JPH03216555A - Method for measuring fructosamine - Google Patents

Abstract

PURPOSE:To shorten a required measuring time and to apply the title method to a manual method and instrumental analysis by incubating a serum specimen in the presence of a water-soluble metal salt prior to adding a color former solution. CONSTITUTION:The first reagent being an alkali buffer solution containing a water-soluble metal salt is added to a serum specimen to incubate the specimen and, next, the second reagent being a color former solution is added to further incubate the specimen to measure absorbancy. Subsequently, various standard serums known in the concn. of fructosamine are used to be operated in the same way and absorbancies are measured to preliminarily prepare a standard calibration curve and the absorbancy value measured with respect to the serum specimen is collated with the calibration curve to measure the concn. of fructosamine in the serum specimen. By this method, the effect of an inhibitor on the measurement can be almost perfectly removed and, when the reagents are properly selected, a required measuring time can be shortened and this measuring method becomes easy even in a case executed by a manual method and can also be applied to instrumental analysis.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for measuring fructosamine, and is used for diagnostic tests for diabetes and for understanding its pathological condition.

(Prior art and its problems) Fructosamine is a reaction product of glucose and protein in blood and is a stable substance. Therefore, glucose concentration can be determined by measuring fructosamine concentration using blood, for example, serum as a sample. Therefore, measuring fructosamine is helpful in diagnosing diabetes and understanding its pathological condition.

In an alkaline environment, this fructosamine becomes an enol form and exhibits a reducing effect, so a method was developed to measure the concentration of fructosamine in serum by utilizing this effect [Japanese Patent Publication No. 1-13062 (1986-1)]
54GIliO)]. This measurement method involves adding an alkaline buffer containing a coloring agent, such as a tetrazolium salt such as nitroblue tetrazolium (NBT), to a serum sample and incubating it. The method uses the reducing action of fructosamine to change the coloring agent mentioned above into formazan dye to develop a color, and measures the absorbance of this liquid to measure the fructosamine concentration, which does not require expensive reagents and is easy to reproduce. Because of its relatively high performance, it has attracted attention and is called the "Baker method" after its inventor, John Rlchard Baker, and has come to be put to practical use as a method for measuring fructosamine. However, this Baker method utilizes the color change associated with the reaction rate, and therefore the first absorbance measurement is performed after mixing the serum sample and the hyperchromic reagent and incubating it for a while, and then incubating it again for a certain period of time. The problem lies in the fact that it is a two-point measurement method in which a second absorbance measurement is carried out after the second measurement, and that high accuracy is required in the measurement timing.

For this purpose, an endpoint measurement method was developed that requires only one absorbance measurement (Japanese Patent Application Laid-Open No. 1825-1825G7).

This method also uses the reducing action of enol-type fructosamine to convert the tetrazolium salt into a colored formazan dye, and then measures the absorbance. If a substance with a reducing effect such as acetone exists as an in-vivo substance or as a result of a drug administered to a subject, and in the end point measurement method, these reducing substances naturally interfere with the measurement of fructosamine. Since they behave as substances, it is necessary to take some measures to prevent them from having a negative effect. As a means for this, the above-mentioned Japanese Patent Application Laid-Open No. 83-182
According to Publication No. 567, it is proposed to pre-process a serum sample before adding a coloring reagent, and the following methods are taught as this pre-processing method.

a) long-term incubation at low temperatures, b) incubation at high temperatures, c) incubation days under alkaline conditions with a pH of 10 or higher, d) desalting treatment, e) oxidation treatment, and f) ) Enzyme treatment (objective of the invention) The object of the present invention is to provide a method for measuring fructosamine that does not require skill in operation and requires short measurement time, and is therefore applicable to both manual and instrumental analysis methods. .

(Means and Effects for Achieving the Object) In order to achieve the above object, the present inventor focused on the end point method disclosed in the above-mentioned Japanese Patent Application Laid-Open No. 11i3-182567, and made extensive efforts regarding sample pretreatment. As a result of repeated studies, we coincidentally found that processing serum samples in the presence of water-soluble metal salts significantly reduced the effects of measurement interfering substances to the point where they could be ignored, and also compared the time required for the processing. The present invention was completed by discovering the shortness of the term.

Therefore, in the method for measuring fructosamine according to the present invention, a first reagent, which is an alkaline buffer containing a water-soluble metal salt, is added to a serum sample and incubated, and then a second reagent, which is an aqueous solution of a color former, is added to the serum sample. After incubation, the absorbance was measured, and the above serum samples were measured using a standard calibration curve prepared in advance by measuring the absorbance in the same manner as above using various standard serums with known fructosamine concentrations. It is characterized by comparing absorbance values.

