Characterization of lipids in three species of sea urchin

Accepted Manuscript Characterization of lipids in three species of sea urchin Xin Zhou, Da-Yong Zhou, Ting Lu, Zhong-Yuan Liu, Qi Zhao, Yu-Xin Liu, Xiao-Pei Hu, Jiang-Hua Zhang, Fereidoon Shahidi PII: DOI: Reference:

S0308-8146(17)31412-7 http://dx.doi.org/10.1016/j.foodchem.2017.08.076 FOCH 21625

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

30 March 2017 17 July 2017 22 August 2017

Please cite this article as: Zhou, X., Zhou, D-Y., Lu, T., Liu, Z-Y., Zhao, Q., Liu, Y-X., Hu, X-P., Zhang, J-H., Shahidi, F., Characterization of lipids in three species of sea urchin, Food Chemistry (2017), doi: http://dx.doi.org/ 10.1016/j.foodchem.2017.08.076

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Characterization of lipids in three species of sea urchin Xin Zhoua, Da-Yong Zhoua,b*, Ting Lua, Zhong-Yuan Liua, Qi Zhaoa,b, Yu-Xin Liua, Xiao-Pei Huc, Jiang-Hua Zhangb, Fereidoon Shahidid a

School of Food Science and Technology, Dalian Polytechnic University, Dalian, PR

China, 116034 b

National Engineering Research Center of Seafood, Dalian, PR China, 116034

c

Beijing Advanced Innovation Center of Food Nutrition and Human Health, China

Agricultural University, Beijing, China, 100083 d

Department of Biochemistry, Memorial University of Newfoundland, St. John's, NL,

Canada, A1B 3X9

Abstract Sea urchin gonad has been regarded as a “healthy” food. Although previous studies have suggested that sea urchin gonad might serve as a potential rich source of long chain omega-3 polyunsaturated fatty acids (n-3 LC-PUFA) enriched phospholipid (PL), the molecular species profile of its PL has rarely been reported. In this study, about

200

molecular

glycerophosphocholine, glycerophosphoinositol,

species

of

glycerophospholipid

glycerophosphoethanolamine,

(GP),

including

glycerophosphoserine,

lysoglycerophosphocholine

and

lysoglycerophosphoethanolamine, in gonads from three species of sea urchin, (Glyptocidaris crenularis, Strongylocentrotus intermedius and Strongylocentrotus nudus.), were characterized using tandem mass spectrometry. Most of the

1

predominant GP molecular species contained PUFA, especially eicosapentaenoic acid (EPA). Meanwhile, the sea urchin lipids contained a high proportion of PL (39.45-50.30% of total lipids) and PUFA (34.47-46.56% of total FA). Among PL, phosphatidylcholine (67.88-72.58 mol%) was dominant. Considering the high level of PUFA enriched GP, sea urchin gonads provide great potential as health-promoting food for human consumption. Keywords: Sea urchin, lipid class composition, phospholipid class composition, molecular species, HPLC-ESI-MS/MS

1. Introduction Sea urchins are marine invertebrates of the phylum Echinodermata which live on the ocean floor. To date more than 800 species of sea urchins have been found. Gonads (also termed “uni” or “roe”), the edible portion of sea urchin, account for approximately 10% of their total weight (La Cruz-García, López-Hernández, González-Castro, Rodríguez-Bernaldo De Quirós & Simal-Lozano, 2000). They are half-moon shaped, yellow-orange in colour, and chiefly composed of moisture, protein, carbohydrate and lipid (Dincer & Cakli, 2007). With distinctive aroma and good taste, sea urchin gonads are expensive and a delicacy in many countries, especially China and Japan. Recently, sea urchin gonad has been regarded as a “healthy” food due to its positive effects on health arising from constituent

lipids, proteins (polypeptides),

polysaccharides, carotenoids, vitamins and minerals (Archana & Babu, 2016;

2

Pozharitskaya et al., 2015). Generally, the lipid content of sea urchin gonad is >20% on a dry weight basis (Zhu et al., 2010; Archana et al., 2016). Previous studies have reported that phospholipid (PL) is the principal lipid (more than 40% of total lipids) in sea urchin gonads (Kalogeropoulos, Mikellidi, Nomikos & Chiou, 2012; Mita & Ueta, 1989). A study by Mita et al. (1989) indicated that the phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) isolated from sea urchins spermatozoa contained a high percentage of polyunsaturated fatty acids (PUFAs) (33.2-67.6% of total fatty acids), in particular arachidonic acid (AA; 20:4n-6) and eicosapentaenoic acid (EPA; 20:5n-3). Moreover, Kostetsky, Sanina and Velansky (2014) characterized 26 species of glycerophosphocholine (GPCho) and 23 species of glycerophosphoethanolamine

(GPEtn)

from

sea

urchin

(Strongylocentrotus

intermedius) gonad. Among them, the molecular species containing long chain omega-3 PUFA (n-3 LC-PUFA) such as 20:5 and 22:6 were predominant. Therefore, sea urchin gonads might serve as a potential rich source of PL enriched PUFAs. However, to the best of our knowledge, there has been no information on simultaneous analysis of fatty acid composition, lipid class composition, PL class composition and glycerophospholipid (GP) molecular species in gonads from different species of sea urchins. Meanwhile, Kostetsky et al. (2014) published the only research findings on characterization of molecular species of two PL classes (GPCho and GPEtn) in sea urchin gonad. Therefore, to provide nutritional and functional information and pave the way for better utilization of sea urchin gonads, detailed characterization of the lipid profile, including the molecular species of more PL

