Influence of Hypoxia and Hypercapnia on Fatty Acid Composition of Lipids in White Muscles of Common Carp Cyprinus carpio

Study of the involvement of fish tissue fatty acids into the organism reactivity caused by an influence of exogenous factors is crucial for investigation of cell mechanisms underlying hypobiotic effect. Gas chromatography was used to estimate the composition and quantities of fatty acids of total lipids in white muscles of the Ukrainian scaly carp species. Twenty eight fatty acids were identified and their quantitative redistribution was revealed under hypoxic-hypercapnic action following a decrease in the environmental temperature (artificial hibernation). The decrease in total content of saturated and an increase in the one of unsaturated fatty acids was mainly due to the ω-3 and ω-6 polyunsaturated acids family. The optimum ω-3/ω-6 ratio under the studied conditions was supposedly maintained due to acyl-lipid ω-3 and ω-6-desaturase activity. Modified content of total lipids fatty acids in carp white muscles was likely a part of the cell mechanism of hypobiosis factors action in the fish organism.

Taking into account the habitat conditions in fish, the lipids play a significant role in biochemical adaptation [14][15][16][17], in particular, this concerns fatty acids (FAs), the most labile component of lipid molecules rapidly responding to exogenous environmental influences [16]. It should be noted that the involvement of fatty acids into adaptation, occurring in fish organism due to the effect of lowered temperature (hypothermia) [20,14,1], leads notably to an increased content of polyene fatty acids (especially ω-3 and ω-6 acids), providing thereby the required viscosity of biological cell membranes and maintaining the thermal homeostasis of the body [8,16]. However, it is necessary to consider the peculiarities of hypoxic-hypercapnic influence jointly with hypothermia on the lipid component of cells of various organs of animals, including fish [10].
Since fatty acids as structural components of biological membranes and energy substrates [7] are involved into the organism reactivity due to the effects of environmental factors [5,11], the study of the fatty acid profile of fish tissues being in hypobiosis, is perspective and relevant.
The research aim was to study the qualitative and quantitative composition of fatty acids of total lipids of Ukrainian scaly white carp under hypoxic-hypercapnic influences when the environmental temperature was reduced.

