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GC-MS analysis of Fatty acids (FAs) of Prosobranch Gastropod species Thais carinifera From Pakistan Coast (North Arabian Sea).

Byline: NUZHAT AFSAR, GHAZALA SIDDIQUI, MUNAWWER RASHEED, VIQAR UDDIN AHMED AND AFSAR KHAN

Summary:

The fatty acids (FAs) composition was analyzed in digestive gland/gonad complex of male and female specimens of Thais carinifera by gas chromatography-mass spectrometry (GC-MS) technique in identification of fatty acids (FAs). Saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA) and steroidal components formed a large proportion of total lipids. Palmitic acid (16:0) and stearic acid (18:0), 11 eicosanoic acid, 5-13- docosadienoic acid and Cholesterol was found in abundance as SFA, MUFA, PUFA and steroidal constituent respectively in both male and female samples. Multi-methyl branched, methyl branched and hydroxy FAs contributed a smaller proportion. Presence of fair amount (~20%) of PUFA along with cholesterol (~24%) in Thais carinifera is unique because of their role in human health. This is the first ever report revealing the FAs and steroidal composition of this species.

Introduction

There are several reports available in the scientific literature concerning FAs distribution of a large number of marine and or fresh water molluscs of commercial importance [1-4]. Phylum mollusca is divided into four major classes Polyplacophora, Gastropoda, Bivalvia and Cephalopoda in which variety of FAs components are found [5, 6]. Gastropoda is the largest class of mollusca and reported to contain 16:0, 18:1n-9, 20:4n-6, 20:5n-3 and 22:5n-3 as the major fatty acids [7]. Marine gastropods have gained commercial importance worldwide, as they are a major part of marine fishery resources. They are rich in PUFA which are not only dietary fats, but are important for human health, because of their use as amelioration product in many cardiovascular diseases [8-10].

Molluscan species have relatively large amounts of sterols other than cholesterols and the gastropods are well known to have large amount of cholesterol among steroidal components. Variety of C27-, C28- and C29- sterols found in marine molluscs [11, 12]. In recent studies steroidal components of marine gastropods gets attention because of their role in molluscan endocrine system. Steroidal molecules have hormonal role, derived from cholesterol and used as hormones to control the reproduction, development and or homeostasis [13-15].

The marine gastropod species Thais carinifera is the commonly occurring species on the Sindh and Balochistan coasts of Pakistan. In Pakistan as such no literature is available regarding the FAs profile of this species. This paper for the first time describe the FAs composition and steroidal consti- tuents of any species of gastropods from Pakistan

Results and Discussion

% Lipid Composition

The total lipid content in the animal tissue (ex-shell) was calculated after separate replicate analysis of females and males. On as is basis it was found ~0.2% (SD+-0.015) in females and ~0.3% (SD+-0.029) in males. In a separate analysis the ex- shell tissue from females and males showed ~23% (SD+-1.115) and ~24% (SD+-1.820) loss on drying respectively as per AOAC method. Thus on ex-shell dry weight basis the total lipid content was found ~74% (SD+-0.67) and ~1.15 (SD+-0.485) respectively.

Fatty acid Profile

The concentration of identified components was calculated using area normalization method over FID response. Lipid components (FAs and steroids) in male and female individuals of T. carinifera were analyzed with gas chromatographic-mass spectrometric technique. FAs (as FAMEs) and steroids of male and female digestive gland/gonad complex were identified by comparing the mass spectra with literature and mass spectral libraries (NIST, Lipid Library). Detailed GC-MS results are shown in Table-1 [23].

Table-1: Fatty acid composition (area % of total lipid) from digestive gland/ gonad complex tissue of Thatis carinifera in male (M) and female (F) individuals. FAs-fatty acids; SFAs- saturated fatty acids; MUFAs- monoenoic fatty acids; PUFAs- polyenoic fatty acids.

###GCMS###Concentrations

###FAs###RT###Male (n=10)###Female (n=10)

###SFAs

n- Tetradecanoic aicd MEl Myristic acid ME###14:0###25.01+-0.03###0.78+-0.26###0.46+-0.16

n- Pentadecanoic acid ME###15:0###26.92+-0.00###0.12+-0.05###0.11+-0.02

n- Hexadecanoic aicd ME/ Palmitic acid ME###16:0###29.45+-0.41###9.97+-2.76###8.81+-2.02

n- Heptadecanoic aicd MEl Margaric acid ME###17:0###30.36+-0.09###0.73+-1.03###0.81+-0.31

n- Octadecanoic acid ME I Stearic acid ME###18:0###32.90+-0.20###6.63+-0.30###7.51+-1.24

n- Nonadecanoic acid ME###19:0###34.74+-0.02###1.62+-1.93###0.58+-0.22

Eicosanoic acid ME / Arachidic acid ME###20:0###36.44+-0.02###0.40+-0.38###1.74+-0.09