3

classes from different species of sea urchin, was deemed necessary. The n-3 LC-PUFA, in particular EPA and docosahexaenoic acid (DHA; 22:6n-3) have been found to possess a wide range of health benefits, such as improving heart disease related outcomes, promoting infant development, decreasing tumour growth and metastasis, inhibiting inflammation, platelet aggregation, hypertension and hyperlipidaemia, and favourably modifying insulin sensitivity (Anderson & Ma, 2009; Riediger, Othman, Suh & Moghadasian, 2009). Therefore, there is a convergence of opinion that daily consumption of 250-1000 mg of EPA/DHA provides health benefits (Kuratko & Salem, 2013). The n-3 LC-PUFA derived from foods is mainly in the triacylglycerol (TAG) and PL forms. Recently, n-3 LC-PUFA in the PL form have captured increasing attention due to their superior bioavailability (Cook et al., 2016; Yurko-Mauro, Kralovec, Bailey-Hall, Smeberg, Stark & Salem, 2015; Köhler, Sarkkinen, Tapola, Niskanen & Bruheim, 2015), higher tissue-delivery capacity (Liu et al., 2014; Rossmeisl et al., 2012; Graf et al., 2010; Cansell, 2010), and better health promoting effects (Batetta et al., 2009; Sampalis, Bunea, Pelland, Kowalski, Duguet & Dupuis, 2003; Ramprasath, Eyal, Zchut & Jones, 2013; Ulven et al., 2011) compared to TAG-containing n-3 LC-PUFA. Therefore, PL-enriched lipids in sea urchin gonad may account for much of its nutritional and healthful functions. Sea

urchin

Glyptocidaris

crenularis,

Strongylocentrotus

intermedius

and

Strongylocentrotus nudus are the most common species widely distributed in East Asian Seas (Shang et al., 2014; Suh et al., 2014; Zhao, Zhou, Tian, Feng & Chang, 2014). Hence, the aim of this study was to investigate the lipid content, fatty acid

4

composition, lipid class composition, PL class composition and GP molecular species belonging to six classes, including GPCho, GPEtn, glycerophosphoserine (GPSer), glycerophosphoinositol

(GPIns),

lysoglycerophosphoethanolamine

lysoglycerophosphocholine (LGPEtn)

in

(LGPCho)

Glyptocidaris

and

crenularis,

Strongylocentrotus intermedius and Strongylocentrotus nudus. This will help to understand the specific health benefits of lipids in sea urchin gonads, as well as to provide theoretical basis for utilization of sea urchin gonads as novel sources of functional foods and to fill the existing knowledge gap in the field.

2. Materials and methods 2.1. Materials Three

species

of

fresh

sea

urchins,

namely

Glyptocidaris

crenularis,

Strongylocentrotus intermedius and Strongylocentrotus nudus, were purchased from a local market in Dalian, Liaoning, China. After dissection, the fresh gonads were collected and lyophilized in a freeze-dryer (2KBTES-55, VirTis Co., Gardiner, NY, USA) for 72 h, subsequently crushed into a powder and stored at –30 oC until use. GP standards were purchased from Avanti Polar Lipids, INC. (Alabaster, AL, USA). Reagents, including methanol, acetonitrile, chloroform and ammonium formate, were HPLC-grade and purchased from Spectrum Chemical Mfg. Corp. (Gardena, CA, USA). Deuterated chloroform (CDCl3) and methanol (MeOD), triethyl phosphate (TEP), cesium carbonate (CsCO3) and D2O were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). All other reagents were of analytical grade and purchased

5

from Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). 2.2. Lipid extraction and sample preparation Total lipids were extracted from samples using a modified version of the methyl tert-butyl ether (MTBE) method (Matyash, Liebisch, Kurzchalia, Shevchenko & Schwudke, 2008). Briefly, 2.0 g of dried sea urchin gonad powder was completely mixed with 3 ml of methanol and 10 ml of MTBE in a conical flask. After stirring the mixture at 30 oC for 1 h, 2.5 ml of deionized water was added. Following adequate mixing, the mixture was centrifuged at 7,800 g for 10 min. The organic layer was subsequently transferred into a centrifuge tube and the precipitate was extracted again according to the aforementioned method. Finally, the organic layers were combined and dried using a stream of nitrogen at 35 oC, to obtain lipids from sea urchin gonads. The recovered lipids were then weighed and stored at –30 oC for further analysis within 2 weeks. The dilute lipid samples used for further analysis were prepared daily, by diluting them using a mixture of methanol and chloroform (1:2, v/v). Before injection for HPLC-MS/MS analysis, the sample solutions were filtered through a 0.22 μm microporous membrane. In order to semi-quantify the same compound in different samples, five GP internal standards, namely phosphatidyl GPCho 12:0/12:0, GPEtn 12:0/12:0 (internal standard for both GPEtn and LGPEtn), GPSer 12:0/12:0, GPIns 8:0/8:0 and LGPCho 10:0 were added to the samples in order to reach a concentration of 0.60, 0.15, 0.875, 0.10 and 0.15 µg/ml, respectively. 2.3. Lipid class composition analysis

6

Lipid class compositions were determined and analysed using an Iatroscan MK-6S thin layer chromatography-flame ionization detection (TLC-FID) analyzer (Iatron Inc., Tokyo, Japan), according to the procedures described in a previous study (Yin et al., 2015). 2.4. Fatty acid composition analysis Fatty acid compositions were determined using an Agilent 7890A GC-5975C MSD (Palo Alto, CA,USA) equipped with an HP-5-MS capillary column (30 m  0.25 mm, 0.25 μm) (Palo Alto, CA, USA), according to the procedures described elsewhere (Yin et al., 2015). 2.5. Quantitation of PL classes by 31P NMR Sample preparation was performed according to Burri, Hoem, Monakhova and Diehl (2016) with some modification. Briefly, 500 mg of lipid samples were dissolved in 1 ml CDCl3/MeOD (2:1, v/v). The addition of 1 ml of 0.2 M CsCO 3-EDTA solution in D2O (pH 7.2-7.6) led to the separation of two layers. Organic and aqueous phases were separated after intense vortexing and centrifugation (7,800 g for 10 min), and then the lower organic phase was collected into an NMR sample tube and subsequently used for

31

P NMR spectrometry.

