Stabilization method of soluble α-klotho in urine

Abstract

To provide a method for stabilizing soluble α-klotho in urine. [Solution] A method for stabilizing urinary soluble α-klotho, characterized by adding a stabilizer to urine.

Description

Technical Field

[0001] The present invention relates to a method for stabilizing soluble α-klotho in urine.

Background Art

[0002] The α-klotho gene is an aging-related gene identified by research on mutant mice with accelerated aging. It has been reported that mice lacking the α-klotho gene show accelerated aging (Non-Patent Document 1), and mice overexpressing the α-klotho gene have an extended average lifespan (Non-Patent Document 2). The α-klotho protein encoded by the α-klotho gene is a single-pass transmembrane protein and functions as a receptor for the phosphaturic hormone fibroblast growth factor-23 (FGF23).

[0003] The α-klotho protein generates soluble α-klotho (short form) by N-terminal cleavage or alternative splicing. Soluble α-klotho is detected in urine, blood, etc. and is expected to be a marker for aging. Blood collection requires pricking the body with a needle, which is painful and also requires a medical professional to perform. On the other hand, urine collection can be easily done by an individual without pain. Therefore, in order to easily confirm the degree of aging progression, it is desirable to use soluble α-klotho in urine as a marker. However, as shown in Non-Patent Document 3, soluble α-klotho in urine is unstable, and after storage at 37°C for 3 hours, its concentration decreases to 20% or less compared to immediately after urine collection.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

[0005] The objective is to provide a method for stabilizing soluble α-klotho in urine. [Means for solving the problem]

[0006] The means for solving the problems of the present invention are as follows: 1. A method for stabilizing urinary soluble α-klotho, characterized by adding a stabilizer to urine. 2. The stabilization method according to 1, characterized in that the stabilizer is one or more selected from the group consisting of DTT, EDTA, thioglycerol (3-mercapto-1,2-propanediol), and cysteine. 3. A urine testing method characterized by detecting the concentration of soluble α-klotho in urine using a urine sample to which a stabilizer has been added. 4. A urine collection kit comprising a urine collection container and a stabilizer, characterized in that it is for measuring the concentration of soluble α-klotho in urine. [Effects of the Invention]

[0007] The stabilization method of the present invention can suppress the decrease in urinary soluble α-klotho concentration, thereby enabling more accurate quantification of urinary soluble α-klotho concentration. The stabilization method of the present invention can suppress the decrease in soluble α-klotho concentration in urine, making it possible to mail urine collected at home or elsewhere to a testing laboratory. Since there is no need to go to a testing laboratory or medical institution to collect urine, the burden on the test subject can be reduced. [Brief explanation of the drawing]

[0008] [Figure 1] A graph showing the results of the soluble α-klotho retention rate in Experiment 1. [Figure 2] A graph showing the results of the soluble α-klotho retention rate in Experiment 3. [Modes for carrying out the invention]

[0009] ·Stabilization method The present invention provides a method for stabilizing urinary soluble α-klotho by adding a stabilizer to urine. The stabilizers are not particularly limited as long as they improve the stability of urinary soluble α-klotho. Examples include DTT (dithiothreitol), EDTA, thioglycerol (3-mercapto-1,2-propanediol), cysteine, histidine, BSA (bovine serum albumin), PEG400, and sucrose. One or more of these can be used in combination. Among these, one or more selected from the group consisting of DTT, EDTA, thioglycerol, and cysteine ​​are preferred because they provide excellent stability for urinary soluble α-klotho. The concentration of the stabilizer can be adjusted within the range that provides its effect; for example, in the case of DTT and EDTA, it is approximately between 0.05% (w / v) and 1.0% (w / v).

[0010] The stabilization method of the present invention preferably results in a residual rate of soluble α-klotho concentration in urine after storage at 20°C for one week (concentration after storage / concentration immediately after urine collection × 100) of 30% or more. More preferably, this residual rate is 40% or more, even more preferably 50% or more, even more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, and even more preferably 95% or more.

