KR102529421B1 - Composition for predicting lactate level in blood - Google Patents

Abstract

The present invention relates to a composition or a kit for predicting blood lactate level comprising a urine-derived CD63 or CD81 detecting substance. The composition or kit for predicting the blood lactate level including the urine-derived CD63 or CD81 detecting substance of the present invention can predict the blood lactate level in a non-invasive manner and provide information related to fatigue caused by exercise.

Description

Composition for predicting lactate level in blood {Composition for predicting lactate level in blood}

The present invention relates to a composition and kit for predicting the level of lactic acid in blood by checking the level of extracellular vesicle markers in urine.

Exosomes are substances in the form of vesicles secreted by cells themselves. Inside, there are lipid, protein, RNA (mRNA, siRNA), DNA, etc. After secretion, it reaches the cells of the target organ through the blood and participates in cell to cell signaling. In addition, due to the nature of its production, it has the information of the secreted cells, so it has recently been proposed to be used as a biomarker for disease diagnosis.

Until now, the most common way to measure fatigue after exercise is to check blood lactate. However, the disadvantage is that blood must be collected from a local area. Exosomes can be identified in various secretions such as blood, tears, breast milk, saliva, and urine. Due to these characteristics, urine-derived exosomes have the advantage of being able to measure changes in the body in a non-invasive manner.

The present inventors confirmed that extracellular vesicles (exosomes) expressing urine-derived exosome markers CD63 and CD81 showed a high quantitative correlation with lactate levels before and after exercise. This confirmed the possibility as a new biomarker that can represent lactic acid in the blood, which is cumbersome to measure.

Korean Patent Publication 10-2019-0088783

The present invention relates to a composition for predicting blood lactate level comprising a substance detecting CD63 or CD81 in urine and a method for predicting blood lactate level, and an object of the present invention is to provide information related to fatigue by non-invasively predicting blood lactate level.

1. A composition for estimating blood lactic acid level comprising a substance for detecting CD63 or CD81 in urine.

2. The composition for predicting blood lactate level according to 1 above, wherein the substance for detecting CD63 or CD81 is an antibody.

3. A kit for predicting blood lactic acid level comprising the composition of any one of 1 to 2 above.

4. A blood lactate level prediction method comprising the step of predicting that the blood lactate level is likely to be high compared to the control group if the level of CD63 or CD81 in urine is higher than that of the control group.

5. A composition for predicting fatigue due to exercise containing a substance for detecting CD63 or CD81 in urine.

The composition or kit for predicting blood lactate level of the present invention measures the level of CD63 or CD81 in urine in a non-invasive manner, and predicts the blood lactate level based on the non-invasive method, thereby providing information related to fatigue caused by exercise. can

Figure 1 schematically shows an experimental plan for measuring urine collection and blood lactic acid before, immediately after, and 1 hour after exercise according to an embodiment of the present invention. (BL: blood lactate; BP: blood pressure; HR; heart rate; REP: assessed perceptual motion)
Figure 2 confirms the relative blood lactate levels and urine-derived concentrations of exosome markers (CD81, CD63, and CD9) before, immediately after, and 1 hour after exercise, and normalized to values before exercise.
Figure 3 shows the correlation between the amount of change in lactate value and the amount of change in exosome marker expression before and after exercise.
Figure 4 shows the correlation between blood lactate and exosome markers (CD81, CD63 and CD9) before and after exercise.
Figure 5 shows the correlation between exosomal markers (CD81, CD63 and CD9) and other factors (RPE, HR, and lean body mass). (RPE: Perceived Exercise; HR: Heart Rate)

The present invention provides a composition for estimating blood lactic acid level comprising a substance for detecting CD63 or CD81 in urine.

CD63 and CD81 are cell surface proteins belonging to the transmembrane 4 superfamily known as the tetraspanin family and are known to form complexes with integrins. In addition, CD63 and CD81 are proteins that are specifically expressed in membranes of exosomes and can be used as exosome markers. The exosome (extracellular vesicle) is a material in the form of a vesicle secreted by cells and contains information on lipids, proteins or nucleic acids, i.e., secreting cells.

The type of material for detecting CD63 or CD81 is not limited as long as it is a material for specifically detecting each protein. For example, it may be an aptamer or an antibody.

The level of lactic acid in the blood means the amount of fatigue substances accumulated in the body during exercise in the blood. When intense exercise for a long time produces a lot of lactic acid and accumulates in the body, causing physical fatigue, which is a factor in fatigue relevance factors. applicable

The present inventors have confirmed that the level of CD63 or CD81 in urine has a high positive correlation with the level of lactate in the blood, and that the increase or decrease in the level of CD63 or CD81 in the urine is positively correlated with the increase or decrease in the level of lactate in the blood. Therefore, by checking the CD63 or CD81 level in urine with the composition of the present invention, it is possible to determine whether the blood lactic acid level is high or low according to the high or low level.

