NMR in Analysis of the Nutritional Value of Lipids from Muscles and Livers of Wild Amazonian Fishes with Different Eating Habits over Seasonal Variation

the Nutritional Value of Lipids from Muscles and of Wild Fishes with Different Eating Habits over Seasonal Variation. Lipid composition of the Amazonian fishes remains unexplored although fishes in general show very high nutritional potential. Endogenous and environmental factors can influence the lipid contents of fishes among which, in the Amazon River, seasonal dynamics influences stand out. Herein, nine most consumed fish species were analyzed and their lipid composition evaluated in terms of effects of tissue from where were extracted, season of the Amazon River and the fish eating habits. Higher amounts of lipids were obtained from livers than dorsal muscles in all studied species. Statistical analysis has shown that Amazonian fishes present different lipid profiles according to their eating habits, which mainly comprises saturated fatty acids to distinguish detritivorous livers, and linolenic acid, cholesterol, polar lipids for carnivorous and piscivorous fish muscles. Furthermore, in Amazonian fish, some very important lipids for human nutrition are present, such as omega 3 and 6 fatty acids whose availability depended on the tissue metabolism and fishes’ eating habit along the seasonal periods. For example, our findings indicate that the piscivorous fish C. monoculus presented higher levels of linoleic acid for liver than linolenic acid and the opposite occurred Abstract: Lipid composition of the Amazonian fishes remains unexplored although fishes in general show very high nutritional potential. Endogenous and environmental factors can influence the lipid contents of fishes among which, in the Amazon River, seasonal dynamics influences stand out. Herein, nine most consumed fish species were analyzed and their lipid composition evaluated in terms of effects of tissue from where were extracted, season of the Amazon River and the fish eating habits. Higher amounts of lipids were obtained from livers than dorsal muscles in all studied species. Statistical analysis has shown that Amazonian fishes present different lipid profiles according to their eating habits, which mainly comprises saturated fatty acids to distinguish detritivorous livers, and linolenic acid, cholesterol, polar lipids for carnivorous and piscivorous fish muscles. Furthermore, in Amazonian fish, some very important lipids for human nutrition are present, such as omega 3 and 6 fatty acids whose availability depended on the tissue metabolism and fishes’ eating habit along the seasonal periods. For example, our findings indicate that the piscivorous fish C. monoculus presented higher levels of linoleic acid for liver than linolenic acid and the opposite occurred for muscles. The omega 6 and 3 fatty acids ratio was influenced by the season dynamic of the Amazon River and availability of food according with each specific eating habit, poiting mainly to the piscivorous fishes as the healthiest fish for human consumption.


