Ganglioside Composition in Beef, Chicken, Pork, and Fish Determined

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Ganglioside Composition in Beef, Chicken, Pork, and Fish Determined Using Liquid Chromatography#High Resolution Mass Spectrometry Bertram Yin Fong, Lin Ma, Geok Lin Khor, Yvonne van der Does, Angela Rowan, Paul McJarrow, and Alastair Kenneth H. MacGibbon J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02200 • Publication Date (Web): 20 Jul 2016 Downloaded from http://pubs.acs.org on July 27, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

Journal of Agricultural and Food Chemistry

1 1

Ganglioside Composition in Beef, Chicken, Pork, and Fish Determined Using Liquid

2

Chromatography‒High Resolution Mass Spectrometry

3 4 5 6 7

Bertram Y. Fong†, Lin Ma†, Geok Lin Khor#, Yvonne van der Does†, Angela Rowan†, Paul

8

McJarrow†, and Alastair K. H. MacGibbon† *

9 10 11 12

†

13

North 4442, New Zealand

14

#

15

Bukit Jalil, 57000 Kuala Lumpur, Malaysia

Fonterra Research and Development Centre, Dairy Farm Road, Private Bag 11029, Palmerston

School of Health Sciences, International Medical University, No 126 Jln Jalil Perkasa 19,

16 17 18 19

ACS Paragon Plus Environment


Page 2 of 37

2 20

ABSTRACT

21

Gangliosides (GA) are found in animal tissues and fluids, such as blood and milk. These

22

sialo-glycosphingolipids have bioactivities in neural development, gastrointestinal tract, and the

23

immune system. In this study, a HPLC‒MS method was validated to characterize and quantitate

24

the GA in beef, chicken, pork, and fish species (turbot, snapper, king salmon, and island

25

mackerel). For the first time, we report the concentration of GM3, the dominant GA in these

26

foods, to range from 0.35 to 1.1 mg/100g and 0.70 to 5.86 mg/100g of meat and fish

27

respectively. The minor GAs measured were GD3, GD1a, GD1b, and GT1b. Molecular species

28

distribution revealed that the GA contained long to very long chain acyl fatty acids attached to

29

the ceramide moiety. Fish GA contained only N-acetylneuraminic acid (NeuAc) sialic acid while

30

beef, chicken, and pork contained GD1a/b species that incorporated both NeuAc and N-

31

glycolylneuraminic acid (NeuGc) and hydroxylated fatty acids

32 33

KEYWORDS: Glycosphingolipids, fatty acids, sialic acid, mammalian tissue, multiple reaction

34

monitoring

35

___________________________________________________________________________

36


Page 3 of 37


3 37

INTRODUCTION

38

Gangliosides (GAs) are a complex family of glycosphingolipids containing a variety of

39

sialic-acid-containing oligosaccharide chains attached to a variety of ceramide structures. The

40

common classification of GAs, and the nomenclature devised by Svennerholm,1 relies on the

41

degree of sialylation and the glycosidic linkages of these oligosaccharide groups. However, GAs

42

have extra complexity because of the variety of fatty acids (FAs) found in the ceramide, giving

43

rise to numerous molecular species within each GA class (Figure 1). In addition, there are many

44

types of sialic acid; however, only two types – N-acetylneuraminic acid (NeuAc) and N-

45

glycolylneuraminic acid (NeuGc) – are commonly incorporated into the oligosaccharide chain

46

along with any modifications, such as O-acetylation. The type of GA, its sialic acid mix, and its

47

FA molecular species profile vary with organism and tissue or fluid.

48

GAs play important biological roles in neurological development, memory formation, and

49

synaptic and transmembrane signal transduction, and are also implicated in regulating the

50

immune system, supporting intestinal maturation, protecting against enteric pathogens, and

51

promoting Bifidobacterium proliferation in the gut of infants.2-4 Different GAs are found in

52

various biological tissues and fluids, and they are part of the milk fat globule membrane in

53

mammalian milk.2, 5 There is growing evidence that dietary GAs, such as those from human

54

breast milk, can have a variety of benefits, which has led to increased research in this area over

55

the last few years.6-10 However, only very few studies have been carried out on the GA

56

quantitation of other food sources such as meat and fish, despite the recognition that they are

57

contributors of GAs and other sphingolipids to the human diet.11

58

The majority of the GA work on animal tissues (including liver) was conducted using thin

59

layer chromatography (TLC)-based methods.12-17 Although most of these studies were restricted

60

to the characterization of total GAs, Nakamura et al.14 and Saito and Rosenberg16 did determine

61

the concentration of a number of GA classes in hog and chicken muscle, respectively, using

62

TLC-based methods.



Page 4 of 37

4 63

To date, only one study reports the GA content in several food products (milk, yogurt, beef,

64

tuna, and egg) and this was used to estimate the GA intake for a small population group.18

65

However, the total GA concentrations in the food products and the dietary intake were

66

determined only as lipid-bound NeuAc (sialic acid) equivalents and NeuGc was not considered

67

as a contributing sialic acid.

68

The significant gaps in the quantitative data for the GAs in foods are probably due to the

69

complex nature of these families of molecules. The traditional methods used to measure them

70

were mainly TLC based, which is laborious. Also, the GA content was estimated using the

71

indirect approach of measuring the lipid-bound sialic acid with no consideration of the variety of

72

sialic acids, their responses to staining, sialic acid modification, or the difference in molecular

73

weight because of the base and the FA length along with the presence of double bonds. With the

74

advancement of high performance liquid chromatography–mass spectrometry (HPLC‒MS),

75

there have been an increasing number of publications on the characterization and absolute

76

quantitation of the GAs in biological fluids, such as blood and milk, using such techniques19-22

77

and in biological samples23-25 but such HPLC‒MS-based methods have yet to be applied

78

systematically to foods.

79

In this study, we validated an HPLC‒high resolution MS-based method to characterize and

80

quantitate the different GA classes in beef, chicken, pork, and fish. This method will provide a

81

direct means of analyzing the GA concentration in foods, as opposed to measuring the GAs

82

indirectly via their sialic acid concentration.

83 84 85

MATERIALS AND METHODS Standards and Chemicals. GA standards [GM1a (bovine brain), GM2 (human brain), GM3

86

(bovine buttermilk), GM4 (egg yolk), GD1a (bovine brain), GD1b (bovine brain), GD3 (bovine

87

buttermilk), GT1b (bovine brain), and GQ1b (bovine brain)] were obtained from Matreya, LLC


Page 5 of 37


5 88

(Pleasant Gap, PA). All solvents were of liquid chromatography grade (Merck, Darmstadt,

89

Germany), except for chloroform, which was analytical grade (ethanol stabilized).

90 91 92

Samples. All food samples were purchased from a local supermarket in Palmerston North, New Zealand, except for the island mackerel, which was sourced from Malaysia. Lipid Extraction. All meat and fish samples (fillet including the skin) were cut into small

93

pieces prior to freeze drying. A sample (20‒30 g) was weighed out and freeze dried. The freeze-

94

dried sample was milled into a powder using a domestic coffee grinder. Approximately 0.1‒0.2

95

g of freeze-dried powder was weighed into a Kimax tube (10 mL) together with water (0.4 mL).

96

The sample was mixed and allowed to rehydrate for 10 min prior to lipid extraction.

97

The lipids were extracted from the samples using a modified Svennerholm and Fredman26

98

extraction protocol, as described previously.24 Briefly, a further volume of water (0.5 mL) was

99

added to the homogenate, followed by methanol (2.7 mL) and chloroform (1.35 mL) at room

100

temperature, and the sample was allowed to rock gently for 30 min on a rocker before

101

centrifugation at 2,000 g for 20 min. The supernatant was transferred to a 15 mL Kimax tube

102

and the pellet was re-extracted using water (0.5 mL) and chloroform:methanol (1:2, 2 mL). After

103

mixing and centrifugation (30 min at 2,000 g), the supernatants were pooled and the pellet was

104

discarded. Potassium chloride (0.01 M, 1.3 mL) was added to the pooled supernatant, vortex

105

mixed, and centrifuged (2,000 g for 30 min). The upper phase was transferred to a 10 mL

106

volumetric flask. Potassium chloride (0.01 M, 0.5 mL) was added to the remaining lower phase

107

and any interfacial fluff (minimal) and was centrifuged (2,000 g, 20 min). The upper phase was

108

pooled into the 10 mL volumetric flask without disturbing any interfacial fluff, and the flask was

109

made up to the mark with methanol (50%). The upper phase was used for GA quantitation.

110

Solid Phase Extraction. Solid phase extraction (SPE) employed a C18 SPE cartridge

111

(Strata, 500 mg/6 mL, 55 µm, 70Å, Phenomenex, Torrence, CA) and the SPE protocol as

112

described by Sørensen.19 The SPE was conditioned with 3 mL of methanol followed 1 mL of

113

60% methanol at a flowrate of 2-3 mL/min. The GA extract (10mL) was diluted with 1mL ACS Paragon Plus Environment


Page 6 of 37

6 114

disodium hydrogen phosphate buffer (30 mM, pH 9.2), followed 4mL of 60% methanol, prior to

115

loading onto the SPE cartridge. The SPE cartridge was washed with 3 mL of 60% methanol and

116

sucked dry under vacuum for 5 seconds. The GAs were eluted with 6 mL of methanol under

117

vacuum. The eluent was dried down under a stream of nitrogen (30 °C) to near dryness, and

118

was made up to a volume of 0.25 mL with 50% aqueous. methanol. The pre-concentrate extract

119

was used for HPLC‒MS2 fragmentation and the characterization of minor level GAs (GD3,

120

GD1a, GD1b, and GT1b).

