JPS6130559B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for measuring kallikrein in human urine, and its purpose is to measure the amount of kallikrein in excreted human urine to treat essential hypertension, primary hyperaldosteronism, The aim is to make it possible to diagnose diseases such as kidney disease.

Until now, there has been no safe and easy method for measuring kallikrein, which is present in minute amounts in human urine, with high sensitivity.

The present inventors conducted intensive studies to develop a practical method for measuring kallikrein in human urine.
In the pH range of 8 to 12, 7-amino-4-methylcoumarin (AMC), which was released by an enzymatic reaction by contacting with prolyl-phenylalanyl-aragin-4-methylcoumarinamide, was measured using a fluorescence method. The present invention was completed based on the discovery that kallikrein, which is present in small amounts in the blood, can be measured with high sensitivity.

The amount of human urine used in the present invention may be as small as about 10 to 100μ when used without diluting.

Note that the peptide used in the present invention is not affected by impurities other than kallikrein contained in human urine.

The pH of human urine is adjusted to 8-12, preferably 10-11.

In the present invention, prolyl-phenylalanyl-arginine-4-methylcoumarin (Pro-Phe-Arg-MCA) is used as a substrate.
It can also be used in the form of addition salts with acids that do not inhibit enzymatic reactions. Among the constituent amino acids, arginine is preferably in the L-form. The imino group of proline may be protected with a protecting group commonly used as a protecting group for peptides.

The enzyme reaction in the present invention is 0.5 to 2 at 30 to 45℃.
It is best to do this for about an hour.

To stop the enzyme reaction, for example, add Trasylol or add an acid such as acetic acid.
This can be done by adjusting the pH to 6.0 or less, preferably about 4.5. By this reaction stopping operation,
It was also confirmed that the measurement of AMC fluorescence intensity was not affected.

There is no particular difficulty in measuring AMC by fluorescence, and any known method may be used. That is,
The fluorescence intensity at 460 nm (excitation at 380 nm) is measured to determine the free generated AMC. liberated
The amount of AMC corresponds to the amount of kallikrene present in urine. The amount of kallikrene can be determined by comparing it with a separately prepared calibration curve.

When kallikrein activity was determined in the urine of normal people, urine of nephritis patients, and urine of Bartter's syndrome patients using the method of the present invention, the activity was compared with that obtained by radioimmunoassay using low molecular weight bovine kininogen. It was compatible. In addition, when urine from a normal person was introduced into a column of "Arginine-Sepharose 4B", two activities, namely, brolyl-phenylalanyl-arginine-MCA hydrolyzing activity and kinine-releasing activity from bovine low-molecular-weight kininogen, were found to be the same. eluted in the image. The fluorescent peptide substrate of the present invention, Pro-Phe-Arg-MCA, is used to measure human urinary kallikrein activity for the purpose of diagnosing diseases caused by it (mainly due to renal dysfunction). It turns out that you can.

The peptide derivative used in the present invention is a safe substance that does not cause problems such as carcinogenicity, and satisfies all aspects such as ease of measurement, stability, and linearity with respect to enzyme amount and contact reaction time. The method of the present invention can be said to be a practical method for measuring kallikrein in human urine.

The present invention will be explained in detail below with reference to Examples.

Example 2 69.0 g (0.2 mol) of N-carbobenzoxy-L-arginine hydrochloride and 17.5 g (0.1 mol) of 7-amino-4-methylcoumarin were dissolved in 300 ml of dimethylformamide, and while stirring at room temperature,
21 g (0.1 mol) of dicyclohexylcarbodiimide was added. After stirring the mixture overnight at room temperature, the produced dicyclohexylurea was filtered off, and the filtrate was concentrated under reduced pressure. Next, 50 ml of methyl alcohol and 500 ml of ethyl acetate were added to the residue, and the precipitated crystals were filtered out to yield 19 g of crude crystals.
I got it. Heat the crystals in 30 ml of dimethylformamide.
and 100 ml of hot methyl alcohol, filter out impurities, and add ethyl acetate to the filtrate.
By adding 400 ml and filtering out the precipitated white crystals, 7-(N
-Carbobenzoxy-L-arginyl)amino-4-methylcoumarin hydrochloride 14.5g (yield 29%)
I got it. The optical rotation [α] ¹⁵ _D of this product was −17.0 (C=2.35 in dimethylformamide), and the elemental analysis values were as follows.

