JPS5961777A - Analytical system of bile acid - Google Patents

Abstract

PURPOSE:To separate and analyze various kinds of bile acids with high accuracy, by a method werein bile acids are fractionated by a column packed with a hydrophobic gel having a specific ion exchange group and fractionated bile acids are subsequently mixed with a reactive reagent solution to be successively sent to a specific immobilized enzyme column, a reaction part, an emitted luminous intensity measuring part. CONSTITUTION:When serum is used as a specimen, a solution of bile acids obtained by desalting at a room temp. and deproteinizing treatment is passed through a column packed with a hydrophilic gel having an ion exchange group such as piperidinohydroxypropyl-Sephadex to be fractionated into three fractions, that is, total unconjugated bile acids, total glycine conjugated bile acids and total taurine conjugated bile acids. Each fraction is sent to an immobilized column 24 of 3a-HSD(3a-hydroxysteroid dehydrogenase) while mixed with NAD, resazurin and a diaphorase containing buffer solution 21 in a specimen injecting part 23 to be reacted in a reaction column 25 and fluorescent luminous intensity emitted by resazurin is measured by a fluorescent luminous intensity measuring meter 26 to enable the separation and quantitative analysis of each bile acids with good accuracy.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a bile acid analysis system. In detail, samples in which various bile acids coexist, for example, total free bile acids, total glycine-bound bile acids, and total taurine-bound bile acids, such as body fluids such as serum, can be easily analyzed with high sensitivity and in a short time. Regarding the water analysis system.

Conventionally, analysis of total bile acids has been carried out, but since there is little information on this, total free bile acids, total free bile acids,
Analysis of total glycine-conjugated bile acids and total taurine-conjugated bile acids is desired.

Figure 1 shows a schematic system diagram of a conventionally used total 110 acid analyzer. A sample is injected by a sample injection pump (1), and then divided into two parts: an analysis line (2) and a reference line (3). The reaction reagent is sent to the analysis system path (2) and the reference system path by reaction reagent injection pumps (4) and (5), respectively. Also, the buffer solution is loose ω(
The liquid is sent to the analytical system path (2) and the 77 lens thread path (3) by the liquid sending pumps (8) and (9). (6) and (7) are pumps for injecting air bubbles between sample sections. The thus injected mixture of sample, reaction reagent, and slow hook solution is heated to 60 to 70°C in heating furnace 4.

After passing through the immobilized enzyme column (11) and the reaction carp/I/(6), the liquid sent to the analysis line (2) is measured for fluorescence intensity using a fluorophotometer. - On the other hand, the liquid sent to the reference system path (3) passes through the reaction coil 0 and then its fluorescence intensity is measured with a fluorophotometer 1. then 2
Total bile acid content is calculated from the two fluorescence data. 0 and 09 are bubble release holes.

In the above-mentioned conventional method, an L/phalerene system is installed for heating and smoking in order to eliminate the influence of substances present in the body fluid of the sample and reacting with the immobilized enzyme, resulting in an increase in size. Another problem is that air bubbles for sample separation must be injected, resulting in a loss of sample size due to air bubble removal, resulting in a decrease in analytical sensitivity.

This defense solves the above four points, and is an analysis system that can further analyze three bile acid groups: total free π-conjugated bile acids, total glycine-conjugated bile acids, and total taurine-conjugated bile acids, A) A sucrose fractionation section filled with a hydrophobic gel having an ion exchange group such as piperidinohydroxypropyl gel, and B) A liquid delivery section for one or more reaction reagent solutions, 3α-■SD
An immobilized enzyme column, a reaction section, and a fluorescence measurement section are connected in this order via a flow path, and an injection section for fractionated bile acid is further provided between the liquid feeding section and the enzyme column. Interpose and have a total battle! Ii! The present invention provides a bile acid analysis system comprising: triglyceride bile acids, total glycine-conjugated bile acids, and total taurine-conjugated bile V1 acids. The analysis system not only allows analysis of three groups of bile acids, but also has the following advantages.

By pre-treating the sample with a hydrophobic gel column containing ion exchange groups, substances that react with enzymes and cause undesirable effects on analysis are removed, eliminating the need for a heating furnace as in the conventional method. The overall reaction time is shortened. Therefore, the analysis time per unit time is 1'! l! The number becomes large. In addition, there is no need to inject air bubbles for sample separation, and there is no sample loss when air bubbles are discharged, increasing sensitivity. There is no need for a reference section, and the analyzer d is simplified.

