REAGENT FOR THE DETERMINATION OF FREE RADICALS

Abstract: The present invention relates to a reagent for the determination of free radicals comprising a chromogen, chosen from the following formula (I) wherein: R1 = CH5-; CH3-; H-; X-, R2 = C2H5-; CH3-; H-; X-, R3 = C2H5-; CH3-; H-; X-, R4 = C2H5-; CH3-; H-; X-, wherein X- is chosen among halides, said chromogen being suitably dissolved in water or other suitable solvent, and a buffer having a pH value between 3 and 6.9.

Description: REAGENT FOR THE DETERMINATION OF FREE RADICALS The present invention relates to a reagent for the determination of free radicals. More particularly, the invention relates to a reagent of the above kind that allows to make the determination of the free radicals in any kind of clinical chemistry analyser, and thus allows to make such determination in any laboratory, thus being possible to use the standard available instruments. As it is well known to those skilled in the art, with the term "free radical" it is meant in the literature any kind of chemical species able to exist independently and containing one or more unpaired electrons on the outermost orbital (atomic and/or molecular). Among the various existing free radicals, that referred to the oxigen has reached in the recent years, in the medical field, always a greater interest in view of the role that they can assume for the determination of a tissue damage of different pathological conditions. In the Angle - Saxon literature, the free radicals are named ROMs (Oxygen Reactive Metabolites), symbolization that will be used in the following of the present specification, and are characterized in having an odd number of electrons in the oxygen outermost orbit, this fact making them chemically reacting with respect to a great number of biological molecules. The main ROMs known in the literature are: the 02* anion superoxide; the H202 hydrogen peroxide, the organic peroxide radical R- 02*; the R-O* alkoxy radical; the OH* hydroxilic radical; the OHX hypoalous acids, wherein X can be halogen and all the relevant amines. During recent studies, numerous clinical conditions have been individuated wherein a role of the ROMs has been invoked and recognised. Particularly, it can be cited the inflammatory and post- ischemia diseases of the alimentary tract, the Central Nervous System disorders due to vascular or traumatic disorders, the atherosclerosis and its cardiac complications, some renal disorders, the arthritis deformans and the alcohol induced disorders in some parts of the organism. However, up today, remarkable difficulties have been encountered in the localisation and determination of the free radicals in the biological samples, so that their patoathogenic role in these diseases have largely deduced on the basis of the effect of antioxidant substances, which, naturally, could have not-specific antiinflammatory effects. Mainly, although not exclusively, up today the methods employed for the determination of the free radicals are the one known as ESR (electron spin resonance), sophisticated technique used only in research laboratories since it is very expensive, or those based on the use of sophisticated instalments, and thus not adoptable in analysis laboratories for the daily standard determinations. In view of the above, it appears extremely important to have a reagent like the one proposed according to the present invention that allows to employ a simple and fast method for the determination of free radicals and of their derivatives in order to allow also the monitoring of antioxidant antiinflammatory therapies by analysis that can be performed in the standard laboratories. The Applicant has determined on an experimental basis that the N,N-diethyl paraphenilendiamine (C 2 H 5 )2-NC 6 H4NH 2 ), already known for the determination of active chlorine within the waters, if employed in a suitable buffer and with particular concentration and pH conditions, thus determining a chromogen reaction, is able to determine by colour formation not only the chlorine, but also all the products having the feature of the unpaired electron in the outer orbit such as the anion superoxide, the hydrogen peroxide, the hydroxyl radicals, the hypoalous acids and all their amines. Similar results have been obtained with products of the same category and that are included in the described and claimed general formula. From the tests performed, it has been thus observed that the reagent according to the invention allows to quantitatively determine the ROMs present in a sample. The developed colour has an absorption spectrum with two peaks, one at 510 nm and one at 550 nm, as it can be noted from the enclosed graph 1. Its colour intensity is directly proportional to the concentration of the free radicals present in the sample and perfectly follows the Lambert and Beer's law, as it is evident also from graph 2, either reading at 510 nm or reading at 550 nm is performed. 