JP2015010938A - Diabetes detection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a diabetes detection technique which detects even a diabetes in an initial stage by high sensitivity, has high specificity to a diabetes, allows a user to operate easily, and uses urine as a sample.SOLUTION: The diabetes detection method in which urine collected from a subject is used as a sample is provided. The sample is urine or derivation matter thereof which is not subjected to refining. Using intensity of fluorescence emitted from a fluorescent material included in the sample in an excitation wavelength of 250-500 nm as an index, presence/absence of a diabetes in the subject is detected. As the sample, urine itself or a diluted matter of the same may be used.

Description

  The present invention relates to a method for detecting diabetes. The method for detecting diabetes according to the present invention can detect the presence or absence of diabetes with high sensitivity, high specificity, and simply.

  In recent years, westernization of eating habits has progressed, and lifestyle-related diseases such as obesity, diabetes, and arteriosclerosis, which are considered to be caused by this, are increasing. Of these, diabetes often causes complications such as sensory palsy, blindness, arteriosclerosis, and the like, and is regarded as a particular problem as a disease that causes a great disability in daily life.

  Diabetes is a complex disease caused by hyperglycemia due to insufficient action of insulin. Diabetes is classified into type 1 and type 2. In type 1 diabetes, pancreatic islets of Langerhans are inflamed, resulting in a decrease or depletion of the ability to secrete insulin, leading to hyperglycemia. On the other hand, type 2 diabetes causes hyperglycemia due to insufficient action of insulin due to other causes. This type 2 diabetes accounts for the majority of Japanese diabetes and is especially regarded as a problem.

  Although the pathogenesis of type 2 diabetes is still unclear, it is thought that the disease is caused mainly by environmental factors, and overeating and obesity are one of the major causes. For example, the amount of insulin secretion in the pancreas increases dramatically due to obesity, resulting in fatigue of the pancreas and conversely the decrease in insulin secretion, resulting in insufficient insulin action and hyperglycemia. Alternatively, insulin receptors decrease due to an increase in fat, resulting in insufficient insulin action and hyperglycemia. On the other hand, excess glucose born from insufficient action of insulin is stored as fat and obesity progresses, and diabetes is closely related to obesity in its onset mechanism. In addition, type 2 diabetes often has no subjective symptoms at the beginning of the onset, and at the time of discovery it is so advanced that it may be difficult to treat. In other words, it is expected that there will be a considerable number of reserves for type 2 diabetes.

  Currently, clinical laboratory items used for detecting diabetes include urine sugar, fasting blood glucose, hemoglobin A1c (HbA1c), blood insulin level, blood / urine C-peptide level (CPR), and the like. In addition, an oral glucose tolerance test (OGTT) is also performed in which blood glucose concentration after ingesting glucose is monitored and its clearance ability is observed.

  In recent years, substances called advanced glycation end products (hereinafter referred to as “AGEs”) have attracted attention in the medical field, and their relationship with various diseases, diseases, aging, etc. has been studied. . And it is known that the generation of AGEs is promoted and accumulated in circulating blood and tissues in a chronic hyperglycemic state. The name AGEs is a generic name, and AGEs include various compounds. As typical AGEs, carboxymethyl lysine (Nε- (carboxymethyl) lysine), pentosidine (Pentosidine), pyralin (Pyrraline), crossline (Crossline) and the like are known.

  AGEs measurement techniques include fluorescence analysis using fluorescence emitted from AGEs (mainly excitation wavelength 370 nm, emission wavelength 440 nm) as an index, absorbance measurement method using brown color change, chemiluminescence method, and the like. (For example, Patent Documents 1 to 3). In addition, precise measurement methods using HPLC and GC / MS are also known (for example, Non-Patent Document 1 and Patent Document 4). Furthermore, measurement methods using ELISA focusing on specific AGEs have also been developed (for example, Patent Documents 5 to 7, Non-Patent Documents 2 and 3).

