JPH0718856B2 - Fractional measurement of blood bilirubin - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for measuring the fraction of blood albumin by high performance liquid chromatography.

(Prior Art) Bililpin is a normal metabolite of hemoglobin, and is contained in a large amount in bile as a free form, a monoglucuronic acid conjugate, or a diglucuronic acid conjugate. The blood level of bilirubin is extremely low, but for example, when liver function declines and jaundice develops, the level increases. Therefore, the measurement of blood bilirubin is one of the typical items in liver function tests.

Blood bilirubin has been conventionally measured by a method of detecting a dye produced by diazo coupling.
This diazo reaction method is the most common method for measuring bilirubin, and in particular Mall using an alcohol as a coupling reagent.
The oy-Evelyn method (J. Biol. Chem., 119 , 481, (1937))
Due to its simplicity, it is widely used in clinical tests.
However, the above-mentioned diazo reaction method has a drawback that the color tone changes depending on the pH during the reaction; or the color intensity varies depending on the reaction time, and thus high-precision measurement cannot be performed. Furthermore, it is not possible to individually measure each bilirubin component in the blood existing as a free form or a glucuronic acid-encapsulated form. Recently, the presence of deltabilirubin ( _Bδ ), which is considered to be strongly covalently bound to albumin, has been newly confirmed in the serum of jaundice patients, and the ratio of this deltabilirubin to total bilirubin reflects liver function. Became known. Therefore, development of a fractional measurement method for each bilirubin component including delta bilirubin is desired.

If the above bilirubin is measured directly by the diazo reaction rather than indirectly, the accuracy will be improved. As such a direct fractionation measuring method, there are various chromatographic methods, particularly a reversed-phase high performance liquid chromatography method (HPLC method). In reversed-phase chromatography, each bilirubin component except B _δ [diglucuronic acid-conjugated bilirubin (BDC), monoglucuronic acid-conjugated bilirubin (BMC), free bilirubin (BU)]
Is not eluted unless the concentration of organic solvent in the mobile phase is more than 50% by volume. However, under such a high concentration of organic solvent, B _δ, which is a form bound to various blood proteins (albumin and globulin) and albumin, aggregates and precipitates, and thus B _δ is not eluted and the number is several. The column is clogged with one measurement.

Since Lauff et al to isolate the B _[delta] in HBLC, are removed by pretreatment of several stages other blood proteins except albumin (such as globulin) [Clinical Chemistry 28, 629, (1
982)]. Although such pretreatment operation enables separation and measurement of B _δ , the pretreatment operation is complicated and B _δ is unstable to light, so it is decomposed during the pretreatment operation to perform high-precision measurement. It has the drawback that it is hard to be discouraged.

(Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional drawbacks, and an object of the present invention is to measure each bilirubin component including deltabiliribine in blood with high precision. To provide a method. Another object of the present invention is to provide a method of fractionating and measuring each of the above-mentioned biliribine components using a reversed-phase high performance liquid chromatography without performing a complicated pretreatment operation.

(Means for Solving Problems) The method for measuring the fraction of bilirubin in blood according to the present invention comprises (a) a (meth) acrylic acid ester-based resin as a raw material, and a hydrophilic property of an organic polymer having a carboxyl group on its surface. A step of introducing a sample containing serum or plasma into a separation column packed with a packing material as a stationary phase to adsorb protein components and bilirubin to the packing material; (b) an aqueous solution having a pH of 2 to 5 to the separation column; Supplying an eluent containing an organic solvent as a main component at a ratio of 40% by volume or less as a mobile phase to elute the albumin fraction, globulin fraction and deltabilirubin, and (c) the separation column (b) Reverse phase high-performance liquid chromatography including the step of supplying an eluent having a higher organic solvent concentration than that of) as a mobile phase and sequentially eluting each bilirubin fraction other than deltabilirubin. It is a fractional measurement method by means of which the above-mentioned objects are achieved.

The method of the present invention is embodied by, for example, the measuring device shown in FIG. This device comprises an eluent tank 11 containing an eluent (mobile phase) 1 and a constant flow pump 12 for the tank 11
The sample introducing device 2 connected to the sample introducing device 2 and the separation column 3 connected to the sample introducing device 2. Since it is necessary to change the organic solvent content of the eluent 1 as described later, in this device, the first eluent tank 11a containing the first eluent 1a made of an aqueous solution such as a buffer solution and A second eluent tank 11b containing a second eluent 1b made of an organic solvent is provided. A concentration gradient device 13 is connected to the constant flow rate pump 12, whereby the first eluent 1a and the second eluent
1b is mixed to the desired degree. Separation column 3 has a detector
41 is connected, and the value detected here is displayed on the recorder 41.

