JPH0630791A - Quantification of saccharified protein - Google Patents

Abstract

PURPOSE:To quickly and easily quantify in high accuracy the saccharified proteins in biological tissues (esp. blood). CONSTITUTION:A specimen of biological tissue origin is treated with hydrazine and then made to react with a color developing reagent to carry out colorimetry, thus accomplishing the objective easy and quick determination of the saccharified proteins in the blood or hair. This method is hardly affected by the coexistent substances in the blood, therefore enabling quantification in high accuracy, besides, being good in color reaction and measurement sensitivity. Specimens to be used are those put to oxidative treatment, with e.g. glucosidase, of the glucose coexisting with the saccharified proteins to be determined. For the color developing reagent, phenylhydrazine is used.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for quantifying glycated proteins in living tissues, particularly blood and hair, and is useful for diagnosing diabetes.

[0002]

2. Description of the Related Art Conventionally, for the purpose of treating and diagnosing diabetes,
There are several known methods for measuring the concentration of glycated protein, which is an index of blood sugar control. Each of these methods is a method for directly or indirectly colorimetrically quantifying a ketoamine-type glycated protein (hereinafter, simply referred to as glycated protein) produced by a protein glycated with glucose through Amadori rearrangement. However, they have various drawbacks in their implementation. For example, the furosine method described in J. Clin. Chem. Clin. Biochem., 19 , 81 (1981), the thiobarbituric acid method described in Diabetes, 29 , 417 (1980), Diabetes, 29 , 1044 (1980) In the affinity column method described in 1), the operation is complicated, the reaction temperature is high, and the reaction time is as long as several tens hours. Further, in order to avoid the influence of coexisting substances in blood, for example, reducing substances other than glycated proteins (glucose, ascorbic acid, glutathione, uric acid, etc.), it is necessary to perform complicated operations such as dialysis. In addition, a calibrator for quantification
This is practically inconvenient because it requires quantification of albumin or total protein.

Japanese Patent Publication No. 1-10662 and Clin. Chi
m. Acta, 127 , 87 (1983), a method for quantification using a nitroblu-tetrazolium salt as a color former (hereinafter referred to as N
BT-reduction method). In this method, the generated formazan is likely to adhere to the measuring instrument, and it is difficult to remove it, and the apparent absorbance of formazan changes due to the lipids and proteins coexisting in the liquid sample. There is. Further, the reactivity is considerably different between the water-soluble standard solution and the serum standard solution, so that the calibration is complicated. Furthermore, since this method utilizes the reducibility of glycated proteins under alkaline conditions, in order to reduce or eliminate the effect of the reducing substance, quantification by a rate assay method or these methods is used. Removal had to be done before or during quantification. J. Biol. Chem., 255 , 7218 (1980) describes a colorimetric assay method using phenylhydrazine (hereinafter simply referred to as the phenylhydrazine method), but it has a weak coloration and has been practically used clinically. Has not reached the end.

[0004]

[PROBLEMS TO BE SOLVED] The present invention provides a method for quantifying glycated proteins in blood simply and rapidly without the above-mentioned drawbacks and difficulties.

[0005]

[Means for Solving the Problems] In view of the above problems, the inventors of the present invention have made diligent studies, and after treating a specimen derived from a biological tissue with hydrazine and then reacting it with a coloring reagent for colorimetric quantification, The present inventors have completed the present invention by finding that the glycated protein of E. coli can be quantified. In the present specification, the sample may be any sample containing a glycated protein, but is preferably a blood sample or a hair sample. The blood sample means blood itself, plasma or serum, etc.
From the viewpoint of measurement accuracy and the like, it is preferable to use serum.
The hair sample means the hair itself or the hair that has been subjected to a treatment such as washing for easy measurement. In addition, as the sample, in order to remove the influence of glucose coexisting with a glycated protein on the present quantification method, the glucose was
The one that has been subjected to an oxidation treatment with glucose oxidase or the like is used. Usually, a blood sample may be incubated in the presence of about 5 to 20 units of glucose oxidase at body temperature for about several minutes to several tens of minutes.