31

P NMR analyses were conducted on

an Avance III 400 MHz Bruker NMR spectrometer (9.4 T) using 5 mm tubes and a quadruple nuclear probe. The number of transients was 4,096 per spectrum acquisition to achieve an optimal signal-to-noise ratio. 31P NMR spectra were acquired with a 9 s inter-pulse delay, 6,488 Hz spectral width, 90 pulse angle (9.5l s), 32 K data points and 1 H decoupling (Waltz 16, decoupling power, 19 dB; pulse width, 13 s). Lock and

7

shimming were performed on CDCl3. PL class quantification data by

31

P NMR was

processed using MestReNova 6.1.1 software. PL class quantification was conducted by comparing the peak area with TEP after integration, normalization was then carried out using the corresponding ratio. 2.6. HPLC-MS analysis The HPLC-MS/MS analysis of lipid samples was performed on a Shimadzu LC-20AVP system (Shimadzu Co. Tokyo, Japan) which was coupled in-line with a hybrid API 4000 Qtrap (AB Sciex, Foster City, CA, USA) quadrupole-linear ion trap (QqLIT) mass spectrometer, as described previously (Yin et al., 2016). The eluent was solvent A [ACN/H2O/100 mM NH4HCO2 (pH 3.2) = 95:2.5:2.5, v/v/v] and solvent B [H2O/100 mM NH4HCO2 (pH 3.2) = 97.5:2.5, v/v] at a flow rate of 0.2 ml/min. The gradient program was conducted as follows: 0-30 min, 0-20% B; 30-31 min, 20-50% B; 31-45 min, 50% B. The lipid sample concentration for analysis of GPCho, GPEtn/LGPEtn, GPSer, GPIns and LGPCho was 30, 400, 1000, 1000 and 1000 µg/ml, respectively, and the corresponding injection volume was 5, 15, 10, 15 and 15 µl, respectively. The HPLC system was coupled in-line with a hybrid API 4000 Qtrap (AB Sciex, Foster City, CA, USA) quadrupole-linear ion trap (QqLIT) mass spectrometer with a Turbo V ESI ionization source interface, and a computer platform equipped with HPLC-MS/MS Solution Analyst software 1.6.1 (AB Sciex, Foster City, CA, USA) which was used for data acquisition and preprocessing. For precursor-ion scanning (PIS) and neutral loss scanning (NLS), the mass spectrometer was operated in the

8

positive ion mode with an electrospray ionization voltage of 5500 V, nebulizer gas and turbo gas settings at 25 and 30 psi, and a turbosource gun temperature of 425 oC. The curtain gas (nitrogen) was set at 10 psi, the collision cell pressure was set at high mode, and the energy for collision-induced dissociation (CID) was set at 60 (for PIS) and 40 V (for NLS), respectively. For enhanced product ion (EPI) scanning, two kinds of injection modes were used. Some of the samples were injected into mass spectrometry through the HPLC, as described above. The others were injected directly into mass spectrometry though a syringe pump. For direct infusion, the sample concentration was 50 µg/ml, and the flow rate was 5-35 µl/min. The mass spectrometer was operated in the negative ion mode with an electrospray ionization voltage of –4500 V, nebulizer gas and turbo gas settings at 25 and 30 psi, and a turbosource gun temperature of 425 oC. The curtain gas (nitrogen) was set at 10 psi, the collision cell pressure was at high mode, and the collision energy was set at 40-55 V. A semi-quantitative method using an internal standard was used to compare the differences in the amount of the same GP molecular species between lipids recovered from gonads of different sea urchin species. The ratio of the intensity of the first-stage MS signal of a GPCho ([M]+), GPEtn ([M+H]+), GPSer ([M+H]+), GPIns ([M+H]+), LGPCho ([M]+) and LGPEtn ([M+H]+) to that of the corresponding internal standard was used to represent its relative intensity. 2.7. Statistical analysis The experiments for determination of lipid content, analysis of fatty acid

9

composition, lipid class composition, PL class composition and semi-quantitative analysis of GP molecular species were performed in triplicate. Date analysis was carried out with SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). The results were denoted as mean ± standard deviation (SD). Differences between means were evaluated by one-way analysis of variance (Student-Newman-Keuls post-hoc test) and independent-samples T text. P values of < 0.05 were considered statistically significant.

3. Results and discussion 3.1. Lipid contents and class compositions The lipid contents of dried gonads of Glyptocidaris crenularis, Strongylocentrotus intermedius and Strongylocentrotus nudus were 37.65, 22.70 and 24.31%, respectively. Though the lipid content of sea urchin gonad should change due to the existing difference in origin and harvesting time, the values were generally higher than 20% on a dry weight basis (Zhu et al., 2010; Archana et al., 2016). Therefore, high lipid contents in sea urchin gonads may account for their nutritional and health functions. 3.2. Fatty acid composition GC-MS analysis indicated that the lipids recovered from gonads of three species of tested sea urchins contained a high proportion of PUFA (34.47-46.56% of total FAs), especially EPA (9.31-19.97% of total FAs) (Table 1S). However, the FA profiles of lipids were significantly different among the various species examined. For example, DHA was only abundant in lipids from Glyptocidaris crenularis but was present in

10

trace amounts in lipids from Strongylocentrotus intermedius and Strongylocentrotus nudus. Actually, the feature of high EPA but low DHA content in lipids has previously been reported in gonads from Strongylocentrotus nudus (Zhu et al., 2010), Stomopneustes variolaris (Archana et al., 2016) and Paracentrotus lividus (Siliani et al., 2016). 3.3. Lipid class composition TLC-FID analysis indicated that the lipids recovered from gonads of sea urchins were composed of PL, TAG, diacylglycerols (DAG), monoacylglycerols (MAG), free fatty acids (FFA) and cholesterols (CHO) (Table 1). Obviously, PL (39.45-50.30% of total lipids) and TAG (39.58-55.08% of total lipids) constituted the major portion of the total lipids. The PL content of the dried gonads of different sea urchin species was in the order of Glyptocidaris crenularis (15.24%), Strongylocentrotus intermedius (11.42%) and Strongylocentrotus nudus (9.59%). In a previous study, Kalogeropoulos et al. (2012) reported that polar lipids (mainly PL) accounted for 45.9% of total lipids in sea urchin eggs (Paracentrotus lividus). However, according to Mita et al. (1989), PL accounted for approximately 80% of total lipids in spermatozoa of sea urchins Hemicentrotus pulcherrimus and Anthocidaris crassispina. 3.4. Phospholipid class composition PL classes were characterized according to their relative chemical shifts compared to triethyl phosphate (TEP, δ=0 ppm) as an internal standard. In this study, seven classes