121

HPLC‒MS Quantitation of GAs. The HPLC separation of the GAs was conducted on an

122

Agilent 1100 series HPLC system (Santa Clara, CA) equipped with a 150 mm x 2.1 mm i.d., 3

123

µm, APS-2 Hypersil hydrophilic column ((Thermo Electron Corporation, Waltham, MA)

124

coupled to a 10 mm x 2.1 mm i.d. APS-2 guard column. The GAs were separated using an

125

acetonitrile:ammonium acetate buffer gradient as described previously.24

126

The eluate from the HPLC system was introduced into an LTQ-Orbitrap mass spectrometer

127

(Thermo Electron Corporation, Waltham, MA) using an electrospray ionization (ESI) probe

128

inlet. Ions were generated and focused using an ESI voltage of ‒3750 V, a sheath gas flow of 30,

129

an auxiliary gas flow of 10, and a capillary temperature of 300 °C. MS data acquisition was

130

carried out with the mass spectrometer scanning in negative ion mode with a resolution of

131

30,000 over a 700−1650 m/z range. The system was calibrated with GA standards obtained from

132

Matreya, LLC (Pleasant Gap, PA). The HPLC run was diverted to waste for the first 9 min and

133

after 18 min. The total run time was 25 min.

134

Calibration Standard Preparation. The HPLC‒MS system was calibrated against

135

commercial GA standards. GA stock standards [GM1a, GM2, GM3, GM4, GD1a, GD1b, GD3, GT1b,

136

and GQ1b] were prepared at 1 mg/mL in methanol (50%), and serially diluted further in methanol

137

(50%) to give a seven-point calibration curve ranging from 0.08 to 10 µg/mL.

138

Identification of GAs Using Accurate Mass. A combination of GA calibration standard

139

retention time and accurate mass was used to identify the GA molecular species in this study. ACS Paragon Plus Environment

Page 7 of 37


7 140

The resolving power of the LTQ-Orbitrap mass spectrometer was used to filter post analysis for

141

known accurate masses of GA species present within each class of GA measured. Three factors

142

were considered when compiling our extract mass lists for filtering post analysis.

143

Sphinosine Group and Acyl FA Side Chains. The exact GA masses extracted were based on

144

sphingosine d18:1 and dihydrosphingosine d18:0, the two most dominant,14 with acyl FA side

145

chains C14:0 to C30:0, and their respective unsaturated and mono-hydroxy to tri-hydroxy FAs.

146

Sialic Acid Moiety. GAs containing NeuAc and NeuGc sialic acids, and their respective O-

147

acetylated moieties, were included in the mass list calculations.

148

Charge State. The number of NeuAc/NeuGc residues present on a GA molecule determines

149

the number of possible charge states that may be observed in the (electrospray) mass spectra of

150

that GA. Where more than one charge state existed for a given class of GA, the charge state(s)

151

observed in the greatest intensity were used for compiling our mass lists. The charge state(s)

152

selected for each GA class were as follows: GM1a, [M–H]–; GM2, [M–H]–; GM3, [M–H]–; GM4,

153

[M–H]–; GD1a, [M–2H]2–; GD1b, [M–2H]2–; GD3, [M–2H]2–; GT1b, [M–2H]2–, [M–3H]3–; GQ1b,

154

[M–2H]2–, [M–3H]3–). GT1b and GQ1b displayed prominent species in their –2 and –3 charge

155

states. Both charged states were included in the extract mass list.

156

MS2 Fragmentation of GA Molecular Species. The identities of the major GA molecular

157

species, particularly those that were observed in the samples but were not present in the

158

calibration standards, were confirmed using HPLC–MS2 experiments. The HPLC–MS

159

conditions were as described above, except that the LTQ-Orbitrap was set to acquire MS2 data in

160

dependent data acquisition mode. The dependent data acquisition mode experiments were

161

performed according to the following parameters: the full MS scan event 1 was carried out with

162

a resolution of 30,000 in the Orbitrap followed by the MS2 scan event, which was carried out in

163

the ion trap (LTQ). The dynamic exclusion time was set at 45 s for the most intense ions, with

164

either collision-induced dissociation or pulsed Q collision-induced dissociation activation type

165

collision and a collision energy of 30 or 25 arbitrary units, respectively. In some cases, specific ACS Paragon Plus Environment


Page 8 of 37

8 166

parent masses were added to the mass list for MS2. The MS2 carbohydrate fragments were

167

labelled using nomenclature as described by Domon and Costello27 and other standard

168

carbohydrate nomenclatures.

169

Accuracy and Repeatability. The accuracy of the method and matrix suppression effects

170

were evaluated using spike recovery studies and standard addition, whereas the repeatability of

171

the method was determined by repeated analysis of several samples in each batch of analyses

172

carried out on different days over the course of this study, using the guidelines from Taylor.28

173

The repeatability coefficient of variation (CV) was determined as the percentage of the standard

174

deviation divided by the mean.

175 176 177

In the standard addition protocol, the samples were spiked with increasing concentrations of a mixed GA standard solution (0.3, 0.6, and 1.2 µg/mL). Statistical Analysis. All statistical analysis was conducted using Minitab (Release 16.2.4,

178

2013, Minitab Inc., State College, PA). Comparison between two groups of data was conducted

179

using paired t-tests.

180 181 182

RESULTS AND DISCUSSION Methodology. The majority of HPLC‒MS methods for GA quantitation are based on

183

multiple reaction monitoring (MRM) MS techniques,7-9, 19, 21, 23 with scope limited to dairy, milk,

184

and blood samples. Although the MRM mode of MS is specific and deemed to be more sensitive

185

than accurate mass extract techniques, MRM methods require prior knowledge of the parent and

186

fragment ions. Whereas MRM methods are easy to set up for known individual compounds,

187

they are more complex for GAs, where multiple molecular species often exist for each GA class

188

and these molecular profile differ between animal types and between tissues in the same animal.

189

In this light the pre-investigatory work required to set up an MRM method makes this method

190

less flexible and more time consuming. In contrast, an accurate mass extract technique using

191

high resolution MS does not require prior knowledge of all the GA molecular species present


Page 9 of 37


9 192

before quantitation can proceed. Accurate mass extraction of parent ions is carried out post

193

analysis, which also has the advantage of allowing the data to be re-mined at later stages as more

194

information becomes available. Furthermore, data on the GA molecular species distribution is

195

immediately available to the analyst.

196

Typical HPLC‒MS extracted ion chromatograms, acquired in the mass range of m/z 700-

197

1650, of the GA standards and the turbot fish sample are presented in Figure 2 and Figure 3,

198

respectively. The GAs were separated on the amino-propyl column based on their sialic-acid-

199

containing oligosaccharide moieties (or class), and were identified by their retention time,

200

against calibration standards, and their accurate mass. In addition, MS2 fragmentation was used

201

to confirm the identification of the GA molecular species that were present in the samples but

202

not in the calibration standards. As there was no on-column separation between the GAs

203

containing either an NeuAc or an NeuGc sialic acid moiety within each class of GA, NeuAc-

204

and NeuGc-containing GA molecular species within each GA class were quantitated together.

205

The extracted ion chromatogram for each of the GA molecular species was generated post

206

analysis using the accurate masses and was calibrated against NeuAc-containing calibration

207

standards using peak areas as NeuGc-containing GA calibration standards were not available. As

208

is common with other GA quantitative methods it was assumed that different acyl FA and sialic

209

acid moieties within each GA class give the same response.23-25

210

The ESI process during MS is prone to matrix effects, typically suppression of ionization.

211

This was evaluated by comparing the standard addition results with the results obtained using

212

the described external standard HPLC‒MS method. The standard addition results for GM3 were

213

not significantly different from the external standard method results across the different sample

214

matrices (beef, chicken, pork, and fish), (Table 1), giving us confidence that any major matrix

215

effects, if present, were insignificant. The accuracy of the method was also evaluated using spike

216

recovery experiments. GM3 was spiked with approximately twice the endogenous level, but

217

GD3, GD1a, GD1b, and GT1b were spiked at approximately 0.1 mg/100g fresh product. The



Page 10 of 37

10 218

recoveries were 101‒104% for GM3, but were 90‒106% for GD3, GD1a, GD1b, and GT1b (Table

219

2). No recovery was determined for GM2, which was detected only in island mackerel. These

220

recoveries are consistent with those (96‒103%) that we reported previously for dairy products22

221

using the same extraction protocol,26 giving us confidence that the protocol was robust.

222

The repeatability of the method, determined as the CV, ranged from 2-10% for GM3.