The present quantification method will be described in detail below. (First step) This step is a step of treating the pretreated sample with hydrazine. For this treatment reaction, hydrazine dissolved in distilled water, alcohols or a mixed solution thereof (hereinafter simply referred to as hydrazine reagent) is used, but the pH of the reaction solution is adjusted in advance. There is a need. The pH at which the reaction proceeds is about 4.0 or more, but it is desirable to carry out the reaction under basic conditions in order to control side reactions as much as possible, and the preferable pH range is about 8.0 to about 12 0.0, more preferably about 9 to 10.0. pH
For adjustment, appropriately, a known acid may be used,
A weak acid such as acetic acid is preferable. The concentration of hydrazine is not necessarily limited, but is preferably about 3 to 10.0.
It is mol / L. The reaction temperature is about 30 to 130 ° C, preferably about 60 to about 110 ° C, particularly preferably about 10
It is 0 ° C. If it is lower than about 30 ° C., the reactivity is lowered, and if it is higher than about 130 ° C., it adversely affects the colorimetric determination in the second step, which is not preferable. The processing time required for this step is about 5 to about 60 minutes, more preferably about 10.
It is about 40 minutes, particularly preferably about 30 minutes.
If the time is less than about 5 minutes, the reaction does not proceed sufficiently, so that accurate quantification is difficult. On the other hand, since the reaction is almost completed by heating for about 60 minutes, further reaction is not preferable from the viewpoint of rapid quantification.

(Second step) After the first step is finished,
In this step, the absorbance of the glucose derivative produced by subsequent reaction with the color-forming reagent is measured and colorimetrically determined. As the coloring reagent, a well-known carbonyl coloring reagent of the general formula, H ₂ N—NH—R, of the hydrazine derivative type can be widely used, and phenylhydrazine, p-nitrophenylhydrazine, phenylhydrazine-p-sulfonic acid, m -Tolylhydrazine, p-tolylhydrazine, 2
Examples thereof include hydrazinobenzothiazole, 2-hydrazinopyridine, and 1-hydrazinophthalazine, with phenylhydrazine being preferred. The reaction temperature varies depending on the type of the color-developing reagent used and is not necessarily limited. For example, when phenylhydrazine is used as the color-developing reagent, it is about 30 to 80 ° C., preferably about 50.
˜70 ° C., particularly preferably about 60 ° C. If the temperature is higher than about 80 ° C, the colorability is lowered, and if the temperature is lower than about 30 ° C, the reactivity is lowered.

The colorimetric determination can be carried out by a commonly used absorbance measuring method. In the present invention, not only the so-called end point method but also the rate assay method can be used depending on the selection of the coloring reagent. The time required to complete the reaction of the second step cannot be unconditionally specified because it depends on the reaction temperature, the type of color-developing reagent used, etc., but if a preferred color-developing reagent is used, it is usually several tens of minutes to
It takes about several hours. Therefore, even when using the endpoint method, a relatively short time, preferably about 0.5 to 3 hours,
The absorbance may be measured after the reaction in the second step. Further, by using a coloring reagent whose absorbance at the initial stage of reaction shows linearity in a time-dependent manner, measurement by a rate assay method within a few minutes to a few tens of minutes after starting the reaction in the second step is also possible. Is. In particular, in this quantification method,
If phenylhydrazine is selected as the coloring reagent, the reaction in the second step is almost completed in about 30 to 60 minutes. Therefore,
Quantification by the endpoint method is possible at any point during that time, but the stability of the coloration of the reaction product, the phenylhydrazone derivative of glucose (D-Glucose-bis-phenylhydrazone), is extremely good. There is no problem with measuring after a few hours have passed. Also, within about 1 to 15 minutes after addition of the coloring reagent, preferably about 5 to about 10
Within minutes, the time course of the absorbance shows a steep linearity, which allows quantification by the rate assay method. The measurement wavelength may vary depending on the type of the color-developing reagent used and cannot be specified unconditionally, but the wavelength at which the generated color-developing substance exhibits the maximum absorbance is usually adopted. In particular,
Approximately 390 nm when using phenylhydrazine
Is preferred.

Conventionally, in the determination of glycated proteins in blood, coexisting substances in blood, particularly reducing substances other than glycated proteins (eg, ascorbic acid, glutathione, uric acid,
Although the influence of pyrirubin, glyceraldehyde, reducing sugars, etc. was an important issue to be investigated, the influence of these interfering substances was extremely low in this quantification method, and a protective measure should be taken especially at the normal level of quantification. Needless to say, it is naturally desirable to incorporate known protective means for the measurement when performing more precise quantification.

