JPS621216B2 - - Google Patents

Description

[Detailed Description of the Invention] Although qualitative and quantitative analysis of individual bile acids and bile acids conjugated with glycine in plasma is important for the diagnosis of liver diseases, there is no effective way to quantify these in trace amounts. There aren't many methods. Various chemical, biochemical, and radiochemical methods have been used so far, but none of them can be said to be sufficient. Recently, analytical methods using high-performance liquid chromatography have been developed, but ordinary bile acids do not exhibit the ultraviolet absorption necessary for high-performance liquid chromatography analysis. For this purpose, there is a method of analyzing bile acids by reacting them with compounds that have ultraviolet absorption, such as phenacyl bromide, p-chlorobenzoyl chloride, o-(p-nitrobenzyl)-N,N'-diisopropylisourea, and naphthyldiazomethane. is used. However, in recent years, with the remarkable development of high-performance liquid chromatography, various highly sensitive detectors have been developed, and in particular, the use of fluorescence detectors is becoming possible for analytical quantification at the picomolar concentration level. Therefore, the present inventors have conducted extensive research into microquantification by converting bile acids into fluorescently labeled compounds, and have discovered that 1-bromoacetylpyrene reacts with bile acids to become a strongly fluorescent substance. The present invention was completed by making microanalysis quantitative determination possible using a fluorescence detector. 1-Bromoacetylpyrene used in the present invention is a new compound having the following chemical structure. 1-Bromoacetylpyrene reacts with bile acids to produce the following fluorescently labeled compound. (In the formula, R represents a residue obtained by removing the carboxyl group from a bile acid.) This reaction involves dissolving the bile acid in an appropriate solvent (generally acetonitrile, acetone, dichloromethane, ethyl acetate, etc.). , add an alkali metal salt such as potassium hydrogen carbonate or potassium carbonate, and add 1
-Proceeds when adding bromoacetylpyrene, but crown ether (18-crown-6, dibenzo-18-crown-6, dicyclohexano-18
-Crown-6, etc.) When coexisting with a phase transfer catalyst, the reaction is more efficient and rapid. The fluorescently labeled compound thus obtained can be analyzed and quantified in trace amounts by making full use of separation techniques such as high performance liquid chromatography and thin layer chromatography. Examples of bile acids include the following compounds. (This exemplified compound No. is referred to in the Examples below.) 1 Glycoursodeoxycholic acid 2 Glycobile acid 3 Glycochenodeoxycholic acid 4 Glycodeoxycholic acid 5 Ursodeoxycholic acid 6 Cholic acid 7 Glycolitocholic acid 8 Chenodeoxycholic acid Acid 9 Deoxycholic acid 10 Lithocholic acid Not only these bile acids but also bile acids conjugated with glycine can be similarly analyzed by fluorescence. Therefore, in the present invention, bile acids also mean glycine-conjugated bile acids. 1-bromoacetylpyrene can be produced as follows. Production example: Pyrene was produced in the literature of Batshamman et al. (Journal of American Chemical Society: 63
2494, 1941) and then selectively brominated the acyl side chain to obtain 1-bromoacetylpyrene. That is, 6.0 g of 1-acetylpyrene and 60 ml of dioxane are charged and dissolved in a 200 ml four-necked flask equipped with a stirrer, thermometer, and dropping funnel, and the mixture is cooled to 10° C. with permanently cold water. On the other hand, a solution of 3.9 g of bromine dissolved in 40 ml of dioxane is placed in a dropping funnel and added dropwise over about 30 minutes while keeping the internal temperature at 10-15°C. After stirring at the same temperature for 10 hours, dioxane was distilled off under reduced pressure. The residue was dissolved in a small amount of methanol, cooled, and the resulting crystals were filtered and dried to yield 6.0 g of 1-bromoacetylpyrene.
get. Yield: 75.6% Recrystallized from toluene, melting point:
Yellow crystals with a temperature of 130.5-131.5°C were obtained. The analysis value (C ₁₈ H ₁₁ OBr) is as follows. C% H% Br% Calculated value 69.89 3.43 24.73 Actual value 66.43 3.38 24.50 The results of mass fragmentometry analysis of this product are shown in FIG. The molecular weight peak was at 322, and it was confirmed that 1-bromoacetylpyrene was produced. The IR spectrum of this product is shown in Figure 2, and the NMR spectrum is shown in Figure 3. Next, examples will be shown. Example: A methanol solution containing potassium hydroxide was added to a certain amount of the 10 bile acid preparations mentioned above (usually 100 microliters of solution with a concentration of 1 micromol/ml) to make it neutral or weakly alkaline, and then 1- Add 50 microliters of an acetonitrile solution containing 0.2 mmol of bromoacetylpyrene and 61.0 ml of dicyclohexyl 18-crown. The mixture was heated to 80°C for 2 hours to complete the reaction. After the reaction, a certain amount (usually 2 microliters) was used for separation and quantification using high performance liquid chromatography. The Shima Ritsu LC3A spectrometer RF is a high-performance liquid chromatography device.
-500LCA type was used, Microbondapak C ₁₈ reverse phase column was used as the separation column, and acetonitrile-methanol-water (A solvent mixture ratio 100-50-70, B solvent mixture ratio 100-50-) was used as the mobile phase. 60,
The experiment was carried out at a solvent mixing ratio of 100-50-40) and a flow rate of 1.0 ml/min. The analytical device is not limited to this device, but can be replaced by a commonly used high-performance liquid chromatography device, and the separation column can also be replaced by a reversed-phase column. A chromatogram analyzed using a bile acid standard is shown in FIG. In the figure, A, B, and C indicate the types of mixed solvents, and the numbers assigned to each peak of the chart correspond to the above-mentioned example numbers, and indicate the bile acid peak indicated by this number. In a similar manner, we analyzed blood bile acids in patients with liver disease who had abnormalities in bile acid metabolism. The results are shown in FIG. In FIG. 5, the meanings of A, B, C and peak numbers are the same as in FIG. 4.

[Brief explanation of the drawing]

Figure 1 shows the mass spectrum of 1-bromoacetylpyrene, Figure 2 shows the IR spectrum of the same substance,
Figure 3 shows the NMR spectrum of the same substance. Figure 4 is a high-performance liquid chromatogram of bile acid standard.
FIG. 5 shows a high performance liquid chromatogram of bile acids in the blood of patients with liver disease.

Claims (1)

[Claims] 1. A bile acid fluorescence analysis method characterized by reacting bile acids with 1-bromoacetylpyrene and detecting the resulting fluorescent substance.