223

However, much higher CVs were measured for the minor GAs [GD3 (4‒20%), GD1a (5‒33%),

224

and GT1b (33%)] because their concentrations were generally close to the LOQ (0.01 mg/100g

225

fresh product equivalent) (Table 1).

226 227

GA Molecular Species in Meat. The GA molecular species in the meats in our study were

228

tentatively identified by d-value (the sum of the carbon atoms and unsaturated bonds attached to

229

the sphingosine base and acyl FAs) (Figure 1), with d18:1 and d18:0 being assumed to be the

230

major and minor sphingosine base FAs, respectively.29

231

The GM3 and GD3 molecular species observed in beef, chicken, and pork samples (Figure 4)

232

were also present in their respective bovine-milk-sourced calibration standards (Figure 2),

233

although their relative distributions were different.

234

The predominant GM3 molecular species observed in beef and pork was d36:1(m/z 1179),

235

which could be tentatively identified as C18:1‒18:0, based on the d18:1 sphingosine being the

236

major sphingoloid base that is commonly reported in mammalian muscle tissue.13,29, 30‒31 Based

237

on the same d18:1 sphingosine, the minor GM3 molecular species were d38:1, d40:1, d41:1, and

238

d42:1 and contained the very long chain acyl FAs C20:0, C22:0, C23:0, and C24:0, respectively.

239

Pork and beef also contained small amounts of GM3 containing d18:0 sphingosine base, as

240

observed by the presence of the d37:0 molecular species containing the odd chain C19:0 acyl FA

241

(Figure 4). The HPLC‒MS GM3 molecular species distribution results for our pork sample are

242

consistent with the TLC‒gas chromatography results reported by Namakura et al.14 in which the

243

GM3 acyl FA composition contained predominantly C18:0 FA (76%), with minor levels of


Page 11 of 37


11 244

C20:0, C22:0, C23:0, and C24:0 FAs. The major sphingosine base was d18:1 (95‒97%),

245

whereas the d18:0 sphingosine base was present in minor proportions (1‒3%). No FA

246

composition data for beef muscle GAs are currently available in the literature for comparison

247

with our observations.

248

In chicken the GM3 dominant molecular species was d34:1 (m/z 1151), which could be

249

tentatively identified as containing d18:1‒16:0 FAs. The minor chicken GM3 molecular species

250

d36:1, d38:1, d40:1, d42:2, and d43:1 could be tentatively identified as containing the long to

251

very long chain acyl FAs C18:0, C20:0, C22:0, C24:1, and C25:0, respectively, also on a d18:1

252

sphingosine base. Similar to beef muscle, no GA molecular species data for chicken muscle

253

could be located.

254

All the GM3 molecular species that were identified across the three meat samples contained

255

the NeuAc sialic acid moiety (Figure 5). However, using TLC methods, Nakamura et al.13,14

256

reported the presence of NeuGc-containing GM3 in hog and bovine skeletal muscle at 4‒7.4%

257

and 21% of the total GAs, respectively. We did not find any NeuGc-containing GM3 in our beef

258

and pork samples. It is possible that either the NeuGc-containing GM3 molecular species were at

259

concentrations below the limit of detection for our methodology or they were inherently absent

260

from our samples because of the tissue origins. No NeuGc-containing GM3 (or GD3) was

261

reported for chicken skeletal muscle by these researchers and, again, we did not detect them in

262

our samples.

263

For meat GD3 molecular species, although d34:1 (d18:1‒16:0, m/z 720 (doubly charged))

264

was the most abundant molecular species, a number of other molecular species were also present

265

in near similar abundance (Figure 4). Like the major GM3 molecular species, and GD3

266

molecular species across the three meat samples were also identified from the MS2 data as

267

containing NeuAc sialic acid moieties. Examples of the MS2 fragmentation of a GD3 molecular

268

species containing an NeuAc sialic acid moiety are provided in Figure 5, showing their

269

distinctive m/z 290 (NeuAc) and m/z 581(NeuAc‒NeuAc, for GD3) fragments.



Page 12 of 37

12 270

GD1a and GD1b were fully resolved using the HPLC separation conditions described (Figure

271

2). Similar to the standards, the GD1a and GD1b mass spectra within each of the meat samples

272

showed that they shared the same parent ion masses. For this reason, the pooled GD1a and GD1b

273

mass spectrum for each of the meat samples is presented in Figures 4G‒4I, as GD1a/b. Accurate

274

mass analysis of the GD1a/b molecular species in these samples indicated that they contained a

275

combination of NeuAc and NeuGc sialic acid moieties (Figures 4G‒4I). Their presence was

276

confirmed using MS2 experiments (Figure 6), in which GD1a shows the NeuAc (m/z 290) and/or

277

NeuGc (m/z 306) fragment ions, and GD1b shows the characteristic NeuAc‒NeuAc (m/z 581) or

278

NeuAc‒NeuGc (m/z 597) MS2 fragment ion. Together with other daughter fragment ions (Figure

279

6), the accurate parent ion mass, and the retention time, tentative identification of the GD1

280

species was made (Figure 4). This is an interesting observation, given that GD3 showed only

281

NeuAc and GD3 is a precursor GA for GD1b biosynthesis. In addition to the different sialic acid

282

moieties, we were also able to tentatively identify in the meat samples some of the GD1a/b

283

containing hydroxylated FAs. These hydroxyl groups were tentatively identified to be located on

284

the acyl FA chain. Based on their accurate mass, they were typically associated with very long

285

chain acyl FAs (d40:0, d41:1, d42:0, d42:1, d42:2, d44:0) containing both NeuAc and NeuGc

286

sialic acid moieties (Figures 4G‒4I). A GA with hydroxylated acyl FAs has not previously been

287

reported in muscle tissues, but 2-hydroxylated acyl FAs are typically found in glucosyl-

288

ceramides and lactosyl-ceramides in various mammalian tissues fluids and are typically

289

associated with very long chain acyl FAs (C22:0, C23:0, and C24:0).12, 32-34 As lactosyl-

290

ceramides are a precursor for GA biosynthesis, the presence of hydroxylated acyl FAs in GAs

291

might be predicted. Skin tissues have also been reported to be rich in ceramides with ω-

292

hydroxylated very long chain acyl FAs with up to 36 carbons that are generally unsaturated with

293

one to two double bonds.35 Although we have tentatively identified the hydroxylated FAs to be

294

associated with the acyl FAs, they could also be associated with the sphingoloid long chain base.

295

Sphingoloid bases with multiple hydroxylated groups have been reported but generally at minor


Page 13 of 37


13 296

levels.29 Future studies will examine the location of these hydroxylated FAs. In this study, these

297

hydroxylated molecular species, and those containing either NeuAc or NeuGc, were quantitated

298

together as either GD1a or GD1b.

299 300

GA concentration in Beef, Chicken, and Pork. Table 1 summarizes the GA concentrations

301

measured across the fish samples. GM3 was the major GA that was found in beef, chicken, and

302

pork, making up 71‒96% of the total GAs in these samples, with the highest concentration (1.1

303

mg/100g) in chicken thigh and the lowest concentration (0.35 mg/100g) in pork mince (Table 1).

304

Data on meat GAs is sparse, with only two studies13, 14 reporting the GA composition in animal

305

muscle. Of these two studies, one14 reported the GM3 concentration of pork (hog) at

306

approximately 1 mg/100g (converted from 8.3 nmol/g using a GM3 molecular weight of 1180)

307

using a TLC‒resorcinol‒HCl method. The other study, published by the same researchers,13

308

reported GM3 as being the major GA in beef, chicken, and pork (hog), making up 71%, 52%,

309

and 34% of the total GA distribution. However, these results13, 14 were based on TLC

310

densitometry responses and were not corrected for GA mass. In this study, GM3 made up 96%

311

of the total GAs in the beef sample, 76‒79% of the total GAs in the two chicken samples and

312

71% of the total GAs in the pork sample.

313

GD3 is the dominant GA in bovine milk,22 but was measured at only a relatively low

314

proportion (4‒10%) of beef GAs and 4% of pork GAs, 0.02‒0.1 mg/100g and 0.02 mg/100g,

315

respectively (Table 1) in this study. Although absolute concentration data on beef GD3 was not

316

available from Nakamura’s TLC densitometry study,13 beef GD3 was estimated at 2% of the

317

total GAs. These researchers also estimated GD3 at 4% of the total GAs in pork, but they did not

318

find any GD3 in their second pork sample.14

319

Compared with pork and beef, chicken contained a significantly higher proportion of GD3, at

320

21% of the total GAs (0.2‒0.3 mg/100g) which is similar to the 16% reported by previously.13

321

However, the GA distribution in chicken changes over its developmental stages, with GD3 (50%



Page 14 of 37

14 322

of the total GAs) being the major GA in thigh and leg muscle during early development and

323

decreasing to ~ 15% (of the total GAs) beyond 90 days postnatal. Conversely, GM3 (60% of

324

total GAs) is dominant only after 20‒90 days postnatal.36

325

GD1a was typically at higher concentrations than GD1b in beef, chicken, and pork (Table 1).

