Impact of Reduced Dietary Levels of Eicosapentaenoic Acid and

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Omics Technologies Applied to Agriculture and Food

Reduced dietary levels of EPA and DHA have a major impact on the composition of skin membrane lipids in Atlantic salmon (Salmo salar L.) ken cheng, Marta Bou, Bente Buyter, Jana Pickova, Emad Ehtesham, Liang Du, Claudia Venegas, and Ali A. Moazzami J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02886 • Publication Date (Web): 25 Jul 2018 Downloaded from http://pubs.acs.org on July 30, 2018

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Journal of Agricultural and Food Chemistry

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Reduced dietary levels of EPA and DHA have a major impact on the composition of

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skin membrane lipids in Atlantic salmon (Salmo salar L.)

3

Ken Chenga, *, Marta Boub, Bente Ruyterb, Jana Pickovaa, Emad Ehteshama, Liang Dub,

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Claudia Venegasc, Ali A. Moazzamia

5

a

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Sciences, Uppsala, Sweden

7

b

8

c

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*Correspondent at Department of Molecular Sciences, Uppsala BioCenter, Swedish

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University of Agricultural Sciences, P.O. Box 7015, 75007 Uppsala, Sweden; Tel: +46 18

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6720 11; E-mail address: [email protected] (K. Cheng)

Department of Molecular Sciences, Uppsala BioCenter, Swedish University of Agricultural

Nofima (Norwegian Institute of Food, Fisheries and Aquaculture Research), Ås, Norway

AVS Chile, Puerto Varas, Chile

ACS Paragon Plus Environment


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Abstract

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Membrane lipids, including sphingolipids and glycerol-phospholipids, are essential in

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maintaining skin barrier function in mammals, but their composition in fish skin and their

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response to diets have not been evaluated. This study investigated the impacts of reducing

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dietary EPA and DHA on membrane lipids in skin of Atlantic salmon, through a 26-week

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feeding regime supplying different levels (0%‒2.0% of drymass) of EPA/DHA. Ceramide,

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glucosylceramide, sphingomyelin, sphingosine, and sphinganine in salmon skin were

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analyzed for the first time. Higher concentrations of glucosylceramide and sphingomyelin,

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and higher ratios of glucosylceramide:ceramide and sphingomyelin:ceramide, were detected

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in the deficient group, indicating interruptions in sphingolipidomics. Changes in the glycerol-

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phospholipid profile in fish skin caused by reducing dietary EPA and DHA were observed.

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There were no dietary impacts on epidermal thickness and mucus cell density, but the

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changes in phospholipid profile suggest that low dietary EPA and DHA may interrupt the

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barrier function of fish skin.

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Keywords:

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sphingolipidomics

ceramide,

DHA,

EPA,

glycerol-phospholipids,


fish

skin

health,

2

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1 Introduction

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The n-3 long-chain polyunsaturated fatty acids (LC-PUFA), mainly eicosapentaenoic acid

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(EPA; 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), have been identified as essential

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fatty acids (EFA) in the diet of Atlantic salmon (Salmo salar L.) for good growth

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performance, health, and final product quality

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used to date in salmon diets, but due to relatively stable fish oil production and growing

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global demand for farmed fish, the aquaculture industry is facing a challenge in meeting the

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demand for fish oil in Atlantic salmon production

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such as algae, krill, and genetically modified plant oils, have been the subject of extensive

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research, but so far this has not yielded an economically and ecologically sustainable solution

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for salmon farming

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currently inevitable. It is important to know the possible impacts of reduced dietary EPA and

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DHA on salmon growth and health. There is still a knowledge gap on Atlantic salmon

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requirements for dietary EPA and DHA under different environmental conditions. Under

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controlled environment in tanks on land, 10g/kg EPA and DHA (1% of feed dry mass) is in

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general considered to be sufficient

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require above 10 g/kg to maintain fish robustness and good health under demanding

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environmental conditions in sea cages.

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The importance of EPA and DHA on fish performance has been studied previously, mostly

47

focusing on the impacts on fish growth, survival and early development, and fatty acid (FA)

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composition in fish liver and muscle

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effects of dietary EPA and DHA on fish skin health 11. As with terrestrial vertebrate skin, fish

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skin acts as the main barrier to the external environment, maintaining homeostasis in the

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organism and protecting against potential physical damage and environmental pathogens 16.

