DE19739120A1 - Determination of the antioxidant capacity of sample - Google Patents

Abstract

The antioxidant capacity of samples is determined by measuring the optical variation of the samples which are heated in the presence of a system which forms temperature dependent free radicals and an indicator which changes its optical properties according to the free radicals formed. The measurement is effected in a new analytical device. The antioxidant capacity of samples is determined by: a) mixing the sample in a plurality of cooled wells arranged in a given x-y frame with a system which forms temperature-dependent free radicals and an indicator which changes its optical properties depending on radicals present; b) bringing the mixture to an initial temperature of 0-20 deg C; c) heating the mixture to a maximum of 90 deg C, preferably over 60 minutes, while continuously recording the change in the optical properties using a conventional reading device; and d) determining the antioxidant capacity by comparing the change with known reference values. An Independent claim is also included for an analytical device comprising a known microtitre plate having flexible wells arranged in an x-y frame and a metallic temperature controllable base having corresponding depressions to receive the wells.

Description

The invention relates to a method for determining the Antioxidant capacity of samples and is used in particular in biochemistry biotechnology, medical and industrial research and used in food chemistry.

Free radicals occur in living nature in a large material Diversity with short, very different lifetimes and different Meaning on. On the one hand, they are dangerous by-products that are in the process of biological oxidation are formed and are able to do almost all to chemically modify biological substance groups and to damage them. On the other hand, they are known for their outstanding responsiveness by nature as helpful tools to combat in the Organism invaded pests by specialized cells, such as Leukocytes and macrophages, formed in the infection control process and used. There is a complicated balance of oxidants formed in the organism and the regulated formation of Antioxidants. Oxidative stress is caused by antioxidants, which result from low molecular weight water or lipid soluble substances such as vitamin C, Glutathione, uric acid, bilirubin, vitamin E, vitamin A u. a. recruit compensated or by enzymatic processes such as superoxide dismutase, Catalase, glutathione reductase and gluthadione peroxidase, ineffective made.

Disorders in the antioxidant balance are considered pathobiochemical Mechanisms of the development of diabetes mellitus and its symptoms (Ceriello A., Bortolotti N., Falleti E., Taboga C., Tonutti L., Crescentini A., Motz E., Lizzio S., Russo A., Bartoli E. Total radical-trapping antioxidant parameters  in NIDMM patients. Diabetes Care 1997; 20: 194-197), for the heavy one Symptoms of sepsis (Dasgupta A., Malhotra D., Levy H., Marcadis D., Blackwell W., Johnston D., Decreased total antioxidant capacity but normal lipid hydroperoxide concentrations in sera of critically ill patients. Life Sci 1996; 60: 4-5 or Cowley H.C., Bacon P.J., Goode H.F., Webster N.R., Jones J.G., Menon D.K. Plasma antioxidant potential in severe sepsis: a comparison of survivors and nonsurvivors. Crit Care Med 1996; 24: 1179-1183), for arteriosclerosis and heart attack (Westhuyzen J. The oxidation hypothesis of atherosclerosis: An update. Annals of Clin and Lab Science (1997); 27: 1-10 or Miller N.J .; Rice-Evans C. A. Antioxidant activity of resveratrol in red wine. Clin-Chem. 1995; 41: 1789 or Mulholland C. W .; Strain J. J. Serum total free radical trapping ability in acute myocardial infarction. Clin-Biochem. 1991; 24: 437-441), for essential problems in the Process of Aging (Butterfield D.A., Howard B.J., Yatin S., Allen K.L., Carney J. M. Free radical oxidation of brain proteins in accelerated senescence and ist modulation by N-tert-butyl-alpha-phenylnitrone. Proc Nat Acad Sci USA 1997; 94: 674-678.), For kidney diseases (Rohrmoser M. M., Mayer G. Reactive oxygen species and glomerular injury. Kidney Blood - Press Res. 1996; 19: 263-269 or McGrath L. T .; Douglas A. F .; McClean E .; Brown J. H .; Doherty C. C .; Johnston G. D .; Archbold G. P. Oxidative stress and erythrocyte membrane fluidity in patients undergoing regular dialysis. Clin-Chim-Acta. 1995; 235: 179-188 or Jackson P .; Loughrey C. M .; Lightbody J. H .; McNamee P. T .; Young I. S. Effect of hemodialysis on total antioxidant capacity and serum antioxidants in patients with chronic renal failure. Clin-Chem. 1995; 41: 1135-1138.), For the development of neurodegenerative processes (Borlongan C. V .; Kanning K .; Poulos S. G .; Freeman T. B .; Cahill D. W .; Sanberg P. R. Free radical damage and oxidative stress in Huntington's disease. J-Fla-Med-Assoc. 1996; 83: 335-341 or Gurney M.E .; Cutting F. B .; Zhai P .; Andrus P. K .; Hall E. D. Pathogenic mechanisms in familial amyotrophic lateral  sclerosis due to mutation of Cu, Zn superoxide dismutase. Pathol-Biol-Paris. 1996; 44: 51-56), and for the development of some types of carcinoma (Dreher D .; Junod A. F. Role of oxygen free radicals in cancer development. Eur-J Cancer. 1996; 32A: 30-38 or Pappalardo G .; Guadalaxara A .; Maiani G.; Illomei G .; Trifero M .; Frattaroli F. M .; Mobarhan S. Antioxidant agents and colorectal carcinogenesis: role of beta-carotene, vitamin E and vitamin C. Tumori. 1996; 82: 6-11 or Kumar K .; Thangaraju M .; Sachdanandam P. Changes observed in antioxidant system in the blood of postmenopausal women with breast cancer. Biochem Int. 1991; 25: 371-380) made.

