Lipid composition and molecular species of phospholipid in oyster Crassostrea lugubris (Sowerby, 1871) from Lang Co Beach, Hue Province, Vietnam

Abstract Oysters are widely distributed worldwide, but are mainly concentrated in tropics and subtropics. Total lipid (TL), fatty acid (FA) composition of TL and polar lipid (PoL) fractions, phospholipid (PL) class, and molecular species composition in soft tissues of Crassostrea lugubris were investigated for the first time from Vietnam. Phosphatidylglycolic acid (PGA) is the new phospholipid class first identified in marine species in general and Crassostrea lugubris in particular. Main eight classes of PL were determined in PoL fraction: diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), ceramide aminoethylphosphonate (CAEP), CAEP with hydroxylated FAs (CAEP‐OH), and lysophosphatidylcholine. PE and PC accounted for approximately 63% of total known PL. Polyunsaturated FAs accounted for more than 30% of TL. Ninety molecular species of glycerophospholipids, including PGA, PE, PC, PS, PI, DPG, and PG, and sphingophosphonolipids (CAEP) were identified in PoL. Alkenyl‐acyl forms of glycerophospholipids were predominated in the molecular species of PGA, PE, and PS. PGA 38:1 (p18:0/20:1), PE 40:6 (p18:0/22:6 and p18:1/22:5), PC 30:0 (14:0/16:0), PS 38:1 (p18:0/20:1), PI 40:5 (20:1/20:4), PG 32:0 (16:0/16:0), DPG 88:24 (22:6/22:6/22:6/22:6), and CAEP 34:2 (d18:2/16:0) were major molecular species.

Dietary supplements of PUFA are mainly in the forms of triacylglycerol (TAG), ethyl ester (EE), and phospholipid (PL) (Witte et al., 2014). These years, due to their better bioavailability, higher tissue-delivery capacity, and enhanced health-promoting effects, PUFAs in the PL form have been increasingly studied (Ghasemifard et al., 2014).
Individual separation of phospholipid molecular species using high-performance liquid chromatography has been widely established (Blank et al., 1984;Patton et al., 1982). In mussel, for example, PC accounted for about 60% of the muscles liver and PL amounted to 53.8% of the sum of all PLs in the gills (Liu et al., 2012). In another study involving various cold-water species, it was found that the PE content may contribute, on average, 24.3, 25.1, and 22.3% to muscles, liver, and gills, respectively (Velansky & Kostesky, 2008). PC, PE, PI, PS, CAEP, PG, and DPG were found, but sphingomyelin (SM) was not detected in the sea worms, mollusks, and arthropods (Kostesky & Velansky, 2009).
Crassostrea lugubris (C. lugubris) (Veneridae genus), also known as oysters, could be found in seashore and estuarine areas and is regarded as a valuable export product with high economic value (Sowerby, 1871). In addition, the oysters could serve as an efficient assimilator of nutrients, causing significant reduction in total nitrogen, total carbon and total phosphorous per hectare (Dame & Libes, 1993;Higgins et al., 2011;Hyun et al., 2013). Oysters are an economically and nutritionally important aquaculture species. As the data on their chemical composition in general and their lipid in particular are limited, our study aims to be the first to completely report the lipid of oyster C. lugubris collected in Lang Co Beach, Hue Province, located in the Central Coast region of Vietnam. The content of total lipid, fatty acids, lipid, and phospholipid classes was presented. We showed that the lipids from oysters contained six classes in which polar lipid occupied approximately a quarter of TL.
Beside the molecular species such as PE, PC, PI, PS, CAEP, PG, and DPG in other research in oysters, mollusks, and marine animals, this research would be the first to identify new molecular species PGA with eight molecular species.

| Lipid extraction
The crushed soft tissues were extracted for TL following a modified Bligh-Dyer extraction procedure (Bligh & Dye, 1959). For 10 g of oyster soft tissues, 30 ml of chloroform/methanol solution (1:2, v:v) was used to extract in 6 hr, at 30°C to afford the homogenate, which was then subjected to filtration to obtain the residue. The residue was then repeatedly extracted in chloroform (20 ml) in 6 hr at 30°C. Afterward, the obtained homogenates were pooled and added with 20 ml of H 2 O to separate the mixture into layers. After evaporating the lower layer, the TL was dissolved in chloroform. Total lipid was extracted with seven repetitions and stored at −5°C.