C ₂₄ H ₂₈ N ₅ O ₅ Calculated value (%) as Cl C57.42, H5.62, N13.96 Actual value (%) C57.61, H5.69, N13.94 7 obtained as above 251 mg (0.5 mmol) of -(N-carbobenzoxy-L-arginyl)amino-4-methylcoumarin hydrochloride was dissolved in a mixture of 50 ml of methyl alcohol, 5 ml of acetic acid and 10 ml of water, and 25 mg of 5% palladium on carbon catalyst was added. The mixture was stirred at room temperature for 3 hours while passing hydrogen gas through the mixture. Next, the reaction mixture was filtered to remove the catalyst, the filtrate was distilled under reduced pressure to remove the solvent, 50 ml of ether was added to the residue, and the precipitated powder was collected by filtration. In this way, 7-L-arginylamino-4-methylcoumarin hydrochloride 1/2 hydrate with a melting point of 275°C (decomposed)
188 mg (yield 100%) was obtained. This substance has an optical rotation [α] ¹⁵ _D +91.9 (C in 25% acetic acid aqueous solution = 1.01) and was analyzed by silica gel thin layer chromatography (n-butyl alcohol:acetic acid:water = 4:1:1). A single spot was given. The elemental analysis values of this product are as follows.

C ₁₆ H ₂₂ N ₅ O ₃ Cl・1/2H ₂ O Calculated value (%) C50.99, H6.15, N18.59 Actual value (%) C51.02, H6.11, N18.41 7- L-Arginylamino-4-methylcoumarin hydrochloride 1/2 hydrate 754 mg (2 mmol)
was dissolved in a mixture of 10 ml of dimethylformamide and 10 ml of water, and carbobenzoxy-L-
Add 1.03 g (2.6 mmol) of N-hydroxysuccinimide ester of phenylalanine. After stirring at room temperature for 15 hours, the mixture was concentrated to dryness. This residue was subjected to silica gel column chromatography (2
× 15 cm), and the main fraction was concentrated to dryness.
By dissolving in 30 ml of acetic acid and freeze-drying, 7-
920 mg of (N-carbobenzoxy-L-phenylalanyl-L-arginyl)amino-4-methylcoumarin hydrochloride was obtained. This one has an optical rotation [α] ^{of 15} _D
-23.4 (C=0.65 in dimethylformamide), and gradually decomposes from 150℃ when heated. The elemental analysis values of this product are as follows.

C ₃₃ H ₃₇ N ₆ O ₆ Calculated value as Cl (%) C61.05, H5.75, N12.95 Actual value (%) C60.57, H5.78, N12.70 Example 2 Obtained in Example 1 1.3 g (2 mmol) of 7-(N-carbobenzoxy-L-phenylalanyl-L-arginyl)amino-4-methylcoumarin hydrochloride was dissolved in 100 ml of methyl alcohol, and 100 mg of palladium on carbon catalyst was added while passing hydrogen gas. After stirring for 3 hours, the catalyst was filtered off and the filtrate was concentrated to dryness. Add 100 ml of ether to this residue and filter the resulting powder to obtain 1.0 g of 7-(L-phenylalanyl-L-arginyl)amino-4-methylcoumarin hydrochloride. This has an optical rotation [α] ¹⁵ _D -33.6 (C= in dimethylformamide
0.52), and when heated, it gradually begins to decompose from 140℃. The elemental analysis values of this product are as follows.

C ₂₅ H ₃₁ N ₆ O ₄ Cl・CH ₃ COOH・H ₂ O Calculated value (%) C54.67, H6.29, N14.17 Actual value (%) C54.86, H6.68, N14.09 Example 3 7-(L-phenylalanyl- obtained in Example 2)
Dissolve 515 mg (1 mmol) of L-arginyl)amino-4-methylcoumarin hydrochloride in 5 ml of dimethylformamide, and add carbobenzoxy-L
- Add 450 mg (1.3 mmol) of N-hydroxysuccinimide ester of broline, stir at room temperature for 15 hours, and then concentrate to dryness. This residue
Chloroform/methyl alcohol/acetic acid (95:
The main fraction is concentrated to dryness. This residue is dissolved in acetic acid and lyophilized to obtain 510 mg of an acetic acid adduct of 7-(N〓-carbobenzoxy-L-prolyl-L-phenylalanyl-L-arginyl)amino-4-methylcoumarin hydrochloride. This material has an optical rotation [α] ¹⁵ _D -53.6 (dimethylformamide, C=0.75), and when heated, it gradually begins to decompose from 160°C. The elemental analysis values of this product are as follows.

C ₃₈ H ₄₄ N ₇ O ₇ Cl・CH ₃ COOH Calculated value (%) C59.58, H6.00, N12.16 Actual value (%) C60.11, H5.98, N12.39 Example 4 Implementation 7-(N〓-carbobenzoxy-L-prolyl-L-phenylalanyl-L-arginyl)amino-4-methylcoumarin hydrochloride obtained in Example 3
200 mg (0.27 mmol) in 50 ml of methyl alcohol
Dissolved in IN-hydrochloric acid 0.27ml and palladium on carbon catalyst.
After adding 20 mg and stirring for 3 hours while passing hydrogen gas, the catalyst was filtered off and concentrated to dryness. Add 50 ml of ether to the residue and filter the resulting powder to obtain 150 mg of 7-(L-prolyl-L-phenylalanyl-L-arginyl)amino-4-methylcoumarin hydrochloride. This material has an optical rotation [α] ²¹ _D -37.5° (C=1.52, 80% DMF) and gradually decomposes from 185°C when heated. The elemental analysis values of this product are as follows.

