JPH0711514B2 - Method for separating glycated albumin - Google Patents

Description

TECHNICAL FIELD The present invention relates to separation of glycated albumin in which separation of albumin in a sample and separation of glycated albumin and non-glycated albumin are performed in the same channel of chromatography. Regarding the method.

In a disease such as diabetes where the blood glucose level is high for a long period of time, various proteins in the living body undergo a non-enzymatic glycation reaction. Serum albumin is one of those proteins.

Conventionally, for the diagnosis of diabetes, blood glucose, urinary glucose, insulin, glucagon measurement, oral glucose tolerance test, etc. have been used, but the results are easily affected by physiological conditions or measurement methods, and the subject is also burdened. There are problems such as. On the other hand, the concentration of glycated proteins in the living body has little attention in recent years because it is less affected by temporary physiological conditions and serves as an index of the average sugar concentration in blood in the past weeks to months.

For example, glycated hemoglobin serves as an average index of blood glucose levels in the past 1 to 3 months, and has a positive correlation with the severity of diabetic patients. Therefore, it has already been clinically applied as a new diagnostic method for diabetes and an index for blood glucose control. There is. In particular, the ratio of glycated hemoglobin to total hemoglobin is said to correlate well with the fasting blood glucose level three months ago.

However, clinically, it is useful to have information on the blood glucose status of several weeks to one month ago, and glycated albumin has a half-life of 17 days, so as an index that can best meet this demand,
Recently, it has been receiving attention from various fields.

That is, glycated albumin responds to a blood glucose state more quickly than glycated hemoglobin, and does not significantly fluctuate under the influence of a temporary physiological condition such as a blood glucose level.

For example, glycated albumin has been suggested to be useful, for example, it is effective in determining whether or not the current or newly modified dietary therapy is suitable for the patient at an early stage. [Kay F. Mcc
farland etal, DIABETS), 28,1011,1979, C. Earl Guthrow etal, Proc.Natl.Acad.Sci.U
SA), 76 , No. 9.4258, 1979] (Prior art) Conventionally, as a method for separating and detecting glycated albumin, ion exchange chromatography using carboxymethyl-type cellulose or the like as a stationary phase [James F.D. , J. Bio Chem (James F.Day etal, JB
Chem.), 254 , No. 3,595, 1979], introducing a dihydroxyboronyl group into a soft gel such as cellulose, and utilizing the interaction between this functional group and glucose.・ AkMallia et al, Analytical Letters
s), 14 (B8), 649-661, 1981], a method utilizing a color reaction represented by the thiobarbituric acid method [Kay F. Mcfarland eta.
l, DIABETES), 28 , 1011, 1979], etc. have been tried.

(Problems to be Solved by the Invention) In all of the above-mentioned conventional techniques, glycated albumin and non-glycated albumin can be separated, but they cannot be separated from other components in the body fluid.

The reason depends on the separation method. In the case of ion exchange chromatography, prealbumin in the body fluid,
This is because there are many substances such as α-globulin that are close to the isoelectric point of albumin.In the case of affinity chromatography and the color-developing method of sugar, most of the proteins except albumin in body fluids are glycoproteins. Because there is.

For some time, for example, a pretreatment of isolating only albumin from the body fluid using a gel to which Cibacron Blue F3G-A, which has a high affinity for albumin, was bound was essential. .

However, conventionally, a method has been employed in which albumin is isolated from a sample, then once taken out of the chromatographic system, concentrated, and then led again to a column capable of separating glycated albumin and non-glycated albumin. That is, the operation of separating albumin from the sample and the operation of separating albumin into glycated albumin and non-glycated albumin are performed as at least two separate operations, which is complicated and requires a long time.

These are the largest unmeasured sources of glycated albumin in clinical laboratories where rapidity, operability, reproducibility and automation are required.

