JPS584556B2 - Bile acid measuring device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring bile acids in blood (serum) and bile.

More specifically, the present invention involves heating a blood or bile sample to immobilize the enzyme, and then adding 3α-HSD and N to the sample.
The present invention relates to a bile acid measuring device that enables the addition of a mixed reagent with AD, mixing and stirring to cause a reaction, and the quantitative fluorescence measurement of the reaction product NADH using a fluorophotometer.

As a conventional method for measuring bile acids in blood and (serum) bile, an automatic measuring method using fluorescence measurement, which eliminates the extraction operation, proposed by Mr. Masashige et al. is known.

This method uses 3α-H SD (
3 a-hydroxysteroid deh
hydrogenase) and NAD (Dip
Hosphopyridine Nucleotide
) was mixed and stirred, and then a mixture of diaphorase and resoazurin was added and stirred, and one of the reaction products, resorufin, a fluorescent substance, was observed using a fluorophotometer. It performs optical quantification and thereby quantitatively measures bile acids by calculation.

Therefore, according to this method, the reaction is complicated and the purity of the reagents is extremely required.

Furthermore, according to this method, since there are a large number of reaction reagents, problems due to mixing of the reagents are likely to occur.

In view of these circumstances, the present inventor has conducted extensive research and discovered that the fluorescent substance NADH is produced during the reaction, and that this N
We have discovered that it is possible to quantitatively measure bile acids by measuring the fluorescence of ADH, and hereby propose a specific device that can actually perform automatic and continuous measurements. A liquid addition tube, a heating coil, a mixing stirring coil, and a fluorescence flow cell of a fluorescence photometer are provided in this order, and the buffer addition tube is further equipped with a sample introduction tube and a pressurized air input tube downstream thereof, and a heating coil is provided. 3 between the coil and the mixing stirring coil
This bile acid measuring device is equipped with a tube for adding a mixed solution of α-HSD and NAD, and is configured so that NADH, a reaction product, can be quantitatively measured by fluorescence using a fluorophotometer.

That is, one of the main features of the bile acid measuring device according to the present invention is the quantitative measurement of bile acids by fluorescence determination of the reaction product NADH.

Therefore, the target reaction product for fluorescence quantification can be obtained using only a mixture of 3α-HSD and NAD as a reagent.
This makes it possible to reduce the number and amount of reagents and the amount of sample, and also simplifies the mechanism for mixing reagents or samples with them.
Furthermore, the reproducibility of quantitative determination is improved.

According to the bile acid measuring device of the present invention, a blood or bile sample is first subjected to heat treatment in order to immobilize its enzymes.

For example, it is placed in a hot bath at about 67° C. for about 5 minutes.

Next, a mixture of 3α-HSD and NAD is added to the sample, mixed and stirred to react, and after a certain period of time, the reaction product NADH is quantified by fluorescence using a fluorophotometer, thereby calculating the amount of bile acids. do.

In fluorescence quantification, for example, a sample in which NADH has been generated is appropriately suctioned into a fluorescence flow cell of a fluorescence photometer, and its NA is measured.
DH is quantified.

The reaction formula for generating NADH is considered to be as follows.

The present invention will be illustrated below based on an example of an embodiment shown in the figures.

In FIG. 1, a bile acid measuring device 1a includes a hot water shaking tank 2a, a heating coil part 11a immersed in this tank, and a blood (serum) sample input tube provided at the entrance of this fill part. 5a and pH 7.4 Tris buffer addition tube 12a
, a mixing stirring coil 13a connected in order from the outlet of the heating coil section 11a, a fluorescent flow cell 7a of the fluorescence photometer 8a, a discharge pipe 10a, and the heating coil section 11a.
and mixing stirring coil 13a. It mainly consists of a reagent addition tube 6a connected between Th.

Note that 14a is a pressurized air injection pipe.

Next, a measuring method using the bile acid measuring device 1a having the above-described configuration will be specifically explained.

First, a sample is introduced through the sample input tube 5a, and the pH is adjusted to 7.4 by the addition of Tris buffer from the Tris buffer addition W12a.Air is input through the pressurized air input tube 14a to distinguish between the samples, and air is introduced into the heating coil section. 11a.

Then, it is heated for 5 minutes at 67°C in the hot water shaking tank 2a and reaches the mixing stirring coil 13a. During this process, NAD and 3α-HSD, which have been adjusted to pH 7.4 with a phosphoric acid buffer, are mixed with the reagent addition tube 6a. Add the mixture.

In this way, the mixed samples are mixed and stirred for several minutes, but the NADH production reaction is completed without mixing of adjacent mixed samples.

Then, fluorescence quantitative measurement of NADH is performed as appropriate at an excitation wavelength of 340 nm and an emission wavelength of 450 nm, and bile acids are determined based on the measured values.

[Brief explanation of drawings]

FIG. 1 is a functional explanatory diagram showing an example of the bile acid measuring device according to the present invention. 1a... Bile acid measuring device, 5a... Sample input tube, 6a
... Reagent addition tube, 7a ... Fluorescence flow cell, 8a ... Fluorescence photometer, 11a ... Warming coil, 12a ... Tris buffer addition tube, 13a ... Mixing stirring coil, 14a ...
Pressurized air input pipe.

Claims (1)

[Claims] 1. A buffer solution addition tube, a heating coil, a mixing stirring coil, and a fluorescent flow cell of a fluorescence photometer are provided in this order, and a sample introduction tube and a pressurized air injection tube are attached to the buffer solution addition tube downstream thereof, and 3α- between the heating coil and the mixing stirring coil
A device for measuring bile acids, which is equipped with a tube for adding a mixture of HSD and NAD, and configured to enable quantitative fluorescence measurement of NADH, a reaction product, using a fluorophotometer.