of

PL,

including

PC,

PE,

PS,

phosphatidylinositol

(PI),

lysophosphatidylcholine (LPC), phosphatidylglycerol (PG) and sphingomyelin (SM),

11

were determined in lipids from sea urchin gonads by comparing their relative chemical shifts with those of the corresponding standards under the same experimental condition. As shown in Table 2, PC (67.88-72.58 mol%) was the dominant component of PL from all three species of sea urchins tested. While PE (9.49-13.19 mol%), LPC (4.37-4.88 mol%), PI (2.89-3.39 mol%), PS (3.03-5.00 mol%) and PG (1.38-1.75 mol%) were also present in smaller amounts compared with PC in their lipids. Moreover, detectable SM (2.13-6.03 mol%) was only present in lipids from Glyptocidaris crenularis and Strongylocentrotus nudus. Previously, Kalogeropoulos et al. (2012) found that the PL in sea urchin (Paracentrotus lividus) was composed of PC (83.7% of total PL), PE (4.9% of total PL), LPC (6.7% of total PL) and PI (1.2% of total PL). Meanwhile, De Quiros, Lopez-Hernandez and Simal-Lozano (2002) reported that the PL in sea urchin (Paracentrotus lividus) was composed of PC (32.1% of total PL), PE (41.5% of total PL), PS (17.9% of total PL), PI (7.7% of total PL) and SM (0.8% of total PL). In addition, Mita et al. (1989) noted that PL in sea urchins Hemicentrotus pulcherrimus and Anthocidaris crassispina was composed of PC (34-49% of total PL), PE (19-21% of total PL), PS (PI) (9.6-19.5% of total PL) and a small amount of LPC (<0.1%). Obviously, the PL class composition of sea urchin lipids vary due to the existing differences in the origin and harvesting time. However, PC was always the dominant PL class present. 3.5. Characterization of glycerophospholipid molecular species GP can be selectively detected by using PIS and NLS modes by employing triple quadrupole masss spectrometry in MS/MS based on the loss of the head group in GP

12

molecules (Pacetti, Boselli, Hulan & Frega, 2005). In this study, GPCho/LGPCho, GPEtn/LGPEtn, GPSer and GPIns were selectively detected by using PIS for m/z 184, NLS for m/z 141, NLS for m/z 185 and NLS for m/z 260 in positive ion mode, respectively. As shown in Fig.1, the above classes of PL all showed visible peaks in the PIS or NLS chromatograms. The measured m/z of the GP were acquired, which coincided with the molecular ion ([M]+) of GPCho/LGPCho, and quasi-molecular ion ([M+H]+) of GPEtn/LGPEtn, GPSer and GPIns, respectively. Common notation for a molecular species of GP follows the format x:y with x and y representing the number of carbons and the number of double bonds of the FAs of GP, respectively (Peterson & Cummings, 2006). As described in previous studies, the x and y of an unknown GP or LGP can be tentatively deduced based on their measured molecular mass according to the formulas we developed previously (Yin et al., 2016; Liu et al., 2017). For an unknown LGP, the measured molecular mass is sufficient for characterization of the molecular species which just contain one FA. In this study, the molecular species of all detected LGPCho and LGPEtn in sea urchin lipids were characterized according to the first stage MS acquired (Tables 2S and 3S). However, for an unknown GP containing two FAs, MS/MS data are needed for characterization of the two individual FAs which are esterified at the sn-1 and sn-2 positions of the glycerol backbone. Our previous studies indicated that the MS/MS information in the negative ion mode is suitable for characterization of molecular species of shellfish GP (Yin et al., 2016; Liu et al., 2017). Therefore, the MS/MS data of the unknown GP were also acquired in negative mode using EPI scanning in this

13

study. The precise structure of an unknown GP could be characterized according to its first-stage MS and MS/MS information. For example, for the unknown GPEtn with measured m/z 794 (quasi-molecular ion ([M+H]+)), phosphatidyl 40:5 could be tentatively deduced based on the measured molecular mass according to the formulas we developed previously (Yin et al., 2016; Liu et al., 2017). Through EPI scanning, the product ions with m/z 792, 506, 309, 303 and 259 were observed in the MS/MS spectrum (Fig.2 and Fig.1S). Among these product ions, fragments at m/z 303 and 309 were identified as the FA anions ([RCOO]–) of 20:4 and 20:1, respectively (Fig.2), which agrees with the GPEtn 40:5 that was deduced by using its first-stage MS data. The sn-2 of GP is usually the preferred position for PUFAs (Peterson et al., 2006). Therefore, the unknown GPEtn with measured m/z 794 was characterized as phosphatidyl GPEtn 20:1/20:4. Furthermore, the fragment at m/z 259 was characterized as [R]– of 20:4 which originates from FA anions ([RCOO]–) of 20:4 due to the loss of carboxyl anion; the fragment with m/z 506 was characterized as [LGPEtn 20:1–H]– which comes from GPEtn 20:1/20:4 due to the loss of fatty acid 20:4; m/z 792 was characterized as [M–H]– (Fig.2). Plasmenyl and plasmanyl GP can also be characterized according to their first-stage MS and MS/MS data. For example, based on the measured m/z 750 (quasi-molecular ion ([M+H]+)) for the unknown GPEtn, plasmenyl 38:5 and plasmanyl 38:6 both can tentatively be deduced. Through EPI scanning, the product ions with m/z 748, 464, 301, 267 and 257 were observed in the MS/MS spectrum (Fig.3 and 1S). Among