INTRODUCTION
The consumption index of fishes by the Amazon inhabitants is one of the highest in the world. [1,2,3,4] It is estimated as > 50 kg of fishes/person/year in the Manaus city and as 510-600 g/day in the countryside. [5] The top ten Amazonian fishes most consumed rankings are 1) Colossoma macropomum, 2) Semaprochilodus insignis, 3) Prochilodus nigricans, 4) Brycon amazonicus, 5) Cichla monoculus, 6) Mylossoma duriventre, 7) Triportheus elongate, 8) Plagioscion squamosissimus, 9) Piaractus brachypomus, and 10) Pseudoplatystoma tigrinum. The Hypophthalmus edentates could be added to this list as a peculiar taste dish mainly at Para State due to its fat amounts. [3,1] Indeed, it is an interesting fact that there is a preference in consumption of a few fish species (100 species), regardless the fact that over 2,500 fish species live in the Amazon River. Also, Amazon inhabitants avoid to consume no scales fish, which are considered as very greasy and harmful to the health [6], in fact they recommend to eat piscivorous or carnivorous fishes when the person is ill. Therefore, they prefer big fat fishes to celebration occasions and some species are consumed with the entire inside content, organs and fish ova as Hypostomus affinis. [7] Fishes meat is considered as a great source of essential fatty acids, such as docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n- 3) recommended to a human diet. [8,9,10] These polyunsaturated and very long chain fatty acids have been shown to have a positive impact on human health since they can prevent many diseases, including cardiovascular, neurological, autoimmune diseases, and cancer. [8,11,9] Amazonian fishes' diet can vary among living plants (herbivorous), plankton (planktivorous), debris (detritivorous), mud (iliophagous), blood (haematovorous), nonfish animals (carnivorous), fish (piscivorous) or both plants and animals (omnivorous).
These feeding niches fluctuate depending on the seasonal dynamics of the river flooding (distributed in low, rising, high, and falling water periods), once the concentration of river's water favours specific predatory hunting and spatial distribution that imposes new biomes, new habitats, and consequent drastic changes in food sources availability. [12,13,14] It is why chemical composition of Amazonian fish may be significantly variable, once besides Amazon River Basin harbours the largest worldwide diversity of freshwater fish, including singular native species, the particular ecology of the area provides a varied nutrient source and a challenging environment that can influence the biochemical pattern of inhabiting fishes. [2] The lipid composition of Amazonian fish, as well as freshwater fish in general, had been reported as fatty acids and cholesterol composition mainly represented by the amount of its bioactive polyunsaturated fatty acids (omega 3 and 6) per species fish and/or place, which could be from farm or wild environment. [15,16,17,18,19,20,21] The amounts of fatty acids found can be varied among the freshwater fish, however usually present the same fatty acids, which are those with 14-22 carbons, comprises saturated and unsaturated chains, presenting the C18 series the most variable in unsaturation possibilities. The total amount of the unsaturated fatty acids can be presented as higher than the saturated fatty acids sum in many cases [16,22], however also the opposite can occur. [17] In the majority of papers, only dorsal muscles' lipids were evaluated, although some works reported on other tissues' lipids, such as liver, eyeball, and brain [20,23,24,18].
While wild freshwater fish lipids reports are focused on the fatty acid profiles, the global lipidomes, which could be influenced by several factors, are poorly characterized.
Also, many works showed and investigated DHA and EPA as the main bioactive polyunsaturated fatty acids in fish, however the precursor of these important fatty acids, which are the omega-3 linolenic acid for DHA, and EPA, and the omega-6 linoleic acid for arachidonic acid (AA, 20: 4n-6) [25,26], are often set aside in the discussion even if those are more abundant than ones with the longer chains [16]. Nevertheless, ratio of linoleic and linolenic acids in fish can indicate their potential to produce EPA, DHA and ARA, besides of suggesting how healthy is that food source since the balance between omega 6 and 3 must be less than 5:1 to be considered healthy. [27] Thus, our aims were to discriminate the lipid differences of the fish species most consumed by Amazonian population according to their eating habits and to seasonal variation of Amazon River, and, also, determine linoleic and linolenic amounts in nine the most consumed fishes by Amazonians.

Sampling
The top nine most consumed species of Amazonian fishes (Colossoma  .S1). Fishes were captured with sizes bigger than that corresponding to their respective sexual maturity during the Amazon flood (08-12 July) and drought (26)(27)(28)(29) periods in 2013.
The recommended protocol for euthanasia, using ice and water at -4 ºC, was

Lipid extraction
The dorsal muscles and livers tissues were submitted to protocols for lipids extraction as described in literature. [28,29,30] Total lipids' extracts were fractionated into three classes of lipids with different polarities as described elsewhere. [29,30] We have extracted 224 lipid samples from 9 fish species (170 samples of total lipids from livers and muscles and 54 samples of lipids classes from muscles).
All lipid samples were stored at -80 °C until analysed.