326

In beef and chicken both GD1a/b were relatively minor GAs, being < 6% of the total GAs (up to

327

0.054 mg/100g) but in pork they were, a significantly higher proportion (16%, 0.08 mg/100g).

328

The GD1a concentration reported for hog muscle14 was 1.19 mg/100g (22% of the total GAs,

329

converted using a molecular weight of 1894), which was about 10 times higher than obtained in

330

this study. This discrepancy may be due to both analytical methodology and sample source.

331

Only relatively small proportions of GD1b (3% of the total GAs, 0.15 mg/100g) were reported by

332

these same researchers. Although the relative proportion of GD1b measured in this study was

333

nearly three times this, at 8% of the total GAs, the absolute concentration was much less, at 0.04

334

mg/100g (Table 1). No GD1a and GD1b concentration data are available in the current literature

335

for either beef or chicken, but the TLC densitometry response (uncorrected) has revealed GD1a

336

and GD1b together to be the second most abundant GA in beef, at 12% of the total GAs.13 No

337

GD1a or GD1b was detected in a chicken muscle sample by these researchers but they were

338

detected in a chicken thigh and leg muscle sample by Saito and Rosenburg36, at 15% of the total

339

GAs.

340 341

GA Molecular Species in Fish. The GM3 in fish was predominantly one molecular species, but

342

the exact molecular species differed across the four fish samples (Figures 3 and 7). The

343

dominant GM3 molecular species was d40:2 (tentatively identified as d18:1‒22:1) in turbot

344

(Colistium mudipinnis), d42:2 (tentatively identified as d18:1‒24:1) in snapper (Chrysophrys

345

auratus) and king salmon (Oncorhynchus tshawytscha), and d34:1 (d18:1‒16:0) in island

346

mackerel (Rastrelliger faughni). The island mackerel GM3 shorter FA chain is similar to that

347

observed for beef, chicken, and pork (Figure 4).


Page 15 of 37


15 348

Fish GD3 molecular species was observed to have an acyl FA composition that was

349

reflective of their GM3 counterpart, except for snapper, where d40:2 was the major GD3

350

molecular species, compared with d42:2 for GM3. In addition, turbot and snapper could contain

351

GD3 molecular species containing FAs with up to three unsaturated bonds [d43:4 (Figure 3),

352

d41:4 (Figure 7)].

353

Fish (except turbot) GD1a/b molecular species distributions were less reflective of GM3 and

354

GD3 (Figures 3 and 7). Furthermore, fish GD1a/b could also contain the molecular species d39:4

355

(d18:1‒21:3), d42:4 (d18:1‒24:3) and d43:4 (d18:1‒25:3) with highly unsaturated acyl FAs

356

(Figures 3 and 7).

357

In contrast to GD1a/b, the GT1b molecular species profiles (d-values) had an acyl FA

358

composition generally reflective of GM3 and GD3 in turbot (Figure 3) and salmon (Figure 7).

359

Snapper, on the other hand contained d-value molecular species (d47:2, d47:0, and d48:1)

360

(Figure 7) that were not observed with other GA classes. These molecular species (d47:2, d47:0,

361

and d48:1) could be tentatively identified as containing the very long chain acyl FAs d18:1‒

362

29:1, d18:1‒29:0 and d18:1‒30:0, respectively. As the fish samples were extracted with the skin

363

on, the presence of such very long chain acyl FAs cannot be ruled out given that FA chains up to

364

C30 have been reported in mammalian skin ceramides, 35 which is important for water proofing.

365

Unfortunately, in negative ion mode, it was difficult to generate secondary fragmentation of the

366

sphingosine base to determine the FA composition. GT1b was not detected in island mackerel.

367

GM2 was only detected in island mackerel, (Figure 7I), with an acyl FA composition that

368

was reflective of its GM3 and GD3 composition, and the d34:1 (d18:1‒16:0) molecular species

369

observed as the major molecular species. GM2 was not detected in any of the other fish or

370

animal muscle samples analysed in this study.

371

Unlike the beef, chicken, and pork samples, the fish GAs detected and measured in this

372

study contained only NeuAc sialic acid (Figure 3). This result is consistent with the total sialic

373

acid studies conducted by both Samraj et al.37 and Li and Fan,38 which showed that fish muscle,



Page 16 of 37

16 374

including salmon and mackerel, contained only NeuAc. However, both NeuAc and NeuGc have

375

been shown in fish liver15 so this could be tissue specific.

376 377

GA concentration in Fish. Table 1 summaries the GA concentration measured across the fish

378

samples. As with the animal samples, fish muscle contained predominantly GM3 (77‒97% of

379

total GAs,0.70‒5.86 mg/100g, Table 1), with the highest total GA concentrations measured in

380

island mackerel.

381

GD3 was present in the fish samples at low proportions of between 2 and 3% of the total

382

GAs (0.02‒0.23 mg/100g). Likewise, the total GD1a/b level in fish was also low (≤ 6% of total

383

GAs) except for turbot, at 14% of total GAs. Like the animal meat samples, GD1a was the major

384

GD1 present (Table 1). GT1b was detected only in small amounts (up to 3% of the total GAs) in

385

turbot, king salmon, and snapper with none in island mackerel. However, island mackerel was

386

the only fish sample analysed in this study that contained GM2, at 4% of the total GAs (0.24

387

mg/100g).

388 389

Molecular Species Diversity Across GA Classes. The acyl FA composition observed for the

390

GM3 molecular species for all the samples analyzed was not retained across the other GA classes

391

for their respective samples, although less so for fish. This observation was unexpected, given

392

that GM3 is the precursor for the biosynthesis of other GAs (Supporting Information Figure 2),

393

and there was an expectation that the acyl FA composition would be retained in subsequent GA

394

classes. This suggests that the glycosyl-transferase enzyme involved in each of the biosynthesis

395

pathways may be selective based on the acyl FA chain, hence resulting in a different molecular

396

species profile for each GA class. Although there is limited understanding of the reasons behind

397

the great diversity in the GA molecular species, recent research has shown that ceramides (GA

398

precursors) with specific acyl FA chain lengths have different biological functions.39 The

399

heterogeneous nature of the GAs across different animal species and the tissue-specific


Page 17 of 37


17 400

expression of GAs make quantitation of the GAs across different samples complex. However,

401

the HPLC‒high resolution MS technique based on accurate mass extract post analysis gives this

402

method great flexibility, such that prior knowledge of the different GA molecular species is not

403

required and allowing for future reanalysis of the data.

404

Although we have characterized and measured GM3, GD3, GD1a, GD1b, GT1b, and GM2 in

405

the samples, and tentatively assigned them with an acyl FA composition, based on a d18:1

406

sphingosine base, the presence of other GAs, such as O-acetylated GAs, in these samples cannot

407

be ruled out. However, they are likely to be present in low concentrations.

408 409

In conclusion, using this method, for the first time, we were able not only to profile and

410

characterize the different GA molecular species present in the beef, chicken, pork, and fish

411

samples but also to quantitate them. Given that these GAs play an important role in various

412

biological processes including brain development, it is important to have a good understanding

413

of not only the concentration but also the type of GA class and their molecular species that are

414

found in the foods consumed. This methodology will enable future studies on dietary GA intake

415

to be conducted for toddlers and young children. This is the first step in understanding what the

416

recommended daily intakes of such complex lipids should be for supporting proper growth and

417

cognitive development. On the other end of the age spectrum, GAs may also be vital for the

418

elderly population, as cognitive decline is a common occurrence. Understanding dietary GA

419

intake in this population group may also provide some insights into their cognitive degeneration.

420

In both population groups, the development of functional foods containing enriched sources of

421

complex lipids requires data studies that are similar to this study.

422

423

ABBREVIATIONS USED



Page 18 of 37

18

Cer, ceramide; CV, coefficient of variation; FA, fatty acid; GA, ganglioside; Gal, galactose;

424 425

GalNAc, N-acetylgalactosamine, Glc, glucose; LOQ, limit of quantitation; MRM, multiple

426

reaction monitoring; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid.

427

Gangliosides (where M, D, T and Q refer to 1 to 4 NeuAc attached)

428

GM4

NeuAcα2,3Gal-Cer

429

GM3

NeuAcα2,3Galß1,4Glc-Cer

430

GM2

GalNAcß1,4(NeuAcα2,3)Galß1,4Glc-Cer

431

GM1

Galß1,3GalNAcß1,4(NeuAcα2,3)Galß1,4Glc-Cer

432

GD3

NeuAcα2,8NeuAcα2,3Galß1,4Glc-Cer

433

GD2

GalNAcß1,4(NeuAcα2,8NeuAcα2,3)Galß1,4Glc-Cer

434

GD1a

NeuAcα2,3Galß1,3GalNAcß1,4(NeuAcα2,3)Galß1,4Glc-Cer

435

GD1b

Galß1,3GalNAcß1,4(NeuAcα2,8NeuAcα2,3)Galß1,4Glc-Cer

436

GT1b

NeuAcα2,3Galß1,3GalNAcß1,4(NeuAcα2,8NeuAcα2,3)Galß1,4Glc-Cer

437

GQ1b

NeuAcα2,8NeuAcα2,3Galß1,3GalNAcß1,4(NeuAcα2,8NeuAcα2,3)Galß1,4Glc-

438

Cer

439 440

AUTHOR INFORMATION *Corresponding author (Tel: +64 6 3504649; Fax: +64 6 356 1476; E-mail:

441

[email protected]),

442 443 444

ACKNOWLEDGMENTS This work was supported by the New Zealand Primary Growth Partnership post-farm

445

gate dairy programme, funded by Fonterra Co-operative Group and the New Zealand Ministry

446

for Primary Industries. The authors would like to thank Maree Luckman for statistical analysis

447

and guidance, and Claire Woodhall for editing the manuscript. The cooperation of Snigdha

448

Misra, Sangeetha Shyam and Megan Chong for the procurement and shipment of the samples

449

from Malaysia is much appreciated.