To measure bilirubin in blood with this measuring device,
First, the first eluent 1a is introduced into the separation column 3 via the constant flow pump 12. This first eluent 1a is an aqueous solution, usually
Various buffers such as acetate buffer are used, and the pH is 2
5, preferably in the range of 3-4. In the neutral to alkaline region where the pH exceeds 5, bilirubin becomes unstable and easily decomposes. Further, when the pH exceeds 12, various proteins in the blood aggregate and precipitate, causing clogging of the column. Next, a sample such as serum or plasma is injected from the sample introduction device 2, mixed with the first elution plate 1a, and supplied to the separation column 3. As a stationary phase, the separation column 3 is filled with an organic polymer-based hydrophilic filler made of (meth) acrylic acid ester-based resin and having a carboxyl group on its surface. This packing material is a highly hydrophilic gel that is used for reverse phase chromatography and does not adsorb proteins nonspecifically. The above (meth) acrylic acid ester-based resin is
An acrylic ester or a methacrylic ester is used as a (co) polymerization component. Bilirubin and serum proteins in the sample are adsorbed on the stationary phase here. First eluent 1a
Flow for an appropriate time, the second eluent 1b is introduced into the system by the concentration gradient device 13, and the first eluent 1a and the second eluent 1b are introduced.
The mixed solution of the eluent 1b is supplied to the separation column 3 as a mobile phase. As the second eluent 1b, an organic solvent compatible with water, usually acetonitrile and / or methanol is used. This organic solvent concentration is the same as that of the entire eluent 1.
It is set below 40% by volume. If the organic solvent exceeds 40% by volume, proteins in blood coagulate and precipitate, resulting in clogging of the column. By introducing such a mobile phase (eluent 1) having an organic solvent concentration of 40% by volume or less into the separation column 3, albumin, globulin and deltabilirubin (albumin-bound bilirubin) are eluted. Normal,
A method of gradually increasing the organic solvent concentration from 0 to 40% by volume is adopted. Next, the ratio of the second eluent 1b to the entire eluent 1 is gradually increased by the concentration gradient device 13. By increasing the concentration of the organic solvent, the bilirubin components other than delta bilirubin, that is, the diglucuronic acid-encapsulated burirubin, the monoglucuronic acid-encapsulated bilirubin and the advantageous bilirubin are sequentially eluted. If there is an isomer of free bilirubin isomerized by light, this isomer is also eluted. Since bilirubin has a tetrapyrrole skeleton, it has four secondary amino groups per molecule. Since this amino group has a positive charge, it may be difficult to be adsorbed by the hydrophilic packing material in the column. Therefore, the bilirubin component may be eluted without being retained in the column, or the separation may be insufficient. In such cases, it is recommended to add a trace amount of alkyl sulfonate to the mobile phase. The addition of alkyl sulfonate neutralizes the charge and increases the hydrophobicity, so the retention on the column is high,
Good separation can be achieved. The bilirubin separated into each component in the separation column 3 is measured by the detector 4 after leaving the separation column 3 and displayed on the recorder 41. For example, an absorptiometer is used as the detector, and the absorption intensity at 450 nm is measured.

(Function) In the method of the present invention, the type of packing material of the stationary phase and the pH of the mobile phase in high performance liquid chromatography were specified, and the organic solvent concentration in the mobile phase was sequentially changed. It is possible to fractionate and measure each bilirubin component including delta bilirubin without coagulation. Since no special pretreatment is required, each bilirubin component is not decomposed or lost, and highly accurate measurement values can be obtained.

(Example) Hereinafter, the present invention will be described with reference to Examples.

Example 1 (A) Preparation of filler gel: 40 ml of 4% by weight aqueous solution of polyvinyl alcohol and 40 g of tetraethylene glycol dimethamellilate, 10 tetramethylolmethane triacrylate
g, methacrylic acid 50 g, toluene 40 g and benzoyl peroxide 1.5 g were added. At 80 ° C with stirring at 400 rpm
After reacting for 10 hours, it was washed with hot water and acetone to obtain a polymeric spherical porous material having a particle size of 8 to 10 μm.

(B) Measurement of bilirubin: Fractional measurement of bilirubin in serum was performed by the measuring device shown in FIG. Separation column 3
As the material, the filler gel obtained in the item (A) was packed in a stainless steel column having an inner diameter of 6 mm and a length of 150 mm. As the first eluent (first mobile phase) 1a, an acetate buffer (pH 3.
0) and acetonitrile as the second eluent (second mobile phase) 1b.

