Fatty acid compositions and atherogenic and thrombogenic indices of commonly consumed marine, brackish and fresh water fishes

Marine fish have gained increasing attention because of rich sources of health beneficiary fatty acids (FA), especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Both of these fatty acids are essential and cannot be synthesized de novo and thus are particularly important for human health (Oksuz et al., 2011). Clinical and epidemiological research suggest that EPA and DHA, found only in fish and other seafood, are extremely beneficial in the prevention of human coronary artery disease (Leaf and Weber, 1998). Increasing the intake of unsaturated FAs, while lowering the consumption of saturated fats cause lowering of blood cholesterol in humans (Kinsella,1987). Of the polyunsaturated FAs (PUFA), omega-3 (n-3) plays a role in preventing heart disease and has anti-inflammatory and anti-thrombotic effects (Conner, 2000). Intake of n-3 FA has been shown to lower the atherogenicity plasma index (API), inhibit the aggregation of plaque, diminish the levels of esterified FA, cholesterol and phospholipids and reduce the size of low-density lipoprotein particles. The nutritional value and therapeutic effects of the wide range of health-beneficial FAs found in fish have led to an increased commercial interest in aquaculture of fish species, particularly for their high content of omega FAs (Garaffo et al., 2011). The objective of this study was to investigate fatty acid profiles, the atherogenicity and thrombogenicity indices of the most commonly consumed fishes in Sri Lanka. We also measured the ratio of unsaturated to saturated fat and n-3/n-6 fatty acid as a possible correlate of consumption of fish or fish oil and the effect that consumption may have on the incidence of coronary heart disease.

Twenty-three species of most commonly consumed fishes in Sri Lanka were collected from the urban and local markets of the Batticaloa district of Eastern Province located at longitude 7.73°N and latitude 81.67°E, Sri Lanka. Muscle tissue from each fish was homogenized using a grinder (Sumeet, Japan). Approximate 3 g samples of homogenized muscle from each fish were weighed in triplicate using an analytical balance (AG204, Mettler, Toledo) and placed in dried conical flasks. Muscle tissue samples were hydrolysed by adding 8 mL of distilled water and 10 mL of concentrated HCL and incubated at 95°C in a boiling bath for 45 minutes. The samples were cooled and transferred to Mojonnier flasks. Fat was serially extracted three times with 25mL volumes of Petrolium ether:diethyl ether (1:1 v/v). The upper phase containing the lipids was evaporated to dryness and weighed for further analysis.
       
Fatty acid methyl esters (FAMEs) were prepared from muscle samples from each species. Samples were extracted with iso-octone/methanol (2:1 v/v) according to Folch et al., (1957). The FAs in the total lipid were esterified into methyl esters by saponification with 0.5 N methanolic NaOH and trans-esterified with 14% BF3 (v/v) in methanol (Paquo, 1998). The FAMEs were analysed in gas chromatograph (GC) (Supelco SP -2330 model, Sigma-Aldrich), equipped with a flame ionization detector (FID) and fitted with a capillary column (30 m, 0.25 mm i.d. and 0.2 μm). Injector and detector temperatures were 250°C and 260°C respectively. The oven program was as follows: 100°C for 5 min, linear temperature gradient to 170°C over 10 minutes, then increased to 190°C over 4 minutes and then held at 190°C for 44 min. Total run time was 45 min. The flow rate of the N₂ carrier gas was 1 ml min^-1. GC analysis of FAMEs was repeated three times for each sample. FAMEs were identified by comparison of peak retention times to those of standards (NU prep check- SD 461, USA). Samples were run in split mode (15:1). Results were expressed as FID response area as relative percentages. The results are given as mean ± SD in Table 1.
 

       
The atherogenicity index (AI) and thrombogenicity (TI) indices were calculated from the fatty acid profile, as proposed by Ulbricht and Southgate, (1991), which relates the profile of FAs with the risk of cardiovascular disorders, using the following equations:
   
       
Where C12 = the percentage of lauric acid in relation to total fatty acid (TFA); C14 = the percentage of myristic acid in relation to TFA and C16 = the percentage of palmitic acid in relation to TFA (Table 3).
   
       
Where: MFSA is monounsaturated fatty acid. Data were analysed statistically using one-way analysis of variance (ANOVA), p <0.05, using SPSS 10.0.

