S. A. Zabelinskii

On Adaptative Changes of Rates of Oxygen Consumption and Lipid Metabolism in Littorina saxatilis at Parasitic Invasions

Effects of parasites as a biotic factor on physiological and some biochemical characteristics of gastropod molluscs Littorina saxatilis are considered. The individuals infected at young age and incapable for reproduction due to parasitic castration have a lower intensity of respiration as compared with non-infected individuals of the same size. Large infected individuals that had time to realize their reproductive potential before pathological changes in hepatopancreas do not differ in respiration from the normal individuals. Comparative analysis of the lipid fraction of liver, particularly of the fatty acid (FA) composition of total lipids and phospholipids, allowed revealing essential differences between the control and infected individuals, as well as between infected individuals of different age groups. The absence of glycogen in the liver of infected L. saxatilis, which is revealed using thin-layer chromatography, indicates functional disturbances, including those in glycogen synthesis. We suggest that the reduction of intensity of metabolism in infected individuals is connected with peculiarities of digestive process at structural changes in the hepatopancreas damaged by sporocysts. In infected molluscs the FA composition relates mainly to parasite tissues than to the liver itself. In this connection, the role of lipids in regulation of enzyme activity and permeability of cell membranes is directed first of all to maintenance of parasite metabolism. The revealed elevated synthesis of stearic acid in infected individuals can be connected with its accumulation in parasite adipose cells. The FA composition of phospholipids in infected individuals had changes that can be directed to realization of barrier function of cell membranes, specifically to restriction of the rate of oxygen diffusion into sporocysts owing to condensation of membranes. Together with adaptive changes in FA composition the ratios saturated FA : unsaturated FA and ω3 : ω6 acids in control and infected individuals were preserved at the constant level, which in any case is connected with maintenance of normal functioning of cell membranes.

Limit of Change in Unsaturation Index of Fatty Acid Composition of Phospholipids at Adaptation of Molluscs to Biogenic and Abiogenic Environmental Factors

Comparative study of fatty acid composition of total phospholipids, as well as of phosphatidylcholine and phosphatidylethanolamine from hepatopancreas and leg muscle was carried out on several representatives of gastropod molluscs (Gastropoda) and on the bivalve mussel Mytilus edulus (Bivalvia). The objects of our study were marine littorins Littorina saxsatilis adapted to different temperature conditions of White Sea and Barents Sea, the freshwater lymnaea Lymnaea stagnalis infested by Trematoda, and mussels from the White and Black Seas. It was shown that depending on the existence conditions of the studied tissue or lipid, the maximal changes occurred in the percentage of saturated acids (4–83%); the percentage of unsaturated acids was less expressed (1–14%) and the changes in unsaturation index (UI), on average, did not exceed 20%. It was suggested that the revealed quantitative restriction of the UI change under the action of various external factors is a limit for maintenance of the membrane fluidity necessary for normal cell viability, specifically in the studied ectothermic molluscs.

Phospholipids composition of cell nuclei of rat’s brain in dynamics of normal ontogenesis and after hypoxia

