# Quantitative Determination of Fatty Acids in Marine Fish and Shellfish from Warm Water of Straits of Malacca for Nutraceutical Purposes

Authors: Nurnadia Abd Aziz; Azrina Azlan; Amin Ismail; Suryati Mohd Alinafiah; Muhammad Rizal Razman
Journal: BioMed Research International
 (2013)

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## Abstract

This study was conducted to quantitatively determine the fatty acid contents of 20 species of marine fish and four species of shellfish from Straits of Malacca. Most samples contained fairly high amounts of polyunsaturated fatty acids (PUFAs), especially alpha-linolenic acid (ALA, C18:3 n3), eicosapentaenoic acid (EPA, C20:5 n3), and docosahexaenoic acid (DHA, C22:6 n3). Longtail shad, yellowstripe scad, and moonfish contained significantly higher (P<0.05) amounts of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and alpha-linolenic acid (ALA), respectively. Meanwhile, fringescale sardinella, malabar red snapper, black pomfret, Japanese threadfin bream, giant seaperch, and sixbar grouper showed considerably high content (537.2–944.1 mg/100g wet sample) of desirable omega-3 fatty acids. The polyunsaturated-fatty-acids/saturated-fatty-acids (P/S) ratios for most samples were higher than that of Menhaden oil (P/S=0.58), a recommended PUFA supplement which may help to lower blood pressure. Yellowstripe scad (highest DHA, ω-3/ω-6=6.4, P/S=1.7), moonfish (highest ALA, ω-3/ω-6=1.9, P/S=1.0), and longtail shad (highest EPA, ω-3/ω-6=0.8, P/S=0.4) were the samples with an outstandingly desirable overall composition of fatty acids. Overall, the marine fish and shellfish from the area contained good composition of fatty acids which offer health benefits and may be used for nutraceutical purposes in the future.


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## Body

## 1. Introduction

Fish and shellfish are widely accepted as highly nutritious and healthy foods. However, people usually think that different types of fish are of similar nutritional value, and fish selections are made only based on availability, freshness, flavor, and other physical factors [1]. Based on the Malaysian Adult Nutrition Survey (2002-2003), the prevalence of daily consumption of marine fish among rural and urban adults were quite high at 51% and 34%, respectively [2]. Therefore, it is crucial to increase the awareness of different nutrient contents of fish and shellfish species by providing complete nutritional value information, especially for fatty acid content, which is associated with various health-related effects.Nutritional analysis of fatty acids can be classified as qualitative and quantitative. Qualitative analysis of fatty acid produces data regarding the fatty acid composition in the form of percentages of total fatty acids (% of total fatty acids). Meanwhile, quantitative analysis is able to quantify the actual amount (weight) of each fatty acid that is present in the food. Quantitative data are often presented in the form of weight of the fatty acid per weight of food or fat (e.g., mg/g oil). Currently, there are limited qualitative fatty acid data on marine fish and shellfish from warm water area; meanwhile, no quantitative data are available especially as a mean to utilize source of natural product for nutraceutical purposes.Reliable analytical data are prerequisite for correct interpretation of findings in nutritional content analysis, since unreliable results may lead to over- or underestimations, false interpretations, and unwarranted conclusions [3]. Thus, validation procedures of analytical methods were also performed to provide reliable quantitative data on the fatty acid contents of various species of local marine fish and shellfish.


## 2. Materials and Methods

### 2.1. Chemicals and Reagents

All chemicals and reagents used for analysis were of analytical and gas chromatography (GC) grade. Menhaden oil, 37 components FAME mix 47885-U (Supelco, Germany), and tridecanoic acid (C13, internal standard, Sigma, USA) were used as standards in the fatty acid content analysis.


### 2.2. Preparation of Sample

A stratified random sampling procedure was used as it was the most suitable method in database work [4]. To ensure representativeness, ten fish landing areas along the Straits of Malacca were identified with the help of Malaysian Fisheries Development Authority (LKIM). The locations are marked as L1 through L10, respectively (Figure 1).Figure 1
Location of sample collection sites.At each of the collection sites, available samples were collected randomly according to species. All samples were from free-roaming fish and shellfish; and they were collected fresh (caught within a period of 0 to 36 hours). All samples were immediately placed on ice, kept cold, and transported in polystyrene boxes to maintain freshness. Upon arrival at Universiti Putra Malaysia, the temperature of ice boxes was checked to ensure samples were still within the range of −4°C to 0°C. Then, samples for nutrient determination were individually measured for total body weight and length. Only samples of a weight within the narrow range for each species were included as primary samples (Table1). Then, the samples were beheaded, gutted, washed, and filleted. These primary samples were placed in sealed plastic bags and frozen at −20°C. The actual degree of freshness of samples during transportation and time of analysis may not be assured as there was no analysis of freshness or quality index of samples was done. However, appropriate precautions were performed to sustain freshness of samples and minimize oxidation throughout the study by performing procedures in chillers’ room (4°C) and under minimal light exposures.Table 1
List of samples with narrow range of weight, length, and fat content.
Category
Common name
Local name
Scientific name
Habitat
Range ofweight (g)
Range oflength (cm)
Fat content (%)
Lean fish (<2% fat)
Hardtail scad
Cencaru
Megalapsis cordyla
Pelagic
100–250
21–28
1.53
±
0.15
a
Golden snapper
Jenahak
Lutjanu sjohnii
Demersal
490–510
30–35
1.29
±
0.41
a
Indian mackerel
Kembung
Rastrelliger kanagurta
Pelagic
50–100
14–20
1.80
±
0.62
a
Indian threadfin
Kurau
Polynemus indicus
Demersal
350–1450
36–59
0.85
±
0.21
a
Malabar red snapper
Merah
Lutjanus argentimeculatus
Demersal
580–760
28–37
1.37
±
1.10
a
Dorab wolfherring
Parang
Chirocentrus dorab
Pelagic
200–900
40–71
1.22
±
0.22
a
Long-tailed butterfly ray
Pari
Gymnura spp.
Demersal
1300–1700
32–36
0.93
±
0.12
a
Large-scale tongue sole
Sebelah/Lidah
Cynoglossusarel
Demersal
50–100
24–32
0.70
±
0.10
a
Spanish mackerel
Tenggiri papan
Scromberomorus guttatus
Pelagic
200–450
30–42
1.05
±
0.06
a
Low fat fish (2–4% fat)
Black pomfret
Bawal hitam
Parastromateus niger
Pelagic
780–1040
33–42
2.33
±
0.11
a
Silver pomfret
Bawal putih
Pampus argentus
Pelagic
100–200
15–25
2.09
±
0.93
a
Sixbar grouper
Kerapu
Epinephelus fasciatus
Demersal
480–750
33–36
3.46
±
3.46
ab
Japanese threadfin bream
Kerisi
Nemipterus japonicus
Demersal
100–230
18–25
2.70
±
0.37
ab
Yellowstripe scad
Selar kuning
Selaroides leptolepis
Pelagic
50–100
16–20
2.12
±
0.50
a
Gray eel-catfish
Sembilang
Plotosus spp.
Demersal
350–600
40–50
3.04
±
0.59
ab
Fourfinger threadfin
Senangin
Eleutheronema tetradactylum
Pelagic
150–300
27–32
2.10
±
0.25
a
Giant seaperch
Siakap
Lates calcarifer
Demersal
700–1000
38–42
2.68
±
0.79
ab
Fringescale sardinella
Tamban
Clupea fimbriata
Pelagic
20–40
13–17
3.00
±
2.40
ab
Medium fat fish (4–8% fat)
Moonfish
Nyior-nyior
Trachinotus blochii
Demersal
400–1400
31–47
6.89
±
2.76
b
High fat fish (>8% fat)
Longtail shad
Terubuk
Hilsa macrura
Pelagic
928
40–45
23.15
±
0.00
c
Shellfish
Cuttlefish
Sotong
Sepia officinalis
—
20–45
12–18
1.35
±
0.28
a
Prawn
Udang putih
Metapenaeus affinis
—
10–20
12–17
1.06
±
0.10
a
Cockles
Kerang
Anadara granosa
—
10–20
2–5
1.93
±
1.28
a
Oyster
Tiram
Ostrea spp.
—
100–300
14–48
1.24
±
0.00
a
1Categories of fish samples were based on the fat content [5].
2Different superscript lowercase letters (a–c) in the same column show significant differences at P<0.05 (Tukey post-hoc test) [5].A small-scale experiment performed independently showed insignificant differences in nutrient contents of samples from different locations. This observation justified that units of primary samples can be combined or composited by geographical locations to minimize the number of analytical measurements and yet represent the contribution of the unit to the estimate of central tendency [4]. Thus, before analysis, three composite samples were prepared by mixing individual samples (n=8–12, whole fillets) of the same weight for each species. Individual samples from L1, L2, L3, and L4 constituted as Composite 1; individual samples from L5, L6, and L7 constituted as Composite 2; while individual samples from L8, L9, and L10 constituted as Composite 3 (Figure 1). All composite samples were analyzed separately and data presented are the mean values of each of the species.


### 2.3. Extraction of Fat

Extraction of fat was done following Bligh and Dyer [6], with slight modifications by Kinsella et al. [7]. Representative samples of fish filets (30 g) were homogenized in a Waring blender for 2 min with a mixture of methanol (60 mL) and chloroform (30 mL). One volume of chloroform (30 mL) was added to the mixture and after blending for additional 30 seconds, distilled water (30 mL) was added. The homogenate was stirred with a glass rod and filtered through Whatman No. 1 filter paper on a Buchner funnel with slight suction. The filtrate was transferred to a separatory funnel. The lower clear phase was drained into a 250 mL round-bottom flask and concentrated with a rotary evaporator at 40°C. To minimize oxidation, the extracted lipids were kept in solvents containing 0.05% butylated hydroxytoluene (B.H.T.), in glass bottles, flushed with nitrogen, and wrapped with aluminium foils to avoid any light exposure. Then, the bottles were stored immediately at −20°C and only being taken out from freezer just before analysis.


