[Preparation of samples for gas-chromatographic determination of fatty acids: direct transesterification of lipids of dry biological sample is preferred in comparison with the methods employing preliminary lipid extraction.]

Different methods of sample preparation and derivatization were compared from the point of view of product yield, speed and convenience of the technique used. Fatty acid determination in absolutely dry objects (biochemical preparations, food protein isolates, lyophilized microbial biomass) may be performed easily with the use of Folch method provided that 4-component system "chloroform/ methanol/water/acetic acid" was employed. Nevertheless, we ould not find any real advantages of classical Folch or Hara-Radin extraction method variants when compared to simple non-extraction technique (which consists in direct trans-esterification of dried biomaterial due to sequential sample treating with sodium methoxide and boron trifluoride methanolic solutions). The letter method, being completely universal, provides considerable increase of fatty esters yield, sample preparation is noticeably simplified and accelerated (becoming much more economical). It's "dry blood spot" variant (using cellulose or, preferably, fluoroplast filter paper disks) seems to be extremely convenient for laboratory routine analysis of liquid biological samples, allowing to exclude not only their liquid-liquid extraction but also the stage of vacuum drying. Unlike the methods of Folch and Hara-Radin, the non-extraction method does not necessarily require the homogenization of the biological material, that is, it's grinding to fragments of micron size. Direct derivatization method provides noticeably better parameters of fatty acids yield even for relatively large particles - 0.2-1.0 mm - of the test material (in comparison with those parameters observed upon extraction of micron size homogenizates by the Folch method in its most advanced modifications).

[The perspective fluoroplastic porous carrier for conservation and transfer of samples of blood plasma: application of "dry drop" technique with the purpose of detection of individual fatty acids.]

It is proposed for preserving samples of biological fluids (blood plasma first of all) and mailing them to analytical laboratory to significantly modify a well-known technique of "dried blood spot" (DBS) at the expense of application of home-made fluoroplastic filtering material type MFFK-G as a porous carrier of fluid biological sample. The synthetic material contains no chemically active molecular fragments and it has four times greater water capacity than filter and chromatographic paper. Thus, fluoroplastic porous disc of mass of 15 mg is suitable to carrying of 70-100 mkl (instead of 15-20 mkl) of fluid. The preliminary impregnation of such a disc with solution of antioxidant - 0.5% - 0.9% 2,6-di-tret-butyl-4-methyl phenol in chloroform - permits to strongly increase stability of higher poly-saturated fatty acids in applied of dry plasma sample or erythrocytic mass on the disc. In that case, time of storage of ready-made disc with dry sample under room temperature increases up to 35-40 days and time of storage in freezer - from 50-60 days to half a year and longer. The storage of dry samples in airtight bottles of vials air-blown with technical propanebutane, also increases time of storage of samples approximately on a degree. This method permits to qualitatively determine content of individual fatty acids in the fraction of etherized and non-etherized fatty acids and also total and free cholesterol in samples of biological fluids.

Effect of Audiogenic Seizures on the Dynamics of Fatty Acid Composition of Hippocampal Dental Gyrus in Krushinsky-Molodkina Rats.

Changes in the fatty acid composition of the hippocampal dentate gyrus in Krushinsky-Molodkina rats with hereditary predisposition to audiogenic seizures were studied in 1, 3, and 14 days after 1 or 5 seizures. Seizure activity changed the content of saturated and monounsaturated as well as polyunsaturated fatty acids at different terms after seizures. After seizures, the content of individual fatty acids changed in different directions. Similar shifts after 1 and 5 seizures were observed only for eicosapentaenoic acid at all observation terms. More pronounced changes in fatty acid composition were observed after 5 seizures. These results can be useful for the development of new approaches to correction of seizure activity.

[THE CHANGES IN FATTY ACIDS COMPOSITION IN THE AUDITORY CORTEX OF RATS AFTER A SINGLE AUDIOGENIC SEIZURE].

