HU187388B - Process and composition for the quantitative determination of substances bound to receptors in the brain and process for preparing the composition - Google Patents

Abstract

The invention relates to a process for the preparation of stable receptor preparations suitable for determining the amount of substances which bind to brain receptors by centrifuging, for 8 to 20 minutes, a homogenate of the brain or part thereof, which has been prepared with an aqueous solution of a water-soluble inert material, at an acceleration of 800 to 1100 g and removing the substance which binds to brain receptors in the preparation from the supernatant liquid, which latter is carried out in such a way that the supernatant liquid is centrifuged at an acceleration of 18,000 to 22,000 g for 10 to 20 minutes, the resulting solid substance is homogenised anew in distilled water, the homogenate is, where appropriate after freezing and thawing, centrifuged at an acceleration of 7000 to 9000 g for 5 to 15 minutes, the supernatant liquid is removed and centrifuged at an acceleration of 35,000 to 45,000 g for 20 to 30 minutes, the resulting solid substance is washed with an aqueous buffer solution with a pH of 6 to 8, and finally a suspension of the solid substance is freeze dried.

Description

The present invention relates to a method and a preparation for quantifying substances that bind to brain receptors (in particular benzodiazepines, β-adrenergic blocking agents and γ-aminobutyric acid). The invention further relates to the preparation of a preparation for use in the definition.

In medicine, it is often necessary to quantify substances that bind to brain receptors in body fluids for diagnostic and / or therapeutic purposes. For example, the γ-amino-butyric acid content of the spinal fluid may suggest certain brain diseases.

It is also known that body fluids usually contain very small concentrations of the analyte (10 " ⁸ -10" ¹² moles), so their quantitative measurement requires special, sensitive methods.

Mass spectrometry, high performance liquid chromatography, and ion exchange fluorometry for the quantification of γ-aminobutyric acid are described in J. Chromat. 118, 395 (1976), Brain Rés. 167, 297 (1979) and Anal. Biochemistry 101, 349 (1980). Although the methods listed are sufficiently sensitive, they have the common disadvantage that the biological sample to be tested cannot be directly used in the measurement but only. after complicated sample preparation (multi-step purification, derivatization, etc.).

A recently developed radioreceptor assay for the quantification of γ-aminobutyric acid [J. Neurochem. 28, 1121 (1977), Brain Rés. 182, 99 (1980), Clinica Chimica Acta 109, 77 (1981)] does not require preparation of the test fluid. However, the disadvantage is that the receptor suspension required for the assay must be freshly prepared from the brains of experimental animals according to known methods. This requires the maintenance of an animal house and a specialized biochemical laboratory, which hospitals and clinics generally do not have. Thus, there is a need for a receptor preparation that can be stored indefinitely in the dry state and can be reliably used to quantify substances that bind to brain receptors.

The preparation of an easy-to-handle, unlimited shelf-life dry receptor preparation is described in U.S. Patent No. 29 02 071. German Federal Publication Document. In the known process, the receptor preparation is prepared by homogenizing animal brain tissue with an aqueous solution of an inert, water-soluble substance, preferably sucrose, and freeze-drying the homogenate.

However, the disadvantage of the known receptor preparation is that it can only be used for quantitative determination of benzodiazepines and, within this, it is not suitable for separate measurement of different types of benzodiazepines. Quantitation of other substances that bind to brain receptors, such as γ-aminobutyric acid and β-adrenergic blocking agents, is not possible with the known receptor preparation.

In our studies, we have found that this deficiency is due to the fact that the known receptor preparation contains the majority of the brain receptors in inactivated form by endogenous substances. By completely removing inactivating endogenous substances, the receptor sites in the brain can be released, thus making the preparation capable of determining any substance that can bind to brain receptors,

The present invention therefore relates to a method of producing a stable receptor preparation for quantifying substances that bind to brain receptors. According to the invention, the brain or brain fragment homogenate prepared with an aqueous solution of a water-soluble inert substance is centrifuged at 800 to 1100 g for 8-20 minutes, the supernatant is separated and centrifuged at 18,000 to 22,000 g for 10 to 20 minutes, the solid obtained is re-homogenized in distilled water, the homogenate is frozen and thawed if desired, then centrifuged at 7000-9000 g for 5-15 minutes, the supernatant is separated, centrifuged at 35,000-45,000 g for 20-30 minutes, The slurry was washed three times with an aqueous buffer solution of pH 6-8, frozen and thawed at least once for the solid and washings, then centrifuged at 35,000-45,000 g for 20-30 minutes and finally freeze-dried.

