Identification of a HydroxymethylfurfuralâLysine Schiff Base and Its

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Food Safety and Toxicology

Identification of a Hydroxymethylfurfural–Lysine Schiff Base and Its Cytotoxicity in Three Cell Lines Ge Wang, Pengzhan Liu, Jun He, Zhao Yin, Shuo Yang, guangwen zhang, Shiyi Ou, Xinquan Yang, and Jie Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04539 • Publication Date (Web): 20 Aug 2019 Downloaded from pubs.acs.org on August 26, 2019

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Journal of Agricultural and Food Chemistry

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Identification of a Hydroxymethylfurfural–Lysine Schiff

2

Base and Its Cytotoxicity in Three Cell Lines

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Ge Wang , Pengzhan Liu , Jun He , Zhao Yin , Shuo Yang , Guangwen Zhang , Shiyi Ou ,

4

Xinquan Yang

5

†

*,＃

†

†

†

†

*,†

Department of Food Science and Engineering, Jinan University, 510632, Guangzhou, Guangdong,

China.

7

‖

8

Guangdong 510641, China.

School of Food Science and Engineering, South China University of Technology, Guangzhou,

‡

Institute of Laboratory Animal Science, Jinan University, Guangzhou 510632, China.

10

§

11

China.

12

§

, Jie Zheng

6

9

‡

‖

College of Traditional Chinese Medicine, Jinan University, Guangzhou 510632, Guangdong,

＃

School of Life Sciences, Guangzhou University, Guangzhou 510006, China.

13 14 15 16 17 18 19 20 21 22 23 24 25

Corresponding Authors

26

*Tel.: +86 20 85226630; E-mail: [email protected] (J.Z.).

27

*Tel: +86 20 39366697; E-mail: [email protected] (X.Y.).

28 29

ACS Paragon Plus Environment


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ABSTRACT

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5-Hydroxymethylfurfural (HMF) can undergo the Maillard reaction with amino acids.

32

However, the safety of the products remains unknown. In this study, an HMF–lysine

33

Schiff base named (E)-N6-((5’-(hydroxymethyl)furan-2’-yl)methylene)lysine (HML)

34

was identified and detected for the first time in baked foods. HML formation

35

significantly decreased the cytotoxicity (IC50, mM) of HMF against GES-1 cells

36

(81.81 vs. 5.02 and 73.76 vs. 2.94 for HML vs. HMF at 24 and 48 h, respectively),

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EA.hy926cells (86.05 vs. 4.85 and 77.22 vs. 0.71, respectively), and Caco-2 cells

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(155.77 vs. 36.84 and 112.70 vs. 18.51, respectively). Exposure of Caco-2 cells to

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HMF at 10.0 mM triggered cell apoptosis of 14.02% (vs. 8.54% in control), whereas

40

exposure to HML at 10-15 mM hardly increased cell apoptosis. Moreover, the

41

absorption capacities of HMF and HML by Caco-2 cells were equivalent (p > 0.05) at

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7.23%-12.57% after incubation at 2 mM for 30–150 min.

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KEYWORDS: 5-Hydroxymethylfurfural, lysine, adduct, Schiff base, cytotoxicity

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INTRODUCTION

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5-Hydroxymethylfurfural (HMF) can be formed through the Maillard reaction,

48

caramelization, and acidic dehydration of carbohydrates.1,2 It occurs at high amounts

49

in thermal processed food, such as cookies (0.5–74.5 mg/kg), breads (up to 410

50

mg/kg), coffee (300–1900 mg/kg), and caramel-containing foods (110–9500 mg/kg),

51

as well as dried fruits (up to 2200 mg/kg).3,4

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HMF has been reported to show beneficial effects on human health, including

53

anti-oxidative, anti-inflammatory, anti-allergic, antihypoxic, anti-sickling, and

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anti-hyperuricemic

55

5-sulfoxymethyfurfural (SMF) by sulfotransferase in animals and humans.6 In a study

56

conducted in a Spanish preadolescent population, HMF was found to be effectively

57

metabolized into SMF in vivo.7 SMF is highly electrophilic and can react with DNA

58

and other macromolecules; in addition, it has been proved to confer genotoxic,

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mutagenic, and carcinogenic effects.5 Its mutagenic effects have been confirmed by

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the European Food Safety Authority,8 who established a threshold of concern of 0.54

61

mg/day for the intake of furan derivatives used as flavoring agents in Europe.9

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Whereas, in a study involving 268 Spanish school pupils, the 24 h dietary intake of

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HMF of the subjects ranged from 3.14 mg/day to 68.86 mg/day (mean: 13.72 mg/day).