| Lipid and phospholipid class analysis
The precoated silica gel plates (6 cm ×6 cm) Sorbfil PTLC-AF-V (Sorbfil, Krasnodar, Russia) was prepared to determine lipid class compositions. The plate was developed in two steps in which full length development using n-hexane/diethyl ether/acetic acid (85:15:1, v:v:v) was performed first, followed by redevelopment with chloroform/methanol (2:1, v:v) for 5% length. Afterward, airdrying was commenced over the plates, followed by spraying with 10% H 2 SO 4 in methanol and heating at 240°C for 10 min (Imbs et al., 2015). These classes of TL were determined by comparison with standards.
Phospholipid class compositions were qualitatively analyzed by two-dimensional thin-layer chromatography (TLC) and quantitatively determined by one-dimensional TLC. First, dissolution of the extracted lipids in chloroform (80 mg/ml) was performed, followed by spotting onto the one-and two-dimensional TLC using the silica gel plates (10 cm ×10 cm). One-dimensional TLC plates were developed with chloroform/methanol/28% aqueous ammonia/benzene ratio of 65:30:5:10 (v:v:v:v). Two-dimensional TLC plates were developed in the first direction with chloroform/methanol/28% aqueous ammonia/benzene ratio of 65:30:5:10 (v:v:v:v). This chromatogram was dried for about 10 min and then developed in the second direction with chloroform/acetone/methanol/acetic acid/water ratio of 70:30:5:5:2 (v:v:v:v:v). Subsequently, one-and two-dimensional TLC plates were air-dried and sprayed with three solutions: ninhydrin, molybdate reagent, and 10% H 2 SO 4 in methanol (Rouser et al., 1970;Skipski et al., 1964). In order to identify phospholipids on TLC plates, authentic standards and the specific spray reagents used earlier were employed.
Grayscale chromatograms were obtained using a flatbed scanner (Epson Perfection 2,400 PHOTO), and their band intensities were evaluated with software (Sorbfil TLC Video densitometer, Krasnodar, Russia) to determine the quantification of lipid classes.

| Polar lipid separation
Polar lipid was separated by TLC plate preparation. Glass-backed precoated ready-made silica gel plates from Merck, Darmstadt F.RG., Cat. No. 5721, 20 × 20 cm × 0.25 mm from Macherey-Nagel were used. All plates used were precleaned by running them in methanol: dichloromethane (2:1, v:v) and dried. The plates were activated before use by heating for 30 min in an oven at 130°C. They were dried for 10 min between migrations, in a vacuum desiccator at 250°C.
After completion of TLC separation on Merck, the spots were located by phosphomolybdic acid and heated in an oven at 100-120°C for at least 2-5 min. The phosphomolybdic acid reagent composed of 5 g phosphomolybdic acid in 100 ml, ethanol plus 1 ml, and 70% perchloric acid (Pucsok et al., 1988). Lipid and polar lipid were first treated with 2% H 2 SO 4 in methanol commenced in 2 hr at 80°C in a screw top vial, followed by purification by TLC development in hexane-diethyl ether (95:5, v:v). GC analysis was employed to analyze fatty acid methyl esters (FAME) with column temperature of 210°C. Identification of FA was carried out by comparing the obtained results with authentic standards and reported the equivalent chain lengths (Christie et al., 1988). Injector and detector temperatures were set at 240°C.

| Fatty acids analysis
Fatty acids were structurally determined by performing GC-MS against the corresponding FAME and subsequently, matching the obtained spectra with the NIST library and FA mass spectra archive (Harrabi et al., 2009; Mass spectrometry of Fatty Acid Derivatives, 2020). The thermal profile of the column was initiated at 160°C, followed by an acceleration at 2°C/min to 240°C that prolonged for 20 min. The injector temperature was set at 250°C.

| Phosphatidylglycolic acid synthesis
The cabbage trans-phospholipase D was used for phosphatidylglycolic acid (PGA) synthesis. Fresh cabbage tissue (1 g) was homogenized with an equal volume of distilled water by using IKA Ultra-Turrax T25 homogenizer with S25N-10G tool. The obtained homogenate was filtrated and centrifuged at 6,000 rpm for 10 min.
The supernatant was used as the enzyme source. 10 mkg of PC (18:0/18:1 diacyl, a16:0/18:1 alkyl-acyl and p18:0/20:4 alkenyl-acyl [Avanti Polar Lipids]) in chloroform was evaporated to dryness in a standard 2-ml vessel and sonicated with 100 mkl of Na-glycolate buffer (2 M, pH 5.6 with 0.08 M CaCl 2 ) for 10 min in an ultrasonic bath. Then, 100 mkl of cabbage extract and 20 mkl of hexane were added, and the mixture was allowed to react for 2 hr at room temperature. The reaction products were extracted by 300 mkl of chloroform and redissolved in 50 mkl of chloroform.
Develosil was used in HILIC mode, and binary gradient was used to perform HPLC separation. The gradient included two solvents, A and B. The solvent A was acetonitrile/water (94:6, v:v) and B was pure water. Both solvents contained 20 mM acetic acid and 10 mM ammonia. The compositional progress of the gradient began at 0% of solvent B, which was then elevated to 20% over 30 min and kept for another 10 min. Afterward, the percentage was allowed to drop to 0% for 7 min. Cumulatively, the whole progress lasted 47 min. The flow rate was 0.2 ml/min. Detection of polar lipids was carried out by HRMS. Authentic standards were used to compare with results using Shimadzu LCMS Solution control and processing software (v.3.60.361). In each polar lipid class, the molecular species were individually quantified the peak areas of ion chromatograms (Boukhchina et al., 2004).