  Of these, the fluorescence measurement method utilizes the fact that many AGEs emit fluorescence. However, since there are AGEs that do not emit fluorescence (for example, pyralin), the fluorescence analysis method does not cover all AGEs. On the contrary, it is considered that measurement is performed including fluorescent substances other than AGEs. Moreover, it is thought that the fluorescent substance which can be detected changes with excitation wavelength and light emission wavelength to select. The fluorescence analysis method is highly sensitive and simple, and is useful when measuring a large number of samples. For example, it is useful when a plurality of urine samples or serum samples are used as a measurement sample in health checkups.

There are reports on the fluorescence analysis of urinary AGEs for the purpose of detecting diabetes (for example, Non-Patent Documents 4 and 5). Here, when the urine sample is subjected to fluorescence analysis, pretreatment such as deproteinization is usually performed.
For example, in the technique described in Non-Patent Document 4, a urine sample is subjected to ultrafiltration as a pretreatment, and a fluorescence analysis is performed after removing a substance having a molecular weight of 10,000 or more. In the technique described in Non-Patent Document 5, a urine sample is subjected to deproteinization treatment with trichloroacetic acid (TCA) as pretreatment.

JP 2001-099838 A JP 2001-021549 A JP 2001-059843 A Japanese Patent Laid-Open No. 7-098317 JP 2004-323515 A JP 2006-31621 A JP 2001-021559 A

MP DE LA MAZA et al., "Fluorescent serum and urinary advanced glycoxidation end-products in non-diabetic subjects", Biol Res. 2007; 40 (2): 203-12. N. Turk et al., "Urinary extraction of advanced glycation endoproducts in patients with type 2 diabetes and various stages of proteinuria", Diabetes Metab. 2004 Apr; 30 (2): 187-92. Takeuchi, M., et al., "Immunological evidence that non-carboxymethyllysine advanced glycation end-products are produced from short chain sugars and dicarbonyl compounds in vivo", Mol Med. 2000 Feb; 6 (2): 114-25. Yanagisawa et al., "Specific Fluorescence Assay for Advanced Glycation End Products in Blood and Urine of Diabetic Patients", Metabolism. 1998 Nov; 47 (11): 1348-53. M. P. de la Maza et al., "Fluorescent advanced glycation end-products (ages) detected by spectro-photofluorimetry, as a screening tool to detect diabetic microvascular complications", Journal of Diabetes Mellitus. 2012 2 (2): 221-226.

  As described above, type 2 diabetes often has no subjective symptoms in the early stage of onset, and at the time of discovery it is quite advanced and often difficult to treat. Therefore, in clinical tests aimed at detecting diabetes, not only the presence or absence of diabetes, but the presence or absence of diabetes at an early stage that cannot be detected normally, the presence or absence of future risk of diabetes, There is a desire to detect whether or not there is. On the other hand, it is also required that high specificity for diabetes is maintained so that non-diabetes is not detected as a false positive. Furthermore, there is a demand for detecting diabetes with the simplest possible operation so that it can be easily applied to mass screening. Accordingly, an object of the present invention is to provide a technology for detecting diabetes that can detect diabetes at an early stage with high sensitivity, has high specificity for diabetes, and is easy to operate.

  In order to solve the above-described problems, the present inventors have advanced research on a highly sensitive, highly specific and simple detection technique for diabetes using urine as a specimen. As a result, it was found that the above problem can be solved by subjecting urine to fluorescence analysis as it is without pretreatment. That is, it is possible to detect diabetes by directly subjecting urine or a diluted urine to fluorescence analysis, and furthermore, it is possible to detect diabetes that has been missed by the conventional method by not performing urine pretreatment. I found. Furthermore, it has been found that high specificity for diabetes is maintained even when pretreatment is not performed, and that diabetes and non-diabetes can be distinguished with high accuracy.

  One aspect of the present invention provided on the basis of the above findings is a method for detecting diabetes using urine collected from a subject as a specimen, and the specimen is urine or urine-derived material that has not undergone purification treatment. A method for detecting diabetes, comprising detecting the presence or absence of diabetes in the subject using as an index the intensity of fluorescence emitted by a fluorescent substance contained in the specimen at an excitation wavelength of 250 to 500 nm.

  The method for detecting diabetes according to the present invention uses urine or urine-derived material that has not undergone purification treatment as a sample, and uses a specific fluorescence intensity emitted from the sample as an index. According to the present invention, diabetes can be detected with high sensitivity and simplicity while maintaining high specificity for diabetes. For example, it is possible to detect diabetes that cannot be detected by fluorescence analysis using a pretreated urine sample as in the prior art.