450 ACS Paragon Plus Environment

Page 19 of 37


19 451 452

SUPPORTING INFORMATION DESCRIPTION Table S1. GA accurate masses used for post analysis to generate extracted ion

453

chromatograms. Figure S1. Schematic of the biosynthetic pathway of GAs. This material is

454

available free of charge via the internet at http://pubs.acs.org.



Page 20 of 37

20 455

REFERENCES

456

1.

457

Neurochem. 1963, 10, 613-623.

458

2.

459

on neonatal brain development. Nutr Rev 2009, 67, 451-463.

460

3.

461

Huang, Y. S.; Sinclair, A. J., Eds. AOCS Press: Champaign, IL., 1998; pp 213-234.

462

4.

463

signaling: ganglioside-protein interactions or ganglioside-driven membrane organization? J.

464

Neurochem. 2013, 124, 432-435.

465

5.

466

human milks: a comparison. J. Dairy Sci. 1990, 73, 223-240.

467

6.

468

Lactational changes in concentration and distribution of ganglioside molecular species in human

469

breast milk from Chinese mothers. Lipids 2015, 50, 1145-54.

470

7.

471

P.; Fong, B. Y., Determination of ganglioside concentrations in breast milk and serum from

472

Malaysian mothers using a high performance liquid chromatography-mass spectrometry-

473

multiple reaction monitoring method. Int. Dairy J. 2015, 49, 62-71.

474

8.

475

A.; Steenhout, P.; Lee, L. Y.; Destaillats, F., Dynamics of human milk nutrient composition of

476

women from Singapore with a special focus on lipids. American journal of human biology : the

477

official journal of the Human Biology Council 2013, 25, 770-9.

Svennerholm, L., Chromatographic separation of human brain gangliosides. J.

McJarrow, P.; Schnell, N.; Jumpsen, J.; Clandinin, T., Influence of dietary gangliosides

Rueda, R.; Gil, A., Role of gangliosides in infant nutrition. In Lipids in Infant Nutrition,

Sonnino, S.; Mauri, L.; Ciampa, M. G.; Prinetti, A., Gangliosides as regulators of cell

Jensen, G.; Ferris, A. M.; Lammi-Keefe, C. J.; Henderson, A., Lipids of bovine and

Ma, L.; Liu, X.; MacGibbon, A. K. H.; Rowan, A.; McJarrow, P.; Fong, B. Y.,

Ma, L.; MacGibbon, A. K. H.; Jan Mohamed, H. J. B.; Loy, S.; Rowan, A.; McJarrow,

Thakkar, S. K.; Giuffrida, F.; Cristina, C.-H.; De Castro, C. A.; Mukherjee, R.; Tran, L.-


Page 21 of 37


21 478

9.

Giuffrida, F.; Elmelegy, I. M.; Thakkar, S. K.; Marmet, C.; Destaillats, F., Longitudinal

479

Evolution of the Concentration of Gangliosides GM3 and GD3 in Human Milk. Lipids 2014, 49,

480

997-1004.

481

10.

482

and human milk gangliosides by matrix-assisted laser desorption/ionization Fourier transform

483

ion cyclotron resonance mass spectrometry. Int J Mass Spectrom 2011, 305, 138-150.

484

11.

485

V.; Merrill, A. H., Sphingolipids in food and the emerging importance of sphingolipids to

486

nutrition. J. Nutr. 1999, 129, 1239-1250.

487

12.

488

chicken liver. J. Biochem. 1986, 100, 553-561.

489

13.

490

Interspecies comparison of muscle gangliosides by two-dimensional thin-layer chromatography.

491

J. Biochem. 1983, 94, 1359-65.

492

14.

493

Miyatake, T.; Suzuki, A.; Yamakawa, T., Gangliosides of hog skeletal muscle. Biochim.

494

Biophys. Acta. 1983, 752, 291-300.

495

15.

496

from fish to mammalian species. Comparative Biochemistry and Physiology B-Biochemistry &

497

Molecular Biology 2001, 129, 747-758.

498

16.

499

and leg muscles. J. Lipid Res. 1982, 23, 3-8.

500

17.

501

globo-series from chicken skeletal muscle. J. Lipid Res. 1991, 32, 499-506.

Lee, H.; An, H. J.; Lerno, L. A.; German, J. B.; Lebrilla, C. B., Rapid profiling of bovine

Vesper, H.; Schmelz, E. M.; Nikolova-Karakashian, M. N.; Dillehay, D. L.; Lynch, D.

Shiraishi, T.; Uda, Y., Characterization of neutral sphingolipids and gangliosides fro

Nakamura, K.; Ariga, T.; Yahagi, T.; Miyatake, T.; Suzuki, A.; Yamakawa, T.,

Nakamura, K.; Nagashima, M.; Sekine, M.; Igarashi, M.; Ariga, T.; Atsumi, T.;

Saito, M.; Kitamura, H.; Sugiyama, K., Liver gangliosides of various animals ranging

Saito, M.; Rosenberg, A., Glycolipids and their developmental patterns in chick thigh

Dasgupta, S.; Chien, J. L.; Hogan, E. L.; Vanhalbeek, H., A Disialoganglioside of the



Page 22 of 37

22 502

18.

Pham, P. H.; Duffy, T.-L.; Dmytrash, A. L.; Lien, V. W.; Thomson, A. B.; Clandinin, M.

503

T., Estimate of dietary ganglioside intake in a group of healthy Edmontonians based on selected

504

foods. J. Food Comp. Anal. 2011, 24, 1032-1037.

505

19.

506

determination of gangliosides GD3 and GM3 in bovine milk and infant formulae. Rapid

507

Commum Mass Spectrom 2006, 20, 3625-3633.

508

20.

509

disialoganglioside GD3 and monosialoganglioside GM3 in infant formulas and whey protein

510

concentrates by ultra-performance liquid chromatography/electrospray ionization tandem mass

511

spectrometry. J. Sep. Sci. 2012, 35, 937-946.

512

21.

513

Gangliosides in Bovine Milk and Colostrum-Based Dairy Products by Ultrahigh Performance

514

Liquid Chromatography-Tandem Mass Spectrometry. J. Agric. Food Chem. 2013, 61, 9689-

515

9696.

516

22.

517

ion-trap mass spectrometric analysis of GD(3) ganglioside in dairy products. Int. Dairy J. 2011,

518

21, 42-47.

519

23.

520

new liquid chromatography/tandem mass spectrometry method for quantification of gangliosides

521

in human plasma. Anal. Biochem. 2014, 455, 26-34.

522

24.

523

Mass Spectrometry for Quantitative Analysis of Gangliosides. Lipids 2009, 44, 867-874.

524

25.

525

gangliosides. J. Chromatogr. B 2014, 947, 1-7.

526

26.

527

gangliosides. Biochim. Biophys. Acta. 1980, 617, 97-109.

Sorensen, L. K., A liquid chromatography/tandem mass spectrometric approach for the

Zhang, J.; Ren, Y.; Huang, B.; Tao, B.; Pedersen, M. R.; Li, D., Determination of

Lee, H.; German, J. B.; Kjelden, R.; Lebrilla, C. B.; Barile, D., Quantitative Analysis of

Fong, B.; Norris, C.; McJarrow, P., Liquid chromatography-high-resolution electrostatic

Huang, Q. Y.; Zhou, X.; Liu, D. T.; Xin, B. Z.; Cechner, K.; Wang, H.; Zhou, A. M., A

Fong, B.; Norris, C.; Lowe, E.; McJarrow, P., Liquid Chromatography-High-Resolution

Garcia, A. D.; Chavez, J. L.; Mechref, Y., Rapid and sensitive LC-ESI-MS of

Svennerholm, L.; Fredman, P., A procedure for the quantitative isolation of brain


Page 23 of 37


23 528

27.

Domon, B.; Costello, C. E., A Systematic Nomenclature for Carbohydrate

529

Fragmentations in FAB-MS MS Spectra of Glycoconjuates. Glycoconjugate J 1988, 5, 397-409.

530

28.

531

Chelsea, Michigan 48118, 1987.

532

29.

533

Sphingolipidomics. Chemical Reviews 2011, 111, 6387-6422.

534

30.

535

High-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid

536

chromatography tandem mass spectrometry. Methods 2005, 36, 207-224.