When carrying out the calibration in this quantification method, a commercially available synthetic saccharified compound such as Np-to is used.
A calibration curve may be prepared using lyl-D-isoglucosamine. By the way, since there are various proteins capable of forming glycated proteins in blood, such as albumin, prothrombin, and globulin, it is unclear whether or not a specific glycated protein is quantified in this quantification method. However, most of glycated proteins in blood are glycated albumin, which is remarkable especially in serum. Therefore, it is considered that the measured values obtained by the present quantification method sufficiently reflect the concentration of glycated albumin in blood. As shown in the examples described below, sufficiently satisfactory results were obtained when this quantification method was performed using a glycated albumin (Glc HSA) solution prepared from human serum albumin as a sample. This is also suggested. Therefore, taking into account the half-life of albumin in blood, the present quantification method is particularly suitable for
It is useful to know the blood glucose control status of a week ago,
It is thought to be useful for treatment and diagnosis of diabetes and the like. In the case of using a hair sample, the literature (Diabetes 34, 571,
1991) and the like, it is also useful to know the past continuous blood glucose control status, the long-term average blood glucose level, the onset time of diabetes, and the like. Reference examples, examples and test examples are shown below, but these do not limit the present invention in any way.

Reference Example 1 (Preparation of Glc HSA Solution) In accordance with the method described in Chem. Pharm. Bull, 40 , 255 (1992), human serum albumin (HSA, human serum albumin,
1g) and D-glucose (2g) in phosphate buffer (20mL, 0.067mo)
It was dissolved in 1 / L, pH 7.4) and incubated at 37 ° C for 2 days. The reaction solution was dialyzed for 2 days at 4 ° C using a dialysis tube. The phosphate buffer, which is the dialysate, was updated daily. Glc HSA concentration was measured by NBT-reduction method and
The HSA preparation (fructosamine value, 1480 μmol / L; albumin, 29 g / L) was dispensed in 1.5 mL aliquots and stored frozen at -20 ° C.

Example (Preparation of Hydrazine Reagent) 3 mL of hydrazine monohydrate (H ₂ N-NH ₂ .H ₂ O) was added to 10
Dissolve in mL of distilled water, add acetic acid to this and add to pH 9.4.
(Final concentration, about 4.0 mol / L) (Preparation of phenylhydrazine solution) 40% acetic acid solution of phenylhydrazine hydrochloride (final concentration, 0.02 mol / L)
L) was prepared. (General quantification method) Sample and hydrazine reagent (0.1m
L, approx. 4.0 mol / L, pH 9.4) at 100 ° C
After heating for 30 minutes at phenylhydrazine solution (0.6m
L, 0.02 mol / L, containing 40% acetic acid) 6
After heating at 0 ° C. for a fixed time (up to 60 minutes), the reaction solution was centrifuged (1400 × g for 10 minutes), and the absorbance obtained at each time was measured at 390 nm for the supernatant obtained. When human serum is used as the sample, the same amount of glucose-oxidase (about 5 units) is added and the temperature is adjusted to 37 ° C.
After 30 minutes of incubation, the reaction was carried out.

Example 1 (Quantification of Glc HSA) According to the above-mentioned general quantification method, a mixed solution of an aqueous solution of Glc HSA (0.1 mL) of various concentrations and a hydrazine reagent (0.1 mL) was heated at 100 ° C. for 10 minutes and then subjected to phenyl. A hydrazine solution (0.6 mL) was added and heated at 50 ° C., and the absorbance at each time was measured. When the reaction product after the reaction in the second step was carried out for 60 minutes was isolated, its UV absorption spectrum (λ _max (in AcOH) _nm (ε): 389.8 (2.5 × 10 ^-4 )) showed that D- It was confirmed to be Glucose-bis-phenylhydrazone.

FIG. 1 shows the change in the absorbance when the reaction of the second step was carried out for a short time. In the initial reaction within about 1 to 5 minutes, the absorbance showed a linear increase in a time-dependent manner, and therefore it is expected that measurement by the rate assay method is possible. The absorbance was measured in the same manner as above except that Glc HSA (185 μmol / L) was used and the temperature in the second step was changed. The results are shown in Figure 2. When the heating temperature is about 50 to 70 ° C, the absorbance is about 6 after the reaction is started.
It increased almost to 0 minutes in a time-dependent manner.