The determination of the antioxidant capacity or antioxidant reactivity as receives diagnostic-analytical parameters in medicine for diagnostics and progress control for a variety of diseases increasingly greater importance.

Wayner and co-workers (Wayner D. D., Burton G. W., Ingold K. U., Barclay L. R., Locke S. J. The relative contributions of vitamin E, urate, ascorbate and proteins to the total peroxyl radical-trapping antioxidant activity of human blood plasma. Biochim Biophys Acta 1987; 924: 408-419) were the first the 1987 a method for determining total radical antioxidant Potentials (TRAP). In a system that consists of one Radical source 2,2'-azo-bis-2-amidinopropane (ABAP), which existed at constant temperature over a constant radical formation rate Oxidized lipid mixture, the oxygen consumption was measured. After Addition of antioxidants has been added to the induction time of the system determined again oxygen consumption, as well as with a reference antioxidant known stoichiometry and reactivity compared and calibrated. In the aftermath, both other radical sources as well other radical indicator systems have been adapted and optimized (Lissi E .; Salim-Hanna M .; Pascual C .; del-Castillo M. D. Evaluation of total antioxidant  potential (TRAP) and total antioxidant reactivity from luminol-enhanced chemiluminescence measurements. Free Radic Biol Med. 1995; 18: 153-158 or Cao G .; Alessio H. M .; Cutler R.G. Oxygen-radical absorbance capacity assay for antioxidants. Free Radic Biol Med 1994; 16: 135-137 or also Candy TEG, M Hodgson, P Jones 1990. Kinetics and mechanisms of a chemiluminescent clock reaction based on the horseradish peroxidase catalysed oxidation of luminol by hydrogen peroxide. J. Chem. Soc Perkin Trans 2: 1385-1388). Other publications on this are for example Uotila J. T .; Kirkkola A. L .; Rorarius M .; Tuimala R. J .; Metsa-Ketela T. The total peroxyl radical-trapping ability of plasma and cerebrospinal fluid in normal and preeclamptic parturients. Free Radic Biol Med. 1994; 16: 581-590 or Whitehead T.P., GHG Thorpe, SRJ Maxwell. Enhanced chemiluminescent assay for antioxidant capacity in biological fluids. Analyte. Chimica Acta 1992; 266: 265-277 or DeLange R. J .; Glazer A.N. Phycoerythrin fluorescence-based assay for peroxy radicals: a screen for biologically relevant protective agents. Anal biochem. 1989; 177: 300-306 or also Thurnham D.I .; Singkamani R .; Kaewichit R .; Wongworapat K. Influence of malaria infection on peroxyl-radical trapping capacity in plasma from rural and urban Thai adults. Br-J-Nutr. 1990; 64: 257-271.

