JPS62226999A - Separation of saccharified albumin - Google Patents

Abstract

PURPOSE:To rapidly and readily separate the titled compound useful for diagnosis of diabetes, etc., by separating albumin from a sample in the first column and separating the albumin without discharging the albumin to the outside of the flow passage system into a saccharified albumin and nonsaccharified albumin in the second column. CONSTITUTION:Saccharified albumin and nonsaccharified albumin in a sample are separated by liquid chromatography. In the process, the albumin is separated from the sample in the first column filled with a gel prepared by linking to a dyestuff, e.g. Cibacron Blue F3G-A, etc., having high affinity for the albumin and then without discharging to the outside of the chromatographic flow passage system led to the second column filled with a copolymer having introduced aminophenylboronic acid, etc., to separate the albumin into the saccharified albumin and nonsaccharified albumin.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention is a method for separating glycated albumin in which the separation of albumin in a sample and the separation of glycated albumin and non-glycated albumin are performed in the same flow path of chromatography. Regarding the method.

In diseases such as diabetes, in which blood sugar levels remain high for a long period of time, various proteins in living organisms undergo non-enzymatic saccharification reactions. Serum albumin/ is one of those proteins.

Conventionally, measurements of blood sugar, urine sugar, insulin, glucagon, oral glucose tolerance tests, etc. have been used to diagnose diabetes, but the results are easily affected by physiological conditions or the measurement method, and it is burdensome for the subject. There are problems such as the amount of time required. In contrast, the concentration of glycated proteins in living organisms has attracted attention in recent years because it is less affected by temporary physiological conditions and serves as an indicator of the average blood sugar concentration over the past few weeks to several months.

For example, glycated hemoglobin is an indicator of the average blood sugar level over the past 1 to 3 months and has a positive correlation with the severity of diabetes, so it has already been clinically applied as a new diagnostic method for diabetes and an indicator of blood sugar control. There is. In particular, it is said that the ratio of glycated hemoglobin to total hemoglobin correlates well with the fasting blood sugar level three months ago.

However, clinically, information on blood sugar status from several weeks to a month ago is useful, and glycated albumin has a half-life of 17
Recently, it has been attracting attention from various quarters as the index that can best meet this demand.

That is, compared to glycated hemoglobin, glycated albumin responds more quickly to blood sugar conditions, and unlike blood sugar levels, it does not fluctuate greatly under the influence of temporary physiological conditions.

For example, the usefulness of glycated albumin has been suggested as being effective in determining at an early stage whether a current or newly changed dietary therapy is appropriate for the patient. [K.F. McFarland, Diapets (Kay
F, Mcfarland etal, DIA-BET8
), 28.1011.1979, C.R.ΦGaslow, Brok Natal Akad Sai.

Ni Nis Ni (0, Fearl Guthrow
etal, Proc.

Natl, Acad, Sci, US&), 76,
A9, 4258.

1979] (Prior Art) Conventionally, methods for separating and detecting glycated albumin include ion-exchange chromatography using carboxymethyl cellulose as a stationary phase; D
ay etal, J.Bio, Ohem, ), 254
.

3.595.1979:), affinity chromatography that introduces dihydroxydyronyl groups into soft gels such as cellulose, and utilizes the interaction between this functional group and glucose [AKM Maria, Analytical.
Letters (A, ni, Malliaetal, &naly
tical Letters), 14 (B 81,
649-661.1981), a method utilizing a color reaction represented by the thionolbituric acid method [Kay F.
-rland etal, DIABFXTFfS)
, 28, 1011.1979] have been attempted.

(Problems to be Solved by the Invention) All of the above conventional techniques can separate glycated albumin and non-glycated albumin, but cannot separate them from other components in body fluids.

The reason varies depending on the separation method. In the case of ion exchange chromatography, prealbumin,
This is because there are many substances that have an isoelectric point close to that of albumin, such as some α-globulins.In the case of affinity chromatography and sugar coloring methods, albumin in body fluids can be removed. This is because.

Therefore, for example, the pigment 0ibacron Blue, which has a high affinity for albumin,
A pretreatment of isolating only albumin from body fluids using a gel bound with P2O-4 or the like was essential.

However, the conventional method was to isolate albumin from a sample, take it out of the chromatography system, concentrate it, and then introduce it to a column that can separate glycated albumin and non-glycated albumin. That is, the operation of separating albumin from a sample and the operation of separating albumin into glycated albumin and non-glycated albumin are performed in at least two separate steps, which are complicated and take a long time. .

