1030-1070 cm -1 The strong absorption in the wavelength range of 1020-1040 cm originates from glucose in the hair sample. -1 The absorption intensity of the S(=O) group is very strong, so depending on the substituent, the absorption intensity of the 1015 cm -1 This has the problem of adversely affecting the measurement of peak intensity. Therefore, an object of the present disclosure is to provide a method for accurately measuring blood glucose concentration without causing pain to a subject. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above problems. As a result, collected hair samples were analyzed by infrared spectroscopy, and the results showed that -1The present inventors have found that by determining the blood glucose concentration based on the absorption area in the range of 0.1 to 1.5, it is possible to indirectly measure the blood glucose concentration without being affected by other compounds or under conditions where the influence of other compounds is small, and have completed the present invention. The present invention will now be described.

[0009] [1] A method for measuring blood glucose concentration in a subject, comprising: collecting hair from the subject; and The method includes a step of analyzing the collected hair by infrared spectroscopy to obtain an infrared absorption spectrum, In the infrared absorption spectrum, 1098 to 1136 cm -1 The method for measuring the blood glucose concentration is characterized by measuring the absorption area in the range included in the above, or the absorbance or transmittance of the peak included in the above range. [2] The method according to [1], wherein the hair is analyzed by infrared spectroscopy using attenuated total reflection. [3] The absorption area is 1476 to 1584 cm -1 The method according to [1] or [2] above, wherein normalization is performed by dividing by the absorption area within the range included in the range, or the absorbance or transmittance of the peak included in the range. [4] 1098~1136cm created in advance -1 The method according to any one of [1] to [3] above, wherein the blood glucose concentration is determined based on an absorption area from a calibration curve showing the relationship between the blood glucose concentration and an absorption area within a range included in the range, or the absorbance or transmittance of a peak included in the range. [5] The method according to any one of [1] to [4] above, wherein the blood glucose concentration is an HbA1c value. [6] The method according to any one of [1] to [5], wherein an area of ​​5 cm or less from the root of the hair is analyzed by infrared spectroscopy. [Effects of the Invention]

[0010] According to the method of the present invention, it is possible to indirectly and accurately measure blood glucose levels from hair samples without causing pain to the subject. Therefore, the method of the present invention makes it possible to measure blood glucose levels easily and continuously, and is extremely useful industrially as it is useful for preventing lifestyle-related diseases such as diabetes. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a graph plotting the ratio of the absorption area in the range of 1098 to 1136 cm −1 to the absorbance at a wavenumber of 1540 cm −1 of a hair sample, and the HbA1c value of the same subject. [Figure 2] FIG. 2 is a graph plotting the ratio of absorbance at 1015 cm-1 to absorbance at 1540 cm-1 wavenumber versus the HbA1c value of the same subject. [Figure 3] FIG. 3 is a graph plotting the ratio of absorbance at 1040 cm −1 to absorbance at 1540 cm −1 wavenumber versus the HbA1c value of the same subject. [Figure 4] Figure 4 shows the second derivative data of the infrared absorption spectrum measured for a subject who dyed his hair. DETAILED DESCRIPTION OF THE INVENTION

[0012] The method for measuring blood glucose concentration according to the present disclosure will be described below, but the present invention is not limited to the following specific examples.

[0013] 1. Hair sample collection process In this step, hair is collected from a subject. The subject is not limited to humans, but also includes non-human animals, as long as the blood glucose concentration is to be measured.

[0014] The non-human animal is not particularly limited as long as it is an animal whose blood glucose concentration should be measured, and examples include pet animals such as dogs, cats, rabbits, hamsters, guinea pigs, and small birds; livestock animals such as cows, pigs, chickens, horses, sheep, goats, ducks, turkeys, and quails; and laboratory animals such as rats, mice, monkeys, dogs, and rabbits.

[0015] Hair is a general term for body hair, and may refer specifically to head hair, but is not limited to head hair as long as it can be collected. When the subject is a human, head hair may be collected, as it is easy to collect. When the subject is a non-human animal, hair other than head hair may be collected, but it is preferable to collect hair from the same position from the viewpoint of comparison and reproducibility.

[0016] Although only one hair may be collected from the subject, two or more and ten or fewer hairs may be collected per measurement from the subject in order to obtain more accurate results by calculating the average value.