This method provides a solid, stable, indefinite receptor preparation that contains all brain receptor sites in the active state, and is generally applicable in the radioreceptor assay to quantify any substance that binds to the brain receptors.

Animal brains (rat, bovine, chicken, etc.) are preferably used as starting materials. A brain homogenate is prepared as described in the German Federal Publication. Preferably, sucrose is added to the aqueous medium as a water-soluble inert substance. The first centrifugation at 800 to 1100 g accelerates to separate the coarse pellet from the brain or brain fragment homogenate; the material containing the receptor preparation is then contained in the supernatant. Second supernatant centrifugation at 18,000-22,000 g acceleration! step 5) isolates the receptor preparation material contaminated with nuclei and mitochondria. The material containing the receptor preparation at this stage contains the vast majority of the active receptor sites even when inactivated by endogenous substances. This solid is then re-homogenized in distilled water. The cell membranes then pop up and endogenous substances that block the active receptor sites are released into the aqueous medium. The homogenization is preferably carried out in cold (4-10 ° C) distilled water. Preferably, at least 15 parts by weight of distilled water are used to re-homogenize one part by weight of the solid. Generally, the greater the amount of water that is re-homogenized, the more effective it is in removing the blocking endogenous substances from the active receptor sites. The upper limit of the volume of distilled water is essentially determined by economic considerations. Typically, 10-20 parts by weight of distilled water are used to re-homogenize one part by weight of a solid.

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The distilled aqueous homogenate may, if desired, be frozen and thawed to aid in the rupture of cell membranes.

The first centrifugation from the distilled aqueous homogenate at 7000-9000 g accelerates to remove nuclei and mitochondria, which do not contain active receptor sites, and their presence would adversely affect the properties of the receptor preparation (e.g., filterability). The supernatant is separated from the supernatant by subsequent centrifugation at 35,000-45,000 g, which is then washed at least once with an aqueous buffer solution of pH 6-8, preferably tris-citrate buffer (pH 7.1). The washing liquid and solid suspension is frozen and thawed at least once and then centrifuged. The resulting solid is suspended in a known amount of saline-containing aqueous buffer. This suspension is freeze-dried.

The stable solid receptor preparation thus prepared can be used to quantify brain receptor binding substances as follows:

To the aqueous solution containing the radiolabeled (usually tritium) test substance is added a suspension of the receptor preparation in aqueous buffer solution at pH 6-8 or in distilled water. The mixture is incubated and the solid is filtered, washed with buffer and assayed for radioactivity in the solid. Subsequently, a known amount of the non-radioactive test substance is added to the receptor preparation containing the radiolabeled test substance, the mixture is incubated, the solid is filtered, washed with buffer solution and its radioactivity is measured again. The latter operation is repeated several times using a variable amount of known non-radioactive test substance. From the measurement results, a concentration / radioactivity calibration line is taken ("concentration" is the concentration of the non-radioactive test substance).

The amount of test substance in body fluid (e.g., spinal fluid) is determined using a calibration line constructed as described above by adding a known amount of body fluid to the receptor preparation containing the radiolabeled test substance, incubating, filtering, washing, measuring radioactivity, and read off the concentration of the measured value from the calibration line.

The invention is further illustrated by the following non-limiting examples.

Example 1

Preparation of a receptor preparation

Male rats of strain CFY were decapitated. Their brains were immediately removed and washed with ice-physiological saline. The cerebellum and rabbit brain, referred to as the bridge, are removed and the residue is homogenized with 15 volumes of 0.32 M aqueous sucrose. The homogenate is centrifuged at 1000 g for 10 minutes, then the supernatant is separated and centrifuged at 20,000 g for 10 minutes. The separated solid was re-homogenized in 15 volumes of cold (+ 4 ° C) distilled water and centrifuged at 8000 g for 10 minutes. The supernatant was collected and centrifuged at 40,000 g for 20 minutes. The resulting solid was homogenized in 50 volumes of 50 mM tris-citrate buffer (pH 7.1) and centrifuged again at 40,000 g. The latter washing step is repeated. The solid was then frozen in 10 volumes of buffer and stored at -20 ° C for 10 hours. The suspension was thawed, diluted 5 times with distilled water and centrifuged again at 40,000 g. This freeze-thaw-centrifuge cycle is repeated three times. After the final thawing, the suspension is freeze-dried and freeze-dried. A powder receptor preparation is obtained which is mixed with buffer solution or distilled water prior to use (measurement). The powder receptor preparation can last for years without any change.

