JP3078881B2 - Method for measuring components in biological samples - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring a component in a biological sample, which is mainly used in the field of clinical testing. More specifically, the present invention relates to a method for measuring a component in a biological sample that avoids the influence of a chelating substance mixed in the biological sample.

[0002]

2. Description of the Related Art Hitherto, the measurement (quantitative and qualitative) of various components in a biological sample has been applied to diagnosis of disease, tracking of prognosis, periodic diagnosis, screening test of health diagnosis, and the like. In such clinical tests, various components in biological samples are measured using various reactions, but some of the reactions used here include chelating substances mixed in the sample. Some are affected. One such example is the measurement of heavy metals in serum. That is,
Measurement of heavy metals in serum is used for diagnosis of various diseases.For example, serum iron is high in aplastic anemia, myelodysplastic syndrome, etc., and serum iron is high in iron deficiency anemia, hemorrhagic anemia, etc. It is known that iron is low. Similarly, serum contents of copper, zinc, magnesium and the like are known to be closely related to various diseases. Atomic absorption spectrometry is used to measure trace amounts of heavy metals in serum, but recently, chelate color formers with a large molecular extinction coefficient have been developed, and heavy metals can be easily measured by colorimetry. Many applications have been made in the measurement of

[0003] The measurement of heavy metals using such a chelate color former is carried out by utilizing the color development, the shift of the absorption wavelength, the change in the degree of coloration, and the like due to the reaction between the chelate color former and the heavy metal. However, chelating substances (eg, citric acid, phosphoric acid, oxalic acid, etc.) in biological samples
When a heavy metal in a sample containing a chelating substance is measured by a measurement method using a chelating colorant, the chelating substance prevents the coloring of the chelating colorant, etc. Causes an error. In addition, in general, the reaction is slowed by the mixture of a chelating substance, a long time is required for measurement, and an automatic analyzer that needs to process many samples in a short time (about 3 to 5 minutes / sample). Is not suitable. Furthermore, in order to prevent coagulation of the collected blood at the time of collecting a blood sample, a blood collection method in which EDTA (ethylenediaminetetraacetic acid), citric acid, or the like is usually added as an anticoagulant is widely used. Is also affected by strong chelating agents such as EDTA and citric acid, causing errors in measured values.
Therefore, a measurement method using a chelate coloring agent cannot be used for a sample collected by this method.

Other examples include metal-requiring enzymes (eg, magnesium, manganese, iron, copper,
Measurement of the activity of an enzyme that requires a metal such as zinc) or measurement of components in a biological sample by an enzyme reaction system using a metal-requiring enzyme. In these cases, a metal is added in advance to the reaction reagent or a metal is added to the reaction system in order to activate the activity of the enzyme. However, in a sample containing a chelating substance as described above, there is a problem that the added metal forms a complex with the chelating substance and is trapped, thereby causing a variation in measured values. In order to avoid this problem, a large excess of metal is added.However, the addition of a large excess of metal causes inhibition of the activity of metal-requiring enzymes, and in a measurement system using an enzyme-coupled system, There is a problem of affecting the activity of other enzymes.

[0005]

As described above, when measuring a component in a sample, if the sample contains a chelating substance, the measuring method using a reaction affected by the chelating substance measures the component to be measured. I can't. The present invention has been made in order to solve the above-mentioned problems of the prior art, and a method for avoiding the influence of a chelating substance mixed in a biological sample, and for accurately and quickly measuring a measurement target component in a biological sample. The purpose is to provide.

[0006]

Means for Solving the Problems In view of the above circumstances, the present inventors have diligently developed a method for avoiding the influence of a chelating substance in a measuring method using a reaction affected by a chelating substance mixed in a biological sample. As a result of the examination, the presence of a metal ion which is not involved in the measurement reaction and can be bound to the chelating substance expected to be mixed in the sample at the time of the measurement is present. In addition, the present inventors have found that a component to be measured in a sample can be measured accurately and quickly without being hindered by a chelating substance, and thus completed the present invention. That is, the measurement method of the present invention is a method for measuring a component in a biological sample, and in a measurement method using a reaction affected by a chelating substance mixed in a sample, the chelating property not involved in the reaction and mixed in the reaction. It consists in performing the measurement in the presence of metal ions which bind to the substance.