Here, the “purification process” refers to a process that changes the composition of the contents of the collected urine. For example, a process for removing some contents and a process for concentrating some contents are included in the purification process. For example, removal of substances having a molecular weight above a certain level (for example, concentration / fractionation by ultrafiltration), removal of proteins and peptides (for example, deproteinization by TCA), removal of some charged molecules (for example, Contact with an ion exchanger) and fractionation treatment by various chromatography are all included in the purification treatment.
On the other hand, the dilution operation only dilutes the entire urine uniformly and does not change the composition of the inclusions, and thus is not included in the purification treatment. Similarly, the operation of uniformly concentrating the entire urine is not included in the purification process. A treatment for adding a stabilizer or the like to the whole urine is not included in the purification treatment unless the composition of the contents is changed.

  A “urine-derived product” that has not undergone purification treatment refers to a product that is not urine itself, but is derived from urine and has not undergone the purification treatment described above. A typical example of the urine-derived material is the urine dilution described above. In addition, a uniform urine concentrate is also included in the urine-derived material.

  Here, “presence / absence of diabetes” is not only the presence / absence of diabetes, but also the presence / absence of early diabetes, which is usually difficult to detect, the presence / absence of the risk of developing diabetes in the future, whether it is a so-called diabetes reserve arm, etc. It is a concept that also includes The presence or absence of the risk of developing diabetes in the future refers to the presence or absence of risk (risk) of having diabetes in the future at the time when diabetes does not occur.

  The fluorescence used as an index in the present invention is fluorescence emitted from a fluorescent substance contained in a specimen at an excitation wavelength of 250 to 500 nm. This excitation wavelength range overlaps with the wavelength conventionally employed in the fluorescence measurement of AGEs. However, the fluorescence used as an index in the present invention does not reflect only AGEs, and covers a wide range including other fluorescent materials, for example, fluorescent materials that have been removed by pretreatment in the prior art.

  Preferably, the intensity of the fluorescence is the intensity of fluorescence at a wavelength 10 to 250 nm larger than the excitation wavelength.

  Preferably, the specimen is urine itself or a urine dilution.

  Preferably, the intensity of the fluorescence is measured, and the obtained measurement value is compared with a reference value.

  According to the present invention, diabetes can be detected with high sensitivity and simplicity while maintaining high specificity for diabetes. For example, it is possible to detect diabetes that cannot be detected by fluorescence analysis using a pretreated urine sample as in the prior art.

(A) is a graph showing the result of measuring fluorescence intensity for urine samples including diabetic and non-diabetic types, and (b) is a dot plot diagram. It is a graph showing the relationship between urinary albumin and fluorescence intensity.

Hereinafter, embodiments of the present invention will be described.
The method for detecting diabetes according to the present invention is a method for detecting diabetes using urine collected from a subject as a specimen, and the specimen is urine or urine-derived material that has not undergone purification treatment, and the excitation wavelength is 250 to 500 nm. The presence or absence of diabetes in the subject is detected using the intensity of fluorescence emitted from the fluorescent substance contained in the specimen as an index. For example, the fluorescence intensity emitted from the specimen is measured with a fluorescence analyzer or the like, the measured value is compared with a reference value, and the presence or absence of diabetes is detected based on the comparison result.

  In the present invention, “urine or urine-derived material that has not undergone purification treatment” is used as a specimen. As described above, the purification treatment refers to a treatment that changes the composition of the contents of the collected urine. That is, in the present invention, urine or urine-derived material that has not undergone pretreatment such as ultrafiltration or protein removal is used.

  As described above, the “urine-derived product” that has not undergone purification treatment refers to a product that is not urine itself, but is derived from urine and has not undergone the purification treatment described above. A typical example of the urine-derived material is a urine dilution.