In addition to these methods, according to the samples to be characterized longer or shorter delay the induction of one by the presence of a signal triggered by a radical source, methods are described that get by with single measurements after a certain time. The disadvantage of the previously known methods is that they time consuming and sequential.

The invention is therefore based on the object Antioxidant capacity of samples in the simplest possible way, quickly and especially without determining time-consuming analyzes.  

The disadvantages of the known solutions according to the invention are thereby circumvented that the analyzes in parallel in sets of pluralities of Mixtures are carried out, each individual measurement in the network of otherwise identical boundary conditions. The Radical formation is synchronized and dosed by programmed temperature increase with suitable inventive Means. The automated evaluation is carried out via carried and otherwise calibrators treated the same as the sample.

The invention will be described below with reference to the drawings Embodiments are explained in more detail.

Show it:

Fig. 1 sample arrangement of machinery mounted on a heatable body analysis vessels (wells) in a 12 x 8 grid of a microtitre plate;

Fig. 2 is a time temperature profile for a defined simultaneous increase of the temperature in the spaced 12 x 8 grid analysis vessels in a period of 8 minutes;

FIG. 3 shows temperature profile of the mixtures in three selected vessels analysis of the microtiter plate;

FIG. 4 shows evaluation results of the analysis;

FIG. 5 shows time course of absorption of ABTS at 620 nm with the free radical source ABAP in all 96 wells of a microtiter plate with different samples over a period of 43 minutes;

Fig. 6 is a time course of the absorption of ABTS at 620 nm with uric acid and catechin as to be examined samples over a period of 43 minutes;

Fig. 7 is a calibration curve of Trolox as Referenzantioxidanz for the colorimetric determination of Antioxidanzkapazität with ABAP / ABTS;

Fig. 8 is a time course of chemiluminescence of luminol with the ABAP as a radical source in a matrix of 6X8 analysis vessels with different samples in a period of 25 minutes;

Fig. 9 Influence of the addition of additional Trolox to the reaction mixture on the course of the chemiluminescence of ABAP / Luminol. (H3 without Trolox, G4 and H4 with Trolox in the reaction mixture);

FIG. 10 is a time course of absorbance at 620 nm of a cell system (resistance of erythrocytes to ABAP in the presence of varying concentrations of Trolox) within a period of 4 hours;

FIG. 11 is a calibration curve of Trolox as Referenzantioxidanz (resistance of a cell system);

FIG. 12 is a time course of chemiluminescence of luminol in the system xanthine-xanthine oxidase;

FIG. 13 is the calibration of the xanthine-xanthine oxidase chemiluminescence method with superoxide dismutase as Referenzantioxidanz;

14 shows time course of chemiluminescence of luminol in the system H ₂ O ₂ / myoglobin as radical images.

FIG. 15 is time-dependent shift of the fluorescence wavelength from Ruby to ABAP addition of (excitation wavelength: 550 nm);

Fig. 16, the influence of different Troloxmengen to the beginning of the time-dependent change in the fluorescence from Ruby to ABAP adding.

In order to determine the antioxidant capacity of samples to be examined, mixtures are produced which, in addition to the samples, contain at least one system which forms free radicals as a function of temperature and an indicator which changes its optical properties as a function of radicals. These mixtures are produced by pipetting into analysis vessels 1 of a microtiter plate 2 known per se. An exemplary sample arrangement of the analysis vessels 1 in a known 12 × 8 grid of the microtiter plate 2 is shown in FIG. 1. The microtiter plate 2 with the analysis vessels 1 is placed on a base body 3 in a positive and non-positive manner. Due to the resilient material, the analysis vessels 1 of the microtiter plate 2 fit into position-corresponding depressions 4 of the metallic base body 3 . Fig. 1 shows the microtiter plate 2 and the base 3 by way of illustration, both in separate and composite representation. The analysis vessels 1 with the mixture batches are initially in the cooled state and are each brought to an essentially identical starting temperature between 0 and approximately 20 ° C. in order to create approximately the same thermal analysis conditions for all mixture batches. From this starting temperature, the mixtures in the analysis vessels 1 are heated uniformly together. For this purpose, the base body 3 can be heated. In Fig. 1 heating spirals 5 are therefore indicated in the base body 3 . Due to the metallic design of the base body 3 and the good heat conduction associated therewith, this is evenly heated, including the vessels 1 which are recorded in the recesses 4 with close thermal contact.

