Component and Content of Lipid Classes and Phospholipid Molecular Species of Eggs and Body of the Vietnamese Sea Urchin Tripneustes gratilla

Sea urchins (Tripneustes gratilla) are among the most highly prized seafood products in Vietnam because of their nutritional value and medicinal properties. In this research, lipid classes and the phospholipid (PL) molecular species compositions from the body and eggs of T. gratilla collected in Hon Tam, Nha Trang, Khanh Hoa, Vietnam, were investigated. Hydrocarbon and wax (HW), triacylglycerol (TG), mono- and diacylglycerol (MDAG), free fatty acid (FFA), sterol (ST), polar lipid (PoL), and monoalkyl-diacylglycerol are the major lipid classes. In PL, five main glycerophospholipid classes have been identified, in which 137 PL molecular species were detected in the body and eggs of T. gratilla, including 20 inositol glycerophospholipids (PI), 11 serine glycerophospholipids (PS), 22 ethanolamine glycerophospholipids (PE), 11 phosphatidic acids (PA), and 73 choline glycerophospholipids (PC). PI 18:0/20:4, PS 20:1/20:1, PE 18:1e/20:4, PA 20:1/20:1, and PC 18:0e/20:4 are the most abundant species with the highest content values of 38.65–48.19%, 42.48–44.41%, 41.21–40.03%, 52.42–52.60%, and 7.77–7.18% in each class of the body–eggs, respectively. Interestingly, PL molecules predominant in the body sample were also found in the egg sample. The molecular species with the highest content account for more than 40% of the total species in each molecular class. However, in the PC class containing 73 molecular species, the highest content species amounted to only 7.77%. For both the body and egg TL samples of the sea urchin T. gratilla, a substantial portion of C20:4n polyunsaturated fatty acid was found in PI, PE, and PC, but C16, C18, C20, and C22 saturated fatty acids were reported at low levels. The most dominant polyunsaturated fatty acid in PI, PE, and PC was tetracosapolyenoic C20, while unsaturated fatty acid C20:1 was the most dominant in PS and PA. To our knowledge, this is the first time that the chemical properties of TL and phospholipid molecular species of the PoL of Vietnamese sea urchin (T. gratilla) have been studied.


Introduction
The study of marine invertebrates has recently increased, resulting in a rapid increase in the number of publications on lipidomics (fatty acids and lipid classes). However, studies on the molecular species of these invertebrates are limited, especially those within Echinodermata such as sea urchins [1]. Approximately 950 species of sea urchins have

Polar Lipid Type and Phospholipid Classes
In marine invertebrates, the PoL usually consists of glycolipids (GLs) and glycerophospholipids (GPLs) with GPL as the largest PL class. In our study, molecular species of PL from the eggs and body of T. gratilla were detected following the previously described HRMS fragmentations of PL standards [8]. Five types of GPLs, including phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidic acids (PA), and phosphatidylcholine (PC), were identified (Figures 1 and 2). Their molecular structures and contents were analyzed using a Shimadzu LCMS-IT-TOF instrument with a Shimadzu LCMS Solution control and processing software (v.3.60.361, Shimadzu, Kyoto, Japan). The polar ends of the structures of the identified GPLs classes, inositol, serine, eth anolamine, and choline were major groups in the polar head contained in their mole cules. The polar ends of the structures of the identified GPLs classes, inositol, serine, ethanolamine, and choline were major groups in the polar head contained in their molecules.