| Mild acidic hydrolysis
This simple method was used for alkenyl forms of phospholipids determination. The sample was evaporated to dryness in a 2-ml vial under a stream of argon. The vial was then inverted over five drops of HCl in a vial cap. After 4 min, the vial was immediately purged with argon for 5 min. Alkyl and acyl bonds are able to withstand this treatment, while alkenyl bond is not (Murphy et al., 1993). Samples were injected into HPLC-HRMS to identify the ion chromatograms.

| Statistical analysis
The difference between mean values was analyzed by one-way analysis of variance (ANOVA), using Excel 2013 software, with seven repetitions. The results were presented as: mean ± SD.

| Lipid class composition
Total lipid was found in an amount of 2.54 ± 0.32% of wet weight of the oysters. Six classes of TL of the oysters were detected, including hydrocarbons and wax (HW), triacylglycerol (TAG), free fatty acids (FFAs), sterol (ST), polar lipid (PoL), and monoalkyl diacylglycerol (MADAG) (Table 1 and Figure S1 in supporting information). The qualitative composition was similar to that of other Bivalvia, zooplankton and coral which have been previously investigated (Abad et al., 1995;Imbs et al., 2010;Nelson et al., 2000;Tran et al., 2019). MADAG claimed a significant share of TL at 13.77%, which was in line with the lipid compositions of cnidarians and coral.
Regarding PoL, this component only represented a relatively low content (22.46% of TL), in comparison with PoL in lipid compositions of Cnidaria, Ctenophora, and clam, which usually occupied more than half of TL.
In particular, FFA content (5.65%) was lower than that of other oyster species, indicating that the collected samples were of high quality. The presence of sterols indicated the widespread of lipid class, which usually acts as membrane constituents, in marine species (Bernsdorff & Winter, 2003;Le Grand et al., 2011;Prato et al., 2010).

| Phospholipid class composition
Resolving capability of two-dimensional TLC technique against polar lipids has been demonstrated in previous studies. Furthermore, quantification of phospholipids by spot analysis and color development without prior elution from TLC adsorbent has been proved to be an efficient and quick routine (Medh & Weigel, 1989;Rouser et al., 1970). The main eight classes of phospholipid were deter-  Figure S2) The composition is similar to that of other previous studies (Lund & Chu, 2002;Yeong et al., 1990;Kraffe et al., 2002). On one-dimensional thin layer, there were six identified classes of phospholipid. PS +CAEP-OH and CAEP +PI did not separate on one way.
The composition of phospholipid classes was determined by one-dimensional TLC (Table 2 and Figure S3). PE and PC are two main classes in phospholipid of oysters, accounting for 32.98 and 29.29% of total known phospholipids, respectively. This result is in agreement with previous studies (Le Grand et al., 2011;Lund & Chu, 2002;Yeong et al., 1990;Kraffe et al., 2002). LPC accounted for the lowest proportion with 3.72%. This lipid is usually a product of  (White, 1973). This is similar to the current DPG percentage of 9.32%. The PS +CAEP-OH and CAEP +PI fractions accounted for 10.35 and 14.33% of total known phospholipids, respectively.

| Fatty acid composition
The fatty acid composition of C. lugubris comprised a total of 26 fatty acids whose carbon atom number ranged from 14 to 22 (Table 3).   Figure S4e). All detected ions of synthetic PGA are summarized in Table 4.

| Molecular species of phosphatidylglycolic acid
This is the first new molecular species to be identified in PL of marine animal in general and oysters in particular. To determine the structure of molecular species of PGA from C. lugubris, the aforementioned data of the HRMS fragmentations of PGA standards (see Figure 1 and

| Molecular species of phosphatidylethanolamine
In the marine oysters, PE included alkenyl-acyl and diacyl forms, but not alkyl-acyl form (Chen et al., 2012). Among PE from C. lugubris, nineteen components were identified and alkenyl acyl glycerophosphoethanolamine (ethanolamine plasmalogen PlsEtn) was abundantly found in the PE composition, accounting for 74.56% of total PE species (Table 5) (Table 5).
To determine the structure of molecular species of PE, PC, PS, PI, and CAEP, we applied the data of the HRMS fragmentations of pre-

| Molecular species of phosphatidylcholine
The results in C. lugubris were similar to hard clams. Different to PE,  Figure S8). Normally, the sn-2 of PL is the preferred position for PUFAs (Tran et al., 2019). Therefore, PC 30:0 was characterized as diacylglycerophosphocholine 14:0/16:0.

| Molecular species of phosphatidylserine
We detected 9 components (Table 5). It is shown that alkenyl-acyl glycerophosphoserine majorly constituted PS profile, which is similar to that of PE. The four components of alkenyl-acyl glycerophosphoserine amounted to 82.32% of total PS species.

| Molecular species of phosphatidylinositol
Among PI of oysters, we determined 12 components (Table 5). In addition, alkenyl acyl glycerophosphoinositol was absent in PI. All ten components PI were diacylglycerophosphoinositol with fatty acids

| CON CLUS IONS
The present study analyzed lipid composition of C. lugubris har-

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
The study's protocols and procedures were ethically reviewed and approved by Vietnam Academy of Science and Technology, Hanoi City, Vietnam.