14

these product ions, fragments at m/z 301 and 257 were identified as FA anions ([RCOO]–) of 20:5 and the incomplete FA anion ([R]–) of 20:5 (Fig.3). For the plasmenyl and plasmanyl GP, the FA anion ([RCOO]–) can only be liberated from the sn-2 position due to the specific linkages at the sn-1 position. Meanwhile, the fragment at m/z 267 can be characterized as specific FA anions ([RCO]–) of vinyl ether 18:0 or alkyl ether of 18:1 which was liberated from the sn-1 position of the plasmenyl and plasmanyl GP (Fig.3). Furthermore, the fragment with m/z 464 was characterized as LGPEtn anion (plasmenyl LGPEtn 18:0 or plasmanyl LGPEtn 18:1) (Fig.3). Therefore, two possible molecular species, including plasmenyl 18:0/20:5 or plasmanyl 18:1/20:5, could be characterized. As described above, the specific FA anions ([RCO]–) of vinyl ether 18:0 and alkyl ether of 18:1 have the same measured m/z. This means that the plasmenyl GPEtn 18:0/20:5 and plasmanyl GPEtn 18:1/20:5 were characterized according to the same first-stage MS and MS/MS data. Obviously, such a possible pair of structures, including a plasmenyl GP x:(y-1) and a plasmanyl GP x:y, can not be differentiated directly by using the first-stage MS and MS/MS data. In this study, all detected GPCho, GPEtn, GPSer and GPIns were characterized based on their first-stage MS and MS/MS data according to a similar structural identification strategy as described above (Tables 4S-7S). Although the fragmentation patterns of GPCho, GPSer and GPIns were slightly different from those of GPEtn, most of them showed the FA anions ([RCOO] –) in MS/MS which are very useful in revealing individual structures of the two FAs of GP (Cui & Thomas, 2009).

15

3.6. GP in lipids recovered from gonads of different sea urchin species In this study, a pair of possible structures including a plasmenyl GP x:(y-1) and a plasmanyl GP x:y corresponding to the same first-stage MS and MS/MS data were counted as one GP. As shown in Tables 8S and 12S, at least 66, 68 and 68 species of GPCho were characterized in lipids recovered from gonads of Glyptocidaris crenularis,

Strongylocentrotus

intermedius

and

Strongylocentrotus

nudus,

respectively. Among them, 20:2/20:5, 20:1/20:5 and 20:2/20:4 were the predominant species of the phosphatidyl subclass, 18:0/20:5 and 22:2/16:1 might be the predominant species of the plasmenyl subclass, and 16:0/20:5, 18:1/20:5 and 22:3/16:1 might be the predominant species of the plasmanyl subclass (Tables 3 and 8S). By contrast, Strongylocentrotus intermedius had the highest amount of the major GPCho molecular species which contained 20:5, which is consistent with the results of FA composition. In an earlier study, Mita et al. (1989) reported that the PC isolated from sea urchins Hemicentrotus pulcherrimus and Anthocidaris crassispina contained a large percentage of 16:0, 20:1, 20:4 and 20:5. Meanwhile, Kostetsky et al. (2014) characterized 26 species of GPCho from sea urchin (Strongylocentrotus intermedius) gonad. Among them, 18:1/20:5, 16:0/20:5, 18:0/20:5 and 18:1/22:6 were dominant. As listed in Tables 9S and 12S, at least 54, 52 and 53 species of GPEtn were present

in

lipids

recovered

from

gonads

of

Glyptocidaris

crenularis,

Strongylocentrotus intermedius and Strongylocentrotus nudus, respectively. Among them, 18:0/20:5, 18:1/20:4, 20:1/20:5, 20:2/20:4 and 20:1/20:4 were the predominant species of phosphatidyl subclass, 18:0/20:4, 22:4/16:0, 18:0/22:5 and 20:0/20:5 might

16

be the predominant species of the plasmenyl subclass, and 18:1/20:4, 22:5/16:0, 18:1/22:5 and 20:1/20:5 might be the predominant species of the plasmanyl subclass (Tables 3 and 9S). In contrast, Strongylocentrotus intermedius had the highest amount of the major GPEtn molecular species which contained 20:5, which is consistent with the results of FA composition. According to Mita et al. (1989), 18:0, 20:1, 20:4 and 20:5 were the dominant FA in the PE isolated from sea urchins Hemicentrotus pulcherrimus, while 16:0, 18:0, 20:4 and 20:5 were the abundant FA in the PE from Anthocidaris crassispina. Meanwhile, Kostetsky et al. (2014) characterized 23 species of GPCho from sea urchin (Strongylocentrotus intermedius) gonad. Among them, 18:0/20:5, 18:0/20:4, 18:1/20:5 and 18:1/20:4 were dominant. In this study, at least 12, 12 and 12 species of GPSer were characterized in lipids recovered from the gonads of Glyptocidaris crenularis, Strongylocentrotus intermedius and Strongylocentrotus nudus, respectively (Tables 10S and 12S). Among them, 20:1/20:2, 20:1/20:1 and 20:1/22:2 were the predominant species of phosphatidyl subclass (Tables 3 and 10S). As described by Mita et al. (1989), 16:0, 18:0, 20:1, 20:4 and 20:5 were the dominant FA in the PS isolated from sea urchins Hemicentrotus pulcherrimus and Anthocidaris crassispina. In this study, at least 23, 26 and 26 species of GPIns were characterized in lipids recovered from gonads of Glyptocidaris crenularis, Strongylocentrotus intermedius and Strongylocentrotus nudus, respectively (Tables 11S and 12S). Among them, 18:0/20:5, 18:0/20:4, 20:0/20:5, 20:1:20:4 and 20:2/20:3 were the predominant species of phosphatidyl subclass (Tables 3 and 11S). By contrast, Strongylocentrotus

17

intermedius had the highest amount of the major GPIns molecular species which contained 20:5, consistent with the results of FA composition. As shown in Tables 2S and 12S, at least 39, 40 and 31 species of LGPCho were characterized in lipids recovered from gonads of Glyptocidaris crenularis, Strongylocentrotus intermedius and Strongylocentrotus nudus, respectively. Among them, phosphatidyl 20:5, 20:4, 20:2 and 22:6 were the predominant species (Tables 3 and 2S). As listed in Tables 3S and 12S, at least 12, 11 and 10 species of LGPEtn were characterized in lipids recovered from gonads of Glyptocidaris crenularis, Strongylocentrotus intermedius and Strongylocentrotus nudus, respectively. Among them, Plasmanyl 18:0, phosphatidyl 18:0, 20:1 and 22:2 were the predominant species (Tables 3 and 3S). As described above, the 31P NMR test does not detect the presence of LGPEtn in lipids recovered from sea urchin gonads, which maybe because of the lower sensitivity of 31P NMR compared to MS detection.