(Means for Solving Problems) As a result of earnest research for overcoming the above problems, the present inventors have found that the glycated albumin has excellent rapidity, operability, reproducibility, and is easy to automate. The inventors have found a method for separating non-glycated albumin and completed the present invention.

That is, the present invention is a method for separating glycated albumin and non-glycated albumin in a sample by liquid chromatography, in which a hydrophilic hydrophilic cross-linked copolymer having an alcoholic hydroxyl group has a dye or an ion having high affinity with albumin. The first column packed with gel with exchange groups bound to it separates albumin from the sample, and dihydrogenates a hard hydrophilic cross-linked copolymer having alcoholic hydroxyl groups without removing it from the chromatographic system. Leading to a second column packed with a gel with a boronyl group attached,
Therefore, the present invention relates to a method for separating glycated albumin, which comprises separating albumin into glycated albumin and non-glycated albumin.

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

The albumin referred to in the present invention refers to both glycated albumin and non-glycated albumin.

The sample referred to in the present invention contains at least either glycated albumin or non-glycated albumin, or both, and examples thereof include body fluids such as blood and urine.

The first column used in the present invention is a column capable of separating albumin from other components in the body fluid, and is a gel to which a dye Cibacron Blue F3G-A (trade name of Ciba Geigy) having a high affinity for albumin is bound. A column packed with
Alternatively, it is a column packed with ion exchange gel.

However, as a carrier for introducing a dye having a high affinity for albumin, it has excellent mechanical strength, chemical stability, and low non-specific adsorption of protein. A hard hydrophilic crosslinked copolymer having a meq / g, a specific surface area of 5 to 1000 m ² / g, and an amount of water that can be retained is 0.5 to 6.0 g / g is preferable, and the most preferable example is JP-A-57-30945. Examples thereof include crosslinked copolymers having an alcoholic hydroxyl group derived from a vinyl alcohol unit described in JP-A No.

The most preferable example of the ion exchange gel is a cross-linked copolymer having an alcoholic hydroxyl group derived from a vinyl alcohol unit and a diethylamino group described in JP-A-60-150839. This cross-linked copolymer is
By the method described in Japanese Patent Application No. 60-1222, albumin and other components in body fluid can be rapidly separated.

The second column used in the present invention is a column capable of separating glycated albumin and non-glycated albumin, and is a column packed with a gel having a dihydroxyboronyl group.

Since the dihydroxyboronyl group specifically binds to compounds having 1,2-cisdiol, affinity chromatography using a gel having this functional group as a stationary phase is effective for separating glycated albumin and non-glycated albumin. Is.

As a stationary phase carrier for introducing a dihydroxyboronyl group, it has excellent mechanical strength, chemical stability, and little non-specific adsorption of protein, and therefore the alcoholic hydroxyl group 1.0 to 14.0 meq / g per weight of the cross-linked copolymer. , Specific surface area 5-1000
A hard hydrophilic cross-linked copolymer having m ² / g and an amount of water that can be retained of 0.5 to 6.0 g / g is preferable. Most preferred example
JP-A-57-30945, JP-A-56-64657 and JP-A-54
The cross-linked copolymer described in JP-A-145036 can be mentioned.

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

In Fig. 1, mobile phase solutions A and B are respectively pumped to the injector, and the sample is sent from the injector together with the mobile phase to the first column to separate albumin and other components in the sample. It Then, by operating the switching valve, only the albumin separated in the first column is fed into the second column, separated into glycated albumin and non-glycated albumin, and fed into the detector.

Further, as shown in FIG. 2, if it is attached to the suction side of a gradient mixer pump often used in high performance liquid chromatography, it is possible to use only one pump.

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

As the mobile phase, at least two types of mobile phases (A liquid and B
It is preferable to prepare a liquid) and switch it during the process.

That is, before injection of the sample, the liquid A was passed through at the time of injection, albumin and other components in the sample were separated in the first column, and only albumin was introduced into the second column,
Switch to B liquid (so-called stepwise method) or use a concentration gradient forming device (gradient mixer) to
A method of continuously converting the liquid to the liquid B (so-called gradient method) can be mentioned.