### 2.4. Analysis of Fatty Acid Content

Lipid samples were converted to their constituent FAMEs following the method used in previous study [8]. Approximately 25 mg (±0.1 mg) of oil was weighed and added with 1.5 mL of NaOH 0.50 M in methanol in a 15 mL capped centrifuge tube. The mixture was heated in a water bath at 100°C for 5 min and then cooled at room temperature. The mixture was added with 2.0 mL of boron trifluoride (BF3, 12%) in methanol and heated again in a water bath at 100°C for 30 minutes. Next, the tube was cooled in running water at room temperature before adding 1 mL of isooctane. It was vigorously stirred for 30 seconds before adding 5.0 mL of a saturated sodium chloride solution to facilitate the phase separation. The esterified sample was placed in a refrigerator and left to rest for better phase separation. After collecting the supernatant, another 1.0 mL of isooctane (containing 0.05% B.H.T. as antioxidant) was added into the tube and stirred. The supernatant was collected and added to the previous fraction. The sample was concentrated to a final volume of 1.0 mL for later injection into the gas chromatograph. As precautions, amber vials were used in order to minimize oxidation during analysis.Analysis of Gas Chromatography (GC). Analysis of methyl esters was performed by a capillary gas chromatograph model Agilent 6890 (USA Agilent Technology) equipped with a split-splitless injector, flame ionization detection (FID) system used to separate and quantify each FAME component. FAMEs were separated using a highly polar HP88 column (Agilent, USA) column (100 m length × 0.25 mm I.D. × 0.2 μm D.F.). Carrier gas was helium at a linear velocity of 30.0 mL/min. Split injection with a split ratio (volume of gas passing to waste: volume of gas passing down the capillary column) of 10 : 1 and 99.9 mL/min split flow was used. The operating conditions were 250°C injection port, 250°C flame ionization detector, and 200°C column temperature. Compounds were identified by comparison of the retention times of 37 components FAME mix 47885-U (Supelco, Germany) and Menhaden oil standards (Supelco, Germany).Quantification of Fatty Acids. The concentration of fatty acids in mg/g of total lipids was measured against tridecanoic acid methyl ester (C13:0, Sigma, USA) as an internal standard. This followed procedures described in a previous study [6] with slight modifications; in which the Empirical Response Factor (Ri) of FID (flame ionization detector) was calculated and used instead of theoretical response factor (CFX). Calculation of the empirical response factor (Ri) was done as described in the literature [9]. The following formulae were used in the quantification of fatty acids: (i)
(1)empiricalresponsefactor(Ri)=(Psi)(WISm)(PsIS)(Wi),where Psi: peak area of individual fatty acids in mixed FAME standard solution, PsIS: peak area of fatty acid internal standard in mixed FAME standard solution, WISm: weight of fatty acid internal standard in mixed FAME standard solution, and Wi: weight of individual FAME in mixed FAME standard solution;(ii)
(2)fattyacid(mg/g)totallipid=(AX)(WIS)(Ri)(1000)(AIS)(WX)(1.04),where AX: peak area of fatty acid, AIS: peak area of internal standard (IS) WIS: weight (mg) of IS added to the sample (mg), WX: sample weight (mg), Ri: empirical response factor, and 1.04: conversion factor necessary to express results in mg fatty acid/g oil (rather than as methyl ester).


### 2.5. Analysis of Method Validation

Linearity Test. Three fatty acid standards, myristic acid, heptadecanoic acid, and linoleic acid were prepared at different concentrations: myristic acid (0.4, 2.0, and 4.0 mg/mL), heptadecanoicacid (0.4, 2.0, and 4.0 mg/mL), linoleic acid (0.5, 1.0, and 5.0 mg/mL). These fatty acid standards were used as they represent both saturated and unsaturated fatty acids present in samples. Calibration curves were formed for each of the compounds. The linearity parameters, which included linear regression (y=mx+c) and the correlation coefficient (R2) were obtained from the linear relationship between the peak area and the concentrations of the fatty acid standards.Precision Test. Two precision tests of repeatability (within-day) and reproducibility (between-day) were performed. Two fatty acid standards were quantified in three randomly selected samples in both tests. For repeatability (within-day precision), four replicates of each sample were analyzed in a single day using the same procedures as in the fatty acid analysis. Data are reported as relative standard deviations (RSD) of four replicates, with minimum and maximum values of both palmitic and linolenic acids quantified in each sample. For reproducibility (between-day precision), samples were analyzed using the same procedures as in the fatty acid analysis on three different days, representing three replicates of each sample. Palmitic acid and linolenic acids were quantified and presented as relative standard deviations (RSD) of three replicates, with minimum and maximum values of each fatty acid.Recovery Test. The recovery of the method was determined using three different concentrations (0.4, 2.0, 4.0 mg/mL) of myristic acid, heptadecanoic acid, and stearic acid standards. Two samples were randomly selected and used for the recovery analysis of each of the fatty acid standards. During the sample preparation, each standard was added together with lipid extract and internal standards and prepared following the same procedures used in normal sample preparation. Recoveries of different standards were performed separately to avoid overlapping of peaks in chromatograms which could lead to biased results. Recoveries of fatty acid standards at different concentrations were determined by comparing the content of the fatty acids in samples with and without the addition of the standard. Data are presented as the percentage of recovery.Statistical Analysis. Data were analyzed using SPSS (Scientific Package of Social Science) version 17.0. The mean, standard deviation (SD), and one-way ANOVA test followed by Tukey post-hoc analysis were performed to compare differences in the mean fatty acid contents of different species of fish and shellfish. Bivariate correlation (Pearson’s r) was performed to explore the relationships between different fatty acid classes in samples.


## 2.1. Chemicals and Reagents

All chemicals and reagents used for analysis were of analytical and gas chromatography (GC) grade. Menhaden oil, 37 components FAME mix 47885-U (Supelco, Germany), and tridecanoic acid (C13, internal standard, Sigma, USA) were used as standards in the fatty acid content analysis.


## 2.2. Preparation of Sample

A stratified random sampling procedure was used as it was the most suitable method in database work [4]. To ensure representativeness, ten fish landing areas along the Straits of Malacca were identified with the help of Malaysian Fisheries Development Authority (LKIM). The locations are marked as L1 through L10, respectively (Figure 1).Figure 1
Location of sample collection sites.At each of the collection sites, available samples were collected randomly according to species. All samples were from free-roaming fish and shellfish; and they were collected fresh (caught within a period of 0 to 36 hours). All samples were immediately placed on ice, kept cold, and transported in polystyrene boxes to maintain freshness. Upon arrival at Universiti Putra Malaysia, the temperature of ice boxes was checked to ensure samples were still within the range of −4°C to 0°C. Then, samples for nutrient determination were individually measured for total body weight and length. Only samples of a weight within the narrow range for each species were included as primary samples (Table1). Then, the samples were beheaded, gutted, washed, and filleted. These primary samples were placed in sealed plastic bags and frozen at −20°C. The actual degree of freshness of samples during transportation and time of analysis may not be assured as there was no analysis of freshness or quality index of samples was done. However, appropriate precautions were performed to sustain freshness of samples and minimize oxidation throughout the study by performing procedures in chillers’ room (4°C) and under minimal light exposures.Table 1
List of samples with narrow range of weight, length, and fat content.
Category
Common name
Local name
Scientific name
Habitat
Range ofweight (g)
Range oflength (cm)
Fat content (%)
Lean fish (<2% fat)
Hardtail scad
Cencaru
Megalapsis cordyla
Pelagic
100–250
21–28
1.53
±
0.15
a
Golden snapper
Jenahak
Lutjanu sjohnii
Demersal
490–510
30–35
1.29
±
0.41
a
Indian mackerel
Kembung
Rastrelliger kanagurta
Pelagic
50–100
14–20
1.80
±
0.62
a
Indian threadfin
Kurau
Polynemus indicus
Demersal
350–1450
36–59
0.85
±
0.21
a
Malabar red snapper
Merah
Lutjanus argentimeculatus
Demersal
580–760
28–37
1.37
±
1.10
a
Dorab wolfherring
Parang
Chirocentrus dorab
Pelagic
200–900
40–71
1.22
±
0.22
a
Long-tailed butterfly ray
Pari
Gymnura spp.
Demersal
1300–1700
32–36
0.93
±
0.12
a
Large-scale tongue sole
Sebelah/Lidah
Cynoglossusarel
Demersal
50–100
24–32
0.70
±
0.10
a
Spanish mackerel
Tenggiri papan
Scromberomorus guttatus
Pelagic
200–450
30–42
1.05
±
0.06
a
Low fat fish (2–4% fat)
Black pomfret
Bawal hitam
Parastromateus niger
Pelagic
780–1040
33–42
2.33
±
0.11
a
Silver pomfret
Bawal putih
Pampus argentus
Pelagic
100–200
15–25
2.09
±
0.93
a
Sixbar grouper
Kerapu
Epinephelus fasciatus
Demersal
480–750
33–36
3.46
±
3.46
ab
Japanese threadfin bream
Kerisi
Nemipterus japonicus
Demersal
100–230
18–25
2.70
±
0.37
ab
Yellowstripe scad
Selar kuning
Selaroides leptolepis
Pelagic
50–100
16–20
2.12
±
0.50
a
Gray eel-catfish
Sembilang
Plotosus spp.
Demersal
350–600
40–50
3.04
±
0.59
ab
Fourfinger threadfin
Senangin
Eleutheronema tetradactylum
Pelagic
150–300
27–32
2.10
±
0.25
a
Giant seaperch
Siakap
Lates calcarifer
Demersal
700–1000
38–42
2.68
±
0.79
ab
Fringescale sardinella
Tamban
Clupea fimbriata
Pelagic
20–40
13–17
3.00
±
2.40
ab
Medium fat fish (4–8% fat)
Moonfish
Nyior-nyior
Trachinotus blochii
Demersal
400–1400
31–47
6.89
±
2.76
b
High fat fish (>8% fat)
Longtail shad
Terubuk
Hilsa macrura
Pelagic
928
40–45
23.15
±
0.00
c
Shellfish
Cuttlefish
Sotong
Sepia officinalis
—
20–45
12–18
1.35
±
0.28
a
Prawn
Udang putih
Metapenaeus affinis
—
10–20
12–17
1.06
±
0.10
a
Cockles
Kerang
Anadara granosa
—
10–20
2–5
1.93
±
1.28
a
Oyster
Tiram
Ostrea spp.
—
100–300
14–48
1.24
±
0.00
a
1Categories of fish samples were based on the fat content [5].
2Different superscript lowercase letters (a–c) in the same column show significant differences at P<0.05 (Tukey post-hoc test) [5].A small-scale experiment performed independently showed insignificant differences in nutrient contents of samples from different locations. This observation justified that units of primary samples can be combined or composited by geographical locations to minimize the number of analytical measurements and yet represent the contribution of the unit to the estimate of central tendency [4]. Thus, before analysis, three composite samples were prepared by mixing individual samples (n=8–12, whole fillets) of the same weight for each species. Individual samples from L1, L2, L3, and L4 constituted as Composite 1; individual samples from L5, L6, and L7 constituted as Composite 2; while individual samples from L8, L9, and L10 constituted as Composite 3 (Figure 1). All composite samples were analyzed separately and data presented are the mean values of each of the species.


## 2.3. Extraction of Fat

Extraction of fat was done following Bligh and Dyer [6], with slight modifications by Kinsella et al. [7]. Representative samples of fish filets (30 g) were homogenized in a Waring blender for 2 min with a mixture of methanol (60 mL) and chloroform (30 mL). One volume of chloroform (30 mL) was added to the mixture and after blending for additional 30 seconds, distilled water (30 mL) was added. The homogenate was stirred with a glass rod and filtered through Whatman No. 1 filter paper on a Buchner funnel with slight suction. The filtrate was transferred to a separatory funnel. The lower clear phase was drained into a 250 mL round-bottom flask and concentrated with a rotary evaporator at 40°C. To minimize oxidation, the extracted lipids were kept in solvents containing 0.05% butylated hydroxytoluene (B.H.T.), in glass bottles, flushed with nitrogen, and wrapped with aluminium foils to avoid any light exposure. Then, the bottles were stored immediately at −20°C and only being taken out from freezer just before analysis.