These are the main reasons why glycated albumin is not currently measured in clinical vehicle inspections, which require speed, operability, reproducibility, and automation.

(Means for Solving the Problems) In order to overcome the above-mentioned problems, the present inventors, as a result of intensive research, have developed a method for producing glycated albumin that is quick, easy to operate, has excellent reproducibility, and is easy to automate. They discovered a method for separating non-glycated albumin and completed the present invention.

That is, the present invention separates albumin from a sample in a first column, guides it to a second column without leaving it outside the chromatography flow path system, and there separates albumin into glycated albumin and non-glycated albumin. The present invention relates to a method for separating glycated albumin by chromatography, which is characterized by separating glycated albumin.

In the present invention, glycated albumin refers to albumin that is covalently bonded to glucose, and non-glycated albumin refers to albumin that is not bonded to glucose.

Furthermore, albumin as used in the present invention refers to both glycated albumin and non-glycated albumin.

The sample referred to in the present invention is one containing at least either glycated albumin or non-glycated albumin, or both, and includes, for example, body fluids such as blood and urine.

The first column used in the present invention is not particularly limited as long as it can separate albumin from other components in body fluids. Preferred examples include a column filled with a gel that binds Dinoclon Blue F3G, a dye with high affinity for albumin, and Ig, which is a large component in serum.
G (molecular weight: approximately 150,000) and albumin (molecular weight: approximately 6.
Examples include packed columns for gel filtration and ion exchange gel-packed columns that can separate 60,000].

However, as a carrier for introducing substances with high affinity for albumin such as Cibacron Blue, and as a stationary phase carrier for gel filtration, it is recommended because it has excellent mechanical strength and chemical stability and has low non-specific adsorption of proteins. Dialcoholic hydroxyl group per weight of crosslinked copolymer 1.0 to 14.0 meq/
A hard hydrophilic crosslinked copolymer having a specific surface area of 5 to 1000 f/1 and a water retention capacity of 0.5 to 6.0 f/g is preferred. As the most preferable column, JP-A-57-3
A crosslinked copolymer having an alcoholic hydroxyl group derived from a vinyl alcohol unit described in Japanese Patent No. 0945 can be mentioned.

Or JP-A-56-64657 and JP-A-59-
The crosslinked copolymers described in JP 145036 are also preferred.

Also, for the same reason, ion exchange gel is also used in JP-A-60-1
A crosslinked copolymer having an alcoholic hydroxyl group derived from a vinyl alcohol unit and a diethylamino group described in Japanese Patent No. 50839 can be mentioned as the most preferred series. This crosslinked copolymer allows albumin to be rapidly separated from other components in body fluids by the method described in Japanese Patent Application No. 1222/1982.

The second column used in the present invention may be a column filled with an ion exchange gel or a gel having a dihydroxyzolonyl group, but is not particularly limited as long as it can separate at least glycated albumin and non-glycated albumin.

Needless to say, when using an ion exchange gel, the slight difference in isoelectric point between glycated albumin and non-glycated albumin is utilized, but the ion exchange group is preferably a cation exchange group, and among them, a carboxyl group, a sulfone group, etc. Acid groups and the like are particularly preferred.

Affinity chromatography using a gel containing this functional group as a stationary phase is also effective for separating glycated albumin and non-glycated albumin, as it specifically binds to compounds with a dihydroxyboronyl group [,1,2-cis diol. It is.

Examples of stationary phase carriers into which these functional groups are introduced include soft gels of polysaccharides such as cellulose and agarose, silica gel, and styrene-divinylbenzene copolymers, which are commonly used as stationary phase carriers for liquid chromatography. etc., but it has 1.0 to 14.0 dialcoholic hydroxyl groups based on the weight of the crosslinked copolymer because it has excellent mechanical strength and chemical stability, and has little nonspecific adsorption of tan and phosphorous substances.
A hard hydrophilic crosslinked copolymer having a specific surface area of 0 meq/f, a specific surface area of 5 to 1000 m''/f, and an amount of water that can be retained of 0.5 to 6.(1/f) is preferred. Examples include crosslinked copolymers described in JP-A No. 57-30945, JP-A-56-64657, and JP-A-54-145036.

The apparatus used in the present invention may be an ordinary liquid chromatography apparatus, but as shown in Fig. 1, it is better to provide a flow path switching valve between the first column and the second column. good. By operating this switching knob, only the albumen separated in the first column can be led to the second column, and the other components can be led out of the flow path system.