We identified nine saturated FAs (SFA), seven monounsaturated FAs (MUFA) and eleven PUFAs in all 23 fishes (Table 1) and total lipids are presented in Table 2. The lipid content of marine and fresh water fishes ranged from1.03% to 20.56%. Amblygaster clupeiodies (20.56%) and Gerres abbreviatus (12.78%) had the highest content of lipid in muscle. Two marine fishes, namely Siganus lineatus and Tricusurus savala had the highest amount of total SFA (52.68 mg g^-1 and 51.84 mg g^-1, respectively). Within SFAs, palmitic acid (C16:0) was the most abundance fatty acids and the highest quantities of which were found in four fishes, S. lineatus (27 mg g^-1), Nibea sp. (26.55 mg g^-1), Wallago attu (26.34 mg g^-1) and T. savala (25.64 mg g^-1). Oleic acid (cis C18:1n9) tended to be the most abundant MUFA, the highest levels being found in two fresh water fishes, Clarius sp. (34.94 mg g^-1) and W attu (33.16 mg g^-1), (Table3). The essential fatty acid DHA (C22:6n3) was the most abundant of 11 PUFAs; four fishes had the highest levels: D acuta (34.73 mg g^-1), T. savala (26.13 mg g^-1), Sphyrenae barracuda (24.08 mg g^-1) and G abbreviatus (24.03 mg g^-1). Among PUFAs, docosadienoic acid (C22:2n6) and adrenic acid (C22:4n6) were identified in 20 fishes. Total PUFAs were significantly lower in fresh water fishes and higher in marine fishes in this study N-3 fatty acid content was the highest in planktivorous fish like A. clupeiodes (26.10 mg g^-1), followed by the carnivorous species, T. savala (25.04 mg g^-1) and G. abbreviatus (22.94 mg g^-1) (Table 3).The n-3 / n-6 ratio was significantly higher in marine fishes (p < 0.05) than in the brackish and fresh water fishes studied the n-3/n-6 ratio is a better index in identifying nutritional value of fish oils of different fishes than n-3 levels alone. AI and TI indices were lower in marine fishes than brackish or fresh water fishes (Fig 1a). Among all fish, G. abbreviatus, which had the very lowest AI (0.01) and TI (0.81), had the highest n-3/ n-6 ratio (Table 3), and thus, consumption of this fish would be expected to be good for cardiovascular patients. S. lineatus (AI=2.68, TI = 1.33) and Stoleophorus commensoni (AI = 2.33, TI = 1.86) had significantly high AI and TI values. Since S. lineatus is a herbivorous fish and S. commensoni is a planktivorous fish, the total SFA content in these fish most influenced their AI and TI values. EPA and DHA level showed significantly higher in marine fishes than brackish and fresh water fishes (Fig1b).
 

 

 

 
The PUFAs were found at high levels in marine fishes whereas the MUFA found high in fresh and brackish water fishes. SFA was high in marine fishes. These results were consistent with those obtained by other researchers (Vlieg and Body, 1988). Pigott and Tucker, (1990) suggested that the n-3/n-6 ratio is a useful indicator for comparing relative nutritional value of fish of different fishes. S. lineatus and A. clupeiodies had high n-3/n-6 fatty acid ratios. It was suggested that ratios of 1:1-1:5 would be in range for a healthy human diet (Osman et al., 2001). All fresh water and marine water fish species studied had n-3/n-6 fatty acid ratios within these recommended values. However, marine fishes had greater n-3/n-6 fatty acid ratios in this study as reported by Hossain, (2011). The ratio of unsaturated FAs (UFA) to SFA ranged from 0.45 to 1.25 in this study. Marine species generally had a ratio greater than one. The essential FAs EPA and DHA were found in all marine fishes and ranged from 23.34 mg g^-1 to 10.7 mg g^-1. Marine water fish species contained high levels of n-3. Similar results obtained by Rasoarahona et al., (2005), fresh water and brackish water fishes had higher n-6 levels and these results agreed with the result obtained by Abouel-Yazeed, (2013). Differences in FAs of marine and fresh water fishes should be considered not only with respect to species habitat, but also based on the natural diet, especially whether a species is herbivorous, omnivorous, planktivorous, or carnivorous (Sargent,1997). Fishes are often classified based on their fat content, according to Bennion, (1980). Based on that classification, lean fish have lower than 5% fat by weight whereas fatty fish have more than 10%. According to another classification scheme described by Greenfield and Southgate (2003), lean fish have <1% total lipid, medium fish have 1-5%, lipids and fatty fish contain more than 5% of lipid. In this study, marine fishes tended to be fatty, with a lipid content of 20.56%, such as herring species. Brackish water and fresh water fish tended to have medium and low lipid content, respectively.
               
In conclusion, this study examined the fatty acid compositions of the fish species most commonly consumed on the eastern coast of Sri Lanka. We found that marine fish were better sources of n-3 FAs, particularly the essential FAs, EPA and DHA, whereas fresh and brackish water fishes were better sources of n-6 FAs. With respect to diet, herbivorous and planktivorous fishes had high levels of n-3 FAs, including EPA and DHA, than their carnivorous counterparts. AI and TI shows high in herbivorous, planktivorous and omnivorous species. Carnivorous species have low AI and TI. These data should be useful to consumers and nutritionists wishing in increase intake of n-3 and n-6 FAs, which have been shown to be associated with ‘heart-healthy’ diets. Understanding the relative lipid profiles of various species of fish will be of use in the application of technological processes for fish preservation, nutritional processing, and value-added development of fish products.