JPS6364439B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an immunoassay of lithocholic acids and lithocholic acid derivatives useful therein. Measuring bile acids in the blood is important for diagnosing various diseases, but the only known method to date is the method of Spenny et al.
Gastroenterology 72 , 305-311 (1977)) is convenient as a radioimmunoassay using a bile acid histamine derivative labeled with ¹²⁵ I as a tracer, but the antibody used has a low titer and cannot be diluted more than 1000 times. Very little usable material is obtained, and its practical value is low. The present inventor developed a method for measuring blood bile acids with higher sensitivity using a lithocholic acid derivative modified at the 3-position, whereas the conventional method uses a substance modified at the 24-position of bile acids. I found it. The compound used in the present invention is represented by the following general formula. (In the formula, R ₁ means OH, histamine, tyramine, tyrosine, radioactive iodine-labeled histamine, radioactive iodine-labeled tyramine, radioactive iodine-labeled tyrosine, protein or polypeptide, and R ₂ means H or a lower alkyl group.) This compound can basically be produced by the method shown in the following reaction formula. (In the formula, R is the same as above, R' is the same as _R1 or its ester, and R'' means lower alkyl.) That is, lithocholic acid () is converted to 2-ethoxy-1 (2H)- It is condensed with glycine ester in the presence of quinoline carboxylic acid ethyl ester (EEDQ) to form glycolytocholic acid ester (), which is oxidized in the presence of an appropriate catalyst to produce the 3-keto form (). A 3-(0-carboxymethyl)oxime () is formed by heating in carboxymethoxylamine hemihydrochloride and pyridine.This is reacted with chlorocarbonate in the presence of a tertiary amine to form a mixed acid anhydride. Then, when reacted with histamine, tyramine, tyrosine ester or their radioiodine-labeled products, or proteins or polypeptides, a compound of formula () is obtained.Hydrolysis of the ester moiety of this yields a compound of formula (). Compounds in which R ₁ in formula () is radioiodine-labeled histamine, tyramine, or tyrosine are useful as tracers for radioimmunoassay, and R′ in formula () or R ₁ in formula () is serum albumin, proteins such as serum globulins, such as bovine serum albumin (BSA), rabbit serum albumin (RSA), bovine serum globulins or polypeptides;
For example, compounds that are polylysine are useful as antigens for antibody production. Other compounds are useful as manufacturing intermediates. To obtain a compound labeled with radioactive iodine, it is possible to label histamine, tyramine or tyrosine ester in advance and then condense it with the compound of formula (); Good too. As a labeling method, it is appropriate to react with radioactive sodium iodide and chloramine T using the most common chloramine T method, and to terminate the reaction with sodium metabisulfite. To obtain antibodies using a compound of formula (), which is a condensate with a protein or polypeptide, as an antigen, the antigen is suspended in Freund's adjuvant, for example, and then incubated with a mammal (rabbit, rabbit, etc.). The antiserum is injected into guinea pigs, sheep, goats, etc., and after an appropriate booster immunization, blood is collected and blood cells are removed to obtain antiserum. For radioimmunoassay (RIA), antiserum diluted to an appropriate concentration is mixed with a buffer solution containing the specimen or standard and a buffer solution containing a labeling substance as a tracer, such as the compound shown in Example 5 below. The generated antigen-antibody reaction product is separated