hexanoic Acid - ACS Publications - American Chemical Society

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Identification and Quantitation of the Lipation product 2-Amino-6(3-methylpyridin-1-ium-1-yl)hexanoic acid (MP-lysine) in Peanuts Martin Globisch, Meike Deuber, and Thomas Henle J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03371 • Publication Date (Web): 06 Aug 2016 Downloaded from http://pubs.acs.org on August 6, 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 35

Journal of Agricultural and Food Chemistry

Identification and Quantitation of the Lipation product 2-Amino6-(3-methylpyridin-1-ium-1-yl)hexanoic

acid

(MP-lysine)

Peanuts

Martin Globisch, Meike Deuber and Thomas Henle*

Institute of Food Chemistry, Technische Universität Dresden, D-01062 Dresden, Germany

*

Corresponding author:

T. Henle Phone: +49-351-463-34647 Fax: +49-351-463-34138 E-mail: [email protected]

1 ACS Paragon Plus Environment

in


1

Abstract

2

The lipid peroxidation product acrolein was semi-quantitated by GC-MS (EI) in unheated and

3

heated peanut oil, respectively, representing a model system for peanut roasting. Depending

4

on the heating time, acrolein levels significantly increased from 0.2 to 10.7 mg/kg oil. As a

5

result of heating Nα-acetyl-L-lysine and acrolein, the pyridinium derivative 2-acetamido-6-(3-

6

methylpyridin-1-ium-1-yl)hexanoic acid (MP-acetyl lysine) was identified. In addition, the

7

lysine derivative 2-amino-6-[5-(hydroxymethyl)-3,6-dihydro-2H-pyridin-1-yl]hexanoic acid

8

was identified after reduction and hydrolysis. After preparation of 2-amino-6-(3-

9

methylpyridin-1-ium-1-yl)hexanoic acid (MP-lysine) as reference material, its amounts were

10

quantitated in acrolein-modified peanut proteins by HPLC-ESI-MS/MS after acid hydrolysis,

11

showing that at low acrolein concentrations, the modification of lysine could be entirely

12

explained by the formation of MP-lysine. Furthermore, for the first time, MP-lysine was

13

quantitated in peanut samples in amounts up to 10.2 mg/kg, showing an increase depending

14

on the roasting time. Thus, MP-lysine might represent a marker to evaluate the extent of food

15

protein lipation by acrolein.

16 17 18

KEYWORDS:

19

peanuts, peanut oil, lipid peroxidation, lipation, acrolein, MP-lysine

20 21


Page 2 of 35

Page 3 of 35


22

INTRODUCTION

23

Roasting of peanuts causes numerous chemical reactions, among which, as a consequence, up

24

to 42% of the essential amino acid lysine is modified.1 Only 10% of this roasting-induced

25

modification could be explained by Maillard reaction products.1 Due to its high amount of fat

26

(48%)2 and unsaturated fatty acids (85%)3, peanut roasting leads to lipid peroxidation

27

reactions, resulting in the formation of highly reactive secondary reaction products.4-6

28

Depending on their structure, these secondary reaction products are able to modify

29

nucleophilic amino acid side chains.5-7 In analogy to glycation reactions between reducing

30

carbohydrates and amino compounds (also referred to as Maillard reaction), the term

31

“lipation” or “lipation reaction” was suggested for the modification of amino acid side chains

32

by lipid peroxidation products.5 Among the secondary products of lipid peroxidation, acrolein

33

represents one of the strongest electrophilic compounds with high reactivity towards

34

nucleophiles.7 In food, formation of acrolein can occur amongst others from glucose via the

35

intermediates deoxyglucosone, hydroxyacetone and 2-hydroxypropanal, from free amino

36

acids methionine and threonine via Strecker degradation as well as from lipids.8 In peanuts,

37

the amounts of free glucose range from 0.01 to 0.08 g/100 g peanut and therefore are a

38

negligible source for acrolein.9 Regarding lipids, linoleic, arachidonic and linolenic acid as

39

well as glycerol are possible precursors for the formation of acrolein.8,10,11 Concerning the

40

amounts of free acrolein in foods, only few data are available. In heated oils (180 °C for 2 h)

41

from coconut, olive, rapeseed, safflower and linseed, 20, 211, 1891, 448 and 1516 µmol

42

acrolein per kg oil were found, respectively.12 In fresh peanut oil, no acrolein was detectable

43

whereas after heating at 145 °C for 2 h, 2.7 µmol/L were quantitated.13 In fruits and

44

vegetables amounts up to 0.9 and 10.5 µmol/kg were found, respectively.14 Alcoholic

45

beverages contain acrolein ranging from below 0.5 to 198.0 µmol/L14-16 and commercial deep

46

fried potato chips, french fries and donuts about 0.3 µmol/kg, each.11 Due to its α,β-



Page 4 of 35

47

unsaturated character, nucleophilic attacks of acrolein can occur at the C1 and C3 of the

48

molecule. A nucleophilic attack of amines at C1 leads to Schiff’ bases that can undergo a

49

Michael reaction involving another molecule of acrolein, followed by oxidation steps and

50

cyclization to an Nɛ-(3-methylpyridinium)derivative (MP-lysine, compound (A) in

51

Figure 1).17 After a Michael reaction of the ɛ-amino group of lysine at C3, a propanal adduct is

52

formed that can undergo a further Michael reaction involving another molecule of acrolein

53

and a subsequent Aldol condensation to form Nɛ-(3-formyl-3,4-dehydropiperidino)lysine

54

(FDP-lysine, compound (B) in Figure 1).10 Following another Michael reaction involving

55

thiols, the corresponding thioether can be formed.18 After incubation of bovine serum albumin

56

and acrolein under physiological conditions (37 °C, 24 h, pH 7.2), 49% and 23% of the loss of

57

lysine could be explained by the formation of FDP- and MP-lysine, respectively.17 In brain

58

samples

59

immunochemically, whereas control brain samples showed no immunoreactivity.19 In oxLDL,

60

0.06 mol MP-lysine/mol LDL were detectable by means of HPLC-ESI-MS/MS using a stable

61

isotope dilution assay.20

62

To the best of our knowledge, no data concerning the amounts of free acrolein in peanut oil

63

heated under peanut roasting conditions as well as no qualitative or quantitative data of

64

acrolein lipation products in food and especially in peanuts are available. The aim of this

65

study, therefore, was to develop methods to semi-quantitate free acrolein in peanut oil by

66

GC-MS (EI) and its lipation product MP-lysine in peanuts by HPLC-ESI-MS/MS. To identify

67

reactive amino acids within the peanut proteins, a raw peanut protein extract was incubated

68

with acrolein and modifications of amino acids were quantitated by amino acid analysis.

69

Possible lipation products between acrolein and the ɛ-amino group of lysine were identified

70

after incubation of Nα-acetyl-L-lysine and acrolein by collision-induced dissociation (CID)

of

patients

with

Alzheimer’s

disease,

FDP-derivatives


were

detected

Page 5 of 35


71

experiments. Following isolation, MP-lysine was quantitated in peanuts by HPLC-ESI-

72

MS/MS for the first time.