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However, unlike human skin, the fish epidermis lacks a keratinised layer (stratum corneum)

1–3

. Fish oil rich in n-3 LC-PUFA has been

4,5

. Alternative sources of n-3 LC-PUFA,

6–9

. As a result, a reduction in n-3 LC-PUFA levels in salmon feed is

1,10–12

. However, a recent study

11

showed that salmon

1,2,13–15

. Very few experiments have investigated the


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and hairs, but it contains a mucus layer and bone tissue-related scales

. The mucus layer

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contains anti-microbial and anti-infection enzymes such as lysozyme, protease, and

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immunoglobulin, which are important for fish skin health 17–19. Their immunological enzyme

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activities have been found to be implicated in fish epidermis histological parameters, such as

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epidermal thickness and mucus cell density

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period, the density of mucus cells, mainly goblet cells, was found to be positively correlated

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with epidermal layer thickness, but negatively correlated with parasite density 20,22.

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The permeability barrier of skin is primarily localized at the stratum corneum in terrestrial

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vertebrates. Ceramide (Cer), composed of a sphingosine (So) and a fatty acid (FA), is the

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main lipid (>50% of total lipid mass) in the stratum corneum

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EFA deficiency results in impaired sphingolipid metabolism, such as conversion of

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sphingomyelin (Sph) and glucosyl-ceramide (GlcCer) into Cer, leading to abnormal

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permeability barrier function in the epidermis of mammals

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the skin of terrestrial vertebrates in structure, many essential functions are shared, such as

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mechanical and chemical barrier formation to maintain osmotic homeostasis 17. To the best of

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our knowledge, only one previous publication has determined the total content of

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sphingolipids, including Sph and GlcCer, in fish skin, in a study on Pacific saury (Cololabis

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saira) using high-performance liquid chromatography (HPLC) coupled with evaporative light

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scattering detection (ELSD) 26. The composition and function of Cer and related sphingolipid

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metabolites, such as Sph, GlcCer, So, and sphinganine (Sa), in fish skin, and their responses

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to dietary treatments, are still unknown.

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Glycerol-phospholipids (GPL), phosphatidylcholine (PC), phosphatidylethanolamine (PE),

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phosphatidylserine (PS), and phosphatidylinositol (PI) are other important types of membrane

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lipids in the epidermis

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fluidity of cell membranes, which is important for signal transduction and substance

20,21

. During a six-week experimental infection

23

. It has been reported that

24,25

. Although fish skin is unlike

27

. The PUFA in GPL are essential components in maintaining the


4

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28,29

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transportation

. Lowered levels of n-6 PUFA, especially arachidonic acid (20:4n-6), and

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elevated levels of monounsaturated fatty acids (MUFA) have been observed in epidermal PC

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and PE in patients with atopic dermatitis

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(Oncorhynchus mykiss) showed that an EFA-deficient diet containing 93.4% saturated FA

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strongly influenced the GPL composition in fish skin, while no changes were detected in the

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permeability to water 29. The function and biosynthesis of GPL in fish skin is still not clear,

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which makes it interesting to determine the FA composition in GPL subclasses in skin when

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feeding diets deficient in EPA and DHA.

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The aim of the present study was thus to investigate the impacts of lowering dietary EPA and

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DHA levels on phospholipids in skin of Atlantic salmon. The composition of sphingolipids

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and FA composition in GPL subclasses (PC, PE, PS, and PI) in skin and epidermal

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histological parameters (epidermal thickness and goblet cell density) were examined. The

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effects of feeding duration were also evaluated.

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2 Materials and methods

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2.1 Fish feed formulation

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Thirteen experimental diets with different levels of EPA and DHA were formulated in the

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study. The feed ingredients are thoroughly described in another paper

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experimental diets were iso-proteic (46.6-47.0%), iso-lipidic (24.6-25.9%) and iso-energetic

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(22.1-22.6 MJ/kg), but contain 0%, 0.5%, 1.0%, 1.5% and 2.0% (of feed dry weight) of either

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only EPA, only DHA, or 1:1 mixture of EPA and DHA (EPA+DHA, Table 1). Among these,

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a diet completely depleted in EPA and DHA (0% EPA+DHA) was used as a negative control

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diet. The experimental diets were fishmeal and fish oil free, but carefully designed to meet

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fish nutritional requirements. Blended poultry oil and rapeseed oil (1:1) which are naturally

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lacking in EPA and DHA, were used as basic lipid sources in the experimental feeds. EPA

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and DHA oil concentrates in the form of triacylglycerol (Croda Chemicals Europe Ltd., East

30

. Moreover, a study on rainbow trout


12

. Briefly, the

5


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Yorkshire, UK) were used to control dietary levels of EPA and DHA. All experimental diets

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were produced by Nofima feed technology center (Bergen, Norway). The measured chemical

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composition and gross energy in the experimental fish feeds are provided in Table S1.