The phospholipid composition of cell nuclei was examined in different animals and at various states of the body differing in the organizational level [1, 2]. However, the composition of lipids of higher vertebrates during ontogeny (prenatal and postnatal), as well as the phospholipid composition of brain cell nuclei at different pathological states, has not been studied sufficiently. For example, hypoxia, which is frequent during different periods of ontogeny and is a stress factor, affects fatty acid composition of lipids [3] and, hence, membrane fluidity [4]. Taking into account the important role of phospholipids (located predominantly in nuclear membranes) in the structural and functional organization of cell nuclei, we studied phospholipids and the unsaturation index of their fatty acids in the cell nuclei of the rat brain during different periods of ontogeny and after hypoxia that occurred during these periods. The study was carried out on 19-day-old rat embryos and young (5and 45-day-old), adult (3.5to 4-month-old) and old (1.4to 1.5-year-old) rats. At these stages of development, part of animals was exposed to hypoxia, and the remaining animals served as a control. The brains of several embryos were pooled into a single sample; there were at least five such samples. The brains of five-day-old rats were subjected to the same procedure. In other age groups, every brain was analyzed separately. There were at least five rats in each group. One-hour hypoxia was induced by passing a nitrogen–oxygen mixture containing 8% of oxygen through a chamber into which pregnant females or animals of the corresponding age groups had been put. During the exposure, room temperature was maintained in the chamber, and the concentrations of oxygen and carbon dioxide were controlled by means of an SF-101 gas analyzer (France). After brain homogenization, cell nuclei were isolated by centrifugation and phospholipids were extracted from them by the standard method [5]. Phospholipids were divided into classes by means of two-dimensional thin-layer chromatography on silica gel [6]. Methyl esters of fatty acids of all phospholipids were analyzed by means of gas–liquid chromatography and identified with the use of a mass-spectrometer (LKB, Sweden). Then, the unsaturation index (the number of double bonds in 100 fatty acid molecules) was calculated.

Peculiarities of the phospholipid and fatty acid composition of erythrocyte plasma membranes of the Black Sea fish

The phospholipid and the fatty acid composition of the main phospholipid families of erythrocyte plasma membranes was studied in two species of cartilaginous fish: the common thrasher (Raja clavata L.) and the common stingray (Dasyatis pastinaca) and in three bony fish species: the scorpion fish (Scorpaena porcus L.), the smarida (Spicara flexuosa Raf.), and the horse mackerel (Trachurus mediterraneus ponticus Aleev). It was shown that in the studied fish, 70.0–80.0% of all membrane phospholipids were composed of phosphatidylcholine and phosphatidylethanolamine. Phosphatidylserine, monophosphoinositide, and sphingomyelin were minor components whose content in the erythrocyte membrane fluctuated from 3.0% to 13.0%. The fatty acid phospholipids composition was represented by a large specter of acids. From saturated acids, basic for plasma membranes are palmitic (C16: 0) and stearic (C18: 0) acids. From unsaturated acids, the larger part belongs to mono-, tetra-, penta-, and hexaenoic acids in fish phospholipids. The calculation of the double bond index and of the unsaturation coefficient showed difference in the deformation ability of erythrocyte membranes of the studied fish.

Effect of Total Hypothermia on the Fatty Acid Composition of Blood Phospholipids of Rats and Ground Squirrels and of the Light Irradiation on Chemical Processes in Lipid Extract

Effect of hypothermia on the fatty acid composition of rat and ground squirrel blood phospholipids is studied. Different reaction of these animals to cooling is revealed; in rats no changes were observed in the fatty acid composition of blood phospholipids, whereas in the winterhibernating ground squirrels there were significant changes in the content of individual fatty acids (FA). The content of monoenic acids in ground squirrels decreased almost by 50%, while the content of saturated acid (C18) and of polyenic acids C18: 2ω6 and C20: 4ω6 rose significantly. Such changes seem to be the mechanism that promotes maintenance of the organism viability under conditions of a decreased level of metabolism, heart rhythm, and body temperature and is evolutionary acquired. At the same time, the observed changes in the content of individual FA do not lead to sharp changes in such integrative parameters as the total non-saturation of phospholipids, which determines liquid properties of chylomicrons and other lipolipoprotein transport particles of the ground squirrel blood. There are studied absorption spectra of blood lipid extracts of rats and ground squirrels under effect of light as well as effect of light upon the FA composition of lipid extracts of these animals. The FA composition of lipid extracts has been established to remain practically constant, whereas the character of changes of spectra under action of light indicates the presence in the extracts of oxidation-reduction reactions. The obtained data allow suggesting that in the lipid extract there occurs cooperation of both the phospholipids molecules themselves and of them with other organic molecules, which makes it possible for fatty acids to participate in processes of transport both of electrons and of protons. This novel role of FA as a participant of the electron transfer might probably be extrapolated to chemical reactions (processes) occurring inside the membrane.