73 74

MATERIALS AND METHODS

75

Materials. Pepsin (EC 3.4.23.1), Pronase E (EC 3.4.24.4), sodium borohydride, methanol and

76

hydrochloric acid (37%) were obtained from Merck (Darmstadt, Germany). Leucine

77

aminopeptidase (EC 3.4.11.1), Prolidase (EC 3.4.13.9), dialysis tubing cellulose membrane

78

(14 kDa MWCO), tetradeuteromethanol, petroleum ether (boiling point 40-60 °C), (E)-but-2-

79

enal, butylated hydroxytoluene, acrolein, nonafluoropentanoic acid and o-(2,3,4,5,6-

80

pentafluorobenzyl)hydroxylamine

81

(Taufkirchen, Germany). Nα-Acetyl-L-lysine and N-benzoylglycyl-L-phenylalanine were

82

obtained from Bachem (Bubendorf, Switzerland). Heptafluorobutyric acid, acetic acid glacial

83

and hexane were obtained from VWR (Darmstadt). Acetonitril was obtained from Fisher

84

Scientific (Schwerte, Germany). Monosodium phosphate, disodium phosphate, sodium

85

sulfate, sodium tetraborate and sodium hydroxide were obtained from Grüssing (Filsum,

86

Germany). Tris-(hydroxymethyl)aminomethane was obtained from Serva (Heidelberg,

87

Germany). Thymol was obtained from Carl Roth (Karlsruhe, Germany). All chemicals were

88

of the highest purity, except otherwise indicated. For all experiments, ultrapure water was

89

used, prepared by an ELGA LabWater Purelab Plus water system (Celle, Germany). For

90

HPLC-ESI-MS/MS measurements, double-distilled water, prepared in the presence of

91

potassium permanganate, was used. Refined peanut oil and roasted commercial peanuts were

92

obtained from a local market. Raw peanuts were obtained from Veggies Delight (Düsseldorf,

93

Germany).

hydrochlorid

were

obtained

94


from

Sigma-Aldrich


95

Preparation of Samples. Raw peanuts with shell were roasted in a laboratory oven ULM 500

96

(Memmert, Schwabach, Germany) for 20 and 40 min at 170 °C, respectively. Afterwards, the

97

samples were shelled, skinned and crushed, using a kitchen machine. Commercially available

98

roasted peanuts were crushed analogously. Protein contents were analyzed by the Kjeldahl

99

method using the factor 5.3 for oilseeds.21 50 g of refined peanut oil were heated in 250 mL

100

round-bottom flasks for 20 and 40 min at 170 °C, respectively.

101 102

Nuclear Magnetic Resonance Spectroscopy (NMR). For 2-amino-6-(3-methylpyridin-1-

103

ium-1-yl)hexanoic acid (MP-lysine), 1H and 13C NMR spectra were recorded using a Bruker

104

Avance 400 instrument (Rheinstetten, Germany) at 400.1 MHz and 100.6 MHz, respectively.

105

Tetradeuteromethanol (MeOH-d4) was used as solvent. All chemical shifts are given in parts

106

per million (ppm) relative to the solvent signal serving as internal standard. The following

107

two-dimensional NMR experiments were performed additionally: correlation spectroscopy

108

(COSY), heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond

109

correlation (HMBC).

110 111

Elemental Analysis. Elemental analysis was performed using an EuroEA3000 (Eurovector,

112

Milan, Italy) to quantitate the product content in the MP-lysine standard. MP-lysine was

113

isolated as salt of nonafluoropentanoic acid and acetic acid, so the molar ratio of cationic

114

pyridinium derivative and acid in the isolated product was unknown. Therefore, the analyzed

115

percentage of nitrogen was compared to the calculated nitrogen percentage. The content of

116

MP-lysine cation is expressed in percent by weight.

117


Page 6 of 35

Page 7 of 35


118

Acid and Enzymatic Hydrolysis. For acid hydrolysis, a ratio of 4 mg protein per 1.0 mL 6 M

119

hydrochloric acid was used. Samples were hydrolyzed under nitrogen for 23 h at 110 °C. For

120

enzymatic hydrolysis the method published elsewhere was used.5,22

121 122

Amino Acid Analysis. Amino acid analyses were performed using a SYKAM S4300 amino

123

acid analyzer (Fürstenfeldbruck, Germany). Amino acids were separated by cationic ion

124

exchange chromatography, using lithium citrate buffer, derivatized by ninhydrin, and detected

125

using a wavelength of 570 nm.23 The amino acids were expressed as valine equivalents

126

referring to the naturally occurring amounts of valine of each sample.

127 128

Semi-Quantitation of Free Acrolein in Peanut Oil by GC-MS (EI). 1.0 g of peanut oil

129

samples and 0.02 mL (48 nmol) of the internal standard (E)-but-2-enal in water were

130

homogenized by vortexing for 1 min. Then 2.0 mL of water and 0.15 mL of a butylated

131

hydroxytoluene solution (30 mg/mL hexane) were added and homogenized for another

132

minute. After centrifugation for 5 min, 5,000 x g and 4 °C, the supernatant was collected, the

133

extraction was repeated analogously and the supernatants were combined and filtrated

134

(Whatman 595 ½). For derivatization (see Fig. I in the Supporting Information), 0.3 mL of a

135

o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochlorid (PFBHA-HCl) solution (20

136

mg/mL water) were added and the samples were incubated for 1 h at 37 °C following

137

homogenization. After adding 1.0 mL of hexane and vortexing for 1 min, the samples were

138

centrifuged for 10 min, 10,000 x g and 4 °C. The supernatant was collected and the extraction

139

was repeated twice. The combined supernatants were dried by evaporation at 35 °C using

140

nitrogen, re-dissolved in 0.5 mL hexane and dried by adding anhydrous sodium sulfate. After

141

filtration (0.45 µm), an aliquot was used for GC-MS (EI) analysis using an Agilent 7890A

142

system, consisting of a 7683 series injector with a sample tray and a 5975C MS detector



143

working in EI mode, all from Agilent Technologies (Böblingen, Germany) and a ZB-5

144

guardian capillary column (30.0 m + 5.0 m guard column, inner diameter = 0.25 mm, 0.25 µm

145

film thickness) from Phenomenex (Torrance, CA). Helium was used as carrier gas with a

146

constant flow of 1.0 mL/min. With the injector temperature set to 250 °C, 1 µL of sample was

147

injected using the pulsed splitless mode. The auxiliary, ion source and quadrupole

148

temperatures were set to 250, 230 and 150 °C, respectively. The initial oven temperature was

149

set to 50 °C and held for 3 min, then raised at 20 °C/min to 300 °C. The post run time was set

150

to 3 min at 300 °C. The mass spectrometer was working in electron impact mode at 70 eV.

151

The solvent delay was set to 8 min. SIM parameters were as follows: for acrolein (anti and

152

syn), quantifier ion m/z 181 and qualifier ion m/z 251, and for but-2-enal (anti and syn),

153

quantifier ion m/z 181 and qualifier ion m/z 265. The dwell time was set to 100 ms.

