J Food Bioact

Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online

Journal website www.isnff-jfb.com

Original Research

Volume 8, December 2019, pages 84-91

Comprehensive study of the lipid from whelk (Chlorostoma rusticum and Neverita didyma) with emphasis on characterization of phospholipid molecular species by shot-gun strategy

Figure 1. Specific detection of glycerophospholipids in lipids recovered from two species of whelks by using direct infusion mass spectrometric approaches. a-d, first-stage MS spectra of glycerophosphocholines (GPCho)/lysoglycerophosphocholines (LGPCho), glycerophosphoethanolamines (GPEtn)/lysoglycerophosphoethanolamines (LGPEtn), glycerophosphoserines (GPSer)/lysoglycerophosphoserines (LGPSer), and glycerophosphatidylinositol (GPIns)/lysoglycerophosphatidylinositol (LGPIns), respectively.

**Table 1.** Class compositions (%) of the lipids recovered from two species of whelks
Species	PL	TAG	DAG	MAG	FFA	CHO
^a,bValues in the same column with different lower-case letters are significantly different at P < 0.05. Abbreviations: PL, phospholipid; TAG, triacylglycerol; DAG, diacylglycerol; MAG, monoacylglycerol; FFA, free fatty acid; CHO, cholesterol.
Chlorostoma rusticum	52.70 ± 0.18^b	31.91 ± 0.18	0.41 ± 0.02^b	0.57 ± 0.04^b	3.71 ± 0.07^a	10.60 ± 0.08^a
Neverita didyma	58.86 ± 0.39^a	32.01 ± 0.47	0.53 ± 0.01^a	0.96 ± 0.09^a	1.11 ± 0.08^b	6.66 ± 0.49^b

Table 1. Class compositions (%) of the lipids recovered from two species of whelks

Species

TAG

DAG

MAG

FFA

CHO

^a,bValues in the same column with different lower-case letters are significantly different at P < 0.05. Abbreviations: PL, phospholipid; TAG, triacylglycerol; DAG, diacylglycerol; MAG, monoacylglycerol; FFA, free fatty acid; CHO, cholesterol.

Chlorostoma rusticum

52.70 ± 0.18^b

31.91 ± 0.18

0.41 ± 0.02^b

0.57 ± 0.04^b

3.71 ± 0.07^a

10.60 ± 0.08^a

Neverita didyma

58.86 ± 0.39^a

32.01 ± 0.47

0.53 ± 0.01^a

0.96 ± 0.09^a

1.11 ± 0.08^b

6.66 ± 0.49^b

**Table 2.** Phospholipid class compositions (mol%) of the lipids recovered from two species of whelks
Species	PC	PE	LPC	PI	PS	PG	PA
^a,bValues in the same column with different lower-case letters are significantly different at P < 0.05. Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; LPC, lysophosphatidylcholine; PI, phosphatidylinositol; PS, phosphatidylserine; PG, phosphatidylglycerol; PA, phosphatidic acid.
Chlorostoma rusticum	52.41 ± 0.42^a	27.67 ± 0.01^b	8.86 ± 0.00^a	nd	nd	11.05 ± 0.02^a	nd
Neverita didyma	50.58 ± 0.23^b	32.73 ± 0.31^a	3.20 ± 0.00^b	2.10 ± 0.01	4.81 ± 0.01	3.76 ± 0.01^b	2.81 ± 0.01

Table 2. Phospholipid class compositions (mol%) of the lipids recovered from two species of whelks

Species

LPC

^a,bValues in the same column with different lower-case letters are significantly different at P < 0.05. Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; LPC, lysophosphatidylcholine; PI, phosphatidylinositol; PS, phosphatidylserine; PG, phosphatidylglycerol; PA, phosphatidic acid.

