JPH0775558B2 - Method for quantifying inorganic phosphorus in biological fluid and reagent for quantifying the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for quantifying inorganic phosphorus in biological fluids and a reagent for quantifying the same, which is mainly used in the field of clinical examination.

[Conventional technology]

Phosphorus forms bone with calcium, but phosphorus is known to be involved in the metabolism of organic compounds in vivo. Phosphorus is usually present in serum as inorganic phosphate, organic phosphates, phospholipids, etc. Among them, inorganic phosphorus is used for the diagnosis of parathyroid function test, nephritis, osteomalacia, etc. in the field of clinical examination. Plays an important role.

The method of measuring inorganic phosphorus has long been a method of precipitating it as magnesium ammonium phosphate by the reaction of magnesium with ammonia and measuring its weight. However, due to the complicated operation, it is currently replaced by molybdenum under acidic conditions. The most commonly used method is to reduce the phosphomolybdate generated by the reaction with an acid and measure the absorbance when the color is developed. Fiske-Sub is a typical example.
There is ba Raw method.

Apart from this, a method for quantifying inorganic phosphorus by an enzymatic reaction has also been reported. The first example is purine nucleoside phosphorylase (PNP) that uses inosine and phosphorus as substrates.
There is a method for measuring a quinone dye produced by a coupling reaction in which the enzymatic reaction of xanthine oxidase and the enzymatic reaction of peroxidase (POD) are combined with the enzymatic reaction of 1. As a second example of the method for quantifying inorganic phosphorus by enzymatic reaction, glyceroaldehyde-3-phosphate (GAP) and nicotinamide adenine dinucleotide oxidized form (NAD) and glyceroaldehyde-3-phosphorus using phosphorus as a substrate. There is a method of measuring the absorbance at ultraviolet wavelength by the enzymatic reaction of acid dehydrogenase (GAPDH).

[Problems to be solved by the invention]

Of these conventional methods, the most general method by reaction with molybdic acid has the drawback of low color development sensitivity, and since a strong acid must be used as a reagent, it is also safe in operation. It's a problem. On the other hand, as researches on enzyme reactions have been promoted in recent years, the method by enzyme reaction has come to be widely used in clinical tests because of its mild composition of reagents and its simple operation and easy automation. However, the enzyme reaction is susceptible to fluctuations due to conditions such as temperature, time, and pH, and it is necessary to consider problems such as the influence of interfering factors existing in the reaction system. In addition, in a quantification method using an enzyme, when there is no indicator substance that can be directly measured in one enzyme reaction and therefore it is not possible to measure, an enzyme reaction is often further combined to develop a coupled enzyme reaction having an appropriate indicator substance. However, in the case of this coupled enzyme reaction, there are different optimum conditions for each enzyme reaction, and there are problems when performing each enzyme reaction in one solution at the same time due to differences in pH, buffer type, concentration, etc. May cause In addition, the non-specificity of the coupled enzyme selected as a combination may rather deteriorate the performance.In addition, the reaction rate may decrease, such as when the reaction equilibrium is not completely biased toward the product side. It is necessary to pay sufficient attention to such complicated problems. In particular, blood, urine, and other biological fluids used in clinical tests contain various components that act as interference factors other than those to be measured, and also show some reactivity with the coupled enzyme reaction system. Often components are also present. In addition to this, when a reagent for measurement is used, an example in which impurities are mixed is often a problem.

From this background, looking at the conventional method for quantifying inorganic phosphorus by enzymatic reaction, first, in the method using the enzyme PNP, which is the first example described above, the POD of the coupling enzyme is non-specific, so However, there is a problem that it is affected by various reducing substances that coexist as an interfering factor, and special measures are required for it. As such reducing substances, various substances such as bilirubin, ascorbic acid, uric acid, glutathione and proteins have been pointed out. Next, the method using the enzyme GAPDH, which is the second example, has the drawback that the substrate GAP contains phosphorus as an impurity, and that GAP itself is unstable and easily decomposed into glyceroaldehyde and phosphorus. There is also a problem when used as a reagent.

It is an object of the present invention to provide a method for quantifying inorganic phosphorus in a biological fluid, which can be used in the field of clinical tests by eliminating the above-mentioned drawbacks, and a reagent for quantifying the same.