154

Calibration was realized by using a commercially available acrolein standard prepared

155

analogously to the samples. But-2-enal is a naturally occurring reaction product of lipid

156

peroxidation, ranging in amounts of 1-2% of the added amount of internal standard but-2-

157

enal. Thus, every sample was measured without the addition of the internal standard (blank)

158

and samples with internal standard were blank-corrected. The limits of detection (LOD) and

159

quantitation (LOQ) in the peanut oil were estimated from the signal-to-noise ratio to 0.26

160

µmol/kg oil and 0.78 µmol/kg oil, respectively. Results are expressed as mean values ±

161

standard deviations of two separate measurements.24

162 163

Incubation of Peanut Proteins with Acrolein. Peanut proteins were isolated from raw

164

peanuts as described elsewhere.6 Approximately 50 mg of the raw peanut protein extract were

165

dissolved in 25 mL of 0.1 M phosphate buffer (pH 7.4) and acrolein was added in following

166

molar ratios related to the sum of relevant reactive amino acids cysteine, histidine, lysine and

167

arginine5: 0.1:1, 0.2:1, 0.5:1, 1:1 and 5:1. Additionally, a blank sample consisting of peanut


Page 8 of 35

Page 9 of 35


168

proteins without acrolein was prepared. The incubation was carried out under nitrogen for

169

24 h at 37 °C while stirring, followed by dialysis (14 kDa MWCO) against deionized water

170

for 24 h at 6 °C. Afterwards, the samples were lyophilized and protein contents were analyzed

171

by the Kjeldahl method using the nitrogen-to-protein calculation factor of 5.3 for oilseeds.21

172

For analysis of amino acid modification rates of cysteine, lysine, and arginine, the samples

173

were hydrolyzed enzymatically and for histidine an acid hydrolysis was performed because of

174

coelutions after enzymatic hydrolysis. For analysis of MP-lysine, 2 mg of the samples were

175

hydrolyzed by 4 mL 6 M hydrochloric acid for 23 h at 110 °C. 0.5 mL of the hydrolyzates

176

were dried at 37 °C in vacuo using a SpeedVac vacuum concentrator (Thermo Fisher

177

Scientific, Waltham, MA), redissolved in 0.13 mL 10 mM nonafluoropentanoic acid in water,

178

0.15 mL 10 mM nonafluoropentanoic acid in acetonitrile and 0.02 mL (3.0 pmol) of internal

179

standard N-benzoylglycyl-L-phenylalanine in methanol:water (50:50, v/v). After filtration

180

(0.45 µmol), samples were subjected to HPLC-ESI-MS/MS analysis. Quantitation was

181

realized using authentic MP-lysine and 3.0 pmol internal standard N-benzoylglycyl-L-

182

phenylalanine in an acid hydrolyzate matrix calibration of the raw peanut protein extract,

183

prepared analogously to the samples.

184 185

Incubation of Nα-Acetyl-L-lysine with Acrolein. For identification of major amino acid side

186

chain reaction products formed between acrolein and Nα-acetyl-L-lysine, 100 mM acrolein

187

and 100 mM Nα-acetyl-L-lysine were incubated for 24 h at 37 °C in 5 mL 50 mM phosphate

188

buffer (pH 7.2) while stirring.10 One mL of the reaction mixture was taken for direct HPLC-

189

ESI-MS/MS analysis. For reduction, 1.0 mL of the reaction mixture was taken and 3.0 mL of

190

a 0.2 M sodium tetraborate solution (pH 9.5) and 2.0 mL of a 1.0 M sodium borohydride

191

solution in 0.1 M sodium hydroxide were added. After reduction for 17 h at room

192

temperature, 1.0 mL of a 6 M and 5.0 mL of a 12 M hydrochloric acid were added, and the



193

sample was hydrolyzed for 23 h at 110 °C. Afterwards, the hydrochloric acid was removed

194

using a water-jet pump at 40 °C. The residue was dissolved in 1.0 mL methanol, filtrated

195

(0.45 µm) and analyzed by HPLC-ESI-MS/MS.

196 197

Preparation of 2-Amino-6-(3-methylpyridin-1-ium-1-yl)hexanoic acid (MP-lysine). In a

198

500 mL round-bottom flask 470 mg Nα-acetyl-L-lysine and 0.368 mL acrolein were heated in

199

250 mL methanol at 75 °C for 4 h under reflux. After evaporation of methanol in vacuo at

200

40 °C, 250 mL of 6 M hydrochloric acid were added and heated for 23 h at 110 °C under

201

reflux. Afterwards, the hydrochloric acid was removed using a water-jet pump at 40 °C and

202

the crude reaction mixture was lyophilized. Purification was realized using a semipreparative

203

HPLC system, consisting of a Smartline solvent manager 5000, a Smartline pump 1000, and a

204

Smartline UV-detector 2500, all from Knauer (Berlin, Germany). For the first purification,

205

20 mg of the crude reaction mixture were dissolved in 1.0 mL methanol and separated after

206

filtration (0.45 µm) using an Eurospher 100-10 C18 column (250 mm x 16 mm) from Knauer

207

at a room temperature of 23 °C, a flow rate of 2.5 mL/min and a detection wavelength of

208

267 nm. A gradient was used with solvent A (water) and B (methanol), each containing

209

11 mM acetic acid and 2 mM nonafluoropentanoic acid. The gradient program started with

210

40% solvent B for 5 min, increased to 74% B within 40 min, to 95% B within 1 min, held at

211

95% B for 13 min, decreased to 40% B within 1 min and held at 40% B for 10 min. Multiple

212

separations were performed, MP-lysine was collected between 20 and 35 min and the solvent

213

was evaporated at 40 °C in vacuo. For further purification, 10 mg of the isolated MP-lysine

214

were dissolved in 1.0 mL methanol, filtered (0.45 µm) and separated using the same eluents, a

215

flow rate of 8.0 mL/min and a gradient increasing from 5% B to 77% B within 55 min, held at

216

77% B for 7 min, decreased to 5% B within 1 min and held at 5% B for 7 min. MP-lysine

217

eluted between 34 and 36 min. Multiple separations were performed and the relevant fractions


Page 10 of 35

Page 11 of 35


218

were pooled. The solvent was evaporated at 40 °C in vacuo. Pure MP-lysine was subjected to

219

HPLC-ESI-MS/MS and one- and two-dimensional NMR analysis. ESI-MS (positive mode),

220

[M]+ m/z 223.1; 1H NMR (400.1 MHz, MeOH-d4): δ 1.54 (m, 4 H, H-8A,B); 1.95 (m, 4 H, H-

221

9A,B); 2.05 (m, 2 H, H-7); 2.54 (s, 3 H, H-12); 3.95 (t, 1 H, J = 6.2 Hz, H-10); 4.58 (t, 2 H, J

222

= 7.6 Hz, H-6); 7.95 (t, 1 H, J = 7.6 Hz, H-5); 8.40 (d, 1 H, J = 8.1 Hz, H-1); 8.77 (d, 1 H, J

223

= 6.6 Hz, H-4); 8.85 (s, 1 H, H-3). 13C NMR (100.6 MHz, MeOH-d4): δ 18.4 (C-12), 22.8 (C-

224

8), 30.8 (C-9), 31.7 (C-7), 53.5 (C-10), 62.3 (C-6), 128.8 (C-5), 141.5 (C-4), 143.1 (C-2),

225

145.6 (C-1), 147.4 (C-3), 171.6 (C-11). MP-lysine eluted chromatographically pure by

226

HPLC-ESI-DAD-MS analysis. Content of cation within the salt based on elemental analysis =

227

24.5%. Yield = 23.5 mg (molar yield = 4.2%).