The solution A referred to here is preferably under conditions under which glycated albumin and a dihydroxyboronyl group are bonded, that is, does not contain a substance having 1,2-cisdiol and has a pH of 7.5 to 9.5. on the other hand,
The solution B preferably has a pH of 2 to 6.5, and may contain a substance having 1,2-cisdiol even at a pH of 7.5 or higher. The concentration of the substance having 1,2-cisdiol is preferably 50 to 500 mM. The salt concentration of both solutions A and B is preferably 10 mM to 1 M, and the type of buffer solution is not particularly limited. Also, ethyl alcohol,
In some cases, it may be preferable to contain a small amount of a water-soluble organic solvent such as methyl alcohol acetonitrile or ethylene glycol.

The shape and size of the stationary phase carrier, the size of the column and the flow rate of the mobile phase used in the present invention are not particularly limited. However,
When importance is placed on speed, the stationary phase carrier is preferably spherical and has a particle size of preferably 1 to 50 μm, more preferably 1 to 10 μm. From the viewpoint of speed, the column size is preferably 10 mm or less in inner diameter and 30 cm or less in length, 2 to 8 mm in inner diameter and 1 to 1 in length.
0 cm is particularly preferred. The flow rate is preferably 0.1-10 ml / min,
More preferably, it is 1 to 10 ml / min.

(Effect of the Invention) In the method for separating glycated albumin of the present invention, in liquid chromatography, albumin is separated from the sample in the first column and the second method is used without separating the albumin from the channel system of the chromatography. The column is characterized in that albumin is separated into glycated albumin and non-glycated albumin there.

Conventionally, in order to separate glycated albumin and non-glycated albumin from a sample containing a component other than albumin, as described above, at least two separate steps were required.
According to the method of the present invention, the glycated albumin and the non-glycated albumin in the sample can be separated by one operation, that is, by only injecting the sample once into the sample injection port, and the respective amounts can be quantified.

According to the method of the present invention, it is possible to analyze glycated albumin and non-glycated albumin easily and quickly in a general clinical laboratory with high reproducibility, and it is also easy to automate.

The present invention largely contributes to the spread of analysis of glycated albumin.

(Examples) Examples of the present invention will be shown below, but the scope of the present invention is not limited by these examples.

Experiment 1 Preparation of Second Column (for Separation of Glycated Albumin and Non-Glycated Albumin) The vinyl alcohol copolymer gel (X; 0.3, hydroxyl group amount; 7.8 meq / g, described in Example 1 of JP-A-57-30945). Amount of water that can be retained (WR); 1.78 g / g, specific surface area; 78 m ² / g, particle size; 9.0 μm) 25 g, epichlorohydrin 116.8 g, dimethyl sulfoxide 250 ml, 10N sodium hydroxide aqueous solution
12.5 ml was added and reacted at 30 ° C. for 20 hours. The introduction amount of the epoxy group was 1.0 meq / g. The epoxy group was quantified by the method described in JP-A-57-190003.

Next, 13 g of aminophenylboronic acid hemisulfate was dissolved in 250 ml of water, and 20 g of the above-mentioned epoxy group-introduced polymer was added to pH 11 and the reaction was carried out at 60 ° C. for 20 hours. Next, 25 mM tris (hydroxymethyl) aminomethane was added to protect the residual epoxy group, and the reaction was carried out at 50 ° C. for 5 hours. The amount of boric acid introduced was 0.28 meq / g. The boric acid was quantified by the following method.

To 1 g of the above-mentioned polymer into which aminophenylboronic acid was introduced
The boron-carbon bond was cleaved by adding 10 ml of 30% hydrogen peroxide solution and reacting for 5 hours. Next, the polymer was spun down and the supernatant was collected. After decarboxylation, boric acid ester was formed by adding sugar, strongly oxidized, and boric acid was quantified by titrating with sodium hydroxide.