[0017] 2.Analysis process In this step, the collected hair is analyzed by infrared spectroscopy to obtain an infrared absorption spectrum. The collected hair may be pretreated for analysis. For example, the hair may be finely ground, and glucose or its derivatives may be extracted using water or other solvents, and the extract may be analyzed. However, for simpler measurements, the collected hair may be analyzed by attenuated total reflection (ATR) without pretreatment.

[0018] Attenuated total reflection (ATR) is a type of infrared spectroscopy that can analyze solid, liquid, paste, and gel samples. Unlike transmission spectroscopy, which requires samples to be thinned to less than 20 μm or tableted with KBr powder, it can analyze solid, liquid, paste, and gel samples. Specifically, the sample is placed in contact with a high-refractive-index prism, which can totally reflect irradiated infrared light. During this total reflection, the infrared light forms an evanescent wave that penetrates slightly into the sample. It interacts with the surface of the sample within a few microns. Specific wavelengths of infrared light are absorbed and attenuated by molecules in the sample, resulting in the attenuation of the attenuated wavelengths, which are then detected as an ATR spectrum. Attenuated total reflection (ATR) is a method for detecting this reflected infrared light and identifying the specific wavelengths of attenuated infrared light, and ultimately the functional groups that attenuate the specific wavelengths.

[0019] Examples of high refractive index prisms include diamond, ZnSe, Ge, etc. In the present invention, any general prism can be used without any particular restrictions, and for example, a high refractive index prism made of diamond can be used from the viewpoint of durability.

[0020] Hair is mainly composed of keratin, a protein derived from dead hair matrix cells, so it may preserve the state of the body in the past. Specifically, analyzing the root of the hair may enable us to ascertain the blood glucose concentration relatively recently. Furthermore, since hair grows approximately 1 cm per month, analyzing a position x cm from the root may enable us to ascertain the blood glucose concentration approximately x months ago.

[0021] The analysis is preferably performed by infrared spectroscopy within a range of 5 cm from the root of the hair sample. As mentioned above, hair samples may contain information about past body conditions, and if the analysis position is 5 cm or less from the root, physical and chemical damage is reduced and past information is more reliably preserved. The position is more preferably 2 cm or less, and even more preferably 1.5 cm or less. There is no particular lower limit to the position, and the vicinity of the root of the hair sample may also be analyzed, i.e., the position may be 0 cm or more.

[0022] It is preferable that multiple hair samples taken from the same subject or multiple hair samples taken from multiple subjects be analyzed at the same position, i.e., at the same length from the root, in order to compare measurement results.

[0023] 3.Analysis process In this step, the infrared absorption spectrum obtained in the previous step 2 is analyzed, and the blood glucose concentration is determined based on the results. The blood glucose concentration is not limited to blood glucose concentration, but can also be HbA1c (hemoglobin A1c) as long as it is an indicator of diabetes. HbA1c is the percentage of glycated hemoglobin (hemoglobin bound to glucose) in red blood cells in the blood. Since the lifespan of a red blood cell is approximately 120 days, HbA1c reflects the average blood glucose level over the past one to two months. Regarding fasting plasma glucose (FPG), a fasting plasma glucose level of 70 to 99 mg / dL is considered normal, 100 to 125 mg / dL is considered borderline, and 126 mg / dL or higher is considered diabetes. Regarding random blood glucose levels and a 75-g oral glucose tolerance test (OGTT), a level of 200 mg / dL or higher is considered diabetes. For HbA1c, a value of 5.6% or less is considered normal, 5.7-6.4% is considered borderline, and 6.5% or more is considered diabetic.

[0024] In this step, in the infrared absorption spectrum obtained in the previous step 2, -1 The absorption area in the range included in this range, or the absorbance or transmittance of the peak included in this range, is calculated. The absorption area is 1098 to 1136 cm -1 Even if the absorption area is in the range of, for example, 1100 to 1130 cm -1 The absorption area may be within a narrower range than the above range. The absorbance peak for determining the absorbance or transmittance may be a peak at a specific wavelength or the maximum peak within this range. -1 The absorption of 1000 kJ / s is thought to be due to the hydroxyl groups contained in glucose and glucose derivatives contained in the hair sample. Examples of glucose derivatives include glycogen and glycosylated proteins to which glucose or sugar chains containing glucose are bound. In the infrared absorption spectrum, the horizontal axis is the wave number (cm -1 ) and the vertical axis represents absorbance (Abs) or transmittance (%T). Absorbance (Abs) = -log 10(Transmittance) The absorption area in the above range is calculated as an integral value in the above range.