In the present invention having the above-mentioned structure, examples of the measuring method using a reaction affected by a chelating substance mixed in a sample include, for example, measurement of a heavy metal using a chelate coloring agent, and measurement of an enzymatic activity of a metal-requiring enzyme. Measurement, measurement of components in a biological sample by an enzyme reaction system using a metal-requiring enzyme, and the like. More specifically, the measurement of heavy metals using a chelating color former includes, for example, Feren-S,
Measurement of iron using nitrosopropylsulfopropylaminophenol (nitroso-PSAP), bathophenanthroline, ferrosin, 2,4,6-tripyridyl-S-triazine, and copper measurement using bathocaproinsulfonic acid, etc. Measurement, measurement of zinc using 1- (2-pyridylazo) -2-naphthol (PAN), dithizone and the like are exemplified.

[0008] The measurement of the enzymatic activity of metal-requiring enzymes includes, for example, oxidoreductases (eg, isocitrate dehydrogenase (EC 1.1.1.41 and 1.1.1.42)) and hydrolytic enzymes [eg, alkaline phosphatase (eg, alkaline phosphatase)]. EC3.
1.3.1) etc.], phosphotransferases [eg creatine kinase (EC 2.7.3.2), pyruvate kinase (EC 2.7.1.4)
0), glucokinase (EC 2.7.1.2), ketohexokinase (EC 2.7.1.3), 6-phosphofructokinase (EC
2.7.1.11), ribokinase (EC 2.7.1.15), xylokinase (EC 2.7.1.17), protein kinase (EC 2.7.1.3)
7) etc.].

Further, examples of the measurement of components in a biological sample by an enzyme reaction system using a metal-requiring enzyme include measurement of organic components and inorganic components using the above-described enzyme reaction of a metal-requiring enzyme. Hexokinase acts on -glucose to produce glucose-6-phosphate, then reacts glucose-6-phosphate dehydrogenase with NADP, measures the resulting NADPH, measures the blood sugar level, magnesium using the above reaction system Urea is reacted with urea to produce ammonia, and then glutamate dehydrogenase is reacted with NAD (P) to produce NAD (P) H
Measurement of urea nitrogen, which measures, is exemplified, but is not limited thereto.

The metal ion used in the present invention which is not involved in the measurement reaction and binds to the mixed chelating substance is not particularly limited as long as it selectively binds to the chelating substance mixed in the sample. Instead, it can be appropriately selected depending on the mixed chelating substance, and for example, calcium ion, magnesium ion, zinc ion, iron (monovalent) ion, tin (monovalent) ion and the like are preferable.
These ions are used as water-soluble salts (eg, hydrochloride, acetate, etc.). The amount of metal ions used is appropriately adjusted depending on the amount of the chelating substance mixed in the sample, etc., but usually, the metal ion concentration in the measurement sample solution is 0.1 mM to 50 m
M, preferably about 0.2 mM to 20 mM, more preferably 0.1 mM.
It is used after being adjusted to about 3 mM to 10 mM. If the amount of metal ions used is less than 0.1 mM, the effect of addition is not recognized, and if the amount exceeds 50 mM, there is no problem, but a sufficient effect can be obtained up to that amount.

In the measurement method of the present invention, the metal ion may be present at the time of measurement, and a metal salt may be added to a reagent solution for measurement, or a metal salt may be added to a sample such as serum. Further, a metal salt may be added at the time of mixing the reagent solution and the sample. Preferably, a method in which a metal salt is added to the measurement reagent solution in advance is used. The measurement method of the present invention can be carried out in the same manner as in the conventional method except that the measurement is carried out in the presence of the above-mentioned metal ion. For example, in the case of a method for measuring a heavy metal using a chelate coloring agent, an appropriate solvent ,
(Purified water, Tris buffer, glycine buffer, etc.) and the above-mentioned metal salts and, if necessary, a solution containing a surfactant, a reducing agent, a stabilizer, etc. are prepared. A sample (which may be diluted if necessary) is mixed, and after adding a reagent solution containing a chelating color former, the mixture is incubated for a predetermined time to obtain a measurement sample solution.
Next, heavy metals can be measured by colorimetric determination according to a conventional method. The measurement can be performed by any of the rate assay method and the end point method.