Fig. 2 shows the temperature profile of the reaction mixture, measured with a thermochromic indicator system [ATS K .; Cumme GA; Hoffmann-Blume E .; Hoppe H .; Horn A. Multiwavelength photometry of thermochromic indicator solutions for temperature determination in multicuvettes. Clin-Chem. 39/2, 251-256 (1993)], over a certain period in all 96 analysis vessels 1 (wells) of the microtiter plate 2 . Fig. 3 shows the temperature profile over time of three selected positions of the microtiter plate (the position positions are shown at the top right in the figure) . From the table in Fig. 3 it can be seen that at an initial temperature of 18 ° C the target temperature after approx. 3 minutes of 42 ° C and is maintained constant.

According to the invention, the radical-forming in the mixtures System formed free radicals depending on the temperature. In the blends is also contain an indicator that changes depending on this Radical formation changes the optical properties of the mixtures. With a reader arrangement known per se (not in the drawing ), the temporal courses of these are shown in all analysis vessels Change in optical properties detected and as follows evaluated:

Determination of the delay time τ

In Fig. 4a, the principle of evaluation of the analytical results is presented τ by determining the delay time. The antioxidant capacity A _{P of} the sample causes, compared to the antioxidant capacity A _{0 of} the reaction mixture without a sample, a time delay in the formation of an optical signal of the indicator which changes its optical properties as a function of the radicals. The antioxidant capacity A _{P of} the sample can be standardized via the comparatively carried determination of the antioxidant capacity A _{S of} a standard. The delay times of the sample (τ _P ), the standard (τ _S ) and the reaction mixture without sample = blank value (τ ₀ ) can be determined from the rising, almost linear part of the curve of the optical signal over time. For practical reasons, it is advantageous to add a defined, small concentration of antioxidants to the reaction mixture in order to ensure that all radicals are bound during the nonlinear part of the radical formation at the beginning of the temperature increase and the change in the optical signal in the blank value in the time-dependent linear part of the Radical formation lies. The antioxidant capacity A _{P of} the sample can then be determined

can be calculated, the antioxidant capacity A _{S of} the standard
A _S = C _S .SF results.
C _S = concentration of the standard
SF = stoichiometric factor of radical formation.

Determination of the integral of the optical signal

In FIG. 4b, the evaluation of the analysis result is shown of the optical signal on the definition of the integral. The integral of the optical signal (extinction; fluorescence; luminescence) of the signal-time curve for the blank value (I ₀ ) for the sample (I _P ) and for the standard (I _S ) is determined. There is a linear relationship between I ₀ / I _P and the antioxidant concentration (Stern Vollmer equation). The antioxidant concentration in the sample can be determined by carrying a standard.

The evaluation of the analysis results to determine the antioxidant capacity, both by determining the delay time ( FIG. 4a) and by determining the integral of the optical signal ( FIG. 4b) is essentially supported by suitable software on an evaluation computer.

Embodiment 1 Colorimetric determination of the antioxidant capacity

The radical generating system is 2,2'-azo-bis-2-amidinopropane (ABAP). This compound produces one even at relatively low temperatures significant amount of peroxyl radicals and reacts with all substances as Antioxidants are important in biological processes.

As an indicator that its optical in the presence of free radicals Changes properties, 2,2'-Azino-bis (3-ethylbenzthiazolin-6-sul fonic acid) (ABTS) is used. The originally colorless solution receives Presence of radicals an intense blue color.