In a preferred embodiment, urine itself or a urine dilution is used as the specimen. The use of urine itself and urine dilution can be performed according to, for example, the fluorescence intensity emitted from the specimen. That is, when the fluorescence intensity of urine is too high, it is preferable to dilute urine to avoid the quenching phenomenon.
The diluting solution used for diluting urine is not particularly limited as long as it does not adversely affect the fluorescence measurement, and water, physiological saline, phosphate buffered saline (PBS), various buffer solutions, etc. are appropriately used. Can be used. The pH of the diluting solution is not particularly limited as long as the fluorescent substance can exist stably, and can be selected from the range of 6.0 to 8.0, for example. The ionic species and the ionic strength in the diluted solution may be anything as long as the fluorescent substance can exist stably. For example, an ionic species or ionic strength that does not denature the protein may be selected.
Moreover, it is preferable that a dilution liquid does not contain a fluorescent substance.

In the present invention, the intensity of fluorescence emitted from the fluorescent substance contained in the specimen at an excitation wavelength of 250 to 500 nm is used as an index. As described above, this excitation wavelength range overlaps with the wavelength conventionally employed in the fluorescence measurement of AGEs. The range of the excitation wavelength is preferably 250 to 450 nm, more preferably 280 to 420 nm, still more preferably 280 to 400 nm, and particularly preferably 300 to 400 nm.
The fluorescence wavelength (emission wavelength) at the time of measuring the fluorescence intensity is not particularly limited as long as the fluorescence can be detected, but it is preferable to select a wavelength that is 10 to 250 nm larger than the excitation wavelength.
As a specific example, when the excitation wavelength is 260 nm, the fluorescence wavelength can be selected from the range of 270 to 510 nm, preferably 300 to 380 nm, more preferably 320 to 370 nm.
When the excitation wavelength is 300 nm, the fluorescence wavelength can be selected from the range of 310 to 550 nm, preferably 350 to 500 nm, more preferably 360 to 480 nm.
When the excitation wavelength is 340 nm, the fluorescence wavelength can be selected from the range of 350 to 590 nm, preferably 380 to 530 nm, more preferably 390 to 520 nm.
When the excitation wavelength is 370 nm, the fluorescence wavelength can be selected from the range of 380 to 620 nm, preferably 4000 to 550 nm, more preferably 420 to 530 nm.
When the excitation wavelength is 400 nm, the fluorescence wavelength can be selected from the range of 410 to 650 nm, preferably 4500 to 600 nm, more preferably 460 to 580 nm.
When the excitation wavelength is 450 nm, the fluorescence wavelength can be selected from the range of 460 to 700 nm, preferably 5000 to 650 nm, more preferably 510 to 630 nm.
When the excitation wavelength is 500 nm, the fluorescence wavelength can be selected from the range of 510 to 750 nm, preferably 5500 to 700 nm, more preferably 560 to 680 nm.

  Furthermore, a combination of “excitation wavelength of 370 nm and fluorescence wavelength of 440 nm” conventionally used in fluorescence measurement of AGEs may be employed.

In the present invention, the intensity of fluorescence emitted from the fluorescent substance contained in the specimen at an excitation wavelength of 250 to 500 nm is used as an index. A typical example of the specific operation is to compare the obtained fluorescence intensity value with a reference value. is there. For example, the fluorescence intensity of a standard substance can be measured at the same time, and the measured value can be used as a reference value.
More specifically, for example, a stable standard solution having a constant concentration is prepared, and the fluorescence intensity of the specimen can be expressed by a relative value when the fluorescence intensity of the standard solution is 100.
A known fluorescent material can be used as the standard material. The fluorescent substance is not particularly limited as long as it can measure fluorescence at the excitation wavelength and emission wavelength. There is no need to be AGEs. A plurality of standard substances may be used.
Specific examples of the fluorescent substance that can be a standard substance include quinine sulfate, sodium fluorescein, rhodamine B, and the like. For example, a 0.1-1 mol / L sulfuric acid solution of quinine sulfate is stable and useful as a standard solution.

In addition, it is also possible to select a standard substance whose reference value is a cut-off value, and to determine non-diabetic when the fluorescence intensity value is lower than the reference value and to determine diabetes when it exceeds the reference value.
In addition, a calibration curve may be prepared using a serially diluted standard substance.