Chlorostoma rusticum

52.41 ± 0.42^a

27.67 ± 0.01^b

8.86 ± 0.00^a

11.05 ± 0.02^a

Neverita didyma

50.58 ± 0.23^b

32.73 ± 0.31^a

3.20 ± 0.00^b

2.10 ± 0.01

4.81 ± 0.01

3.76 ± 0.01^b

2.81 ± 0.01

**Table 3.** Fatty acid compositions (%) of the lipids recovered from two species of whelks
Fatty acid	Chlorostoma rusticum	Neverita didyma
^a,bValues in the same line with different lowercase letters are significantly different at P < 0.05. nd, not detected. Abbreviations: SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; AI, atherogenicity index; TI, thrombogenicity index.
C14:0	4.04 ± 0.09^a	2.39 ± 0.04^b
C15:0	1.42 ± 0.13^a	0.65 ± 0.11^b
C16:1n-7	6.28 ± 0.25^a	5.38 ± 0.30^b
C16:0	23.36 ± 0.41^a	16.14 ± 0.33^b
C17:1n-7	0.51 ± 0.06	nd
C17:0	3.12 ± 0.48^a	1.86 ± 0.07^b
C18:4n-3	0.46 ± 0.04	nd
C18:2n-6	3.34 ± 0.25^a	1.65 ± 0.23^b
C18:1n-9	26.58 ± 0.56^a	7.38 ± 0.33^b
C18:0	3.77 ± 0.32^b	10.68 ± 0.97^a
C20:4n-6	4.08 ± 0.20^a	2.47 ± 0.45^b
C20:5n-3	3.17 ± 0.06^b	11.00 ± 0.73^a
C20:2n-9	1.66 ± 0.19^a	0.89 ± 0.12^b
C20:1n-9	9.73 ± 0.64^b	12.78 ± 0.49^a
C20:1n-7	nd	4.94 ± 0.26
C22:6n-3	0.40 ± 0.03^b	11.10 ± 0.08^a
C22:4n-6	0.61 ± 0.09^b	1.12 ± 0.13^a
C22:5n-3	1.77 ± 0.27^b	2.67 ± 0.35^a
C22:2n-9	4.60 ± 0.45^b	6.78 ± 0.44^a
C22:2n-6	1.61 ± 0.21	nd
SFA	35.21 ± 0.16^a	32.06 ± 0.22^b
MUFA	43.10 ± 0.21^a	30.48 ± 0.13^b
PUFA	21.69 ± 0.12^b	37.68 ± 0.18^a
n-3/n-6	0.60 ± 0.03^b	4.73 ± 0.16^a
AI	0.68 ± 0.05^a	0.42 ± 0.04^b
TI	0.70 ± 0.08^a	0.30 ± 0.01^b

Table 3. Fatty acid compositions (%) of the lipids recovered from two species of whelks

Fatty acid

Chlorostoma rusticum

Neverita didyma

^a,bValues in the same line with different lowercase letters are significantly different at P < 0.05. nd, not detected. Abbreviations: SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; AI, atherogenicity index; TI, thrombogenicity index.