Example 2

Plot of calibration curve for radioreceptor assay

To record the calibration curve, use the following solutions or solutions. use suspensions:

Ia solution: dissolved 0.7435 g NaCl, 0.0186 g KC1,0,0144 g _CaCl2, 0.012 g of MgSO ₄ 100 ml of double-distilled water

I. b. solution: 1 g bovine serum albumin in 10 ml I.a. dissolved in solution

II. the. solution 1 mg of γ-aminobutyric acid in 1000 μ 1000 of solution V.

Il.b. solution: 10 μΐ Il.a. and 10 ml of solution V.

II.c. solution: 100 μΐ II.b. and 900 μΐ of solution V.

H. D. solution: 750 μΐ II.c. and 250 μΐ of solution V.

Il.e, solution: 500 μΐ II.c. and 500 μΐ of solution V.

II F solution: 250 μΐ II.c. and 750 μΐ of solution V.

II. g. solution: 100 μΐ II.c. and 900 μΐ of solution V.

III. slurry: 0.2 g of the receptor preparation prepared in Example 1 is suspended in 3 ml of double-distilled water and diluted with 17 ml of solution V.

ARC. solution: ³ Hy-aminobutyric acid solution with 40 nCi / 500 μΐ activity (solvent: pH 4 aqueous hydrochloric acid solution)

Solution V: 50 mM Tris-citrate buffer solution (pH 7.1) (a) Determination of total binding capacity (C _c ):

The following solutions, respectively. slurry in ice:

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500 μΐ IV. solution, μΙ I.b. solution, μΐ solution V, and 500 μΐ III. suspension

The mixture was incubated for 20 minutes at 4 ° C and then filtered through a Whatman GF / C microporous glass filter. The solid was mixed with 10 ml of a liquid scintillation solution of known composition, and the suspension was left in the refrigerator for 1 hour, shaken and the radioactivity of the suspension determined. The radioactivity thus obtained is denoted C _c ; this is the total amount of γ-aminobutyric acid that the preparation can bind.

(b) gamma-amino-butyric depression radiolabeled non-radioactive gamma-aminobutyric acid (C Determination _x values)

Proceed as in (a), but instead of 10 μΐ solution V, 10-10 μΐ II.c., Il.d., Il.e., Il.f., and Il.g. using a solution. The measured radioactivity values are denoted by C '.

(c) Determination of Non-specific binding of y-aminobutyric acid content _(Cb value):

Proceed as in (a), but instead of 10 μΐ solution V, 10 μΐ Il.a. using a solution. In this case, the total amount of specifically bound prepara ³ Η-γ-aminobutyric acid is clamped nonradioactive γ-amino-butyric acid. By measuring the radioactivity of the sample, a correction factor is obtained which represents the amount of non-specifically bound ³ -aminobutyric acid. This correction value (C _b ) is subtracted from the values of C _c and C _x for the calibration line.

The measurement results are reported in Table I below.

Table 1

Solution sign Inactive γ-amino butyric concentration established by them nM Stroke dpm c _c -c _b c _x -c _b Il.a. 10 ⁵ 160 II.c. 10 2049 2.15 Il.d. 7.5 2367 1.84 Il.e. 5.0 2780 1.55 Il.f. 2.5 3284 1.30 Il.g. 1.0 3754 1.13 V 4221 1.00

Plotting C _c -C _b / C _x -C _b as a function of the concentration of inactive γ-aminobutyric acid gives a calibration line.

To determine the concentration of γ-amino-butyric acid in body fluid, proceed as follows: Instead of a buffer solution (Solution V) or an inactive solution of γ-aminobutyric acid of known concentration, add 10 μΐ body fluid, measure the radioactivity of the preparation and read the concentration of γ-aminobutyric acid from the calibration line. ⁴

Example 3

Quantification of other substances that specifically bind to brain receptors

3.1. ^{Determination of β-} diazepam binding mg The lyophilized receptor preparation of Example 1 was suspended in a mixture of 0.9 ml of aqueous tris-citrate buffer pH 7.1 and 0.1 ml of double-distilled water. To the suspension was added 2 nM ³ H-diazepam, incubated for 30 minutes at 4 ° C, the solid was filtered through a Whatman GF / C microporous glass filter and washed eight times with 2.5 ml of ice-tris-citrate buffer. Radioactivity was measured as in Example 2. Thus, the total binding capacity (Cc) of the preparation is determined. The calibration line is truncated by the clamping method described in Example 2 and the amount of non-specifically bound ³ H-diazepam (correction factor Cb) is determined as described in Example 2.