We found small but statistically significant increase in the number of stable to peroxide oxidation saturated and monounsaturated fatty acids in the auditory cortex of KM rats in comparison with control Wistar ones. The levels of fatty acids in the cells of the auditory cortex of KM rats were studied at different times (1 h, 1 day, 3 days and 14 days) after a single audiogenic seizure. The changes in fatty acids composition in auditory cortex of KM rats were found already in time point 1 h after convulsion, the maximal decrease of fatty acids levels was observed at 3 days after convulsion. These data suggest that the fatty acids pool in this time was depleted. Finally, we found the recovery of the better part of fatty acids in the auditory cortex of KM rats to 14 day after convulsion. These results can be used for development of new approaches to eliminate brain damage after seizures.

Correction of Mitochondrial Enzyme Activities in the Skeletal Muscles of Old Rats in Response to Addition of Olive Oil to the Ration.

Activities of mitochondrial electron transport chain enzymes NADH-CoQ oxidoreductase (complex I), cytochrome C-oxidase (complex IV), and citrate synthase were measured by spectrophotometry in m. quadriceps femoris homogenate from old rats receiving olive oil with the ration. Reduced activities of complexes I and IV in old animals were restored to the level of young animals after 6-week consumption of olive oil. Activity of citrate synthase did not change with age. Positive effect of olive oil on fatty-acid composition of the muscle tissue in old animals was demonstrated. The content of summary monounsaturated fatty acids, reduced with aging, and of summary polyunsaturated ones, increasing with age, were restored in old rats to the levels virtually not differing from the levels of young animals.

Changes in fatty acid composition of thymus cells, liver, blood plasma, and muscle tissue in mice with solid Ehrlich carcinoma.

The fatty acid composition of thymus cells, liver, blood plasma, muscle tissue, and tumor focus has been studied in mice with solid Ehrlich carcinoma. The tumor growth in the mice was associated with an increase in the total content of monounsaturated fatty acids in all organs and tissues studied and with a decrease in the total amount of polyunsaturated fatty acids in all tissues except blood plasma. The tumor tissue was characterized by increased levels of monounsaturated fatty acids in comparison with their levels in organs and tissues of intact animals. In the thymus of tumor-bearing mice, the contents of myristic and palmitic saturated fatty acids, which are associated with activation of the T-cell immunity, were increased. The most expressed and considerable changes in the fatty acid composition during tumor growth were observed in the muscle tissue of the animals. A possible role of changes in the fatty acid composition in the investigated organs and tissues of tumor-bearing mice in the organism's response to tumor growth is discussed.

Dietary supplementation of old rats with hydrogenated peanut oil restores activities of mitochondrial respiratory complexes in skeletal muscles.

The effect of dietary supplementation of old rats (26-33 months) with hydrogenated peanut oil on the activity of mitochondrial enzymes in skeletal muscles has been studied. The activities of NADH-coenzyme Q1 oxidoreductase, cytochrome c oxidase, and citrate synthase were determined spectrophotometrically in muscle homogenates. The activities of respiratory complexes I and IV were shown to significantly decrease with the age compared to the activity of the same enzymes in young animals, while the activity of citrate synthase was virtually unchanged. The fatty acid composition of muscle homogenates of old rats differed from that of young animals by a reduced content of myristic, oleic, linoleic, and α-linolenic acids and enhanced content of dihomo-γ-linolenic, arachidonic, and docosahexaenoic acids. Per oral supplementation of the old rats with hydrogenated peanut oil completely restored the activity of complex IV and increased the activity of complex I to 80% of the value observed in muscles of young animals, reducing the content of stearic, dihomo-γ-linolenic, arachidonic, eicosapentaenoic, docosapentaenoic, and docosahexaenoic acids relative to that in the groups of old and young rats. The content of oleic and linoleic acids increased relatively to that in the group of the old rats, as well as young animals. The possible mechanisms of the restoration of the activity of the respiratory enzymes under the administration of hydrogenated peanut oil are discussed.

Influence of seizures on lipids of homogenate and neuronal and glial nuclei of rat neocortex.

Lipid composition of homogenate and neuronal and glial nuclei of the brain cortex of Wistar rats was studied under normal conditions and after seizures induced by injection of picrotoxin. Seizures increased contents of lysophosphatidylcholine, sphingomyelin, and total phospholipids in the homogenate. In neuronal nuclei contents of total phospholipids, sphingomyelin, phosphatidylcholine, and phosphatidylserine decreased, and contents of free fatty acids and lysophosphatidylcholine increased. In glial nuclei content of total phospholipids decreased and content of free fatty acids increased. The role of changes in the lipid composition of the neocortex cells during seizures and the involvement of lipid messengers in signal mechanisms are discussed.

