JPS6359464B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for separating and quantifying hemoglobin in blood, particularly catabolic hemoglobin, for purposes such as diagnosis.

Adult hemoglobin (hereinafter abbreviated as Hb) is a protein with a molecular weight of approximately 68,000, and exists in the form of four hemes bound to one globin protein. However, under alkaline pH (PH8.6) conditions, normal human Hb
When electrophoresed, in addition to Hb, which accounts for 90 to 95%, that is, HbA ₀ , there are HbF and HbA ₁ c, whose mobility is slightly slower than that of HbA ₀ , and HbA ₁ a, which is further delayed.
HbA ₁ b, etc. have been isolated and their existence has been recognized. Furthermore, the presence of HbA ₁ d, HbA ₁ e, etc. has also been confirmed from the results of more precise electrophoresis tests. These abnormal hemoglobins have a different primary protein structure compared to normal hemoglobin (HbA ₀ ), or have sugars attached to HbA ₀ . It has been found that the separation and quantification of these catabolic hemoglobins is important as a pathological indicator for various diseases; for example, checking for increases and decreases in HbA _1c is essential for the treatment of diabetes. Also, the component called HbA ₂ is hereditary like HbF, and abnormal hemoglobin called acetylated hemoglobin (Ac, Hb) is produced by taking drugs with acetyl groups, such as aspirin. It has become clear that the separation and quantification of these catabolic hemoglobins is becoming increasingly important from a medical standpoint.

However, up to now, no method has been proposed that can accurately separate and quantify catabolic hemoglobin as described above.

In view of the above-mentioned current situation, the present invention has been made for the purpose of providing a method for separating and quantifying catabolic hemoglobin with high sugar content and in a short time. 5 to 90% by weight of a polymerizable monomer (A) having a polymerizable double bond and one or more amino groups, having two or more polymerizable double bonds in the molecule and a hydrophobic parameter of 2.3 or less 10 to 95% by weight of a crosslinking polymerizable monomer (B), a monomer (C) having one polymerizable double bond in the molecule and having a hydrophobic parameter of 2.3 or less and different from the above polymerizable monomer (A). A method for separating catabolic hemoglobin, characterized in that it is carried out by liquid chromatography using as a stationary phase a hydrophilic ion exchanger copolymerized with a monomer mixture of 0 to 85% by weight.

In the present invention, a hydrophilic ion exchanger formed by copolymerizing a monomer mixture with a specific composition is used as a stationary phase in liquid chromatography. In order to use the above-mentioned copolymerized ion exchanger as a stationary phase, that is, a filler, for example, the copolymer particles obtained by copolymerization may be made to have the same particle size and used as they are as a filler. In the case of bulk polymerization, a pulverized copolymer may be used with uniform particle size, or the surface of an appropriate core material such as a glass bulb may be coated with the above copolymer by an appropriate method. Good too.

However, the copolymer constituting the filler of the present invention is
5 to 90% by weight of the polymerizable monomer (A) having one polymerizable double bond and one or more amino groups in the molecule, and two or more polymerizable double bonds in the molecule and The crosslinked polymerizable monomer (B) with a hydrophobicity parameter of 2.3 or less is 10 to 95% by weight, and the above polymerizable monomer (A) has one polymerizable double bond in the molecule and has a hydrophobicity parameter of 2.3 or less. ) different monomer from
A monomer mixture containing (C) from 0 to 85% by weight is copolymerized, and this copolymerization includes suspension polymerization,
Conventional polymerization methods such as emulsion polymerization, solution polymerization, and bulk polymerization can be employed, but suspension polymerization is preferred because the polymer particles can be used as they are as a filler. Additionally, if a small amount of an organic solvent is added during suspension polymerization, which dissolves the monomer but does not dissolve the polymer, the resulting copolymer particles will become porous, resulting in a packing that increases the contact area with the mobile phase. agent is obtained.

Here, the hydrophobic parameter is the logarithm of the solubility ratio of a certain compound in an aqueous octyl alcohol system, and is a value specific to that compound. Although this parameter can be determined by experiment, the contribution of each fragment in the molecule (hydrophobic fragment constant) can be determined by calculation, and the hydrophobic parameter value of the compound is calculated as the sum of these. can.

For information on how to calculate hydrophobicity parameter values, see
“The Hydrophobic Fragmental” by RFRekker
"Constant" (Elsevier Scientific Publishing)
Co., Ltd. (published in 1977), Chapter 3, and the parameter values in the present invention are based on this calculation method.