228 229

Quantitation of MP-lysine in Roasted Peanuts. For quantitation of MP-lysine in peanuts,

230

60 mg of crushed peanut samples were hydrolyzed by 4.0 mL hydrochloric acid at 110 °C for

231

23 h. 1.0 mL of the hydrolyzate were dried at 37 °C in vacuo using a SpeedVac vacuum

232

concentrator, redissolved in 0.13 mL 10 mM nonafluoropentanoic acid in water, 0.15 mL 10

233

mM nonafluoropentanoic acid in acetonitrile and 0.02 mL (3.0 pmol) of internal standard N-

234

benzoylglycyl-L-phenylalanine in methanol:water (50:50, v/v). After filtration (0.45 µm),

235

samples were subjected to HPLC-ESI-MS/MS analysis. Quantitation was realized, using

236

authentic MP-lysine and the addition of 3.0 pmol internal standard N-benzoylglycyl-L-

237

phenylalanine in a hydrochloric acid hydrolyzate matrix calibration of raw peanuts, prepared

238

analogously to the samples. The LOD and LOQ were 1.5 and 4.6 µmol/kg peanut,

239

respectively. Results are expressed as mean values ± standard deviations of two separate

240

measurements.24

241



242

HPLC-ESI-MS/MS Analysis. The HPLC system consisted of a degasser, a pump, an

243

autosampler and a diode array detector, all from Agilent Technologies 1200 Series

244

(Böblingen, Germany). A Triple Quad LC/MS 6410 from Agilent Technologies was used for

245

MS/MS-measurements. For identification of major reaction products between acrolein and

246

Nα-acetyl-L-lysine, 10 µL of samples were injected and separated using an Eurospher 100-5

247

C18 column (250 x 3.0 mm; Knauer) at 30 °C and a flow rate of 0.38 mL/min. A gradient was

248

used with solvent A (water) and B (acetonitrile), each containing 2 mM heptafluorobutyric

249

acid and 11 mM acetic acid. The gradient increased from 2% B to 70% B within 50 min, to

250

90% B within 15 min, isocratic elution at 90% B for 10 min, decreased to 2% B within 3 min

251

and isocratic elution at 2% B for 10 min. First, full scan analysis and then CID experiments

252

(product ion scans) were performed. Full scan analysis were performed from 5.1 to 78.0 min

253

in positive mode, scan ranges were from m/z 80 to 1000, scan time 200 ms, fragmentor

254

voltage 135 V, gas temperature 300 °C, gas flow 11 L/min and nebulizer pressure 15 psi. CID

255

experiments were performed for the unhydrolyzed sample from 14 to 25 min with precursor

256

ions m/z 264.9 for the acetylated MP-lysine-derivative and from 5 to 25 min with precursor

257

ion m/z 243.1 for the reduced FDP-derivative after reduction and acid hydrolysis. Product ion

258

scan ranges were from m/z 60 to 300 with scan times of 200 ms, fragmentor voltages of 135 V

259

and collision voltages of 20 eV and 15 eV for the MP- and the FDP-derivative, respectively.

260

For quantitation of MP-lysine in modified peanut proteins and in peanut samples, 35 and 50

261

µL of samples, respectively were separated using a Zorbax SB-C18 column (50 x 2.1 mm)

262

from Agilent Technologies at 30 °C and a flow rate of 0.25 mL/min. A gradient was used

263

with solvent A (water) and B (acetonitrile), each containing 10 mM nonafluorpentanoic acid.

264

Gradient increased from 2% B to 27% B within 20 min, increased to 90% B within 5 min,

265

held at 90% B for 10 min, decreased to 2% B within 2 min and held at 2% B for 5 min.

266

Measurements were performed using the multiple reaction monitoring (MRM) mode in


Page 12 of 35

Page 13 of 35


267

positive mode, gas temperature was set to 300 °C, gas flow to 11 L/min and nebulizer

268

pressure to 15 psi. Internal standard N-benzoylglycyl-L-phenylalanine was analyzed from 20

269

to 32 min with a mass transition from m/z 327.1 to 166.1 for quantitation using fragmentor

270

and collision voltages of 90 V and 4 eV and a dwell time of 200 ms, respectively. Mass

271

transition from m/z 327.1 to 105.1 was used for qualification using fragmentor and collision

272

voltages of 90 V and 34 eV and a dwell time of 200 ms, respectively. MP-lysine was also

273

analyzed from 20 to 32 min with a mass transition from m/z 223.1 to 84.1 for quantitation

274

using fragmentor and collision voltages of 129 V and 11 eV and a dwell time of 100 ms,

275

respectively. Mass transition from m/z 223.1 to 94.1 was used for qualification using

276

fragmentor and collision voltages of 129 V and 20 eV and a dwell time of 100 ms,

277

respectively.

278 279

RESULTS AND DISCUSSION

280

Semi-Quantitation of Free Acrolein in Heated Peanut Oil Samples.

281

Due to the fact that (E)-but-2-enal was used as internal standard, acrolein was semi-

282

quantitated in the oil samples by GC-MS (EI), following a derivatization step by PFBHA-HCl

283

(see Fig. I in the Supporting Information). Heating of peanut oil represents a suitable model

284

system for roasting peanuts in which the quantitation of acrolein would not have been

285

possible due to possible reactions of the internal standard with nucleophilc compounds. The

286

amounts of free acrolein are given in Figure 2. Depending on the roasting time, the amounts

287

increased from 0.3 to 19.1 µmol/100 g oil or 0.2 to 10.7 mg/kg oil. This is in good agreement

288

with recently published data: For peanut oil heated for 2 h at 145 °C and 200 °C, 2.7 µM and

289

24 µM acrolein were found, respectively.13 Based on an average density of peanut oil of

290

0.915 g/mL,26 this corresponds to 0.3 and 2.6 µmol/100 g oil, which is comparable to the data

291

of the present work. According to the literature, no acrolein was detectable in the unheated



Page 14 of 35

292

peanut oil,13 indicating that the oil analyzed in the present work was in an advanced stage of

293

the lipid peroxidation where the decomposition of primary products already has been started.

294

In heated (180 °C for 2 h) coconut, olive, rapeseed, safflower and linseed oils 1.1, 11.8, 106.1,

295

25.1 and 85.7 mg acrolein/kg oil were found.12 Thus, the data found in the present work are

296

within the range published in literature for different oils.

297 298

Amino Acid Modifications in Modified Peanut Proteins.

