Lipid Class Specific Quantitative Analysis of n-3 Polyunsaturated

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Lipid Class Specific Quantitative Analysis of n-3 Polyunsaturated Fatty Acids in Food Supplements Laura Kutzner, Annika I. Ostermann, Thade Konrad, Dieter Riegel, Stefan Hellhake, Jan Philipp Schuchardt, and Nils Helge Schebb J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03745 • Publication Date (Web): 14 Nov 2016 Downloaded from http://pubs.acs.org on November 20, 2016

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Journal of Agricultural and Food Chemistry

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Lipid Class Specific Quantitative Analysis of n-3 Polyunsaturated Fatty Acids

2

in Food Supplements

3

Short title: Lipid Class Pattern in n-3 PUFA Food Supplements

4 5 6

Laura Kutzner 1, #, Annika I. Ostermann 1, #, Thade Konrad2, Dieter Riegel2, Stefan Hellhake2,

7

Jan Philipp Schuchardt3 and Nils Helge Schebb1, 2*

8 9 1

10

Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany

11 2

12 3

13

Institute of Food Chemistry, University of Wuppertal, Wuppertal, Germany

Institute of Food Science and Human Nutrition, Leibniz University Hannover, Hannover Germany

14 15 16

*Corresponding author: Tel: +49 511 856 7780; Fax: +49 511 856 7409; E-mail:

17

[email protected]

18 19

#

These authors contributed equally to this work.

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List of abbreviations: ALA alpha-linolenic acid AO algae oil CE cholesterol ester DHA docosahexaenoic acid DPA docosapentaenoic acid EE (fatty acid) ethyl ester ES pharmaceutical ethyl ester product EPA eicosapentaenoic acid FFA free fatty acid FA fatty acid FAME fatty acid methyl ester FO fish oil GC-FID gas chromatography-flame ionization detection IS internal standard KO krill oil MG monoglyceride MUFA monounsaturated fatty acid MTBE methyl tert-butyl ether PL phospholipid PO plant oil PUFA polyunsaturated fatty acid SFA saturated fatty acid rTG re-esterified triglyceride TG triglyceride


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Supplementation products containing n-3 PUFA from marine sources serve a large

46

market. While the amount of eicosapentaenoic acid and docosahexaenoic acid in the

47

products is provided by the manufacturer, no or little information is available on their

48

lipid pattern. Therefore, we quantitatively analyzed the fatty acid pattern in the lipid

49

fractions

50

supplementation

51

chromatography. Twelve products from the European and US market containing fish,

52

krill, algal or plant oil were analyzed. Total n-3 PUFA content ranged between 64 (fish

53

oil) over 42 (algal oil) to 17 (krill oil) g/ 100 g fat. Based on the n-3 PUFA containing

54

lipid class, the supplements can be separated dominantly in ethyl ester, re-esterified

55

triglyceride, triglyceride and phospholipid containing products. Algae based products

56

contained natural triglycerides, krill oils a complex mixture of phospholipids,

57

triglycerides and free fatty acids and fish oil products either ethyl esters, re-esterified

58

triglycerides or triglycerides. Even products of the same class and source showed

59

distinct differences in their lipid pattern. A specification of the lipid composition of n-3

60

PUFA products will allow distinguishing the different (qualities of) supplements.

triglycerides,

phospholipids,

products

by

means

ethyl of

esters solid

and

phase

free

fatty

extraction

acids and

in gas



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Introduction

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The intake of polyunsaturated fatty acids (PUFA) affects human health. In the western diet,

63

large amounts of n-6 PUFA, particularly linoleic acid (18:2 n-6 from soy, safflower and corn

64

oil) are consumed. On the contrary, the intake of n-3 PUFA, particularly of the long chain n-3

65

PUFA eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3), is

66

low in non-fish eaters 1-3. However, a growing body of evidence suggests beneficial effects of

67

the intake of EPA and DHA on human health. For instance, these n-3 PUFA lower blood

68

triglyceride levels and are incorporated into phospholipids in the membrane of cells, thereby

69

affecting fluidity and lipid rafts

70

inflammatory effects and reduces the risk for cardiovascular diseases

71

these effects are at least partly mediated by a modulation of the endogenous pattern of

72

eicosanoids and other oxylipins 1. Aside from a reduction of the formation of pro-inflammatory

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oxylipins such as series 2 prostaglandins (PG, e.g. PGE2), EPA and DHA can give rise to

74

anti-inflammatory, cardio protective lipid mediators such as resolvins or epoxy-PUFA 6, 7.