In the present invention, it is preferable to perform concentration correction such as creatinine correction in the same manner as other clinical markers using urine as a specimen.
As a method for measuring creatinine in urine, a known method can be used as it is. For example, organic chemical measurement methods, ultraviolet absorption methods, enzymatic measurement methods, mass spectrometry, and the like can be used. Among these, the ultraviolet absorption method is simple because urine or a diluted urine can be used as a measurement sample as it is.
Other than correction by creatinine, correction may be made by specific gravity of urine.

  The fluorescence intensity of the specimen can be measured using a known fluorescence analyzer. For example, the fluorescence intensity of the specimen can be measured by using a spectrofluorometer. Further, by using a fluorescence microplate reader, the fluorescence intensity of a plurality of specimens placed in each hole of the microplate can be measured simultaneously.

Furthermore, a microanalysis technique using a microdevice (microfluidic device, μTAS) can also be applied. For example, a micro device including a sample introduction unit, a micro channel communicating with the sample introduction unit, and a sample storage unit communicating with the micro channel and functioning as an optical cell is prepared. Then, a sample (for example, urine dilution that has not undergone purification treatment) is introduced from the sample introduction unit, and is accommodated in the sample accommodation unit via the microchannel. Then, the fluorescence intensity emitted by the sample stored in the sample storage unit (optical cell) is measured with a fluorescence analyzer.
At this time, a micro device provided with two sample storage units (optical cells) may be employed, and one sample storage unit may be used for fluorescence intensity measurement and the other sample storage unit may be used for creatinine concentration measurement. As the creatinine measurement method at this time, for example, the above-described ultraviolet measurement method (220 to 250 nm) can be used.
By applying the microdevice to the present invention, it is possible to perform fluorescence measurement with a very small amount of specimen (measurement sample). Furthermore, by reducing the optical path length (cell thickness, liquid thickness) of the optical cell, it is possible to use urine itself as a measurement sample without diluting even a thick urine. The thickness of the cell at this time can be, for example, about 100 to 500 μm.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

(1) Correlation between blood glucose level and blood HbA1c level The urine fluorescent substance measurement according to the present invention was performed on the urine of 114 adult males whose blood glucose level and blood HbA1c level were known, and the correlation was examined. . The distribution of blood glucose levels (mg / dL) of the 114 patients was a minimum value 83, a maximum value 271, an average value 117.25, and a standard deviation 27.016. The distribution of the blood HbA1c value (%) was a minimum value of 4.4, a maximum value of 9.9, an average value of 5.706, and a standard deviation of 1.0562.

Urine was diluted 10 to 200 times with PBS to prepare a specimen (measurement sample) for fluorescence measurement. 200 μL of each measurement sample is directly placed in each hole of a fluorescence measurement microplate (Greiner bio-one, 762077) without pretreatment, and fluorescence intensity is measured with a microplate reader (Spectra Max Gemini EM; Molecular Devices, Sunnyvale, Calif.). (Excitation wavelength: 370 nm, fluorescence wavelength: 440 nm) was measured. Separately, the creatinine concentration of each urine sample was measured using L type Wako CRE · M (Wako Pure Chemical Industries, Ltd.). The obtained fluorescence intensity value was divided by the creatinine concentration value to obtain a corrected fluorescence intensity value (AU / g creatinine).
As a result, the distribution of the corrected fluorescence intensity values was a minimum value 46, a maximum value 1276, an average value 196.33, and a standard deviation 158.067.

Statistical processing was performed with SPSS software (IBM). As a result, the Pearson correlation coefficient between the corrected fluorescence intensity value and the blood glucose level was 0.260, and the significance (both sides) was 0.05. The Pearson correlation coefficient between the corrected fluorescence intensity value and the blood HbA1c value was 0.281, and the significance (both sides) was 0.02.
From the above, the fluorescence intensity of urine (excitation wavelength: 370 nm, fluorescence wavelength: 440 nm) was correlated with blood glucose level and blood HbA1c value.

(2) Discrimination between diabetes and non-diabetes by fluorescence intensity The following measurements were performed using urine of 393 adult males (22 diabetic types, 371 non-diabetic types (healthy people)) as samples. The breakdown of the 22 diabetics is
(A) 15 people using anti-diabetic drugs (b) Not using anti-diabetes drugs but fasting blood glucose is 126 mg / dL and HbA1c meets 6.1% or higher diabetes diagnostic criteria People: 7 people.