[Means for solving problems]

As a result of intensive studies to solve these problems and achieve the above-mentioned object, the present inventors have found that an enzymatic reaction by a polynucleotide phosphorylase and a pyruvate kinase [Pyruvate kinas]
e, E C2.7.1.40 (PK)] and the enzymatic reaction of lactate dehydrogenase (Lactate de-hydrogenase (LDH)) are combined to eliminate the drawbacks of the above-mentioned coupled enzyme reaction system. The inventors have found that inorganic phosphorus in biological fluids can be quantified with high sensitivity, and further researches have completed the present invention.

The present invention is characterized in that (1) a biological fluid sample is reacted with a reagent comprising an enzyme reaction system based on PK and an enzyme reaction system based on LDH, and then reacted with a reagent comprising an enzyme reaction system based on polynucleotide phosphorylase. Method for quantifying inorganic phosphorus in biological fluid, (2) biological fluid characterized by comprising a reagent comprising an enzymatic reaction system by PK and an LDH, and a reagent comprising an enzymatic reaction system by polynucleotide phosphorylase The present invention relates to a reagent for quantifying inorganic phosphorus in a product.

That is, the present invention relates to the following formulas (1), (2) and (3) (1) Enzymatic reaction with polynucleotide phosphorylase: polyadenylic acid (RNA) n ₊₁ + 0-phosphate (RNA) n + adenosine-5 ′ -Diphosphate (ADP) (2) PK enzymatic reaction: ADP + phosphoenolpyruvate (PEP) Adenisin-5'-triphosphate (ATP) + pyruvate (3) LDH enzymatic reaction: pyruvate + nicotine Amidoadenine dinucleotide reduced form (NAD
H) + H lactic acid + nicotinamide adenine dinucleotide oxidized form (NA
D) The present invention relates to a method for quantifying inorganic phosphorus in a biological fluid, which uses a coupled enzyme reaction in which the above three enzymatic reactions are combined, and a reagent for quantifying the same.

In the present invention, the biological fluid to be quantified is not particularly limited as long as it contains inorganic phosphorus, and examples thereof include blood (especially serum), urine, and cerebrospinal fluid, and the origin of the animal is not particularly limited. .

In the present invention, in the above-mentioned coupled enzyme reaction, the inorganic phosphorus in the biological fluid is used as a substrate in place of 0-phosphate and the reaction proceeds, and the inorganic phosphorus in the biological fluid is measured by measuring the decrease amount of NADH which is an indicator. Can be quantified. NADH is a widely known indicator substance in coupled enzyme reactions.
It can be easily measured by measuring the absorbance in the ultraviolet region, especially at a wavelength of 340 nm.

The coupled enzyme reaction used in the present invention is hardly affected by the reducing substance as an interfering factor like the enzyme reaction by POD described above. Further, in this coupled enzyme reaction, it can be predicted that ADP and pyruvic acid coexisting in the biological fluid will also react simultaneously as substrates, but in the present invention, the biological fluid sample is preliminarily analyzed by PK and the enzyme reaction system by LDH. Since ADP and pyruvic acid can be eliminated by mixing with a reagent consisting of and reacting with them, the influence of interference by these can be avoided. Furthermore, in the present invention, it is not necessary to stop the enzymatic reaction because the rate of decrease of NADH is measured, and therefore the operation step of adding the reaction stop solution can be omitted. Since the rate of decrease of NADH can be measured with high sensitivity, the amount of sample can be reduced.

Although the present invention is constituted from such findings, the method for quantifying inorganic phosphorus in a biological fluid according to the present invention can be summarized as follows. First, a biological fluid sample is used for the enzymatic reaction system with PK and LDH.
Is mixed with the first reagent consisting of the enzyme reaction system to react, and then the second reagent consisting of the enzyme reaction system consisting of polynucleotide phosphorylase is added and reacted to measure the amount of change in absorbance at ultraviolet wavelengths. . At this time, instead of the biological fluid sample, a standard solution containing a known concentration of inorganic phosphorus is similarly measured as a sample, and the inorganic phosphorus in the biological fluid can be quantified by comparing with this. In addition, in this measurement, what was measured using purified water as a sample is used as a blank.