Next, examples of the polymerizable monomer (A) preferably used in the present invention include dimethylaminoethyl methacrylate, diethylaminoethyl methacrylate, etc., and those having a hydrophobic parameter of 2.3 or less are preferably used.

Moreover, the crosslinking polymerizable monomer (B) used in the present invention
Examples include tetramethylolmethane triacrylate (hydrophobicity parameter = -0.73, below the parameter values are shown in parentheses), tetramethylolmethane trimethacrylate (0.59), tetramethylolmethane diacrylate (-1.70), and tetramethylolmethane. Dimethacrylate (0.82), Tetramethylolmethanetetraacrylate (0.24), Tetramethylolmethanetetramethacrylate (0.20), Trimethylolethane triacrylate (0.88), Trimethylolethane trimethacrylate (2.20), Dipentaerythritol hexaacrylate (-0.09) ), and alkylene glycol diacrylates (or methacrylates) such as polyethylene glycol diacrylate (or methacrylate) such as tetraethylene glycol dimethacrylate (-0.30) and polypropylene glycol diacrylate (or methacrylate) are also used. I can do it. or,
A monomer (C) different from the monomer (A) can be used in the present invention up to a quantitative range of 85% by weight if necessary. 0.88),
2-Hydroxyethyl methacrylate (-
0.41), glycidyl methacrylate (-0.48)
NN-dimethylacrylamide, 2-hydroxyethyl acrylate, glycidyl acrylate, etc. can also be used.

In the present invention, as mentioned above, the polymerizable monomer
A monomer mixture consisting of (A) 5 to 90% by weight and a crosslinking polymerizable monomer (B) 10 to 95% by weight, or a monomer mixture in which the above monomer (C) is added up to 85% by weight as necessary, is copolymerized. However, the resulting copolymer itself becomes an ion exchanger exhibiting ion exchange action due to the presence of the polymerizable monomer (A) having an amino group in the molecule. In the monomer mixture, if there is too little monofunctional monomer (A) having an amino group, the ion exchange ability will decrease, so the monomer (A) needs to be at least 5% by weight, and it is also necessary to have at least 2 monomers (A). If the amount of the cross-linked polymerizable monomer (B) having a polymerizable double bond is too small, the density of the cross-linked bonds in the copolymer will decrease, making it difficult to withstand high pressure, especially when used as a packing material for high-performance liquid chromatography. Since a problem arises in mechanical strength for drilling, the monomer (B) is required to be in an amount of 10% by weight or more. In addition, polymerizable monomer 2 other than the polymerizable monomer (A)
The monomer (C) having a heavy bond is a polymerizable monomer (A)
and the crosslinking polymerizable monomer (B) can be included in the mixed monomer in such a range that the proportion used does not become less than the required amount.

The monomers (B) and (C) contained in the monomer mixture produced by copolymerizing the hydrophilic ion exchanger used in the present invention have a hydrophobicity parameter of 2.3 or less; This is based on the knowledge of the present invention that a packing material made of an ion exchanger copolymerized with Since divinylbenzene, which has been commonly used as a monomer, has a high hydrophobicity parameter of 3.8, the effects of the present invention cannot be achieved even if an ion exchanger containing divinylbenzene as a copolymerization component is used as a filler.

In addition, the hydrophobicity parameters of the polymerizable monomer (A), the cross-linking polymerizable monomer (B), and the monomer (C) added as necessary are 0 or less or on the negative side when analyzing the substance to be analyzed in the present invention. This is preferable in terms of preventing adsorption to the filler.

The above-mentioned hydrophilic ion exchanger is packed into a column by a conventional method and used for the separation of the present invention by liquid chromatography, and exhibits analytical behavior based on an ion exchange mechanism.

In the separation according to the present invention, the column filled with the above-mentioned hydrophilic ion exchanger is connected to a liquid chromatograph, preferably a high-performance liquid chromatograph. This can be done by injecting into a chromatograph and flowing the eluent. The optimal elution conditions should be determined depending on the type of catabolic hemoglobin to be separated and the type of column packing material used, but generally an aqueous solution with a pH of 5.0 to 10.0 and an ionic strength of 0.01 is used as the eluent. Moreover, various catabolic hemoglobins can be well separated and quantified by changing the pH and ionic strength during the separation operation.
Further, detection is preferably performed using visible light with a wavelength of 415 nm.