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A diet resembling a commercial diet with 2.2% 1:1 mixture of EPA and DHA (BioMar,

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Trondheim, Norway) was included as a commercial-type control (CC), in which 26%

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fishmeal and 9.8% fish oil were used. The main purpose of using the CC was to set a

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benchmark for growth.

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The FA composition in all diets were described in Bou et al.

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18:3n-3, the precursor of EPA and DHA in biosynthetic pathway was kept at the same level

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(about 4.7% of total FA) in all diets. The EPA and/or DHA dietary groups contained

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increased content of EPA and DHA as it was designed, and 0% EPA+DHA diet had little

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EPA (0.05% of total FA) and DHA (0.08% of total FA).

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2.2 Experimental design

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The feeding trial conditions are described in detail in Bou et al. 12. In brief, Atlantic salmon

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with mean initial body weight of 52.8 g were randomly distributed into 33 tanks, and 70

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fish/tank (2 tanks/diet for the 0.5, 1.0, and 1.5 % dietary groups, 3 tanks/diet for the CC, 0%

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EPA+DHA, and 2.0% dietary groups; Table 1), and reared at Nofima Institute in

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Sunndalsøra, Norway, for 26 weeks. All tanks (1 m2 surface area, 0.6 cm water depth) were

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supplied with 15 L/min seawater (33 g/L salinity) at ambient temperature. The water

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temperature varied between 6.3 °C and 13.8 °C and the oxygen saturation level was kept

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above 85%. Prior to the experiment, the fish were fed a commercial diet (Skretting,

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Stavanger, Norway) and treated with light to induce smoltification. Feed ration was 15−20%

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higher than assessed feed intake and was supplied by automatic belt feeders.

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Skin samples for lipid analysis were collected twice, following the same sampling

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procedures, when fish reached a body weight of 182.9 ± 69.3 g (referred to as 200 g), after 19


12

. Importantly, the content of

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weeks of feeding, and when they reached a body weight of 379.7 ± 96.5 g (referred to as 400

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g), after 26 weeks of feeding. Five fish were randomly selected from each tank and killed

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using overdose MS 222 (0.05−0.08 g/L). Skin samples with mucus and scales from the right

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fillet were dissected from the dorsal fin to the caudal fin, pooled by tank, and homogenized in

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dry ice. The homogenate was kept at -40 °C, with the bags left open until the dry ice

133

evaporated, and thereafter stored at -80 °C until analysis. Skin covering the white muscle

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from the Norwegian Quality Cut from the left fillet of fish were used for histology analysis.

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Samples were randomly taken at termination of the experiment (at 400 g, after feeding for 26

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weeks, n=5 fish per tank), cut into sizes (approximately 0.5 cm2) suitable for histological

137

analysis, and fixed in 10% buffered formalin. The experimental procedure was in accordance

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with National Guidelines for Animal Care and Welfare published by the Norwegian Ministry

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of Education and Research.

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2.3 Sphingolipidomics analysis using LC-QToF MS

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Sample preparation

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Fish skin samples from eight groups (CC, 0% EPA+DHA, 0.5% EPA, 0.5% DHA, 0.5%

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EPA+DHA, 2.0% EPA, 2.0% DHA, and 2.0% EPA+DHA) at stage 200 g and 400 g were

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subjected to sphingolipidomics analysis, using methods described elsewhere 31,32. In brief, the

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homogenized, pooled skin samples made out of five fish from each tank were analyzed three

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times. The homogenate containing an internal standard cocktail (0.15 nmol of C17

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sphingosine, C17 sphinganine, C17 sphingosine-1-phospate, C17 sphinganine-1-phosphate,

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C12 sphingomyelin, C12 ceramide, C12 glucosyl(β)-ceramide, C12 lactosyl(β)-ceramide and

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C12 ceramide-1-phosphate; sphingolipid mix II, LM-6005, Avanti Polar lipids, Alabaster,

150

AL, US) were extracted twice, using 3 mL chloroform:methanol (1:2, v/v) each time, under

151

sonication in a water-bath for 30 min at room temperature. The extract was centrifuged (1800

152

g, 20 min) at room temperature and the supernatant was collected.


