JPS60152947A - Continuous quantitative analysis of uric acid and inorganic phosphorus - Google Patents

Abstract

PURPOSE:To simply perform the indirect quantitative analysis of the concn. of inorg. phosphorus by measuring the concn. of uric acid in a specimen in a polarographic manner by an enzyme electrode and reacting a solution to be examined with a solution mixture of inosine, PNP and XOD to detect uric acid and H2O2. CONSTITUTION:A specimen is injected in a reaction cell chamber 20 from an injection port 22 and diluted by a buffer solution under stirring while uric acid in the specimen is brought into contact with an immobilized uricase film 40 to generate H2O2. Whereupon, the current value of an H2O2 electrode rises in proportion to the amount of uric acid and, therefore, quantitative analysis can be performed. On the other hand, a solution mixture containing inosine, PNP and XOD in a predetermined concn. is injected in the specimen and reacted with inog. phosphorus in the specimen to generate uric acid and H2O2 in the cell chamber 20. In this case, because the current value of the electrode 10 again increases, the concn. of inorg. phosphorous is quantitatively measured and, after the cell chamber 20 is washed with the buffer solution 50, measurement is performed repeatedly and continuously. By this method, a reaction time can be shortened and accuracy is enhanced while continuous quantitative analysis can be performed.

Description

[Detailed Description of the Invention] [Field to which the invention pertains] This invention relates to a polarographic method for measuring uric acid and inorganic phosphorus concentrations in a test solution using a water bath enzyme and an immobilized enzyme membrane [Prior art and its problems] Clinical examination The determination of uric acid in the field is based on the advanced production of uric acid in the body (
It is considered important as an indicator of abnormalities in renal excretion (such as gout) and abnormal renal excretion. In addition, ζ-kouric acid is of interest in relation to metabolic disorders. Quantification of inorganic phosphorus in the field of clinical testing is mainly necessary to understand the dynamics of bone and mineral deposits.Furthermore, in the field of environmental measurement, it is an important measurement item as an indicator of eutrophication of water quality.

This type of method for quantifying uric acid and inorganic phosphorus is generally required to be accurate, simple, and rapid, and in the case of blood in particular, there is a strong desire to measure whole blood without the need for centrifugation. .

There is no known method for continuously quantifying uric acid and inorganic phosphorus in the same reaction cell using an enzyme 1L electrode.

Methods for quantifying uric acid can be broadly classified into two types: reduction methods and enzyme methods. A typical reduction method is a method in which phosphotungstic acid is reduced and a blue pigment is determined colorimetrically. In the case of the enzymatic method, uric acid is treated with uric acid degrading enzyme urease (EC
I, 7, 3.3) and determine the concentration of uric acid from the difference in absorbance in the ultraviolet region, and other reductases that can colorimetrically compare the hydrogen peroxide produced when uricase acts. A method of conjugating and easily quantifying in the visible region is known.

These methods have the disadvantage that positive errors occur due to uric acid-like compounds ζ, and negative errors occur due to ascorbic acid, glutathione, etc. coexisting in the sample. Furthermore, there are disadvantages in that colored samples such as body fluids cannot be used as they are, and the time required for measurement is long.

In recent years, an enzyme electrode method using immobilized enzymes has become known as a method for eliminating these drawbacks.

Updike et al. (Nature, 214.886 (1
967)] The concept of an enzyme electrode has been reported and its application to uric acid has also been carried out, but there is no known method for continuously converting uric acid and inorganic phosphorus.

Fiske-8ubba Ro is a method for quantifying inorganic phosphorus.
w method (J, Biol, Chem, 66, 375 (192
The reduction coloring method described in 5)) is common, but this method has drawbacks such as color stability and sensitivity. In recent years, enzymes (purine nucleoside phosphorylases) have reduced these effects.

A colorimetric method using xanthine oxidase and peroxidase (xanthine oxidase and peroxidase) has been used, but 1) it is not economical because the three enzymes used are disposable, and 2) colored samples such as body fluids cannot be used as is. ,3)
There is a problem that the measurement takes a long time. Using the substrate inosine, the enzyme brine nucleoside phosphorylase, and the enzyme xanthine oxidase, H2O2 generated in the presence of inorganic phosphorus is directly quantified using an IJ rack using a hydrogen peroxide electrode coated with an H702 selectively permeable membrane. A method for analyzing the 411J concentration of
.

Compared to the colorimetric method, this method has the advantages of being able to analyze colored samples and shortening the measurement time. In phosphorus measurement, the detection system must have highly accurate and stable characteristics because the amount of H2O2 produced is small; 3) H2
Since a cellulose acetate membrane is used as the 02 selectively permeable membrane, there is a problem that needs to be solved, such as a high fiF property that causes clotting when whole blood samples are measured over a long period of time.