Docosanoic acid ME / Behenic acid ME###22:0###39.33+-0.00###0.08+-0.11###0.55+-0.78

MUFAs

9- hexadecenoic acid ME/ Palmitoleic acid ME###16:1 n-7###28.84+-0.26###1.39+-0.29###1.17+-0.23

9c-octadecenoic acid ME/ Oleic acid ME###18:1 n-9###32.29+-0.08###1.76+-2.48###0.75+-0.99

11c-octadecenoic acid ME/ Vaccenic acid ME###18:1 n-7###32.48+-0.02###1.31+-0.30###3.50+-2.50

11-eicosenoic acid ME###20:1 n-9###35.82+-0.27###11.50+-0.20###13.71+-1.34

9-Eicosenoic acid ME###20:1 n-11###36.07+-0.10###0.91+-0.21###0.84+-0.00

11-docosenoic acid ME###22:1 n-11###39.21+-0.03###0.88+-0.06###2.21+-0.11

PUFAs

9,12-octadecadienoic aicd MEl Linoleic acid ME###18:2 n-6###31.93+-0.54###1.12+-1.58###0.94+-1.27

5, 8, 11, 14- eicosatetraenoic acid ME/ Arachidonic acid###20:4n-6###35.24+-0.02###3.29+-1.71###3.09+-3.29

11,14-eicosadienoic acid ME###20:2 n-6###35.56+-0.30###2.60+-0.98###2.64+-1.54

11,14,17-eicosatrienoic acid###20:3 n-3###37.60+-0.02###0.42+-0.14###0.23+-0.14

Eicosa-5,8,11,14,17-pentaenoic acid ME / EPA###20:5 n-3###38.67+-0.04###3.42+-1.37###4.23+-2.17

5,13-docosadienoic acid ME###22:2 NMID###38.94+-0.07###9.36+-0.51###8.06+-3.34

Multi- methyl branched FA

4,8,12-trimethyl tetradecenoate-ME###26.35+-0.53###0.23+-0.15###0.21+-0.06

Methyl- branched FAs

9-methyl tetradecanoic aicd ME###27.13+-0.02###0.46+-0.52###0.46+-0.17

14-methyl Pentadecanoic acid MEl iso###28.51+-0.15###0.34+-0.23###0.18+-0.13

14-methyl hexadecanoic acid ME / anteiso###30.55+-0.02###0.45+-0.64###0.86+-0.93

13-methyl hexadecanoic acid ME###31.10+-0.03###1.63+-0.35###1.17+-1.65

7-methyl-octadecanoic acid MEl iso###34.23+-0.02###0.66+-0.23###0.56+-0.65

Hydroxy FAs

3-hydroxy tetradecanoic acid ME###40.21+-0.05###0.97+-0.36###0.71+-0.73

Steroidal components

5-hydroxy-(5) Cholestane-3-one###50.06+-0.11###0.57+-0.80###0.74+-0.76

Cholestane-3,5-diol 5-acetate, (3,5)###51.54+-0.13###0.01+-0.00###0.05+-0.07

Cholest-5-en- 3 -ol (Cholesterol)###51.72+-0.07###24.52+-0.92###23.02+-3.67

Ergost-22-en-3-one###53.04+-0.19###1.87+-2.15###3.79+-1.99

Cholesta-3,S-dien-7-one (3,5)###53.53+-0.51###1.15+-1.62###1.17+-1.38

22-ketocholesterol###57.13+-3.24###3.97+-5.61###3.54+-3.06

-sitosterol###61.57+-3.42###0.59+-0.28###0.88+-1.25

Miscellaneous

Triglycerides (unknown Acyl chain)###38.43+-0.03###3.80+-1.03###0.13+-0.18

Hexacosane###40.87+-0.00###0.55+-0.30###0.67+-0.47

SAs###-###20.31+-6.82###20.55+-4.17

MUFA###-###17.74+-1.42###22.17+-2.02

PUFA###20.21+-6.29###19.18+-5.08

Multi- methyl branched FAs###-###0.23+-0.15###0.21+-0.06

Methyl- branched FAs###3.53+-1.05###3.22+-3.52

Hydroxy FAs###0.97+-0.37###0.72+-0.73

Steroidal components###-###32.65+-1.68###33.17+-0.57

Miscellaneous###4.35+-1.32###0.79+-0.65

RT retention times on DB- 5 capillary column

A total of 36 compounds were chromatographed and identified, comprising of 8 SFAs, 5 MUFAs, 6 (PUFAs), 1 multi-methyl branched fatty acid, 5 methyl branched fatty acids, 1 hydroxy fatty acid and 7 steroidal components. SFA, PUFAs and steroidal components contributed a large proportion of total lipids. Smaller amount of miscellaneous components (triglycerides with unknown acyl chain and hexacosane) were also found.