C14:0

4.04 ± 0.09^a

2.39 ± 0.04^b

C15:0

1.42 ± 0.13^a

0.65 ± 0.11^b

C16:1n-7

6.28 ± 0.25^a

5.38 ± 0.30^b

C16:0

23.36 ± 0.41^a

16.14 ± 0.33^b

C17:1n-7

0.51 ± 0.06

C17:0

3.12 ± 0.48^a

1.86 ± 0.07^b

C18:4n-3

0.46 ± 0.04

C18:2n-6

3.34 ± 0.25^a

1.65 ± 0.23^b

C18:1n-9

26.58 ± 0.56^a

7.38 ± 0.33^b

C18:0

3.77 ± 0.32^b

10.68 ± 0.97^a

C20:4n-6

4.08 ± 0.20^a

2.47 ± 0.45^b

C20:5n-3

3.17 ± 0.06^b

11.00 ± 0.73^a

C20:2n-9

1.66 ± 0.19^a

0.89 ± 0.12^b

C20:1n-9

9.73 ± 0.64^b

12.78 ± 0.49^a

C20:1n-7

4.94 ± 0.26

C22:6n-3

0.40 ± 0.03^b

11.10 ± 0.08^a

C22:4n-6

0.61 ± 0.09^b

1.12 ± 0.13^a

C22:5n-3

1.77 ± 0.27^b

2.67 ± 0.35^a

C22:2n-9

4.60 ± 0.45^b

6.78 ± 0.44^a

C22:2n-6

1.61 ± 0.21

SFA

35.21 ± 0.16^a

32.06 ± 0.22^b

MUFA

43.10 ± 0.21^a

30.48 ± 0.13^b

PUFA

21.69 ± 0.12^b

37.68 ± 0.18^a

n-3/n-6

0.60 ± 0.03^b

4.73 ± 0.16^a

0.68 ± 0.05^a

0.42 ± 0.04^b

0.70 ± 0.08^a

0.30 ± 0.01^b

**Table 4.** Major molecular species of glycerophosphocholines, glycerophosphoethanolamines, glycerophosphatidylserines, glycerophosphatidylinositols and their lyso- counterparts in lipids recovered from two species of whelks. The ratio of the intensity of the first-stage MS signal of a glycerophospholipid in the positive ion mode to that of the corresponding internal standard was used to represent its relative amount for comparison
Classes	Measured m/z ([M]⁺) or ([M+H]⁺)	Molecular species	Relative intensity
^a,bValues in the same line with different lower-case letters are significantly different at P < 0.05. The number labeled following the quantitative values indicated that the corresponding molecular species which lists in the second column of the table were present. [M]⁺ for glycerophosphocholines and lysoglycerophosphocholines; [M+H]⁺ for glycerophosphoethanolamines, lysoglycerophosphoethanolamines, glycerophosphatidylserines, lysoglycerophosphatidylserines, glycerophosphatidylinositols and lysoglycerophosphatidylinositols. Abbreviations: GPCho, glycerophosphocholine; GPEtn, glycerophosphoethanolamine; GPSer, glycerophosphatidylserine; GPIns, glycerophosphatidylinositol; LGPCho, lysoglycerophosphocholine; LGPEtn, lysoglycerophosphoethanolamine; LGPSer, lysoglycerophosphatidylserine; LGPIns, lysoglycerophosphatidylinositols.
GPCho	760	Phosphatidyl 17:0/17:1 Phosphatidyl 16:0/18:1	2.75 ± 0.07^a	1.29 ± 0.08^b
	766	Plasmanyl 16:1/20:4 Plasmenyl 16:0/20:4 Plasmanyl 16:0/20:5	0.28 ± 0.01^b	2.97 ± 0.01^a
	780	Phosphatidyl 16:0/20:5	1.28 ± 0.07^b	2.73 ± 0.02^a
	782	Phosphatidyl 16:0/20:4	1.69 ± 0.02^a	1.29 ± 0.08^b
	792	Plasmanyl 16:0/22:6	0.19 ± 0.01^b	2.9 ± 0.02^a
	808	Phosphatidyl 18:1/20:4 Phosphatidyl 16:0/22:5	1.15 ± 0.13^b	2.16 ± 0.09^a
GPEtn	738	Phosphatidyl 16:0/20:5	2.48 ± 0.08^a	1.57 ± 0.01^b
	752	Plasmanyl 18:0/20:5 Plasmanyl 16:0/22:5	1.03 ± 0.08^b	1.24 ± 0.04^a
	766	Phosphatidyl 16:0/22:5 Phosphatidyl 18:1/20:4 Phosphatidyl 18:0/20:5 Phosphatidyl 16:1/22:4	3.1 ± 0.03^b	4.11 ± 0.04^a
	768	Phosphatidyl 18:0/20:4	2.35 ± 0.09^b	2.59 ± 0.03^a
	772	Phosphatidyl 16:0/22:2 Phosphatidyl 18:1/20:1 Phosphatidyl 18:0/20:2	1.79 ± 0.06^b	2.32 ± 0.02^a
	786	Phosphatidyl 20:4/20:5 Plasmanyl 20:1/20:1 Plasmenyl 20:0/20:1	1.32 ± 0.04^b	3.01 ± 0.04^a
	792	Phosphatidyl 20:1/20:5 Phosphatidyl 18:0/22:6	0.62 ± 0.02^b	2.03 ± 0.07^a
GPSer	810	Phosphatidyl 18:0/20:5 Phosphatidyl 18:1/20:4	1.01 ± 0.08^b	3.86 ± 0.02^a
	812	Phosphatidyl 16:0/22:4	1.10 ± 0.06^b	2.41 ± 0.06^a
	818	Phosphatidyl 18:0/20:1 Plasmanyl 18:2/22:6 Plasmenyl 18:1/22:6	0.89 ± 0.06^b	1.43 ± 0.04^a
	844	Phosphatidyl 18:0/22:2 Phosphatidyl 20:1/20:1 Plasmanyl 22:4/20:5 Plasmenyl 22:3/20:5	1.53 ± 0.07^b	3.23 ± 0.09^a
GPIns	885	Phosphatidyl 18:0/20:5 Phosphatidyl 18:1/20:4	1.22 ± 0.09^b	5.83 ± 0.02^a
	887	Phosphatidyl 18:0/20:4	3.57 ± 0.06^b	4.50 ± 0.07^a
LGPCho	496.6	Phosphatidyl 16:0	0.05 ± 0.00^b	0.14 ± 0.01^a
	522.6	Phosphatidyl 18:1	0.03 ± 0.00^b	0.12 ± 0.01^a
	542.6	Phosphatidyl 20:5	0.01 ± 0.00^b	0.20 ± 0.01^a
	568.6	Phosphatidyl 22:6	0.01 ± 0.00^b	0.28 ± 0.00^a
LGPEtn	500.3	Phosphatidyl 20:5	0.08 ± 0.03^b	0.12 ± 0.02^a
	506.3	Phosphatidyl 20:2	0.11 ± 0.03	0.15 ± 0.01
	508.4	Phosphatidyl 20:1	0.71 ± 0.02^a	0.40 ± 0.11^b
	528.4	Phosphatidyl 22:5	0.34 ± 0.07^a	0.22 ± 0.05^b
	534.4	Phosphatidyl 22:2	1.65 ± 0.25^a	0.71 ± 0.00^b
LGPSer	526.4	Phosphatidyl 18:0	0.12 ± 0.02^b	0.45 ± 0.01^a
	552.4	Phosphatidyl 20:1	0.96 ± 0.03^a	0.35 ± 0.04^b
	572.3	Phosphatidyl 22:5	0.15 ± 0.01^a	0.09 ± 0.01^b
	578.4	Phosphatidyl 22:2	1.73 ± 0.02^a	0.52 ± 0.06^b
LGPIns	579.2	Phosphatidyl 18:4	0.27 ± 0.09^b	1.02 ± 0.11^a
	599.1	Phosphatidyl 18:1	0.43 ± 0.33^b	0.50 ± 0.18^a
	601.6	Phosphatidyl 18:0	0.63 ± 0.05^a	0.50 ± 0.18^b
	619.4	Phosphatidyl 20:5	0.27 ± 0.09^b	1.02 ± 0.11^a