The biological sample according to the present invention includes blood,
Serum, plasma, urine, cerebrospinal fluid and the like can be mentioned. Particularly, serum and plasma are used as samples, and are suitably used for measurement of heavy metals using a chelating color former or a metal-requiring enzyme, and measurement of metal-requiring enzyme activity. In addition, the measurement method of the present invention is suitable for measurement using an automatic analyzer because the measurement is completed in a short time (generally, about 1 to 10 minutes). In the practice of the present invention, depending on the measurement method and the like, a surfactant (eg, lauryl sulfate, Triton, etc.), a reducing agent (eg, ascorbic acid, thioglycolic acid, etc.), a stabilizer, You may add the usual additives, such as.

[0013]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. Example 1 Measurement of Serum Iron Using Feren-S A reagent solution having the following composition was prepared. First reagent Bis-Tris 100mM lithium lauryl sulfate 7.0% Triton X-405 1.2% thiourea 65.7mM ascorbic acid 0.5% calcium chloride 5mM acetic acid qs pH 6.1 Second reagent Feren-S 5mM When normal human serum was measured, a serum sample (4 samples) whose measured values did not show abnormalities and a serum sample (4 samples) showing abnormalities were selected (see Table 1). Sample 20μl and first reagent 400
Take the μl in a cuvette, incubate at 37 ° C for 5 minutes, measure the absorbance after 5 minutes (measurement wavelength: 600 nm), and then
0 μl was added, the temperature was kept at 37 ° C., and the absorbance was measured again 5 minutes later. As a control, the same operation was performed using purified water instead of the sample. Further, the amount of change in absorbance in an iron standard solution (500 μg / dl) was determined, and the concentration of iron in each serum sample was calculated from the amount of change in absorbance. For comparison, the same sample was measured in the same manner using the first reagent to which calcium chloride was not added. Furthermore, the measurement was also performed on a commercially available reagent using nitroso-PSAP as a chelating color former. Table 1 shows the results. As shown in Table 1, according to the ferren-S method (the method of the present invention) to which calcium chloride was added, a good measurement value was obtained even for a sample showing a low value.

[0014]

[Table 1]

Example 2 Measurement of Unsaturated Iron Binding Ability (UIBC) The following reagent solutions were prepared. First reagent Tris 500 mM potassium chloride 0.5% Triton X-405 0.2% iminodiacetic acid 1 mM thiourea 65.7 mM ferrous ammonium sulfate 0.015 mM hydroxylamine hydrochloride 36 mM calcium chloride 10 mM pH 8.5 second reagent ferren-S 10 mM iminodiacetic acid 5 mM hydrochloric acid Hydroxylamine 360 mM Transferrin from iron (Seikagaku Corporation) 700 mg / dl
Was added to physiological saline containing 1 mM so as to be 1 mM to prepare a sample having a high UIBC value. Calibration was confirmed using a solution obtained by diluting this sample with EDTA · 2Na 1 mM solution in 10 steps. The measurement was performed by the same operation as in Example 1. The UIBC value is determined by measuring how much the absorbance decreases compared to the control (purified water) because excess iron is added in advance and the amount of transferrin in the sample that can be taken in is measured. In this case, if iron is excessively removed by the chelating substance (EDTA · 2Na) mixed in the sample, the UIBC value becomes large. As a comparison, a commercially available Feren-S method without addition of calcium chloride,
The same sample was measured by the ferrozine coloring method and the nitroso-PSAP method. The result is shown in FIG. In FIG. 1, ●
Is the ferrite-S method with calcium chloride (the method of the present invention),
○ indicates the ferrite-S method without addition of calcium chloride, フ ェ indicates the ferrozine coloring method, and △ indicates the nitroso-PSAP method. As is clear from FIG. 1, the method of the present invention exhibited better calibration than the method without calcium chloride.

Example 3 Measurement of Human Serum and Plasma UIBC Values Using the same reagents as in Example 2, the same procedure as in Example 2 was used.
The UIBC value was measured using human serum and plasma as samples. Table 2 shows the results. As shown in Table 2, a sample showing an abnormality by another method was well measured by the method of the present invention.