3 In case the sample to be analized is comprised of a biologic sample (blood, plasm, serum, liquor, etc.), the ROMs determination by the method employing the reagent according to the invention can be made either with the end read of the colour development (end Point) or with the predetermined time range (Fixed Point), and with a continued kinetic. This is an aspect that greatly differentiates the reagent according to the present invention with respect to all the systems presently known and that make the same particularly advantageous. In fact, being it possible to use the reagent according to the invention with any analysis method, it is possible to use the same in any laboratory without the need of providing particular apparatuses, but even allowing to use the already available ones. The method proposed by the Applicant and employing the reagent according to the invention particularly, but not solely, provides, and thus this cannot be considered as a limitation, the use of two separated reagents A and B, wherein A represents the chromogen and B a suitably buffered solution . The two reagents are mixed with the suitable proportions so as to make a sole reagent as working reagent. In anycase, the two reagents can be used also in two following steps. It is therefore specific object of the present invention a reagent for the determination of free radicals comprising a chromogen, chosen from the following formula (I) wherein: R1 = CH5-; CH3-; H-; X- R2 = C2H5-; CH3-; H-; X- R3 = C2H5-; CH3-; H-; X- R4 = C2H5-; CH3-; H-; X- wherein X- is chosen among halides, said chromogen being suitably dissolved in water or other suitable solvent, and a buffer having a pH value between 3 and 6.9 Preferably, said chromogen and buffer are mixed with a proportion between 1:20 and 1:300, this ratio baing variable for different solution proportions of the chromogen and of the buffer. According to a first preferred embodiment of the reagent according to the invention, said chromogen comprises 4-amino- paraphenylen-N,N-diethylamine, or its salts. In a second preferred embodiment of the reagent according to the invention, said chromogen can comprise 4-amino-paraphenylen-N,N- diethylamine sulphate (C10H16N2H2SO4) or its salts. Still according to the invention, in a third preferred embodiment, said chromogen can comprise 4-diamine-paraphenylen-N,N- diethylenanilina oxalate (C10H16N2C2H2O4) or its salts. Further, in the reagent according to the invention, said chromogen can comprise 4-amino-paraphenylen-N,N-dimethylamine or its salts. Always according to the invention, said chromogen can comprise 4-amino-paraphenylen-N,N-dimethylamine sulphate or its salts. Further, according to the invention, said chromogen can comprise 4-diamine-paraphenylen-N,N-dimetylamine oxalate or its salts. Preferably, said buffer will have, according to the invention, a pH value between 4 and 5. Now, some preparation examples for the two reagents making the reagent according to the invention are given in the following. Said examples must not be in any way intended as limitative of the scope of the invention, since they are only exemplicative of some possible solutions. It must be further underlined how some ratios given between chromogen and buffer can be modified in function of the solubility of the same chromogen and buffer. REAGENT A - Chromogen Example A Components Doses relevant to 1 ,000 ml of A reagent NN diethyl para phenyl diamine sulphate 1 - 200 gr H 2 0 up to 1,000 ml Example B 5 Components Doses relevant to 1 ,000 ml of A reagent Nn diethyl para phenylen diamine 1 - 150 gr CH3-COOH up to 1,000 ml Example C Components Doses relevant to 1 ,000 ml of A reagent NN diethyl para phenylen diamine oxalate 1 - 190 gr H20 bi-distilled up to 1 ,000 ml REAGENT B - Buffer .Example 1 (can be used with the chromogen according to the examples A and C) Acetate buffer between pH 3 and pH 6.9 Example 2 (can be used with the chromogen