NMR analysis
5 mg of lipid samples were dissolved in 600 µL of 99.8% deuterated chloroform (CDCl3; Cambridge Isotope Laboratories, Inc., USA) and transferred to NMR tubes (5 mm). 1 H and 13 C NMR analyses were conducted using a Bruker AVANCE 600 MHz spectrometer equipped with a Triple Band Inverse (TBI) probe. Used conditions for qualitative and quantitative analysis were as those described previously. [30]

Data processing
The obtained 1 H-NMR spectra (170) from total lipids were referenced to tetramethyl silane, aligned and had their phase corrected, then binned (0.04 ppm) using The levels of linolenic and linoleic acids were calculated from the quantitative 1 H-NMR data according to the method previously reported by others [31,13], where the concentrations of fatty acids are expressed in molar percentages according to the equations 1 and 2.

Equations
) Where ALn and AL are the areas of the bis-allylic proton peaks for linolenic (ω-3) and linoleic (ω-6) acids, respectively, and AG is the area of the proton peaks of glyceryl group.
The studied species of Amazonian fishes presented higher lipid amounts in livers than in muscles, irrespective of the seasonal period. This is coherent with the fact that liver is a highly metabolic organ. [32] However, the exception were two species B.
amazonicus and H. edentates, which presented lower lipid amounts in livers than in muscles.
For the H. edentates, Carvalho et al. showed that this fish does not deposit fat, instead the lipids are diffused in the muscular tissue. [33] This fish as well the other studied Amazonian fish species experienced an important change in total lipid amounts of dorsal muscles and livers in response to seasonal dynamics of Amazon river (flood and drought) among the eating habits. These findings are in line with previous studies reporting an influence of different feeding habits on the total lipid content in fishes and also agree with the popular knowledge. For example, H. edentates is a planktivorous fish and it used as a dish which tastes fat, while piscivorous or carnivorous fishes are preferred and recommend to ill people because those are considered no greasy and good to the health. [18,7] An interesting fact is that lipids from carnivorous and piscivorous fish did not suffer an apparent influence of season dynamics and the food availability did not affect total lipids contents. However, distribution of the lipids (types) in studied seasons is an issue to debate. Liver and muscle also may contain different proportions of lipids when classified per their polarities. The lipid class profiles response to the seasonal dynamic in dorsal muscles for the studied Amazonian fishes are illustrated in Figure 2. The higher amounts of phospholipids were found in the dorsal muscle of piscivorous fishes at flood period when compared with samples from the drought period, while the opposite pattern was found for carnivorous specie. The opposite was observed for neutral lipids. Only piscivorous and the P. nigricans (detritivorous) had their phospholipids increased in flood periods, all the other fish species had the neutral lipids increased in flood periods instead of phospholipids. For instance, the abundance of food sources for piscivorous habits occurs during the drought period, enabling fat storage as neutral lipids. [34] Figure 2. Lipid classes (mg/g of oil) of Amazonian fishes with five different eating habits extracted from the dorsal muscle at A) flood or B) drought period of Amazon River. P.s., Plagioscion squamosissimus (carnivorous); S.i., Semaprochilodus insignis and P.n., Prochilodus nigricans (detritivorous); B.a., Brycon amazonicus; T.e., Triportheus elongatus and C.ma. Colossoma macropomum (omnivorous); C. mo., Cichla monoculus and P.t., Pseudoplatystoma tigrinum (piscivorous); H.e., Hypophthalmus edentates (planktivorous).