Effect of habitat and motor activity of molluscs on fatty acid composition of triglycerides and phospholipids

A comparative analysis of fatty acids (FA) in neutral lipids and phospholipids of digestive gland and pedal muscle has been performed in molluscs from various ecological groups differing by belonging to sea or fresh water, trophic types or the associated motor activity. In freshwater pulmonary gastropods Lymnaea stagnalis and Lymnaea ovalis and marine prosobranchial molluscs Buccinum undatum and Littorina littorea the total content of ω3-acids in phospholipids of the studied tissues differed more than twice, predominantly due to the combined effect of temperature and salinity of the habitat. The lower viscosity of cell membranes in marine species (ω3/ω6 < 1) is determined to the greatest degree by the presence of eicosapentaenoic acid that accounts for 22–25% of the FA sum in marine species. Comparison of the molluscs by their trophic belonging has revealed the presence of linoleic acid in triglycerides in digestive glands of phytophages (8–12%), but the practically complete absence of this acid in the predator B. undulatum (< 0.8%). By mobility, L. littorea inhabiting the high-low tide littoral was inferior to freshwater pulmonary gastropods and to the marine predator, as it stops moving twice a day during the low tide. In phospholipids of pedal muscle of this mollusc the amount of long-chain polyunsaturated C: 22 FA was 3–6 times lower than that in other studied species, which might possibly indicate the role of these acids in functioning of the pedal muscle contractile tissue. On the whole, use of the FA characteristics as the parameters determining belonging to certain ecological group requires a certain caution due to a complex action of biotic and abiotic factors on the animal metabolism. The exception is the ω3/ω6 ratio in total phospholipids of fresh water and marine gastropods.

Participation of π-Electrons of Phospholipid Molecules in Absorption of Ultraviolet Light in the Range of 260–280 nm

The UV-spectra (230–260 nm) of the rat brain lipid extract and of individual extract lipid fractions obtained on a column with silica gel (10 µm) were studied. It was found out that hydration of the lipid extract led to a decrease of the absorption intensity of the UV-spectrum by 70% as compared with the initial intensity. Addition of silica gel to the lipid extract decreases twice the UV-spectrum intensity, whereas the repeated addition of silica gel does not decrease intensity of the UV absorption, with no changes of the amount of phospholipids in the lipid extract. Some lipid fractions isolated from the column shift the UV-spectra towards the shorter wavelength region, while the fractions containing phosphatidylcholine shift the spectrum towards the longer wavelength region. It has been established that the phosphatidylcholine fractions containing different amounts of polyenic acids differ by the UV-spectrum intensity. It was concluded that chromophore groups of polyunsaturated acids of phospholipids participated in absorption of energy in the range of 260–280 nm, which lead to excitation of valence electrons of multiple (double) bonds. Energy of such electrons can be used in interactions with other molecules, in particular, for energy transfer inside the membrane monolayer.

The fatty acid composition of phospholipids and absorption spectra of lipid extracts from lamprey, frog, and rat erythrocytes

The work studies the content and fatty acid composition of phospholipids as well as the absorption spectra of lipid extracts from red blood cells of poikilothermal and homoiothermal animals at different evolutionary levels. The objects of study include two poikilothermal species, the river lamprey (Lampetra fluviatilis) that uses oxygen dissolved in water, and the common frog (Rana temporaria) that consumes oxygen both from water and from air. A homoithermal animal is the white rat (Rattus rattus) that inhabits the terrestrial-aerial environment. The animals are studied in winter and spring. The phospholipid content in lamprey blood plasma is found to be twice higher than that in its erythrocytes. In the frog and the rat, the ratio is reverse. Determination of the fatty acid lipid composition of red blood cell phospholipids suggests that membranes in the lamprey are denser than in the frog. As for the fatty acids in the erythrocyte fraction of rat blood, they appear to be less diverse, with a double prevalence of saturated acids over unsaturated ones and devoid of long chain (C22) ω3 fatty acids. All of this results in a lower degree of unsaturation and a denser packing of fatty acids in the membrane structures of rat erythrocytes. The mechanism of reversible binding of O2 molecules to hemoglobin in erythrocytes is discussed. Presumably, the mechanism of interaction between molecules of O2 and molecules of water prevents the exchange interaction of electrons of the hemoglobin iron atoms with an oxygen molecule. This is confirmed by our obtained absorption spectra, which show that in the lipid extract almost totally devoid of water the heme isolated from erythrocytes is converted to hemin.