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Both n-6 and n-3 PUFA are essential nutrients and mammals cannot transform one into the

76

other. However, the long chain n-3 PUFA EPA and DHA can be synthesized endogenously

77

from alpha-linolenic acid (ALA, 18:3 n-3), found in land plants such as flaxseed

78

conversion rates are low in healthy subjects without nutritional deficiencies 8. Therefore, the

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main source of EPA and DHA is the diet. Especially fatty cold water fish such as salmon or

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mackerel are rich in these FA

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EPA and DHA with minor importance 3. However, fish and consequently n-3 PUFA intake is

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low in western countries such as the US and Germany

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products are available as food supplements on the European and the US market. Most

84

products are oil-filled capsules with a high content of DHA and EPA. Additionally, other

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products such as emulsions or gums can also be found in supermarkets and pharmacies.

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The main natural sources for DHA and EPA comprise marine products, such as krill, algae

87

and fish

88

purpose, but also includes by-catch and by-products from fishery and fish meal production 12.

89

Among other methods, fish oil is mostly produced from the raw material by the wet rendering

12

1, 4

1, 2, 9

. Furthermore, EPA and DHA intake shows anti1, 4, 5

. It is believed that

2, 4

, though

. Other nutritional sources only contribute to the intake of

1, 10, 11

. Several n-3 PUFA containing

. Raw material for the production of fish oil is fish specifically caught for this


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process, which includes cooking and pressing of the raw fish. The raw oil is dewatered and

91

refined yielding purified fish oil

92

esters (EE) allowing further purification and concentration by distillation or urea complexation

93

14, 15

94

In order to generate the “more natural” binding form of the FA, several manufactures (trans-)

95

re-esterify the EE to triglycerides (TG)

96

contain several n-3 PUFA per molecule, which rarely occurs naturally. Apart from these

97

highly processed and concentrated products, purified oils from other marine sources than

98

fish are used directly for food supplements, e.g. krill oils. Krill oils contain n-3 PUFA in TG

99

and a major portion as polar phospholipids (PL)

13

. Usually the lipids are trans-esterified to fatty acid ethyl

. The resulting DHA- and EPA-EE are directly used in supplements and pharmaceuticals.

14, 15

. However, it should be noted that these TG

16, 17

. In addition, vegetarian alternatives for 12

. It should be noted that

100

food supplements containing n-3 PUFA EPA and DHA are algae

101

several oils from land plants like flaxseed are sold alongside the marine n-3 PUFA products,

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which may serve as source for ALA

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DHA 19.

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The total amount of n-3 PUFA and the content of other relevant PUFA are declared on the

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label of the supplements as requested by European and US law. Despite the relevance of n-

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3 PUFA for nutrition and their economic importance (annual world market volume about 25

107

billion $US in 2011

108

supplements is available or provided by the manufacturer. New studies suggest that the

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bioavailability of n-3 PUFA differs between the lipid classes in which the n-3 PUFA are bound

110

20

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food supplements, i.e. EE, TG, PL and free fatty acids (FFA). We do not only show the

112

results from twelve exemplary products from both the German as well as the US market, but

113

also provide an optimized method for the determination of the quantitative distribution of n-3

114

PUFA in the lipid classes. This will readily enable other researchers and institutions to

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expand this analysis to a larger, more representative set of n-3 PUFA supplements.