## 2.4. Analysis of Fatty Acid Content

Lipid samples were converted to their constituent FAMEs following the method used in previous study [8]. Approximately 25 mg (±0.1 mg) of oil was weighed and added with 1.5 mL of NaOH 0.50 M in methanol in a 15 mL capped centrifuge tube. The mixture was heated in a water bath at 100°C for 5 min and then cooled at room temperature. The mixture was added with 2.0 mL of boron trifluoride (BF3, 12%) in methanol and heated again in a water bath at 100°C for 30 minutes. Next, the tube was cooled in running water at room temperature before adding 1 mL of isooctane. It was vigorously stirred for 30 seconds before adding 5.0 mL of a saturated sodium chloride solution to facilitate the phase separation. The esterified sample was placed in a refrigerator and left to rest for better phase separation. After collecting the supernatant, another 1.0 mL of isooctane (containing 0.05% B.H.T. as antioxidant) was added into the tube and stirred. The supernatant was collected and added to the previous fraction. The sample was concentrated to a final volume of 1.0 mL for later injection into the gas chromatograph. As precautions, amber vials were used in order to minimize oxidation during analysis.Analysis of Gas Chromatography (GC). Analysis of methyl esters was performed by a capillary gas chromatograph model Agilent 6890 (USA Agilent Technology) equipped with a split-splitless injector, flame ionization detection (FID) system used to separate and quantify each FAME component. FAMEs were separated using a highly polar HP88 column (Agilent, USA) column (100 m length × 0.25 mm I.D. × 0.2 μm D.F.). Carrier gas was helium at a linear velocity of 30.0 mL/min. Split injection with a split ratio (volume of gas passing to waste: volume of gas passing down the capillary column) of 10 : 1 and 99.9 mL/min split flow was used. The operating conditions were 250°C injection port, 250°C flame ionization detector, and 200°C column temperature. Compounds were identified by comparison of the retention times of 37 components FAME mix 47885-U (Supelco, Germany) and Menhaden oil standards (Supelco, Germany).Quantification of Fatty Acids. The concentration of fatty acids in mg/g of total lipids was measured against tridecanoic acid methyl ester (C13:0, Sigma, USA) as an internal standard. This followed procedures described in a previous study [6] with slight modifications; in which the Empirical Response Factor (Ri) of FID (flame ionization detector) was calculated and used instead of theoretical response factor (CFX). Calculation of the empirical response factor (Ri) was done as described in the literature [9]. The following formulae were used in the quantification of fatty acids: (i)
(1)empiricalresponsefactor(Ri)=(Psi)(WISm)(PsIS)(Wi),where Psi: peak area of individual fatty acids in mixed FAME standard solution, PsIS: peak area of fatty acid internal standard in mixed FAME standard solution, WISm: weight of fatty acid internal standard in mixed FAME standard solution, and Wi: weight of individual FAME in mixed FAME standard solution;(ii)
(2)fattyacid(mg/g)totallipid=(AX)(WIS)(Ri)(1000)(AIS)(WX)(1.04),where AX: peak area of fatty acid, AIS: peak area of internal standard (IS) WIS: weight (mg) of IS added to the sample (mg), WX: sample weight (mg), Ri: empirical response factor, and 1.04: conversion factor necessary to express results in mg fatty acid/g oil (rather than as methyl ester).


## 2.5. Analysis of Method Validation

Linearity Test. Three fatty acid standards, myristic acid, heptadecanoic acid, and linoleic acid were prepared at different concentrations: myristic acid (0.4, 2.0, and 4.0 mg/mL), heptadecanoicacid (0.4, 2.0, and 4.0 mg/mL), linoleic acid (0.5, 1.0, and 5.0 mg/mL). These fatty acid standards were used as they represent both saturated and unsaturated fatty acids present in samples. Calibration curves were formed for each of the compounds. The linearity parameters, which included linear regression (y=mx+c) and the correlation coefficient (R2) were obtained from the linear relationship between the peak area and the concentrations of the fatty acid standards.Precision Test. Two precision tests of repeatability (within-day) and reproducibility (between-day) were performed. Two fatty acid standards were quantified in three randomly selected samples in both tests. For repeatability (within-day precision), four replicates of each sample were analyzed in a single day using the same procedures as in the fatty acid analysis. Data are reported as relative standard deviations (RSD) of four replicates, with minimum and maximum values of both palmitic and linolenic acids quantified in each sample. For reproducibility (between-day precision), samples were analyzed using the same procedures as in the fatty acid analysis on three different days, representing three replicates of each sample. Palmitic acid and linolenic acids were quantified and presented as relative standard deviations (RSD) of three replicates, with minimum and maximum values of each fatty acid.Recovery Test. The recovery of the method was determined using three different concentrations (0.4, 2.0, 4.0 mg/mL) of myristic acid, heptadecanoic acid, and stearic acid standards. Two samples were randomly selected and used for the recovery analysis of each of the fatty acid standards. During the sample preparation, each standard was added together with lipid extract and internal standards and prepared following the same procedures used in normal sample preparation. Recoveries of different standards were performed separately to avoid overlapping of peaks in chromatograms which could lead to biased results. Recoveries of fatty acid standards at different concentrations were determined by comparing the content of the fatty acids in samples with and without the addition of the standard. Data are presented as the percentage of recovery.Statistical Analysis. Data were analyzed using SPSS (Scientific Package of Social Science) version 17.0. The mean, standard deviation (SD), and one-way ANOVA test followed by Tukey post-hoc analysis were performed to compare differences in the mean fatty acid contents of different species of fish and shellfish. Bivariate correlation (Pearson’s r) was performed to explore the relationships between different fatty acid classes in samples.