[0011] • Urine test method The present invention provides a urine testing method that detects the concentration of soluble α-klotho in urine using a urine sample to which a stabilizer has been added. The method for detecting soluble α-klotho concentration in urine is not particularly limited, and commercially available kits such as "Human soluble α-Klotho Assay Kit-IBL (manufactured by Immunobiological Laboratories Co., Ltd.)" can be used. The urine testing method of the present invention uses a urine sample to which a stabilizer has been added, thereby suppressing the decrease in soluble α-klotho concentration in the urine over time. Since the urine testing method of the present invention does not require testing immediately after urine collection, urine collected at home or elsewhere can be mailed to a hospital or testing facility for testing.

[0012] Urine collection kit The stabilization method and urine testing method of the present invention preferably use a urine collection kit having at least a urine collection container and a stabilizer. More preferably, the urine collection kit has the stabilizer sealed inside the urine collection container so that the stabilizer can be added to the urine sample at an appropriate concentration.

[0013] The stabilizer is not particularly limited as long as it improves the stability of soluble α-klotho in urine. For example, DTT (dithiothreitol), EDTA, thioglycerol (3-mercapto-1,2-propanediol), cysteine, histidine, BSA (bovine serum albumin), PEG400, sucrose, etc. can be mentioned, and one or more of them can be mixed and used. Among these, one or more selected from the group consisting of DTT, EDTA, thioglycerol, and cysteine are preferable because they are excellent in the stability of soluble α-klotho in urine. The concentration of the stabilizer can be adjusted within the range where the effect is exhibited. For example, it is about 0.05% (w / v) or more and 1.0% (w / v) or less with respect to a predetermined amount of urine volume in the urine collection container. More preferably, it is 0.1% (w / v) or more and 0.5% (w / v) or less.

[0014] The urine collection container is not particularly limited as long as it has a shape and material that do not cause alteration of urine components used for urine tests conducted in hospitals, inspection institutions, etc., and can be used. The amount of urine that can be collected in the urine collection container is not particularly limited as long as it is an amount capable of preparing a urine specimen. For example, it is 10 ml or less. The urine collection container may be in a form that can directly collect urine, or in a form that transfers the urine collected in a urine collection cup or the like. Further, it is preferable that the urine collection container is provided with graduations so that a predetermined amount of urine can be collected. Furthermore, it is preferable that the urine collection container can be sealed so that the collected urine container can be mailed. In addition to the urine collection container and the stabilizer, the urine collection kit can include an outer box, an instruction manual describing inspection procedures and precautions, a urine collection cup, a vinyl bag with a clip, a urine test request form for recording information such as the name, date of birth, and gender of the subject, a return envelope, etc.

[0015] The urine test method of the present invention may be either therapeutic or non-therapeutic. Therapeutic means to be used in combination with treatment, examination, etc. at a medical institution, and non-therapeutic means a concept that does not include medical acts, that is, a concept that does not include methods of operating on, treating, or diagnosing a human. More specifically, it is a concept that does not include methods of operating on, treating, or diagnosing a human by a doctor or a person under the doctor's instruction.

[0016] In the present invention, the timing of urine collection is not particularly limited, but it is preferable to collect urine before taking the first meal of the day, and the first urine collected immediately after waking up (early morning) is particularly preferable. Since the concentration of urinary components is easily affected by diet, water intake, sweating, etc. and varies greatly depending on the urine volume at that time, creatinine correction to obtain the ratio with the simultaneously measured creatinine value can also be performed.

Examples

[0017] As an ethical consideration, this test was reviewed by an ethics review committee regarding the appropriateness of conducting the test, and when conducting this clinical trial, the purpose of the test was fully explained to the subjects in advance, and written consent to participate was obtained based on their free will.