In Figure 1, the mobile phases, liquid 2 and liquid B, are each pumped to the injector, and the pump is ink x /7.
The sample is sent to the first column along with the mobile phase from the sample, and albumin and other components in the sample are separated. Then, by operating the switching knob, only the albumin separated in the first column is sent to the second column, where it is separated into glycated albumin and non-glycated albumin and sent to the detector.

Further, as shown in FIG. 2 used in the practical example, if a gradient mixer often used in high performance liquid chromatography is attached to the suction side of the pump, the number of pumps can be reduced to one.

Next, the mobile phase used in the method of the present invention will be described.

Even if a column packed with a gel into which a functional group such as an ion exchange group or a dihydroxyboronyl group is introduced is used as the 20th column, at least two types of mobile phases (liquid A and liquid B) are prepared, and It is preferable to switch.

That is, before and during sample injection, N solution is passed through the sample, albumin and other components in the sample are separated in the first column, and only albumin is introduced into the second column.
Examples include a method of continuously converting from liquid K to liquid B (so-called gradient method) using a concentration gradient forming device (gradient mixer).

The N solution referred to herein is preferably under conditions for bonding glycated albumin and dihydroxybonyl groups, that is, it does not contain a substance having 1°2-cis diol and has a pT-T of 7.5 to 9.5. On the other hand, the pH of liquid B is preferably 2 to 6.5;
Even if it is 7,5 or more, it is sufficient if it contains a substance having 1,2-cis diol. The concentration of this substance having 1,2-cis diol is preferably 50 to 500 rr+M. The salt concentration of both solutions A and B is preferably 10rr+IJ to IM, and the type of buffer solution is not particularly limited. In other words, it may be preferable to contain a small amount of a water-soluble organic solvent such as ethyl alcohol, methyl alcohol acetonitrile, or ethylene glycol.

The shape and dimensions of the stationary phase carrier, the dimensions of the column, and the flow rate of the mobile phase used in the present invention are not particularly limited. Tatashi,
When speed is important, the stationary phase carrier is preferably spherical, and its particle size is preferably 1 to 50 μm, particularly preferably 1 to 10 μm. The dimensions of the column were determined from the viewpoint of speed, with an inner diameter of 10 mm.
m+ or less, preferably 30 m or less in length, inner diameter 2-8 m,
A length of 1 to 10 crn is particularly preferred. Flow rate is 0.1~1
0 me/min is preferable, more preferably 1 to 10 me/min.
It's a minute.

(Effects of the Invention) The method for separating glycated albumin of the present invention is such that in liquid chromatography, albumin is separated from a sample in the 10th column, and then introduced into the 20th column without leaving the chromatography flow path system. It is characterized by separating albumin into glycated albumin and non-glycated albumin.

Conventionally, in order to separate glycated albumin and non-glycated albumin from a sample containing components other than albumin, at least two separate operations were required, as described above.
According to the method of the present invention, glycated albumin and non-glycated albumin in the sample can be separated by one operation, that is, by injecting the sample once into the sample injection port, and it is also possible to quantify each of them.

According to the method of the present invention, glycated albumin and non-glycated albumin can be easily and quickly analyzed in general clinical laboratories with good reproducibility, and automation is also easy.

121 The present invention greatly contributes to the popularization of glycated albumin analysis.

(Executive Examples) Examples of the present invention are shown below, but the scope of the present invention is not limited to these Examples.

Experiment 1 Preparation of second column (for separation of glycated albumin and non-glycated albumin) Vinyl alcohol copolymer gel (X: 0.3, amount of hydroxyl groups: 7. 8 meq/7, amount of water that can be held: 1.
78 t25F, epichlorohydrin 116.8f,
Dimethyl sulfoxide 250d, ION sodium hydroxide aqueous solution 12.5m/! was added and reacted at 30°C for 20 hours. The amount of epoxy group introduced is 1.0 meq/f
Met. The method for quantifying epoxy groups is described in Japanese Patent Application Laid-open No. 1986-
The method described in Japanese Patent No. 190003 was used.

Next, aminophenyl lronic acid hemiVt acid salt 13 y
The above epoxy group-introduced polymer 201 was added to the solution dissolved in f 250 me water and adjusted to pH 11, and the mixture was heated at 60°C for 2 hours.
The reaction was carried out for 00 hours. Next, 25 mM tris(hydroxymethyl)amitumekun was added to protect the remaining epoxy groups, and the reaction was carried out at 50° C. for 5 hours. The amount of boric acid introduced was 0.28 meq/S'.