## 3. Results and Discussion

### 3.1. Fatty Acid Content in Samples

Data from this study are reported in the form of milligrams per 100 grams of wet muscle. Tables2, 3, and 4 show the sample content of saturated, monounsaturated, and polyunsaturated fatty acids, respectively.Table 2
Saturated fatty acids (SFAs) content of samples.
Category
Samples
Saturated fatty acids (SFA) content (mg/100 g wet sample)
8:0
12:0
14:0
15:0
16:0
17:0
18:0
24:0
∑SFA
Lean fish(<2% fat)
Hardtail scad
42.2ab
1.4abcd
69.8a
18.6abc
531.7a
45.2a
nd
nd
708.8
Golden snapper
152.9bcd
0.7abc
9.6a
8.5ab
203.7a
21.5a
1.8a
20.1abcd
418.7
Indian mackarel
139.0abcd
0.7abc
64.1a
9.5ab
313.7a
22.8a
35.7bcd
2.3ab
587.8
Indian threadfin
98.9abcd
0.9abc
10.3a
6.8a
134.7a
17.7a
4.8ab
33.4bcde
307.4
Malabar red snapper
112.8abcd
0.9abc
28.8a
5.7a
373.7a
31.8a
nd
4.3ab
557.9
Dorab wolfherring
72.2abc
1.4abcd
39.3a
7.6a
395.9a
21.1a
nd
nd
537.4
Long-tailed butterfly ray
140.1abcd
nd
4.6a
2.9a
129.8a
11.3a
nd
nd
288.8
Large-scale tongue sole
80.7abc
0.3ab
7.5a
5.4a
95.1a
15.2a
1.5a
36.8cde
242.4
Spanish mackarel
86.3abcd
0.4ab
16.1a
5.9a
183.7a
9.5a
nd
21.0abcde
322.8
Low fat fish(2–4% fat)
Black pomfret
214.8d
nd
46.9a
18.0abc
549.5a
48.5a
6.9ab
50.4def
935.0
Silver pomfret
40.0ab
1.1abc
102.4a
15.5ab
590.2a
38.6a
11.2abc
79.7f
878.7
Sixbar grouper
186.1cd
1.6abcd
65.6a
19.8abc
775.6a
62.1a
6.4ab
191.6g
1308.7
Japanese threadfin bream
34.0ab
5.2de
102.6a
28.2abcd
996.5a
36.8a
8.0ab
nd
1211.2
Yellowstripe scad
101.1abcd
1.7abcd
69.5a
19.9abc
560.8a
65.7a
nd
10.9ab
829.6
Gray eel-catfish
35.3ab
3.9bcde
159.7a
24.3abc
717.8a
359.0a
40.9cd
nd
1340.8
Fourfinger threadfin
52.9ab
2.5abcd
127.2a
33.7bcd
677.0a
31.9a
7.0ab
21.1abcde
953.4
Giant seaperch
79.5abc
4.4cde
102.5a
15.5ab
928.2a
76.7a
8.0ab
52.4ef
1267.2
Fringescale sardinella
21.5a
2.2abcd
242.0a
43.1cd
822.2a
108.1a
33.5bcd
nd
1272.8
Medium fat fish (4–8% fat)
Moonfish
383.9e
7.2e
335.3a
53.4d
1308.0a
431.6a
31.1abcd
21.9abcde
2572.4
High fat fish (>8% fat)
Longtail shad
20.3a
21.1f
2805.4b
113.8e
12542.6b
2034.5b
82.5e
nd
17620.2
Shellfish
Cuttlefish
93.7abcd
0.7abc
30.9a
9.3ab
394.5a
7.0a
nd
33.0bcde
569.1
Prawn
139.9abcd
0.3ab
7.0a
11.4ab
125.3a
24.5a
nd
nd
308.4
Cockles
37.0ab
2.8abcd
69.9a
12.4ab
359.2ª
130.3ª
9.5abc
22.2abcde
643.3
Oyster
30.2ab
0.5abc
41.4ª
10.0ab
325.8ª
80.3a
62.6de
13.0abc
563.7
1Categories of fish samples were based on the fat content [5].
2Different superscript lowercase letters (a–g) in same column show significant differences at P<0.05 (Tukey post-hoc test).
3nd: not detected.Table 3
Monounsaturated fatty acids (MUFAs) content of samples.
Category
Samples
Monounsaturated fatty acids (MUFAs) content (mg/100 g wet sample)
14:1
15:1
16:1
17:1
18:1 n9c
∑MUFA
Lean fish (<2% fat)
Hardtail scad
2.5abc
1.5ab
7.5abc
45.7ef
63.9ab
121.2
Golden snapper
2.7abc
4.6abc
40.7de
6.6ab
39.5a
94.0
Indian mackarel
1.8abc
7.5c
116.4f
10.4ab
158.7ab
294.8
Indian threadfin
1.0ab
1.3ab
3.3ab
14.1abc
18.5a
38.3
Malabar red snapper
1.0ab
1.0ab
4.4ab
16.0abc
119.5ab
141.8
Dorab wolfherring
1.0ab
0.6a
1.1a
20.1abcd
123.1ab
145.9
Long-tailed butterfly ray
1.6abc
0.8a
1.9ab
7.7ab
72.2ab
84.6
Large-scale tongue sole
0.6a
1.4ab
nd
10.6ab
20.8a
33.4
Spanish mackarel
0.4a
0.3a
2.8ab
13.8abc
53.2ab
70.6
Low fat fish (2–4% fat)
Black pomfret
1.8abc
4.1abc
13.6abcd
20.6abcd
98.8ab
138.8
Silver pomfret
1.6abc
1.2ab
30.9bcde
36.0cdef
81.5ab
151.1
Sixbar grouper
4.5bcd
15.7d
116.0f
nd
151.0ab
287.2
Japanese threadfin bream
6.9de
1.5ab
105.3f
28.8bcdef
144.7ab
287.2
Yellowstripe scad
2.8abc
1.9ab
43.4e
49.9f
188.8ab
286.8
Gray eel-catfish
11.3f
5.7bc
nd
47.4ef
103.7ab
168.1
Fourfinger threadfin
5.1cd
2.4ab
35.3cde
43.2def
241.5ab
327.4
Giant seaperch
3.6abcd
2.6ab
43.4e
25.2bcde
155.3ab
230.0
Fringescale sardinella
3.5abcd
3.5abc
53.0e
51.3f
192.0ab
303.3
Medium fat fish (4–8% fat)
Moonfish
9.5ef
14.8d
nd
81.5g
398.5b
504.2
High fat fish (>8% fat)
Longtail shad
10.7ef
nd
458.1g
36.1cdef
2554.4c
3059.3
Shellfish
Cuttlefish
0.8ab
2.62ab
4.0ab
17.2abc
89.5ab
114.2
Prawn
0.7a
1.67ab
7.4abc
16.9abc
87.8ab
114.5
Cockles
2.9abc
13.52d
nd
75.9g
303.8ab
396.1
Oyster
0.7ab
2.65ab
nd
25.1bcde
53.9ab
82.4
1Categories of fish samples were based on the fat content [5].
2Different superscript lowercase letters (a–g) in same column show significant differences at P<0.05 (Tukey post-hoc test).
3nd: not detected.Table 4
Polyunsaturated fatty acids (PUFA) content of samples.
Category
Samples
Polyunsaturated fatty acids (PUFA) content (mg/100 g wet sample)
18:2 n6t
18:2 n6c
18:3 n6
18:3 n3
20:3 n3
20:5 n3
22:6 n3
∑PUFA
Lean fish(<2% fat)
Hardtail scad
22.5a
30.8a
1.6a
117.3a
nd
18.9a
196.0cdefg
387.0
Golden snapper
105.2ab
9.6a
nd
365.6ab
nd
7.3a
18.6ab
506.3
Indian mackarel
23.1a
29.3a
nd
53.9a
7.3ab
53.6a
23.3ab
190.5
Indian threadfin
33.2a
5.2a
nd
271.7a
nd
23.7a
82.2abcde
415.9
Malabar red snapper
86.1ab
60.4a
nd
335.0ab
9.2ab
24.1a
209.9defg
724.7
Dorab wolfherring
36.3a
6.4a
nd
204.0a
11.6ab
23.9a
54.3abcd
336.5
Long-tailed butterfly ray
11.2a
15.1a
nd
337.5ab
nd
2.7a
9.0a
375.5
Large-scale tongue sole
25.9a
4.3a
nd
111.6a
2.5ab
8.3a
113.4abcde
266.2
Spanish mackarel
18.2a
19.7a
nd
178.9a
nd
27.7a
69.8abcde
314.2
Low fat fish(2–4% fat)
Black pomfret
16.8a
45.1a
29.06c
272.8a
nd
73.5a
277.1fg
714.3
Silver pomfret
104.7ab
49.5a
10.91b
136.2a
6.1ab
116.3a
148.0abcdefg
571.6
Sixbar grouper
139.2ab
119.3a
nd
645.5b
nd
100.9a
197.7cdefg
1202.5
Japanese threadfin bream
81.6ab
51.0a
nd
112.2a
nd
258.7a
293.0g
796.5
Yellowstripe scad
78.6ab
113.6a
nd
338.1ab
7.5ab
97.1a
782.05h
1417.0
Gray eel-catfish
257.4ab
133.6a
nd
185.0a
nd
145.8a
88.8abcde
810.7
Fourfinger threadfin
148.3ab
48.7a
nd
114.3a
nd
96.2a
53.1abc
460.6
Giant seaperch
142.5ab
94.4a
nd
245.6a
215.7d
139.5a
95.4abcde
933.0
Fringescale sardinella
33.2a
164.2a
nd
69.3a
31.0c
211.5a
225.4efg
734.6
Medium fat fish (4–8% fat)
Moonfish
637.7ab
215.3a
nd
1046.8c
257.6e
176.7a
122.6abcdef
2456.7
High fat fish (>8% fat)
Longtail shad
2254.1b
1327.4b
nd
68.9a
437.5f
2041.8b
168.7bcdefg
6298.4
Shellfish
Cuttlefish
13.4a
17.5a
nd
251.1a
14.9b
121.9a
85.0abcde
503.8
Prawn
16.3a
31.3a
nd
320.4ab
nd
48.7a
45.3abc
461.9
Cockles
71.5ab
55.0a
nd
18.6a
nd
343.0a
123.4abcdef
611.4
Oyster
62.0a
35.9a
nd
100.7a
nd
54.0a
82.8abcde
335.4
1Categories of fish samples were based on the fat content [5].
2Different superscript lowercase letters (a–h) in same column show significant differences at P<0.05 (Tukey post-hoc test).
3nd: not detected.Based on the previous findings, the studied fish samples can be categorized into lean-, low-, medium-, and high-fat fish based on the fat content [5]. Among the lean-fish, hardtail scad was high in saturated fatty acids (SFAs), with palmitic acid as the highest (531.7 mg/100 g wet sample) (Table 2). The monounsaturated fatty acid (MUFA) of the fish was 121.2 mg/100 g wet sample; with fairly high oleic acid (C18:1n9c) and heptadecenoic acid (C17:1), at 63.9 and 45.7 mg/100 g wet sample, respectively (Table 3). For PUFA, hardtail scad contained quite high amounts of DHA (C22:6n3), at 196.0 mg/100 g wet sample, followed by ALA (C18:3n3) at 117.3 mg/100 g wet sample (Table 4). Meanwhile, malabar red snapper was higher in PUFA, characterized by high DHA (C22:6n3) at 209.9 mg/100 g wet sample, followed by ALA (C18:3n3) at 335.0 mg/100 g wet sample (Table 4). For SFA, palmitic acid was the dominant fatty acid (373.7 mg/100 g wet sample), followed by caprylic acid at 112.8 mg/100 g wet sample (Table 2). The MUFA content was 141.8 mg/100 g wet sample, with fairly high oleic acid (C18:1 n9c) at 119.5 mg/100 g wet sample (Table 3).Among the low-fat fish, yellowstripe scad contained a very high PUFA content, with significantly higher (P<0.05) levels of DHA (782.1 mg/100 g wet sample) compared to other samples (Table 4). The DHA amount was about 2.7 times higher than Japanese threadfin bream (2nd highest DHA, 293.0 mg/100 g wet sample); and about 86.6 times higher than long-tailed butterfly ray (lowest DHA, 9.0 mg/100 g wet sample). Additionally, yellowstripe scad also had a high ALA (C18:3n3) level at 338.1 mg/100 g wet sample. For SFA and MUFA, the most dominant fatty acids were palmitic acid (560.8 mg/100 g wet sample) and oleic acid (188.8 mg/100 g wet sample), respectively (Tables 2 and 3).The medium-fat fish, moonfish, showed palmitic acid as the most prominent fatty acid (1308.0 mg/100 g wet sample) (Table2). This was followed by α-linolenic acid (C18:3n3) at 1046.8 mg/100 g wet sample, which was significantly higher (P<0.05) compared to the others (Table 4). Other ω-3 fatty acids, such as eicosatrienoic acid (C20:3n3), EPA (C20:5n3), and DHA (C22:6n3) were at 257.6, 176.7, and 122.6 mg/100 g wet sample, respectively. Its ω-6 fatty acid content, linolelaidic acid (C18:2n6t, 637.7 mg/100 g wet sample) and linoleic acid (C18:2n6c, 215.3 mg/100 g wet sample), were the second highest after level in longtail shad (linolelaidic acid at 2254.1 mg/100 g and linoleic acid at 1327.4 mg/100 g wet sample), respectively. Meanwhile, for MUFA, the most dominant fatty acid was oleic acid (C18:1n9c), at 398.5 mg/100 g wet sample. Moonfish also contained significantly higher (Tukey post-hoc test, P<0.05) of heptadecenoic acid (C17:1, 81.5 mg/100 g wet sample) compared to others (Table 3).The high-fat fish, longtail shad also showed palmitic acid (C16:0) as the dominant fatty acid (12542.6 mg/100 g wet muscle, Table2). For MUFA, longtail shad contained significantly higher (Tukey post-hoc test, P<0.05) of palmitoleic acid (C16:1, 458.1 mg/100 g wet sample) and oleic acid (C18:1n9c, 2554.4 mg/100 g wet sample) compared to others (Table 3). While for PUFA, the fish contained significantly higher (Tukey post-hoc test, P<0.05) of EPA (C20:5 n3, 2041.8 mg/100 g wet sample) and eicosatrienoicacid (C20:3n3, 437.5 mg/100 g wet sample) compared to others (Table 4). Besides, longtail shad was also high in ω-6 fatty acids; with linolelaidic acid (C18:2n6t) at 2254.1 mg/100 g wet muscle, and linoleic acid (C18:2n6c) at 1327.4 mg/100 g wet muscle.As in most of fish samples, shellfish including cuttlefish, cockles, and oyster also showed palmitic acid (C16:0) as the most prominent fatty acid (Table2). Meanwhile, prawn showed caprylic acid (C8:0) as the highest fatty acid (139.9 mg/100 g wet sample), followed by palmitic acid (C16:0) at 125.3 mg/100 g wet sample. For MUFA, cockles contained higher oleic acid (C18:1n9c, 303.8 mg/100 g wet sample) compared to other shellfish (Table 3). While for PUFA, cockles were found to be higher in both EPA (C20:5n3, 343.0 mg/100 g wet sample) and DHA (C22:6n3, 123.4 mg/100 g wet sample), compared to other shellfish (Table 4). However, the content of alpha-linolenic acid (C18:3n3) in cockles (18.6 mg/100 g wet sample) was the lowest among all samples; with cuttlefish (251.1 mg/100 g wet sample), prawn (320.4 mg/100 g wet sample), and oysterr (100.7 mg/100 g wet sample).