by a BF separation method such as the two-antibody method or the dextran charcoal method, and the radioactivity of one of the two is measured, and the value for the standard is determined. A standard curve is created from this, and the amount of lithocholic acids in the sample is determined from this. The antiserum produced using the protein or polypeptide condensate of the present invention hardly cross-reacts with taurolithocholic acid and is suitable for measuring glycolithocholic acid. In this case, although an esterified carboxyl group of the glycine moiety can be used, a carboxylic acid type carboxyl group is considered to be more preferable. On the other hand, in a compound used as a tracer, if the carboxyl group of glycine is esterified, the sensitivity will decrease, which is not preferable. Next, an example will be given and explained. Example 1 2.3 g of glycine methyl ester hydrochloride was suspended in 140 ml of dry ethyl acetate containing 2 ml of triethylamine and stirred at room temperature for 30 minutes. This includes EEDQ3.46
g and 3.8 g of lithocholic acid were added thereto, stirred at room temperature for 30 minutes, and then heated and perfused for 3 hours. The reaction solution was filtered, and the filtrate was washed with 1N hydrochloric acid and an aqueous sodium bicarbonate solution, and then dried over anhydrous sodium sulfate. The solvent was distilled off, and the residue was purified by silica gel column chromatography (solvent: chloroform) to obtain 3.0 g of lithocholylglycine methyl ester (:R″=methyl). Obtained by recrystallizing from ethanol-water. The melting point of the colorless needle crystals was 163-165°C. NMR (pyridine-d ₅ ) δ: 0.58 (3H, s, 18-CH ₃ ) 0.90 (3H, s, 19-CH ₃ ) 3.60 (3H, s, COOCH ₃ ) 3.88 (1H, m, 3-H) 4.30 (2H, d, N-CH ₂ ) 9.10 (1H, t, NH) Elemental analysis Calculated value as C ₂₇ H ₄₅ O ₄ N C 72.44, H 10.13, N 3.13 Experimental value C 72.64, H 10.18, N 3.09 Example 2 Drying 2.7 g of chromium trioxide under ice cooling with pyridine 27
ml to make a pyridine chromate complex, and to this 2.7 g of the compound () obtained in Example 1.
was added and stirred at room temperature for 4 hours. Ice water was added to the reaction solution, neutralized with 1N hydrochloric acid, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the solvent was distilled off. The residue was purified by silica gel column chromatography (solvent: chloroform), and lithocholylglycine methyl ester-3-one (:
R'' = methyl) was obtained. The melting point of colorless needle crystals recrystallized from methanol was 141-143°C. Example 3 650 mg of the compound () obtained in Example 2 and carboxymethoxylamine (hemihydrochloride) were obtained. ) was added to 15 ml of pyridine and heated to 80°C for 3 hours. After distilling off the solvent, it was purified by silica gel column chromatography (solvent: chloroform: methanol = 50:1) to obtain lithocholylglycine methyl ester-3-
600 mg of (0-carboxymethyl)oxime (V:R″=methyl) was obtained.The melting point of colorless needle crystals recrystallized from methanol-water was 156-158°C.Example 4 Compound obtained in Example 3 () 155mg, tyrosine ethyl ester 61mg, hydroxybenzotriazole
80 mg of dicyclohexylcarbodiimide and 67 mg of dicyclohexylcarbodiimide were added to 3 ml of dry tetrahydrofuran, stirred for 1 hour under ice cooling, and then stirred at room temperature for 24 hours. Distill the solvent,
The residue was purified by silica gel column chromatography to obtain lithocholylglycine methyl ester-3-
(0-carboxymethyl)oximutyrosine ethyl ester (:R'=tyrosine ethyl ester,
R''=methyl) 70 mg was obtained. NMR (CDCl ₃ ) 0.62 (3H, s, 18-CH ₃ ) 0.95 (3H, s, 19-CH ₃ ) 1.40 (3H, t, C-CH ₃ ) 3.04 ( 2H,d,