In the present invention, the above-mentioned water-soluble metal salt brings metal ions into the system, and examples of the metal ions include cobalt, nickel, chromium, manganese, iron, copper, zinc, etc. Cobalt is preferred, and its salts include, for example, cobalt oxalate. A typical example of the alkaline buffer is carbonate buffer, and its pn range should be about 10-14 from the viewpoint of expressing a reducing effect by converting fructosamine, which is the substance to be measured, into the enol form. is preferred. Reagent I is a so-called "pretreatment solution" for serum samples, and in the present invention, since it contains a water-soluble metal salt as described above, some amount of this metal salt is precipitated during incubation. Therefore, a suspending agent may be contained, and examples of the suspending agent include sodium potassium tartrate, sodium citrate, and ethylenediaminetetraacetate.

The incubation time required for pretreatment of serum samples is approximately 1-15 minutes.

The coloring agent in the second reagent is formed by the reaction between glucose and protein in the serum sample, and is reduced by fructosamine, which becomes an enol form due to the alkaline environment caused by the alkaline buffer in the first reagent. It can be used as long as it develops a color, and tetrazolium salts such as p
Examples include -iodonitrotetrazolium (INT), nitro blue tetrazolium (NBT), tetrazolium burble, and tetrazolium violet. The second reagent may contain a solubilizer to prevent precipitation of the formazan dye formed when the coloring agent is a tetrazolium salt, and a preservative to prevent deterioration. The above solubilizer is Trl
Examples of the preservative include surfactants such as ton I can give an example. After the second reagent is added, further incubation is performed, during which fructosamine in the sample serum is reduced to enol form because the surrounding environment is alkaline due to the alkaline buffer as described above. As a result, the above-mentioned color former, such as a tetrazolium salt, is reduced to a formazan dye, and the solution becomes colored. Although this color reaction takes a long time to reach a plateau, the incubation time required for the reaction to be practically completed is about 5 to 20 minutes.

Next, the absorbance of this reaction solution is measured.

The measurement wavelength depends on the type of coloring agent selected, but the maximum absorption wavelength of the formazan dye produced in the case of tetrazolium salt is around 500 na+ (INT530
nm, NTB 530 nm, etc.), and this maximum absorption wavelength is selected.

The absorbance measured in this way is used as a standard calibration curve, which is prepared by using various serum samples with known fructosamine concentrations as samples, operating as described above, measuring absorbance, and examining the relationship between fructosamine concentration and absorbance in advance. The fructosamine concentration of the above-mentioned sample serum can be determined by comparing it with the graph or other materials.

In the first step of the measurement method according to the present invention, the first reagent, which is an alkaline buffer containing a water-soluble metal salt, is added to the serum sample and incubated to remove a reducing substance, such as ascorbin, which may be present in the serum sample. This is to suppress or eliminate acids and the like from acting as measurement interfering substances, and corresponds to pretreatment of serum samples. Regarding the pretreatment of serum samples, Japanese Patent Application Laid-Open No. 1983-1997 introduced in the "Prior Art" section of this specification
As mentioned above, "incubation under alkaline conditions" is shown as one of the measures in Publication No. 3-1B2511i7. However, as shown in later test examples, when reducing substances such as ascorbic acid are present, simply incubating the serum sample in the presence of an alkaline buffer for a short period of time does not eliminate the effect on absorbance measurements. However, it should be noted that the influence of interfering substances can only be sufficiently eliminated when water-soluble metal salts are present during pretreatment as in the present invention.

Although it is unclear what mechanism of action this water-soluble metal salt uses to suppress the effects of interfering substances, it is presumed that the dissociated metal ions play some role.

(Examples, etc.) Next, the present invention will be described in more detail and specifically with reference to Examples and Test Examples.

Husband a) Preparation of reagents I) First reagent: Prepared by combining the following solutions.

300+aM carbonate buffer (pll 10.3), 2.5
mM cobalt sulfate and 0.3% sodium potassium tartrate.

11) Second reagent: Prepared by combining the following solutions.

3.0mM INT aqueous solution, 0.05% NaN* and 1O% Tr1ton X-100.

b) Add 11 of the first reagent to 100μ1 of the manipulated serum sample) 37°C
Incubate for 5 minutes at . Next, 11 times of the second reagent was added, and after further incubation at 37°C for 5 minutes, the absorbance of the solution was measured at a wavelength of 500 nm, and the absorbance value was compared with the standard calibration curve to determine the fructosamine in the serum sample. Find the concentration.