Molecular Species of Phosphatidylinositol (PI)
In total, 20 molecular species were found in the phosphatidylinositol (PI) class from the PoL in both samples of the sea urchin T. gratilla (Table 2) 11.49%, and 9.36% content in the eggs and 12.64%, 9.89%, and 6.96% content in the body, respectively. PI 16:0/20:4, PI 18:0e/20:4, PI 18:0/20:3, and PI 20:1/20:5 were in the third highest group with the contents ranging from 2.17% to 4.82%. The other species presented low contents that were less than 1% (see Table 2 for detail). The polar ends of the structures of the identified GPLs classes, inositol, serine, ethanolamine, and choline were major groups in the polar head contained in their molecules.
The structure of PI can be characterized according to their MSand MS/MSdata. Signals of negative quasi-molecular ions [M -H]were observed in the HRMS spectra of all components of the formula species of PI ( Figure 3A and Table 2).
For example, a negative quasi-molecular ion [M -H] − at m/z 885.5562 for PI 18:0/20:4 was detected and assigned for a molecular formula of C 47 H 83 O 13 P with a calculated value of 885.5571 and a different value of 0.0009 ( Figure 3B,C). This was the strongest signal (highest peak) in the HPLC-HR/MS of total molecular species of the PI class, with a retention time (Rt) of 17.939 min ( Figure 3A,B). From the MS 2− spectrum of the ions [M -H] − of this PI 38:4 ( Figure 3D), one signal corresponding to one carboxylate anion of 20:4 was detected at m/z 303.2308 (calc. for C 20 H 32 O 2 ). Furthermore, the above observation was supported by a peak appearing at m/z 581.3085 which was calculated for C 27 H 50 O 11 P − ( Figure 4B). For the structure of PI, the fatty acid (FA) anion ([RCOO]-) was liberated from the sn −1 position due to the alkenyl linkages at the sn −2 position ( Figure 4). Thus, the peak that appeared at m/z 283.2636 could be assigned for the loss of the fatty acid (C18:0) anion (C 18 H 35 O 2 − ) at sn −1 in the molecular species of PI 38:4, and this observation was also supported by a signal corresponding to C 29 H 46 O 11 P − at m/z 599.316 ( Figures 3D and 4B).  The structure of PI can be characterized according to their MSand MS/MSdata. Signals of negative quasi-molecular ions [M − H] -were observed in the HRMS spectra of all components of the formula species of PI ( Figure 3A and Table 2).     Figure 3D), one signal corresponding to one carboxylate anion of 20:4 was detected at m/z 303.2308 (calc. for C20H32O2). Furthermore, the above observation was supported by a peak appearing at m/z 581.3085 which was calculated for C27H50O11P − ( Figure 4B). For the structure of PI, the fatty acid (FA) anion ([RCOO]-) was liberated from the sn −1 position due to the alkenyl linkages at the sn −2 position ( Figure 4). Thus, the peak that appeared at m/z 283.2636 could be assigned for the loss of the fatty acid (C18:0) anion (C18H35O2 − ) at sn −1 in the molecular species of PI 38:4, and this observation was also supported by a signal corresponding to C29H46O11P − at m/z 599.316 ( Figures 3D and 4B).     The ion peak at m/z 315.0481 corresponded to a fragmentation of a partial structure glycerol phosphatidylinositol unit ( Figure 4C), which could mean the loss of two fatty acid units (C18:0) and (C20:4) at sn −1 and sn −2 in the molecular species PI 38:4. From the observation above, the molecular species of PI 38:4 was therefore characterized as diacylglycerol phosphatidylinositol 18:0/20:4, and its structure is presented in Figure 4A. The ion peak at m/z 315.0481 corresponded to a fragmentation of a partial structure glycerol phosphatidylinositol unit ( Figure 4C), which could mean the loss of two fatty acid units (C18:0) and (C20:4) at sn −1 and sn −2 in the molecular species PI 38:4. From

Molecular Species of Phosphatidylserine (PS)
In the phosphatidylserine (PS) class from the PL of both samples of the Vietnamese sea urchin T. Gratilla, a total of 11 PS molecular species have been identified ( Figure 5 and Table 3). The ion peak at m/z 315.0481 corresponded to a fragmentation of a partial structure glycerol phosphatidylinositol unit ( Figure 4C), which could mean the loss of two fatty acid units (C18:0) and (C20:4) at sn −1 and sn −2 in the molecular species PI 38:4. From the observation above, the molecular species of PI 38:4 was therefore characterized as diacylglycerol phosphatidylinositol 18:0/20:4, and its structure is presented in Figure 4A.

