JPS6148867B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for separating and quantifying glucose-bound hemoglobin in a sample, particularly in blood.

Measuring blood sugar levels (glucose concentration in the blood) has traditionally been widely used as a diagnostic method for diabetes, but since blood sugar levels are directly affected by diet and exercise, multiple tests are required to determine diabetes. . Treatment guidelines for diabetes need to express the state of long-term diabetes control, but in this sense, it is insufficient to simply indicate the blood sugar level at the time of blood sampling.

In recent years, new diabetes diagnostic methods have been developed that solve these problems. In other words, while hemoglobin in red blood cells circulates in the body, it non-enzymatically reacts with glucose in the blood to form glucose hemoglobin (hereinafter abbreviated as G-Hb).
It was found that G-Hb expressed the time-averaged concentration of glucose in the blood over a long period of time, and a diagnostic method based on the measurement of G-Hb was developed. A method using a cation exchange resin has been proposed for measuring G-Hb, but this method is easily affected by other cations, is difficult to reuse, and has poor separation of G-Hb and hemoglobin A. It has the following disadvantages.

The present invention was achieved as a result of intensive research aimed at providing a better measurement method in view of the above-mentioned current situation in G-Hb measurement. formula; (In the formula, R ₁ and R ₂ are hydrogen or methyl groups, and n is 3 to
100 parts by weight of a compound represented by x methylolalkyl y (meth)acrylate (where x and y are integers and x≧y≧3)
A liquid chromatography packing material made of a porous polymer obtained by aqueous suspension polymerization of a mixture consisting of 5 to 60 parts by weight in the presence of an organic solvent that dissolves the mixture but does not dissolve the polymer is fixed. A method for quantifying glucose-bound hemoglobin, characterized in that it is carried out by liquid chromatography in which water with a pH of 3.0 to 9.0 is used as a phase and as an eluent.

General formula used in the present invention The compound represented by the formula is an acrylic ester or methacrylic ester of glycol, in which R ₁ and R ₂ are hydrogen or a methyl group, and n is an integer of 3 to 18, such as triethylene glycol diacrylate, triethylene Examples include glycol dimethacrylate, tetraethylene glycol diacrylate, tetraethylene glycol dimethacrylate, nonaethylene glycol diacrylate, nonaethylene glycol dimethacrylate, tetradecaethylene glycol diacrylate, and tetradecaethylene glycol dimethacrylate.

In the compound represented by the above general formula, n is 1 or 2
When n is too hydrophobic, it cannot be applied to hydrophilic samples, and when n is larger than 19, the particles become soft and lack mechanical strength as a packing material for high performance liquid chromatography. 3 to 18, preferably 4 to 14. The x methylol alkyl y (meth)acrylate used in the present invention has at least three methylol groups and three or more Compounds having an acrylate or methacrylate group, such as tetramethylolmethanetetraacrylate, tetramethylolmethanetetramethacrylate, tetramethylolmethane triacrylate, tetramethylolmethane trimethacrylate, trimethylolmethane triacrylate, trimethylolmethane trimethacrylate, Examples include methylolpropane triacrylate, trimethylolpropane trimethacrylate, and the like, and tetramethylolmethanetetraacrylate, tetramethylolmethane triacrylate, and tetramethylolmethane trimethacrylate are preferably used. The compound represented by the above general formula and the methylolakyl y (meth)acrylate are made into a mixture, but if the amount of x methylolakyl y (meth)acrylate added is small, the resulting porous polymer will be soft and porous. If too much, phenomena such as adsorption and distribution occur, resulting in a decrease in resolution. Therefore, 5 to 60 parts by weight, preferably 10 to 40 parts by weight, is added to 100 parts by weight of the compound represented by the above general formula.