The samples to be analyzed are pipetted into the individual wells of the cooled, heatable microtiter plate. The assignment of the individual wells of the microtiter plate are shown in the legend of FIG. 5. In some wells, Trolox, a water-soluble vitamin E derivative with known concentration and antioxidant capacity, is presented as a standard, or water as a blank value.

Then, with a 96-fold pipette (hedgehog), 200 µl of cooled, freshly prepared substrate (1.64 mg ABTS, 54 mg ABAP and 128 µl 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox (376 µM in Water)), dissolved in 20 ml buffer (2 ml 50 mM phosphate buffer pH 7.4 and 18 ml 0.9% NaCl), added, the microtiter plate shaken, to 37 ° C heated and with a known microtiter plate reader at 620 nm at certain time intervals and a total duration of in the example measure the specified 43 min.

The different course of the absorption as a function of time in all 96 wells of the microtiter plate is shown in FIG. 5. There is a clear gradation of the delay time in the individual wells, which passes until the start of the increase in absorption. During this time, the radicals formed are "caught" by the antioxidants used, and there is no change in the optical properties of the ABTS. The dependence of the delay time on the amount of Trolox used as the reference antioxidant gives a linear function, as shown in the calibration curve in FIG. 7. The exact starting point of the reaction can be determined precisely by adding a defined amount of Trolox to the reaction solution by considering the time obtained for the blank value, which only contains the amount of Trolox of the reaction solution, as the starting point of the reaction (see also Example 2).

With the help of the calibration curve created, the antioxidant capacity of investigating samples with the antioxidant capacity of Trolox as Default value can be compared.

In FIG. 6, the absorption curve with time varying amounts of the example of a) urea and b) catechin is shown. The position of the individual wells in the microtiter plate is shown in the overview at the top right in FIG. 6.

Embodiment 2 Determination of the antioxidant capacity with chemiluminescence

The radical-generating system is analogous to the first application example 2,2'-azo-bis-2-amidinopropane (ABAP). As an indicator that is radical dependent changes optical properties, Luminol is used.

The samples to be analyzed are placed in the wells of a cooled Pipetted microtiter plate. In one or more wells is water or Sodium chloride solution (0.9%). These wells that are not samples contain, serve for the exact determination of the starting point of the reaction (Blank value).  

The reaction solution consists of 12.5 ml of 0.1 M glycine buffer (pH 8.6) 15.6 µl 10 mM luminol and 34 mg ABAP and is brought to a temperature in the Chilled range from 40 to 150 C.

Finally, 10 to 20 µl of 300 to 500 µM are added to this solution Given 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox).

100 to 200 µl of the above reaction solution, which has a temperature between 4 ° C and 15 ° C are pipetted into the samples in the wells.

Due to the cooling of samples and reaction solution is none significant consumption of antioxidants.

The plate is placed in a microtiter plate reader known per se and heated to 37 ° C. In a period of 25 minutes is set in The chemiluminescence of the indicator luminol is measured and time intervals expressed as arbitrary luminescent units per second (RLU / sec). The reaction starts during the heating phase. In this time it will additionally added Trolox consumed. The first signal appears in the Wells with blank samples.

Fig. 8 shows the temporally different start of chemiluminescence in the entirety of the analysis vessels with different samples described in the legend. In the wells B1 to B6 different amounts of 376 µM Trolox are carried as reference antioxidant, to which the other samples are related in their antioxidant capacity.

The measurement results of the different samples differ both at the start of chemiluminescence as well as in intensity of the recorded signal.

FIG. 9 shows the effect of adding additional Trolox to the reaction mixture for determining the exact starting point of the reaction. The upper curve H3 was obtained without adding Trolox to the reaction solution. It is characterized by a comparatively flat increase in chemiluminescence from time "0", which is caused by the temperature profile in the heating phase.

In contrast, curves G4 and H4 included Trolox in the Reaction mixture. Until this additional Trolox is "used up" As expected, no chemiluminescence was observed, and the curve moves to the "zero value".