The analytical system according to this invention is a biliary tract such as a biological sample.
” Suitable for analysis of test sample 1 containing multiple components of iso.

In the analysis system of the present invention, depending on the sample, for example, in the case of blood oil, desalination and protein removal treatments are performed using conventional methods. For example, when bile acids are preserved by passing them through an Amberlite
The solution was allowed to cool for 61 minutes, washed with distilled water, and then eluted with ethanol. The combined eluate was treated with a hydrophobic gel containing ion-exchange groups to obtain three fractions containing free, glycine-conjugated, and taurine-conjugated II acids (F and G fractions, respectively). and T fraction). This fractionation method was developed by Ichihara et al.
no-hydroxypropyl 8ephade
x, LIl-26 (PLiP Lll-20) (S
A chemically modified version of ephadex LII-20. ]
Column chromatography using

Each fraction thus obtained is analyzed using the following enzymatic reaction.

NAD N ADII Resorufin Resazurin Trial *I Middle I]
d juice mll'i enzyme 8 a -1, I S D (3α
-hydroxysteroiddehydrogen
NALI, which is oxidized and allowed to coexist with NALI), is simultaneously converted to NAI)H. In the presence of diaphorase, N A D II is oxidized to N A IJ, and the coexisting rezurin simultaneously produces resorufin, which emits fluorescence. Bile acids are analyzed by determining the fluorescence intensity of the fluorophore 'hJ'R.

FIG. 2 shows a schematic system diagram of an embodiment of the analysis system of the present invention ('@l water-gel column is not shown). In other words, the sample injection part (NAI), resazurin, and diafarase-containing buffer solution Qυ is sent with the pump (c).
c), a free form pre-fractionated with a hydrophobic gel column,
Inject samples of the glycine- and taurine-conjugated fractions of each bile acid. Immobilized enzyme column (3
α-ILsD) (e) and the reaction coil (c) were allowed to pass through the reaction coil (c) to complete the reaction, and the total bile acids in each fraction were analyzed by measuring the fluorescence intensity at 4111 with a fluorophotometer (1). be done.

Furthermore, FIG. 8 shows a 411'J schematic diagram (IT! aqueous gel column not shown) of a modified example of the bile acid analyzer of the present invention. That is, a buffer solution Ct1 containing resazurin, diafuofuse, and 1-filament (3α-T18D)
) is fed with pump C'12, and while feeding M buffer solution c (east) containing NAT) with pump Q41, samples of each fraction are injected from the sample injection section 2 as in the previous example. Then, the reaction coil is (7) to complete the reaction, and the total bile acids in each fraction are analyzed by measuring the fluorescence intensity in a fluorometer (c3I).

[Brief explanation of drawings]

Fig. 1 is a general system diagram of a conventional bile acid analyzer, Fig. 2 is a schematic system diagram of five embodiments of the bile acid analysis system of the present invention, and Fig. 3 is a schematic system diagram of the present invention. (11, (4), (5), (8), (9)-
(e), e...liquid pump, (6), (7).
... Air bubble injection pump, (2) ... Analysis system line,
(3)...Reference system line, OQ...Heating furnace,
01), (:?A...immobilized enzyme column,
0ri, (c), 0)...reaction coil, υ・°・NA
I, N buffer solution containing resazurin and diaphorase, 6D...°resazurin, diaphorase and 8α-E
[SI) Buffer solution containing fermented rice, Fig. 1 Fig. 2

Claims (1)

[Claims] 1. A) A bile acid fractionation section filled with a hydrophobic gel having an ion exchange group such as piperidinohydroxypropyl gel, and B) A liquid delivery section for one or more reaction reagent solutions, 8α-1181
) Immobilized enzyme column, reaction part and fluorescence intensity 71ill
The fixed parts are connected in this order via a flow path, and an injection part for fractionated bile acids is interposed between the liquid feeding part and the enzyme column, and total free bile acids and total free bile acids are separated. A bile acid analysis system configured to analyze glycine-conjugated bile acids and total taurine-conjugated bile acids.