JPH09318626A - Method and device for measuring lactic acid in urine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and a device for measuring lactic acid concentration in urine by using an optical means without any pre-treatment. SOLUTION: For lactic acid in urine, a wavelength having a 0.5 or over, preferably 0.9 or over of a correlation between concentration of lactic acid water solution and absorbance thereof in a visible or near infrared wavelength region is selected as the measured wavelength intrinsic of a lactic acid component. A light visible or within the range of a near infrared wavelength is emitted from a light source device 11, divided into specified wavelengths by a spectral device 12 controlled by a computer 16, a urine test piece in a urine sample setting part is irradiated with this light and a transmission light and a reflection light from the urine test piece are received by a light receiving element set in a detecting device 14 and a light receiving signal is outputted. The outputted light receiving signal is fetched through a signal processing interface 15 into the computer 16, subjected to numerical value calculation and then lactic acid concentration is measured.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention provides a non-invasive measuring method and device by urine measurement as means for obtaining a physical fatigue index and a health index using lactic acid as a parameter.

[0002]

BACKGROUND ART Lactic acid is mainly produced in skeletal muscle, erythrocyte, brain, skin and intestine as an end metabolite of anaerobic glycolysis (1200-1500 mmol / day), and most of it is TCA cycle in liver and kidney. It is also used as a substrate for gluconeogenesis. Pathological conditions that cause an imbalance between lactate production and utilization are reflected as abnormal blood lactate levels. In addition, it is known that lactic acid accumulates due to glycolysis in the body, and the amount of lactic acid accumulated and the degree of fatigue are highly correlated, so in the fields of sports medicine and exercise physiology, as a management index for training,
The concentration of lactic acid in blood carried from the muscle to the blood is measured.

Lactic acid metabolism in diabetes generally shows an increasing tendency. In addition, the more severe the dehydration, acidosis, and hyperglycemia, the higher the lactic acid level.

As a test for lactic acid, measurement of lactic acid in blood has been clinically performed conventionally. An enzymatic method using lactate dehydrogenase is generally used as a method for measuring blood components, and a method for measuring urinary components using an enzyme has been proposed as a non-invasive method for measuring lactate concentration. (JP-A-5-3798). Also, in a non-invasive blood lactate measurement method, enzymes such as lactate oxidase and lactate dehydrogenase are used as reagents.

Methods for measuring urinary components include a reagent method, a test strip method, a chemiluminescence method, an immunoassay method, an enzyme method and a chromatography method.

As a method for optically measuring the components in urine, a urine sample is irradiated with light of a specific wavelength and the absorbance is measured.
A method for quantifying the concentration of urinary components by a multivariate regression analysis method has been devised (Japanese Patent Application No. 7-70849).

[0007]

[Problems of the prior art] In these conventional methods, the pretreatment for sample preparation and the measurement operation itself are very complicated and the measurement time is long, and they are easily affected by contaminants.
Further, there is a problem that an expensive light source and a spectroscopic device are required, the size of the entire device is increased, and the price is expensive.

The test paper used in the test paper method is often one in which the reaction portion of cellulose containing a reactive reagent is fixed to a plastic indicator piece with an adhesive or the like and dried. If the reaction part contains moisture, a reaction will occur between the reagents, and it will also deteriorate due to high temperature and light, and the sensitivity will often decrease.Therefore, the test paper container should be sealed and stored at high temperature. , It is necessary to use within the expiration date. The test strip method can measure items such as pH, protein, glucose, ketone bodies, bilbilin, occult blood, unobilinogen, nitrite, and bacterial infection within 1 minute, but the reaction of the reagent part is In addition to the inhibitory substances, it is affected by the reaction temperature and the conditions of the test strip, etc.

The enzymatic method is used for measuring sugar. For example,
Glucose oxidase / peroxidase (GOD-
(POD) dye system, a method of measuring the color development associated with redox reaction by a reflection system, or a method of amperometrically measuring anodizing current with a GOD-immobilized enzyme electrode and converting the current value to a concentration (biosensor, etc.) ) Is done. The method using glucose oxidase is highly specific to glucose and is simple, but the GOD reaction is a redox reaction, and the reaction is suppressed by various endogenous and exogenous oxidation and reduction substances. There is also a risk of false negatives and false positives.

In the chromatography method, it is difficult to say that the cost performance is excellent because an expensive apparatus such as a gas chromatogram or a liquid chromatogram is used.

The measurement of lactic acid concentration is generally performed on blood. However, when lactic acid is used as a diabetes marker, the subject will repeatedly perform sample extraction over a long period of time, and blood is extracted as a sample. Subject will be physically distressed.