299

Due to its high reactivity towards nucleophils,7,10,17 reactions of acrolein and amino acid side

300

chains (lipation reactions)5 may contribute to the loss of lysine occurring as a consequence of

301

peanut roasting.1 To investigate the ability of acrolein to modify amino acid side chains

302

within the peanut proteins, extracted peanut proteins of raw peanuts and acrolein were

303

incubated at 37 °C for 24 h in phosphate buffer (pH 7.4) as previously described for the

304

incubation with other secondary products.5,6,17,29,30 Incubations were performed in different

305

molar ratios between acrolein and the sum of the reactive amino acids cysteine, histidine,

306

lysine, and arginine and amino acid decreases were analyzed after enzymatic hydrolysis,

307

except for histidine (see Materials and Methods), because of the instability of certain possible

308

reaction products toward acid hydrolysis (Figure 3). The decreases are given relative to the

309

amounts in the blank sample. Modifications of lysine and arginine increased from 7% and 3%

310

in the 0.5:1 sample to 79% and 8% in the 5:1 sample, respectively. Cysteine and histidine

311

modifications increased from 12% and 11% in the 0.5:1 sample to 18% and 45% in the 5:1

312

sample, respectively. Therefore, the amino acid side chain modification by acrolein is,

313

compared to that of 4-hydroxynon-2-enal (4-HNE),6 much higher and less selective.

314

Concerning peanut proteins, the order of reactivity at low concentrations is as follows (the

315

0.1:1

316

lysine > cysteine = histidine > arginine and at high concentrations: lysine > histidine >

sample

cannot

be

interpreted

due

to


statistical

uncertainties):

Page 15 of 35


317

cysteine > arginine. Thus, lysine should represent the preferred reaction partner of acrolein

318

within the peanut proteins.

319 320

Identification of Major Reaction Products of Acrolein and Nα-Acetyl-L-lysine.

321

For identification of major reaction products of acrolein and the ε-amino group of lysine,

322

acrolein and Nα-acetyl-L-lysine were incubated in equimolar amounts in methanol for 4 h at

323

75 °C. Lipation products were identified by HPLC-ESI-MS/MS analysis in CID experiments

324

(product ion scans) before hydrolysis and after reduction and hydrolysis (Figure 4). Besides

325

unreacted Nα-acetyl-L-lysine, showing an m/z of 189.1 [M+H]+, the m/z 265.1 indicated the

326

presence of the acetylated MP-lysine [M]+, which was identified by its fragment ions in a CID

327

experiment (Figure 4A). The loss of -42 u was indicative for a deacetylation step, resulting,

328

after loss of NH3 and CO (-45 u), in the fragment ion with m/z 178.1 [M]+. As a result of a

329

nucleophilic attack of the α-amino group to the ɛ-carbon of the lysine moiety, the pyridinium

330

moiety with m/z 94.1 [M+H]+ was split off and the typical lysine fragment ion with m/z 130.1

331

[M+H]+ was explainable. The second typical lysine fragment ion with m/z 84.1 [M+H]+

332

resulted after losses of CO and H2O (-46 u) from the ion with m/z 130.1.31 Formation of the

333

MP-derivative occurs initially by a nucleophilic attack of the ɛ-amino group of lysine at C1 of

334

acrolein leading to a Schiff’ base that subsequently undergoes a Michael reaction involving

335

another molecule of acrolein, oxidation steps and cyclization to the MP-derivative.17 After

336

hydrolysis of the reaction mixture with 6 M hydrochloric acid for 23 h at 110 °C, the retention

337

time was compared to that of the synthesized MP-lysine standard, confirming the presence of

338

MP-lysine in the reaction mixture. Furthermore, the formation of the acetylated FDP-

339

derivative, showing an m/z of 283.1, was assumed. However, a direct identification was not

340

possible because not all of the resulting fragment ions were explainable. This might have been

341

due to co-eluting substances showing a monoisotopic mass of m/z 283.1, too. To remove



342

interfering substances, a reduction and acid hydrolysis was performed resulting in the

343

expected monoisotopic mass of m/z 243.1 [M+H]+ of the reduced and deacetylated FDP

344

derivative 2-amino-6-[5-(hydroxymethyl)-3,6-dihydro-2H-pyridin-1-yl]hexanoic acid. The

345

identification was realized by a CID experiment (Figure 4B). The fragment ion with m/z 225.1

346

was indicative for a cyclisation und loss of water (-18 u). After a further loss of CO and NH3

347

(-45 u), the fragment ion with m/z 180.1 resulted. A loss of -45 u from the mother ion was

348

indicative for a direct loss of CO and NH3, leading to the fragment ion with m/z 198.1. As a

349

result of a nucleophilic attack of the α-amino group to the ɛ-carbon of the lysine moiety, the

350

tetrahydropyridin moiety with m/z 114.1 [M+H]+ was split off, leading to the typical lysine

351

fragment ion with m/z 130.1 [M+H]+. After losses of CO and H2O (-46 u), the fragment ion

352

with m/z 84.1 [M+H]+ resulted.31 Formation of the FDP-derivative occurs initially by a

353

Michael reaction of the ɛ-amino group of lysine to the C3 of acrolein leading to an

354

Nɛ-propanal-derivative that undergoes a second Michael reaction followed by an Aldol

355

condensation to the FDP-derivative.10 Representing a relatively stable and advanced lipation

356

end product, further studies were performed focusing on MP-lysine. For the isolation, Nα-

357

acetyl-L-lysine was incubated with a 2-fold molar excess of acrolein to induce the formation

358

of the pyridinium derivative. After acid hydrolysis, the product was isolated by

359

semipreparative HPLC with UV detection, using an RP-18 column. Identification was

360

performed by means of HPLC-ESI-MS/MS and one- and two-dimensional NMR

361

spectroscopy. Figure 5 shows the structure of 2-amino-6-(3-methylpyridin-1-ium-1-

362

yl)hexanoic acid (MP-lysine) and the relevant HMBCs. Interestingly, no heteronuclear

363

multiple bond correlation between H-5 and C-1 were detectable which is consistent with the

364

literature.17 In the correlation spectroscopy (COSY) experiment, the correlation between H-5

365

and H-1 was visible (see Fig. II in the Supporting Information).

366


Page 16 of 35

Page 17 of 35


367

Quantitation of MP-lysine in Modified Peanut Proteins.

368

MP-lysine was quantitated after acid hydrolysis by HPLC-ESI-MS/MS (MRM mode) using a

369

peanut protein matrix calibration. The quantitated amounts of MP-lysine and the explainable

370

loss of lysine due to the formation of MP-lysine compared to the lysine level in the blank

371

sample are given in Figure 6. In the blank sample MP-lysine was detectable but below the

372

LOQ of 4.6 µmol/kg or 1.8 µmol/100 g protein. The amounts of MP-lysine increased from the

373

0.1:1 (0.63 mmol/100 g protein) to the 1:1 sample (6.6 mmol/100 g protein) and decreased to

374

the 5:1 sample (1.89 mmol/100 g protein), representing lysine blockages of 25.1, 212.3 and

375

71.5 mmol/mol lysine, respectively. At low concentrations of acrolein, 96% of the

376

modification of lysine were explainable by the formation of MP-lysine, whereas at higher

377

concentrations of acrolein the explainable loss of lysine decreased, reaching a minimum of

378

10% in the 5:1 sample. This indicates that at low concentrations of acrolein, MP-lysine

379

represents the major reaction product, whereas at higher concentrations other lipation

380

products became relevant. Possible reaction products might be cross-linking lipation products,

381

FDP-derivatives or Aldol condensation reactions, leading to a lesser amount of free acrolein.