3.2. Determination of y-Aminobutyric acid-dependent ³ H-diazepam binding:

A 3.1. Example 2, however, 2 x 10 ⁵ moles of γ-aminobutyric acid were also added to the incubation mixture.

3.3. Determination of Ca ¹⁺ -dependent ³ H-γ-Aminobutyric Acid Binding mg of the lyophilized receptor preparation of Example 1 was suspended in 0.9 ml Tris-HCl buffer solution, pH 7.4, and 0.1 ml double-distilled water. . To the suspension were added calcium chloride (concentration 250 mM in suspension), ³ -Hyminobutyric acid (concentration 5.4 nM in suspension) and 1,2,3,6-tetrahydropyridine-4-carboxylic acid (isoguvacin). Isoguvacin, at a concentration of 40 μΜ in the suspension, inhibits the specific binding of Ca ²⁺ -independent ³ Hy-aminobutyric acid, thereby increasing the sensitivity of the assay. After incubation at room temperature for 30 minutes, the solid is filtered off and washed at room temperature with Tris-HCl buffer (pH 7.4). Radioactivity was measured as in Example 2; the correction factor (C _b ) is determined by the method described in Example 2.

3.4. Determination of ³ H-Dihydroalprenolol:

mg of the lyophilized receptor preparation of Example 1 are suspended in 1 ml of Tris-HCl buffer, pH 8.0. To the suspension was added 2 nM ³ H-dihidroalprenololt, incubated at room temperature for 30 minutes, then the solid filtered and washed at room temperature in Tris-HCl buffer (pH 8.0). Radioactivity was measured as in Example 2; the correction factor (C _b ) is determined by the method described in Example 2.

The amount of specifically bound ³ H ligands (C _c -C _b ) is shown in Table 2.

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Table 2

The number of the example Specifically bound ³ H ligands fmol / ml 3.1. 111 3.2. 231 3.3. 22 3.4. 19

Patent claims

Claims (7)

CLAIMS 1. A method of making a stable receptor preparation for quantifying brain receptor-binding substances, comprising centrifuging a brain or brain fragment homogenate prepared with an aqueous solution of a water-soluble inert substance at 800 to 1100 g for 8 to 20 minutes and supernatant at 18,000 to 22,000 g. pelleted by centrifugation for 20 minutes at 10-20, the resulting solid was rehomogenized in distilled water, then centrifuged at 7000-9000 g for 5-15 minutes and the supernatant was collected, and from 35,000 to 45,000 g for 20 to 30 minutes centrifugation at ₂₅ is separated from the preparation base material, characterized in that this material was washed three times with pH, the aqueous buffer solution between 6 and 8 and a mixture of solid and wash slurry - 10 and - freezing temperature of from 30 ° C _{to 30} for at least 8 hours, thawed, after thawing 3 After centrifugation at 5,000-45,000 g for 20-30 minutes, this freeze-thaw centrifugation cycle is preferably repeated twice, and finally the suspension is freeze-dried under vacuum. 2. Azl. A process according to claim 1, wherein the washing comprises Tris-citrate buffer solution (pH 7.1). 3. Azl. A method according to claim ⁵ is characterized such that said freezing - 15 and -25 ° C, preferably Continue at -20 ° C for 10 hours. The process according to claim 1, characterized in that the post-thawing centrifugation is carried out at an acceleration of 38,000 to 42,000, preferably 40,000 g. 5. Receptor preparation according to any one of claims 1 to 4, which can be stored at 4 ° C for 1 year and at room temperature for 1 month, characterized in that it is a white, dry, powdery substance with a free water content of 0.5-5%; suspended in distilled water or buffer, suspension ratio: 2 mg / ml to 6 mg / ml; the resulting suspension is translucent and does not settle (stable); the preparation has a protein content of 15-25%; the preparation has a content of γ-aminobutyric acid of less than 20 pmol / g. 6. A method for quantifying cerebral receptor binding agents by using radioactive ligands that specifically bind to cerebral receptors, and reducing the amount thereof in a concentration-dependent manner, characterized in that the receptor preparation in the assay is 1-5. Use of a material according to any one of claims 1 to 5. 7. A method for determining the concentration of γ-aminobutyric acid in the spinal fluid based on the reduction of the amount of ^3- Hy-aminobutyric acid specifically bound to the receptor by the spinal fluid, characterized in that the preparation of the receptor for the assay is as described in claims 1-5. Use of a material according to any one of claims 1 to 5.