Example 2 (Correlation with NBT-reduction method) A clinical sample (serum, n = 35) was quantified as a sample, and at the same time, NBT-disclosed in Japanese Patent Publication No. 1-10662.
The correlation with the reduction method was obtained. (Test method) The present invention : sample (0.05 mL) and glucose oxidase
Zeze solution (5 units, 0.05 mL) at 37 ℃
After the incubation for 1 minute, the hydrazine reagent (0.1 mL) was added according to the general quantification method described above, and the mixture was heated at 100 ° C. for 30 minutes. Further, a phenylhydrazine solution (0.6 mL) was added, the mixture was heated at 60 ° C. for 60 minutes, and the absorbance of the supernatant obtained by centrifugation was measured. NBT-reduction method : A mixture of a sample (0.05 mL) and a coloring reagent (nitromol-tetrazolium chloride (0.48 mmol / L) -containing 0.2 mol / L carbonate buffer, pH 10.3, 1 mL) was incubated at 37 ° C. 10 minutes and 1 after
After 5 minutes, the absorbance at 546 nm was measured using the reagent blank as a control. The glycation degree (μmol / L) of the protein was determined by the following formula. Saccharification degree _{= (△ E S / △ Ec} ) × standard solution glycation degree (although, △ Es, △ Ec is 10 samples, standard solutions, respectively
The difference in absorbance after 15 minutes is shown. ) The results are shown in FIG.
The correlation coefficient is r = 0.92 (y = 1.2x−7.1, SD =
63.9, n = 35), and both results showed good correlation.

Example 3 (Quantification of Glycated Protein in Blood in Diabetic Patients and Normal Subjects) (Target) Normal subjects: men and women (age 2
2-52 years old, n = 32) Diabetic patient: Patient determined to be diabetic based on WHO standard (age 47-75 years old, n = 32) (Preparation of specimen) Blood collected before meals (blood coagulation inhibitor is not After the addition) solidified, centrifuge (1400 xg, 10
And the supernatant serum was stored at -20 ° C. (Quantitative method) A mixture of a sample (0.05 mL) and a glucose-oxidase solution (5 units, dissolved in a 0.2 mol / L phosphate buffer solution, 0.05 mL) was incubated at 37 ° C for 30 minutes. The hydrazine reagent (0.1 mL) was added according to the above general quantification method, and the mixture was heated at 100 ° C. for 30 minutes. Furthermore, phenylhydrazine solution (0.6m
L) and heated at 60 ° C. for 60 minutes, and then centrifuged (1
The absorbance of the obtained supernatant was measured at 400 xg for 10 minutes. (Results) The results are shown in FIG. 7. A significant difference was observed when comparing the average values of glycated protein concentration in blood between a normal person and a diabetic patient. Example 4 (Quantification of Glycated Protein in Hair in Diabetic Patients and Normal Persons) (Target) Normal persons: men and women who were certified as healthy by a group physical examination (age 2
2-52 years old, n = 32) Diabetic patient: Patients judged to be diabetic based on WHO standard (age 47-75 years old, n = 32) (Preparation of specimen) Ethanol at 60 ° C and distilled water (1 mL each) ) Suspended the hair fragment (about 10 mg, 1 mm or less) washed with distilled water (0.1 mL). (Quantitative method) A hydrazine reagent (0.1 mL) was added to distilled water suspension (0.1 mL) according to the above-mentioned general quantitative method, and heated at 100 ° C for 30 minutes. Further, a phenylhydrazine solution (0.6 mL) was added, the mixture was heated at 60 ° C. for 60 minutes, and then centrifuged (1400 × g, 10 minutes) to measure the absorbance of the obtained supernatant. (Results) The results are shown in FIG. A significant difference was observed when comparing the average values of glycated protein concentration in hair between a normal person and a diabetic patient. Therefore, it is suggested that the quantification method of the present invention is useful for diagnosing diabetes even when human hair is used as a sample. Test Example 1 (Comparison of color developability) The color developability was compared with the phenylhydrazine method described in the above literature. (Sample) Glc HSA solution (180 μmol / L, 0.1 m
L) (Test method) According to the method of the present invention, a mixed solution of a sample and a hydrazine reagent was heated at 100 ° C for 30 minutes, then a phenylhydrazine solution was added and the mixture was heated at 60 ° C to measure the absorbance at 390 nm. It was measured. Phenylhydrazine method: A mixture of a sample and distilled water (0.1 mL) is left at room temperature for 30 minutes, then a phenylhydrazine solution (0.6 mL) is added and heated at 60 ° C. to give 390 n.
Absorbance was measured at m. The results are shown in Fig. 4. This quantitative method showed significantly stronger coloration than the phenylhydrazine method.