PK in the first reagent is 0.05 to 500 U / ml, preferably 0.2 to
LDH in an amount of 15 U / ml is 0.05-50 U / ml, preferably 0.2-20
It is blended in U / ml. The first reagent contains PEP, NADH,
Inorganic salts (eg magnesium salt, potassium salt, etc.)
May be blended, and a buffering agent and the like are further blended. Examples of magnesium salts include magnesium chloride, and examples of potassium salts include potassium chloride. The magnesium salt is usually 0.2 to 50 mM, preferably 1 to 10 mM, and the potassium salt is usually 2 to 50 mM.
It is preferable to add 0 mM, preferably 10 to 100 mM. An example of the buffering agent is, for example, a triethanolamine buffer solution, and the compounding amount thereof is usually 10 to 500 mM, preferably 50 to 200 mM, and pH 7.4 to 7.8 is preferable.

The second reagent contains a polynucleotide phosphorylase, and may further contain an inorganic salt (eg, magnesium salt, potassium salt, etc.), or a buffering agent or the like. Examples of magnesium salts include magnesium chloride, and examples of potassium salts include potassium chloride. Magnesium salt is usually 0.2-50m
It is preferable to add M, preferably 1 to 10 mM, and the potassium salt is usually to add 500 mM, preferably 10 to 100 mM. As the buffer solution, those similar to those exemplified in the first reagent are exemplified, and the blending amount thereof is usually 10 to 500 mM, preferably 50 to 200 mM. The amount of the polynucleotide phosphorylase may be appropriately selected depending on the origin of the enzyme. For example, in the case of M.lysodeiktiscus, it is usually 2 to 500 U / ml, particularly 10 to 200 U / ml, and E. coli-derived. In this case, it is usually preferable to contain 0.5 to 200 U / ml, particularly 2 to 50 U / ml.

The substrate for the initial reaction, polyadenylic acid, may be contained in either the first reagent or the second reagent. This is because the coupling enzyme reaction used in the present invention proceeds when the second reagent is mixed with the first reagent. For example, when the amount of polyadenylic acid contained in the first reagent is 0.2 to 1
An example of the amount of polyadenylic acid when it is contained in the second reagent is 0.2 to 10 mM.

The blending amount or blending ratio of each component in the first reagent and the second reagent is not limited to the above amount,
It is needless to say that the above blending amount is merely an example of a preferred embodiment, and may be out of the above range of the amount ratio, or the amount ratio of each component may be increased or decreased in the ratio of the above amount. .

When the method of the present invention is carried out in a clinical test, the operating method differs depending on the use conditions.
Take 0.1-0.02 ml, add 2.0-0.5 ml of the first reagent consisting of PK-based enzyme reaction system and LDH-based enzyme reaction system, mix and react at 25-37 ° C for 2-10 minutes, then Add 0.5 to 0.05 ml of the second reagent consisting of the enzyme reaction system with folylase and heat it at 25 to 37 ° C to react it. After 1 to 3 minutes and 4 to 7 minutes after reacting with water, the wavelength 34
The absorbance at two wavelengths having a main wavelength of 0 nm or a wavelength of 340 nm is measured, and the change in absorbance over time is obtained. Note that this may automatically measure the amount of change in absorbance. Here, as a sample, a biological fluid sample (referred to as S), a standard solution containing inorganic phosphorus with a known concentration (referred to as ST), and purified water (referred to as B) were measured, respectively, and (SB) / (ST -The concentration of inorganic phosphorus in the biological fluid is calculated by (B) x (concentration of standard solution). In this case, S and ST may be automatically measured against B. In this way, inorganic phosphorus in biological fluid can be quantified by the method of the present invention.

The present invention also relates to reagents for quantifying inorganic phosphorus in biological fluids. The reagent according to the present invention is composed of the first reagent and the second reagent, and is usually a kit in which a standard solution containing a known concentration of inorganic phosphorus and a control for quality control are added to the two reagents. Preferably.

[Operation and effect of invention]

The method for quantifying inorganic phosphorus in a biological fluid according to the present invention eliminates various problems that are likely to occur in the above-described coupled enzyme reaction, and exhibits excellent effects on a special reagent called biological fluid. is there. Furthermore, this method has an advantage that it can be applied to an automatic analyzer, which has been increasing in clinical examinations in recent years, because it has a small number of operation steps and a short reaction time.