At the time when the additional Trolox is completely used up, the Reaction temperature reached, and thus a steep increase in Chemiluminescence enables.

The additional Trolox in the reaction mixture is more practical Meaning and facilitates the evaluation of the measurement results. It catches them Radicals go away at the start of the reaction, if not a constant one Radical formation, due to the temperature profile. If the Trolox in the reaction mixture is also the reaction temperature reached and you get a sharp and precisely evaluable starting point the reaction.

Embodiment 3 Determination of the antioxidant capacity of a cell system (erythrocytes)

The colorimetric determination of the antioxidant capacity of a cell system is also carried out with 2,2'-azo-bis-2-amidinopropane (ABAP) as radical-forming system. The erythrocyte suspension itself is called a indicator used to change its optical properties. The measured Absorption wavelengths are 620 and 550 nm.

One subject was given 5 ml of blood with a syringe called heparin Contains anticoagulant, removed and centrifuged at 4 ° C for 10 minutes (2,000 Xg). Then the plasma is carefully drawn off with a pipette.  

After removing the interlayer from leukocytes and others Cells, the remaining erythrocytes are 3 times with 15 ml 0.9% Washed NaCl solution and centrifuged to remove any remaining plasma to remove completely.

40 µl of the erythrocytes are made up of 10 ml of a solution 1 ml 0.1 M potassium phosphate buffer (pH 7.4) and 9 ml 0.9% NaCl solution given. The temperature of the solution is between 4 ° C and 10 ° C. This 0.4% erythrocyte solution is called "Suspension 1" in the following. called.

The hemolysate is washed by adding 40 µl of the erythrocytes to 10 ml of distilled water at a temperature between 4 ° C and 10 ° C obtained and hereinafter referred to as "Suspension 2".

6-Hydroxy-2,5,7,8-tetramethylchroman-2-car is used as the standard antioxidant bonic acid (Trolox) in a concentration of 6.4 mg in 25 ml 0.9% NaCl solution used.

The samples to be examined are pipetted into a microtiter plate. The Reaction solution ("ABAP") consisting of 136 mg ABAP in 3 ml of chilled 0.9% NaCl solution, must be freshly prepared immediately before the experiment and finally pipetted into the wells.

Differences in total volumes are due to 0.9% NaCl solution balanced.

FIG. 10A shows the resistance of erythrocytes to ABAP as a time-dependent course of the absorption at 620 nm.

In the top two curves (A9 and A10) is the time Absorption curve of the erythrocyte suspension with and without the addition of ABAP shown. The curve A10 shows the stability of the pure Erythrocyte suspension without the addition of ABAP over the entire Period of measurement. Curve A9 represents the reaction of ABAP with the Erythrocyte suspension. By the radicals released by ABAP  the membrane is destroyed, hemolysis sets in, and there is a Approaching curve A11, which shows the effect of ABAP on the Hemolysate (suspension 2) shows. The radicals formed cause them Formation of methaemoglobin. In contrast, curve A12 illustrates the Stability of the hemolysate over the entire period of the measurement.

Fig. 9b and 9c show the protective effect of antioxidants on the example of Trolox in the form of a later onset of hemolysis or methemoglobin formation.

As shown in Fig. 11, there is a linear relationship between the amount of Trolox used and the time that elapses before the onset of hemolysis.

Embodiment 4 Xanthine-xanthine oxidase-luminol chemiluminescence method for Determination of superoxide radical scavengers

Hypoxanthine and the enzyme xanthine oxidase form peroxide radicals. As Luminol is also used as an indicator.

If you add the enzyme superoxide dismutase (SOD) to the reaction solution, the radical formation depends on the amount added reduced and the intensity of chemiluminescence decreases.

The individual wells of a cooled microtiter plate are spatially separate the different samples and each Pipette 2 µl xanthine oxidase (20 units / ml in 20 ml 0.9% NaCl). Thereon 100 µl of reaction solution are made up in each analytical vessel from 9 ml 0.1 M glycine buffer pH 7.4 with 1 ml 10 mM hypoxanthine and 15 ul 10 mM luminol, given.  