Test Example 2 (Coloring stability) (Sample) Glc HSA solution (360 or 720 μmol / L, 0.1 m)
L) (Test method) According to the above-mentioned general quantification method, a mixed solution of a sample and a hydrazine reagent was heated at 100 ° C for 30 minutes, and then a phenylhydrazine solution was added and heated at 60 ° C for 60 minutes. The reaction solution was centrifuged, and the supernatant was stored at room temperature (22-23
After standing at (° C.), the absorbance was measured. Results are shown in FIG.
Since the change in absorbance after 10 to 60 minutes after leaving was about 0.4 to 2.2% with respect to the initial value, it can be seen that the coloration stability in this quantitative method is extremely high.

Test Example 3 (Effect of Pretreatment with Glucose Oxidase) (Sample) Test Sample: Glucose (27.8 mmol / L) and Glucose Oxidase (0 to 20.8 units, 50 μm)
L) containing Glc HSA solution (740 μmol / L, 5
0 μL) Control sample: Glc HSA solution in which distilled water was added to the test sample instead of glucose (Test method) Each sample was incubated at 37 ° C. for 30 minutes, and then according to the general quantification method described above. The mixture of the sample and the hydrazine reagent was heated at 100 ° C. for 30 minutes, then a phenylhydrazine solution was added, and the mixture was heated at 60 ° C. to measure the absorbance. Using the absorbance value of the test sample as OD (test) and the absorbance value of the control sample as OD (control), the effect of pretreatment with glucose oxidase was compared with the control ratio (%) ((OD
(test) / OD (control)) × 100 (%), (OD (tes
t): absorbance value of test sample, OD (control): absorbance value of control sample) are shown in FIG. When glucose oxidase was about 5 units or more, the control ratio was almost 100%, and it was found that the glucose in the sample was oxidized and the influence on the coloration system was almost eliminated. Therefore, in the case of a human serum sample, pretreatment with glucosoxidase (about 5 units or more) may be performed, and preferably about 30 ° C at about 30 ° C.
Incubate for about a minute.

Test Example 4 (Measurement Accuracy) Simultaneous and day-to-day reproducibility was determined by this quantification method. (Sample) 4 to 6 types of samples obtained by diluting a Glc HSA (1440 μmol / L) solution with distilled water. (Test method) Any reproducibility was measured and determined according to the above-mentioned general quantitative method. However, the simultaneous reproducibility was measured by reacting the frozen sample (n = 10) at the same time, and the daily reproducibility was measured by the frozen sample (n = 10). , One each day 10
It was examined by reacting and measuring for a day. Each reproducibility (CV,%) was calculated from the ratio (%) of the error to each average value. The results are shown in Table 1.

[Table 1] Simultaneously and with respect to reproducibility by day difference, when the sample concentration was about 10 μmol / L or more, the CV value was about 10% or less, and high measurement accuracy was obtained.

Test Example 5 (Accuracy) (Sample) A sample prepared by adding 25 μL of Glc HSA solution of various concentrations to the pooled normal human serum (25 μL). (Test method) A mixed solution of the sample (50 μL) and glucosoxidase (5 units, 50 μL) was incubated at 37 ° C. for 30 minutes, and then quantified according to the general quantification method (OD _A ). On the other hand, as a blank, a sample obtained by adding the same amount of distilled water to the pulled normal human serum instead of the Glc HSA solution was used to similarly determine the absorbance (OD
_B ). Similarly, Glc H was added to distilled water instead of human serum.
The absorbance obtained from the sample to which the SA solution was added (OD _C ),
The absorbance obtained by adding the same amount of distilled water to the distilled water instead of the Glc HSA solution is defined as (OD _D ). The recovery rate is [(O
_{D A -OD B) / (OD} C -OD D)] results obtained as × 100 (%) shown in Table 2.