In addition, as described above, the blood lactic acid level is a fatigue-related factor, and when the blood lactic acid level increases, fatigue due to exercise increases. Since the composition of the present invention can predict the blood lactic acid level, it can be used for predicting fatigue due to exercise. can

In addition, the present invention provides a kit for predicting blood lactic acid level comprising the composition for predicting blood lactic acid level.

In addition to the material for detecting the CD63 or CD81 protein described above, the kit may further include a composition, solution or device that is one or more other components suitable for the assay method for determining protein levels that the kit utilizes.

The kit includes an antibody that can specifically bind to the protein to be measured, a secondary antibody conjugate conjugated with a label that develops color by reaction with the substrate, a color-developing substrate solution that will react with the label, a washing solution, and an enzyme reaction. It may contain a stop solution and the like, and may be manufactured in a number of separate packaging or compartments containing reagent components to be used, but is not limited thereto. As the substrate, a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, and glass slide glass may be used, and the color-developing enzyme may be peroxidase, alkaline phosphatase ( alkaline phosphatase), etc. can be used, FITC, RITC, etc. can be used as a fluorescent substance, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or o -Phenylenediamine (OPD), tetramethyl benzidine (TMB), etc. may be used.

The kit may include essential components required to perform ELISA in order to implement various ELISA methods such as sandwich ELISA.

The type of kit may be, for example, an immunochromatography strip kit, a Luminex assay kit, a protein microarray kit, an ELISA kit, or an immunological dot kit.

The kit may be a kit for implementing Western blot, immunoprecipitation assay, complement fixation assay, flow cytometry, or protein chip, and may further include additional components suitable for each assay method.

In addition, the present invention provides a method for predicting blood lactate level.

The blood lactate level prediction method of the present invention includes predicting that the blood lactate level is higher than that of the control group when the level of CD63 or CD81 in urine is higher than that of the control group.

The control group may be the level of CD63 or CD81 before exercise, that is, the basal level of the protein. In addition, since there may be differences in the basal level of lactate in the blood and the level that increases with increasing fatigue, the control group may be the CD63 or CD81 level before exercise in the subject to be measured. Alternatively, the control group may be the level of CD63 or CD81 in urine in any condition of the subject to be measured, and in this case, the difference between the condition at the time of measurement and the level of lactate in the blood in any condition can be predicted.

As described above, the level of CD63 or CD81 shows a high positive correlation with the level of lactate in the blood, and if the level of CD63 or CD81 in the urine is higher than that of the control group, it can be predicted that the level of lactate in the blood is higher than that of the control group. If it is low, it can be predicted that the blood lactate level is low.

In addition, as described above, when the blood lactate level increases, fatigue due to exercise increases, and since the method of the present invention can predict the blood lactate level, it can also be used to predict fatigue due to exercise.

The present invention provides a composition for predicting fatigue due to exercise comprising a substance for detecting CD63 or CD81 in urine.

CD63, CD81 and substances detecting them are as described above.

Fatigue due to exercise means the degree to which the body feels fatigue due to energy depletion and accumulation of lactic acid after exercise. In this case, fatigue is judged to be high. In addition, the higher the intensity of exercise, the higher the degree of fatigue. The intensity of exercise can be expressed in terms of Perceived Exercise (RPE), percentage of maximum heart rate, and maximum oxygen intake.

The Rating of Perceived Exertion (RPE) is a highly reliable and practical indicator of exercise intensity representing subjective feelings when exercising on a numeric scale from 6 to 20.

Hereinafter, the present invention will be described in detail by examples. The following examples are intended to illustrate the present invention, but the content of the present invention is not limited to the following examples.

Example

1. Experiment method

All experimental procedures were approved by the Institutional Review Board of Seoul National University (IRB No. 2009/003-028) in accordance with the Helsinki Declaration of World Society standards. All subjects were informed orally and in writing about the procedure and purpose of the study, and their participation was confirmed.

(1) Subject

Nine healthy males aged 24.4±1.3 years old were recruited from Seoul National University in response to an advertisement, and males with a history of cardiovascular disease, musculoskeletal disease, or psychological disorder were excluded. Subject characteristics are shown in Table 1. All participants were encouraged to fast for at least 9 hours prior to the test, were also informed of the study procedure and the risks of the 20 m shuttle run test, and written informed consent was provided to the participant prior to the test.

(2) Urine sample collection and preparation

Participants (n=9) were asked to collect urine in a sterile container (VWR) immediately after waking up (first), after the 20 m shuttle run test (second), and 1 hour (third) later. After collection, samples were filtered once through Minisart® (Sartorius stedim) and then frozen at -80 °C for further analysis.