4. Conclusion Dried gonads of Glyptocidaris crenularis, Strongylocentrotus intermedius and Strongylocentrotus nudus contained 37.65, 22.70 and 24.31% lipids, respectively. The lipids contained a high proportion of PUFA (34.47-46.56% of total FAs), especially EPA (9.31-19.97% of total FAs). These lipids were composed of TAG, DAG, MAG, PL, FFA and CHO. Among which, PL (39.45-50.30% of total lipids) and TAG (39.58-55.08% of total lipids) were dominant. For PL, PC (67.88-72.58 mol%) was the major component. At least 206, 209 and 200 GP molecular species were

18

characterized for Glyptocidaris crenularis, Strongylocentrotus intermedius and Strongylocentrotus nudus, respectively. Results indicated that most of the dominant molecular species of GP contained EPA. According to the amount of the major molecular species containing EPA, Strongylocentrotus intermedius was the best fit species for nearly all classes of GP. Therefore, lipids in sea urchin gonads may account for their nutritional and health beneficial functions.

Acknowledgement This work was financially supported by “Public Science and Technology Research Funds Projects of Ocean (201505029)”, “Project of Distinguished Professor of Liaoning Province (2015-153)”, “Program for Liaoning Excellent Talents in University (LR2015006), “Liaoning Provincial Natural Science Foundation of China (2015020781)”, and Supported by Program for “Dalian High-Level Innovative Talent (2015R0007)”.

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25

Figure legends Fig.1. Specific detection of glycerophospholipids in lipids recovered from gonads of three

species

of

sea

urchins

by

using

high-performance

liquid

chromatography-electrospray ionization-tandem mass spectrometry. a-e, liquid chromatograms for glycerophosphocholines (GPCho), glycerophosphoethanolamines (GPEtn) and lysoglycerophosphoethanolamines (LGPEtn), glycerophosphoserines (GPSer), glycerophosphatidylinositols (GPIns), and lysoglycerophosphocholines (LGPCho), respectively. f-j, first-stage MS spectrum of GPCho, GPEtn and LGPEtn, GPSer, GPIns and LGPCho, respectively. Fig.2. Fragmentation pathways of phosphatidyl glycerophosphoethanolamine (GPEtn) 20:1/20:4 in MS/MS. Fig.3. Fragmentation pathways of plasmenyl glycerophosphoethanolamine (GPEtn) 18:0/20:5 and plasmanyl glycerophosphoethanolamine (GPEtn) 18:1/20:5 and several specific anions ([RCO]–) from the fatty acids of plasmenyl or plasmanyl glycerophospholipids in MS/MS.

26

4.0e7 2.0e7

10

20

30 Time, min

40

792.7 758.7 820.7 732.7

796.7 818.5 746.6

5000.0

700 800 m/z, Da

900

790.6 768.6

1.0e4

LGPEtn

794.6 752.6 738.6 778.6 810.5 744.6 813.6

580.4

424.2 482.5 582.6 0.0 400 500 600

1000

4.0e6 3.0e6 2.0e6

700 m/z, Da

800

10

20

30 Time, min

900

1000

Max. 3.9e4 cps.

2.5e4 2.0e4

0.0

844.7 624.5 810.6 872.6 626.4 600

894.7

788.8 700

800 m/z, Da

900

1000

Fig.1. Specific detection of glycerophospholipids in lipids recovered from gonads of three species of sea urchins by using high-performance liquid chromatography-electrospray ionization-tandem mass

spectrometry.

a-e,

liquid

glycerophosphoethanolamines glycerophosphoserines

chromatograms

(GPEtn)

(GPSer),

and

for

glycerophosphocholines

lysoglycerophosphoethanolamines

glycerophosphatidylinositols

(GPCho), (LGPEtn),

(GPIns),

and

lysoglycerophosphocholines (LGPCho), respectively. f-j, first-stage MS spectrum of GPCho, GPEtn and LGPEtn, GPSer, GPIns and LGPCho, respectively.

27

2.5e6 2.0e6 1.5e6 14.90

1.0e6

10

20

30 Time, min

+NL (260.00): 8.034 to 18.113 min fro...

h

3.0e4

5000.0

3.0e6

0.0

50

870.7

1.5e4

12.55

5.0e5 40

3.9e4 3.5e4

1.0e4

3.9e6 3.5e6

28.78

+NL (185.00): 17.966 to 30.090 min fro...

g

764.6

5000.0

860.7

5.0e6

0.0

50 Max. 2.2e4 cps.

1.5e4

624.4 600

40

766.6

2.2e4

In te n s ity , c p s

780.6 622.7

30 Time, min

2.0e4

832.6 1.5e4

20

+NL (141.00): 15.045 to 21.034 min fro...

f

808.6

2.0e4

10

TIC of +NL (260.00): from Sample 1 (H...

c

23.38

1.0e6

0.00

Max. 2.6e4 cps.

2.5e4

In te n s ity , c p s

4.00e6

50

806.6

0.0

6.00e6

2.00e6

+Prec (184.00): 16.944 to 22.933 min f...

1.0e4

8.00e6

Max. 6.6e6 cps.