The above standard calibration curve was created by using various standard serum samples with known fructosamine concentrations as samples, and by performing the above procedure on each standard serum sample to investigate the relationship between fructosamine concentration and absorbance in advance. be.

(Correlation with Baker's method) The fructosamine measurement reagent according to Baker's method disclosed in the Patent Publication I-T3OE2 is commercially available from Nippon Roche Co., Ltd.; Fructosamine was measured manually on 50 specimens,
In addition, fructosamine was measured in these serum samples using the method of the present invention described in the "Procedure" section above, and the correlation between both methods was determined. The results are as shown in Figure 1, indicating that there is a good correlation. It turned out (regression formula Y: 0.873x
+0.25).

Furthermore, as a result of investigating the simultaneous reproducibility of the method of the present invention, CV:
It was 2.0%.

(Effects of reducing substances on absorbance) Reducing substances that may exist in the serum sample and may interfere with the measurement of fructosamine by the end-point method include ascorbic acid, glutathione,
There are pirirubin, uric acid, glyceraldehyde, dihydroxyacetone, etc., but especially ascorbic acid is vitamin C.
It is a substance that often exists in serum derived from ingested foods and various drugs administered.

Therefore, ascorbic acid was selected as the interfering substance, and a 22+ag/dl aqueous solution thereof was prepared. Take 3ml and make 200mM
carbonate buffer (p[1 10.3) or 2.5mM cobalt sulfate solution 1.01 and then operated as in the previous examples, i.e. incubated for 5 minutes at 37°C, then Add 1.01 of 2 reagents and further heat at 37°C.
After incubation for 5 minutes, the absorbance at a wavelength of 500 n+s was measured. The samples used and the results are as follows. The influence of ascorbic acid, which is a measurement interfering substance, cannot be removed by the alkaline carbonate buffer, but can be ignored if cobalt sulfate, a water-soluble metal salt, is present. It was found that the reduction was as low as possible.

Sample I: (ascorbic acid solution 0.21 + carbonate buffer) 10th 2nd reagent sample 2: (ascorbic acid solution 0.31 + carbonate buffer) ÷ 2nd reagent sample 3: (ascorbic acid solution 0.21 + cobalt sulfate carbonate buffer Solution) 10th Second Reagent Sample 4: (Ascorbic Acid Solution 0.31 + Cobalt Sulfate Carbonate Buffer) 10th Second Reagent Sample 5 (Blank Sample): (Purified Water 0.21 + Cobalt Sulfate Carbonate Buffer) 2nd Reagent (Invention) (Effect) The method for measuring fructosamine according to the present invention employs the so-called "end points method", so that only one absorbance measurement is required and no special skill is required for the measurement operation. The method for measuring fructosamine according to the present invention is basically similar to the conventional method, in which fructosamine, which is a reaction product between glucose and protein in a serum sample, is converted into an enol form in an alkaline environment. It is based on the method of coloring a coloring agent through the reducing action of the coloring agent and measuring the absorbance of this coloring solution.
If ascorbic acid, etc., which has a reducing effect, is present in the serum sample, these substances will naturally interfere with the reducing effect of enol-type fructosamine and prevent accurate measurement of fructosamine. It is necessary to eliminate the influence of substances, and according to the present invention, the influence of interfering substances on measurements can be eliminated by incubating the serum sample in the presence of a water-soluble metal salt prior to adding the coloring agent solution. It can be almost completely eliminated. In the method of the present invention, if the reagents are appropriately selected, the incubation time required for this so-called pretreatment of the serum sample can be reduced to about 5 minutes, and the incubation time required after adding the coloring agent can also be reduced to about the same level. Therefore, the method of the present invention is easy to carry out manually,
It also becomes possible to apply it to instrumental analysis.

[Brief explanation of drawings]

FIG. 1 is a graph showing the results of measuring fructosamine in serum by the method of the present invention and Baker's method, and examining the correlation between the two methods.

Claims (1)

[Claims] (1) Adding a first reagent, which is an alkaline buffer containing a water-soluble metal salt, to a serum sample and incubating it, then adding a second reagent, which is an aqueous solution of a coloring agent, further incubating, and then measuring the absorbance; On the other hand, the absorbance value measured for the above serum sample is compared with a standard calibration curve prepared in advance by performing the same operation as above and measuring the absorbance using various standard serums with known fructosamine concentrations. A method for measuring fructosamine.