382

Maybe at higher concentrations of acrolein the reaction remains on the level of the

383

Schiff’base, representing the precursor of MP-lysine. These results are comparable to recently

384

published results investigating the formation of 2-pentylpyrrol lysine (2-PPL) as a result of

385

modifying peanut proteins with 4-HNE.6

386 387

Quantitation of MP-lysine in Peanuts.

388

Analogous to the modified peanut proteins, MP-lysine was quantitated in the peanut samples

389

by HPLC-ESI-MS/MS (MRM mode) after acid hydrolysis using a peanut matrix calibration.

390

Figure 7 shows representative HPLC-ESI-MS/MS-chromatograms of a hydrolyzed peanut

391

sample (170 °C, 20 min) and the MP-lysine standard in a peanut hydrolyzate matrix. The



392

quantitated amounts of MP-lysine in raw, roasted and commercial peanut samples are given in

393

Figure 8. In the raw peanuts, MP-lysine was detectable, but amounts were below the LOQ of

394

4.6 µmol/kg peanut or 1.8 µmol/100 g protein. Depending on the roasting time, an increase of

395

MP-lysine from 2.2 to 19.4 µmol/100 g protein was quantitated, corresponding to 1.1 and

396

10.2 mg/kg peanut, respectively. Compared to the amounts of 2-PPL in the samples roasted

397

for 20 and 40 min,6 levels of MP-lysine were 20- to 111-fold higher, respectively. Therefore,

398

MP-lysine should represent an even better marker for the lipation status of food proteins than

399

2-PPL. In the commercial sample, 0.5 µmol MP-lysine/100 g protein, or 0.3 mg/kg peanut

400

were found. A possible explanation for the lower amounts of MP-lysine in the commercial

401

sample compared to the sample roasted at 170 °C for 20 min in laboratory scale might have

402

been different stages of lipid peroxidation of the respective used raw peanuts for roasting. The

403

explainable loss of lysine due to formation of MP-lysine for the 20 and 40 min samples were

404

0.05% and 0.13% and lysine blockages were 0.1 and 0.6 mmol/mol lysine, respectively. To

405

the best of our knowledge, this is the first direct quantitation of MP-lysine in food. In oxLDL,

406

0.06 mol MP-lysine/mol LDL were detected by means of HPLC-ESI-MS/MS, using a stable

407

isotope dilution assay contributing to 1% of loss of lysine.20

408

In conclusion, our results show that acrolein is formed as a result of heating peanut oil, being

409

a model system of roasting peanuts, depending on the heating time. The reaction with peanut

410

proteins leads primarily to a decrease of lysine. Cysteine, histidine and arginine were

411

modified, too, but to a lesser extent. MP-lysine and, following reduction and hydrolysis, FDP-

412

lysine in its reduced form were identified as major lipation products. At low concentrations of

413

acrolein, the formation of MP-lysine accounts for 96% of lysine modifications within a

414

modified peanut protein extract. Furthermore, to the best of our knowledge, MP-lysine was

415

quantitated for the first time directly in peanuts. Increased MP-lysine levels were detectable

416

depending on the roasting time. Due to the fact that its amounts were 20- to 111-fold higher


Page 18 of 35

Page 19 of 35


417

than the levels of 2-PPL,6 MP-lysine represents a suitable marker to evaluate the lipation

418

status of food proteins. However, the contribution of MP-lysine to the observed loss of lysine

419

was small. Regarding the abundance of secondary products that might be formed when

420

peanuts are roasted, a large number of lipation products are possible contributing in sum to

421

the observed loss of lysine. In addition, reactions of free radicals with amino acid side chains

422

might be of interest.32-34 Further studies are needed to quantify MP-lysine in other food

423

samples, especially with lower amounts of ω-3 and ω-6 fatty acids, and other lipation

424

products to clarify the loss of lysine.

425 426

Disclosure

427

The authors declare no financial interest.

428 429

Abbreviations used:

430

MP-lysine, 2-amino-6-(3-methylpyridin-1-ium-1-yl)hexanoic acid (Nɛ-(3-methylpyridinium)

431

lysine); FDP-lysine, 2-amino-6-(5-formyl-3,6-dihydro-2H-pyridin-1-yl)hexanoic acid (Nɛ-(3-

432

formyl-3,4-dehydropiperidino)lysine); MWCO, molecular weight cut-off; GC-MS (EI), gas

433

chromatography with mass spectrometry after electron-impact ionization; HPLC-ESI-

434

MS/MS, high performance liquid chromatography with electrospray ionization and tandem

435

mass spectrometry; DAD, diode array detector; CID, collision-induced dissociation; MRM,

436

multiple reaction monitoring; 4-HNE, 4-hydroxynon-2-enal; LOD, limit of detection; LOQ,

437

limit of quantitation; COSY, correlation spectroscopy; HSQC, heteronuclear single-quantum

438

coherence;

439

pentafluorobenzyl)-hydroxylamine;

440

acid (2-pentylpyrrole lysine)

HMBC,

heteronuclear

multiple-bond 2-PPL,

correlation;

PFBHA,

o-(2,3,4,5,6-

2-amino-6-(2-pentyl-1H-pyrrol-1-yl)hexanoic

441



442

Associated content

443

Fig. I, Derivatization reaction of acrolein by o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine

444

(PFBHA); Fig. II, COSY spectrum of 2-Amino-6-(3-methylpyridin-1-ium-1-yl)hexanoic acid

445

(MP-lysine) showing the correlation between H-1 and H-5.

446 447

Acknowledgment

448

We thank Karla Schlosser, Department of Food Chemistry, TU Dresden, for performing the

449

amino acid analysis, Stephen Schulz, Department of Inorganic Chemistry, TU Dresden, for

450

performing the elemental analysis and Sivathmeehan Yogendra, Department of Inorganic

451

Chemistry, TU Dresden, for performing the NMR analysis.

452 453

References

454

1. Wellner, A.; Nußpickel, L.; Henle, T. Glycation compounds in peanuts. Eur. Food Res.

455

Technol. 2012, 234, 423-429.

456

2. Souci, S. W.; Fachmann, W.; Kraut, H. Food composition and nutrition tables, 7th edition.

457

MedPharm Scientific Publishers, Stuttgart, Germany 2008, 1175.

458

3. Maguire, L. S.; O'Sullivan, S. M.; Galvin, K.; O'Connor, T. P.; O'Brien, N. M. Fatty acid

459

profile, tocopherol, squalene and phytosterol content of walnuts, almonds, peanuts, hazelnuts

460

and the macadamia nut. Int. J. Food Sci. Nutr. 2004, 55, 171-178.