22.62

6.6e6 6.0e6

In te n s ity , c p s

0.0

TIC of +NL (185.00): from Sample 1 (X...

b

In te n s ity , c p s

6.0e7

Max. 1.0e7 cps.

17.41

1.00e7

1.6e4 1.4e4

In te n s ity , c p s

TIC of +NL (141.00): from Sample 1 (H...

a In te n s ity , c p s

In te n s ity , c p s

Max. 8.9e7 cps.

18.71

8.9e7 8.0e7

In te n s ity , c p s

TIC of +Prec (184.00): from Sample 1 (...

1.2e4 1.0e4 8000.0 6000.0 587.3

85

4000.0 2000.0 601.5 621.3 0.0 600

699.3 765.5779 700

800 m/z, Da

O H2C O

H2N H2C

C H2

O

P

O

HC O

C

C19H37

OH

CH2

O -

O

-

[(LGPEtn 20:1)–H]–, m/z 506

c du

n t io

O

C

C H2

O

[RCOO] 20:1, m/z 309

C19H37

product ion O H2C

C19H37 –

O H2C

H3N

C

pro

loss of [RCOO]‒ 20:4

+

O

P

HC O

O

CH2

C

C19H31

-

O

C

C19H31

loss of [COO] ‒

‒‒C19H31

O

O

[RCOO]– 20:4, m/z 303

[R]– 20:4, m/z 259

OH

[GPEtn 20:1/20:4 +H]+, m/z 794

Fig.2. Fragmentation pathways of phosphatidyl glycerophosphoethanolamine (GPEtn) 20:1/20:4 in MS/MS.

28

-

O

C H

C H

C16H33

[RCO]– for vinyl ether 18:0, m/z 267

or -

O

C H

O

H2 C

C H

C16H33

O

[RCO]–

on

i uct

H2 C

H2 C

C16H31

for alkyl ether 18:1, m/z 267

d pro

or H2C

H2 C

C16H31

product ion

+

O

H3N H2C

C H2

O

HC

P

O

O

C

CH2

C19H29

-

O

O

loss of [COO] ‒ C

C19H29

O

[RCOO]– 20:5, m/z 301

‒‒C19H29

[R]– 20:5, m/z 257

OH

plasmenyl GPEtn 18:0/20:5

or

+ , m/z 750

+H

plasmanyl GPEtn 18:1/20:5

O

C H

O

H2 C

loss of [RCOO]‒ 20:5

O H2C

C H2

O

C16H33

or H2C

H2N

C H

P

HC O

H2 C

C16H31

OH

CH2

-

O

[(LGPEtn alkyl ether 18:1)–H]–, m/z 464

Fig.3. Fragmentation pathways of plasmenyl glycerophosphoethanolamine (GPEtn) 18:0/20:5 and plasmanyl glycerophosphoethanolamine (GPEtn) 18:1/20:5 and several specific anions ([RCO]–) from the fatty acids of plasmenyl or plasmanyl glycerophospholipids in MS/MS.

29

Table 1 Different lipid class compositions (%) of the lipids recovered from gonads of three species sea urchins. Samples

PL

TAG

DAG

MAG

FFA

CHO

1

40.47±1.71b

53.37±1.35b

0.51±0.02a

0.11±0.02c

1.83±0.26b

3.76±0.08b

2

50.30±1.21a

39.58±0.81c

0.57±0.07a

0.26±0.04a

2.32±0.13a

6.94±0.37a

3

39.45±0.54b

55.08±0.76a

0.47±0.04a

0.22±0.01b

1.49±0.04c

3.92±0.05b

a-c

Values in the same column with different lower-case letters are significantly

different at P < 0.05. Samples 1-3 are lipids recovered from gonads of Glyptocidaris crenularis,

Strongylocentrotus

intermedius

and

Strongylocentrotus

nudus,

respectively. Abbreviations are: PL, phospholipid; TAG, triacylglycerol; DAG, diacylglycerol; MAG, monoacylglycerol; FFA, free fatty acid; CHO, cholesterol.

30

Table 2 Different phospholipid class compositions (mol%) of the lipids recovered from gonads of three species of sea urchins. Sample

PC

PE

LPC

PI

PS

PG

SM

1

72.58±0.71a

10.20±0.28b

4.37±0.50a

2.89±0.18b

4.29±0.40b

1.38±0.23b

2.13±0.26b

2

71.78±0.42a

13.19±0.07a

4.88±0.17a

3.39±0.17ab

5.00±0.21a

1.75±0.06a

nd

3

67.88±0.30b

9.49±0.09c

4.78±0.16a

3.22±0.18a

3.03±0.16c

1.41±0.07b

6.03±0.23a

a-c

Values in the same column with different lower-case letters are significantly

different at P < 0.05. Samples 1-3 are lipids recovered from gonads of Glyptocidaris crenularis, Strongylocentrotus intermedius and Strongylocentrotus nudus, respectively. Abbreviations are: PC, phosphatidylcholine; PE, phosphatidylethanolamine; LPC, lysophosphatidylcholine; PI, phosphatidylinositol; PS, phosphatidylserine; PG, phosphatidylglycerol; SM, sphingomyelin; nd, not detected.

31

Table 3 Major

glycerophosphocholines,

glycerophosphatidylserines, lysoglycerophosphocholines

glycerophosphoethanolamines, glycerophosphatidylinositols,

and

lysoglycerophosphoethanolamines

in

lipids

recovered from gonads of three species of sea urchins. The ratio of the intensity of the first-stage MS signal of a glycerophospholipid in positive ion mode to that of the corresponding internal standard was used to represent its relative amount for comparison. Measured