### 3.2. Fatty Acids of Fish and Shellfish from Local and Other Countries

Most of previous local studies have focused on qualitative aspects of fat content in various samples of marine and freshwater origin samples [10–15]. A previous study found very high percentage of PUFA; with omega-3 PUFA (29.7–48.4%), other PUFA (27.7–40.0%), and omega-6 PUFA (11.0–20.0%) in ten common fish species with the current study [9]. Meanwhile, percentages of SFA and MUFA were quite low; at 3.63–11.4% and 1.37–9.12%, respectively. This study found hexadecadienoic acid as the most prominent fatty acid (18.1–24.9%) in six samples, hexadecatrienoic acid (C16:3n4) in two samples, and hexadecadienoic acid (C16:2) in other two samples [10]. Another local study reported PUFA as the dominant group of fatty acid in seven species, and SFA as the dominant fatty acid group in another six species of their fish samples [11]. Palmitic acid (C16:0) was the most prominent fatty acid (17.6–32.1%) in eight samples; meanwhile, DHA (C22:6n3) was the highest fatty acid (19.3–24.0%) in another five samples [11]. Same findings were reported by other study of 16 species of local pelagic fish; with palmitic acid (C16:0) as the most prominent fatty acid (20.9–34.5%) in nine species, meanwhile, DHA (C22:6n3) was the highest fatty acid (26.0–29.8%) in another seven species [12]. The current study, however, focuses on the quantitative aspect of samples fat thus allow limited comparison be made with these previous local data. However, there were a few studies focused on the quantitative aspects of fatty acids in other cold water species of marine and freshwater fish and shellfish performed in other countries. The comparisons made with the previous findings would be useful in giving better overview of the content of fatty acids in local marine fish and shellfish.One of the previous quantitative studies of fatty acids had reported theω-3 and ω-6 fatty acids content in different types (wild, farmed, supermarket, and feed) of salmon from North America and Europe [16]. Linoleic acid (C18:2n6) were at 67, 647, 604, and 1719 mg/100 g muscles for wild salmon, farmed salmon, supermarket salmon, and salmon feed [16]; which were higher than most of the samples (4.3–215.3 mg/100 g wet sample) in current study (Table 4). However, longtail shad is comparable in linoleic acid content with salmon feed, at 1327.4 mg/100 g wet sample [16]. This previous study also reported low γ-linolenic acid (C18:3n6) at 3 mg/100 g (wild salmon), 14 mg/100 g (farmed salmon), 13 mg/100 g (supermarket salmon), and 40 mg/100 g (salmon feed) [16]. Only three samples in current study were found to contain this fatty acid; hard tail scad (1.6 mg/100 g), silver pomfret (10.9 mg/100 g), and black pomfret (29.1 mg/100 g), which were comparable with their findings [16]. For α-linolenic acid (C18:3n3), most samples in current study contained about 100–300 mg/100 g of this fatty acid; which were higher than wild salmon (50 mg/100 g), comparable with farmed salmon (181 mg/100 g) and supermarket salmon (168 mg/100 g), meanwhile were lower when compared to salmon feed (505 mg/100 g) [16].Most samples in current study contained lower EPA (C20:5n3, 2.7–343.0 mg/100 g wet sample) compared to wild salmon (414 mg/100 g), farmed salmon (1079 mg/100 g), supermarket salmon (969 mg/100 g), and salmon feed (2638 mg/100 g) [16]. Only longtail shad was found to contain comparable amount of EPA with salmon feed; at 2638 mg/100 g wet sample [16]. It is really interesting to find such a high level of EPA in longtail shad, as high intake of this fatty acid had been related with protective effects to the occurrence of asthma, coronary heart problems and many other diseases [17–19]. Meanwhile, compared to DHA values (629–2633 mg/100 g) reported by the previous study [16], the content of this fatty acid in most samples of the current study were lower (9.0–277.1 mg/100 g wet sample). However, yellowstripe scad (782.1 mg/100 g wet sample) was found to contain slightly higher DHA content compared to wild salmon (629 mg/100 g) [16].Data from National Nutrient Data by United States Department of Agriculture (USDA) showed Greenland halibut, farmed catfish, wild catfish, farmed salmon, and wild salmon containedEPA+DHA at 1177.6, 177.6, 236.5, 2147.1, and 1840 mg/100 g muscles [20]. Generally, most samples in the current study contained EPA+DHA amounts (11.8–551.7 mg/100 g wet sample); which were lower compared to Greenland halibut, farmed salmon, and wild salmon; but comparable with farmed and wild catfish. However, longtail shad (2210 mg/100 g wet sample) was found to contain EPA+DHA at comparable amount with farmed and wild salmon [20]. This could be due to the high fat content of the fish (23.2% fat) [5].The current study is novel as it provides new quantitative findings for warm water species of fish and shellfish. Moreover, the findings are of necessary representativeness, which resulted from systematic sampling procedures performed. Data from the current study is also highly important as it represents samples from the Straits of Malacca, which aligns the west coast of Peninsular Malaysia, the main contributor of marine landings production in the country which produced 50.16% of the total marine landings production of Malaysia and 67.34% of the marine landings production of peninsular areas (Department of Fisheries Malaysia, 2007).


### 3.3. Ratios of Polyunsaturated/Saturated (P/F) andω-3/ω-6 Fatty Acids

Overall, seventeen species of samples contained SFA as the dominant group of fatty acid (Table5), with palmitic acid (C16:0) as the highest fatty acid quantified in most of samples (Table 2). The high amount of palmitic acid is due to its function as a key metabolite in fish, and the level is not influenced by the diet [21]. Meanwhile, seven of the samples (golden snapper, Indian threadfin, malabar red snapper, long-tailed butterfly ray, large-scale tongue sole, yellowstripe scad, and prawn) contained PUFA as the dominant group of fatty acid. Generally, the PUFA to SFA (P/S) ratios of most of the samples of this study (Table 5) were above the value for Menhaden oil (0.58); as suggested by Food and Drug Association (FDA) as PUFA supplement [22]. Besides, ratios exceeding 0.50 have also been shown to lower blood cholesterol level [23].Table 5
Polyunsaturated/saturated fatty acids (P/S) andω-3/ω-6 ratio of samples.
Classification
Samples
SFA
MUFA
PUFA
PUFA/SFA
ω
-
3
ω
-
6
ω
-
3
/
ω
-
6
Lean fish(<2% fat)
Hardtail scad
708.8
121.2
387.0
0.5
332.1
54.8
6.1
Golden snapper
418.7
94.0
506.3
1.2
391.5
114.8
3.4
Indian mackarel
587.8
294.8
190.5
0.3
138.1
52.4
2.6
Indian threadfin
307.4
38.3
415.9
1.4
377.5
38.4
9.8
Malabar red snapper
557.9
141.8
724.7
1.3
578.3
146.5
3.9
Dorab wolfherring
537.4
145.9
336.5
0.6
293.7
42.7
6.9
Long-tailed butterfly ray
288.8
84.6
375.5
1.3
349.3
26.3
13.3
Large-scale tongue sole
242.4
33.4
266.2
1.1
235.9
30.2
7.8
Spanish mackarel
322.8
70.6
314.2
1.0
276.3
37.9
7.3
Low fat fish(2–4% fat)
Black pomfret
935.0
138.8
714.3
0.8
623.3
91.0
6.8
Silver pomfret
878.7
151.1
571.6
0.7
406.6
165.1
2.5
Sixbar grouper
1308.7
287.2
1202.5
0.9
944.1
258.5
3.7
Japanese threadfin bream
1211.2
287.2
796.5
0.7
663.9
132.6
5.0
Yellowstripe scad
829.6
286.8
1417.0
1.7
1224.8
192.2
6.4
Gray eel-catfish
1340.8
168.1
810.7
0.6
419.6
391.1
1.1
Fourfinger threadfin
953.4
327.4
460.6
0.5
263.6
197.0
1.3
Giant seaperch
1267.2
230.0
933.0
0.7
696.2
236.9
2.9
Fringescale sardinella
1272.8
303.3
734.6
0.6
537.2
197.4
2.7
Medium fat fish (4–8% fat)
Moonfish
2572.4
504.2
2456.7
1.0
1603.7
853.0
1.9
High fat fish (>8% fat)
Longtail shad
17620.2
3059.3
6298.4
0.4
2716.9
3581.5
0.8
Shellfish
Cuttlefish
569.1
114.2
503.8
0.9
472.9
30.9
15.3
Prawn
308.4
114.5
461.9
1.5
414.3
47.6
8.7
Cockles
643.3
396.1
611.4
1.0
484.9
126.5
3.8
Oyster
563.7
82.4
335.4
0.6
237.5
97.9
2.4
1Categories of fish samples were based on the fat content [5].Among the lean fish, hardtail scad and Indian mackerel contained fairly high SFA but fairly low PUFA; which resulted in low P/S ratio of 0.6 and 0.3, respectively. Meanwhile, other lean fish showed higher P/S ratios, between 0.6 and 1.4. For low-fat fish, four-finger threadfin showed low P/S ratio of 0.5. Other low-fat fish showed P/S ratio between 0.6 and 1.7. While for the medium fat fish, moonfish contained quite high P/S ratio of 1.0. The high-fat fish, longtail shad contained higher SFA content compared to PUFA that resulted in low P/S ratio of 0.4. Overall, most of samples contained favorable ratio of fatty acids, which was beneficial for PUFA intake and lowering blood cholesterol [22, 23].There is no specific trend in levels of saturation or unsaturation relative to the fat content of samples. On the other hand, a very high positive correlation was found between fat content of samples with all three classes of fatty acids; SFA (Pearson’sr=0.989, P=0.001), MUFA (Pearson’s r=0.984, P=0.001), and PUFA (Pearson’s r=0.983, P=0.001). This suggested that increment in fat content is followed by increment in the content of all forms of fatty acids: SFA, MUFA, and PUFA in the samples.The −3 :ω-6 ratio has been suggested as a useful indicator for comparing relative nutritional values of fish oils. The ratio is also expressed in the form of ω-3/ω-6. It has been suggested that an −3 : ω-6 ratio of 1 : 1 to 1 : 5 (or ω-3/ω-6 between 0.2 and 1.0) would constitute a healthy human diet [22]. High ω-3 : ω-6 ratio is preferred, as excess ω-6 fatty acids can counteract the health benefits of ω-3 fatty acid intake [24].Overall, long-tailed butterfly ray and cuttlefish had very high ratios ofω-3/ω-6, at 13.3 and 15.3, respectively (Table 5). However, the high ratio was not contributed to by high ω-3 fatty acids, but by the low content of ω-6 fatty acids. These fish contained 349.3 and 472.9 mg/100 g wet samples of ω-3 fatty acids, respectively, which were within the common range of other samples. Meanwhile, their ω-6 fatty acids contents were only 26.3 mg/100 g wet sample for long-tailed butterfly ray, and 30.9 mg/100 g wet sample for cuttlefish. The same reason applied to the fairly high ratio of ω-3/ω-6 at 9.8, 8.7, 7.8, and 7.3 for another four samples; namely, Indian threadfin, prawn, large-scale tongue sole, and Spanish mackerel. These samples contained ω-3 fatty acids between 235.9 and 414.3 mg/100 g wet samples; meanwhile their ω-6 fatty acids were below than 40 mg/100 g wet sample. While for yellowstripe scad, the high ratio of ω-3/ω-6 was contributed by its high content of ω-3 fatty acids (1224.8 mg/100 g wet sample) compared to ω-6 fatty acids (192.2 mg/100 g wet sample). In contrast, longtail shad contained high content of ω-6 fatty, resulted in fairly low ratio of −3 : ω-6 (0.8), which was the lowest among all samples.


### 3.4. Validation Procedures

A few validation analyses were performed to ensure the reliability of the method used for fatty acid analysis in this study; these measures included linearity, precision (repeatability and reproducibility), and recovery tests. Linear relationships were observed for myristic acid (C14:0) standard (linearity parameters:y=221.25x-23.04; R2=0.999), heptadecanoic acid (C17:0) standard (linearity parameters: y=7552x-1060; R2=0.9976), and linoleic acid (C18:2n6) standard (linearity parameters: y=325.23x+18.996; R2=0.9985).The precision test assessed both repeatability (within-day) and reproducibility (between-day) tests. Table6 shows the RSD values for the repeatability test of fatty acids; these values ranged between 1.4–3.6% for palmitic acid and 0.2–1.9% for linolenic acid. Meanwhile, Table 7 shows the RSD values for the reproducibility test, which ranged between 0.9–2.4% for palmitic acid, and 0.16–1.86% for linolenic acid. The RSD values in this study were lower compared to most RSD values (3.1–13.3%) as reported previously [25], but were higher when compared with excellent RSD values (<2.0%) reported by another study [26]. However, from overall, the RSD values in all repeatability and reproducibility tests were satisfactory and showed that the methods used were reliable to produce precise data for multiple determinations of fatty acids (saturated and unsaturated) in fish and shellfish samples, both in a single-day and multiple-day determinations.Table 6
Repeatability (within-day precision) data of fatty acids (n=4).
Sample
n
Palmitic acid (C16:0)
Linolenic acid (C18:3 n3)
Mean of fatty acid content (mg/g oil)
SD
RSD (%)
Minimum
Maximum
Mean of fatty acid content (mg/g oil)
SD
RSD (%)
Minimum
Maximum
Gray eel-catfish
4
321.06
4.60
1.43
315.91
326.43
55.40
0.19
0.34
55.23
55.66
Giant seaperch
4
334.48
12.04
3.59
319.45
345.48
92.50
1.73
1.87
91.00
94.02
Cockles
4
146.36
3.00
2.05
142.12
149.19
109.22
1.86
1.70
107.52
111.33Table 7
Reproducibility (between-day precision) data of fatty acids (n=3).
Sample
n
Palmitic acid (C16:0)
Linolenic acid (C18:3 n3)
Mean of fatty acid content (mg/g oil)
SD
RSD (%)
Minimum
Maximum
Mean of fatty acid content (mg/g oil)
SD
RSD (%)
Minimum
Maximum
Gray eel-catfish
3
319.27
3.48
1.09
315.91
322.85
55.31
0.09
0.16
55.23
55.40
Giant seaperch
3
339.49
8.19
2.41
330.15
345.48
91.99
1.71
1.86
91.00
93.96
Cockles
3
147.77
1.25
0.85
146.84
149.19
108.51
1.48
1.36
107.52
110.22Meanwhile, the percentages of recoveries for different concentrations of myristic acid were between 90.3–105.6% in golden snapper and 106.8–119.4% in cockles (Table8). Meanwhile for heptadecanoic acid, the percentages of recoveries were between 87.1–111.9% in golden snapper and 112.8–115.9% in cockles. For stearic acid, the range of 93.4−108.8% in cockles. Overall, the recovery percentages were satisfactory, at about 90–120%, which is comparable to the values (most showed 80–120%) reported by previous studies [25, 27]. This shows that the methods used in the analysis are highly accurate for determinations of fatty acid contents at various concentrations in fish and shellfish samples.Table 8
Recovery rates of fatty acids in samples.
Fatty acids standards
Concentrations (mg/mL)
Samples
Recovery (%)
Myristic (C14:0)
0.4
a
105.61
b
106.84
2.0
a
105.40
b
119.35
4.0
a
90.34
b
114.80
Heptadecanoic (C17:0)
0.4
a
94.63
b
114.13
2.0
a
111.92
b
112.84
4.0
a
87.09
b
115.92
Stearic (C18:0)
0.4
a
116.15
b
106.75
2.0
a
105.40
b
108.81
4.0
a
110.39
b
93.39