Table 4. Major molecular species of glycerophosphocholines, glycerophosphoethanolamines, glycerophosphatidylserines, glycerophosphatidylinositols and their lyso- counterparts in lipids recovered from two species of whelks. The ratio of the intensity of the first-stage MS signal of a glycerophospholipid in the positive ion mode to that of the corresponding internal standard was used to represent its relative amount for comparison

Classes

Measured m/z ([M]⁺) or ([M+H]⁺)

Molecular species

Relative intensity

Chlorostoma rusticum

Neverita didyma

^a,bValues in the same line with different lower-case letters are significantly different at P < 0.05. The number labeled following the quantitative values indicated that the corresponding molecular species which lists in the second column of the table were present. [M]⁺ for glycerophosphocholines and lysoglycerophosphocholines; [M+H]⁺ for glycerophosphoethanolamines, lysoglycerophosphoethanolamines, glycerophosphatidylserines, lysoglycerophosphatidylserines, glycerophosphatidylinositols and lysoglycerophosphatidylinositols. Abbreviations: GPCho, glycerophosphocholine; GPEtn, glycerophosphoethanolamine; GPSer, glycerophosphatidylserine; GPIns, glycerophosphatidylinositol; LGPCho, lysoglycerophosphocholine; LGPEtn, lysoglycerophosphoethanolamine; LGPSer, lysoglycerophosphatidylserine; LGPIns, lysoglycerophosphatidylinositols.