The total amount of SFA (area % of total lipid) was found almost similar in females (20.55%) and males (20.31%). The major fatty acids were palmitic acid (16:0) and stearic acid (18: 0) in both sexes in same order. Other than these nonadecanoic acid (19: 0) contributed 1.62% in males and 0.58% in females. Whereas, arachidic acid (20: 0) was 1.74% in females and 0.40% in males. In females 0.55% of behenic acid (22: 0) was found and a smaller proportion (0.08%) was detected in males.

The concentration of total MUFA was found slightly higher in females. (22.17% in females followed by 17.74% in males). Large proportion MUFAs were composed of 11- eicosenoic acid (20:1n-9) which constituted 13.71% in females and 11.50% in males. Vaccenic acid (18:1n-7) contributed 3.50% in females and 1.31% in males.

11-docosenoic acid (22:1n-11) formed 2.21% of MUFA in females followed by 0.88% in males. In contrast Oleic acid (18:1n-9) was found more (1.76 %) in males as compare to females (0.75%).

Total PUFAs contributed 20.21% in males and 19.18% in females. A non-methylene interrupted dienoic 5, 13-docosenoic acid (22:2 NMID) constituted a large proportion of total PUFA, with 9.36% in males and 8.06% in females. Other than this EPA (20:5n-3) contributed 4.23% in females and 3.42% in males, arachidonic acid 3.29% in males and 3.09% in females, whereas 11, 14-eicosadienoic acid (20:2n-6) was found 2.64% in females and 2.60% in males.

Considerable amount of methyl-branched FAs was found in digestive gland/ gonad complex of both sexes with 3.53% in males and 3.22% in females The major contributors were 13-methyl hexadecanoic acid (1.63% in males and 1.17% in females) followed by 7-methyl-octadecanoic acid (0.66% in males and 0.56% in females).

A smaller proportion (~0.2%) of a multi- methyl branched FA was also found and identified as 4, 8,12-trimethyl tetradecenoate along with a hydroxy FA 3-hydroxy tetradecanoic acid, which amounted to 0.97% in males and 0.71% in females.

Steroidal and other Components

Steroidal components formed almost 1/3rd of the total lipids (33.17% in females and 32.65% in males). Cholesterol was detected in fair amount, contributing 24.52% in males and 23.02% in females which contributed ~75% and ~69% of the steroidal components in males and females respectively. 22- ketocholesterol was next with 3.97% in males and 3.54% in females. Ergost-22-en-3-one formed 3.79% in females and 1.87% in males. Smaller proportion of b-sitosterol was identified in females (0.88%) males 0.59%.

In addition 0.67% and 0.55% hexacosane was also found in females and males respectively. Some unknown triglycerides were also observed. The MS of these compound reflected typical pattern of triglycerides but acyl portion remained unidentified. Their percentages were 3.80 and 1.30 in males and females respectively.

As such no considerable variation was found in composition of lipids profile among males and females. However, variability was recorded in fractions of fatty components of males and females like behanic acid was found 86.4% in excess in females as compare to males. 76.9% variation was present in arachidic acid among females and males.

64.4% variation was observed in component of nonadecanoic acid, which was higher in males than females The major SFA which found in both male and female individuals were palmitic (C16:0) and stearic (C18:0) found in digestive gland/ gonad complex. According to Ackman [1] palmitic acid (C16:0) is the principal fatty acid at all evolutionary and trophic levels. Total SFA concentrations were found almost in equal amount in males and females. Higher concentrations of SFA and MUFA have been recorded in mesogastropod species Bellamya bengalensis and Pila globosa from Indian waters and suggested that these are because of thermal adaptations [16]. Higher concentrations of 16:0, 18:0, 14:0 SFA and 18:1, 16:1 MUFA have been reported in Telescopium telescopium from India [10]. 16:0, 18:0, 14:0 were also recorded in T. carinifera. 16:0 and 18:0 found chiefly whereas 14:0 was found in lesser extent among SFAs in this species. Whereas the total content of MUFA was slightly higher in females than males.

11-eicosenoic acid (20:1 n-9) was found in abundance among MUFA in T. carinifera. Other than that vaccenic acid (18: 1 n-7), palmitoleic acid (16:1 n-7) and oleic acid (18:1 n-9) were chiefly present. From Red and Mediterranian Seas concentrations of 18:1 n-9 and 18:1 n-7 and 20:1 n-9 were reported in eight marine gastropod species [4]. They found high concentrations of 18:1 n-9 in Nassa serata, Nassarius albescens, Nodiolittorina subnodosa, Littorina scarab, Planaxis sulcata, Monodonta turbinate, Gibula cineraria and Littorina neritoides.