[Influence of experimental epileptogenesis on memory: the role of lipids in cognitive disorders]. / Vliianie éksperimental'nogo épileptogeneza na protsessy pamiati: rol' lipidov v mekhanizmakh kognitivnykh narushenii.

Learning and memory disorders accompanying epileptogenesis were studied in rats with the use of two experimental models of epilepsy, picrotoxin kindling and kainic treatment. Rise of exploratory activity and decrease in animal's capability for experimental extinction of a response were characteristic of the initial stage of epileptogenesis. It was suggested that a dysfunction of brain hippocampal system can be responsible for cognitive disorders. To reveal their mechanisms, lipid contents were determined in the neocortex and hippocampus in appropriate periods after exposure to epileptogenic factors. Long-term changes in hippocampal lipid spectrum were found five days after the exposure to kainic acid. In particular, after sodium valproate treatment (the compensation of kainic effects), the total content of phospholipids in hippocampus was decreased. The hippocampal sphingomyelin level dropped as a result of picrotoxin kindling. The sphingomyelin changes suggest some recovery processes in hippocampal cells and point to an adaptive role of membrane lipids in the mechanisms of the damaging epiptogenous effects.

Synthesis of lipids in isolated nuclei from rat thymus and liver cells.

Synthesis of lipids was studied in isolated nuclei from rat thymus and liver cells. On incubation of the isolated nuclei with [2-14C]acetate and [1-14C]glycerol, the label was intensively incorporated into phospholipids and with a significantly lower intensity into fatty acids and cholesterol. Only trace amounts of radioactivity were detected in the lipids of chromatin prepared from isolated thymus nuclei after their incubation, and this suggested that lipids were mainly synthesized on the nuclear membrane. On the preincubation of thymus tissue homogenate with [2-14C]acetate and the subsequent isolation of the nuclei and chromatin, the radioactivity of chromatin lipids was comparable to the radioactivity of nuclear lipids. The findings suggested that in the isolated nuclei the newly synthesized lipids were not transported into chromatin from the nuclear membrane. The specific radioactivities of individual phospholipids and fatty acids were different in the isolated nuclei and in nuclei obtained from preincubated homogenate. Mechanisms of lipid synthesis in isolated nuclei and causes of the different radioactivities of lipids in the isolated nuclei and in the nuclei obtained from the preincubated homogenate are discussed.

[Nonmonotonous metabolic response of mammalian cells and tissues to ionizing radiation]. / Nemonotonnost' metabolicheskogo otveta kletok i tkanei mlekopitaiushchikh na vozdeistvie ioniziruiushchei radiatsii.

Changes in the activity of ornithindecarboxylase in various tissues and in the amount of catecholamine in rat hypothalamus by the action of acute and chronic ionizing radiation were studied. A nonmonotonous relationship between the metabolic parameters of animal tissues and cells and the radiation dose was revealed. It was assumed that the nonmonotonous character of the dose-response dependence results from the nonmonotonous time course of the metabolic response to irradiation. It was also assumed that living systems have the property of responding to stress agents by nonmonotonous changes in metabolism. In the case of acute irradiation, this response manifests itself as oscillations of metabolic parameters about the control. The oscillations occur with a particular amplitude and periods, which vary with radiation dose, and damp out with time. As a result, in a fixed time interval, the dose-response curve may be nonmonotonous. Reverse dose-response relationships are also possible. In the case of chronic irradiation, the metabolic and functional parameters oscillate throughout irradiation time, and a modification of the response occurs. A prolong exposure to ionizing radiation causes strong changes in the metabolism of lipids of cell membranes, organelles and chromatin, as well as in the functional properties of some mammalian cells and tissues. The necessity of constructing quantitative models for explaining the nonmonotonous dose-response dependence is discussed.

Nuclear and chromatin lipids: metabolism in normal and gamma-irradiated rats.