12

2, 4, 18

, but do not contain relevant amounts of EPA and

), no or only little information about the lipid composition of the food

. Therefore, we evaluated the quantitative pattern of n-3 PUFA in the major lipid classes in



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Material and Methods

117

Chemicals:

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Chloroform (uvasolv grade), ethanol (uvasolv grade) and ammonium acetate (p.a.) were

119

obtained from Merck (Darmstadt, Germany). n-Hexane (HPLC grade) and methyl tert-butyl

120

ether (HPLC grade) were purchased from Carl Roth (Karlsruhe, Germany). Methanol (HPLC

121

grade) was purchased from J.T.Baker (Deventer, Holland). Acetic acid (Acros Organics) was

122

obtained from Fisher Scientific (Nidderau, Germany). Methyltricosanoate (FAME 23:0, >98%)

123

and cholesteryl heptadecanoate (CE 17:0,>98%) were obtained from Santa Cruz

124

Biotechnology (SantaCruz, CA, USA). Dihenarachidoyl-sn-glycero-3-phosphocholine (PL

125

21:0, >99%) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Ethyl

126

tricosanoate (EE 23:0) was obtained from Cayman Chemicals (Ann Arbor, MI, USA).

127

Pentadecanoic acid (FFA 15:0, >98%), methyl pentacosanoate (FAME 25:0, >99%, used as

128

internal standard (IS) 2) and all other chemicals were purchased from Sigma Aldrich

129

(Schnelldorf, Germany).

130 131

n-3 PUFA supplementation products:

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Six n-3 PUFA products were obtained on the German market: Algal oil Ovega3 Life

133

Vegetarian (Energybalance AG, Muralto, Switzerland; AO1; purchased via Amazon

134

Germany), fish oil Omega3 Loges (Dr. Loges + Co. GmbH, Winsen (Luhe), Germany; FO1;

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purchased at a local pharmacy, Hannover, Germany), Doppelherz Omega3 fish oil

136

concentrate (Queisser Pharma, Flensburg, Germany; FO2; purchased via Meddox mail-order

137

pharmacy, Limburg, Germany), pure NKO krill oil (Neptune Technologies & Bioressources

138

Inc., Quebéc, Canada; KO1; purchased at Tresorix, Ruggell, Liechtenstein), Superba krill oil

139

(KALA Health BV, Den Haag, Netherlands; KO2; purchased via the KALA Health online

140

shop) and ethyl ester oil Omacor (Abbott GmbH & Co. KG, Wiesbaden, Germany; ES;

141

purchased at a local pharmacy, Hannover, Germany). Six n-3 PUFA products were obtained

142

on the US market (purchased at CSV Pharmacy, Davis, CA, USA): Super DPA Menhaden

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Fish Oil (Swanson Health Products, Fargo, USA; FO3), OceanBlue Omega 3 Super 6 ACS Paragon Plus Environment

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Concentrated Fish Oil (Sancilio & Company, Inc., Riviera Beach, USA; FO4), plant oil

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Vegetarian Omega 3-6-9 (Rexall Sundown Inc., Boca Raton, USA; PO), algal oil Vegetarian

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Omega 3 (Rexall Sundown Inc.; AO2), Adult Gummies Fish Oil (Nature Made Nutritional

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Products, Mission Hills, USA; Gum) and fish oil containing Omega Squeeze’n’Go (Rexall

148

Sundown Inc.; Emul).

149 150

Determination of total fat content, cholesterol, overall fatty acid pattern and peroxide value:

151

The total fat content of all supplementation products was determined by gravimetry after lipid

152

extraction according to Weibull-Stoldt performed as rapid microextraction

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KO1 and KO2 the total fat content was determined after lipid extraction according to

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Twisselmann using a mixture of cyclohexane and ethanol (1:1) as extraction solvent

155

cholesterol content was determined after alkaline hydrolysis of the lipid extract

156

(Twisselmann) by means of gas chromatography with flame ionization detection (GC-FID) as

157

described

158

the extracts were determined by GC-FID after trans-esterification of the lipids to fatty acid

159

methyl esters (FAME) calculated as g FA/100 g fat (lipid extract). For each product, two

160

independent extractions were carried out. The peroxide value (PV) was determined by

161

iodometry as described

162

potassium iodide and a starch solution was added. The formed iodine was titrated with

163

sodium thiosulfate.