Molecular Species of Phosphatidylserine (PS)
In the phosphatidylserine (PS) class from the PL of both samples of the Vietnamese sea urchin T. gratilla, a total of 11 PS molecular species have been identified ( Figure 5 and Table 3).      P) followed next with 10.43% and 7.31% in the eggs and 14.60% and 11.32% in the body, respectively. Except for PS 20:1/20:4 with a content of up to 9.67% in the eggs, the other PS molecular species presented the contents that were under 9%. There were two species, PS 38:5 and PS 20:1/18:1, which presented only 0.50% and 0.60% in body sample, respectively (see Table 3 for detail).

Molecular Species of Phosphatidylethanolamine (PE)
For phosphatidylethanolamine (PE), 22 molecular species were found (Table 4 and Figure 6A). Among these 22 components, 3 presented high content (over 10%) including PE 18:1e/20:5, PE 18:1e/20:4, and PE18:1e/20:2, accounting for 59.75% in the eggs and 60.31% in the body of total PE species. The remaining species presented low contents that were under 10%. There was no variation in the identified molecular species and little variation in the content of each composition between the two samples (eggs and body). In this study, the letter "e" denotes the head attached to the glycerol molecule with an ether bond.

Molecular Species of Phosphatidic Acids (PA)
In the phosphatidic acid PA class, 11 molecular forms were identified (Table 5 and

Molecular Species of Phosphatidic Acids (PA)
In the phosphatidic acid PA class, 11 molecular forms were identified (Table 5 and Figure 7). Among the received signals, the negative ion signal [M -H]had the highest intensity at m/z 755.5570 in both the eggs and body TL samples of sea urchin T. gratilla, and the signal corresponding to the molecular species occupied the highest concentration in this PA class ( Figure 7A,B). lost a neutral fragment C20H36O (C19H37COOH-H2O); and the signal at m/z 445.2713 corresponds to the molecular ion losing a neutral fragment, the fatty acid C20H38O2 ( Figure 7C).

Molecular Species of Phosphatidylcholine (PC)
From the two samples (eggs and body) of sea urchin T. gratilla, 73 signals were identified in the composition of the phosphatidylcholine (PC) class (Table 6), corresponding to 73 molecular species (Table 5 and Figure 8A). Among them, the positive   Figure 7C).

Molecular Species of Phosphatidylcholine (PC)
From the two samples (eggs and body) of sea urchin T. gratilla, 73 signals were identified in the composition of the phosphatidylcholine (PC) class (Table 6), corresponding to 73 molecular species (Table 5 and Figure 8A Figure 8D). In addition, the chemical structure of the PC 16:0/20:4 was consistent with the other data. Therefore, the identified PC molecular species was identified as PC 16:0/20:4.

Material
A wild-caught sample of Cau gai vang T. gratilla (Linnaeus, 1758) was collected by divers in shallow water using specialized tools from the intertidal zone to a depth of about 70 m, on 17 November 2016, from Hon Tam Island, Nha Trang, Khanh Hoa, Vietnam (12 • 10 33.3" N, 109 • 14 34.8" E). Its scientific name was examined by Dr. Nguyen An Khang, Nha Trang Institute of Oceanography, Vietnam Academy of Science and Technology. After collection, the samples were stored in an insulated container and kept at a temperature of 0 to 4 • C while being transported by airplane to the laboratory in Hanoi. The total time of shipment was 2.5 h. A voucher specimen was deposited at the Institute of Natural Products Chemistry, VAST.

Extraction of Total Lipid
The sea urchins were cut in half and washed with distilled water. Eggs were separated from the body using a spoon. The eggs (TG-E) and body (TG-B) materials were immediately homogenized with a blender under cool conditions for 2 min before extracting the total lipid by using the Bligh and Dyer method [9]. Briefly, the eggs and body (each 300 g) materials were extracted with 900 mL of CHCl 3 -MeOH (v/v = 1/2), the solid:liquid ratio being 1:3 (g/mL), and then sonicated for 2 h. Afterward, 300 mL of CHCl 3 and 600 mL of distilled water were added to the mixture for partitioning it. When partitioned, the lower layer (containing lipid) was separated, and the residue (upper layer) was extracted twice using continuous sonication for 2 h. Thus, the total extraction time was 6 h for one sample and the procedure was repeated three times. The combined lipid extract solution was dehydrated via anhydrous Na 2 SO 4 and was evaporated to give a total lipid extract. The total lipid content was calculated as a percentage of lipid quantity compared to the fresh sample weight. Finally, the lipid extract was stored in pure CHCl 3 at −20 • C. Lipid samples used for further analyses were prepared daily by diluting them in a mixture of CHCl 3 and MeOH (v/v = 1:2).