according to the example B) Sodium hydroxide from 0.001 N and 1 N Example 3 (can be used with the chromogen according to the examples A and B) Posphate buffer between pH 3 and pH 6.9 Example 4 (can be used with the chromogen according to the examples A and B) Pipes buffer between pH 3 and pH 6.9 Example 5 (can be used with the chromogen according to the examples A and B) Epes buffer between pH 3 and pH 6.9 Will be in the following described for illustrative, but not limitative purposes a method for the preparation of the reagent according to the invention. Working reagent preparation The Reagent A and the Reagent B are mixed with a ratio between 1:300 and 1:100, preferably 1:200. For the chromogen according to example B, the preferred ratio will be 1 :40. Using method with end reaction 1 ml of working reagent (A+B) are mixed with 5 ml of biologic sample (bloob, serum, plasm, liquor, etc.). The solution is left within thermostat at 37°C for 1 hour. The reading is performed between 480 and 570 nm. The test is executed parallelly using a sample having a known concentration and applying the classic formula: C sample = (A sample / A standard) x C standard. Use method with Fixed Time reaction The reaction temperature is equal to 37°C. 1 ml of working reagent is mixed with 20 ml of sample. The reading occurs after 30M The initial absorbance is comprised between 480 and 570 nm. An incubation for 5' at 37°C is realized. It then read the absorbance again. The calculation is made with the fornula: C sample = ( final A - initial A) sample / (final A - initial A) standard x c standard. Use method with continuous kinetic The reaction temperature is of 37°C (35°C - 30°C - 20°C) (really the analysis temperature is not a fundamental parameter). The reading is made between 480 and 570 nm. The buffer volume is 0,1 ml, the serum volume is 10 1, and the chromogen volume is 10 I. Now the reaction rate remains constant for many minutes, allowing the kinetic determination of the ROMs. Provoking the variation of D.O/minute of a sample having a known concentration, the concentration of the ROMs within the sample is determined as follows: Cone (sample): D.O/minute sample x Cone. Standard D.O/minute standard The present invention has been described for illustrative, but not limitative purposes according to its preferred embodiments, but it is to be understood that modifications and/or changes can be introduced by those skilled in the art without departing from the relevant scope as defined in the enclosed claims.

Claims: CLAIMS 1. Reagent for the determination of free radicals characterized in that it comprises a chromogen, chosen from the following formula (I) wherein: R1 = CH5-; CH3-; H-; X- R2 = C2H5-; CH3-; H-; X- R3 = C2H5-; CH3-; H-; X- R4 = C2H5-; CH3-; H-; X- wherein X- is chosen among halides, said chromogen being suitably dissolved in water or other suitable solvent, and a buffer having a pH value between 3 and 6.9 2. Reagent according to claim 1, characterized in that said chromogen and buffer are mixed with a proportion between 1:20 and 1:300. 3. Reagent according to claim 1 or 2, characterized in that said chromogen comprises 4-amino-paraphenylen-N,N-diethylamine (C10H16N2), or its salts. 4. Reagent according to claim 1 or 2, characterized in that said chromogen comprises 4-amino-paraphenylen-N,N-diethylamine sulphate (C10H16N2H2SO4) or its salts. 5. Reagent according to claim 1 or 2, characterized in that said chromogen comprises 4-diamine-paraphenylen-N,N-diethylenanilina oxalate (C10H16N2C2H2O4) or its salts. 6. Reagent according to claim 1 or 2, characterized in that said chromogen comprises 4-amino-paraphenylen-N,N-dimethylamine or its salts. 7. Reagent according to claim 1 or 2, characterized in that said chromogen comprises 4-amino-paraphenylen-N,N-diMethylamine sulphate or its salts. 8. Reagent according to claim 1 or 2, characterized in that said chromogen comprises 4-diamine-paraphenylen-N.N-dimetylamine oxalate or its salts. 9. Reagent according to one of the preceding claims, characterized in that said buffer has a pH value between 4 and 5. 10. Reagent for the determination of free radicals according to each one of the preceding claims, substantially as illustrated and described.

Inventors: CARRATELLI MAURO, CARRATELLI, MAURO
Applicants: CARRATELLI MAURO, DIACRON SRL, CARRATELLI, MAURO, DIACRON S.R.L.
Application year: 1995
Main classification: G01N31
IPC codes: G01N31