[Formula]) 3.74 (3H, s, COOCH ₃ ) 4.02 (2H, d, N-CH ₂ ) 4.12 (2H, dd, O-CH ₂ -C) 4.46 (2H, s, O-CH ₂ -COO) 4.84 (1H, dd, N-CH-C) 6.14 (1H, t, NH) 6.80 (6H,

[Formula]) 50 mg of this product was dissolved in 2 ml of tetrahydrofuran, 2.5 ml of a 1% aqueous methanol solution of sodium hydroxide was added thereto, and the mixture was stirred at room temperature for 24 hours. The reaction solution was neutralized with 1N hydrochloric acid, ice water was added, the precipitated crystals were collected by filtration, and lithochorylglycine-3-(0-
Carboxymethyl) oximutyrosine (:R ₁
= tyrosine) 30 mg was obtained. NMR (pyridine-d ₅ ) 0.59 (3H, s, 18-CH ₃ ) 0.82 (3H, s, 19-CH ₃ ) 3.55 (2H, d,

[Formula]) 4.10 (2H, d, N-CH ₂ ) 4.92 (2H, s, O-CH ₂ -COO) 5.36 (1H, dd, N-CH-C) 7.34 (6H,

[Formula]) 7.92 (1H, d, NH) 8.96 (1H, t, NH) Example 5: Synthesis of tracer Ethanol solution of compound () obtained in Example 4 (1
mg/ml) 20 μl and 1 mci of Na ¹²⁵ I to chloramine T
10μl of 1/15M phosphate buffer (6mg/ml, PH7.4)
was added and stirred for 1 minute at room temperature, and then a 1/15M phosphate buffer solution of sodium metabisulfite (12
mg/ml, PH7.4) was added to stop the reaction.
The solvent was distilled off, the residue was dissolved in a small amount of ethanol, and subjected to silica gel thin layer chromatography (chloroform:methanol = 3:1 solution adjusted to pH 3 with 1N hydrochloric acid), and the Rf0.24 portion was scraped off and extracted with ethanol. . Example 6: Synthesis of BSA condensate 150 mg of the compound () obtained in Example 3 was dissolved in 2.74 ml of anhydrous tetrahydrofuran, and 0.0677 ml of tri-n-butylamine was added thereto, and after cooling to -15°C, isobutyl chloroformate was added. Add 0.037ml -
Stirred at 15°C for 15 minutes. This was mixed with a solution of 397 mg of bovine serum albumin (BSA) cooled to 0°C (10.47 mg of water).
ml, a mixed solvent of 10.47 ml of tetrahydrofuran, and 0.33 ml of 1N sodium hydroxide). After stirring for 1 hour in a cool dark place, adjust the pH to 9.0 with 1N sodium hydroxide, stir at 0℃ for 3 hours, and then dialyze against cold water (4℃) for 24 hours (cellulose tube 36/3
2, Visking Company) and adjusted to pH 4.5 with 1N hydrochloric acid, which turned cloudy. The mixture was left in a cool, dark place for 12 hours, and centrifuged (2000 rpm, 30 minutes). 30 ml of water was added to the resulting precipitate to suspend it, and a small amount of saturated aqueous sodium bicarbonate solution was added to dissolve the precipitate. This was dialyzed against cold water for 6 hours and then lyophilized to form a lithocholylglycine methyl ester-3-(0-carboxymethyl)oxime BSA condensate (:R'=BSA,
Example 7: Production of antibodies The BSA condensate obtained in Example 6 was injected intradermally (0.5 mg/mouse) into New Zealand white rabbits together with complete Freund's adjuvant, and about 1 month later. Two months later, booster immunizations (0.5 mg/mouse each) were performed in the same manner, and blood was collected 14 days after the final booster to obtain antiserum.The antibody titer of this antiserum was
35.000 times dilution or more, and the cross-reactivity with bile acids other than glycolithocholic acid is higher than that of glycocholic acid.
0.05%, glycodeoxycholic acid 0.05%, taurolithocholic acid 0.2%, and lithocholic acid 0.9%. Example 8: A 0.02M borate buffer (PH8.0) containing 0.1% gelatin was used for RIA assay of glycolithocholic acid. 100μl of labeled antigen solution obtained in Example 5 (approx. 35000cpm)
Antiserum obtained in Example 7 (40,000 to 50,000 times diluted)
100 μl and standard glycolithocholic acid (0-
After adding 100 μl of a buffer containing 1000 ng) or a test specimen, 1000 μl of the buffer was further added and the mixture was incubated at room temperature for 2 hours. Next, 500 μl of dextran thiacol solution (50 mg of dextran T-70 and 0.5 g of charcoal dissolved in 100 ml of buffer)
was added and left at room temperature for 10 minutes, centrifuged at 3000 rpm at 4°C, and the radioactivity of the supernatant was measured. A standard curve prepared based on the measured values for standard glycolithocholic acid is shown in the drawing.

[Brief explanation of drawings]

The drawing is a standard curve.

Claims (1)

[Claims] 1. General formula A lithocholic acid derivative represented by However, during the ceremony
R ₁ means histamine, tyramine, tyrosine or a radioactive iodine labeled product thereof, OH, protein or polypeptide, and R ₂ means H or lower alkyl. 2. A tricholic acid derivative in claim 1, wherein R ₁ is bovine serum albumin and R ₂ is methyl. 3. A lithocholic acid derivative in claim 1, wherein R ₁ is bovine serum albumin and R ₂ is H. 4. A lithocholic acid derivative in claim 1, wherein R ₁ is radioactive iodine-labeled tyrosine and R ₂ is H.