[0012]

The reagent method, test strip method, and enzyme method require reagents, test strips, or enzymes that are consumables, and the storage stability of the reagent before use and the disposal after use. There is also the problem of. The blood-based measurement method requires blood collection, which is associated with pain and risk of infection during blood collection. Furthermore, the operation is complicated, and errors such as the amounts of reagents and samples added during operation may occur, and there is also the drawback that interference with other components such as ascorbic acid, which is not intended for measurement, causes interference. Although the reagent method and the enzyme method can perform quantification, the multicomponents cannot be measured at one time, and the test strip method allows simultaneous measurement of multiple components, but has a disadvantage that only semiquantification is possible.

The chromatography method requires an expensive apparatus, and when the column performance is deteriorated, the column must be replaced, which causes a problem of high cost.

In view of solving the above problems in the conventional method,
The present invention eliminates the need for consumables such as reagents, test strips, and enzymes, and further, the storage stability of these consumables before use, the problem of disposal after use, the error-prone operation and other components It is an object of the present invention to provide a method for quantitatively measuring multiple components at the same time by eliminating problems such as interference action.

By subjecting urine to the measurement of lactic acid concentration, the subject does not have any pain at the time of extracting the sample, so that the mental distress of patients with low tolerance to physical pain such as young children and the elderly can be relieved. It

[0016]

Means for Solving the Problems In measuring the concentration of lactic acid contained in urine according to the present invention, the absolute value of the correlation coefficient between the concentration of the aqueous lactic acid solution in the visible or near infrared region and the absorbance is determined. At least one wavelength included in the wavelength region of 0.5 or more, preferably 0.9 or more is selected as a measurement wavelength unique to the lactic acid component, and a urine sample is irradiated with visible light or near infrared light, The lactic acid concentration measuring method is characterized by measuring the absorbance at the measurement wavelength selected in 1. and quantifying the lactic acid concentration in urine.

Further, the present invention provides a lactate concentration measuring device suitable for realizing the above method. That is, the present invention
A light emitting means for irradiating a urine sample with light having a specific wave number in the visible or near-infrared region, and a light receiving signal for receiving the light transmitted through the sample and outputting a light receiving signal corresponding to the absorbance of a component contained in the sample A lactate concentration measuring apparatus comprising: a light receiving unit; and an arithmetic processing unit for calculating an absorbance from a light receiving signal from the light receiving unit to quantify the lactate concentration.
The measuring apparatus is characterized by including arithmetic processing means including a multivariate regression analysis method.

Absorbance A at wavelength λj for a component
The correlation Rj between and of the concentration is given by the following equation.

[Equation 1] In the above equation, A _ij is the absorbance of the component at the wavelength λ _j in the i-th sample, and C _i is the concentration of the component in the i-th sample.

As a wavelength to be measured, a wavelength range having a strong absorption for water is avoided, and a high transmittance for water is 250.
00-5280 cm ^-1, or 4980-4000 cm
^-1 wavelength range. Especially, the preferable measurement wavelength for lactic acid is 6300 to 5 in terms of wave number.
Selected from 400 cm ^-1 and 4800~4200cm ^-1.

[0020]

Light is irradiated to the action sample when measuring its absorbance, transmitted light intensity at wavelength j _{I tj} has LAMBERT-B
It is expressed by the following equation according to EER's law. I _tj = I _oj exp (-Σα _kj C _k L) ... = I _oj T _j (1) where I _tj is the transmitted light intensity at the wavelength j and I _oj is the incident light intensity α _kj at the wavelength j. The extinction coefficient C _k of the k component at the wavelength j is the concentration of the k component in the solution k = 1, 2, ... K, where K is the number of the components in the solution T _j is the transmittance at the wavelength j L is the cell length Is. The absorbance A j at the wavelength j is represented by A _j = −log T _j = −log (I _tj / I _oj ) = LΣ (α _kj C _k ) (2), ignoring the reflection at the interface between the cell and the solution. .

From equation (2), the unknowns are C _k (k = 1,
2, ..., K), it is possible to calculate the concentration of each component by measuring the absorbance with K independent variables and solving the simultaneous equations. If the data analysis is performed using a multivariate regression analysis method such as a principal component regression analysis method (PCR method) or a partial least squares method (PLS method), the concentration can be obtained with higher accuracy.

In the multivariate regression analysis method, since regression analysis can be performed using a large amount of absorbance information at one time, quantitative analysis can be performed with higher accuracy than single regression analysis. The multiple regression analysis is most frequently used, but a large number of samples are required, and when the correlation between the absorbances at each wavelength is high, the accuracy of the quantitative analysis becomes very low. On the other hand, the principal component regression analysis method, which is a multivariate regression analysis method, can aggregate the multi-wavelength absorbance information into principal components that are uncorrelated with each other, and can also eliminate unnecessary noise data, thus achieving high quantification. Analytical accuracy can be obtained. Further, since the partial least squares method can also use the data of the sample concentration when extracting the main component, it is possible to obtain a high quantitative analysis accuracy as in the principal component regression analysis method.