Materials and methods
Experiments were carried out in the Ukrainian scaly carps (Cyprinus caprio L.) weighing 250-270 g, which were obtained from Ivanivka Fish Processing Plant of the Kyiv region. The fish were caught in autumn and were kept in a 2,000 dm 3 pool for three days to adapt. The animals were divided into two groups (n = 5 each): 1 (control) -active fish; 2 (experimental) -fish under an artificial hypobiotic state (the influence of hypoxia, hypercapnia, with the decrease of environmental temperature) [12]. For the experiment the fish were placed into a closed glass aquarium with a water temperature of 8…10°C, supplied with the gas mixture of carbon dioxide and oxygen in 1:1 ratio for half an hour at a blowing rate of 150-200 cm 3 /min (at 50-100 dm 3 water). Under conditions of lowered temperature and increasing hypoxia-hypercapnia fish gradually switched to a hypobiotic state (suspended animation).
Fish of groups 1 (control) and 2 (at the 6 th and 24 th hour of exposure to artificial hypobiosis) were dissected and the white muscle tissues were extracted. Homogenization and extraction of lipids were performed поступово переходили у гіпобіотичний стан (знижена життєдіяльність).
with chloroform methanol mixture according to the Folch method [4]. The fatty acid methyl esters were prepared according W.W. Christi [2] and analyzed by means of Trace GC Ultra gas chromatograph (Thermo Scientific, USA). The separation was carried out with a high-polar capillary chromatographic column SPTM-2560 (Supelco, USA). Acids were identified using the standard mixture of methyl esters of fatty acids 37 Compone FAME Mix (Supelco) [3]. For quantification of individual FAs, the method of area normalization was used and the relative content of FAs was represented as the percentage to their total amount.
The obtained results were processed by the method of variation statistics using Student's t-criterion and Excel software (Microsoft, USA). Changes were considered as statistically significant at p < 0.05.
Investigation of the FA spectrum of total lipids of carp white muscles under hypoxic-hypercapnic effect found no qualitative changes in respect of the control of a pool of FA lipids, however, there was a redistribution of their content versus the control (Table 1). There was a decrease in total amount of UFAs, in particular due to a reduced content of С 14:0 , С 15:0 , С 16:0 , С 17:0 , С 18:0 , C 20:0 , C 21:0 , C 22:0 , C 23:0 , C 24:0 acids to the 6 th and 24 th hour of hypoxic-hypercapnic effect in average by 41.7 and 65.9%, respectively, relative to the control (Fig. 1). This may be due to their expenditure as an energy substrate [18,5].
Total content of UFAs was increased due to a rise in the level of MUFAs and PUFAs if compared with the control (Fig. 1). Under hypoxic-hypercapnic effect the unsaturation ratio (FAs/UFAs ratio) decreased to the 6 th and 24 th hour if compared to the control and was 0.23 and 0.12, respectively.
It is known that PUFAs are precursors of biologically active substances [8,1]. Arachidone ω-6 PUFA derivatives are a series of thromboxanes and leukotrienes that enhance the permeability of the membrane and cause inflammation, and the ω-3 PUFA metabolites, which are anti-platelets and anti-inflammatory agents, contribute to the stabilization of membranes. Therefore it is important to maintain the physiological ratio of ω3/ω6 PUFAs.
The results of our studies indicate that at the 6 th and 24 th hours of hypoxic-hypercapnic exposure on fish organism the ratio of (ω-3/ω-6) in case of white muscle lipid PUFAs decreased if compared with the control by 11.5 and 14.0%, respectively, along with a more significant rise in the content of ω-3 and ω-6 fatty acids. This dynamics may be related to desaturation and fatty acid elongation [20].
It is known that a change in the unsaturation degree of fatty acids (especially at the expense of PUFAs of ω-3 and ω-6 families) can occur due to the participation of acyl-lipid ω-3 and ω-6-desaturases, which perform the desaturation of fatty acids at positions 3 and 6, respectively [19]. Their activity is evidenced by the change in the values of desaturation indices (C 22:6ω3 / C 18:3ω3 and C 20:4ω6 /C 18:2ω6 ratio). The ratio C2 0:4ω6 / C 18:2ω6 , which shows the conversion rate of linoleic acid to arachidone one, for carp white muscles increased, and to the 6 th and 24 th hours of exposure was 0.36 and 0.42 respectively, whereas in the control group this index was 0.28. The ratio C 22:6ω3 /C 18: 3ω3 , which reflects the level of metabolism of the family ω-3 acids, decreased, and at the 6 th and 24 th hours of exposure was 3.77 and 2.88 respectively, while in the control group that was 4.76.
Consequently, the changes in activity of acyl-lipid ω-3 and ω-6-desaturase are observed in lipids of white carp muscles after hypoxic-hypercapnic effects. Similar changes in the activity of desaturases under artificial hypobiosis are found in other organs of fish [20,13,10]. The activity of these highly specific enzymes under the influence of external factors is likely aimed at maintaining an optimal (ω-3 / ω-6) ratio by control of PUFAs content.
Thus, since PUFAs are directly involved into regulation of the majority of cellular processes, the observed changes in the ω-3 and ω-6 families PUFA spectra under hypoxic-hypercapnic effect (artificial hypobiosis) can be considered as mobilization of body adaptive responses.

Conclusions
The performed studies of the FA spectrum of carp white muscles total lipids indicated a redistribution of the content of fatty acids under hypoxic-hypercapnic effect following a decrease in temperature (artificial hypobiosis), which led to a reduction of the saturated and an increase of unsaturated fatty acids content. The ω-3 and ω-6 PUFAs underwent the most prominent changes, in particular docosahexaenoic, eicosapentaenoic and arachidonic acids, characterized by a high metabolic activity. It is assumed that the optimal ω-3/ω-6 ratio could be maintained by acyl-lipid ω-3 and ω-6-desaturases, and this is a manifestation of biochemical adaptation. The revealed modifications of the content of FA lipids of white carp could be explained by the involvement of FAs into systems of reactivity of an organism under the effect of hypobiosis factors, that provided an optimal performance of all metabolic processes. модифікації вмісту ЖК ліпідів білих м'язів коропа можна пояснити залученням ЖК до систем реактивності організму за дії гіпобіотичних чинників, що забезпечує оптимальну роботу всіх метаболічних процесів.