461

4. Liu, X.; Jin, Q.; Liu, Y.; Huang, J.; Wang, X.; Mao, W.; Wang, S. Changes in volatile

462

compounds of peanut oil during the roasting process for production of aromatic roasted

463

peanut oil. J. Food Sci. 2011, 76, 404-412.


Page 20 of 35

Page 21 of 35


464

5. Globisch, M.; Schindler, M.; Kressler, J.; Henle, T. Studies on the reaction of trans-2-

465

heptenal with peanut proteins. J. Agric. Food Chem. 2014, 62, 8500-8507.

466

6. Globisch, M.; Kaden, D.; Henle, T. 4-Hydroxy-2-nonenal (4-HNE) and its lipation product

467

2-pentylpyrrole lysine (2-PPL) in peanuts. J. Agric. Food Chem. 2015, 63, 5273-5281.

468

7. Esterbauer, H.; Schaur, R. J.; Zollner, H. Chemistry and biochemistry of

469

4-hydroxynonenal, malondialdehyde and related aldehydes. Free Radic. Biol. Med. 1991, 11,

470

81-128.

471

8. Stevens, J. F.; Maier, C. S. Acrolein: Sources, metabolism, and biomolecular interactions

472

relevant to human health and disease. Mol. Nutr. Food Res. 2008, 52, 7-25.

473

9. Vercellotti, J. R.; Sanders, T. H.; Chung, S.-Y.; Bett, K. L.; Vinyard, B. T. Carbohydrate

474

metabolism in peanuts during postharvest curing and maturation. Dev. Food Sci. 1995, 37,

475

1547-1578.

476

10. Uchida, K.; Kanematsu, M.; Morimitsu, Y.; Osawa, T.; Noguchi, N.; Niki, E. Acrolein is

477

a product of lipid peroxidation reaction - Formation of free acrolein and its conjugate with

478

lysine residues in oxidized low density lipoproteins. J. Biol. Chem. 1998, 273, 16058-16066.

479

11. Ewert, A., Granvogl, M., Schieberle, P. Isotope-labeling studies on the formation pathway

480

of acrolein during heat processing of oils. J. Agric. Food Chem. 2014, 62, 8524-8529.

481

12. Ewert, A.; Granvogl, M.; Schieberle, P. Development of two stable isotope dilution assays

482

for the quantitation of acrolein in heat-processed fats. J. Agric. Food Chem. 2011, 59,

483

3582-3589.



484

13. Casella, I. G.; Contursi, M. Quantitative analysis of acrolein in heated vegetable oils by

485

liquid chromatography with pulsed electrochemical detection. J. Agric. Food Chem. 2004, 52,

486

5816-5821.

487

14. Feron, V. J.; Til, H. P.; de Vrijer, F.; Woutersen, R. A.; Cassee, F. R.; van Bladeren, P. J.

488

Aldehydes: Occurrence, carcinogenic potential, mechanism of action and risk assessment.

489

Mutat. Res. 1991, 259, 363-385.

490

15. Miller, B. E.; Danielson, N. D. Derivatization of vinyl aldehydes with anthrone prior to

491

high-performance liquid-chromatography with fluorometric detection. Anal. Chem. 1988, 60,

492

622-626.

493

16. Panosyan, A. G.; Mamikonyan, G. V.; Torosyan, M.; Gabrielyan, E. S.; Mkhitaryan, S.

494

A.; Tirakyan, M. R.; Ovanesyan, A. Determination of the composition of volatiles in cognac

495

(brandy) by headspace gas chromatography-mass spectrometry. J. Anal. Chem. 2001, 56,

496

945-952.

497

17. Furuhata, A.; Ishii, T.; Kumazawa, S.; Yamada, T.; Nakayama, T.; Uchida, K. N-epsilon-

498

(3-methylpyridinium)lysine, a major antigenic adduct generated in acrolein-modified protein.

499

J. Biol. Chem. 2003, 278, 48658-48665.

500

18. Furuhata, A.; Nakamura, M.; Osawa, T.; Uchida, K. Thiolation of protein-bound

501

carcinogenic aldehyde - An electrophilic acrolein-lysine adduct that covalently binds to thiols.

502

J. Biol. Chem. 2002, 277, 27919-27926.

503

19. Calingasan, N. Y.; Uchida, K.; Gibson, G. E. Protein-bound acrolein: A novel marker of

504

oxidative stress in Alzheimer's disease. J. Neurochem. 1999, 72, 751-756.


Page 22 of 35

Page 23 of 35


505

20. Maeshima, T.; Honda, K.; Chikazawa, M.; Shibata, T.; Kawai, Y.; Akagawa, M.; Uchida,

506

K. Quantitative analysis of acrolein-specific adducts generated during lipid peroxidation-

507

modification of proteins in vitro: Identification of Nτ-(3-propanal)histidine as the major

508

adduct. Chem. Res. Toxicol. 2012, 25, 1384-1392.

509

21. Matissek, R.; Steiner, G.; Fischer, M. Lebensmittelanalytik, 4th edition. Springer-Verlag,

510

Berlin, Heidelberg, Germany 2010, 103-109.

511

22. Henle, T.; Walter, H.; Klostermeyer, H. Evaluation of the extent of the early Maillard-

512

reaction in milk-products by direct measurement of the Amadori-product lactuloselysine. Z.

513

Lebensm. Unters. Forsch. 1991, 193, 119-122.

514

23. Henle, T.; Walter, H.; Krause, I.; Klostermeyer, H. Efficient determination of individual

515

Maillard compounds in heat-treated milk products by amino acid analysis. Int. Dairy J. 1991,

516

1, 125-135.

517

24. Chung, S.-Y.; Champagne, E. T. Allergenicity of Maillard reaction products from peanut

518

proteins. J. Agric. Food Chem. 1999, 47, 5227-5231.

519

25. Funk, W.; Dammann, V.; Donnevert, G. Qualitätssicherung in der Analytischen Chemie.

520

Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany 2005.

521

26. Krist, S. Lexikon der pflanzlichen Fette und Öle. Springer Verlag, Wien, Austria 2013.

522

27. Abraham, K.; Andres, A.; Palavinskas, R.; Berg, K.; Appel, K. E.; Lampen, A.

523

Toxicology and risk assessment of acrolein in food. Mol. Nutr. Food Res. 2011, 55,

524

1277-1290.



525

28. Collin, S.; Osman, M.; Delcambre, S.; El-Zayat, A. I.; Dufour, J.-P. Investigation of

526

volatile flavor compounds in fresh and ripened Domiati cheeses. J. Agric. Food Chem. 1993,

527

41, 1659-1663.

528

29. Ishino, K.; Wakita, C.; Shibata, T.; Toyokuni, S.; Machida, S.; Matsuda, S.; Matsuda, T.;

529

Uchida, K. Lipid peroxidation generates body odor component trans-2-nonenal covalently

530

bound to protein in vivo. J. Biol. Chem. 2010, 285, 15302-15313.