[Purpose of the invention]

The present invention aims to eliminate the above-mentioned drawbacks and provide a continuous analysis method for uric acid and inorganic phosphorus using an enzyme electrode that is rapid, simple, highly selective, and capable of measuring whole blood and colored samples. purpose.

[Key points of the invention]

A sample is injected into a reaction cell filled with a buffer solution, and uric acid contained in the sample is first detected with an oxygen electrode.Next, a mixed reagent for inorganic phosphorus measurement is injected, and inorganic phosphorus coexists with inosine. Enzyme purine nucleoside phosphorylase (
PNP, EC2,4゜2.1) and first converted into hypoxanthine and ribose-1-phosphate, and then the generated hypoxanthine was treated with the enzyme xanthine oxidase (XOD, ECI, 2,3) in the presence of 02. .2) was converted into uric acid and hydrogen peroxide, and the uric acid and hydrogen peroxide produced by the hydrogen peroxide electrode covered with the immobilized uricase membrane installed in the reaction cell were detected by the polarographic method, and the uric acid and hydrogen peroxide prepared in advance were detected. This is achieved by quantifying the inorganic phosphorus concentration from the calibration curve obtained. The inorganic phosphorus concentration can also be quantified by detecting the oxygen consumed and the uric acid produced during the reaction using an oxygen electrode covered with an immobilized uricase membrane installed in the reaction cell.

The continuous determination method for uric acid and inorganic phosphorus according to the present invention is based on the following measurement principle.

Uric acid is detected by measuring H 2 O 2 or O 2 consumed by the subsequent reaction proceeding within the immobilized uricase membrane using a polarographic method.

Uric acid + 02 + H20 Tomokakodanriba no ta copy 1 Allantoin + H2O2 + CO2 (1) After quantifying uric acid, inorganic phosphorus is determined using the following reaction. Inorganic phosphorus has a pH
7,35+7) In the buffer solution, most of HPO4 exists, and the next reaction proceeds by brine nucleoside phosphorylase in the presence of the substrate inosine.

Inon + npo knee - hypoxanthine + liposu 1
-Phosphoric acid (2) Hypoxanthine is converted to uric acid and hydrogen peroxide by xanthine oxidase according to the following formula.

Hypoxanthine +202 + 2 H20-1 → Uric acid +
H2O2 (3) The present invention polarographically detects the generated uric acid and H2O2 or the generated uric acid and the consumed 02, and determines the degree of inorganic phosphorus anchorage at the indirect point from the amount of change in the reaction current after a certain period of time. be.

The unique feature here is that the uric acid and hydrogen peroxide produced during the quantification of inorganic phosphorus after the quantification of uric acid, or the uric acid produced and the oxygen consumed, are collected using a hydrogen peroxide electrode coated with an immobilized uricase membrane. Alternatively, using an oxygen electrode (
3) In addition to detecting hydrogen peroxide produced or oxygen consumed according to the formula, uric acid produced at the same time is detected using an immobilized uricase membrane-electrode method with excellent selectivity.

By using an immobilized enzyme membrane, the present invention has not only made it possible to measure uric acid and inorganic phosphorus continuously, but also made it possible to detect uric acid when measuring inorganic phosphorus concentration. The identified uricase membrane is less susceptible to the effects of coexisting substances.
Furthermore, since the electrodes and enzyme layer are protected from contaminants such as blood cells and proteins, it is based on the idea that stable output can be obtained over a long period of time without being affected by interfering substances.

[Embodiments of the invention]

Figure 1 shows the configuration of the uric acid and inorganic phosphorus detection section.

Here, 10 is a hydrogen peroxide electrode, 12 is its lead wire, 1
4 is a temperature compensation thermistor, 16 is its lead wire, 18
indicates a reaction cell, and 2o indicates a reaction cell chamber filled with a buffer solution. 22 is an injection port for sample and reagent solution;
4 is a diaphragm for stirring inside the reaction cell, and 26 is a stainless steel pipe. The reaction cell chamber 2o has a constant volume (0.4 ml) and is maintained at a constant temperature (27 to 37° C.) by means not shown.

Next, FIG. 2 shows an example of a hydrogen peroxide electrode acidified with an immobilized uricase membrane used in the present invention. Here, 30 is a platinum electrode, 34 is an electrode, and 32 is an insulating layer between these two electrodes. 36 is OIJ song carrying guide, 38 is o
The ring 4o is an immobilized enzyme membrane of uricase, which is closely attached to the surfaces of the platinum electrode 3o and the electrode 34. Here, a constant voltage in the range of +0.6 to 0.8 V is applied to the platinum electrode 3o with respect to the electrode 34. On the other hand, the hydrogen peroxide electrode 10 shown in FIG.
By applying a constant voltage in the range of 0.6 to 0.8
It can also be used as an oxygen '(5) electrode.