537

31.

538

sphingolipids by liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue

539

imaging mass spectrometry (TIMS). Biochimica Et Biophysica Acta-Molecular and Cell Biology

540

of Lipids 2011, 1811, 838-853.

541

32.

542

Et Biophysica Acta-Molecular and Cell Biology of Lipids 2010, 1801, 405-414.

543

33.

544

milk fat globule membrane. Biochemical and Biophysical Research Communications 1979, 88,

545

1217-22.

546

34.

547

1964, 5, 103-8.

548

35.

549

Febs Letters 2010, 584, 1907-1913.

550

36.

551

chicken embryonic liver. J. Lipid Res. 1982, 23, 9-13.

552

37.

553

K.; Gregg, C. J.; Bingman, A. E.; Secrest, P.; Diaz, S. L.; Varki, N. M.; Varki, A., A red meat-

Taylor, K. J., Quality Assurance of Chemical Measurements. Lewis Publishers, Inc:

Merrill, A. H., Jr., Sphingolipid and Glycosphingolipid Metabolic Pathways in the Era of

Merrill, A. H.; Sullards, M. C.; Allegood, J. C.; Kelly, S.; Wang, E., Sphingolipidomics:

Sullards, M. C.; Liu, Y.; Chen, Y.; Merrill, A. H., Jr., Analysis of mammalian

Hama, H., Fatty acid 2-Hydroxylation in mammalian sphingolipid biology. Biochimica

Bouhours, J. F.; Bouhours, D., Galactosylceramide is the major cerebroside of human

Sambasivarao, K.; McCluer, R. H., Lipid components of gangliosides. J. Lipid Res.

Sandhoff, R., Very long chain sphingolipids: Tissue expression, function and synthesis.

Saito, M.; Rosenberg, A., Sialosylgalactosylceramide (GM4) is a major ganglioside in

Samraj, A. N.; Pearce, O. M. T.; Laeubli, H.; Crittenden, A. N.; Bergfeld, A. K.; Banda,



Page 24 of 37

24 554

derived glycan promotes inflammation and cancer progression. Pro Natl Acad Sci USA 2015,

555

112, 542-547.

556

38.

557

HPLC-FLD. Open Journal of Preventive Medicine 2014, 4, 57-63.

558

39.

559

ceramides. Prog Lipid Res 2012, 51, 50-62.

Li, H.; Fan, X., Quantitative analysis of sialic acids in Chinese conventional foods by

Groesch, S.; Schiffmann, S.; Geisslinger, G., Chain length-specific properties of

560

561


Page 25 of 37


25 562

FIGURE CAPTIONS

563

Figure 1. The structure of GM3 d40:2 consisting of a ceramide, with a d18:1 sphingosine base

564

and a C22:1 fatty acid, and sugar groups; glucose, galatose and sialic acid.

565

Figure 2. Extracted HPLC‒MS traces of GA calibration standards and their respective

566

molecular species profiles in negative ion mode. Molecular species are identified using their d-

567

values. The ganglioside structures are represented by symbols as follows, ceramide (Cer),

568

glucose (●), galactose (○), GalNAc (□) and NeuAc (

569

Figure 3. Extracted HPLC‒MS traces in negative ion mode of GAs (A) GM3, (B) GD3, (C)

570

GD1a/b , and (D) GT1b in turbot fish, followed by their respective mass spectra (E-H), and the

571

MS2 fragmentation spectra of the d40:2 major molecular species (I-L). Fragment ions are

572

annotated using carbohydrate fragment ion nomenclature devised by Domon and Costello.27

573

Where possible, the abbreviated sugar residue description is also provided. The NeuAc fragment

574

(B1α, B1α”) from multiple regions of GD1a/b or GT1b is labelled as B1.

).

575 576

Figure 4. Mass spectra in negative ion mode of GA [(A-C) GM3, (D-F) GD3, and (G-I) GD1]

577

molecular species profiles for (A,D,G) beef, (B,E,H) chicken, and (C,F,I) pork. The molecular

578

species were tentatively identified using their d-values (the sum of carbon atoms and the number

579

of double bonds attached to the dihydroxy sphingosine base and their fatty acid moieties). All

580

GD1a/b species were tentatively identified as containing NeuAc sialic acid except those denoted

581

by an asterisk, which also contained NeuGc sialic acid. Some molecular species were tentatively

582

identified as containing extra hydroxyl (‒OH) groups.

583

Figure 5. Examples of MS2 fragmentation (in negative ion mode) of some key GM3 and GD3

584

molecular species found in beef, chicken, and pork from Figure 1. Only identified fragments

585

have been labelled.



Page 26 of 37

26

Figure 6. MS2 fragmentations (in negative ion mode) of some key GD1a/b molecular species found in beef, chicken, and pork, showing the presence of fragments containing NeuAc and NeuGc sialic acid moieties. 586

Figure 7. Mass spectra (in negative ion mode) of (A-C) GM3, (D-F) GD3, (G-I) GD1a/b, and

587

(J,K) GT1b molecular species found in (A,D,G,J) snapper, (B,E,H,K) king salmon, and (C,F,I,L)

588

island mackerel. (L) GM2, but not GT1b, was detected in island mackerel.


Page 27 of 37


27 Table 1. GA Concentration in Measured Various Animal Meat and Fish Samples. The GA Results Determined Using Standard Addition Are Also Provided for Beef, Chicken, Pork, and Turbot Fish. sample

n

GM3 mg/100g fresh product (CV,%) 0.35 ± 0.02a (6) 0.34 ± 0.01a

GD3 mg/100g fresh product (CV,%) 0.02 ± 0.002 (10)

GD1a mg/100g fresh product (CV,%) 0.08 ± 0.01 (13)

GD1b mg/100g fresh product (CV,%) 0.04 ± 0.00

GT1b mg/100g fresh product (CV,%) ND

total GAs mg/100g fresh product (CV,%) 0.49

0.02 ± 0.003 (15)

< LOQb

< LOQb

ND

0.48

0.05 ± 0.01 (20) < LOQb

< LOQb

ND

0.95

< LOQb

ND

0.95

0.01 ± 0.00

ND

1.44

0.04 ± 0.00

0.03 ± 0.01 (33)

0.94

pork mince (10% fat)

external standard

6

(Sus scrofa domesticus)

standard addition

2

beef mince (10% fat)

external standard

6

(Bos taurus)

standard addition

4

0.46 ± 0.03a (6) 0.41 ± 0.04a

4

0.80 ± 0.05 (6)

0.1 ± 0.01 (10) 0.20 ± 0.02 (10)

beef blade steak (Bos taurus) chicken breast

external standard

6

(Gallus gallus domesticus)

standard addition

2

0.75 ± 0.04a (5) 0.79 ± 0.02a

4

1.10 ± 0.10 (9)

0.3 ± 0.02 (7)

0.72 ± 0.02a (3) 0.80 ± 0.03a

0.02 ± 0.004 (20)

chicken thigh (with skin) (Gallus gallus domesticus) turbot fish

external standard

6

(Colistium nudipinnis)

standard addition

4

0.03 ± 0.01 (33) 0.13 ± 0.02 (15)

c

island mackerel 2 5.86 ± 0.09 (2) 0.23 ± 0.01 (4) 0.38 ± 0.02 (5) 0.01 ± 0.002 ND (Rastrelliger faughni) (20) king salmon 2 1.09 ± 0.10 (9) 0.03 ± 0.002 < LOQb < LOQb < LOQb (Oncorhynchus tshawytscha) (7) snapper 2 0.70 ± 0.07 0.02 ± 0.003 0.01 ± 0.003 0.03 ± 0.003 < LOQb (Chrysophrys auratus) (10) (15) (30) (10) a Superscript letters denote that paired t-test results between the external standard and standard addition were not significantly different (p > 0.05). b

c

GA levels above the limit of detection (0.004 mg/100g fresh product) but below the limit of quantitation (LOQ).

GM2 was measured at 0.24 ± 0.04 mg/100g.

ND: not detected.


