JPH0635966B2 - Analytical reagent useful for detecting thiol compound and detection method using the same - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to analytical reagents and methods for measuring thiol compounds in liquid systems, including thiol compounds that are transiently generated during the reaction process. More specifically, the analytical reagent and method of the present invention relate to the colorimetric analysis of thiol compounds and can be used to measure the presence of a thiol compound or the presence of a reagent or enzyme that causes it.

Background of the Invention Thiol compounds occur naturally and can also be prepared by chemical reactions. Such a reaction has the following formula: Examples thereof include reduction reaction and biochemical reaction of disulfide.

Many methods are known for the detection of compounds containing thiol groups. A particularly useful and often cited method is described by GK Ellman in Arch. Biochem. Biophys.
Volume 70-77 (1959). This method produces 3,3'-dithio-bis-6-, which produces the yellow anion of 3-mercapto-6-nitro-benzoic acid.
It is based on the thiol reaction with nitrobenzoic acid (Ellman's reagent).

However, one drawback of the Ellman method is that the reduced disulfides produce only a yellow shade. It is usually considered much more desirable to have a tint in the red or blue region, and thus
It may be more suitable by visual measurement of thiols.

Accordingly, it is an object of the present invention to provide a reliable, accurate and inexpensive analytical reagent and method for the colorimetric analysis of thiol compounds, where the color provided by the reagent and method is in the red or blue region. That is.

Other objects of the invention will be apparent to those skilled in the art upon reading the specification and claims.

Detailed Description of the Preferred Embodiments The reagents and methods of the present invention relate to the following reaction schemes.

2R-SH + 2Fe ³⁺ → RSS-R + 2Fe ^{2+ In} this reaction, the thiol compound interacts with iron in the +3 oxidation state, reducing the ion to the +2 state,
This oxidizes the thiol compound to form a disulfide. The resulting Fe ²⁺ ions can form a complex with a suitable ligand to cause a color change. In this method, the molar reduction rate of Fe ³⁺ (molar extinc
and the color change obtained by the chromogenic ligand: Fe ²⁺ complex is directly related to the amount of thiol compound initially present in the system.

Numerous methods are known for the detection of Fe ²⁺ ions with complex ligands suitable as chromophoric complexes. Examples of suitable ligands for this purpose include ferroins,
There may be mentioned a compound selected from the group consisting of cuproins and teloins. Particularly preferred complex ligands are hydrazones and their tautomeric azo forms, tetrazoylpyridines, pyridylquizolines, bis-isoquinolines, imines, phenanthrolines, bipyridines,
Terpyridines, vidazines, pyridyldiazines,
Pyridylbenzimidazoles, diazyltriazines, o-nitroanilines, phenols, tetrazines, triazines, pyridines, phenazines, quinoxalines, benzimidazoles, substituted methyl or phenyl-2-pyridylketone oximes And the like [Smith, Analyt. Chem., 26, 1534-1538 (1954); Schil
t et al., Talanta, 15, 475-478 (1968); Schilt et al., Talan.
ta, 15, 1055-1058 (1968); and Schilt et al., Talant.
a, 16,448-452 (1969)]. For a description of other complex ligands, see Blandamer et al., J. Chem. Soc., Dalton 1001-1008 (1.
978), Case J. Org. Chem., 31, 2398-2400 (1966)
And also in British Patent Application No. 701,843. Of course, complex ligands other than those shown here can also be used.

When incorporating a complex ligand into the test strip template and using embodiments of the present invention, it is advantageous to also have hydrophobic or ion-exchange functional groups. This improves the binding of the complex ligand to the matrix, which prevents the specimen from "bleeding". Examples of hydrophobic groups that can be used include long chain alkyl or aralkyl groups. Polymer binding of complex ligands is also contemplated.

According to the invention, the test reagent is a liponamide (liponamide)
can be used to detect thiols such as ic acid amide), thiocholine, glutathione or coenzyme A, or thioglycosides. Furthermore,
A thiol precursor can also be detected, such as when a thioester, thioether, disulfide or thioacetal is produced in the test system.

The test reagent of the present invention is a thioether hydrolase,
It is suitable for assaying enzymes that cleave thio compounds such as thioesterases or thioglycosidases.
Furthermore, esterases such as cholinesterase (CHE) can also be detected. A common substrate for CHE is acetylcholine, but this enzyme can also cleave acetyl- and butyrylthiocholine, and the test reagent can detect the free thiol group produced by the cleavage. The present invention also provides a liponamide dehydrogenase in the presence of reduced nicotinamide adenine dinucleotide (NADH).
It can also be used with biochemical reactions where free thiol groups are generated by redox reactions such as the reduction of liponic acid using as a catalyst.

Therefore, the test reagent of the present invention can be used for analysis of a reaction by NADH. Typical examples of the enzyme by NADH include lactate dehydrogenase, alcohol dehydrogenase, glucose dehydrogenase,
Glycerol aldehyde dehydrogenase, glycerol phosphate dehydrogenase, malate dehydrogenase and the like can be mentioned. NADH is also a glutamate oxal acetate transaminase (EC 2.
It may also be the end product of a multi-step enzymatic reaction, as in the case of analysis of 6.11), glutamate pyruvate transaminase (EC 2.6.12) or creatinine kinase (EC 2.7.32).

NADH also includes lactate, glucose, malate,
It may be a reaction product in the analysis of a substrate such as urea. The use of the test reagents of the invention in the analysis of reactions with NADH yields 2 moles of Fe ²⁺ per mole of NADH, and in some cases the chromophoric complex has a very high extinction coefficient which results in a substantial sensitivity. Improve to.