## 3.1. Fatty Acid Content in Samples

Data from this study are reported in the form of milligrams per 100 grams of wet muscle. Tables2, 3, and 4 show the sample content of saturated, monounsaturated, and polyunsaturated fatty acids, respectively.Table 2
Saturated fatty acids (SFAs) content of samples.
Category
Samples
Saturated fatty acids (SFA) content (mg/100 g wet sample)
8:0
12:0
14:0
15:0
16:0
17:0
18:0
24:0
∑SFA
Lean fish(<2% fat)
Hardtail scad
42.2ab
1.4abcd
69.8a
18.6abc
531.7a
45.2a
nd
nd
708.8
Golden snapper
152.9bcd
0.7abc
9.6a
8.5ab
203.7a
21.5a
1.8a
20.1abcd
418.7
Indian mackarel
139.0abcd
0.7abc
64.1a
9.5ab
313.7a
22.8a
35.7bcd
2.3ab
587.8
Indian threadfin
98.9abcd
0.9abc
10.3a
6.8a
134.7a
17.7a
4.8ab
33.4bcde
307.4
Malabar red snapper
112.8abcd
0.9abc
28.8a
5.7a
373.7a
31.8a
nd
4.3ab
557.9
Dorab wolfherring
72.2abc
1.4abcd
39.3a
7.6a
395.9a
21.1a
nd
nd
537.4
Long-tailed butterfly ray
140.1abcd
nd
4.6a
2.9a
129.8a
11.3a
nd
nd
288.8
Large-scale tongue sole
80.7abc
0.3ab
7.5a
5.4a
95.1a
15.2a
1.5a
36.8cde
242.4
Spanish mackarel
86.3abcd
0.4ab
16.1a
5.9a
183.7a
9.5a
nd
21.0abcde
322.8
Low fat fish(2–4% fat)
Black pomfret
214.8d
nd
46.9a
18.0abc
549.5a
48.5a
6.9ab
50.4def
935.0
Silver pomfret
40.0ab
1.1abc
102.4a
15.5ab
590.2a
38.6a
11.2abc
79.7f
878.7
Sixbar grouper
186.1cd
1.6abcd
65.6a
19.8abc
775.6a
62.1a
6.4ab
191.6g
1308.7
Japanese threadfin bream
34.0ab
5.2de
102.6a
28.2abcd
996.5a
36.8a
8.0ab
nd
1211.2
Yellowstripe scad
101.1abcd
1.7abcd
69.5a
19.9abc
560.8a
65.7a
nd
10.9ab
829.6
Gray eel-catfish
35.3ab
3.9bcde
159.7a
24.3abc
717.8a
359.0a
40.9cd
nd
1340.8
Fourfinger threadfin
52.9ab
2.5abcd
127.2a
33.7bcd
677.0a
31.9a
7.0ab
21.1abcde
953.4
Giant seaperch
79.5abc
4.4cde
102.5a
15.5ab
928.2a
76.7a
8.0ab
52.4ef
1267.2
Fringescale sardinella
21.5a
2.2abcd
242.0a
43.1cd
822.2a
108.1a
33.5bcd
nd
1272.8
Medium fat fish (4–8% fat)
Moonfish
383.9e
7.2e
335.3a
53.4d
1308.0a
431.6a
31.1abcd
21.9abcde
2572.4
High fat fish (>8% fat)
Longtail shad
20.3a
21.1f
2805.4b
113.8e
12542.6b
2034.5b
82.5e
nd
17620.2
Shellfish
Cuttlefish
93.7abcd
0.7abc
30.9a
9.3ab
394.5a
7.0a
nd
33.0bcde
569.1
Prawn
139.9abcd
0.3ab
7.0a
11.4ab
125.3a
24.5a
nd
nd
308.4
Cockles
37.0ab
2.8abcd
69.9a
12.4ab
359.2ª
130.3ª
9.5abc
22.2abcde
643.3
Oyster
30.2ab
0.5abc
41.4ª
10.0ab
325.8ª
80.3a
62.6de
13.0abc
563.7
1Categories of fish samples were based on the fat content [5].
2Different superscript lowercase letters (a–g) in same column show significant differences at P<0.05 (Tukey post-hoc test).
3nd: not detected.Table 3
Monounsaturated fatty acids (MUFAs) content of samples.
Category
Samples
Monounsaturated fatty acids (MUFAs) content (mg/100 g wet sample)
14:1
15:1
16:1
17:1
18:1 n9c
∑MUFA
Lean fish (<2% fat)
Hardtail scad
2.5abc
1.5ab
7.5abc
45.7ef
63.9ab
121.2
Golden snapper
2.7abc
4.6abc
40.7de
6.6ab
39.5a
94.0
Indian mackarel
1.8abc
7.5c
116.4f
10.4ab
158.7ab
294.8
Indian threadfin
1.0ab
1.3ab
3.3ab
14.1abc
18.5a
38.3
Malabar red snapper
1.0ab
1.0ab
4.4ab
16.0abc
119.5ab
141.8
Dorab wolfherring
1.0ab
0.6a
1.1a
20.1abcd
123.1ab
145.9
Long-tailed butterfly ray
1.6abc
0.8a
1.9ab
7.7ab
72.2ab
84.6
Large-scale tongue sole
0.6a
1.4ab
nd
10.6ab
20.8a
33.4
Spanish mackarel
0.4a
0.3a
2.8ab
13.8abc
53.2ab
70.6
Low fat fish (2–4% fat)
Black pomfret
1.8abc
4.1abc
13.6abcd
20.6abcd
98.8ab
138.8
Silver pomfret
1.6abc
1.2ab
30.9bcde
36.0cdef
81.5ab
151.1
Sixbar grouper
4.5bcd
15.7d
116.0f
nd
151.0ab
287.2
Japanese threadfin bream
6.9de
1.5ab
105.3f
28.8bcdef
144.7ab
287.2
Yellowstripe scad
2.8abc
1.9ab
43.4e
49.9f
188.8ab
286.8
Gray eel-catfish
11.3f
5.7bc
nd
47.4ef
103.7ab
168.1
Fourfinger threadfin
5.1cd
2.4ab
35.3cde
43.2def
241.5ab
327.4
Giant seaperch
3.6abcd
2.6ab
43.4e
25.2bcde
155.3ab
230.0
Fringescale sardinella
3.5abcd
3.5abc
53.0e
51.3f
192.0ab
303.3
Medium fat fish (4–8% fat)
Moonfish
9.5ef
14.8d
nd
81.5g
398.5b
504.2
High fat fish (>8% fat)
Longtail shad
10.7ef
nd
458.1g
36.1cdef
2554.4c
3059.3
Shellfish
Cuttlefish
0.8ab
2.62ab
4.0ab
17.2abc
89.5ab
114.2
Prawn
0.7a
1.67ab
7.4abc
16.9abc
87.8ab
114.5
Cockles
2.9abc
13.52d
nd
75.9g
303.8ab
396.1
Oyster
0.7ab
2.65ab
nd
25.1bcde
53.9ab
82.4
1Categories of fish samples were based on the fat content [5].
2Different superscript lowercase letters (a–g) in same column show significant differences at P<0.05 (Tukey post-hoc test).
3nd: not detected.Table 4
Polyunsaturated fatty acids (PUFA) content of samples.
Category
Samples
Polyunsaturated fatty acids (PUFA) content (mg/100 g wet sample)
18:2 n6t
18:2 n6c
18:3 n6
18:3 n3
20:3 n3
20:5 n3
22:6 n3
∑PUFA
Lean fish(<2% fat)
Hardtail scad
22.5a
30.8a
1.6a
117.3a
nd
18.9a
196.0cdefg
387.0
Golden snapper
105.2ab
9.6a
nd
365.6ab
nd
7.3a
18.6ab
506.3
Indian mackarel
23.1a
29.3a
nd
53.9a
7.3ab
53.6a
23.3ab
190.5
Indian threadfin
33.2a
5.2a
nd
271.7a
nd
23.7a
82.2abcde
415.9
Malabar red snapper
86.1ab
60.4a
nd
335.0ab
9.2ab
24.1a
209.9defg
724.7
Dorab wolfherring
36.3a
6.4a
nd
204.0a
11.6ab
23.9a
54.3abcd
336.5
Long-tailed butterfly ray
11.2a
15.1a
nd
337.5ab
nd
2.7a
9.0a
375.5
Large-scale tongue sole
25.9a
4.3a
nd
111.6a
2.5ab
8.3a
113.4abcde
266.2
Spanish mackarel
18.2a
19.7a
nd
178.9a
nd
27.7a
69.8abcde
314.2
Low fat fish(2–4% fat)
Black pomfret
16.8a
45.1a
29.06c
272.8a
nd
73.5a
277.1fg
714.3
Silver pomfret
104.7ab
49.5a
10.91b
136.2a
6.1ab
116.3a
148.0abcdefg
571.6
Sixbar grouper
139.2ab
119.3a
nd
645.5b
nd
100.9a
197.7cdefg
1202.5
Japanese threadfin bream
81.6ab
51.0a
nd
112.2a
nd
258.7a
293.0g
796.5
Yellowstripe scad
78.6ab
113.6a
nd
338.1ab
7.5ab
97.1a
782.05h
1417.0
Gray eel-catfish
257.4ab
133.6a
nd
185.0a
nd
145.8a
88.8abcde
810.7
Fourfinger threadfin
148.3ab
48.7a
nd
114.3a
nd
96.2a
53.1abc
460.6
Giant seaperch
142.5ab
94.4a
nd
245.6a
215.7d
139.5a
95.4abcde
933.0
Fringescale sardinella
33.2a
164.2a
nd
69.3a
31.0c
211.5a
225.4efg
734.6
Medium fat fish (4–8% fat)
Moonfish
637.7ab
215.3a
nd
1046.8c
257.6e
176.7a
122.6abcdef
2456.7
High fat fish (>8% fat)
Longtail shad
2254.1b
1327.4b
nd
68.9a
437.5f
2041.8b
168.7bcdefg
6298.4
Shellfish
Cuttlefish
13.4a
17.5a
nd
251.1a
14.9b
121.9a
85.0abcde
503.8
Prawn
16.3a
31.3a
nd
320.4ab
nd
48.7a
45.3abc
461.9
Cockles
71.5ab
55.0a
nd
18.6a
nd
343.0a
123.4abcdef
611.4
Oyster
62.0a
35.9a
nd
100.7a
nd
54.0a
82.8abcde
335.4
1Categories of fish samples were based on the fat content [5].
2Different superscript lowercase letters (a–h) in same column show significant differences at P<0.05 (Tukey post-hoc test).
3nd: not detected.Based on the previous findings, the studied fish samples can be categorized into lean-, low-, medium-, and high-fat fish based on the fat content [5]. Among the lean-fish, hardtail scad was high in saturated fatty acids (SFAs), with palmitic acid as the highest (531.7 mg/100 g wet sample) (Table 2). The monounsaturated fatty acid (MUFA) of the fish was 121.2 mg/100 g wet sample; with fairly high oleic acid (C18:1n9c) and heptadecenoic acid (C17:1), at 63.9 and 45.7 mg/100 g wet sample, respectively (Table 3). For PUFA, hardtail scad contained quite high amounts of DHA (C22:6n3), at 196.0 mg/100 g wet sample, followed by ALA (C18:3n3) at 117.3 mg/100 g wet sample (Table 4). Meanwhile, malabar red snapper was higher in PUFA, characterized by high DHA (C22:6n3) at 209.9 mg/100 g wet sample, followed by ALA (C18:3n3) at 335.0 mg/100 g wet sample (Table 4). For SFA, palmitic acid was the dominant fatty acid (373.7 mg/100 g wet sample), followed by caprylic acid at 112.8 mg/100 g wet sample (Table 2). The MUFA content was 141.8 mg/100 g wet sample, with fairly high oleic acid (C18:1 n9c) at 119.5 mg/100 g wet sample (Table 3).Among the low-fat fish, yellowstripe scad contained a very high PUFA content, with significantly higher (P<0.05) levels of DHA (782.1 mg/100 g wet sample) compared to other samples (Table 4). The DHA amount was about 2.7 times higher than Japanese threadfin bream (2nd highest DHA, 293.0 mg/100 g wet sample); and about 86.6 times higher than long-tailed butterfly ray (lowest DHA, 9.0 mg/100 g wet sample). Additionally, yellowstripe scad also had a high ALA (C18:3n3) level at 338.1 mg/100 g wet sample. For SFA and MUFA, the most dominant fatty acids were palmitic acid (560.8 mg/100 g wet sample) and oleic acid (188.8 mg/100 g wet sample), respectively (Tables 2 and 3).The medium-fat fish, moonfish, showed palmitic acid as the most prominent fatty acid (1308.0 mg/100 g wet sample) (Table2). This was followed by α-linolenic acid (C18:3n3) at 1046.8 mg/100 g wet sample, which was significantly higher (P<0.05) compared to the others (Table 4). Other ω-3 fatty acids, such as eicosatrienoic acid (C20:3n3), EPA (C20:5n3), and DHA (C22:6n3) were at 257.6, 176.7, and 122.6 mg/100 g wet sample, respectively. Its ω-6 fatty acid content, linolelaidic acid (C18:2n6t, 637.7 mg/100 g wet sample) and linoleic acid (C18:2n6c, 215.3 mg/100 g wet sample), were the second highest after level in longtail shad (linolelaidic acid at 2254.1 mg/100 g and linoleic acid at 1327.4 mg/100 g wet sample), respectively. Meanwhile, for MUFA, the most dominant fatty acid was oleic acid (C18:1n9c), at 398.5 mg/100 g wet sample. Moonfish also contained significantly higher (Tukey post-hoc test, P<0.05) of heptadecenoic acid (C17:1, 81.5 mg/100 g wet sample) compared to others (Table 3).The high-fat fish, longtail shad also showed palmitic acid (C16:0) as the dominant fatty acid (12542.6 mg/100 g wet muscle, Table2). For MUFA, longtail shad contained significantly higher (Tukey post-hoc test, P<0.05) of palmitoleic acid (C16:1, 458.1 mg/100 g wet sample) and oleic acid (C18:1n9c, 2554.4 mg/100 g wet sample) compared to others (Table 3). While for PUFA, the fish contained significantly higher (Tukey post-hoc test, P<0.05) of EPA (C20:5 n3, 2041.8 mg/100 g wet sample) and eicosatrienoicacid (C20:3n3, 437.5 mg/100 g wet sample) compared to others (Table 4). Besides, longtail shad was also high in ω-6 fatty acids; with linolelaidic acid (C18:2n6t) at 2254.1 mg/100 g wet muscle, and linoleic acid (C18:2n6c) at 1327.4 mg/100 g wet muscle.As in most of fish samples, shellfish including cuttlefish, cockles, and oyster also showed palmitic acid (C16:0) as the most prominent fatty acid (Table2). Meanwhile, prawn showed caprylic acid (C8:0) as the highest fatty acid (139.9 mg/100 g wet sample), followed by palmitic acid (C16:0) at 125.3 mg/100 g wet sample. For MUFA, cockles contained higher oleic acid (C18:1n9c, 303.8 mg/100 g wet sample) compared to other shellfish (Table 3). While for PUFA, cockles were found to be higher in both EPA (C20:5n3, 343.0 mg/100 g wet sample) and DHA (C22:6n3, 123.4 mg/100 g wet sample), compared to other shellfish (Table 4). However, the content of alpha-linolenic acid (C18:3n3) in cockles (18.6 mg/100 g wet sample) was the lowest among all samples; with cuttlefish (251.1 mg/100 g wet sample), prawn (320.4 mg/100 g wet sample), and oysterr (100.7 mg/100 g wet sample).