The microtiter plate is placed in a microtiter plate reader known per se brought, shaken and heated to 37 ° C. In a period of 15 minutes will be given at predetermined intervals Chemiluminescence measured.

The measured course of chemiluminescence as a function of time is plotted in FIG . As can be seen from the legend, different amounts of SOD with different activities were examined as standard. A significant decrease in the intensity of chemiluminescence can be seen in samples with higher activity compared to those with lower activity.

If one plots against the SOD activity based on the Stern-Volmer equation I ₀ / I _i , where I _{0 is} the integral of chemiluminescence in the absence and I _{i is} the integral of chemiluminescence in the presence of SOD, a linear is obtained Dependence. ( Fig. 13). This can be used to determine the antioxidant capacity of samples to be examined.

Embodiment 5 Reaction of H ₂ O ₂ with myoglobin

H ₂ O ₂ / myoglobin is used as the radical-forming system and luminol is used as an indicator that changes its optical properties (chemiluminescence) depending on the radical.

The reaction mixture consists of 12.5 ml 0.1 M glycine buffer pH 8.6 with 75 µl H ₂ O ₂ (6 µl 30% H ₂ O ₂ to 10 ml distilled water), 250 µl myoglobin (1 mg in 1 ml 0.9% NaCl) and 15.6 µl 10 mM luminol.

1 to 5 µl of the are added to the wells of the cooled microtiter plate examining samples or the standard pipetted. For this, 200 µl given the above reaction solution and in one  known microtiter plate readers at 37 ° C chemiluminescence measured.

Fig. 14 shows the time profile of the chemiluminescence in individual wells of the microtiter plate. The samples are named in detail in the legend.

The antioxidant capacity of samples is determined using the measured delay time with a known standard, such as. B. Trolox.

Embodiment 6 Fluorimetric determination of the antioxidant capacity

The radical-forming substance is 2,2'-azo-bis-2-amidinopropane (ABAP); as Indicator that, depending on the radical, its optical properties (fluorescence) changes, a dye consisting of poly (benzyldimethylvinyl-ben cylammonium chloride) and sulforhodamine 101 (Ruby).

The reaction solution consists of 170 mg ABAP and 100 µl Ruby as Fluorescence indicator in 20 ml buffer solution (1 part 50 mM phosphate buffer pH 7.4 and 9 parts 0.9% NaCl) and must be used immediately before use freshly prepared (temperature: 4-15 ° C).

The samples to be analyzed are placed in the wells of a microtiter plate pipetted. Water or a NaCl solution is used to determine the blank value used.

200 µl each are placed in the individual wells with the samples to be analyzed pipetted the reaction solution. The plate is then immediately in one in itself known microtiter plate reader heated to 37 ° C, and at 570 nm with an excitation wavelength of 550 nm at certain time intervals Fluorescence measured.  

A solution of Ruby in water as a solvent fluoresces at one Wavelength of approx. 604 nm (excitation wavelength: 550 nm). In the the wavelength of 570 nm considered later is only a small fluorescence to observe.

As shown in FIG. 15, after addition of ABAP, the fluorescence wavelength shifts time-dependent in the direction of smaller wavelengths with a simultaneous increase in intensity, which suggests that the indicator is influenced by ABAP.

The start of the time-dependent shift in fluorescence can be achieved by adding antioxidants, such as. B. Trolox (388 µM). The influence of different amounts of Trolox is shown in Fig. 16. The fluorescence was measured at 570 nm (excitation wavelength: 550 nm). The dependence of the time shift on the amount of Trolox results in a linear relationship (not shown) and is also suitable for determining the antioxidant capacity of samples to be examined via the delay time.