23

21

. For products

22

. The

following derivatization with 1-(trimethylsilyl)imidazole. The overall FA pattern in

24

. In brief, the capsule content (~ 0.5 g) was dissolved in acidic

164 165

Separation of lipid classes by solid phase extraction (SPE):

166

The capsule content or the extracts from methyl tert-butyl ether (MTBE)/methanol

167

extraction 25 of the products Gum and Emul was fractionated by SPE into four lipid classes:

168

ethyl esters (EE), triglycerides (TG), free fatty acids (FFA) and phospholipids (PL).

169

Separation was carried out according to Kaluzny et al.

170

detail in the supplementary information. In brief, the lipid extract was dissolved in chloroform

171

and separated using two Supelclean LC-NH2 columns (100 mg, 1 mL, Sigma Aldrich), which

26

with modifications as described in



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were washed with two column volumes of each elution solvent prior to sample loading. The

173

neutral lipid fraction, containing EE/CE/TG was eluted with 1.5 mL chloroform/isopropanol

174

(2/1 (v/v)). Free fatty acids and the polar lipid fraction were eluted with 2 mL diethyl

175

ether/acetic acid (98/2 (v/v)) and 3 mL methanol, respectively. The neutral lipid fraction was

176

reconditioned in hexane and subjected to a second column; the CE/EE fraction was eluted

177

with 4 mL hexane and the TG fraction with 4 mL hexane/diethyl ether/dichloromethane

178

(89/1/10 (v/v/v)).

179

For quantification an IS was added for each lipid class (10 µL containing 0.75 mM FA

180

equivalent concentration) prior SPE: ethyl tricosanoate (EE 23:0), glyceryl trinonadecanoate

181

(TG 19:0), pentadecanoic acid (FFA 15:0), 1,2-dihenarachidoyl-sn-glycero-3-phosphocholine

182

(PL 21:0). FAME 25:0 served as IS 2 (added after SPE separation) and was utilized to

183

calculate the recovery rates of IS 1 as described

184

triplicate and results are calculated as mean ± standard deviation.

27, 28

. All separations were carried out in

185 186

Gas chromatographic quantification of fatty acids as fatty acid methyl esters:

187

FA were quantified by means of GC-FID on a 6890 series instrument (Agilent, Waldbronn,

188

Germany) as described

189

chloride in methanol (1:9) at a temperature of 95°C for 1 h

190

separated on a 30 m x 0.25 mm FAMEWAX column (0.25 µm film, Restek, Bad Homburg,

191

Germany) within 24 min. The carrier gas was helium (1.5 mL/min, constant flow) and the

192

oven temperature was ramped from 140°C to 210°C within 7 min, to 230°C within 10 min and

193

held at 230°C for 7 min. Absolute FA content and relative FA pattern were calculated using

194

odd chain IS (see above) based on response factors 25, 30.

25

. In brief, lipid extracts/fractions were trans-esterified utilizing acetyl 29

. The resulting FAME were


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Results

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The total fat content of the products is shown in Tab. 1 and ranged for the capsules between

197

65.4 ± 0.2 g/100 g (FO4) and 75.5 ± 0.5 g/100 g (PO). Lowest fat content was observed for

198

the product Gum (4.2 ± 0.3 g/100 g); highest fat content was observed for the product Emul

199

(80.19 ± 0.05 g/100 g). The cholesterol content was 1.4 ± 0.1 g/100 g fat and 0.99 ± 0.01

200

g/100 g fat for the products KO1 and KO2, respectively. Tab. 1 also shows the overall FA

201

profile of the products. The pattern of saturated (SFA), monounsaturated (MUFA), n-3 and n-

202

6 PUFA varied considerably between products from different sources, while similar patterns

203

were observed for products of the same source (Fig. 1, Tab. 1).