6.48 1.12 0.76


Page 28 of 37

28

Table 2. Recovery (%) of GA after Spiking into the Sample. sample pork mince (10% fat) beef prime mince (10% fat) chicken breast turbot fish (Colistium nudipinnis)

n 3 4 3 4

GM3 103 ± 3 101 ± 3 104 ± 9 102 ± 6

GD3 99 ± 8 94 ± 10 93 ± 3 101 ± 4

GD1a 103 ± 4 100 ± 4 101 ± 6 106 ± 3

GD1b 92 ± 4 91 ± 4 92 ± 1 100 ± 5


GT1b 94 ± 4 93 ± 6 90 ± 6 99 ± 2

Page 29 of 37


29

Ceramide

Glucose

Figure 1


Galactose

Sialic acid

5e+3

0 1e+5

GM4 0

5 10 3e+4

4e+4

2e+4

0 8e+5

GD1a 6e+4

4e+5

2e+5

0 8e+5 GD3 0 5e+4

6e+5 4e+4

4e+5 3e+4

2e+5 2e+4

0 6e+5 GT 1b

0 4e+5

3e+5 GQ1b

2e+5 8e+3

1e+5 4e+3

0 15

Time (min) 20 25 890

1e+4

4e+5

2e+5 2e+4

8e+3

0

790 800

2e+4

1e+4

1040

900

2e+4 720 740

1040 1060

810

1150

0

GD1b 1080

4e+4

2e+4

910 920

760

1080

0 820 830

m/z

Figure 2


1249.8120 (d41:1)

1550

1200

1120

930

1e+4

1200

1277.8440 (d43:1)

1400 1263.8270 (d42:1)

1410.8419 (d38:1)

1500

1222.58621 (d38:1) 2[M-2H]

1e+5 1221.7822 (d39:1)

1572.8929 (d38:1)

1544.8620 (d36:1)

2e+3

1235.7970 (d40:1)

5000

775.9484 (d42:2) 776.9564 (d42:1) 783.9677 (d43:1)

1e+4

931.4898 (d38:1)

2e+5

1208.5711 (d36:1) 2[M-2H]

6e+5

4e+3

1117.7704 (d43:0)

15000

769.9472 (d41:1)

3e+5

1209.7799 (d38:0)

1350

1115.7523 (d43:1)

8e+3

1382.8103 (d36:1)

2e+4

762.9399 (d40:1)

8e+4 2e+4 1179.7336 (d36:1)

4e+4

1207.7659 (d38:1)

6e+4

1077.0374 (d38:1)

GM3

1089.7437 (d41:0)

1450

1103.7576 (d42:0)

0 5e+5

917.4746 (d36:1)

4e+5 2e+4

749.9340 (d38:0) 755.9326 (d39:1)

2e+4

748.9259 (d38:1)

GM2

1063.0223 (d36:1)

8e+4

814.7186 (d38:1) 3[M-3H]

0 1e+5

1151.7030 (d34:1)

10000

734.9095 (d36:1)

GM 1

720.8947 (d34:1)

6e+4

Intensity

20000

805.3758 (d36:1) 3[M-3H]

Intensity

Journal of Agricultural and Food Chemistry Page 30 of 37

30

25000

0 1600

2e+4 0 1450 1500

1250 1300

1160 1200

940

1225

950

0 780 800

1100 1120

1250

3e+3

5

2e+3

10

C 5e+4

GD1a

GD1b

0

15 Time (min) 20

20 25

D GT1b

1e+3

25

4e+3 4e+4

3e+4

2e+4

1e+4

860

6e+3

4e+3

2e+3

1020 880

0 1040 1060

6e+4

G

900

1e+4

8e+3

H

750

920

760 770

940 960

0 0

1080 1100

780

980

1120

790

1140

800

0

768.9333 (d41:2 770.9308 (d41:0) 775.9406 (d42:2) 780.9328 (d43:4) 784.9454 (d43:0)

Intensity 250

200

K

150

100 50

1000 180

160

140

L

120

100 80

60

40

20

200

200

m/z

0

Figure 3


400

0 400

500

600

600

800

1000 m/z

800

1000

1500

1304.1771 (0, 2 X3 )

1233.5164 (Y1 )

942.35693 (Y2 )

780.5163 (Y1 )

762.0204 [M-2H]2-

618.5025 (Y0 )

-

581.1588 [(NeuAc-NeuAc)-H20-H]

290.0130 [NeuAc-H20-H]- (B1)

0 1000

1200

1589.9156 [M-NeuAc-H]-

740 1000

1307.5948 [M-2NeuAc-H]-

730 2000

1126.7465 (Gal-GalNAc-GalNeuAc-NeuAc-H)

720

J

944.4088 [M-2H]2-

710

4000

1248.7855 (d41:2) 1251.7803 (d40:0) 1261.8003 (d42:2) 1271.7846 (d43:3) 1279.8106 (d43:0)

-

762.6729 (Y1-H20) 780.5163 (Y1 )

1000

1400

1215.3685 [M-H20-H]1233.5936 [M-H]-

1189.4852 (1,3 X1)

1012.0654 (0, 2X1)

942.3974 (Y2)

618.5025 (Y0 )

290.0130 [NeuAc-H20-H] (B1)

800

1890.3517 [M-NeuAc-H]-

6e+3 0 600

1598.5676 [M-2NeuAc-H]-

8e+3 700

3000

400

833.8568 (Glc-Gal-NeuAc-NeuAc)

0

200

1378.9830

-

1e+4

0

618.5025 (Y0 )

2e+4

1300


4e+4

500

1307.9830 [M-3NeuAc-H]- (Y3a')

15 25

F 1250

1000

944.5958 [M-NeuAc-2H]2-

-

GD3

I


3e+4

1200

290.0330 [NeuAc-H20-H]- (B1)

1e+3 1150 1500

511.3790 (NeuAc-GalNac)

10 20

1100

951.4966 (41:2) 958.5049 (d42:2) 963.4968 (d43:4)

0

2000

364.2633

2e+3 15

E 1219.7582 (d39:2) 1233.7710(d40:2)

4e+4

290.0530 [NeuAc-H20-H]- (B1)

5 5e+4

1205.7417(d38:2)

0

754.9180 (d39:2) 761.9270 (d40:2)

5e+4

1104.0524 (d42:2)

10 25

747.9118 (d38:2)

2e+3 20

1097.0457 (d41:2)

5 15

1077.0298 (d38:1) 1084.0399 (d39:1) 1090.0372 (d40:2)

10

903.4514 (d34:1)

5e+4

917.4668 (d36:1) 922.4596 (d37:3) 931.4813 (d38:1) 938.4886 (d39:1) 944.4906 (d40:2)

1e+4 1123.6632 (d32:1)

1e+5

1063.0145 (d36:1) 11068.0058(d37:3)

2e+4

1048.9982 (d34:1)

B 2e+5

720.8876 (d34:1)

3e+4

706.8729 (d32:1)

GM3

713.3432(d33:1)

5 2e+5

875.4206(d30:1) 882.4287 (d31:1) 889.4364 (d32:1)

0 Intensity

A

1034.9802 (d32:1)

Intensity

Page 31 of 37 Journal of Agricultural and Food Chemistry

31

1200

1200 1400

1600

2000

Intensity 2e+3

880

G

2e+3

1e+3

3e+3

5e+2

900 920 940 m/z 960 980

0 1000 6e+3

4e+3

2e+3

880 900

8e+3

H

920 940 m/z 960

Figure 4.


2e+3

F

0 780

0 0

980 1000

1e+3

800 700

4e+3

3e+3

I

2e+3

1e+3

880

5e+2

720

900 920

2e+3

740

940 m/z 960

1200

760

776.9555 (d42:1)

775.9405 (d42:2)

1150 762.9329 (d40:1)

748.9177 (d34:1)

1100

734.9020 (d36:1)

720.8864(d34:1)

1300

984.5042 (d42:0)* (+2 OH- )

Intensity

1179.7236 (d 36:1)

1151.6928 (d 34:1)

Intensity

1277.7953 (d 43:1)

1261.8014 (d 42:2)

1235.7865 (d 40:1)

1207.7548 (d 38:1)

1263.8134 (d42:1)

1235.7855 (d40.1)

1207.7549 (d38:1)

1195.7212 (d37:0)

1179.7228 (d36:1)

1151.6939 (d34:1)

2e+3

975.5103(d43:0)*

760

783.9372 (d 43:1)

755.9413 (d 42:2)

762.9344 (d 40:1)

8e+3 4e+3

966.5125 (d42:2)*

740

1250 6e+3

954.5034 (d40)* (+ OH-) 958.5150 (d42:2)

720

748.9189 (d 38:1)

0

C

939.4900 (d38:1)* 945.5055 (d40:1)

700

734.9027 (d 36:1)

1e+4

8e+3

925.4748 (d35:2)* 931.4899 (d38:1)

800 2e+4

720.8879 (d 34:1)

Intensity

1263.8160 (d42:1)

1249.8003 (d41:1)

1235.8015 (d40:1)

1221.7835 (d39:1)

1207.7552 (d38:1)

1195.7177 (d37:0)

1165.7112 (d35:1) 1179.7244 (d36:1)

1e+5

903.4505 (d34:1) 911.4588 (d35:0) 917.4762 (d36:1)

790

E 1200

Intensity

Intensity 3e+4

1150

996.5271 (d 44:2)* (+2 OH-) 998.4935 (d 44:0) (+3 OH-)

780

1100

984.4960 (d 42:0)* (+2 OH-)

770

1300

968.5096 (d 41:1)* (+ OH-) 970.4793 (d 40:0)* (+2 OH-)

760

2e+4

958.5052 (d 42:2)

0 4e+4

954.4853 (d 40:0), (+ OH-)

750 1250

6e+4

940.4696 (d 38:0)*

740

8e+4

931.4882 (d 38:1)

730 776.9478 (d42:1)

0

B

917.4733 (d 36:1)

720

1e+5

903.9516 (d 34:1)

710

Intensity

2e+3

-

4e+3 1200

984.4961 (d42:0)* (+2 OH )

6e+3

762.9334 (d40:1)

1150

-

2e+4

962.4841 (d40:0)* (+ OH )

4e+4

1151.6950 (d34:1)

6e+4

-

1100

734.9020 (d36:1)

8e+4

954.4867(d40:0) (+ OH )

D 720.8874 (d34:1)

A

925.4639 (d36:1)*

700

903.4523(d34:1)

intensity 1e+5

911.4483(d35:0) 917.4673 (d36:1)

Intensity


32

1e+4

0 1250 1300

0 780

980

800

1000

Intensity 80

60

40

20

200 400 600 800

0 1000 1200 400

200

1200

2 D. MS of GM3 m/z 1249.7 (d41:1) from beef 140 200 400 600 800 1000 2 F. MS of GD3 m/z 762.8 (d40:1) from beef

120

100

80

60

40

20

200 400

m/z

Figure 5.

