SU1532869A1 - Method of determining antioxidant activity of chemical compounds - Google Patents

Abstract

The invention relates to experimental and clinical medicine, in particular to medical biophysics, clinical pharmacology, biochemistry, and pathophysiology, and can be used to determine the pro- and antioxidant activity of chemical compounds. In order to increase accuracy while simultaneously expanding the functionality of the method by simultaneously determining the prooxidant activity, levels of spontaneous and latex-induced 1.7 µm chemiluminescence of neutrophils in the presence and absence of the test compounds are determined (and similar), while neutrophil chemiluminescence levels decrease in the presence of chemical compounds relative to the original determine the antioxidant activity of the studied chemical compounds, and with increasing levels of milyuminestsentsii neutrophils in the presence of the test compounds relative to the original - prooxidant activity. Table 1.

Description

The invention relates to experimental and clinical medicine, in particular to medical biophysics, clinical pharmacology, biochemistry, pathophysiology, oncology and immunology, and can be used for medical and biological research to determine the pro- and antioxidant activity of various chemical compounds.

The purpose of the invention is to improve the accuracy and selectivity of the method while expanding its functionality.

The method is carried out as follows.

Siliconized glass vials with buffer consisting of physiological concentrations of NaCl, KC1, KHjjPO.,., NazHP04, HEPES, L-glutamine and glucose and used to isolate cells, add 1 ml of solution a containing 50 µg of luminol, 0 , 1 ml of neutrophils (/ ml), investigated chemical compounds. Chemiluminescence (XM) registration is carried out immediately after the addition of drugs for 1-4 minutes on an LS-1800 liquid scintillation counter (Westap). For induction of ChL, 0.01 ml of latex (particle diameter 1.7 µm) is added to the incubation medium and again

register CL neutrophils. At the same time, a substance that reduces spontaneous CL is referred to as antioxidants, and that increases it is referred to prooxidants. In the case of a decrease in the CHL of activated neutrophils with unchanged or increased CL in intact neutrophils, it is established that the antioxidant activity is associated with q suppression of phagocytosis.

Example 1. Hepgarinized venous blood was defended for 30 min at 37 ° C, the plasma was layered on a double gradient of ficollvelographin (q - 1.077, q 1.093). After 30 min of centrifugation at 1500 rev / min at the border of two gradients, a ring of neutrophils was formed, which were removed, washed twice in a buffer containing physiological concentrations of NaCl, KC1, KKgPO, HEPES, L-glutamine and glucose (pH 7, four); centrifuged with an acceleration of 1500 g for 10 min. The cells were then resuspended, brought to a concentration of 106 / ml and used to determine chl. The viability of neutrophils was assessed by trypan exclusion (blue and in all experiments it was 99-100%.

Known compounds were used as prooxidants: μM HgOg (0.01 ml of 3% solution), 1 mM NADPH-Ng Glutathione (I mm) and medium molecular peptides (SMP) of blood were used as antioxidants in an amount of 20-40 μg / ml. In addition, chemical compounds with previously unknown oxidative activity were investigated: acridine orange (0.002 mg / ml), solcoseryl (200 µg / ml) and alcoholic plasma extract (200 µg / ml). To identify the optimal regimes of induced CL, various activators were used — polystyrene monodisperse latexes 00.5; 1.0; 1.7; 2.11; 2.65 μm, latex 0 1.7 μm with sorbed IgG, rheumatoid diagnostics, (latex with sorbed u-globulin and opsonized zymosan (Sigma).

CL was measured in counts / min. The intensity of the flash was estimated as the ratio of intact neutrophils to conventional lymphocytes in conventional units, the total amount of the flash was estimated throughout the study period, the average value of the intensity of the flash was evaluated in conventional units.

5 0 5 0

five

50

The results indicate that polystyrene latex (ph, 1 µm) is the most optimal stimulator of neutrophilic HL, since the maximum burst of CL is noted in 1-2 minutes of recording and the amplitude of the flash is greater than in other activators.

l

Fe ions substantially stimulate spontaneous and induced CL by 5.7 and 1.6 times, respectively: NADPH 1.35 and 1.6 times; NgOe stimulates spontaneous CL in 13.6 times, slightly reducing induced CL; Glutathione reduces spontaneous and induced CL by 3 times; acridine orange 4 and 6 times, respectively; Spm blood 6 and 7 times; solcoseryl 1.5 and 3 times; alcoholic blood extract in Zi80 times.

Example 2. The table shows data on the effect of various chemical compounds on spontaneous and latex-induced CL neutrophils isolated from another blood sample,

The light sum of the luminescence was estimated as the average result of the CL intensity over the observation period (cpm).

The pro- or antioxidant activity of chemical compounds was estimated as a percentage as the ratio of the light sum of neutrophil luminescence in the presence of the studied chemical compounds to the light sum of luminescence of intact or latex-activated neutrophils.

The proposed technical solution expands the functionality of the method by determining not only antioxidant, but also prooxidant activity (the effect of Fe14 ions, Н20г, СМ blood from batted animals). Expanding the functionality of the method and increasing its selectivity are also demonstrated by the possibility of a differentiated approach to studying the effect of chemical compounds on spontaneous CL in intact neutrophils and CL induced by latex, i.e. conjugated with phagocytosis.

The ability to detect chemical compounds whose antioxidant activity is associated with the suppression of phagocytosis (by examining the levels of spontaneous and latex-induced human neutrophil CL, in the case of decreased CL of activated neutrophils with unchanged or increased CL of intact neutrophils) is a significant advantage of this method. This makes it possible to select drugs with anti-inflammatory effects, in particular, drugs that affect phagocytosis.

Claims (2)

1. A method for determining the antioxidant activity of chemical compounds by introducing the chemical compound under investigation into a biological object, induction of free radical oxidation and evaluating antioxidant activity by the difference in the intensity of chemiluminescence in the presence of and Absence of test compound, characterized in that, in order to increase accuracy while simultaneously expanding the functionality of the method due to simultaneous determination of prooxidant activity, human neutrophils are used as a biological object, the chemiluminescence of neutrophils is measured and, with a decrease in chemiluminescence in the presence of the Test compound, the antioxidant is measured activity, with an increase - prooxidant activity. 2. A method according to claim 1, characterized in that, in order to increase the selectivity of the method, human neutrophils activated with latex are used, and in the case of decreasing chemiluminescence of activated neutrophils against the background of unchanged or increased chemiluminescence of intact nitrophils, it is established that the antioxidant activity of chemical compounds are associated with the suppression of phagocytosis. Editor O.Spesyvyh Compiled by I.Gul Eva Tehred L. Oliynyk Proofreader E. Lonchakova Order 8094 / S1 Circulation 789 VNIIPI State Committee for Inventions and Discoveries at the State Committee on Science and Technology of the USSR 113035, Moscow, Zh-35, Raushsk nab. 4/5 Subscription