Effect of Stress Actions on Some Hematological and Biochemical Parameters of Rat Blood and on Energetic Intermolecular Interactions in Lipid Extract under Effect of Light Radiation

Comparative study has been carried out on effects of the three-day long starvation, running, and their combination on morphological parameters of rat blood, lipid metabolism, and activity of blood Na,K-ATPase. Different effects of these stress factors on the blood erythrocyte composition have been shown. Starvation is accompanied by the most pronounced release of stored erythrocytes into blood, which results in a significant decrease of both the total amount of reticulocytes and in the complete absence of reticulocytes of I stage of maturity (the youngest). The running on a treadmill led to a significant increase in the total amount of blood reticulocytes and to a multifold increase of immature reticulocytes (RC-1 and RC-2), which can indicate some stress of the bone marrow erythroid stem line. The curve of acid resistance of blood reticulocytes has shown the animals to experience the greatest stress at a combination of starvation and running. Starvation and running also produced different effects on blood lipid characteristics. The content of triacylglycerides (TAG) in blood rose by 40% at starvation and decreased by 30% at running, a similar tendency being found for the atherogeneity index. The fatty acid composition of blood phospholipids at running and its combination with starvation practically did not differ from control. A change of Na,K-ATPase activity, which is so typical for reactions of various kinds of stress sharply fell at starvation (by 22%), but increased at running (by 13%) and decreased markedly at combination of these actions. The absorption spectra of the whole blood lipid extracts of the rats subjected to various stress actions showed that organic substance with coupled bonds, which absorbs light within diapason of 360–620 nm, is extracted from the blood (at different amount depending on the kind of action). The absorption of light in diapason of 400–410 nm has been found to belong to the Soret band of ferroheme and ferriheme. The shift of Soret band indicates an electron transitions in the iron cation. By the change and disappearance of Soret band it is possible to judge about the processes occurring in the lipid extract. Disappearance of the Soret band from the lipid extract indicates formation in it of the steady radicals as a result of the ferriheme disintegration due to accumulation of energy in porphyrin, which does not seem to occur in the blood cell membranes. The iron atom in the ferriheme molecule is known to accept electron and to yield a part of energy, probably to porphyrin. Then ferroheme yields electron and becomes ferriheme with an excess of the energy in porphyrin. Hence, at admission of the next electron to the iron atom the porphyrin molecule is to get rid of the energy obtained earlier to prevent its disintegration. The heme is possible to be an accumulator and distributor of energy in tissue.

Significance of electron interactions of fatty acids of phospholipid molecules in the organism adaptation to habitat temperature

Data in the fatty acid composition of muscle tissue phospholipids of some representatives of gastropod molluscs (Gastropoda) have been presented for the first time. In the lake phytophagues Lymnaea stagnalis and Lymnaea ovata the long-chained C22-acid was not detected, where-as in the predator common whelk Buccinum undatum, C22:6ω3 was present. Comparison of absorption spectra (240–720 nm) of lipid extracts of the studied invertebrates and of rat has been performed. The obtained data are discussed from the point of view of participation of π-electrons of phospholipid fatty acid molecules in adaptation of membranes to the habitat temperatures, which arises owing to interelectron attraction and to the process of formation of Cooper’s pairs.