[0023]

DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 shows the outline of a measuring device used in the present invention. The light source device 11 generates light in the visible and near-infrared wavelength regions, and the spectroscopic device 1 controlled by the computer 16
2, light having a specific wavelength is separated. The dispersed light is
The urine sample is guided to the urine sample installation unit 13 in which the cell enclosing the urine sample is installed. By detecting the light transmitted or diffusely reflected from the urine sample by the detection element in the detection device 14, the absorbance of the component in the urine sample with respect to the specific wavelength can be measured. The output of the detection element is converted into a digital value by the signal processing interface 15 and stored in the storage medium of the computer 16, and a calibration curve of the absorbance and the lactate concentration is obtained by the numerical calculation processing.

In order to measure the lactic acid concentration with the device shown in FIG. 1, first, the spectral wavelength of the spectroscopic device 12 is changed from j = 1 to n while leaving the cell in the urine sample setting part 13 empty, and the transmission at that time is changed. The light quantity I _oj (j = 1, 2, ..., N) is measured.
Next, a urine sample is put in the cell, the spectral wavelength of the spectroscopic device 12 is similarly changed from j = 1 to n, and the transmitted light intensity I _tj (j = 1, 2, ..., At each wavelength that has passed through the cell). n) is measured. Data analysis is performed based on these I _oj and It _j to obtain each component concentration C _k (k = 1, 2, ... K).

FIG. 2 shows an absorption spectrum in the near infrared region obtained by putting an aqueous lactic acid solution into a cell. Sample has a concentration of 1
Four kinds of aqueous solution samples of 00, 200, 300 and 400 mM. Bands where the absorbance changes in proportion to the concentration are found everywhere, which characterizes the absorption spectrum of lactic acid. If these bands are represented by wave numbers, 6300-
It is 5400 cm ^-1 , 4800-4200 cm ^-1 .

The calibration curve obtained by plotting the absorbance at a characteristic absorption wavelength of 4361.5 cm ^-1 against the lactic acid concentration is shown in FIG. The linearity of the calibration curve in FIG. 2 is 0.999967 when expressed by the correlation coefficient R,
It shows that it is excellent as a calibration curve.

FIG. 4 shows the correlation coefficient R ² between the absorbance and the concentration calculated for each wave number from the absorption spectrum of FIG. Many have high correlation coefficients in a wave number band obtained by performing the multivariate analysis operation such as PCR or PLS method at high wavenumber region the correlation coefficient, high precision calibration formula of lactic acid is obtained .

[0028]

INDUSTRIAL APPLICABILITY In the present invention, the wavelength of light having a high correlation coefficient between the concentration and the absorbance of lactic acid to be measured is selected as the measurement wavelength by irradiating the object to be measured with visible light or near-infrared light, and the absorbance is measured. Is measured and quantitatively analyzed by the multivariate regression analysis method, so that lactic acid in the measurement sample can be quantitatively measured, and consumables such as reagents and test papers are not required, and use of such consumables There is no problem of later disposal.

[Brief description of drawings]

FIG. 1 is a block diagram schematically showing a lactate concentration measuring device.

FIG. 2 is a diagram showing an absorption spectrum of an aqueous lactic acid solution in a near infrared region.

FIG. 3 is a diagram showing a calibration curve obtained by plotting the absorbance of an aqueous lactic acid solution at an absorption wave number of 4361.5 cm ⁻¹ against the concentration.

FIG. 4 shows the correlation coefficient R between the absorbance and the concentration calculated for each wave number from the absorption spectrum of FIG.

[Explanation of symbols]

 11 light source device 12 spectroscopic device 13 urine sample installation unit 14 detection device 15 signal processing interface 16 computer

Claims (4)

[Claims] 1. When measuring the concentration of lactic acid contained in urine, the absolute value of the correlation coefficient between the concentration of the aqueous lactic acid solution in the visible or near infrared region and the absorbance is 0.5 or more, preferably 0. Select at least one wavelength included in the wavelength region of .9 or more as a measurement wavelength unique to the lactic acid component, irradiate a urine sample with visible light or near infrared light, and measure the absorbance at the selected measurement wavelength. A method for measuring lactic acid concentration, which comprises quantifying the lactic acid concentration in urine. 2. The wavelength range for selecting the lactic acid concentration measurement wavelength is 6300 to 5400 cm ⁻¹ , 4800 in terms of wave number.
To 4200 cm ^−1.
The method for measuring the concentration of lactic acid according to 1. 3. A light emitting means for irradiating a urine sample with light having a specific wave number in the visible or near-infrared region, and light absorption corresponding to the absorbance of a component contained in the sample by receiving the light transmitted through the sample. A lactate concentration measuring apparatus comprising: a light receiving unit that outputs a light receiving signal; and an arithmetic processing unit that calculates the absorbance from the light receiving signal from the light receiving unit and quantifies the lactic acid concentration. 4. The lactate concentration measuring apparatus according to claim 3, wherein the arithmetic processing means for quantifying the lactate concentration includes a multivariate regression analysis method.