204 205

The amount of SFA was low in fish oil based products. The lowest amount was found in the

206

pharmaceutical product ES with 0.74 ± 0.06 g/100 g fat and the highest in the product Gum

207

with 27.0 ± 0.4 g/100 g fat. MUFA were also lowest in product ES (0.40 ± 0.03 g/100 g fat)

208

and highest in the algal oil product AO2 with 25.1 ± 0.1 g/100 g fat. In most of the marine

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products only low amounts of n-6 PUFA were found (1.22 ± 0.05 – 3.72 ± 0.05 g/100 g fat),

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while the plant based product PO contained 21.1 ± 0.2 g/100 g fat.

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The content of n-3 PUFA varied considerably between the products from 17.2 ± 0.6 g/100 g

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fat (KO1) to 81 ± 2 g/100 g fat (ES; Tab. 1, Fig. 1). The lowest amount of n-3 PUFA in fat was

213

found in the krill oils (KO1 and KO2) and the products Gum and Emul (17.2 ± 0.6 – 29.2 ± 0.2

214

g/100 g fat). The other fish oil based products showed higher n-3 PUFA content with 51.17 ±

215

0.05 – 67.5 ± 0.3 g/100 g fat. The vegetarian products showed intermediate n-3 PUFA

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amounts: in algae oil based products 41 ± 1 – 42.9 ± 0.2 g/100 g fat were determined; in the

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plant oil based product the n-3 PUFA amount was 49.8 ± 0.4 g/100 g fat.

218

The main n-3 PUFA in the supplements were EPA and DHA, which make up >90% of the n-3

219

PUFA in most of the products (Tab. 1). The absolute amount of EPA and DHA in g per

220

capsule also differed between the products (Fig. 2), varying from 0.03 g/gum (Gum) to 0.79

221

g/capsule (ES). Only the product PO did not contain significant amounts of EPA or DHA, as

222

the main n-3 PUFA in this product is ALA (49.6 ± 0.4 g/100 g fat). The product FO3 9 ACS Paragon Plus Environment


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contained aside from EPA and DHA 9.8 ± 0.2 g/100 g fat DPA (docosapentaenoic acid,

224

22:5n-3). The amount of DPA in all other products was lower (0.26 ± 0.01 – 4.18 ± 0.03

225

g/100 g fat, Tab. 1).

226 227

If the lipid content and FA pattern were provided by the manufacturer, it was mostly in good

228

agreement with the obtained results (Tab. 1). Discrepancies were only observed for the krill

229

oil based product KO1 and for the fat content of the product FO4.

230

The lipid pattern of the products is shown in Fig. 3 and in detail in the supplementary

231

information (Tab. S1). The relative distribution of the lipid classes is shown separately for

232

total FA (Fig. 3A), n-3 PUFA (Fig. 3B), n-6 PUFA (Fig. 3C) as well as EPA, DHA and ALA

233

(Fig. 3D-F).

234 235

FA in algal oil (AO1 and AO2) were only found in the TG fraction. In the other vegetarian

236

product containing n-3 PUFA obtained from flaxseed and sunflower oil (PO) FA were also

237

found solely in the TG fraction (Fig. 3A). Similarly, FO1 and FO2 and the oil extract of Gum

238

and Emul contained mainly TG. It should be noted that products FO1 and FO2 contained a

239

portion of FA bound in mono- and diglycerides, which were not taken into account for the

240

calculation of the relative lipid class pattern. Minor amounts (1-5 %) of total FA in the TG

241

containing fish oils FO1 and FO2 and of the krill oils were found in the EE/CE fraction. In

242

order to differentiate between EE and CE a direct GC analysis (without derivatization) was

243

performed of the mixed EE/CE fraction. In this way the main FA EPA and DHA were

244

identified to be EE (>98% of EPA and DHA for products FO1 and FO2, >90% of EPA and

245

DHA for products KO1 and KO2). By contrast, the fish oils FO3 and FO4 as well as ES

246

contained predominantly EE (>95%). For TG and EE containing oils, the distribution of n-3

247

PUFA in the lipid fraction correlated to the distribution of total FA (Fig. 3A and B).

