Ether Phospholipids of Scallops

Andrew MacKenzieIndustrial Research Limited, Lower Hutt, NZ

Yutaka ItabashiHokkaido University, Hakodate, Japan

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Scallop Phospholipids

• Seafood can be a commercially viable source of omega-3 rich phospholipids (e.g. krill1)

• Omega-3 fatty acids may be more readily absorbed as dietary phospholipids2

• Ether phospholipids (e.g. plasmalogens) may be important factors in brain health (e.g. Alzheimer’s)3

• Aim: to investigate commercial scallop species as sources of omega-3 rich ether phospholipids

Pecten yessoensis

1. C.Watkins (2007) inform 18:588-592

2. M. Cansell et al (2003) Lipids 38:551-559

3. X. Han et al (2001) J Neurochem 77:1168-80

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• Difficult to separate by chromatography

• Can distinguish and quantify by 31P NMR but standards not always available

Diacyl-Phosphatidyl ethanolamine (PE)

Ethanolamine plasmalogen (EPLAS)

1-alkyl-, 2-acyl- phosphatidyl ethanolamine (AAPE)

Alkyl-acyl-Phospholipids (AAPL)

CH3(CH2)xCOCH

O

CH2OPOCH2CH2NH3

O

O-

CH2OCH CH(CH2)nCH3

+

CH2O(CH2)nCH3

CH3(CH2)xCOCH

O

CH2OPOCH2CH2NH3

O

O-

+

CH2OC(CH2)nCH3

O

CH3(CH2)xCOCH

O

CH2OPOCH2CH2NH3

O

O-

+

-7.0 -7.5 -8.0 ppm

14.014.5 ppm

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31P NMR of Scallop Flesh LipidPC

AAPCPI

PS

LPC*

PE

EPLAS

AAPE

CL

CAEP

-6.5 -7.0 -7.5 -8.0 ppm

14.014.5 ppm

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31P NMR of Deacylated Scallop LipidGPC

LAAPCGPIGPS

GPE

LEPLAS

LAAPE

DCL

CAEP

LAAPS

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Phospholipid classes of Scallop Meat (mol%)

58706074wt% PL (lipid)

0.70.70.60.7wt% lipid (sample)

15.118.720.019.1CAEP

2.52.13.52.3CL

4.43.05.65.2PI

2.92.01.14.0AAPS

4.83.40.95.2PS

7.16.59.06.5AAPE

13.814.117.510.5EPLAS

12.47.712.08.5PE

6.48.011.310.1AAPC

30.634.119.328.8PC

J (fresh)J (frozen)C (frozen)NZ (frozen)

NZ = Pecten novaezelandiae, J = Pecten yessoensis, C = Chlamys farreri

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Phospholipid classes of Scallop Organs (mol%)

-2.9---PG

4.2

33

10.7

2.8

4.0

-

2.6

4.4

20.9

11.4

7.1

36.0

Ovaries

58147270wt% PL (lipid)

0.74.81.50.7wt% lipid (sample)

15.57.514.515.1CAEP

2.63.14.42.5CL

3.56.02.34.4PI

2.0--2.9AAPS

5.13.64.94.8PS

4.24.10.97.1AAPE

21.816.116.413.8EPLAS

7.710.615.712.4PE

10.74.84.36.4AAPC

26.641.136.530.6PC

VisceraHepat.TestesMeat

Samples from fresh Pecten yessoensis. Hepat. = Hepatopancreas

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Confirmation of AAPS in Scallop

[M-H]-

[M-serine]-

[M-serine-DHA]-

[M-serine-DHAketene]-

18:0alkyl-22:6acyl-PS

[DHA]-

MS/MS –ve mode

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LC-MS of Scallop AAPC

(25) 16:0alkyl-22:6acyl-PC

[M+H]+

[M-N(CH3)3+Na]+[M-22:6+2Na]+

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LC-MS of Scallop Meat AAPC

plus 7 other identified molecular species (< 2%) [22 total]