Although differences between periods may explain the seasonal availability of food sources, but they don't explain detritivorous species lipids distribution once this type of fish shows quite variable diet. In this way, a distinct seasonal difference in muscle lipid class amount was observed between the two detritivorous fishes: S. insignis presented an increase of neutral lipid content balanced with the decrease of phospholipids in the flood period compared to drought period, while P. nigricans showed the opposite pattern. These findings may be explained by another fact, as the distinct seasonal migratory habits could be the causes of differences in lipids distribution. The S. insignis migrates for dispersion in rising water periods and P. nigricans in falling water periods. High amounts of neutral lipids may be required for fish migration, as their energy source. It was reported that dispersal migration of S. insignis is characterized by the accumulation of lipids (neutral lipids) while foraging in the flooded forest. [35] Also, it is noteworthy to state that even within a trophic category, there is enough diversity in the diet among the Amazonian fishes, or that it is difficult to classify them by eating habits. The two species S. insignis and P. nigricans are considered as detritivorous, but they also consume genipap fruit at flood period. [36] Wild Amazonian fishes' lipids identification Typical 1 H NMR spectra for lipids extracted from the fish with characteristic eating habits are shown in Figure S3. Lipids were assigned according to reported NMR data. [37,30,38,39] The assigned peaks are displayed in Table 1. [40]. dynamics of the Amazon River and eating habits (Fig. 3), we were able to see and determine endogenous lipid patterns and their correlation with ecological behaviours. The PLS-DA using the 1 H NMR spectral data as a matrix showed that the tissue metabolism ( Fig. 3A) influenced the lipid profile of the studied Amazonian fishes more than the seasonal periods (Fig. 3B). This is very obvious when we correlate the studied seasonal periods with each tissue (Fig. 3C). In fact, we expected a big difference between the tissues as their specific functions can lead to different lipid composition. However, the seasonal periods had an important role in distinguish the lipid profile of the studied Amazonian fishes (Fig. 3B), showing that the chemical composition of Amazonian fishes may depend on the seasonal dynamics, which correlates with the type and amount of available food in Amazon river. [18] Also, the lipid profiles of samples under study were important for separating groups of fishes with different eating habits ( Fig. 3D) despite of the studied seasonal periods for each tissue.
The most pronounced lipid profiles differences can be seen in their variable importance projection (VIP) in Figure S4, which revealed general fatty acids, linoleic and linolenic acids besides cholesterol and/or polar lipids (phosphocholine, sphingomyelin or saccharolipids) as having higher intensity in muscle samples than liver.
These differences are assigned, as described in Table 1, by the following NMR signals: 1.24, 5.34, 2.81 and 2.77 ppm (Fig. S4A). When we observe the season from which the fish samples came from, the flood period presented general fatty acids, while the drought period showed cholesterol and choline based lipids as the main difference between the seasons (Fig. S4B). These results are confirmed by the features for muscle in both season periods with cholesterol or polar lipids (phosphocholine, sphingomyelin or saccharolipids), and cholesterol or polar lipids for drought period independent from the tissue type (Fig. S4C). However, the lipid influence of the eating habits without considering the environmental factors does not present useful information to distinguish the samples, once the only general fatty acids was observed (Fig. S4D).
Separation of season sample type, i.e. drought or flood regardless of tissue factor and considering different eating habits, have found some singularity to the eating habits comparison (see Fig. S5