In the present invention, aqueous suspension polymerization is carried out in the presence of an organic solvent that dissolves the mixture but does not dissolve the polymer.The organic solvent dissolves the mixture but does not dissolve the polymer. All organic solvents can be used, such as aromatic hydrocarbons such as toluene, xylene, diethylbenzene, dodecylbenzene, saturated hydrocarbons such as hexane, heptane, octane, decane, isoamyl alcohol, hexyl alcohol, octyl alcohol, etc. The amount of alcohol added is not limited in any way, but it is preferably used in an amount of 15 to 200 parts by weight, more preferably 20 to 150 parts by weight, per 100 parts by weight of the mixture. The organic solvent is uniformly dissolved in the mixture, and since the mixture is subjected to aqueous suspension polymerization, it exists dispersed in the resulting polymer particles, and after the polymerization is completed, the organic solvent is removed from the particles. By removing it, a porous polymer is obtained.

Therefore, by using various organic solvents having different compatibility with the above mixture, the pore size of the porous polymer can be arbitrarily changed. For the aqueous suspension polymerization, any known method may be used. For example, the mixture and the radical generating catalyst are dissolved in the organic solvent, and the resulting solution is mixed with a suspension polymerization stabilizer such as polyvinyl alcohol or calcium phosphate. Add to the dispersed aqueous phase with stirring for 50 min.
This is done by heating to ~100°C.

The above radical generating catalyst is a catalyst that generates radicals as a reaction initiator. Examples of the catalyst include organic peroxides such as benzoyl peroxide and cumene oxide, and inorganic peroxides such as hydrogen peroxide, potassium persulfate, and ammonium persulfate. Any known radical generating catalyst can be used, such as peroxide, azo compounds such as azobisisobutyronitrile, and azobisisobutyramide.

The polymer particles polymerized by the above-mentioned aqueous suspension polymerization are dried by heating or the like to release the organic solvent in the particles, thereby forming a porous polymer, which is used as a filler for liquid chromatography. In order to use the porous polymer as a packing material for liquid chromatography, it is preferable that the particle size of the porous polymer be uniform and within the range of 3 to 40 μm.

In the present invention, the above-mentioned porous polymer is used as a stationary phase in a packing material for liquid chromatography, but in order to use the above-mentioned porous polymer as a packing material, it is necessary to use finer particles among the synthetic polymer particles obtained by polymerization. Particles preferably having a diameter of 8 to 12μ obtained by removing coarse particles are preferably dispersed in ion-exchanged water, and the ion-exchanged water is pumped into a stainless column using a constant flow pump to fill the stainless steel column. G-Hb is separated using a liquid chromatography method using a column prepared by the company.

As is well known, hemoglobin is a protein with a molecular weight of 64,450, and normal human hemoglobin is composed of two aggregates of two subthieins, α and β. When glucose binds to hemoglobin, it becomes G-Hb, which increases the hydrophilicity of hemoglobin, and in liquid chromatography using the packing material of the present invention, G-Hb is eluted in a shorter time than normal hemoglobin. For this reason, it is possible to quantify G-Hb in the present invention. When actually performing chromatography, the packing material made of the granular synthetic polymer of the present invention is used as a stationary phase, and the eluent is a buffer such as acetic acid-sodium phosphate or phosphoric acid-sodium phosphate system. and the PH is
Elution is performed in the range of 3.0 to 9.0, preferably 5.0 to 9.0. Furthermore, the shape of the chromatogram obtained can be improved by changing the pH during elution or changing the elution rate during elution.

The method for quantifying G-Hb of the present invention is as described above, and in particular, since a specific porous polymer is used as a stationary phase, G-Hb can be separated easily and accurately, and it can be repeated repeatedly. It can also be used effectively in the diagnosis of diabetes, and has great utility as a clinical test.

Examples of the present invention will be described below.

