[The comparison of two chemiluminescent models for assessing anti-oxidative activity of blood serum of patients with liver pathology].

The comparative assessment was carried out concerning anti-oxidation activity of blood serum of patients with liver pathology using two chemiluminescent techniques with different models of free radical oxidation: «Hb-Н2О2-luminol¼ и «ABAP-luminol¼. The reliable but low correlation of results was established (r=0,798) related mainly to difference in mechanisms of initiation of free radicals and effect of blood serum on initiation process. This effect is stronger manifested in model «Hb-Н2О2-luminol¼. The discrepancy of results of measurement is more expressed in patients with anomalous higher content of bilirubin in blood. Thereupon, oxidation model «ABAP-luminol¼ is to be considered as a more preferable for clinical practice.

[The antioxidant activity of components of blood serum of donors and recipients before liver transplantation].

The antioxidant activity is implemented in human blood serum by ascorbic acid, uric acid, amino acids, glucose, mono unsaturated fatty acids (in the first instance Ω-9 oleic acid), essential polyenoic fatty acids, thiol groups of albumins and proteins, tripeptide and pigment of bilirubin. The antioxidant activity of blood serum of donors and recipients before liver transplantation was determined The input of particular biochemical analytes into liver transplantation was determined too. The antioxidant parameters were detected using technique of termo-induced chemiluminescence under application of set of corresponding reagents. The analysis of antioxidant activity of blood serum in donors and recipients with hepatic pathology revealed in vivo a significant disorder in the syndrome of compensatory anti-inflammatory defense. Under hepatic pathology, absence of endogenous ascorbic acid, deficiency of exogenous ascorbic acid and disorder of antioxidant activity the uric acid and bilirubin become the major hydrophilic acceptors of active forms of oxygen and inhibitors of oxidative processes in vivo. In patients with physiological level of bilirubin the uric acid provides 40%-80% of antioxidant activity. In case of high hyperbilirubinemia in recipients only 9.6%. It is possible to consider hyperiricosuria under aphysiological processes as a nonspecific test of activation of biological reaction of inflammation, syndrome of compensatory anti-inflammatory defense and test of disorder of biological function of endoecology. To activate the syndrome of compensatory anti-inflammatory defense it is very important to decrease both hyperiricosuria and compensatory function of uric acid as an acceptor of active forms of oxygen by force of prolonged intake of optimal amount of ascorbic acid.

[Status of the water-soluble component of the antioxidant defense system in the conditions of 520-day isolation].

In the 520-d chamber experiment within the international project Mars-500 blood samples of 6 male test-subjects of 28 to 39 years of age were analyzed for water-soluble antioxidants: total bilirubin and uric acid; in addition, total antioxidant capacity of blood plasma was determined. Maximal values of these parameters were associated with the most stressful periods of the experiment, i.e. adaptation to the life in isolation and confinement, simulation of the egress onto Martian surface, and change of the diet. On attainment of the homeostatic equilibrium the parameters stabilized on levels slightly lower relative to baseline (pre-isolation) values. Therefore, dynamics of the water-soluble antioxidants reflected adequately the homeostatic reactions to and compensation by organism of the effects of the 520-day life in isolation and confinement.

[Antioxidative system of the organism and thermo-initiated chemiluminescence method for quantitative evaluation of its state].

Evolutionary stages of the development of an antioxidative system within the human organism are shortly considered and the technique for measurement of blood plasma parameters, which reflect the state of antioxidative homeostasis, based on thermo-initiated chemiluminescence of azo compounds in the presence of luminal, is described. Parameters, suitable for quantitative estimation of the degree of oxidative stress and the efficiency control of causal and adjuvant antioxidative therapy in the course of primary and secondary prevention of civilization illnesses are recommended. A special attention is focused on sports activities as the most accessible and effective way of achieving the specified preventive purposes.

[Photochemiluminescent study of the antioxidant activity in biological systems. Mathematical modeling]. / Fotokhemiliuminestsentsiia kak metod izucheniia antioksidantnoi aktivnosti v biologicheskikh sistemakh. Matematicheskoe modelirovanie.

The mathematical modeling of the kinetics of riboflavin photo-chemiluminescence (PCL) in the presence of antioxidants, superoxide dismutase and ascorbic acid, has been performed. A specially developed computer program "Kinetic Analyzer" was used for the modeling. The PCL intensity of was taken as directly proportional to the superoxide concentration, because lucigenin had been added to the system. It was found, that the experimental curves of PCL virtually coincide with those calculated in the case of the following set of reactions (reaction rate constants, M-1.s-1 are given in brackets): hv + RH-->R. + .O2- (2,3 x 10(-4) s-1); RH + .O2(-)-->R. + H2O2 (1000); RH-->...(0,005 s-1); .O2- + .O2(-)-->... + phi OTOH (2 x 10(5) M-1 s-1); SOD + .O2(-)-->SOD H2O2 (1 x 10(8)); ASC + .O2(-)-->...(2 x 10(7)). Here RH is, .O2(-)--superoxide, R.--riboflavin radical, SOD--superoxide dismutase, ASC--ascorbate.