The data on nuclear and chromatin lipid metabolism are reviewed. The amount of neutral lipids and phospholipids in nuclei of rat thymus, liver and neocortex neuron as well as the amount of lipids in rat thymus and liver chromatin are described. The metabolic responses of nuclear and chromatin lipids from thymus to different doses and dose rates of gamma-irradiation of rats are discussed. In most cases, the nuclear and chromatin lipid responses are distinct. Changes in nuclear and chromatin lipid metabolism in response to gamma-irradiation are suggested to connect with the signal transduction pathway and the regulation of the transcriptional and replicative chromatin activity. The influence of beta-carotene and picrotoxin on rat liver nuclear lipids and neocortex neuronal nuclear lipids, respectively, was analyzed. The possible involvement of the lipid traffic in the chromatin lipid responses to gamma-irradiation and other agents is suggested.

Effect of picrotoxin-induced seizures on lipid composition of cortical tissue homogenate and its nuclear fraction in rats.

The content of lipids in rat cortical tissue homogenate and fraction of neuronal nuclei was determined under normal conditions and after picrotoxin-induced seizures. Changes in lipid composition of homogenate and nuclear fraction differed considerably. In homogenate, the content of sphingomyelin, lysophosphatidylcholine, and total phospholipids increased, while the content of free fatty acid remained unchanged; in the nuclear fraction the total phospholipid content decreased, while the concentration of free fatty acids increased.

Nonmonotonous Changes in Metabolic Parameters of Tissues and Cells under Action of Ionizing Radiation on Animal.

A nonmonotonous relationship between changes of metabolic parameters of tissues and cells of animal and radiation dose were discussed. Under acute irradiation of animals the nonmonotonous dose-response curve for metabolic parameters of tissues and cells were found. The nonmonotonous dose-response curves of metabolic and functional tissues and cells parameters were also revealed upon chronic irradiation of animals at a low dose-rate. The nonmonotonous shape of dose-response curves may be explained on the basis of nonmonotonous kind of the time-course of metabolic response after irradiation. Living cells were supposed to possess a fundamental property in response to action of different stress agents by nonmonotonous changes of cell metabolism. This response was damping in time oscillation of the value of metabolic parameters around the normal level. Amplitudes and periods of oscillations in these changes of metabolic parameters could be observed. In case of chronic irradiation at a low dose-rate the metabolic and functional parameters showed some modified oscillation during irradiation. The nonmonotonous type of changes in metabolic and functional parameters of tissue and cell by chronic low dose-rate irradiation threw some new light on the peculiarities of biological effects of chronic irradiation.

Activation of lipid metabolism of nuclei and chromatin in thymocytes of rats subjected to chronic low dose intensity gamma-irradiation.

Contents and radioactivity (as incorporation of [2-14C]acetate) of thymocyte nuclear and chromatin lipids were investigated in rats subjected to chronic gamma-irradiation in a dose 10 Gy at the intensity of 12.9 cGy/day. The incorporation of label into total fraction of lipids of nuclei and chromatin and into total phospholipid fraction of thymocyte nuclei increased. However, in the whole cells differences in radioactivity of these fractions were not found. The amount of fatty acids in thymocytes from irradiated rats was decreased but synthesis of fatty acids was activated. The amount of fatty acids in nuclei and chromatin remained unchanged compared with control, whereas radioactivity was significantly higher than in the whole cells. In irradiated thymocytes the amounts of phosphatidylserine in nuclei and phosphatidylserine and phosphatidylinositol in chromatin were increased. The incorporation of [2-14C]acetate into proteins increased in thymocytes of irradiated rats. The role of lipid metabolism in genome functioning is discussed.

[Difference in metabolic pools of cholesterol and free fatty acids of rat thymocyte chromatin and nuclear membranes]. / Razlichie metabolicheskikh pulov kholesterina i svobodnykh zhirnykh kislot khromatina i iadernoi membrany timotsitov krys.

Incorporation of [2-14C]acetate into cholesterol and free fatty acids of nuclear membrane, chromatin, and nuclei of rat thymocytes have been studied in 7 and 45 min of cell incubation. Specific radioactivity of the lipids from the nuclear membrane was lower than that from chromatin. Basing on the results obtained, it should be concluded that the pools of lipids from the nuclear membrane and the chromatin are different.