JPS61155847A - Quantitative determination of urea nitrogen and creatinine - Google Patents

Abstract

PURPOSE:To determine quantitatively and quickly urea and creatinine in one conductivity cell by adding successively creatinine deiminase and urease to a liquid to be inspected, detecting the change quantity of the conductivity in each stage and calculating the difference thereof. CONSTITUTION:A platinum electrode 1 is provided to a measuring cell 4. A sample liquid and buffer soln. are put into the cell 4 through a feed port 6. The liquid to be inspected and creatinine deiminase soln. are first injected into the cell and the conductivity by the NH<+>4 formed by enzyme reaction is measured by a measuring instrument 11, by which the creatinine is determined and the change quantity X of the conductivity thereof is calculated. The urease soln. is then put into the cell and the change quantity of the conductivity by the formed NH<+>4 and HCO<->3 is detected. The urea nitrogen is determined by subtracting the quantity X from the quantity Y. The two different enzyme reactions are continuously effected in one conductivity cell and the change quantity of the conductivity is detected and calculated and therefore the quick analysis of the creatinine and urea nitrogen by a simple measuring method is made possible.

Description

Detailed description of the invention [Technical field to which the invention pertains] This invention relates to oxygen urease (g, c, 3.s, t, s
) and creatinine diminase (E, C, 3, 5, 4
, 2) to quantify urea nitrogen and creatinine in a test liquid by conductivity measurement.

[Conventional technology and its problems]

In connection with this type of conductivity measuring method, the applicant previously proposed a urea nitrogen analyzer and a method for quantifying creatinine in Japanese Patent Applications No. 58-61284 and No. 59-7671, respectively. There is.

The former is equipped with two conductivity measurement electrodes covered with a porous membrane, and introduces the enzyme urease, which can decompose urea to produce ammonia and carbon dioxide, into a conductivity measurement cell chamber filled with a buffer solution. Furthermore, a test solution containing urea is introduced into this measurement cell chamber, and the amount of change or rate of change in the conductivity of the buffer solution containing this test solution is measured to quantify urea nitrogen in the test solution. It is something.

In the latter method, creatinine is decomposed by adding creatinine diminase to the sample, and the amount or rate of change in conductivity due to the generated ammonia is detected using a conductivity measuring electrode with a porous membrane. This is for quantifying creatinine.

Both use a reaction solution that specifically decomposes urea or creatinine in the test solution, so urea or creatinine can be determined quickly and accurately without being affected by coexisting substances in the test solution. Furthermore, since the conductivity measurement electrode is equipped with a porous membrane, it has the advantage of being able to perform stable measurements over a long period of time.

However, in recent years, the creatinine concentration and urea nitrogen concentration in biological fluids have become important as indicators of renal function in the field of clinical testing, and a simpler and faster quantitative method is required.

[Purpose of the invention]

The present invention was developed in view of the above, and involves continuously conducting 12 enzyme reactions in one measurement cell chamber, and calculating the concentrations of urea nitrogen and creatinine from the amount of change in conductivity caused by each reaction. It is intended to be measured using a temperature measuring electrode.

[Key points of the invention]

According to the present invention, this purpose includes a step of detecting the amount of change in electrical conductivity by adding creatinine diminase to the test solution, and a step of detecting the amount of change in electrical conductivity by adding urease to the test solution. By sequentially performing the process of detecting the amount of change in the same cell at different times, and subtracting the amount of change in conductivity due to the process that started earlier from the amount of change in conductivity due to the process that started later, This is achieved by determining the amount of change in conductivity during the initial process alone.

[Embodiments of the invention]

FIG. 1 is a system diagram of an analyzer that is an embodiment of the present invention. In FIG. 1, a platinum electrode for measuring conductivity is attached to a measurement cell 3, and a porous membrane 2 is attached to the lower end of the platinum electrode 1.
is installed. The conductivity measurement cell chamber 3 is filled with a buffer solution, which is stirred by vibrating a silicon diaphragm 5 using an air pump (not shown) or the like. A fixed amount of the test liquid and the enzyme solution are injected from the ocean inlet 6. The measurement cell chamber 4 has a constant volume (for example 0.4 m),
The temperature is maintained at a constant temperature of 20 to 40° C. by a temperature adjusting means (not shown).

When the creatinine diminase solution and the test liquid are injected into the measurement cell chamber 4, the next enzymatic reaction proceeds.

+NH4" + OH- Therefore, the amount or rate of change in conductivity in the measurement cell chamber 4 due to ammonia ions (NH4) generated by the reaction (1) is detected, and creatinine is measured. Furthermore, when the urease solution is injected, The next enzymatic reaction proceeds.

Urine IA+3H20 sentence 1:j-2NH4+HCOa-+
OH"" ...(2) Ammonia ion (NH4) produced by the reaction of (2).

The amount of change in conductivity or rate of change due to hydrogen carbonate ions (HCOs-) is detected, and urea nitrogen is measured. That is,
Using the platinum electrode 1, the amount or rate of change in the conductivity in the measurement cell chamber 4 due to each reaction is measured by the conductivity measuring device 11, and by performing predetermined calculations in the calculator 12, urea nitrogen and creatinine are determined. The concentration is displayed on the display 13.

The conductivity measuring device 11 used in the present invention may be a Kohlrausch type four-sided bridge or a linear bridge type, and the voltage applied between the conductivity measuring electrodes is an alternating current voltage (preferably 0.05 mV) to prevent polarization. 53G(z, IV
degree) is desirable. In addition to platinum, platinum-platinum black, stainless steel, or the like can be used for the conductivity measuring electrode.

Figure 2 shows the measurement cell chamber 4 when using the measurement system shown in Figure 1.
FIG.