## 3.2. Fatty Acids of Fish and Shellfish from Local and Other Countries

Most of previous local studies have focused on qualitative aspects of fat content in various samples of marine and freshwater origin samples [10–15]. A previous study found very high percentage of PUFA; with omega-3 PUFA (29.7–48.4%), other PUFA (27.7–40.0%), and omega-6 PUFA (11.0–20.0%) in ten common fish species with the current study [9]. Meanwhile, percentages of SFA and MUFA were quite low; at 3.63–11.4% and 1.37–9.12%, respectively. This study found hexadecadienoic acid as the most prominent fatty acid (18.1–24.9%) in six samples, hexadecatrienoic acid (C16:3n4) in two samples, and hexadecadienoic acid (C16:2) in other two samples [10]. Another local study reported PUFA as the dominant group of fatty acid in seven species, and SFA as the dominant fatty acid group in another six species of their fish samples [11]. Palmitic acid (C16:0) was the most prominent fatty acid (17.6–32.1%) in eight samples; meanwhile, DHA (C22:6n3) was the highest fatty acid (19.3–24.0%) in another five samples [11]. Same findings were reported by other study of 16 species of local pelagic fish; with palmitic acid (C16:0) as the most prominent fatty acid (20.9–34.5%) in nine species, meanwhile, DHA (C22:6n3) was the highest fatty acid (26.0–29.8%) in another seven species [12]. The current study, however, focuses on the quantitative aspect of samples fat thus allow limited comparison be made with these previous local data. However, there were a few studies focused on the quantitative aspects of fatty acids in other cold water species of marine and freshwater fish and shellfish performed in other countries. The comparisons made with the previous findings would be useful in giving better overview of the content of fatty acids in local marine fish and shellfish.One of the previous quantitative studies of fatty acids had reported theω-3 and ω-6 fatty acids content in different types (wild, farmed, supermarket, and feed) of salmon from North America and Europe [16]. Linoleic acid (C18:2n6) were at 67, 647, 604, and 1719 mg/100 g muscles for wild salmon, farmed salmon, supermarket salmon, and salmon feed [16]; which were higher than most of the samples (4.3–215.3 mg/100 g wet sample) in current study (Table 4). However, longtail shad is comparable in linoleic acid content with salmon feed, at 1327.4 mg/100 g wet sample [16]. This previous study also reported low γ-linolenic acid (C18:3n6) at 3 mg/100 g (wild salmon), 14 mg/100 g (farmed salmon), 13 mg/100 g (supermarket salmon), and 40 mg/100 g (salmon feed) [16]. Only three samples in current study were found to contain this fatty acid; hard tail scad (1.6 mg/100 g), silver pomfret (10.9 mg/100 g), and black pomfret (29.1 mg/100 g), which were comparable with their findings [16]. For α-linolenic acid (C18:3n3), most samples in current study contained about 100–300 mg/100 g of this fatty acid; which were higher than wild salmon (50 mg/100 g), comparable with farmed salmon (181 mg/100 g) and supermarket salmon (168 mg/100 g), meanwhile were lower when compared to salmon feed (505 mg/100 g) [16].Most samples in current study contained lower EPA (C20:5n3, 2.7–343.0 mg/100 g wet sample) compared to wild salmon (414 mg/100 g), farmed salmon (1079 mg/100 g), supermarket salmon (969 mg/100 g), and salmon feed (2638 mg/100 g) [16]. Only longtail shad was found to contain comparable amount of EPA with salmon feed; at 2638 mg/100 g wet sample [16]. It is really interesting to find such a high level of EPA in longtail shad, as high intake of this fatty acid had been related with protective effects to the occurrence of asthma, coronary heart problems and many other diseases [17–19]. Meanwhile, compared to DHA values (629–2633 mg/100 g) reported by the previous study [16], the content of this fatty acid in most samples of the current study were lower (9.0–277.1 mg/100 g wet sample). However, yellowstripe scad (782.1 mg/100 g wet sample) was found to contain slightly higher DHA content compared to wild salmon (629 mg/100 g) [16].Data from National Nutrient Data by United States Department of Agriculture (USDA) showed Greenland halibut, farmed catfish, wild catfish, farmed salmon, and wild salmon containedEPA+DHA at 1177.6, 177.6, 236.5, 2147.1, and 1840 mg/100 g muscles [20]. Generally, most samples in the current study contained EPA+DHA amounts (11.8–551.7 mg/100 g wet sample); which were lower compared to Greenland halibut, farmed salmon, and wild salmon; but comparable with farmed and wild catfish. However, longtail shad (2210 mg/100 g wet sample) was found to contain EPA+DHA at comparable amount with farmed and wild salmon [20]. This could be due to the high fat content of the fish (23.2% fat) [5].The current study is novel as it provides new quantitative findings for warm water species of fish and shellfish. Moreover, the findings are of necessary representativeness, which resulted from systematic sampling procedures performed. Data from the current study is also highly important as it represents samples from the Straits of Malacca, which aligns the west coast of Peninsular Malaysia, the main contributor of marine landings production in the country which produced 50.16% of the total marine landings production of Malaysia and 67.34% of the marine landings production of peninsular areas (Department of Fisheries Malaysia, 2007).