Relative intensity

m/z

Molecular

Glyptocidaris

Strongylocentrotus

Strongylocentrotus

([M]+) or

species

crenularis

intermedius

nudus

Plasmanyl

0.84±0.11c

3.88±0.23a

3.05±0.25b

1.51±0.20c

6.23±0.59a

4.30±0.36b

1.24±0.22c

3.27±0.36a

2.38±0.29b

1.06±0.25c

8.87±0.61a

5.59±0.33b

Classes

([M+H]+)d GPCho

766

16:0/20:5 780

Phosphatidyl 16:0/20:5

792

Plasmanyl 18:1/20:5 Plasmenyl 18:0/20:5

794

Plasmanyl

32

18:0/20:5 796

Plasmanyl

0.54±0.05b

3.20±0.10a

3.23±0.08a

2.19±0.42c

4.28±0.24a

3.22±0.46b

2.04±0.44b

4.63±0.24a

4.44±0.51a

1.41±0.25c

6.78±0.21a

4.83±0.34b

1.36±0.20c

12.14±0.96a

9.63±0.95b

0.75±0.12b

6.45±0.69a

6.88±0.25a

22:3/16:1 Plasmenyl 22:2/16:1 Plasmanyl 18:0/20:4 806

Phosphatidyl 18:1/20:5

808

Phosphatidyl 18:1/20:4 Phosphatidyl 18:0/20:5

832

Phosphatidyl 20:2/20:5

834

Phosphatidyl 20:1/20:5 Phosphatidyl 20:2/20:4

836

Phosphatidyl 20:1/20:4 Phosphatidyl

33

20:0/20:5 GPEtn

750

Plasmanyl

0.51±0.05c

1.10±0.08a

0.86±0.04b

0.87±0.05c

1.90±0.19a

1.39±0.05b

2.83±0.14a

1.00±0.11b

0.66±0.06c

4.00±0.25b

4.82±0.35a

1.64±0.04c

1.81±0.07b

2.14±0.21a

1.38±0.04c

18:1/20:5 Plasmenyl 18:0/20:5 752

Plasmanyl 18:1/20:4 Plasmenyl 18:0/20:4 Plasmanyl 22:5/16:0 Plasmenyl 22:4/16:0

764

Phosphatidyl 18:1/20:5 Phosphatidyl 16:1/22:5

766

Phosphatidyl 18:0/20:5 Phosphatidyl 18:1/20:4

768

Phosphatidyl

34

18:0/20:4 778

Plasmanyl

0.85±0.05b

1.10±0.13a

0.58±0.06c

2.20±0.19c

7.11±0.06a

3.60±0.12b

1.53±0.17c

6.19±0.10a

3.77±0.28b

0.62±0.02b

1.04±0.06a

1.05±0.03a

0.29±0.02b

1.47±0.11a

1.52±0.14a

0.56±0.03b

1.46±0.09a

0.41±0.04c

18:1/22:5 Plasmenyl 18:0/22:5 Plasmanyl 20:1/20:5 Plasmenyl 20:0/20:5 792

Phosphatidyl 20:1/20:5 Phosphatidyl 20:2/20:4

794

Phosphatidyl 20:1/20:4

796

Phosphatidyl 20:2/20:2 Phosphatidyl 20:0/20:4

798

Phosphatidyl 20:1/20:2

GPSer

836

Phosphatidyl

35

20:1/20:5 838

Phosphatidyl

0.32±0.04c

1.76±0.10a

0.80±0.10b

0.15±0.01c

2.19±0.06b

2.78±0.11a

0.42±0.00c

2.43±0.06b

2.57±0.01a

0.94±0.07c

5.99±0.28a

5.39±0.04b

1.57±0.17a

1.00±0.07b

1.01±0.09b

3.04±0.19c

8.05±0.14a

6.03±0.04b

2.08±0.10c

6.71±0.45a

5.57±0.04b

0.61±0.09b

2.75±0.08a

2.64±0.13a

0.72±0.09c

2.60±0.12b

3.04±0.03a

20:1/20:4 842

Phosphatidyl 20:1/20:2

844

Phosphatidyl 20:1/20:1

870

Phosphatidyl 20:1/22:2

GPIns

883

Phosphatidyl 18:1/20:5

885

Phosphatidyl 18:0/20:5

887

Phosphatidyl 18:0/20:4

911

Phosphatidyl 20:1/20:5 Phosphatidyl 20:2/20:4

913

Phosphatidyl 20:0/20:5 Phosphatidyl

36

20:1/20:4 Phosphatidyl 20:2/20:3 LGPCho

542.4

Phosphatidyl

1.76±0.26a

1.56±0.08a

0.91±0.11b

1.01±0.19a

1.19±0.08a

1.26±0.20a

0.49±0.06c

1.76±0.22a

0.78±0.09b

2.32±0.44a

0.11±0.01b

0.13±0.03b

0.04±0.00a

0.03±0.00b

0.05±0.00a

0.05±0.00a

0.03±0.01b

0.03±0.00b

0.04±0.00a

0.04±0.00a

0.04±0.00a

0.03±0.00b

0.04±0.00b

0.07±0.01a

20:5 544.2

Phosphatidyl 20:4

548.2

Phosphatidyl 20:2

568.2

Phosphatidyl 22:6

LGPEtn

468.4

Plasmanyl 18:0

482.4

Phosphatidyl 18:0

508.4

Phosphatidyl 20:1

534.6

Phosphatidyl 22:2

a-c

Values in the same line with different lower-case letters are significantly different at

P < 0.05. [M+H]+

d

for

[M]+ for glycerophosphocholines and lysoglycerophosphocholines; glycerophosphoethanolamines,

37

lysoglycerophosphoethanolamines,

glycerophosphatidylserines and GPCho,

glycerophosphocholine;

glycerophosphatidylserine;

glycerophosphatidylinositols. Abbreviations are: GPEtn,

GPIns,

glycerophosphoethanolamine;

glycerophosphatidylinositol;

lysoglycerophosphocholine; LGPEtn, lysoglycerophosphoethanolamine.

38

GPSer, LGPCho,

- Total lipids were recovered from gonads of three species of edible sea urchin - The lipids contained a high proportion (34.47-46.56%) of PUFAs, especially EPA - Phospholipid accounted for 39.45-50.30% of total lipids - Phosphatidylcholine (67.88-72.58 mol%) was the dominant phospholipid class - About 200 glycerophospholipid molecular species were characterized

39