Influnces on Lipids composition and distribution in Amazonian fishes
Many factors can influence on the fish lipid profile such as season, tissue, food and eating behaviour (Fig. 4). As related in literature the season has an effect on the lipids profile of fish. [41] In the drought period livers were separated in distinct groups in which only planktivorous fish presented slight similarity to those from omnivorous habit (Fig.  4A), while muscle from this period had similarity between omnivorous and detritivorous ( Fig. 4B). At the flood period, the liver presented distinct groups (Fig. 4C), and the muscles showed similarity between planktivorous and omnivorous again as at drought period, and between piscivorous and carnivorous (Fig. 4D).  Figure 5. These results revealed that at drought periods general fatty acids and saturated fatty acids were found in livers (Fig. 5A), with more intensity of response of saturated fatty acids for detritivorous fish, while muscles showed besides of general fatty acids, the linolenic acid and cholesterol or polar lipids (phosphocholine, sphingomyelin or saccharolipids) with higher concentration for carnivorous and piscivorous habit (Fig. 5B). At flood periods general fatty acids were found in both tissues, livers and muscles as important lipids that distinguish between eating habits (Fig.

Changes in linolenic and linoleic acids amounts
Amounts of the omega-3 linolenic acid and the omega-6 linoleic acid for all lipid samples are presented in Figure 6 as well the samples' omega-3 and omega-6 ratios based on amounts of these acids. [44] Many good nutritional habits are based on use of the two serving portion per week of 250-500 mg of EPA+DHA that are consider healthy. [45].
Lipids isolated from muscles from Amazonian planktivorous and omnivorous fishes presented coherent amounts of linoleic acid that were similar to other planktivorous and omnivorous species, respectively, near 6% to planktivorous and 4% to omnivorous as compared to Vasconi et al. work. [16] However, the livers' lipids showed higher amounts (near 8% to planktivorous, 6% to omnivorous or 10% for B. amazonicus in flood season) [16], while linolenic amounts were lower even in liver samples [16], although similar species had lower contents of these as reported in other works [19,46]. As well, the Amazonian carnivorous fish showed higher amounts of linoleic acid than other carnivorous species, such as those from South African Cape hake. [17] Therefore, even rich in linoleic acid, the lipidome of the studied Amazonian fishes can be recognized as potentially great nutritional sources. Even potentially detrimental, linoleic acid is also the precursor of lipoxins (lipid mediators with a relevant role on the resolution of inflammation) and an adequate omega-6/omega-3 ratio can be required for proper immune response. [47] Importance of the omega-6/omega-3 ratio in the diet has been addressed in human nutrition and is considered as healthy when ratio is The P. nigricans species (detritivorous fish) has an ideal omega-6/omega-3 ratio only when the liver is evaluated as regardless of the season. Thus, as fish consumption is based mostly on the muscle tissue, in fact it is not recommended to eat this fish as a health source, unless liver-based food can be explored. However, the other detritivorous fish studied here is S. insignis, have shown optimal results for lipids from liver and muscle at drought season, point to a health fish for this period. The difference between the two detritivorous species was already explained by the distinct seasonal migratory habits, and they can be supported by the agreement with the above mentioned S. insignis´s ,an increase of phospholipids content balanced with the decrease of neutral lipids at drought period [35]. It is possible to relate these findings with eating habits, since this fish usually have more food abundance due to the increase of the flooded area, which contributes to the accumulation of energy reserves in the form of triglycerides rich in omega-6, during the flooding period. Thus, during the drought season the accumulation ends up being lower from omega-6 fatty acids, which contributes to the reduction of omega-6/omega-3 ratio. Furthermore, species with ideal omega-6/omega-3 ratios in both tissues and periods of the year are C. monoculus and P. tigrinum (piscivorous fish), which means they are great fishes for consumption at any time of the year, as they often accumulate omega-3 fatty acids in large amounts in their tissues to balance the omega 6 amounts. While the carnivorous fish evaluated, the P. squamosissimus specie presents an ideal omega-6/omega-3 ratio at any time of the year only when the animal's muscle is evaluated, being considered an excellent source of omega-3 lipids for human consumption, since the human consumption is based on the muscle tissue. Indeed, once more the popular knowledge showed itself accurate since Amazonian population recommends piscivorous and carnivorous fish as a healthy food source to ill people.
Our data indicate the Amazonian´s fish as beneficial lipid source once corroborates with the idea of that diets enriched in fish oil omega-3 is good for health.
[48] This is based on omega 6 and 3 ratio but also based on the fact that fatty Amazon Thus, our findings suggest that Amazonian fishes have a lipidome with a high nutritional potential to benefit human health, which can vary according to tissue origin (mainly in amount) and is generally sensitive to the eating habits of the species (mainly in quality) and individually sensitive to seasonal changes. The observed high amounts of omega-3 with a significant contribution of omega-6 (mainly linoleic acid) reflect an ideal balance between these fatty acids, which associated with omega-6/omega-3 ratio < 5:1 might be a perfect combination to promote human health. This is a relevant finding, considering that local population consumes high amounts of almost all parts of these fishes (including muscle, head, eyeball, liver, and ova) and that these are acquired in a low cost, favoring the worldwide consumption.