248

For all marine products the n-3 PUFA occurred dominantly as EPA and DHA (Tab. 1). Thus,

249

the relative distribution of EPA and DHA correlated in general to that of n-3 PUFA (Fig. 3B,

250

Fig. 3 D-E). Interestingly, the amount of n-3 PUFA in TG is not representative for the EPA 10 ACS Paragon Plus Environment

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content of FO3: Only 3% of n-3 PUFA was found in the TG fraction while 32% of EPA was

252

detected as TG. With respect to the lipid binding form, there were remarkable differences

253

between the EE containing fish oil products: In FO3 32% of EPA was found as TG while FO4

254

contained EPA only as EE.

255

PL were not found in the fish, plant and algal oil based products. However, the majority of n-3

256

PUFA (67-71%) in krill oil was bound in PL. While the patterns of total FA and n-3 PUFA

257

were similar in all products, the distribution of total FA in krill oil (Fig. 3A) was different to that

258

of n-3 PUFA (Fig. 3B). Total FA (dominantly 14:0, 16:0, 16:1 n-7, 18:1 n-9) were mostly

259

found as TG (46-63%), whereas only 33-46% were bound in PL. Consistently, the n-6 PUFA

260

in krill oil were mainly bound in TG (54-60%), whereas only 36-39% were found in the PL

261

fraction (Tab. S1).

262

The krill oils (KO1 and KO2) were the only products containing relevant amounts of FFA:

263

8-13% of n-3 PUFA and 3-7% of total FA. All other tested products contained 96%), though at lower EPA and DHA concentrations than in the

288

pharmaceutical product (Tab. 1). Both EE containing supplementation products showed

289

distinct differences in the lipid pattern, as FO3 consisted dominantly of DHA (Tab. 1) and

290

contained a relevant portion of EPA (32%) as TG and about 1.5% as FFA. In the dominantly

291

EPA containing product FO4 >99% of all FA were present as EE.

292

The almost identically presented (in terms of labeling and advertisement) fish oil

293

supplements FO1 and FO2 contained the major part of FA as TG. Because of the high

294

content of n-3 PUFA in these TG, which is by far higher than that of fish-fat

295

these TG are so called re-esterified TG (rTG) generated via EE by trans-esterification. This

296

“more natural” binding form is a common n-3 PUFA enriched lipid product

12

it is likely that

14, 15

. The 12

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assumption that FO1 and FO2 contain rTG is further supported by the finding that 3-4% of

298

FA in these products were found in the EE fraction. The identity of at least 98% of both EPA

299

and DHA as EE in this fraction was confirmed by direct GC analysis and quantification of

300

EPA and DHA as EE (Fig. S4). Because EE do not occur naturally, trace amounts of EE

301

resulting from incomplete re-esterification may serve as indicator for rTG in addition to the

302

occurrence of mono- and diglycerides.

303

The analyzed n-3 PUFA enriched gummies and emulsions contain n-3 PUFA from fish oils.

304

All FA, including n-3 PUFA were found in the TG fraction. No residual EE were found and the

305

content of n-3 PUFA, EPA and DHA corresponded to that of non-concentrated fish oil

306

Thus, it can be assumed that these products contain natural TG made from purified and

307

refined fish oil.

308

Algae are the primary producer of (marine) long chain n-3 PUFA. Thus, algal products are

309

the (only relevant) vegetarian source of EPA and DHA

310

comparable fat content to the fish oil products, with a lower concentration of n-3 PUFA

311

(Tab. 1, Fig. 1) because of a higher content of SFA, MUFA and n-6 PUFA. Both tested

312

products contained FA only as TG. Moreover, the observed pattern of SFA and MUFA, n-6

313

PUFA and n-3 PUFA (EPA and DHA) was within the range of algal fat of different species

314

e.g. Schizochytrium 12. Based on this finding and the lack of EE it can be assumed that these

315

products contain purified and refined algal oil containing natural TG.