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Since the amount of Sph in the skin samples was much higher than that of the other

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sphingolipids measured (Cer, So, Sa, and GlcCer), the content of Sph was determined

155

separately. Skin extract (0.25 mL×2) was transferred to two tubes, one with C12:0 Sph

156

internal standard (0.17 nmol; Avanti Polar lipids, Alabaster, AL, US) and one without.

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Sample solvent was evaporated under nitrogen and re-dissolved in 0.5 mL ethanol. The

158

remaining skin extract (5.2 mL) was used for quantification of the other sphingolipids. After

159

evaporation, samples were re-dissolved in 1 mL ethanol. All samples were centrifuged at 12

160

000 g for 20 min at 4 °C before analysis.

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Liquid chromatography-mass spectrometry analysis

162

Liquid chromatography-mass spectrometry (LC-MS) was carried out on an HP1100 LC

163

system (Hewlett-Packard, Palo Alto, CA) coupled to an electrospray ionization-

164

quadropole/time of flight mass spectrometer (ESI-QTOF MS; Bruker maXis Impact; Bruker

165

Daltonik GmbH, Bremen, Germany). System integrity was controlled by HystarTM software

166

(Bruker Daltonik GmbH). A sodium formate solution (4 µL formic acid, 20 µL 1 M NaOH,

167

100 mL H2O, and 100 mL 2-propanol) was used as the MS calibrant to correct for any mass

168

drift in the analyte. The spectra were acquired in positive ionization mode scanning within a

169

50-1500 m/z range.

170

Analyte separation was performed on a hydrophilic interaction chromatograph (Atlantis silica

171

HILIC column, particle size 3 µm, 2.1 mm×150 mm, Waters, Wexford, Ireland). The

172

injection volume was 10 µL and the column temperature was maintained electronically at 30

173

°C. The mobile phase consisted of: eluent A, with 1% v/v formic acid, and 10 mM

174

ammonium formate in MS grade water; and eluent B, with 0.1% v/v formic acid in

175

acetonitrile, at a constant flow rate of 0.25 mL/min. The programmed eluent gradient started

176

initially with 95% A to 5% A for 0.5 min, ramping to 60% A for 10 min, which was held for

177

4.5 min by further ramping to 5% A for 2 min and held for 15 min before the next run. A


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plasma reference and a sphingolipid standard mixture (sphingolipid mix II, LM-6005, Avanti

179

Polar lipids, Alabaster, AL, US) were run three times throughout the analysis as a quality

180

control to check the stability of the instruments. The MS raw data were calibrated

181

automatically and converted to mzXML files using Compass DataAnalysis software (Bruker

182

Daltonik GmbH). Peak height gave good linearity when comparing QToF responses to a

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standard Cer C17:0 (Larodan AB, Solna, Sweden) at different concentrations (0.1-1 µg/mL).

184

Therefore, peak height for the compounds of interests was calculated by Mzmine software

185

(version 2.15) based on their assigned m/z and retention time. The concentrations of

186

sphingolipids were determined against known amounts of internal standards and expressed in

187

nmol/g tissue. The contribution from overlapping signals from the

188

compounds was accounted for when relevant.

189

2.4 Fatty acid analysis of glycerol-phospholipids using TLC and GC-FID

190

Sample preparation

191

Total lipid in fish skin samples (2 g, from five fish per tank) was extracted with 50 mL

192

chloroform:methanol (2:1, v/v) containing 0.07% (w/v) butylated hydroxytoluene as

193

antioxidant and 6 mL NaCl (0.9%), according to the method described by Folch et al. 33. The

194

organic phase was collected and dried under a stream of nitrogen. The GPL fraction was

195

separated from the other lipid classes, such as triacylglycerol, diacylglycerol, and free FA, by

196

thin layer chromatography (TLC; silica gel 20×20 cm plates, Merck, Darmstadt, Germany)

197

using a mixture of petroleum ether, diethyl ether, and acetic acid (113:20:1, v/v/v) as mobile

198

phase, using the method described by Bou et al.