Urine was diluted 10 to 200 times with PBS to prepare a specimen (measurement sample) for fluorescence measurement. 200 μL of each measurement sample is directly placed in each hole of a fluorescence measurement microplate (Greiner bio-one, 762077) without pretreatment, and fluorescence intensity is measured with a microplate reader (Spectra Max Gemini EM; Molecular Devices, Sunnyvale, Calif.). (Excitation wavelength: 370 nm, fluorescence wavelength: 440 nm) was measured.
Separately, the creatinine concentration of each urine sample was measured using L type Wako CRE · M (Wako Pure Chemical Industries, Ltd.). The obtained fluorescence intensity value was divided by the creatinine concentration value to obtain a corrected fluorescence intensity value (AU / g creatinine). The results are shown in FIGS. 1 (a) and (b).

The average value ± standard deviation of the corrected fluorescence intensity values (AU / g creatinine) was 171 ± 132 in the non-diabetic type (371 patients). On the other hand, the above (a) diabetic type (15 persons) is 260 ± 181, the above (b) diabetic type (7 persons) is 344 ± 413, and the above (a) and (b) are combined (22 persons) 287 ±. 269.
And between non-diabetic type and diabetic type (a), between non-diabetic type and diabetic type (b), between non-diabetic type and diabetic type (a) and (b), A significant difference (P <0.05) was observed in the corrected fluorescence intensity values.

(3) Examination of excitation wavelength and fluorescence wavelength For each specimen in (2) above, the fluorescence intensity (AU / g creatinine) is measured by shaking the excitation wavelength in the range of 260 to 500 nm and the fluorescence wavelength in the range of 300 to 700 nm. A significant difference test was performed between the diabetic group (22 cases) and the non-diabetic group (371 cases). The results are shown in Table 1.
That is, a significant difference was observed in the fluorescence intensity at least in the excitation wavelength range of 260 to 500 nm (P <0.05, bold portion). As for the fluorescence wavelength, at least the excitation wavelength plus a wavelength of about 20 nm could be sufficiently employed.

(4) Relationship between urinary albumin and fluorescence intensity Urinary albumin was measured for each specimen of (2) above, (N) 20 mg / g creatinine or less (353 cases), (Mi) 21-200 mg / g creatinine (36 Example, microalbuminuria), (Ma) 201 mg / g creatinine or higher (4 cases, macroalbuminuria). About each group, the fluorescence intensity (AU / g creatinine) measured by said (2) was compared. The results are shown in FIG.

The mean value ± standard deviation of the fluorescence intensity (AU / g creatinine) was 170 ± 144 for (N), 229 ± 120 for (Mi), and 361 ± 223 for (Ma). And the significant difference was recognized between (N) and (Mi) and between (N) and (Ma) (P <0.05).
Thus, in the method of the present invention, the higher the urinary albumin, that is, the higher the severity of diabetes, the higher the fluorescence intensity. According to the method of the present invention, it was possible to detect diabetes depending on the severity.

  In the method described in Non-Patent Document 4, urine is subjected to ultrafiltration as a pretreatment, and albumin is removed in advance. And no significant difference is recognized among three groups of N group, Mi group, and Ma group (for example, Fig. 2B, Fig. 3). In this method, there is a possibility that a severely diabetic patient whose urinary albumin is detected may be missed, and it is considered difficult to detect diabetes according to the severity.

Claims (4)

A method for detecting diabetes using urine collected from a subject as a specimen,
The specimen is urine or urine-derived material that has not undergone purification treatment,
A method for detecting diabetes, comprising detecting the presence or absence of diabetes in the subject using as an index the intensity of fluorescence emitted by a fluorescent substance contained in the specimen at an excitation wavelength of 250 to 500 nm.   The method for detecting diabetes according to claim 1, wherein the fluorescence intensity is the fluorescence intensity at a wavelength 10 to 250 nm larger than the excitation wavelength.   The method for detecting diabetes according to claim 1 or 2, wherein the specimen is urine itself or a diluted urine.   The method for detecting diabetes according to any one of claims 1 to 3, wherein the fluorescence intensity is measured, and the obtained measurement value is compared with a reference value.

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