Here, a polycarbonate membrane with a pore diameter of 0.01 Sun and a thickness of 7 μm was used as the porous membrane 2, and the solutions used were 0.01 M phosphate buffer solution (PH70),
．． Urea nitrogen dissolved in 05M phosphate buffer solution, creatinine mixed solution, urease solution 2.5”F/d,
10 sv/- of L/atinine diminase solution,
Measurements were performed at 25±0.01°C.

In Figure 2, creatinine diminase solution 25μ
When t is injected into the measurement cell chamber 4 (Il), shadows of electrolytes etc. in the creatinine diminase solution! The conductivity increased from #l, but when stirring was sufficient, a constant value was obtained about 5 seconds after injection. After the conductivity reached a constant value, the urea nitrogen concentration dissolved in 0.05M monophosphate buffer solution was 50 capes/dt.

50 μ of sample solution with creatinine concentration of 100 q/dL
Immediately after injection, the conductivity increased rapidly due to the influence of the electrolyte in the sample solution, and then gradually increased (ΔS1). This gradual increase in electrical conductivity is due to the reaction between creatinine and creatinine diminase to generate ammonia ions (NH4) according to reaction formula (1).

Furthermore, when 20 μt of urease solution is injected into the measurement cell chamber 4 (Step 3), ammonia ions (
NH4'')eAn increase in electrical conductivity (Δ82) due to the generation of bicarbonate ions (HCO3-) was observed.Here,
8i includes the increase in conductivity according to the reaction equation (1) in the time Δt2 as a positive error, but the amount of change in conductivity in the time Δtl, calculated from the formula (, Jx/xt
t×Δt2), and the calculation unit 12 calculates the group 2
By subtracting from the equation (2), it is possible to determine the amount of change in conductivity due to reaction formula (2) alone. -2 After measurement, the liquid feed pump 14 is operated and a constant flow of -1 (6sd
/JOIE) to clean the inside of the measurement cell chamber 4 by sending a buffer solution 15, and the extruded liquid is washed away by 16, and the conductivity in the measurement cell chamber 4 returns to the level before measurement in about 30 to 60 seconds. You can now return to the previous position and perform the next measurement.

Figure 3 is a calibration curve using the analyzer shown in Figure 1, showing the relationship between the change in conductivity q (Δtl) and creatinine concentration (straight line a) for 1 to 5 minutes (Δtl) after injection of the sample solution, and the relationship between the creatinine concentration (straight line a) and the relationship between creatinine concentration (straight line a) after injection of the urease solution. Relationship between change in conductivity (Δ82) and urea nitrogen concentration for 5 to 30 seconds (Δt2) (straight line b
) is shown. Although the amount of change in conductivity due to creatinine was small, both showed a good linear relationship. Therefore, by preparing test tS for each of urea nitrogen and creatinine in advance, unknown concentrations of urea nitrogen and creatinine can be easily quantified.

Note that in the above embodiment, a certain period of time (Δtl.

Although the amount of change in conductivity (Jt, -z) at Δ12) is detected, it is also possible to quantify urea nitrogen and creatinine in the test liquid by detecting the rate of change in conductivity after a certain period of time. Further, the porous membrane used in the above example had a pore size of 0.
It is a polycarbonate membrane with a thickness of 015μ and a thickness of 7μ.
Cellulose acetate membranes, polyvinyl chloride membranes, porous ceramic membranes, etc. may also be used. Pore diameter is 0.00
A porous membrane with a pore size of 1 to 0.1 μm and a thickness of 0.5 to 1000 μm can be used, but preferably has a pore size of 0.
．． 015-0.1 μm, thickness 2-10 μm.

Further, in the above example, urease is added after adding creatinine diminase to the test solution and detecting the amount of change in conductivity due to ammonia produced. However, the present invention is not limited to this, and the present invention is not limited to this. Creatinine diminase may be added after detecting the amount of change in conductivity due to ammonia and hydrogen carbonate produced by adding urease to the solution.

〔Effect of the invention〕

As is clear from the above description, according to the present invention, two different enzymatic reactions are allowed to proceed continuously in one conductivity measurement cell, and one set of changes or rates of change in conductivity due to each reaction is obtained. urea nitrogen and creatinine are detected using conductivity measurement electrodes, and urea nitrogen and creatinine are quantitatively measured using a simple measurement system, which is essential for diagnosing kidney function. , the effect of being able to perform analysis simply and quickly can be obtained.

[Brief explanation of drawings]

Fig. 1 is a system diagram of an analyzer showing an embodiment of the present invention, Fig. 2 is a time-course characteristic diagram of electrical conductivity showing experimental results according to an embodiment of the present invention, and an rs diagram is a diagram of the analyzer shown in Fig. 1. It is a calibration curve diagram of urea nitrogen and creatinine. 1... Platinum electrode for conductivity measurement, 2... Porous membrane, 3
... Measuring cell, 4... Tolerance measurement cell room, 11...
- Conductivity measuring device, 12... Arithmetic unit, 13... Display device.

Claims (1)

[Scope of Claims] 1) A step of detecting the amount of change in conductivity due to the addition of creatinine diminase to the test solution, and a change in conductivity due to the addition of urease to the test solution. By sequentially performing the process of detecting the amount in the same cell at different times, and subtracting the amount of change in conductivity due to the process that started earlier from the amount of change in conductivity due to the process that started later, A method for quantifying urea nitrogen and creatinine, characterized by determining the amount of change in conductivity in a single process. 2) In the method according to claim 1, after the step of adding creatinine diminase is performed first,
A method for quantifying urea nitrogen and creatinine, comprising a step of adding urease.