199

plates were sprayed with 2% 2,7-dichlorofluorecin in 96% ethanol. Lipid classes were

200

identified under ultraviolet (UV) light at 366 nm. The GPL bands were scraped off the plates

201

and soaked in a mixture of chloroform, methanol, acetic acid, and water (50:39:1:10, v/v/v/v)

202

for 4 hours at -40 °C, to elute GPL from silica gel. The GPL fractions were collected after

13

C isotope of other

12

and Thomassen et al.13. After drying, the


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adding 0.5 mL NaCl (0.9%), centrifuged twice at 700 g for 10 min, and dried under a stream

204

of nitrogen 12.

205

The different types of GPL (PC, PE, PS, and PI) were isolated by the second TLC procedure

206

using chloroform:methanol:acetic acid:water (100:75:6:2, v/v/v/v) 12,34. The GPL classes were

207

revealed by spraying with 2% 2,7-dichlorofluorecin in 96% ethanol and detected under UV

208

light at 366 nm by comparing with an external standard (Nu-chek Prep, Elysian, MN, USA).

209

The GPL bands were then separately scraped off the TLC plates and trans-methylated to FA

210

methyl esters (FAME) with benzene, methanolic HCl, and 2,2-dimethoxypropane (10:10:1,

211

v/v/v) overnight at room temperature

212

methylation. Tricosylic acid (C23:0, Nu-chek Prep, Elysian, MN, USA) was used as an

213

internal standard.

214

Gas chromatography-flame ionization analysis

215

The FAME were analyzed using a gas chromatograph (Hewlett Packard 6890, Palo Alto, CA,

216

USA) equipped with an auto-injector in split mode (HP 7683, Agilent, Avondale, PA, USA),

217

a BPX70 capillary column (SGE Victoria, Australia, 60 m length, 0.25 mm internal diameter,

218

0.25 µm thickness film), and a flame ionization detector (Hewlett Packard 6890) 13. Helium

219

was the carrier gas, with a constant flow of 20 mL/min. Both the injector and the detector

220

temperature were set at 270 °C. The oven temperature was initially held at 50 °C for 1.2 min,

221

then ramped at 4 °C/min to 170 °C, at 0.5 °C/min to 200 °C, and to the final temperature of

222

240 °C at a rate of 10 °C/min. The individual FA were identified by comparing the retention

223

time with external standards (Nu-chek Prep, Elysian, MN, USA). Peak areas were integrated

224

using HP ChemStation to calculate the relative FA content.

225

2.5 Skin histological analysis

226

Histopathological evaluation was performed on the skin of fish from the eight treatments

227

(CC, 0% EPA+DHA, 1.0% EPA, 1.0% DHA, 1.0% EPA+DHA, 2.0% EPA, 2.0% DHA, and

35

. Samples were neutralized with 6% NaHCO3 after


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2.0% EPA+DHA; n=10 per dietary group). Paraplast-embedded skin samples were

229

microtome-cut (5 µm) and stained with standard hematoxylin and eosin (Merck KGaA,

230

Darmstadt, Germany). Stained slides were examined using a standard light microscope

231

(Nikon Optiphot, Japan). Images were captured by means of a Micropublisher camera and

232

QCapture software using a 40X objective. Samples were firstly subjected to a blinded

233

evaluation on histopathology which means that the identity of samples were unrevealed,

234

followed by a second evaluation after decoding the samples which provide a description per

235

dietary group, to ensure the observation are unbiased. Epidermal thickness and goblet cell

236

numbers/100 µm were evaluated using Image J (NIH, Bethesda, MD, USA).

237

2.6 Data analysis

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Statistical Analysis System (SAS 9.3, SAS Institute, Cary, NC, USA) was used for univariate

239

data analysis within the experimental groups. The FA data in percentages were square root-

240

arcsine transformed before test. Data normality distribution (Anderson-Darling test) and

241

homoscedasticity (Bartlett’s test or Levene’s test) were checked. If the tests were failed, the

242

initial data were log-transformed and re-tested. General Linear Model was used for statistical

243

comparisons. For comparison of sphingolipid concentrations, two-way ANOVA was used

244

with diet and sampling time as fixed factors. For comparison of FA composition in GPL

245

fractions, data from different sampling times (at 200 g and 400 g, after feeding for 19 and 26

246

weeks, respectively) were analyzed separately using one-way ANOVA. For evaluation of the

247

histological parameters, one-way ANOVA was conducted. If the data did not satisfy the test

248

of distribution or homoscedasticity, Mann-Whitney test was applied as a non-parametric test.

249

Furthermore, Tukey’s test was employed as a post hoc test against a predefined significance

250

level (P

Impact of Reduced Dietary Levels of Eicosapentaenoic Acid and

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