[0025] Keratin, a protein that is the main component of hair, contains a large amount of cysteine ​​and is bound by numerous disulfide bonds between molecules. These disulfide bonds are reduced by permanent waving to form thiol groups (-SH), which are generally reoxidized to disulfide bonds by subsequent oxidation treatment. However, during this process, the thiol groups may be oxidized to sulfinyl groups [-S(=O)-]. Furthermore, residual thiol groups may be oxidized to sulfinyl groups during oxidation treatment for hair dyeing. The absorption wavelength of thiol groups is 2550-2600 cm -1 The absorption wavelength of the CS single bond is 500-750 cm -1 The absorption wavelength of the sulfoxide group (-S(=O)-) is 1030 to 1070 cm -1 The absorption wavelength of the sulfinic acid group (-S(=O)OH) is 1150-1300 cm -1 The absorption wavelength of the disulfide group (-SS-) is 500 to 550 cm -1 The absorption area is calculated from 1098 to 1136 cm -1 Since the absorption wavelength range of the fluorophores does not overlap with that of the fluorophores, it is possible to measure the amount of glucose more accurately.

[0026] The absorption area in the above range is 1476 to 1584 cm -1 Alternatively, normalization may be performed by dividing by the absorption area in the range included in the range, or by the absorbance or transmittance of the peak included in the range, i.e., the peak of the amide bond. Because the main component of hair is keratin, which is a protein, normalization by the absorbance of the peak of the amide bond or the like makes it possible to compare data from other hairs of the same subject or data from other subjects.

[0027] In addition, in advance, in the infrared absorption spectra of hair samples from a number of other subjects, the range of 1098 to 1136 cm -1It is preferable to determine the absorption area within the range included in the range, or the absorbance or transmittance of the peak included in the range, and to actually measure the glucose concentration in a blood sample from the same subject, accumulate a large amount of data on the absorption area, absorbance, or transmittance and the blood glucose concentration, and create a calibration curve. This calibration curve makes it possible to predict and indirectly measure blood glucose concentrations without invasive measurements such as blood sampling. Of course, the measurement conditions for the absorption area and blood glucose concentration for creating the calibration curve are the same or approximately the same as the measurement conditions for the absorption area and blood glucose concentration of the subject. Furthermore, the slope of the calibration curve showing the relationship between the absorption area and blood glucose concentration is sufficiently large, allowing for more accurate measurements. [Example]

[0028] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples, and it is possible to carry out the invention by making appropriate modifications within the scope of the above and below-described aims, and all such modifications are included in the technical scope of the present invention.

[0029] Example 1 (1) Infrared spectroscopy analysis This experiment was conducted with the approval of the Morimoto Memorial Clinic Institutional Review Board (IRB). The purpose and objectives of the study were fully explained to the subjects, and after obtaining their consent, they provided hair samples as clinical samples. Five hairs were cut from the roots of each of the 46 subjects' heads. After collection, the hair was analyzed as is, without any pretreatment such as washing or drying. Some subjects had dyed hair, but as many people generally dye their hair, this experiment did not distinguish between dyed and undyed hair. For spectroscopic measurements, a Fourier transform infrared spectrophotometer ("Cary 630 FTIR" manufactured by Agilent) was used, and the hair samples were analyzed by infrared spectroscopy using the attenuated total reflection method (ATR). Specifically, a crystal ("single reflection diamond ATR attachment" manufactured by Agilent) was placed on the hair sample, and infrared light was irradiated at a position approximately 1 cm from the cutting position (root). -1 The infrared absorption spectrum was acquired by integrating reflected light 32 times within the range. The obtained spectral data was preprocessed using the savgol_filter function included in the Python library Scipy with the following parameters: window_length = 11 polyorder = 3 deriv = 2 By the above processing, a second derivative spectrum was obtained while suppressing noise.