It has proved to be advantageous to provide Fe ³⁺ complexes bound to thiol substrates and possible complexing reagents are EDTA, HEDTA, citric acid, malic acid, lactic acid such as alanine, glycine, and Amino acids such as glutamine, eg 18-crown-6, phenylaza-15-crown-5, benzo-15-crown-5.
And crown ethers such as dibenzopyridino-15-crown-5, or triazinophanes and cryptates.

The concentration ratio of thiol: Fe ³⁺ should be at least 1: 1 and preferably about 1: 5. A ratio of 1:10 is also preferred for the fastest reaction, but a ratio of 1:20 is particularly preferred.

The test reagent or test system of the present invention can be used to measure the analyte in the cell. In addition to Fe ³⁺ ions and complex ligands, the test reagents include enzymes, substrates,
It may also contain all reagents necessary for a particular assay, such as coenzymes, effectors, antigens, antibodies and the like.
These test reagents can contain non-reactive substances such as buffering agents, wetting agents and stabilizing agents.

As described above, NADH and NADPH can be used as coenzymes to measure enzymes that generate thiol groups, such as liponamide dehydrogenase and glutathione reductase. Reagent formulations can be prepared from the enzymes, reagents and the substances already mentioned and can be mixed as solutions or powders or in the form of tablets or lyophilizates. If the reagent formulation is not already in solution, it can be dissolved in water or other suitable solution, by which the reagent solution is prepared. If the reagent formulation consists of the individual components, these are mixed with one another. After mixing the sample (eg substrate solution, enzyme solution, serum, plasma or urine) with a portion of the reagent mixture, the resulting color is measured with a photometer. The specific concentration, or substrate concentration, is then calculated from the molar extinction coefficient and the amount of reagent or sample added. Both dynamic and endpoint measurements are possible.

Fe ³⁺ / ligand system 1. one or more reagents or other enzymes required for specific parameter detection; Buffer system; 3. 3. Wetting agent; 4. an activator; and It can also be impregnated onto an absorbent reagent carrier (eg, paper, fleece, etc.) together with other auxiliaries. For this, one or more impregnation solutions can be prepared in the form of aqueous solutions, organic solutions or mixed solutions, depending on how well the reagents or auxiliaries are dissolved.

Absorbent or swellable supports, preferably paper or absorbent fleeces of glass or plastic, are impregnated or sprayed with these solutions and then dried. The reagent carrier produced in this manner is used as a rapid diagnostic agent for directly measuring a liquid sample, that is, a body fluid such as blood, urine or saliva, or the content of food such as fruit juice or milk. You can also The liquid is thereby applied directly to the reagent carrier or the carrier is immersed in the liquid for a short time. Semi-quantitative analysis is possible by arranging the comparative colors against the colors thus generated. Quantitative analysis can be performed with a reflectance photometer.

It is also possible to incorporate the test reagents according to the invention into a carrier matrix prepared from a casting solution. Examples which may be mentioned here are cellulose, cellulose derivatives, gelatin, gelatin derivatives or plastics such as polyurethane and acrylamide. Here, it is advantageous to add the test reagents and, where appropriate, other necessary reagents directly to the casting solution. Thereby, the test device including the carrier and the reagent can be manufactured by one operation.

A reagent solution can be obtained by eluting the above-described reagent from the absorbent carrier with water or a buffer or serum. The substrate or enzyme can then be measured in the photometer cell as described above.

Suitable buffers for the test reagents described are phosphate, citrate, borate, and GOOD buffers with alkali metal or ammonium counterions. However, other systems can be used. A pH value of 6 to 10 is desired although pH 6.5 to 7.5 is preferable.

Wetting agents are particularly preferred anionic and cationic wetting agents which undergo an ionic interaction with the compounds according to the invention. Non-ionic wetting agents which activate the enzyme can also be used, sodium lauryl sulphate, sodium dioctyl sulfosuccinate and alkylaryl polyether alcohols being preferred.

Effectors that can be used are known for particular enzymatic reactions.

Suitable other auxiliaries are the usual thickeners, solubilizers, such as those known in the corresponding tests with other chromogens.
Examples include emulsified goods, optical brighteners, and fixed media.

Example Analysis of thiols with FeCl ₃ and complex ligands To detect NADH in the test system described,
The following reagent components were placed in the cell.

After measuring the reagent blank value, the reaction was started by adding 20 μ of NADH solution. The maximum absorbance was measured at 515 nm. Dynamic measurements showed a stable end point (1% change in absorbance within 20 minutes) after a reaction time of only 1 hour.

In order to test the potency and linearity, NADH concentrations in the range of 1-10 mmol / ml were determined in batch tests. 5
The difference in the absorbance measured at 15 nm is shown in Table 1.

The colors obtained with various complex ligands and the corresponding maximum absorbances are shown in Table 2.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication location G01N 33/68 7055-2J (56) Reference JP-A-61-153564 (JP, A) Japanese analysis Analytical Chemistry Handbook by the Society of Chemistry Maruzen 1972 430-433

Claims (2)

[Claims] 1. A complex which can be selectively formed with iron in the +3 oxidation state and at least as much as the amount of the thiol compound to be detected, and +2 in the oxidation state, whereby the iron and the iron can be selectively formed. A reagent for an analytical test for colorimetrically analyzing a thiol compound in a liquid sample or a substance related to the presence of the thiol compound, which comprises a ligand that exhibits a color change based on the complex formation. 2. Iron consisting of +3 oxidation state and +2
Can form a complex selectively with iron in the oxidation state of
A thiol compound in a liquid sample, characterized in that the liquid sample is combined with a liquid which also comprises a ligand which causes a color change by complex formation with such iron ions in the +2 oxidation state, or A method for analyzing a substance related to the presence of a thiol compound.