Example 1 In a 2L separable flask equipped with a condenser, stirrer, thermometer, and dropping funnel, 400ml of 4% by weight polyvinyl alcohol aqueous solution, 90g of tetraethylene glycol dimethacrylate, 10g of tetramethylolmethane triacrylate, and 1.5g of benzoyl peroxide are added. A mixed solution consisting of: Furthermore, 100 g of toluene was added. Next 400r.
The temperature was raised to 80°C while stirring at pm, the reaction was carried out for 10 hours, and the mixture was cooled. After cooling, the polymerization product is separated and washed with hot water and acetone to reduce the particle size to 5-20μ.
A porous spherical polymer of m was obtained. Particles of 8 to 12 μm obtained by removing fine particles and coarse particles were dispersed in 80 ml of ion-exchanged water, and ion-exchanged water was added at 1.6 ml/min to a stainless steel column (diameter 7.9 mm, length 25 cm) using a constant flow pump. It was filled by pressure feeding at a speed of . The obtained packed column was connected to a high performance liquid chromatograph (manufactured by Shimadzu Corporation, Model LC2). Next, attach the column of the high performance liquid chromatograph mentioned above.
The following separation operation was performed while maintaining the temperature at 25°C. After collecting 2 ml of blood from a normal person, it is converted into hevarin to prevent coagulation.
After blood cell plasma separation by centrifugation at °C, the red blood cells were washed three times with 10 ml of physiological saline.

Next, 1.5 volumes of distilled water and 0.5 volumes of toluene were added to the washed red blood cells and shaken vigorously to cause hemolysis.
This was centrifuged at 4°C at 3000 rpm for 15 minutes, and the supernatant of the hemolyzed layer _was cooled at ₄ °C with phosphate-cyanide buffer ( _{Na2H2PO4.H2O4.59g} /l,
Dialysis was performed for 24 hours (using a dialysis tube manufactured by Nippon Irika Plastics) against Na ₂ HPO ₄ 1.19 g/l KCN 0.65 g/l). This dialyzed hemolysate was used as a liquid chromatograph test solution, and 1 μl of it was injected.

0.1N phosphate buffer is used as the eluent for liquid chromatography, and the chromatography is started at pH 6, and the pH is increased just before the large peak of normal hemoglobin appears.
The chromatogram obtained by switching to 7 is the first
As shown in the figure. This chromatogram shows 415 as a detector.
Visible light with a wavelength of mm was used. The assignment of each peak was determined by liberating glycose from hemoglobin with oxalic acid and then quantifying glycose using the glycose oxidase method.

As a result, it was found that peaks 2, 3, and 4 were glucose-bound hemoglobin, and peak 5 was normal hemoglobin.

The ratio of the sum of the areas of peaks 2, 3, and 4 to the total area (the sum of peaks 1 to 5) was 0.065, which was within the range of 5 to 8% of the G-Hb amount of a normal person.

Example 2 Example 1 except that the monomers used were 80 g of nonaethylene glycol dimethacrylate and 20 g of tetramethylolmethane trimethacrylate.
A porous spherical polymer produced under the same polymerization conditions and sphere-splitting operation was similarly filled, and a blood sample solution treated in the same manner as in Example 1 was heated to pH 6 using the same equipment and buffer as in Example 1. Elution was performed at 5.

The obtained chromatogram was similar to FIG. 1 except that peak 5 was slightly broader, and the measured value of the G-Hb amount was almost the same as in Example 1.

[Brief explanation of the drawing]

FIG. 1 shows the chromatogram obtained in Example 1. Peaks 2, 3, 4...Glucose-bound hemoglobin, Peak 5...Normal hemoglobin.

Claims (1)

[Claims] 1. Detection of glucose-bound hemoglobin is performed using the general formula; (In the formula, R ₁ and R ₂ are hydrogen or methyl groups, and n is 3 to
100 parts by weight of a compound represented by x methylolalkyl y (meth)acrylate (where x and y are integers and x≧y≧3)
A liquid chromatography packing material made of a porous polymer obtained by aqueous suspension polymerization of a mixture consisting of 5 to 60 parts by weight in the presence of an organic solvent that dissolves the mixture but does not dissolve the polymer is fixed. A method for quantifying glucose-bound hemoglobin, characterized in that it is carried out by liquid chromatography, in which water with a pH of 3.0 to 9.0 is used as a phase and as an eluent.