Further, FIG. 3 shows an example of a detection device for uric acid and inorganic phosphorus.

Here, 52 is the detection section shown in FIG. 1, to which the buffer solution 50 is fed by a liquid feed pump 54. 56 is a waste liquid reservoir. connecting the electrode to a polarographic device 58;
Furthermore, it is connected to the output recording section 6°.

An embodiment of the method of the present invention will be described below with reference to FIGS. 1 to 3.

Example 1 A hydrogen peroxide electrode 10 was equipped with an immobilized uricase membrane 4°. 0.1 M Tris buffer (p
H7, 35) was used. Inosine, the substrate used in the inorganic phosphorus determination step, was 3.88 mM, and PNP was 1.3
8 units/rr]7. %X OD is 13.8uni
The Sandoku reagent was mixed and prepared to have a concentration of t/rnl. Solution Lt 0. IM to IJ buffer (p
H7, 35) was used. Here, inosine, PNP and υ
For XOD, commercially available reagents can be used.

When the sample is injected into the reaction cell chamber 20 yen from the injection port 22,
The mixture is stirred by a silicon diaphragm 24 and uniformly mixed with a buffer solution in the reaction cell chamber 20. Thus, the uric acid in the sample comes into contact with the immobilized uricase membrane 40 and is oxidized to generate H2O, thereby causing the current value of the hydrogen peroxide electrode 10 to rise and temporarily reach a steady state.

At this time, since the increase in flow rate is proportional to t acid ti', it can be quantified.

Then) -91 constant density inosine, PN'P and XOD
When the mixed reagent solution consisting of The current value of the coated hydrogen peroxide electrode 10 increases again. From the increase or increase rate ζ after a certain period of time, it is possible to quantify the amount of tflIJn'a fart. After quantitative determination, buffer solution 50 is added to reaction cell chamber 2.
The waste liquid is sent to a waste liquid reservoir 56 by a liquid feed pump 54. When the current value returns to the value before sample injection due to cleaning, a new sample and mixed reagent can be sequentially injected again, thus making it possible to perform repeated and continuous measurements. Injection of samples and reagents can also be performed automatically if an automatic sampler is attached.

Example 2 Example 1. Figure 4 shows the current changes when uric acid and inorganic phosphorus were actually continuously quantified in the same reaction cell chamber. Uric acid 8.2 mg/c1 l and inorganic phosphorus 4.7 m
Upon injection of 20 μl of the sample containing g/dl (time point 100), the electrode current value rose sharply and reached a steady state after about 15 s. After 30 seconds, 20 μl of a mixed reagent consisting of inosine, PNP, and XOD was injected (time point 1).
02), an increase in the reaction current was again observed after about 15 seconds, and a linear increase was observed after 20 seconds (time point 104). After the reagent was injected, the interior of the 3-minute wave ζ reaction cell chamber 20 began to be washed with the buffer solution 50 (time point 106), and 30 seconds later, the current returned to the value before sample injection.

If a calibration curve is prepared in advance using uric acid and inorganic phosphorus standard solutions or the device is calibrated, uric acid can be determined from Δ11 in FIG. 4, and inorganic phosphorus can be determined from Δ12 in FIG. 4 and displayed.

The calibration curve for uric acid prepared by the above method is shown in FIG. 5, and the calibration curve for inorganic phosphorus is shown in FIG. 6.

At an inorganic phosphorus concentration of 2.5 mg/d 1 or more, the relationship deviated from a linear relationship, but up to 10 mg/d 7 a concentration dependence with good reproducibility was shown.

Example 3 The measurement time (time points 104 to 106) for quantifying inorganic phosphorus shown in Example 2 was changed to 40 seconds, 1 minute 40 seconds, and 2 minutes 40 seconds.
10 consecutive measurements were performed using a solution with an inorganic phosphorus concentration of 5 mg/dl, and the simultaneous reproducibility was examined. The obtained C and V values are 235 chi and 1.47, respectively.
The ratio is 1.26, which is a good result, and it can be seen that sufficient accuracy can be obtained even with a measurement time of 40 seconds. From this, it is clear that continuous analysis of uric acid and inorganic phosphorus concentrations can be performed within 2 to 3 minutes after sample injection.

Example 4 Further, 100 consecutive measurements using whole blood samples were attempted, but no decrease in output due to contamination of the immobilized uricase membrane was observed, indicating that the device could withstand long-term use.

Example 5 To investigate the effect of an immobilized uricase membrane on inorganic phosphorus measurement The reaction current was measured by attaching an acetyl cellulose membrane with a thickness of 6 μm to the hydrogen electrode 10. Electrode activity and film thickness correction were performed using a standard hydrogen peroxide aqueous solution (120 ppm). Comparing the reaction currents obtained, it was found that by using the immobilized uricase membrane, a value twice the reaction current obtained with the acetylcellulose membrane was obtained, and the accuracy was improved by measuring the generated uric acid and H2O2. I understand.