[Table 2] Since the recovery rate was close to 90 to 100%,
In this quantification method, it can be seen that the reaction progresses selectively and quantitatively with high accuracy.

Test Example 6 (Influence of coexisting substances) The influence of serum components and other chemical substances on the present quantification method was examined. (Sample) Normal human serum (1 m) containing 0.1 mL of an aqueous solution of each substance (however, in the case of uric acid, an alkaline solution)
Samples were prepared in addition to L). The final concentration of added substance in each sample is 2-10 times the normal concentration. On the other hand, the one to which only the same amount of distilled water was added was used as a control. (Test method) Each sample was measured three times according to the above-mentioned general quantification method, and each average value (sample: X ₁ , control:
Control ratio (%) obtained from X ₂ ) [((X ₁ −X ₂ ) / X ₂ ).
× 100] is shown in Table 3.

[Table 3] Regardless of which substance is added, the control ratio is within several%, and it is presumed that this control method will not be affected if it is within about 2-3 times the upper limit of the normal blood concentration. Further, since the simultaneous reproducibility (CV) when measuring only the control was about 8%, the maximum concentration when the control ratio reached 8% was obtained. The results are shown in Table 4.

[Table 4] For typical sugars and proteins, the highest concentration at which the control ratio reached 8% was well above the normal concentration. Therefore, at the normal level, it is estimated that these coexisting substances have almost no effect on the present quantification method.

[0022]

INDUSTRIAL APPLICABILITY According to the present invention, glycated proteins in living tissues, particularly blood or hair, can be quantified rapidly and easily under mild conditions. This quantification method has high sensitivity, good color stability, and no problems such as adsorption of coloring substances.
In addition, it is highly reproducible and highly accurate and accurate quantification is possible.
It is a highly practical quantitative method suitable for automatic analysis by the rate assay method.

[Brief description of drawings]

FIG. 1 shows the relationship between the reaction time in the second step and the absorbance when Glc HSA at various concentrations was quantified by this quantification method.

FIG. 2 is a graph showing that Glc was changed by changing the reaction temperature in the second step.
The relationship between the reaction time and the absorbance in the second step when HSA is quantified by this quantification method is shown.

FIG. 3 shows the correlation between the present quantification method and the NBT-reduction method when glycated protein was quantified using human serum as a sample.

FIG. 4 shows the difference in color development between the present quantification method and the phenylhydrazine method when quantifying Glc HSA. The horizontal axis represents the reaction time of the second step.

FIG. 5 shows the coloration stability of Glc HSA when quantified by this quantification method. The horizontal axis represents the time elapsed after the reaction of the second step was performed for 60 minutes.

FIG. 6 is a graph showing glucose oxidase levels when Glc HSA was quantified by this quantification method in the presence of glucose.
The effect of pretreatment with ze is shown. The vertical axis represents the control ratio to the blank containing no glucose, and the horizontal axis represents the units of glucose oxidase.

FIG. 7 shows results of comparative measurement of blood glycated protein concentrations by the present quantification method in normal subjects and diabetic patients.

FIG. 8 shows results of comparative measurement of glycated protein concentrations in hair by the present quantification method in normal subjects and diabetic patients.

Claims (8)

[Claims] 1. A method for quantifying glycated protein, which comprises subjecting a sample obtained by oxidizing glucose coexisting with glycated protein to hydrazine, and then reacting it with a color former for colorimetric quantification. 2. The quantification method according to claim 1, wherein the sample is a blood sample or a hair sample. 3. The quantification method according to claim 1, wherein the oxidation treatment is carried out with glucose-oxidase. 4. The treatment with hydrazine has a pH value of about 8.0.
The quantification method according to claim 1, which is carried out at about 12.0. 5. The treatment with hydrazine comprises about 30 to about 13.
The quantification method according to claim 1, which is performed at 0 ° C. 6. The method according to claim 1, wherein the color former is phenylhydrazine. 7. The reaction with phenylhydrazine is carried out by about 30 to
The quantification method according to claim 6, which is performed at about 80 ° C. 8. The quantification method according to claim 1, wherein the colorimetric quantification is performed by a rate assay method.