## 3.3. Ratios of Polyunsaturated/Saturated (P/F) andω-3/ω-6 Fatty Acids

Overall, seventeen species of samples contained SFA as the dominant group of fatty acid (Table5), with palmitic acid (C16:0) as the highest fatty acid quantified in most of samples (Table 2). The high amount of palmitic acid is due to its function as a key metabolite in fish, and the level is not influenced by the diet [21]. Meanwhile, seven of the samples (golden snapper, Indian threadfin, malabar red snapper, long-tailed butterfly ray, large-scale tongue sole, yellowstripe scad, and prawn) contained PUFA as the dominant group of fatty acid. Generally, the PUFA to SFA (P/S) ratios of most of the samples of this study (Table 5) were above the value for Menhaden oil (0.58); as suggested by Food and Drug Association (FDA) as PUFA supplement [22]. Besides, ratios exceeding 0.50 have also been shown to lower blood cholesterol level [23].Table 5
Polyunsaturated/saturated fatty acids (P/S) andω-3/ω-6 ratio of samples.
Classification
Samples
SFA
MUFA
PUFA
PUFA/SFA
ω
-
3
ω
-
6
ω
-
3
/
ω
-
6
Lean fish(<2% fat)
Hardtail scad
708.8
121.2
387.0
0.5
332.1
54.8
6.1
Golden snapper
418.7
94.0
506.3
1.2
391.5
114.8
3.4
Indian mackarel
587.8
294.8
190.5
0.3
138.1
52.4
2.6
Indian threadfin
307.4
38.3
415.9
1.4
377.5
38.4
9.8
Malabar red snapper
557.9
141.8
724.7
1.3
578.3
146.5
3.9
Dorab wolfherring
537.4
145.9
336.5
0.6
293.7
42.7
6.9
Long-tailed butterfly ray
288.8
84.6
375.5
1.3
349.3
26.3
13.3
Large-scale tongue sole
242.4
33.4
266.2
1.1
235.9
30.2
7.8
Spanish mackarel
322.8
70.6
314.2
1.0
276.3
37.9
7.3
Low fat fish(2–4% fat)
Black pomfret
935.0
138.8
714.3
0.8
623.3
91.0
6.8
Silver pomfret
878.7
151.1
571.6
0.7
406.6
165.1
2.5
Sixbar grouper
1308.7
287.2
1202.5
0.9
944.1
258.5
3.7
Japanese threadfin bream
1211.2
287.2
796.5
0.7
663.9
132.6
5.0
Yellowstripe scad
829.6
286.8
1417.0
1.7
1224.8
192.2
6.4
Gray eel-catfish
1340.8
168.1
810.7
0.6
419.6
391.1
1.1
Fourfinger threadfin
953.4
327.4
460.6
0.5
263.6
197.0
1.3
Giant seaperch
1267.2
230.0
933.0
0.7
696.2
236.9
2.9
Fringescale sardinella
1272.8
303.3
734.6
0.6
537.2
197.4
2.7
Medium fat fish (4–8% fat)
Moonfish
2572.4
504.2
2456.7
1.0
1603.7
853.0
1.9
High fat fish (>8% fat)
Longtail shad
17620.2
3059.3
6298.4
0.4
2716.9
3581.5
0.8
Shellfish
Cuttlefish
569.1
114.2
503.8
0.9
472.9
30.9
15.3
Prawn
308.4
114.5
461.9
1.5
414.3
47.6
8.7
Cockles
643.3
396.1
611.4
1.0
484.9
126.5
3.8
Oyster
563.7
82.4
335.4
0.6
237.5
97.9
2.4
1Categories of fish samples were based on the fat content [5].Among the lean fish, hardtail scad and Indian mackerel contained fairly high SFA but fairly low PUFA; which resulted in low P/S ratio of 0.6 and 0.3, respectively. Meanwhile, other lean fish showed higher P/S ratios, between 0.6 and 1.4. For low-fat fish, four-finger threadfin showed low P/S ratio of 0.5. Other low-fat fish showed P/S ratio between 0.6 and 1.7. While for the medium fat fish, moonfish contained quite high P/S ratio of 1.0. The high-fat fish, longtail shad contained higher SFA content compared to PUFA that resulted in low P/S ratio of 0.4. Overall, most of samples contained favorable ratio of fatty acids, which was beneficial for PUFA intake and lowering blood cholesterol [22, 23].There is no specific trend in levels of saturation or unsaturation relative to the fat content of samples. On the other hand, a very high positive correlation was found between fat content of samples with all three classes of fatty acids; SFA (Pearson’sr=0.989, P=0.001), MUFA (Pearson’s r=0.984, P=0.001), and PUFA (Pearson’s r=0.983, P=0.001). This suggested that increment in fat content is followed by increment in the content of all forms of fatty acids: SFA, MUFA, and PUFA in the samples.The −3 :ω-6 ratio has been suggested as a useful indicator for comparing relative nutritional values of fish oils. The ratio is also expressed in the form of ω-3/ω-6. It has been suggested that an −3 : ω-6 ratio of 1 : 1 to 1 : 5 (or ω-3/ω-6 between 0.2 and 1.0) would constitute a healthy human diet [22]. High ω-3 : ω-6 ratio is preferred, as excess ω-6 fatty acids can counteract the health benefits of ω-3 fatty acid intake [24].Overall, long-tailed butterfly ray and cuttlefish had very high ratios ofω-3/ω-6, at 13.3 and 15.3, respectively (Table 5). However, the high ratio was not contributed to by high ω-3 fatty acids, but by the low content of ω-6 fatty acids. These fish contained 349.3 and 472.9 mg/100 g wet samples of ω-3 fatty acids, respectively, which were within the common range of other samples. Meanwhile, their ω-6 fatty acids contents were only 26.3 mg/100 g wet sample for long-tailed butterfly ray, and 30.9 mg/100 g wet sample for cuttlefish. The same reason applied to the fairly high ratio of ω-3/ω-6 at 9.8, 8.7, 7.8, and 7.3 for another four samples; namely, Indian threadfin, prawn, large-scale tongue sole, and Spanish mackerel. These samples contained ω-3 fatty acids between 235.9 and 414.3 mg/100 g wet samples; meanwhile their ω-6 fatty acids were below than 40 mg/100 g wet sample. While for yellowstripe scad, the high ratio of ω-3/ω-6 was contributed by its high content of ω-3 fatty acids (1224.8 mg/100 g wet sample) compared to ω-6 fatty acids (192.2 mg/100 g wet sample). In contrast, longtail shad contained high content of ω-6 fatty, resulted in fairly low ratio of −3 : ω-6 (0.8), which was the lowest among all samples.


## 3.4. Validation Procedures

A few validation analyses were performed to ensure the reliability of the method used for fatty acid analysis in this study; these measures included linearity, precision (repeatability and reproducibility), and recovery tests. Linear relationships were observed for myristic acid (C14:0) standard (linearity parameters:y=221.25x-23.04; R2=0.999), heptadecanoic acid (C17:0) standard (linearity parameters: y=7552x-1060; R2=0.9976), and linoleic acid (C18:2n6) standard (linearity parameters: y=325.23x+18.996; R2=0.9985).The precision test assessed both repeatability (within-day) and reproducibility (between-day) tests. Table6 shows the RSD values for the repeatability test of fatty acids; these values ranged between 1.4–3.6% for palmitic acid and 0.2–1.9% for linolenic acid. Meanwhile, Table 7 shows the RSD values for the reproducibility test, which ranged between 0.9–2.4% for palmitic acid, and 0.16–1.86% for linolenic acid. The RSD values in this study were lower compared to most RSD values (3.1–13.3%) as reported previously [25], but were higher when compared with excellent RSD values (<2.0%) reported by another study [26]. However, from overall, the RSD values in all repeatability and reproducibility tests were satisfactory and showed that the methods used were reliable to produce precise data for multiple determinations of fatty acids (saturated and unsaturated) in fish and shellfish samples, both in a single-day and multiple-day determinations.Table 6
Repeatability (within-day precision) data of fatty acids (n=4).
Sample
n
Palmitic acid (C16:0)
Linolenic acid (C18:3 n3)
Mean of fatty acid content (mg/g oil)
SD
RSD (%)
Minimum
Maximum
Mean of fatty acid content (mg/g oil)
SD
RSD (%)
Minimum
Maximum
Gray eel-catfish
4
321.06
4.60
1.43
315.91
326.43
55.40
0.19
0.34
55.23
55.66
Giant seaperch
4
334.48
12.04
3.59
319.45
345.48
92.50
1.73
1.87
91.00
94.02
Cockles
4
146.36
3.00
2.05
142.12
149.19
109.22
1.86
1.70
107.52
111.33Table 7
Reproducibility (between-day precision) data of fatty acids (n=3).
Sample
n
Palmitic acid (C16:0)
Linolenic acid (C18:3 n3)
Mean of fatty acid content (mg/g oil)
SD
RSD (%)
Minimum
Maximum
Mean of fatty acid content (mg/g oil)
SD
RSD (%)
Minimum
Maximum
Gray eel-catfish
3
319.27
3.48
1.09
315.91
322.85
55.31
0.09
0.16
55.23
55.40
Giant seaperch
3
339.49
8.19
2.41
330.15
345.48
91.99
1.71
1.86
91.00
93.96
Cockles
3
147.77
1.25
0.85
146.84
149.19
108.51
1.48
1.36
107.52
110.22Meanwhile, the percentages of recoveries for different concentrations of myristic acid were between 90.3–105.6% in golden snapper and 106.8–119.4% in cockles (Table8). Meanwhile for heptadecanoic acid, the percentages of recoveries were between 87.1–111.9% in golden snapper and 112.8–115.9% in cockles. For stearic acid, the range of 93.4−108.8% in cockles. Overall, the recovery percentages were satisfactory, at about 90–120%, which is comparable to the values (most showed 80–120%) reported by previous studies [25, 27]. This shows that the methods used in the analysis are highly accurate for determinations of fatty acid contents at various concentrations in fish and shellfish samples.Table 8
Recovery rates of fatty acids in samples.
Fatty acids standards
Concentrations (mg/mL)
Samples
Recovery (%)
Myristic (C14:0)
0.4
a
105.61
b
106.84
2.0
a
105.40
b
119.35
4.0
a
90.34
b
114.80
Heptadecanoic (C17:0)
0.4
a
94.63
b
114.13
2.0
a
111.92
b
112.84
4.0
a
87.09
b
115.92
Stearic (C18:0)
0.4
a
116.15
b
106.75
2.0
a
105.40
b
108.81
4.0
a
110.39
b
93.39


## 4. Conclusion

Overall, most of the marine fish and shellfish samples contained desirable compositions of fatty acids with a fairly high amount ofω-3 fatty acids, a suitable ratio of ω-3/ω-6 fatty acids, and preferable P/S ratios which were higher than the level in the recommended PUFA supplement, Menhaden oil. Three samples were identified as being outstanding in their desirable overall composition of fatty acids: yellowstripe scad (highest DHA, ω-3/ω-6=6.4, P/S = 1.7), moonfish (highest ALA, ω-3/ω-6=1.9, P/S = 1.0), and longtail shad (highest EPA, ω-3/ω-6=0.8, P/S = 0.4). These findings showed that marine fish and shellfish from warm water area contain a good composition of fatty acids and could provide many health benefits if consumed regularly. These reliable and representative data are very useful to develop a nutritional database of marine fish and shellfish from warm water area and as reference to people for intake locally and globally. Additionally, the findings also showed that some identified species of fish and shellfish from this area may have possible value in terms of future manipulation for various nutraceutical purposes. However, further studies should be developed on this matter.



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*Source: 284329-2012-12-23.xml*