2.116:0alk/16:0742.653.6

4.718:0alk/22:6842.644.5

2.816:0alk/22:5816.640.2

4.416:0alk/14:0714.638.3

3.317:0alk/22:6828.637.5

6.116:0alk/22:5816.636.0

22.216:0alk/22:6814.632.0

10.716:0alk/20:5788.629.0

4.715:0alk/22:6800.627.3

6.3MeO16:0alk/22:6844.526.7

5.416:0alk/18:4762.626.3

2.1MeO16:0alk/20:5818.624.3

5.416:1alk/22:6812.5

7.614:0alk/22:6786.623.3

2.314:0alk/20:5760.621.4

(%)Molecular species[M+Na]+RT (min)

2.1%16:0

4.4%14:0

57.0% DHA

8.9% 22:5

16.7% EPA

9.4% 18:4

Fatty Acid

0.0%20:1

8.1%16:1

4.7%18:0

10.1% MeO16:0

55.2% 16:0

4.7%15:0

11.2%14:0

Alkyl group

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Sample Preparation for GC analysis of

ethers and fatty acids

AAPL

Prep TLC

AAPE

+ EPLASAAPS AAPC

LiAlH4, Si column

Fatty alcohol Glycerol ether

BSTFA

GC, GC-MS analysis FAlc GE cetyl alcohol

FAlc

GE

2methoxy-GE

1:3 ether:pet ether 9:1 ether:ethanol

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Glycerol Ethers of Phospholipid ClassesNZ Scallop Meat

0.0

10.0

20.0

30.0

40.0

50.0

AAPC AAPS AAPE

%

14:0

16:0

17:0

18:0

MeO16:0

16:1

18:1

20:1

Japanese Scallop Meat

0.0

10.0

20.0

30.0

40.0

50.0

AAPC AAPS AAPE

%

14:0

16:0

17:0

18:0

MeO16:0

16:1

18:1

20:1

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Glycerol Ether and Fatty Acid Profiles

• Glycerol Ethers

– Glycerol ether profiles are not the same for the different phospholipid classes

– 1-O-(2-methoxyhexadecyl)glycerol, (MeO16:0) mostly associated with AAPC

– 20:1-GE mostly in AAPS and AAPE, higher proportion in Japanese and Chinese scallops

• Fatty Acids

– High LC-PUFA content in alkyl-acyl-PL classes (up to 75%)

– DHA is the major fatty acid in most fractions (up to 55%)

– EPA the major fatty acid in Japanese scallop AAPE/EPLAS fraction (33%)

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Conclusions (Analysis)

• 31P NMR is a rapid quantitative technique for the analysis of phospholipids. Can distinguish and measure AAPL

• LC-MS for qualitative profiling of AAPC

• AAPS positively identified by MS/MS

• Care must be taken for isolation of glycerol ethers (significantly higher polarity of 2-methoxy-16:0 glycerolether)

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Conclusions (Scallop AAPL)

• AAPL are a major part of scallop phospholipids

• Profiles of scallop flesh AAPL classes were similar for three scallop species obtained from different regions

• Organs exhibit different phospholipid class profiles

• Major glycerol ethers differ between species (e.g. 20:1 in Japanese scallop)

• AAPS present in scallop meat and viscera (not in ovaries, testes or hepatopancreas)

• 2-methoxy-16:0 in AAPC (minor in AAPE, AAPS)

• High LC-PUFA content in AAPL

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Acknowledgements

• Lipid Group at IRL: Dr Mikhail Vyssotski, Dr Eduard Nekrasov, Dr Kirill Lagutin, Dawn Scott

• Herbert Wong, NMR (IRL). Yinrong Lu, MS (IRL)

• Professor Yutaka Itabashi and research group (Hokkaido University)

• Foundation for Science, Research and Technology, New Zealand. C08X0709 “High Value Lipids”

• Japan Society for the Promotion of Science/Royal Society of New Zealand