Further, the reagent for quantifying inorganic phosphorus in a biological fluid according to the present invention does not contain impurities that itself influence the measurement, and is used as a concrete object for carrying out the method of the present invention in daily clinical tests. It can be used in. Further, in consideration of preservation by the user, this reagent can be made into a form that can be preserved for a long period of time, such as freeze-drying, so that a way to supply this reagent as a product is also open.

Thus, the present invention can be provided as being useful in the field of clinical examination.

〔Example〕

Examples of the present invention will be shown below, but the present invention is not limited thereto.

Example 1 Two types of reagents according to the present invention were prepared according to the following compositions.

First reagent PK 0.9 U / ml LDH 8.0 U / ml PEP 0.87 mM NADH 0.21 mM Polyadenylic acid 1.2 mM Magnesium chloride 5.0 mM Potassium chloride 50 mM Triethanolamine buffer 100 mM (pH
7.6) Second reagent Polynucleotide phosphorylase M.lysodeiktiscus (derived) 50 U / ml Magnesium chloride 5.0 mM Potassium chloride 50 mM Triethanolamine buffer 100 mM (pH 7.
6) (Example 2) Using the reagents prepared in Example 1, the inorganic phosphorus in the biological fluid was quantified by the following operating method.

Take 50 μm of serum as a sample and add 0.9 ml of the first reagent 37
The reaction was carried out at ℃ for 5 minutes, then 0.1 ml of the second reagent was added and the reaction was carried out at 37 ℃. Then, the absorbance at a wavelength of 340 nm was measured after 3 minutes and 5 minutes with water as a control, and the amount of change in absorbance per 2 minutes was obtained (this is designated as S). 5.0m as a sample
Similarly, the amount of change in absorbance per 2 minutes was calculated for the standard solution containing g / dl of inorganic phosphorus and purified water (ST and B, respectively).
And).

The concentration of inorganic phosphorus in serum was calculated by the following formula.

Inorganic phosphorus (mg / dl) = (SB) / (ST-B) × 5.0 At this time, the serum was diluted in 1/5 steps, and the concentration of inorganic phosphorus was plotted against each dilution rate. As shown in, it was found that the concentration of inorganic phosphorus was proportional to the dilution ratio of serum.

(Example 3) When the polynucleotide phosphorylase in the second reagent of Example 1 was replaced with that derived from Escherichia coli and the amount thereof was set to 10 U / ml, inorganic phosphorus in the biological fluid was changed as in Example 2. It turned out that it can be quantified. At this time, when a calibration curve was prepared by changing the concentration of the standard solution, good linearity as shown in FIG. 2 was obtained.

(Example 4) In order to examine the reactivity of the polynucleotide phosphorylase by the method of the present invention with inorganic phosphorus, the Km value was measured. The measurement conditions are the same as those of the reagent composition in which the first reagent and the second reagent of Example 1 were mixed in the ratio of Example 2. As a result, Km = 1.3 × 10 ⁻³ M was obtained. This Km value shows that the reactivity is excellent even for the sample containing a low concentration of inorganic phosphorus in carrying out the present invention.

[Brief description of drawings]

FIG. 1 shows the concentration of inorganic phosphorus in the diluted serum obtained in Example 2, in which the vertical axis represents the concentration of inorganic phosphorus and the horizontal axis represents the serum dilution rate. FIG. 2 shows the calibration curve obtained in Example 3, in which the vertical axis represents the amount of change in absorbance per 2 minutes and the horizontal axis represents the concentration of the standard solution.

Claims (2)

[Claims] 1. A biological fluid sample is reacted with a reagent consisting of an enzyme reaction system based on pyruvate kinase and an enzyme reaction system based on lactate dehydrogenase, and then reacted with a reagent composed of an enzyme reaction system based on polynucleotide phosphorylase. A method for quantifying inorganic phosphorus in a biological fluid, which comprises: 2. An inorganic substance in a biological fluid, which comprises a reagent comprising an enzyme reaction system based on pyruvate kinase and an enzyme reaction system based on lactate dehydrogenase, and a reagent composed of an enzyme reaction system based on polynucleotide phosphorylase. Reagent for quantitative determination of phosphorus.