First, while the first eluent 1a was supplied to the separation column 3 at a rate of 1.0 ml / min by the constant flow pump 12, the sample was supplied to the separation column 3 via the sample introduction device 13. Serum of a jaundice patient was used as a sample. Three minutes after the addition of the sample, the concentration gradient device 13 (built into the liquid chromatograph system LC-4A (manufactured by Shimadzu Corporation)) was operated, and the mixture of the first eluent 1a and the second eluent 1b became the eluent. The (mobile phase) 1 was supplied to the separation column 3. The concentration of the second eluent 1b in the entire eluent 1 was gradually increased at a rate of 4% by volume / minute. As the detector 4, an absorptiometer (UVIDEC 100-
VI; manufactured by JASCO Corporation) was used to measure the absorption intensity at 450 nm. The chromatogram displayed on the recorder 41 is shown in FIG. As a comparison, the same operation was performed using 10 μm serum of a healthy person. The obtained chromatogram is shown in FIG. 2 (B).

Comparing FIGS. 2 (A) and (B), peaks a to d of substances which are not contained in the serum of healthy subjects are found in the serum of jaundice patients.
Is confirmed. When these peaks were identified, they were confirmed to be delta bilirubin (B _δ ), diglucuronic acid-conjugated bilirubin (BDC), monoglucuronic acid-conjugated bilirubin (BMC) and free bilirubin (BU), respectively.

Example 2 The procedure of Example 1 was repeated except that the pH of the first eluent 1a was set to 4.0, and the bilirubin in the serum of jaundice patients was measured. The obtained chromatogram is shown in FIG. In FIG. 3, the peaks a to d are the same as in Example 1, and a ₀ is
It is B _δ which was partially eluted in a state where the pH of the eluent 1 was high.

Comparative Example 1 Bilirubin in the serum of a jaundice patient was measured in the same manner as in Example 1 except that the pH of the first eluent 1a was 8.0. The obtained chromatogram is shown in FIG. In FIG. 4, bilirubin is not sufficiently separated into each component and appears as one peak f. e peak is eluent 1
Is a bilirubin mixture eluted at a high pH.

Comparative Example 2 As the first eluent 1a, the same acetate buffer solution as in Example 1 (pH 3.
The procedure of Example 1 was repeated except that a mixed solution of 0) and acetonitrile (volume ratio 1: 1) was used, and an attempt was made to measure bilirubin in the serum of a jaundice patient. However, the serum protein coagulated in the separation column 3 and the column internal pressure increased, so that the measurement was impossible.

(Effects of the Invention) According to the present invention, the fractional measurement of bilirubin in blood can be performed with high accuracy without complicated pretreatment.
This method is extremely useful as a liver function test method, for example, because bilirubin in blood, particularly delta bilirubin, which is an important indicator of liver function, is accurately measured.

[Brief description of drawings]

FIG. 1 is a flow diagram showing an embodiment of a measuring apparatus used in the method of the present invention; FIGS. 2 (A) and 2 (B) are
Chromatograms obtained by measuring sera of jaundice patients and sera of healthy individuals by the method of the present invention respectively; FIG. 3 is a chromatogram obtained by measuring sera of jaundice patients by the other methods of the present invention; and FIG. , PH different from the method of the present invention
2 is a chromatogram obtained by measuring the serum of a jaundice patient using the eluent of. 1 ... Eluent, 2 ... Sample introduction device, 3 ... Separation column, 4 ...
… Detector, 13… Concentration gradient device.

Claims (3)

[Claims] 1. A separation column containing (a) a (meth) acrylic acid ester-based resin as a material, and an organic polymer-based hydrophilic filler having a carboxyl group on its surface as a fixed layer is filled with serum or plasma. A step of introducing a sample containing the adsorbed protein component and bilirubin to the packing material, (b) the separation column containing an aqueous solution of pH 2 to 5 as a main component
A step of supplying an eluent containing an organic solvent at a ratio of 40% by volume or less as a moving phase to elute the albumin fraction, globulin fraction and deltabilirubin, and (c) the separation column from the step (b) Is a method for measuring the fractionation of bilirubin in blood by reverse phase high performance liquid chromatography, which comprises a step of supplying an eluent having a high organic solvent concentration as a mobile phase and sequentially eluting each bilirubin fraction other than deltabilirubin. 2. The measuring method according to claim 1, wherein the concentration of the organic solvent in the eluent in the items (b) and (c) is gradually increased. 3. The measuring method according to claim 1, wherein the bilirubins other than the delta bilirubin are diglucuronic acid-conjugated bilirubin, monoglucuronic acid-conjugated bilirubin and free bilirubin.