[0017]

[Table 2]

Example 4 Measurement of Serum Magnesium by Enzymatic Method The following reagent solution was prepared. First reagent Tris 100 mM β-NADP 0.7 mM hexokinase 0.45 units / ml glucose-6-phosphate dehydrogenase 0.75 units / ml ATP 12.5 mM pH 8.5 Second reagent Tris 100 mM glucose 50 mM calcium chloride 5 mM pH 8.5 As a sample, EDTA What was added to serum so that 2Na was 200 mg / dl at the maximum, and diluted with the same amount of purified water added to serum was used. To 7 μl of this sample, 320 μl of the first reagent is added, the temperature is kept constant at 37 ° C. for 5 minutes, then 80 μl of the second reagent is added, and the change in average absorbance per minute at a wavelength of 340 nm is determined from 1.5 minutes. The same operation is performed as a blind test using purified water instead of the sample. The same operation was performed for the magnesium standard solution (5 mg / dl).
From the average change in absorbance, the magnesium concentration in the sample was calculated. As a comparison, magnesium was measured for the same sample by the same operation using a reagent solution obtained by removing calcium chloride from the second reagent. Table 3 shows the results. As shown in Table 3, the method using a reagent to which calcium chloride was not added was affected by EDTA · 2Na and the measured value was reduced, but the method using the reagent to which calcium chloride was added (the method of the present invention) was not used.・ 2Na
Good measurement value without being affected by

[0019]

[Table 3]

Example 5 Measurement of Serum Alkaline Phosphatase (ALP) Activity The following reagent solutions were prepared. First reagent Ethylaminoethanol 100 mM Magnesium chloride 0.65 mM Calcium chloride 0.45 mM Hydrochloric acid Appropriate amount pH 9.9 Second reagent p-Nitrophenyl phosphate disodium 76 mM Serum prepared in Example 4 was used as a sample. Take 6 μl of this sample, mix with 320 μl of the first reagent, incubate at 37 ° C. for 5 minutes, then add 80 μl of the second reagent, and after 2 minutes,
The average change in absorbance per minute at 405 nm was determined. As a blind test, the same operation was performed using purified water instead of the sample. For the measured value, the activity value was determined from the molecular extinction coefficient of p-nitrophenol based on the following formula. As a comparison, the same sample was subjected to the same operation using a reagent solution obtained by removing calcium chloride from the first reagent.
LP activity was measured. Table 4 shows the results. Table 4
As shown in the above, the method using a reagent without the addition of calcium chloride is affected by EDTA · 2Na and the measured value is reduced, but the method using the reagent with calcium chloride (the method of the present invention) uses EDTA · 2Na. Good measurement value without being affected by

[0021]

[Table 4]

[0022]

As described above, according to the measurement method of the present invention, the influence of a chelating substance mixed in a sample can be avoided. Can be measured with high accuracy, the measurement can be completed in a short time, and the present invention can be applied to automatic analysis.

[Brief description of the drawings]

FIG. 1 is a diagram showing a calibration curve according to the measurement method of the present invention.
In FIG. 1, ● represents the ferrite-S method with calcium chloride added (the method of the present invention);
S method, □ indicates ferrosin color development method, Δ indicates nitroso-PSAP
Show the law.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 33/90 C12Q 1/32 C12Q 1/42 C12Q 1/48

Claims (3)

(57) [Claims] 1. Chelate coloration of heavy metals in biological samples
In the measurement method using a reaction which is affected by a chelating substance mixed in a sample, the method is performed in the presence of a metal ion which does not participate in the reaction and binds to the mixed chelating substance. A method for measuring heavy metals in a biological sample, comprising performing measurement. 2. A heavy metal in a biological sample is converted to a metal-requiring enzyme.
Is a method for measurement using
Measurement method using reactions affected by toxic substances
And a chelating substance that does not participate in the reaction and is mixed
The measurement is performed in the presence of the binding metal ion.
Of heavy metals in biological samples. 3. The enzyme activity of a metal-requiring enzyme in a biological sample.
Is a method of adding the required metal and measuring the
Use reactions affected by mixed chelating substances
In the measurement method, clean particles that do not participate in the reaction and are mixed
The measurement in the presence of metal ions that bind to
Enzyme activity of metal-requiring enzymes in biological samples
Measurement method.