[0018] (Measurement of absorbance of soluble α-klotho) Soluble α-klotho was measured using Human soluble α-Klotho Assay Kit-IBL (manufactured by Immunological Biomarkers Laboratory Co., Ltd.). Recombinant human soluble α-Klotho (12000 pg / mL) was used as a standard product (6000 pg / mL to 93.75 pg / mL) in a 96-well ELISA antibody plate, and 100 μL / well of stabilized human urine (dilution ratio 2 times) diluted with a diluent was added and incubated at room temperature for 60 minutes. After washing 5 times with a washing solution, 100 μL / well of a labeled antibody solution (dilution ratio 30 times) diluted with a dissolution solution for the labeled antibody was added and incubated at room temperature for 3 hours. After washing 4 times with a washing solution, 100 μL / well of TMB substrate solution was added. After 30 minutes, 100 μL / well of a stop solution was added, and the absorbance at 450 nm was measured.

[0019] "Experiment 1" SDS was prepared at 4% (w / v) in demineralized water. Arginine was prepared at a concentration of 10% (w / v) using desalted water. Sodium bicarbonate was prepared at a concentration of 10% (w / v) using desalinated water. Sucrose was prepared at a concentration of 10% (w / v) using desalinated water. PEG400 was prepared at 10% (w / v) in desalinated water. BSA was prepared at 10% (w / v) in desalinated water. EDTA was prepared at a concentration of 2% (w / v) using desalinated water. DTT was prepared at a concentration of 2% (w / v) using desalinated water.

[0020] A 10% solution of the stabilizer prepared above was added to the first morning urine sample collected from one subject on the day of the study. In addition, histidine was directly added to this first morning urine sample to a total concentration of 1% (w / v) and ascorbic acid to a total concentration of 10% (w / v).

[0021] The absorbance of soluble α-klotho was measured immediately after urine collection and after storage at 20°C for one week with the addition of a stabilizer. The remaining percentage of soluble α-klotho was calculated from the ratio to the absorbance immediately after urine collection. In addition, the remaining percentage of soluble α-klotho was calculated for urine samples without stabilizer as a negative control. The results are shown in Figure 1.

[0022] DTT, EDTA, histidine, BSA, PEG400, and sucrose all showed retention rates of 35% or more, with DTT having a retention rate of approximately 100% and EDTA a retention rate of 75%.

[0023] "Experiment 2" For DTT and EDTA, which showed high retention rates in Experiment 1, 1% (w / v), 2% (w / v), and 5% (w / v) solutions were prepared using desalinated water. To the first morning urine sample collected from one subject on the same day, 10% of the stabilizer solution prepared above was added, and the residual rate of soluble α-klotho was calculated in the same manner as in Experiment 1. DTT showed a survival rate of approximately 100% in all cases, while EDTA showed a survival rate of over 78% in all cases.

[0024] "Experiment 3" DTT and EDTA, which showed high retention rates in Experiments 1 and 2, contain thiol groups, while the other agents do not. Therefore, the stabilizing ability of compounds containing thiol groups was investigated. DTT was prepared at a concentration of 1% (w / v) using desalinated water. Glutathione was prepared in desalted water at concentrations of 1% (w / v) and 5% (w / v). Cysteine ​​was prepared at 1% (w / v) and 5% (w / v) concentrations using desalinated water. Thioglycerol was prepared in 1% (w / v) and 5% (w / v) concentrations using desalted water.

[0025] To a first-morning urine sample collected from one subject on the same day, 10% of the stabilizer solution prepared above was added. The residual rate of soluble α-klotho after storage at 20°C for one week and two weeks was calculated in the same manner as in Experiment 1. The results are shown in Figure 2.

[0026] Glutathione has a thiol group, but its persistence was poor. Cysteine ​​and thioglycerol exhibited excellent stability, and in particular, cysteine ​​0.1%, cysteine ​​0.5%, and thioglycerol 0.5% showed a retention rate of over 50% even after two weeks.

Claims

1. A method for stabilizing urinary soluble α-klotho, characterized by adding a stabilizer to urine.

2. The stabilization method according to claim 1, characterized in that the stabilizer is one or more selected from the group consisting of DTT, EDTA, thioglycerol (3-mercapto-1,2-propanediol), and cysteine.

3. A urine testing method characterized by detecting the concentration of soluble α-kloth in urine using a urine sample to which a stabilizer has been added.

4. A urine collection kit comprising a urine collection container and a stabilizer, characterized in that it is for measuring the concentration of soluble α-klotho in urine.

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