[A simple method of measuring superoxide dismutase activity using photochemiluminescence]. / Prostoi metod izmereniia aktivnosti superoksiddismutazy s pomoshch'iu fotosensibilizirovannoi khemiliuminestsentsii.

At present, photochemical detection of SOD-activity is used in most cases, where superoxide production is based on the NADPH oxidase activity or autooxidation of some substances, for instance, adrenaline. The shortcomings of these assays are the requirement of stable pH and temperature and need long-term experiments and sophisticated preliminary work. For this reason a novel method to determine SOD activity have been proposed, based on the photochemiluminescence (PCL), where riboflavine is used as a photosensitizer and lucigenin as a free-radical detector. Minimal concentrations of the system components have been selected, at which PCL intensity became virtually independent on the concentration and was sufficient for proper measurements: 25 nmole/ml of riboflavine, 3 nmole/ml of lucigenin and 250 nmole/ml of methionine. Under this conditions, the amount of SOD causing two-fold inhibition of PCL was 10 +/- 0.3 ng of SOD (Sigma, USA). Antioxidants occurring in the blood changed the PCL intensity by 5% at following concentrations: ascorbic acid--above 3.6 microM, uric acid--above 0.3 microM, glutathione--above 4.0 microM. Taking into account the amount of these antioxidants in whole blood and erythrocytes, it has been calculated that the antioxidants containing in the sample do not influence the results of SOD assay, even without erythrocytes washing. The proposed method was compared with a procedure offered by Calbiochem. The amounts of SOD causing two-fold decrease of PCL in our method and that of Calbiochem, were 10 and 40 ng (Sigma, USA), and standard deviations 0.45 and 0.96%, respectively. The SOD activity from 107 healthy donors (76 males and 31 females, 40 smokers and 67 non-smokers) has been determined and was found to be 15.1 +/- 0.4 ng SOD/1 x 10(9) erythrocytes.

The antioxidant properties of lycopene.

The antioxidant properties of the carotenoid lycopene were compared in three different model oxidative systems. In egg yolk liposomes, in the presence of 2.5 mM FeSO4 and 200 mM ascorbate, lycopene, alpha-tocopherol, and beta-carotene inhibited the accumulation of lipid peroxidation products reacting with 2-thiobarbituric acid (TBARS) in a dose-dependent mode, with the concentration of half-inhibition being 80, 30 and 130 mM, respectively. In the liposomes subjected to illumination with a He-Ne laser (632.8 nm) at a dose of 10.5 J/cm2, in the presence of 32.5 micrograms/ml hematoporphyrin derivatives (Fotogem, NIOPIC, Russia) TBARS accumulated, and this effect was inhibited by lycopene, alpha-tocopherol, and dihydroquercetin with approximately equal efficiencies (the half-inhibition concentrations were 10(-5) mM). In both systems studied, sodium azide at a concentration of 10 mM inhibited the TBARS accumulation by no more than 20%. Apparently, the inhibitory action of not only alpha-tocopherol, but also beta-carotene and lycopene was the result of their antiradical action, rather than quenching of the singlet oxygen in an aqueous medium. The introduction of lycopene, as well as beta-carotene in liposomes subjected to Fe(2+)-induced lipid peroxidation decreased the chemiluminescence (CL) intensity at the stage of CL slow flash, with no essential influence on the lag period. These data suggest that the effect of lycopene on lipid peroxidation was the result of its interaction with free radicals rather than chelating ferrous ions. The antiradical activity of lycopene was also confirmed by the method of luminol photochemiluminescence (PCL). Lycopene increased the PCL lag period (L) and decreased the PCL amplitude (A), which implies its antiradical and SOD-like activity in this system.

Evidence for a direct interaction of superoxide anion radical with carnosine.

Using photochemiluminescence, the interaction between carnosine and superoxide anion was measured directly. Carnosine at physiological concentrations decreased the amplitude of luminol chemiluminescence like superoxide dismutase (SOD) did, and prolonged the lag-period of the chemiluminescence similar to the effect of ascorbic acid. From the interaction of nitro blue tetrazolium with superoxide anion generated by the xanthine oxidase system, the constant for interaction of carnosine with 02-. was calculated to be 10(5) M-1.sec-1. The possible biological significance of the quenching of superoxide anion by carnosine is discussed.

[Antioxidant properties of UV-irradiated blood plasma as determined by the photochemiluminescence method]. / Antiokislitel'nye svoistva UF-obluchennoi plazmy krovi i ee komponentov, otsenivaemye metodom fotokhemiliuminestsentsii.