Example 6 Reduction when uric acid and inorganic phosphorus were continuously determined according to Example 2 by using the hydrogen peroxide electrode 10 of Example 1 and applying 10.60 V to the platinum electrode 30 to act as an oxygen electrode. Figure 7 shows the changes in current.

Upon injection of 20 μm of sample (time point 200), the electrode current decreases rapidly and reaches a steady state after 30 seconds.

After 30 seconds, 20 μl of mixed reagent is injected (time point 202).
), a decrease in reduction current was observed again after about 15 seconds.

In this way, similarly to Example 2, the uric acid concentration can be analyzed from Δ11 in FIG. 7, and the inorganic phosphorus concentration can be analyzed from Δ12.

〔Effect of the invention〕

As can be seen from the above description, according to the present invention, since the detection section is configured using the enzyme electrode method, continuous determination of uric acid and inorganic phosphorus within the same reaction cell is possible. Furthermore, inorganic phosphorus is reacted with inosine, PNP, and XOD to enzymatically generate uric acid and hydrogen peroxide, which are then quantified using a hydrogen peroxide electrode or an oxygen electrode coated with an identified uricase membrane. This resulted in a doubling of the reaction current, thereby making it possible to shorten the reaction time and improve accuracy. Furthermore, since an immobilized enzyme membrane is used, stable output can be obtained even for long-term whole blood measurements. Furthermore, since inorganic phosphorus is quantified using the enzyme electrode method, it has become possible to analyze uric acid and inorganic phosphorus continuously, quickly and easily, and without being affected by coexisting substances.

[Brief explanation of the drawing]

Fig. 1 is a plan view of the detection part of the device a♀ used in the method of the present invention, Fig. 2 is a cross-sectional view showing the structure of a superheated hydrogen electrode for polarography, and Fig. 3 is a plan view of the detection part of the device a♡ used in the method of the present invention. Fig. 4 is a diagram showing the output of the polarographic device when a hydrogen peroxide electrode is used, that is, the state of the constant-ζν method of the present invention, and Fig. 5 is a diagram showing the state of the constant-ζν method of the present invention. Lines showing the relationship between the output of the polarographic device of the invention and uric acid concentration, Figure 6 is a gauze diagram showing the relationship between the output of the polarographic device of the present invention and inorganic phosphorus concentration, and Figure 7 is a polarographic graph when an oxygen electrode is used. FIG. 3 is a diagram showing the output of the device, that is, the state of the tray method of the present invention. 10. Hydrogen peroxide'1L pole, 18 Reaction cell, 20...
Reaction cell chamber, 22 injection port, 30 - platinum electrode, 34 electrode, 40 immobilized uricase, 5° buffer, 52 - detection section, 58 polarographic device, 60 - output recording section. '-゛8cho1no1.゛, τλ-11 noro; 9, -m-bar 1st Figure Ya Z sensai 3 flash θ l 2 3 4 Thread [Gosaki Seki (now) Sai 4 (2) 0 to 20, 30 4θ deflection (笥Jrokuρ) Oj Sen0, f tQUfishets°rin) / gl /li/l) Sairo ki

Claims (1)

[Claims] 1) In a method for quantifying uric acid and inorganic phosphorus in a sample, a sample solution is introduced into a reaction cell equipped with an enzyme electrode consisting of an immobilized uricase membrane and a hydrogen peroxide electrode and filled with a buffer solution. Then, the uric acid concentration in the sample was measured polarographically using the enzyme electrode, and then a mixed solution containing brine nucleoside phosphorylase, inosine, and xanthine oxidase was introduced into the reaction cell, reacted with the test solution, and sequentially 1. A continuous method for quantifying uric acid and inorganic phosphorus, characterized in that the produced uric acid and hydrogen peroxide are polarographically detected using the enzyme electrode, and the inorganic phosphorus concentration is indirectly determined. 2) In the method for quantifying uric acid and inorganic phosphorus in a sample, a sample solution is introduced into a reaction cell equipped with an enzyme electrode consisting of an immobilized uricase membrane and an oxygen electrode and filled with a buffer solution, and the enzyme electrode Measure the uric acid concentration polarographically, then introduce a mixed solution containing purine nucleondophosphorylase, inosine, and xanthine oxidase into the reaction cell and react with the test solution, successively producing uric acid and dissolved enzyme. A method for continuously quantifying uric acid and inorganic phosphorus, characterized in that the amount of decrease is polarographically detected using the enzyme electrode to indirectly quantify the inorganic phosphorus concentration.