GPCho

760

Phosphatidyl 17:0/17:1
Phosphatidyl 16:0/18:1

2.75 ± 0.07^a

1.29 ± 0.08^b

766

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

0.28 ± 0.01^b

2.97 ± 0.01^a

780

Phosphatidyl 16:0/20:5

1.28 ± 0.07^b

2.73 ± 0.02^a

782

Phosphatidyl 16:0/20:4

1.69 ± 0.02^a

1.29 ± 0.08^b

792

Plasmanyl 16:0/22:6

0.19 ± 0.01^b

2.9 ± 0.02^a

808

Phosphatidyl 18:1/20:4
Phosphatidyl 16:0/22:5

1.15 ± 0.13^b

2.16 ± 0.09^a

GPEtn

738

Phosphatidyl 16:0/20:5

2.48 ± 0.08^a

1.57 ± 0.01^b

752

Plasmanyl 18:0/20:5
Plasmanyl 16:0/22:5

1.03 ± 0.08^b

1.24 ± 0.04^a

766

Phosphatidyl 16:0/22:5
Phosphatidyl 18:1/20:4
Phosphatidyl 18:0/20:5
Phosphatidyl 16:1/22:4

3.1 ± 0.03^b

4.11 ± 0.04^a

768

Phosphatidyl 18:0/20:4

2.35 ± 0.09^b

2.59 ± 0.03^a

772

Phosphatidyl 16:0/22:2
Phosphatidyl 18:1/20:1
Phosphatidyl 18:0/20:2

1.79 ± 0.06^b

2.32 ± 0.02^a

786

Phosphatidyl 20:4/20:5
Plasmanyl 20:1/20:1
Plasmenyl 20:0/20:1

1.32 ± 0.04^b

3.01 ± 0.04^a

792

Phosphatidyl 20:1/20:5
Phosphatidyl 18:0/22:6

0.62 ± 0.02^b

2.03 ± 0.07^a

GPSer

810

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

1.01 ± 0.08^b

3.86 ± 0.02^a

812

Phosphatidyl 16:0/22:4

1.10 ± 0.06^b

2.41 ± 0.06^a

818

Phosphatidyl 18:0/20:1
Plasmanyl 18:2/22:6
Plasmenyl 18:1/22:6

0.89 ± 0.06^b

1.43 ± 0.04^a

844

Phosphatidyl 18:0/22:2
Phosphatidyl 20:1/20:1
Plasmanyl 22:4/20:5
Plasmenyl 22:3/20:5

1.53 ± 0.07^b

3.23 ± 0.09^a

GPIns

885

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

1.22 ± 0.09^b

5.83 ± 0.02^a

887

Phosphatidyl 18:0/20:4

3.57 ± 0.06^b

4.50 ± 0.07^a

LGPCho

496.6

Phosphatidyl 16:0

0.05 ± 0.00^b

0.14 ± 0.01^a

522.6

Phosphatidyl 18:1

0.03 ± 0.00^b

0.12 ± 0.01^a

542.6

Phosphatidyl 20:5

0.01 ± 0.00^b

0.20 ± 0.01^a

568.6

Phosphatidyl 22:6

0.01 ± 0.00^b

0.28 ± 0.00^a

LGPEtn

500.3

Phosphatidyl 20:5

0.08 ± 0.03^b

0.12 ± 0.02^a

506.3

Phosphatidyl 20:2

0.11 ± 0.03

0.15 ± 0.01

508.4

Phosphatidyl 20:1

0.71 ± 0.02^a

0.40 ± 0.11^b

528.4

Phosphatidyl 22:5

0.34 ± 0.07^a

0.22 ± 0.05^b

534.4

Phosphatidyl 22:2

1.65 ± 0.25^a

0.71 ± 0.00^b

LGPSer

526.4

Phosphatidyl 18:0

0.12 ± 0.02^b

0.45 ± 0.01^a

552.4

Phosphatidyl 20:1

0.96 ± 0.03^a

0.35 ± 0.04^b

572.3

Phosphatidyl 22:5

0.15 ± 0.01^a

0.09 ± 0.01^b

578.4

Phosphatidyl 22:2

1.73 ± 0.02^a

0.52 ± 0.06^b

LGPIns

579.2

Phosphatidyl 18:4

0.27 ± 0.09^b

1.02 ± 0.11^a

599.1

Phosphatidyl 18:1

0.43 ± 0.33^b

0.50 ± 0.18^a

601.6

Phosphatidyl 18:0

0.63 ± 0.05^a

0.50 ± 0.18^b

619.4

Phosphatidyl 20:5

0.27 ± 0.09^b

1.02 ± 0.11^a