531

30. Ichihashi, K.; Osawa, T.; Toyokuni, S.; Uchida, K. Endogenous formation of protein

532

adducts with carcinogenic aldehydes - Implications for oxidative stress. J. Biol. Chem. 2001,

533

276, 23903-23913.

534

31. Shek, P. Y. I.; Zhao, J.; Ke, Y.; Siu, K. W. M.; Hopkinson, A. C. Fragmentations of

535

protonated arginine, lysine and their methylated derivatives: Concomitant losses of carbon

536

monoxide or carbon dioxide and an amine. J. Phys. Chem. A 2006, 110, 8282-8296.

537

32. Vogt, W. Oxidation of methionyl residues in proteins - tools, targets, and reversal. Free

538

Radic. Biol. Med. 1995, 18, 93-105.

539

33. Özben, T. Free radicals, oxidative stress, and antioxidants - pathological and physiological

540

significance. Springer Science and Business Media, New York, USA 1998.

541

34. Stadtman, E. R.; Levine, R. L. Free radical-mediated oxidation of free amino acids and

542

amino acid residues in proteins. Amino Acids 2003, 25, 207-218.

543 544


Page 24 of 35

Page 25 of 35


545

FIGURE LEGENDS

546

Figure 1.

547

Formation pathways of (A) Nɛ-(3-methylpyridinium)lysine and (B) Nɛ-(3-formyl-3,4-

548

dehydropiperidino)lysine.17

549 550

Figure 2.

551

Semi-quantitation of free acrolein in the unheated peanut oil and the samples heated at 170 °C

552

by GC-MS (EI).

553 554

Figure 3.

555

Decreases of amino acids cysteine, histidine, lysine and arginine in extracted raw peanut

556

proteins modified by acrolein for 24 h at 37 °C in 0.1 M phosphate buffer (pH 7.4), analyzed

557

by amino acid analysis after enzymatic hydrolysis for cysteine, lysine and arginine and acid

558

hydrolysis for histidine. Decreases are presented relatively to the amounts of the blank

559

sample.

560 561

Figure 4.

562

Identification of lipation products by HPLC-ESI-MS/MS after incubation of Nα-acetyl-L-

563

lysine and acrolein in equimolar amounts in phosphate buffer (pH 7.2) for 24 h at 37 °C. The

564

HPLC-ESI-MS/MS-scan chromatogram (top) shows peaks of unreacted Nα-acetyl-L-lysine

565

(m/z 189.1) and MP-acetyl-lysine (m/z 265.1, A). (Bottom) Product ion patterns after

566

performing CID experiments of the MP-acetyl-lysine and reduced FDP-lysine (m/z 243.1, B)

567

after reduction and hydrolysis. Explanations of the fragment ions are given in the text.

568 569



570

Figure 5.

571

Structure of MP-lysine, showing the observed relevant heteronuclear multiple bond

572

correlations (HMBCs).

573 574

Figure 6.

575

Formation of MP-lysine (A) due to modification of extracted raw peanut proteins by acrolein

576

after incubation at 37 °C for 24 h in 0.1 M phosphate buffer (pH 7.4), analyzed after acid

577

hydrolysis by HPLC-ESI-MS/MS and explainable loss of lysine due to MP-lysine formation

578

(B) compared to the lysine level in the blank sample, analyzed by amino acid analysis. n. q. -

579

not quantifiable

580 581

Figure 7.

582

Representative HPLC-ESI-MS/MS chromatogram (MRM mode) of MP-lysine quantitation in

583

a peanut sample, roasted at 170 °C for 20 min (top, black) and an MP-lysine calibration

584

standard in a peanut hydrolyzate matrix (bottom, grey) with the mass transitions

585

m/z 223.1 94.1 for MP-lysine and m/z 327.1 166.1 for the internal standard

586

N-benzoylglycyl-L-phenylalanine, respectively.

587 588

Figure 8.

589

Quantitated amounts of MP-lysine in raw, roasted (170 °C) and commercial peanut samples,

590

analyzed by HPLC-ESI-MS/MS after acid hydrolysis. n. q. - not quantifiable

591


Page 26 of 35

Page 27 of 35


FIGURES O +

O

H2N

OH

NH2

- H2O

N

R

+

N

R

N H

O +

O R

R

O

O

O N R - H2O oxidation R N

O

- H2O R N O

(A)

(B)

Figure 1.



Acrolein [µmol/100 g peanut oil]

25

20

15

10

5

0

unheated

20 min

40 min

Figure 2.


Page 28 of 35

Page 29 of 35


Decreases of amino acids [%]

80

60

cysteine histidine lysine arginine

40

20

0 0.1:1

0.2:1

0.5:1

1:1

5:1

10:1

Ratio acrolein:sum of potentially reactive amino acids Figure 3.



Page 30 of 35

m/z 243.1 (B) 8

Intensity [counts]

4x10

m/z 283.1

3x108

m/z 265.1 (A)

m/z 189.1 8

2x10

after reduction and acid hydrolysis

1x108

0 10

20

30

40

50

60

70

Retention time [min] (A) 84.1 - 42, - 93, - 46

80 60

100 Relative intensity [%]

Relative intensity [%]

100

(B)

- 42, - 129 - 42, - 93

94.1

- 42, - 45 265.1 - 42

130.1

40 20

178.1 223.1

0

100

150

200 m/z

250

300

- 113, - 46 84.1

- 129 - 113 243.1

80 60

130.1

- 18, - 45 - 45

40

198.1 - 18

114.1

20 0

225.1 180.2

100

150

Figure 4.


200 m/z

250

300

Page 31 of 35


Figure 5.



A

B 100 Explainable loss of lysine [%]

MP-lysine [mmol/100 g protein]

7 6 5 4 3 2 1 0

n. q.

80 60 40 20 0

5:1 blank 0.1:1 0.2:1 0.5:1 1:1 Ratio acrolein:sum of potentially reactive amino acids

5:1 0.1:1 0.2:1 0.5:1 1:1 Ratio acrolein:sum of potentially reactive amino acids

Figure 6.


Page 32 of 35

Page 33 of 35


15000

Peanut sample

MP-lysine

m/z 223.1 m/z 327.1

94.1 166.1

20000

10000 Internal standard

5000

10000

0 Calibration standard MP-lysine

F

-5000 5000 Internal standard

-10000

0

-15000 22

23

24

25

26

27

28

29

30

Retention time [min]

Figure 7.


31

32

Intensity [counts]

Intensity [counts]

15000

MP-lysine [µmol/100 g protein]


20 15

3 2 1 0

n. q. raw

20 min

commercial sample

40 min

Figure 8.


Page 34 of 35

Page 35 of 35


TOC graphic + protein-NH2

acrolein Acrolein [µmol/100 g peanut oil]

25

LIPATION PRODUCTS

20

acid hydrolysis

15

10

LC-ESI-MS/MS

5

unheated

20 min

40 min

MP-lysine [µmol/100 g protein]

0 20 15

3 2 1 0

n. q. raw

heating peanut oil

20 min

40 min

commercial sample

MP-lysine


hexanoic Acid - ACS Publications - American Chemical Society

Recommend Documents