(3) Quantification of urine-derived exosomes

Physical and biological properties of exosomes in urine samples were assessed using Exoview R-100 and CD81, CD63 and CD9 antibody-immobilized chips, labeling agents, washing solutions (Solution A and B) and blocking agents (Nanoview Bioscience). Characterized using the Exoview tetraspanin kit. First, 35 ul of the sample was dropped on the Exoview tetraspanin chip and incubated overnight (16 hours) at room temperature. After the incubation process, the sample-loaded chip was repeatedly washed three times with 1 ml of Solution A. Subsequently, in order to analyze (colocalization of) tetraspanin on the surface of the exosome, 250 ul of a mixture of CD81 antibody (Alexa Fluor 555), CD63 antibody (Alexa Fluor 647) and CD9 antibody (Alexa Fluor 488) was used to label the exosomes on the chip and incubated for 1 hour. In this case, the labeled antibody (Ab) was diluted in a mixture of solution A and blocking solution (1:600). Finally, the chip was rinsed with 1 ml of A and B solutions and dried at room temperature. The exosome-captured chips were scanned with Exoview R-100.

(4) Blood lactate measurement

Using a disposable lancet (Accu-chek softclix plus lancing device & softclix, Roche®), 5 μl of blood from the fingertip was absorbed onto an enzyme-coated electrode test strip (Arkray, Japan). , immediately after, and one hour later, checked with a Lactate Pro2® (LT-1730, Arkray) device All equipment was cleaned and operated according to the manufacturer's instructions Blood samples were analyzed within 30 seconds of blood collection.

(5) 20m shuttle driving test

Participants participated in continuing to run between two cones 20 m apart in response to a recorded beep. Before conducting the test, participants confirmed that a beep sound could be clearly heard at the end of the line. After warming up for 10 minutes, the subject started running back and forth at an initial speed of 8 km/h. The speed was gradually increased according to the test grade, and the total number of shuttles was checked when the participant could not respond to the warning sound more than 2 times. The test was conducted between 09:00 am and 11:00 am at Seoul National University Gymnasium.

2. Experiment method

(1) Participant characteristics

The anthropometric characteristics (age, height and weight) and body composition (body fat mass and lean mass) of the subjects (n=9) who participated in the shuttle driving test are shown in Table 1. As a result of the shuttle driving test, that is, the total number of shuttles at the time when the test ended because it could not respond to the warning sound more than twice was 81.3±16.3.

variable (n=9) anthropometric characteristics age (years) 24.4±1.3 height (cm) 179.0±5.7 Weight (kg) 73.6±5.7 body composition Fat mass (%) 12.6±4.2 Lean mass (kg) 36..8±3.2 20m Shuttle Running Result repeat 81.3±16.3

(2) Relative blood lactate levels and relative concentrations of urine-derived exosome markers before, immediately after, and 1 hour after exercise

Blood lactate levels increased immediately after exercise compared to before exercise and returned to pre-exercise levels 1 h after exercise (Figure 2A). In urine immediately after exercise, the exosome marker CD81 increased immediately after exercise and returned to pre-exercise levels 1 h after exercise (Figure 2B). In the urine immediately after exercise, the exosome marker CD63 increased immediately after exercise and returned to the pre-exercise level 1 hour after exercise (Fig. 2C). In the urine immediately after exercise, the exosome marker CD9 increased immediately after exercise and returned to the pre-exercise level 1 hour after exercise (Fig. 2D).

(3) Urine before exercise, right after exercise, and 1 hour after exercise

In urine samples, it was confirmed that the exosome marker CD9 and lactate concentration did not show a significant correlation, whereas the exosome markers CD81 and CD63 had a high quantitative correlation with lactate concentration, respectively. Similarly, there was no significant correlation between the change in blood lactate and the change in the amount of exosome marker CD9 (p>0.05), but the change in blood lactate (red dot on x-axis: change in lactate value before and after exercise; black dot: exercise The greater the change in lactate value immediately after exercise and 1 hour after exercise), the greater the change in exosome markers CD63 (p<0.01) and CD81 (p<0.05) in urine (red dots on y-axis: change in exosome marker expression before and after exercise) ; Black dots: change in exosome marker expression right after exercise and 1 hour after exercise) was confirmed to increase significantly (FIG. 3). As a result of analyzing the correlation between lactate in blood before and after exercise and exosome markers in urine, the expression levels of CD63 and CD81 had individual diversity at the basal level, but the exosome marker CD63 and exosome markers in urine The amount of CD81 expression has a significant quantitative correlation with the amount of lactate in blood (FIG. 4).

(4) Correlation between urine-derived exosome markers, perceived exercise (RPE), and heart rate (HR) before, immediately after, and 1 hour after exercise

In the urine sample, it was confirmed that the exosome markers CD81, CD63, and CD9 had a significant quantitative correlation with the perceived exercise degree (RPE), respectively, and a trend of quantitative correlation with heart rate (HR) was confirmed (FIG. 5) .

Claims (5)

A composition for predicting blood lactic acid level comprising a substance for detecting CD63 or CD81 in urine.
The composition for predicting blood lactate level according to claim 1, wherein the material for detecting CD63 or CD81 is an antibody.
A kit for predicting blood lactic acid level comprising the composition of any one of claims 1 to 2.
A method for predicting blood lactate level comprising predicting that the blood lactate level is likely to be high compared to the control group when the level of CD63 or CD81 in urine is higher than that of the control group.
A composition for predicting fatigue due to exercise comprising a substance for detecting CD63 or CD81 in urine.

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