[0030] For the spectrum after the above processing, the wave number is 1015 cm -1 and 1040cm -1 Absorbance at 1098-1136cm -1 The area of ​​absorption (AUC) in the range was calculated. In addition, to correct for measurement errors and variations in sample volume for each hair sample, the wavenumber of 1540 cm, which corresponds to the stretching of amide bonds, was used because the main component of hair is protein. -1 Each value was normalized by the absorbance at 0.05°C, and the average value of the results for five hair samples per subject was calculated.

[0031] (2) Analysis of results The wavenumber of the hair sample is 1540 cm -1 Absorbance at 1098-1136cm -1 Figure 1 shows a graph plotting the ratio of absorption area in the range of 1540 cm and the HbA1c value of the same subject. The HbA1c value was obtained from the subject's health checkup. For comparison, the absorption area ratio of 1540 cm was also plotted. -1 Absorbance at 1015cm -1and 1040cm -1 The graphs plotting the absorbance ratios at 1000 and 10000 and the HbA1c values ​​of the same subjects are shown in Figures 2 and 3, respectively. Furthermore, an approximate curve was obtained using a spreadsheet software (Microsoft Excel), and the correlation coefficient r and the coefficient of determination R 2 was calculated.

[0032] As shown in Figures 2 and 3, the wavenumber is 1015 cm -1 The slope of the fitted curve showing the relationship between absorbance and HbA1c value at 1040 cm is -0.123. -1 The slope of the fitted curve showing the relationship between absorbance and HbA1c value was 0.2424, which was almost parallel to the X-axis and could not be used as a calibration curve. The correlation coefficients r were -0.0026 and 0.14, respectively, and the wavenumber was 1015 cm. -1 and 1040 cm -1 No significant correlation was observed between absorbance and HbA1c values. 2 were 0.0007 and 0.02, respectively, and could not be said to be reliable calibration curves. In contrast, 1098-1136cm -1 The slope of the fitted curve showing the relationship between the area of ​​absorption (AUC) and HbA1c value in the range of 1098 to 1136 cm is 0.6423, which means that it has a slope that can be used as a calibration curve. -1 A positive correlation was observed between the area of ​​absorption (AUC) and HbA1c values ​​in the range of . Furthermore, the coefficient of determination R 2 is 0.52, which can be said to be a reliable calibration curve in terms of analyzing complex samples such as biological samples. From these results, the hair length is 1098-1136cm -1 It was shown that blood glucose concentration can be indirectly measured by measuring the absorption area in the range of 1000 nm to 1000 nm.

[0033] Figure 4 shows the second derivative data of the infrared absorption spectrum measured for a subject who dyed his hair. The second derivative shows how the rate of change is changing, so 1098~1136cm -1For the absorption peak at 1040cm -1 Although both regions are absorption regions derived from the hydroxyl groups of aliphatic alcohols, this difference is observed because the disulfide bonds of the keratin in the hair are oxidized and further cleaved by the oxidation process during hair dyeing, and the absorption peak due to the sulfinyl group (-S(=O)-) is strong, resulting in a sharp and strong absorption peak at 1040 cm. -1 This is thought to be because the absorption peak of the sulfinyl group overlaps with the absorption peak of the In this way, 1098~1136cm -1 By measuring the absorption area, it is possible to measure the blood glucose concentration more accurately, especially in subjects who have permanent waves or dyed hair.

Claims

1. 1. A method for measuring blood glucose concentration in a subject, comprising: collecting hair from the subject; and The method includes a step of analyzing the collected hair by infrared spectroscopy to obtain an infrared absorption spectrum, In the infrared absorption spectrum, 1098 to 1136 cm -1 The method for measuring the blood glucose concentration is characterized by measuring the absorption area in the range included in the above, or the absorbance or transmittance of the peak included in the above range.

2. 10. The method of claim 1, wherein the hair is analyzed by infrared spectroscopy using attenuated total reflectance.

3. The absorption area is 1476 to 1584 cm -1 The method according to claim 1, wherein normalization is performed by dividing the absorption area in the range included in the range, or the absorbance or transmittance of the peak included in the range.

4. Pre-created 1098-1136cm -1 2. The method of claim 1, wherein the blood glucose concentration is determined based on the absorption area from a calibration curve showing the relationship between the blood glucose concentration and the absorbance or transmittance of a peak included in the range.

5. The method of claim 1, wherein the blood glucose concentration is an HbA1c value.

6. The method according to claim 1, wherein an area of ​​5 cm or less from the root of the hair is analyzed by infrared spectroscopy.

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