316

The land plant product (PO) was the only product that contained relevant amounts of ALA

317

(49.6 ± 0.4 g/100 g fat), whereas the ALA content in all other products was below 2 g/100 g

318

fat. Moreover, the content of n-6 PUFA was clearly higher for PO (21.1 ± 0.2 g/100 g fat)

319

compared to the products from marine sources (1.8 ± 0.1 g/100 g fat – 3.72 ± 0.05 g/100 g

320

fat) (Tab. 1). Due to the FA composition of PO and the presence of all FA in the TG fraction,

321

it contains most likely natural TG. However, as conversion rates of ALA to long chain n-3

322

PUFA are low in healthy subjects on a western diet, the product PO cannot be considered as

323

relevant source for EPA and DHA 8.

324

The krill oil based supplements differed considerably from fish and algal oil based products.

325

The n-3 PUFA content in fat was with 17-19% lowest among the supplements tested

31

12

.

. The tested products showed a



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326

(Tab. 1). While the other products consisted mainly of one lipid class, the lipid pattern of the

327

krill products was diverse, comprising PL (33-46% of total FA), TG (46-63% of total FA) and

328

FFA (3-7% of total FA). This indicates that the content is only little processed natural krill fat,

329

which has not been refined, enriched and/or chemically modified. Consistently, the oil was

330

cloudy and had a significant odor. A minor portion of total FA (1%) was found in the EE/CE

331

fraction. In earlier reports 2±3 to 27±9% of lipids are described as steroid esters for E.

332

crystallorophias

333

used for human consumption

334

content for the krill oil based products of 1.4 ± 0.1 g/100 g fat (KO1) and 0.99 ± 0.01 g/100 g

335

fat (KO2) one could assume that a significant portion of n3-PUFA-CE is present in the krill oil

336

products. However, more than 90% of both EPA and DHA in the EE/CE fraction could be

337

identified as EE by GC-FID analysis of the underivatized EE/CE fraction (Fig. S4). A

338

contamination of the krill oil products with (non-natural) EE during the production process is

339

the most likely explanation for this finding.

340

It is highly interesting that the dominant portion of EPA and DHA in krill products were bound

341

in polar lipids (Fig. 3B, Fig. 3D-E), while – as expected for a natural oil – the majority of FA

342

was bound in TG (Fig. 3A). These findings are in line with a previous study of krill oil, where

343

n-3 PUFA were mainly found in PL, while the majority of SFA was bound to TG 34. Regarding

344

the total FA, we detected 46-63% as TG and 33-46% as PL in the krill oil products. Castro-

345

Gómez found about equal amounts of FA in TG and PL (49 and 44%)

346

1-3% in TG and 20-33% of FA in PL

347

8-36% in TG and 53-57% in PL

348

range of variation between the levels can be explained by differences between krill species

349

and gender, and variations during season, diet or maturity

350

products, in which FFA are removed during refining/purification, krill oil products contained a

351

relevant amount (3-7%) of FFA (Tab. S1). The concentration of FFA can be regarded as

352

quality marker for these oils, as the content increases rapidly between the harvest and

353

freezing/processing and with the storage temperature of the raw material

354

the shelf life, the high FFA content of up to 8% of n-3 PUFA for KO1 could be highly

32

. In other krill species, such as E. superba, one of the krill species mainly 33

, no or little steroid esters were found 17, 32. With a cholesterol

32

34

35

, while Gigliotti found

, Araujo 12-30% in TG and 19-81% in PL

and Phleger 2-54% in TG and 36-96% in PL

17

16

, Bottino

. The large

17, 36

. In contrast to the other

13

. With respect to


Page 15 of 28


355

problematic for the stability of the krill oil product because free PUFA are prone to

356

(aut)oxidation leading to rancidity. A common parameter for the determination of the degree

357

of oxidation is the peroxide value (PV), giving an estimation of the primary lipid oxidation

358

products (hydroperoxides).

359

oils and 10 for native oils.

360

Lipid Class Specific Quantitative Analysis of n-3 Polyunsaturated

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