The instruments used for this analysis are all made of quartz.

The hydroxyl group density of the copolymer into which aminophenylboronic acid was introduced was 9.2 meq / g (after cleavage of dihydroxyboronyl group), and the amount of water that could be retained was 1.70 g / g. The particle size and specific surface area were the same as before the introduction.

The amount of water that can be retained and the specific surface area are described in JP-A-57-
It was determined by the method described in Japanese Patent No. 30945.

Example 1 The above aminophenylboronic acid-introduced copolymer was used to obtain an inner diameter of 7.6 m.
It was packed in a m- and 100-mm-long stainless steel column, and used as the second column.

As the first column (column for separating albumin from the sample), Asahipak ES-502N [Asahi Kasei Co., Ltd., trade name, alcoholic hydroxyl group derived from vinyl alcohol unit described in JP-A-60-150839] is used. Cross-linked copolymer with diethylamino group, inner diameter 7.6mm, length 100mm
Packed in a stainless steel column of the above] was used. The outline of the flow path system of the high performance liquid chromatograph used is as shown in FIG. As shown in the figure, a flow path switching valve was provided between the first column and the second column, and by this operation, only the albumin separated in the first column was put in the second column and other components. To lead to the outside of the flow path system.

The detailed conditions of chromatography are shown below.

Sample: Serum of healthy person (5 μl) Mobile phase: (A solution) 250mH ammonium acetate ＋ 50mM magnesium chloride ＋ 500mM sodium chloride ＋ 2% ethanol (pH8.5) (B solution) 100mM tris (hydroxymethyl) aminomethane ＋ 200mM sorbitol ＋ 50mM EDTA ( (pH 8.5) (Note) The flow path was switched, and albumin was introduced into the second column, and 8 minutes later, the solution A was switched to the solution B.

Gradient mixer: JASCO Corporation, trade name GP-A40 Pump: JASCO Corporation, trade name TRIROTAR-V (flow rate 0.5 ml / min) Detector: JASCO Corporation, trade name Fluorescence detector FP −210 Temperature: 35 ° C Figure 4 shows a chromatogram obtained from the first column only, using the above liquid A as the mobile phase [However, the detector is UV (Jasco Corporation, trade name UVIDEC 100-IV). ,wavelength
280 nm)]. As can be seen from FIG. 4, albumin and other components in serum can be separated in a short time.

Under the same conditions, the cone fractions I, II, III, IV
Was confirmed not to be mixed in the albumin fraction.

In the first column, albumin elutes in 6-12 minutes. Therefore, the flow path was switched by the time program as shown in FIG. 3 (however, a and b are the states of the switching valve shown in FIG. 2).

(Results) FIG. 5 shows a chromatogram obtained by such a combination of the flow channel switching and the first column and the second column. Glycated albumin and non-glycated albumin are highly separated.

In addition, when each peak was separated and sampled and the specific color of the sugar was developed by the thiobarbituric acid method, it was confirmed that the peak was albumin bound to sugar and the peak was albumin not bound to sugar. did.

Experiment 2 Preparation of Second Column (for Separation of Glycated Albumin and Non-Glycated Albumin) The vinyl alcohol polymer gel (X; 0.4, hydroxyl group amount: 4.2 meq / g, described in Example 2 of JP-A-57-30945). WR;
An epoxy group was introduced into 25 g of 2.48 g / g, specific surface area; 120 m ² / g, particle size; 9.6 μm) by the same method as in Example 1. The introduction amount of the epoxy group was 0.95 meq / g.

Next, 20 g of aminophenylboronic acid hemisulfate was dissolved in 400 ml of water and adjusted to pH 11, 20 g of the above epoxy group-introduced polymer was added, and a boronyl group was introduced in the same manner as in Example 1. Next, the residual epoxy group was protected by the same method as in Example 1. The amount of boric acid introduced is 0.45 meq /
It was g. The hydroxyl group density of the copolymer introduced with aminophenylboronic acid was 4.8 meq / g, and WR was 2.42 g / g. The particle size and specific surface area were the same as before introduction.