ACS Paragon Plus Environment 600 800

m/z 1000

-

600

1235.7495 [M-NeuAc-H] (Y3)

800

-

1600 -

1179.7722 [M-NeuAc-H] (Y3)

888.4603 [M-2NeuAc-H] (Y2)

-

735.0123 [M-2H]

2-

1800

944.5053 [M-2NeuAc-H] (Y2)

1000

2-

1200

-

-

-

1151.7078 [M-NeuAc-H] (Y3)

-

2-

860.5734 [M-2NeuAc-H] (Y2)

721.0252 [M-2H]

581.0749 [2NeuAc-H]

536.7307 (Y0)

20

762.8301 [M-2H]

1400 581.1108 [2NeuAc-H]

1200

290.1490 [NeuAc-H]

40

732.3531

0 60

-

0 80

290.1490 [NeuAc-H]

Intensity

100

-

-

1179.7789 [M-H]

120

-

1000 Intensity

-

140

581.1428 [2NeuAc-H] 620.3694 (Y0)

800 1151.4955 [M-H]

888.6679 [M-NeuAc-H] ( Y2)

726.8030 [M-Hex-NeuAc-H] (Y1)

564.6516 [M-2Hex-NeuAc-H] (Y0)

336.5716

-

2 A. MS of GM3 m/z 1179.8 (d36:1) from pork

290.1257 [NeuAc-H]

600

-

400 -

200 400 600 800 1000 2 C. MS of GM3 m/z 1151.5 (d34:1) from chicken

Intensity

200

-

200 860.4360 [M-NeuAc-H] ( Y2)

1200

1249.6851 [M-H]

400

-

600

958.6159 [M-NeuAc-H] ( Y2)

800

698.3844 [M-Hex-NeuAc-H] (Y1)

10

536.4629 [M-2hex-NeuAc-H] (Y0)

20

408.1899

30

290.0682 [NeuAc-H] (B1)

40

778.7771 [M-Hex-NeuAc-H20-H] (Y1)

100 -

1000

290.0282 [NeuAc-H] (B1)

50

634.6345 [M-2Hex-NeuAc-H] (Y0)

0

-

Intensity 60

290.1127 [NeuAc-H] (B1)

Intensity


33

2 B. MS of GD3 m/z 721.0 (d34:1) from pork

0 200 400 600 800 1000 2 D. MS of GD3 m/z 735.0 (d36:1) from chicken 1200

0 1200

1200

1400

0 1400

200

150

100

50

0 200 400

250

600

0 800 1000 1200 1400 1600 m/z 50

30

20

10

0 200 400 600

40

800 1000

747.4907

1200

1200 1400

Intensity

1336.5126 (Y2α')

970.3803 (Y2β - B1α')

0

1600

40

20

1400

D. MS2 for GD1b m/z 925.55 (d36:1) from beef d 7

0

0 200

200

6

5

4

3

2

1 400

m/z

Figure 6.


400

600

600

800

800

m/z

1677.6537 [M-NeuAc-2H]

1662.9182 [M-NeuGc-H]

1371.1302 [M-(NeuAc+NeuGc)-H]

0

2984.8369 [M-2H] 1122.4702

655.1985 (NeuAc-Gal-GalNAc) 725.2111753.0759 831.4360

306.0586 (NeuGc)

290.0526 (NeuAc)

80

-

1000

2 E. MS for GD1a m/z 984.5 (d42:0) beef

e

1678.8748 [M-NeuAc-H]

800

60

2 F. MSf for GD1b m/z 984.5 (d42:0) beef 902.4890 951.7296 2984.4490 [M-2H] 1030.8717

600

896.4900 940.4160 [M-2H-H20]2958.5100 [M-2H]2-

100

615.7296 (NeuAc+NeuGc+H20)

400

597.2180 (NeuAc+NeuGc)

200

563.4926 (B1α' - H 0) 2 580.9960 (B1α')

C. MS2 for GD1b m/z 958.5 (d42:2) from pork

c

Intensity

0

1398.9877 (Y3β − NeuAc)

1200

365.2612 (Gal-GalNAc)

290.1149 (NeuAc)

10

1542.5532 (Y2α'' -H20) 1561.6600 (Y2α'')

1253.3721 (Y2β ) Intensity

1181.9064

20

1253.8434 (Y2α)

B. MS2 for GD1a m/z 998.5 (d47:5) from Chicken b 30

925.3836 [M-2H]2-

1000

40

705.4937

800

50

597.0471 (B1α')

0

306.1629 (B1α'')

600

Intensity

400 936.4963 [M-2H-H20]2954.4929 [M-2H]2-

25

1706.9444 [M-NeuAc-H]-

200 709.0954 726.0886 (Y1-2H) 729.2063 (Y1) 790.3869

A. MS2 for GD1a m/z 954.5 (d40:0) from chicken

a

1483.8480 1544.9579 (Y3β )

5

1050.8145 (Y2β − NeuAC)

10

888.3132 (Y1) 954.3987 976.8137 998.6851 [M-2H]2-

15

290.2999 (NeuAc)

20

589.5417

Intensity 30

290.1656 (NeuAc)

300

187.1945

Intensity


34

1000 1200 1400 1600

1000 1200 1400 1600 1800 2000

Intensity 5e+2

1020

3e+2

2e+2

0

1040 1060 1080 940

1100

m/z 960

6e+2

J

4e+2

3e+2

1120 980

2e+2

1e+2

0 1140 1160 1180 5e+3

1000

1020

1e+4

8e+3

6e+3

720

4e+3

H

3e+3

920 940

1040

740

2e+3

1060

760

960

2e+3

K

1e+3

1080

780

980 1000

5e+2

0 1100 1120 1140

Intensity

4e+3

0

6e+3

3e+3

F

2e+3

800 700

1e+4

1020 840

2e+3

1e+3

8e+2

4e+2

m/z 1300

Figure 7.

ACS Paragon Plus Environment 860

734.9089 (d36:1)

1e+3

720

880

1350

4e+3

2e+3

740

I

9e+3

6e+3

2e+3 900 920

L

1400 m/z

940

775.9476 (d42:2)

1200

769.8797(d41:1)

1150

761.9319(d40:2) 762.9381 (d40:1)

739.9001(d37:3)

1e+4 720.8928 (d34:1)

1100

943.4044 (d39:4)

E 1300

916.4761 (d35:3)

700

1250

Intensity

1261.8010 (d42:2)

Intensity

1271.7871 (d43:4) 1279.8114 (d43:0)

760

960

1450

1261.8115 (d42:2)

1249.8129 (d41:1)

1233.7808 (d40:2) 1235.7963 (d40:1)

1189.7185 (d37:3)

1179.7334 (d36:1)

1151.7025 (d34:1)

1123.6700 (d32:1)

2e+3

1482.9002 (d43:0)

G 800

1200 4e+3

1464.8904 (d42:2) 1466.9030 (d42:1)

4e+2 780

0

6e+3

1436.8579 (d40:2) 1438.8738 (d40:1)

6e+2 5e+3

C

1392.7975 (d37:3)

1e+3

0 920 760

1e+4

8e+3

1354.7798 (d34:1)

1e+2

900 740

2e+4

1326.7529 (d32:1)

0 1150

Intensity

720 1e+4 1104.5553 (d42:2)

6e+3

B

Intensity

1e+3 1100

979.5418(d45:2)

2e+3 1300

958.5046 (d42:2)

3e+3 1250

Intensity

0

963.4976 (d43:4)

4e+3 2e+4

1104.5553 (d42:2)

880 1200 1261.8067 (d42:2)

A

Intensity

700 1233.7747 (d40:2)

1e+4

Intensity

1150

790.9540 (d44:1)

1100

1141.5778 (d47:0) 1147.0700 (d48:1)

5e+2

D 761.9477d40:2)

2e+3 1151.7000 (d34:1)

4e+3

766.9401 (d41:4)

Intensity 6e+3

958.5095 (d42:2) 963.5026 (d43:4)

5e+3

956.4958 (d42:4)

Intensity 8e+3

1139.0653 (d47:2)

Intensity


35

1e+4

0 1250 1300

0

2e+4

780 800

3e+3

0 980 1000

0 1500


Page 36 of 37

36

For Table of Contents Only


Page 37 of 37


80x45mm (298 x 298 DPI)


Ganglioside Composition in Beef, Chicken, Pork, and Fish Determined

Recommend Documents