The determination of boric acid was carried out by the following method.

Polymer 12 incorporating the above aminophenylboronic acid
The boron-carbon bond was cut by adding 10% 30% hydrogen peroxide solution and reacting for 5 hours. Next, the polymer was spun down and the supernatant was collected. This is decarburized c1
After N, the boric acid ester was formed by adding sugar, strongly oxidized, and the boric acid was quantified by titration with sodium hydroxide.

All instruments used in this analysis were made of quartz.

The hydroxyl group density of the aminophenylboro/acid-introduced copolymer was 9.2 meq/7 (after dihydroxyboronyl group cleavage), and the amount of water that could be retained was 1.70 meq/7.

Furthermore, the particle size and specific surface area were the same as before introduction.

The amount of water that can be retained and the specific surface area are determined according to Japanese Patent Application Laid-open No. 57
It was determined by the method described in Publication No.-30945.

Example row 1 The above aminophenylboronic acid-introduced copolymer was prepared with an inner diameter of 7.
A stainless steel column with a length of 6 m and a length of 100 mm was packed to serve as a second column.

As the first column (column for separating albumin from a sample), N5ahipak BS-502N [Mukasei Kogyo Co., Ltd., trade name, alcoholic acid derived from vinyl alcohol units described in JP-A-60-150839] was used. A crosslinked copolymer having a hydroxyl group and a diethylamino group was
．． A stainless steel column with a length of 6 m and a length of 100° C. was used. FIG. 2 shows an outline of the channel system of the high performance liquid chromatograph used. As shown in the figure, a flow path switching pulp is provided between the first column and the second column, and by this operation, only the albumin separated in the first column is transferred to the second column, and the others are transferred to the second column. The components were guided out of the channel system.

Detailed conditions for chromatography are shown below.

Sample: Healthy human serum (5μt) Mobile phase: (1'-'tL) 250mH ammonium acetate + 50mM magnesium chloride + 500mM sodium chloride + 2% ethanol (pH 8,53 (Solution B J 100mM) Lis(hydroxymethylj aminometh/ + 200mM sorbitol + 50 rr+M BDT & (pH 8,5) (Note) After switching the flow path and introducing albumin into the second column, the N solution was switched to the B solution 8 minutes later.

Gradient mixer Ni Nippon Bunko Kogyo Co., Ltd. Product name P-
A40 Pump Nihon Bunko Kogyo Co., Ltd. Product name TR, IROTAB, -V (Flow rate 0.5 mt/min
+ Detector Nihon Bunko Kogyo Co., Ltd. Trade name Fluorescence detector FP-210 Temperature = 35°C Figure 4 shows a chromatogram obtained only from the first column using the above solution as a mobile phase. As shown in Figure 4, albumin and other components in serum can be separated in a short time.

In addition, the cone fractions I and It under the same conditions.

It was confirmed that II and iV were not mixed in the albumin fraction.

In the first column, albumin elutes in 6-12 minutes. Therefore, the flow path was switched according to the time program shown in FIG. 3 (where ■ and @ indicate the switching shown in FIG. 2).

(Results) The chromatogram obtained by switching the flow path and the combination of the first column and the second column is
Shown in 1. Glycated albumin and non-glycated albumin can be separated to a high degree.

In addition, when each peak was separated and collected and specific coloring of the shield was performed using the thiobarbic acid method, it was confirmed that the peak (■) was albumin bound to the shield, and the peak (■) was albumin not bound to sugar.

[Brief explanation of drawings]

Figure 1 is a cross-sectional diagram of the straw channel system of the iVlam liquid chromatograph used in the present invention, Figure 2 is a perspective diagram of the channel system of the high performance liquid chromatograph used in the examples, and Figure 3 is a diagram of the channel system of the high performance liquid chromatograph used in the examples. Fig. 4 shows the chromadogsim in which albumin was separated from the serum of a healthy person using the No. 10 column used in the present invention, and Fig. 5 shows the separation of glycated albumin and non-shielded albumin from the serum of a healthy person by an uninvented method. It is an isolated chromadogsim. Figure 2 Figure 3 Figure 4 Figure 5

Claims (1)

[Claims] In a method for separating glycated albumin and non-glycated albumin in a sample by liquid chromatography, a first step is performed.
The method is characterized in that albumin is separated from the sample using a column, and the albumin is led to a second column without leaving the chromatography flow path system, where albumin is separated into glycated albumin and non-glycated albumin. Method for separating glycated albumin.