In addition, the elution order of catabolic hemoglobin generally does not change much, but by changing the elution conditions during the elution operation, specific catabolic hemoglobin can be made to appear as a good chromatographic peak,
It is also possible to make other catabolic hemoglobins and normal hemoglobin appear as clustered peaks that are not well separated, so that the target catabolic hemoglobin can be quantified effectively in a short time.

The present invention is a method for separating catabolic hemoglobin as described above, in which a hydrophilic ion exchanger copolymerized with a monomer mixture having a specific composition is used as a stationary phase, and the stationary phase has both separation performance and strength. Because of its excellent properties, it can be used as a packing material for high performance liquid chromatography, and the present invention has the excellent effect of allowing catabolic hemoglobin to be separated and quantified with high accuracy and in a short time.

Examples of the present invention will be described below.

Example 1 Into two separable flasks equipped with a cooler, a stirrer, a thermometer, and a dropping funnel, 400 ml of a 4% by weight aqueous polyvinyl alcohol solution, 40 g of tetraethylene glycol dimethacrylate, 10 g of tetramethylolmethane triacrylate, and diethylaminoethyl were added. Methacrylate 50g, toluene 40g
A mixture of 1.5 g of benzoyl peroxide and 1.5 g of benzoyl peroxide was supplied. Next, the temperature was raised to 80° C. while stirring at a stirring speed of 400 rpm, reaction was carried out for 10 hours, and the mixture was cooled. After cooling, the polymerization product was separated from the mother liquor and washed with hot water and acetone to obtain a spherical polymer having a particle size of 5 to 20 microns. The 8-12 micron particles obtained by removing fine particles and coarse particles were dispersed in 80 ml of ion-exchanged water, and the ion-exchanged water was poured into a stainless steel column (diameter 7.9 mm, length 30 cm) using a high-pressure constant flow pump. It was filled by pumping at a rate of 1.6 ml/min.

The resulting packed column was then processed using a high-performance liquid chromatograph (product name: Shimadzu DuPont High-Performance Liquid Chromatograph).
830 model) and performed the following analysis operations.

Analysis was carried out by injecting 10 μl of a hemoglobin solution prepared by mixing 50 μl of blood from a normal person and a diabetic patient with 1 c.c. of ion-exchanged water and hemolyzing the sample using a microsyringe. As an eluent
A mixture of 0.05M Tris-HCl and 0.01M sodium chloride aqueous solution is used as Solution A (PH7.8), and A as Solution B.
An aqueous solution adjusted to 0.05M by adding NaCl to the solution was used.

Elution was started with 100% solution A, and solution B was increased at a rate of 2%/1 minute. The obtained chromatogram is shown in FIG. Note that a 415 nm visible light detector was used for detection.

Next, the A ₁ c and A ₀ components were separated by ion exchange column chromatography using a liquid chromatography packing material (Biorex-70, manufactured by BioRad (USA)), which is well known in this field. Fractionation was carried out for about 2 hours or more, and high performance liquid chromatography analysis according to the present invention was performed on each fractionated component under the above conditions to identify peaks 1 and 2 in the chromatogram shown in Fig. 1. ₁ c and A ₀ were found to correspond to peaks 2 and 1, respectively. The amount of HbA ₁ c determined by the baseline method was 3.5% in normal human blood and 6.2% in diabetic patient blood.

Example 2 36 g of nonaethylene glycol dimethacrylate, 5 g of tetramethylolmethane triacrylate, and 50 g of dimethylaminoethyl methacrylate were used as monomers, and 40 g of toluene was used as an organic solvent.
A packing material and a packed column were prepared in the same manner as in Example 1, except that G. It was done.

[Brief explanation of the drawing]

FIG. 1 shows a chromatogram of human blood obtained in Example 1. A... Chromatogram of normal human blood, B... chromatogram of diabetic patient blood, 1... HbA ₀ peak, 2... HbA ₁ c peak.

Claims (1)

[Claims] 1 5 to 90% by weight of a polymerizable monomer (A) having one polymerizable double bond and one or more amino groups in the molecule, hydrophobic and having two or more polymerizable double bonds in the molecule 10 to 95% by weight of a crosslinking polymerizable monomer (B) with a property parameter of 2.3 or less, one polymerizable 2 in the molecule
A monomer (C) having a heavy bond and a hydrophobic parameter of 2.3 or less and different from the above polymerizable monomer (A)0
1. A method for separating catabolic hemoglobin, which is carried out by liquid chromatography using a hydrophilic ion exchanger as a stationary phase, which is a copolymerized monomer mixture containing ~85% by weight.