C ₃₀ H ₃₉ N ₇ O ₅ Cl ₂・4H ₂ O Calculated value (%) C50.00, H6.75, N13.61 Actual value (%) C49.97, H6.39, N14.11 Example 5 Using normal human urine, an experiment was conducted to determine the rate of AMC production from L-prolyl-L-phenylalanyl-L-arginine-4-methylcoumarinamide (substrate).

The results regarding reaction time and urine amount are shown in FIGS. 1 and 2, respectively.

The experimental method was as follows.

0.1MTris containing 0.15M sodium chloride
Add 0.5 ml of HCl buffer (pH 8.0) to 5-100 μl of urine at 37°C.
Mix with. After 5 minutes, 5μ of 10mM of the above substrate was added, and the intensity of the generated MCA fluorescence at each time was measured. Fluorescence intensity at 460nm (380nm
excitation). Fluorescence intensity of 10 μM of 0.1% dimethyl sulfoxide (DMSO) solution of AMC
Calculate as 1.0.

Example 6 Measurement of optimal pH It is thought that it is best to perform the enzyme reaction at a pH of about 8.0 (Y.Matsuda, K.Miyazaki, H.
Moriya, Y. Fujimoto, Y. Hojima, and, C.
Moriwaki, J.Biochem, 80 , 671-679, 1976) conducted an experiment to determine the preferred pH range.

0.5ml Britton Robinson
An enzymatic reaction was carried out with 20μ of normal human urine and 5μ of L-prolyl-L-phenylalanyl-L-arginine-4-methylcoumarinamide (10mM) at 37°C for 60 minutes in a wide range buffer solution (Robinson).
Pure urine kallikrein 10μ under the same conditions for min
(A280=0.0067) Contrasted with the reacted one. The results are shown in Figure 3.

In the figure, the urine measured from a normal person is marked with a circle.
Measurement results using pure enzyme are indicated by a circle.

Example 7 0.5ml of 01MTris-HCl buffer (pH 8.0)
(containing 0.15M NaCl) 20μ of normal human urine or human urine of Bartter's syndrome patient
20μ and 10μ Trasylol
(5000U/ml) or 20μ of 10% acetic acid were mixed.
After adding 5 µm of 10mML-prolyl-L-phenylalanyl-L-arginine-4-methylcoumarinamide to this, the fluorescence intensity at each time was determined. The results when trasylol was used and the results when 10% acetic acid were used are shown in Figures 4 and 5, respectively.

In Figure 4: 〓 Bartter syndrome patient's urine 20μ 〓 Normal person's urine 20μ 〓 Bartter syndrome patient's urine 20μ + Trasylol (50U) 〓 Normal person's urine + Trasylol (50U) In Figure 5 〓 Normal person's urine 20μ 〓 Normal Human urine 20μ + 10% acetic acid 20μ Example 8 350ml of normal human urine was dialyzed against 0.02M Tris-HCl buffer (pH 8.0) for one day and equilibrated with the same buffer. x 11cm). After washing the buffer, 500
Linear gradient elution was performed using 0.02M Tris-HCl buffer (pH 8.0) and the same buffer containing 0.5M NaCl. 10ml
The fractions were fractionated, and the fraction numbers are indicated by arrows. The results are shown in FIG.

〓 is the activity to hydrolyze prolyl-phenylalanyl-arginine-MCA 〓 is the activity to liberate kinin from bovine low-molecular-weight kininogen Example 9 Kallikrein in the urine of normal people, the urine of patients with glomerulonephritis, and the urine of Bartter syndrome patients The amount was measured by both the method of the present invention and the radioimmunoassay method, and the results are shown in FIG.

[Brief explanation of the drawing]

Figures 1 and 2 show the results of Example 5, and Figure 3 shows the results of Example 6.
4 and 5 show the results of Example 7, FIG. 6 shows the results of Example 8, and FIG. 7 shows the results of Example 9.

Claims (1)

[Claims] 1. Contact 10-100μ of human urine with prolyl-phenylalanyl-L-arginine-4-methylcoumarinamide at a pH range of 10-11 to perform an enzymatic reaction, then stop the enzymatic reaction with trasylol or acid, A method for measuring kallikrein in human urine, which comprises measuring freely produced 7-amino-4-methylcoumarin by a fluorescence method.