Example 2 The above aminophenylboronic acid-introduced copolymer was used to obtain an inner diameter of 6.0 m.
It was packed in a stainless steel column having a length of m and a length of 100 mm to obtain a second column.

As the first column (column for separating albumin from the sample), an anion exchange gel (X; 0.32, hydroxyl amount; 4.9 meq / g, described in Example 1 of JP-A-60-150839) was used.
Ion exchange capacity; 0.5 meq / g, amount of water that can be retained (WR); 1.
9 g / g, particle size; 9.0 μm) was used to fill a stainless steel column having an inner diameter of 7.6 mm and a length of 50 mm. The outline of the flow path system of the high performance liquid chromatograph used is as shown in FIG. As shown in the figure, a flow path switching valve was provided between the first column and the second column, and by this operation, only albumin separated in the first column was put in the second column and other components were Was guided to the outside of the flow path system.

The chromatographic conditions are shown below.

Sample: (I) Serum from healthy person diluted 20-fold with physiological saline (10 μl) Mobile phase: (Solution A) 50 mM glycine + 150 mM magnesium chloride (pH 8.5) (Solution B) 100 mM Tris (hydroxymethyl) Aminomethane + 100 mM sorbitol + 50 mM EDTA + 100 mM NaCl (pH8.5) Pump: JASCO Corporation, trade name 880-PU (2 units) (Flow rate: 1.0 ml / min) Detector: JASCO Corporation, trade name FP-210 Temperature: 40 ℃ (Note) 2.5 minutes after injecting the sample, switch the valve,
Albumin was introduced into the second column, and after 3.5 minutes, the solution A was switched to the solution B.

(Results) FIG. 6 shows a chromatogram obtained only from the first column using the above liquid A as a mobile phase [however, the detector is
UV (Nippon Bunko Kogyo Co., Ltd., trade name UVIDEC 100-IV, wavelength 280
nm)]. In the chromatogram of FIG. 6, it was confirmed by electrophoresis that the albumin fraction substantially contains only albumin. Further, FIG. 7 shows a chromatogram obtained by combining the first and second columns using the above chromatography conditions. When the respective peaks were separated and collected and subjected to a thiobarbituric acid reaction, it was confirmed that the A peak was albumin to which sugar was not bound and the B peak was albumin to which sugar was bound.

[Brief description of drawings]

FIG. 1 is a flow chart of the liquid chromatograph used in Example 2, FIG. 2 is a flow chart of the liquid chromatograph used in Example 1, and FIG. Time program, FIG. 4 is a chromatogram obtained by separating albumin from serum of a healthy person using the first column in Example 1, and FIG. 5 is a graph of glycated albumin and non-glycated albumin from serum of a healthy person in Example 1. The separated chromatogram, FIG. 6, shows the first in Example 2.
Chromatogram obtained from the column of
3 is a chromatogram obtained by separating glycated albumin and non-glycated albumin from serum of a healthy person in Example 2.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP 61-14572 (JP, A) JP 58-223061 (JP, A) JP 58-135454 (JP, A)

Claims (1)

[Claims] 1. A method for separating glycated albumin in a sample by liquid chromatography, wherein a hard hydrophilic cross-linked copolymer having an alcoholic hydroxyl group is bound with a dye or an ion-exchange group having a high affinity for albumin. In the first column packed with gel, albumin was separated from the sample, and dihydroxyboronyl was added to the hard hydrophilic cross-linked copolymer having alcoholic hydroxyl groups without removing it from the chromatographic flow channel system. A method for separating glycated albumin, which comprises leading to a second column packed with a group-bonded gel, and separating albumin into glycated albumin and non-glycated albumin there.