Reference list

1

Analysis tubes (wells)

2nd

Microtiter plate

3rd

Basic body

4th

Indentations

5

Heating coils
A _P

Antioxidant capacity of the sample
A ₀

Antioxidant capacity of the reaction mixture without sample
A _S

Antioxidant capacity of a standard
τ delay time
τ _P

Sample delay time
τ _S

Standard delay time
τ ₀

Delay time of the reaction mixture without sample
C _S

Concentration of the standard
SF stoichiometric factor of radical formation
I ₀

Integral of the optical signal for the reaction mixture without sample addition
I _P

Integral of the optical signal for the sample
I _S

Integral of the optical signal for standard

Claims (19)

1. A method for determining the antioxidant capacity in samples, characterized in that mixtures are produced with the samples in a plurality of cooled analysis vessels arranged in a predetermined xy grid, which in each case continue

• a) from at least one system which forms free radicals as a function of temperature, and
• b) at least one indicator which changes its optical properties depending on the radical,

consist that the mixtures in the cooled analysis vessels are first brought to an essentially identical analysis starting temperature between 0 and about 20 ° C. and then, preferably in a period of up to 60 minutes, evenly to a temperature of up to about 90 ° C are heated so that during the heating of the mixtures in the analysis vessels with a reader arrangement known per se in the mixtures the time course of the change in the optical properties is detected, and that the antioxidant capacity is determined from this change by comparison with known reference values. 2. The method according to claim 1, characterized in that at least in one of the analysis vessels has a substance with a defined Antioxidant capacity in a reference mixture of the same Temperature increase is exposed like that in its antioxidant capacity mixtures to be determined and that the antioxidant capacity of the determining mixtures by changing their optical properties compared to the change in optical properties in the Reference mixture is determined.   3. The method according to claim 1, characterized in that as System dependent on temperature forming free radicals 2,2'-azo-bis-2-ami dinopropane (ABAP) is used. 4. The method according to claim 1, characterized in that the system of mixtures forming free radicals depending on the temperature Xanthine oxidase with hypoxanthine is formed as a substrate. 5. The method according to claim 1, characterized in that H ₂ O _{2 is} used as the temperature-dependent free radical-forming system. 6. The method according to claim 1, characterized in that as an indicator, which changes its optical properties depending on the radical, luminol is used. 7. The method according to claim 1, characterized in that as an indicator, which changes its optical properties depending on the radical, 2,2'-azino-bis (3-ethyl benzthiazoline-6-sulfonic acid) (ABTS) is used. 8. The method according to claim 1, characterized in that as an indicator, which changes its optical properties depending on the radical, one Erythrocyte suspension is used. 9. The method according to claims 1, 3 and 6 and 7 characterized thereby records that in all reaction mixtures of an analytical approach defined small compared to the sample and the standard Antioxidant concentration is set.   10. The method according to claim 1, characterized in that as an indicator, which changes its optical properties depending on the radical, (benzyldimethylvinylbenzylarnmonium chloride) and sulforhodamine 101 (Ruby) is used. 11. The method according to claim 1, characterized in that the Temperature of the mixtures is increased suddenly. 12. The method according to claim 1, characterized in that the Temperature of the mixtures is increased continuously. 13. The method according to claim 1, characterized in that the Temperature of the mixtures with concave temperature-time function increased becomes. 14. The method according to claim 1, characterized in that the Temperature of the mixtures with convex temperature-time function increased becomes. 15. A device for performing the method according to claim 1, characterized in that the xy grid of a known microtiter plate ( 2 ) arranged analytical vessels ( 1 ) consist of compliant material which is non-positively and positively in a metallic, the analytical vessels ( 1 ) the position-corresponding depressions ( 4 ) and the temperature-adjustable base body ( 3 ) are used. 16. The apparatus according to claim 14, characterized in that the base body ( 3 ) is in thermal contact with means ( 5 ) for tempering them. 17. The apparatus according to claim 15, characterized in that the means for tempering the base body ( 3 ) from electrical heating elements, for example one or more in or on the base body ( 3 ) attached heating spirals ( 5 ). 18. The apparatus according to claim 15, characterized in that the means for tempering the base body ( 3 ) from fluid media, for. B. water, which flows through the base body ( 3 ) exist. 19. The apparatus according to claim 14, characterized in that for colorimetric measurements on the mixtures, the analysis vessels ( 1 ) consist of translucent material and the depressions ( 4 ) in the base body ( 3 ) are designed as continuous bores.