Analysis of Polar Lipid Classes
Polar lipid classes composition was first analyzed via thin-layer chromatography (TLC) using silica gel plates (Sorbfil, Krasnodar, Russia) and a solvent system of dichloromethane/ diethyl ether/NH 3

Analysis of Molecular Species of Phospholipids
The molecular species of polar lipids (phospholipids) from eggs and body materials were detected using previously described HRMS fragmentations of PL standards [10,11]. The high-performance liquid chromatography/high-resolution tandem ion trap-time of the flight mass spectrometry with a Shimadzu LCMS-IT-TOF instrument (Shimadzu, Kyoto, Japan) was used to analyze the molecular species of PL. The LCMS-IT-TOF equipped with two LC-20AD pump units, a high-pressure gradient forming module, 9CCTO-20A column oven, SIL-20A auto sampler, CBM-20A communications bus module, DGU-20A3 degasser, and a Shim-Pack diol column (50 mm × 4.6 mm ID, 5 µm particle size) was operated both at positive and negative ion modes during each analysis at electrospray ionization (ESI) conditions. The ion source temperature was 200 • C, the range of detection was m/z 200-1600, and the potential in the ion source was −3.5 and 4.5 kV for negative and positive modes, respectively. The drying gas (N2) pressure was 200 kPa. The nebulizer gas (N2) flow was 1.5 L/min. The mobile phase condition for separation of PoL was performed using a binary gradient consisting of solvent mixture A: n-hexane/2-propanol/acid formic/(C 2 H 5 ) 3 N (82:17:1:0.08, v/v/v/v) and mixture B: 2-propanol/H 2 O/acid formic/(C 2 H 5 ) 3 N (85:14:1:0.08, v/v/v/v).
The gradient started at 5% of mixture B, and its percentage increased to 80% over 25 min. This composition was maintained for 1 min before being returned to 5% of mixture B for more than 10 min and maintained at 5% for another 4 min (total run time of 40 min). The flow rate was 0.2 mL/min. Polar lipids were detected via high-resolution mass spectrometry (HRMS) and compared against authentic standards using a Shimadzu LCMS Solution control and processing software (v.3.60.361, Shimadzu, Kyoto, Japan). Individual molecular species within each PL class was measured by calculating the peak areas on extracted ion chromatograms [12].

Conclusions
Through experimentation to determine the total lipid and lipid classes of both eggs and body material from sea urchin T. Gratilla, this study found that the total lipid of eggs was much higher than that of the body sample (4.41% vs. 1.32% on wet weight base, respectively). The seven lipid classes of total lipid were hydrocarbon and wax (HW), triacylglycerol (TAG), free fatty acids (FFA), sterol (ST), polar lipid (PoL), and monoalkyl-diacylglycerol (MADAG). Among these, the proportions of TAG accounted for the highest amount from approximately 76% to 78%. The two lowest classes were HW and MADAG with percentages of 1.1% and 2.3% of the total lipid for eggs and body materials, respectively.
To our knowledge, this is the first study to determine the total lipid, lipid classes, fatty acid compositions, and phospholipid molecular species of sea urchins in general and T. gratilla in particular. The five types of phospholipids were identified as PI, PS, PE, PA, and PC with 137 molecular species in total for each sample. In both body and eggs, PoLs of the sea urchin T. gratilla, C20:4n was the most abundant polyunsaturated fatty acid in PI, PE, and PC classes, while C16, C18, C20, and C22 saturated fatty acids were less common. The most dominant polyunsaturated fatty acid in PI, PE, and PC was tetracosapolyenoic C20, while unsaturated fatty acid C20:1 was the most dominant in PS and PA classes. To our knowledge, this is the first time that the chemical properties of TL, especially the phospholipid molecular species of the PoL in the Vietnamese sea urchin (T. gratilla), have been studied.  Data Availability Statement: Study data are available from the corresponding authors upon reasonable request.