Almost equal concentrations of total PUFA were recorded in current study in male and female samples of T. carinifera. PUFA are dietary products and commercial importance of PUFAs is obvious because of their role in human health. PUFA, especially 20:5 n-3 and 22:6 n-3 attributed to have a definite beneficial effect in deferring the cardiovascular incidences and are also used in amelioration of certain cardiovascular diseases 9, 10 and 17]. Considerable amount of EPA (20:5 n-3) was found in both sexes of T. carinifera. 22:2 NMID was among the chief PUFA found in males and females.

Varying amount of NMI FAs frequently occur in marine and freshwater gastropods [18-22]. High concentrations of 20:5 n-6, 20:5 n-3 and 18:2 (linoleic) in Telescopium telescopium and suggested that the high and low concentrations of Arachidonic acid (20:4 n-6) is related to high and low salinity respectively[10]. In T. carinifera 20:5 n-3 and 20:4 n-6 is found fairly and 20: 2 n-6 is found fairly in large amount as compare to 18:2 n-6.

Considerable amount of multi-methyl branched, methyl-branched and hydroxy FAs were present in T. carinifera. Branched FAs in other marine gastropods (Nassa serata, Nassarius albescens, Nodiolittorina subnodosa, Littorina scarab, Planaxis sulcata, Monodonta turbinate, Gibula cineraria and Littorina neritoides) have been reported [4], however hydroxy FAs have not been previously reported in any marine gastropod species. Fairly large concentration of steroidal components was also found.

Steroidal components found largely in samples of T. carinifera. Cholesterol contributed a larger proportion of steroidal component in both males and females of T. carinifera. It is well known fact that gastropods contain large amount of cholesterol (63 to 99%) of total sterols) and comparatively minor concentrations of other sterols [12]. Teshima and Kanazawa [11] have reported the substancial amount of 22-dehydrocholesterol, brassicasterol, desmosterol, campesterol, 24- methylenecholesterol and b-sitosterol in gastropod species Conomurex luhuanus, Charonia sauliae, Cymatiidae sp and Murex asianus from Japan. In T. carinifera b-sito-sterol contributed a smaller proportion of total sterols.

Experimental

Animals Material

Prosobranch neogastropod species Thais carinifera was collected in June 2007 from Sonmiani Bay, Balochistan situated along the Pakistan coast at 25deg 25' N, 66deg 35' E in the northwest of Karachi. The samples were hand picked from the low tide mark during ebb tides.

Extraction

Soft tissue of the gastropod species was extracted out by breaking the shell. Two replicate samples were prepared from the digestive gland / gonad complex of five male and female snails in each sample, which were analyzed for FAs by Gas Chromatography Mass Spectrometry (GC-MS) method.

Extraction was done following the method of Folch et al., (1957). Fresh frozen animal tissue (~1.6 g) of species (T. carinifera) was homogenized with mortar-pestle by adding 10 ml of chilled chloroform/methanol (2:1, v/v). Cold extract was decanted in glass vials and evaporated under N2 stream. Organic phase was extracted with 10 ml of n- hexane. Hexane layer was collected using a small separatory funnel, dried over anhydrous sodium sulfate and evaporated to dryness using N2 stream. The residue (0.019g from males and 0.024g from females) obtained was mixed with saturated solution of sodium methoxide (prepared from Na-metal and dried methanol) and kept for 24 hrs in a freezer for methylation. The mixture is then shaken with 10 ml of n-hexane, dried with anhydrous sodium sulfate and subjected to FAME (fatty acid methyl esters) analyses on GC-MS.

GC and GCMS Analysis

A gas chromatograph (Agilent 6890N) coupled with Jeol JMS-600H mass spectrometer and equipped with a DB5-MS[?] capillary column (30m, 0.32mm ID and 0.22 um df) was used, with helium carrier gas at flow rate of 1.8 ml/min. Injector was kept at 260 degC, while MS was operated at 250 degC and 70ev ionization. Column temperature was kept at 50 degC for 2 minutes and then raised to 260 degC with ramping of 5 degC/min. Final temperature was kept for 20 min. 1 uL of sample diluted in diethyl ether was injected. The concentrations were calculated using Area Normalization Method on FID responses.

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1Center of Excellence in Marine Biology University of Karachi, Karachi-75270, Pakistan., 2Department of Chemistry, University of Karachi, Karachi-75270, Pakistan., 3HEJ Research Institute of Chemistry, ICCBS, University of Karachi, Karachi-75270, Pakistan, Present Address: Faculty of Marine Sciences, Lasbela University of Agriculture, Water and Marine Sciences (LUAWMS), Uthal, Balochistan.
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Author:Afsar, Nuzhat; Siddiqui, Ghazala; Rasheed, Munawwer; Ahmed, Viqar Uddin; Khan, Afsar
Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Geographic Code:9PAKI
Date:Jun 30, 2012
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