UV-irradiation of human serum albumin, tryptophan, and histidine resulted in products formation showing antiradical activity, as detected by increased latent period in development of luminol photochemiluminescence. UV-irradiation of ascorbic acid decreased its antiradical activity. Under UV-illumination, antiradical activity of blood plasma decreased rapidly followed by a gradual increase of antiradical activity. Apparently, the former effect (decrease of antiradical activity) is a result of photolysis of natural blood antioxidants, while subsequent increase of antiradical activity is a consequence of the accumulation of plasma protein photolysis products.

Photochemiluminescent detection of antiradical activity; IV: testing of lipid-soluble antioxidants.

A new method for quantification of antiradical properties of pure lipid-soluble antioxidants and for measurement of integral antioxidant capacity in the lipid phase (ACL) of polycomponent systems, such as blood plasma or tissue homogenates, is developed. It is based on an antioxidant-sensitive inhibition of a photo-induced, chemiluminescence accompanied autoxidation of luminol. The sensitivity of the photochemiluminescent (PCL) assay lies within nmol quantities of substances, the measuring range for alpha-tocopherol is between 0.1 and 3 nmol. The interassay variability of the method is lower than 5%, the intraassay variability <2%. The antioxidant efficiency of gamma-tocopherol was found to be 43% of alpha-tocopherol. The results of the PCL measurements on pure antioxidants and on lipid extracts from blood plasma were compared with the level of, 'vitamin E' (VE) determined as a sum of alpha- and gamma-tocopherol by HPLC. Very good coincidence of both methods was observed for pure substances (r = 0.998, P<0.001). The ACL of human blood plasma was found to be 27.98 +/- 0.68 mumol equivalents of alpha-tocopherol/l (mean +/- mean error, n = 142), it is approximately 25% more than the concentration of VE found in the same samples (22.09 +/- 0.59 mumol/l). In this case, the correlation of both parameters was lower. r = 0.811, P<0.001. The animal experiments showed that synthetic antioxidants may not only increase the value of ACL of blood plasma but in the same time reduce the concentration of biological antioxidants, e.g. VE drastically. The prooxidant activity of synthetic antioxidants in vivo or the replacing of structured alpha-tocopherol from its position can be the cause. This important circumstance has to be considered during the testing of new antioxidants for clinical application.

Photochemiluminescent detection of antiradical activity: II. Testing of nonenzymic water-soluble antioxidants.

A method for testing and quantification of nonenzymic antioxidants, such as ascorbic acid and uric acid, and of polycomponent systems, e.g., blood plasma, is described. This method is based on a photo-induced, chemiluminescence-accompanied, and antioxidant-inhibitable autoxidation of luminol. The sensitivity of the assay lies in a region of nanomolar quantities of substances. The interassay variability of the method is lower than 5%, the intraassay variability is 2%. The mean values of an integral antioxidant capacity (AC) of human blood plasma, measured with this method as a duration of the chemiluminescence inhibition, were between 10 and 20 arbitrary Units (10 arb.U. = 1 min) per 1 microliter, and showed the age-dependent patterns with maximal values (+/- SD) with newborns (n = 7): 20.8 +/- 4.1 arb.U. and aged persons (46 +/- 9.4 years old, n = 16): 15.4 +/- 5.2 arb.U., and minimal with children (7.3 +/- 3 years old, n = 10): 9.9 +/- 2.6 arb.U. AC of young people (28.7 +/- 6.5 years old, n = 22) was 12.1 +/- 2.7 arb.U. AC of six tested animal species was lower as that of humans, with maximal values with guinea pigs: 8.5 arb.U. (mean value) and spontaneously hypertensive rats: 8.6 arb.U. The lowest values were registered with minipigs: 1.4 arb.U. Mice (strain 17) showed 3.8, Lewis and Wistar rats accordingly 4.6 and 6.2 arb.U.

Photochemiluminescent detection of antiradical activity. I. Assay of superoxide dismutase.

The luminol sensitized photogeneration of superoxide radicals was combined with their luminol-dependent chemiluminescent detection. The inhibition of chemiluminescence by superoxide dismutase (SOD) versus enzyme concentration in reciprocals was strongly linear. The amount of SOD notable for 50% inhibition was between 115 and 500 ng for different enzyme preparations.

[Apolipoprotein B of plasma lipoproteins incorporated in liposomes: immunological properties and organ distribution when administered to rabbits]. / Apolipoprotein B plazmennykh lipoproteidov, vstroennyi v liposomu: immunologicheskie svoistva i raspredelenie mezhdu organami pro vvedenii kroliku.

Apolipoprotein B (apo B) isolated from low density lipoproteins (LDL) was built in phospholipid-cholesterol liposomes, with the lipid/protein ratio being equal to 33:1. Such liposomes preserved their integrity, whereas the constituent apo B retained its antigenic properties. After intravenous injection to rabbits the pattern of apo B liposome distribution among organs was similar to that of LDL. Apo B liposomes may be used for goal-oriented transport of some substances to organs and tissues whose cells have specific receptors for apo B-containing lipoproteins.