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Moreover, SMF was detected in 70 out of 268 subjects with a mean level of 0.33 nM,

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which is almost three times higher than that observed in mice 90 min after acute

66

intake of HMF.7 Thus, the adverse effect of high HMF contents in foods remains a

67

public concern.

effects.5

However,

HMF


can

be

converted

to


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HMF contains a reactive aldehyde functional group that can react with amino

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acids or polyphenols to form adducts.10‒13 The reaction products, including these

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adducts and the other intermediates formed between HMF and amino acids, may bury

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HMF exposure level. Previous researches proved that the reaction intermediates

72

released HMF during the storage and digestion of foods. Michalak et al. found that

73

HMF content increased by 28% and 46% in sweetened cacao products and by 11%

74

and 30% in biscuits for infants after 12 months of storage at 4 °C and 25 °C,

75

respectively.14

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1-dicysteinethioacetal−5-hydroxymethylfurfural (DCH), one of the HMF−cysteine

77

adducts, released HMF during digestion and metabolism in rats.15 These findings

78

indicate that the exposure level would be greatly underestimated by the analysis of

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HMF content in freshly produced foods.

Our

research

found

that

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HMF can also react with amino acids via the Maillard reaction,16,17 but the risk

81

of the formed products is unknown. Lysine (Lys) is an essential amino acid present in

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comparable amount in potato (347.9 μg/g) and cereal products (up to 37 μg/g).18,19

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Moreover, it has been widely applied as a nutrient supplement in cereal foods since

84

195520 and recently as an inhibitor of acrylamide formation in thermal processed

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foods.21 The reactive amino group on Lys could react with HMF to form Schiff bases

86

during the thermal processing or digestion of foods. However, the toxicity of the

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HMF–Lys Schiff bases remains unknown, which might increase potential risk for the

88

consumption of thermal processed foods, especially those containing a high amount of

89

or enriched with Lys.


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For the first time, we prepared a standard compound corresponding to the Schiff

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base formed between HMF and Lys, and found that it occurs in four types of baked

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foods. On the basis of this finding, its cytotoxicity against three cell lines was

93

measured. The apoptosis of Caco-2 cells induced by the treatment of HMF and HMF–

94

Lys Schiff base was compared. Moreover, the absorption and transformation of the

95

Schiff base were investigated within Caco-2 cells.

96 97

MATERIALS AND METHODS

98

Materials and Chemicals. 5-Hydroxymethyfurfural (98%) and L-lysine (Lys,

99

99%) were purchased from J & K Scientific Ltd. (Beijing, China). Human colon

100

(Caco-2) cells (ATCC HTB-37TM) were obtained from the American Type Culture

101

Collection (Rockville, MD, USA). Human gastric epithelial (GES-1) cells and human

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vascular endothelium (EA.hy926) cells were obtained from Shanghai Enzyme

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Research Biotechnology Co., Ltd. (Shanghai, China). Penicillin–streptomycin, fetal

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bovine serum (FBS), and Dulbecco’s modified Eagle’s medium (DMEM) were

105

purchased from Thermo Fisher Scientific Inc. (MA, USA). Trypsin–EDTA was

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purchased from Biosharp Co., Ltd. (Hefei, China). PBS was obtained from Boster

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Biological Technology Co., Ltd. (CA, USA). Propidium iodide staining (PI) and

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Annexin V-FITC were purchased from BestBio Co., Ltd. (Shanghai, China). Samples

109

of food, including biscuits, cookies, cakes, and breads, were bought from the local

110

market.



111

Effect of pH, Temperature, Reaction Time, and Substrate Concentration on

112

HMF Elimination. The reactions between HMF and Lys were performed in 25 mL

113

screw thread stainless steel test tubes. A final volume of 4 mL aqueous-base reaction

114

mixture was heated at a respective temperature in an oil bath for a respective duration.

115

Different reaction conditions were investigated individually while keeping the other

116

conditions constant as follows: concentrations of HMF and Lys, 50 and 100 mM,

117

respectively; reaction temperature, 160 ℃; reaction duration, 15 min; and pH, 11. To

118

investigate the effect of pH on the elimination of HMF, we adjusted the pH of the

119

reaction medium with 0.1% HCl (v/v) and 0.1% NaOH (v/v) to different levels of 1, 3,

120

5, 7, 9, 11, and 13. The effect of reaction temperature was tested at 80 ℃, 100 ℃,

121

120 ℃, 140 ℃, 160 ℃, and 180 ℃, and that of reaction duration at 10, 15, 20, 25,

122

and 30 min. The concentration ratio of HMF: Lys was set as 2:1, 1:1, 1:2, 1:3, and 1:4,

123

where equivalent of 1 refers to 50 mM in concentration, to investigate their effects on

124

the elimination of HMF. After the reaction, the tube was placed immediately in an ice

125

bath to terminate the reaction. Then, the reaction mixture was diluted to 10 mL with

126

distilled water and filtered through a 0.45 μm syringe filter for HPLC analysis of

127

HMF.

128

Separation, Purification, and Identification of HMF–Lys Schiff Base. HMF–

129

Lys Schiff base was prepared for food analysis and cellular experiments by reacting

130

HMF and Lys under optimized reaction conditions. A mixture of 50 mM HMF and

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100 mM Lys in distilled water with pH 11 was kept at 160 °C for 25 min to form the

132

HMF–Lys Schiff base. The reaction was then terminated with an ice bath. The


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mixture was concentrated with a rotary evaporator at 40 °C and then purified

134

successively with macroporous resin HP-20 (Sinopharm Chemical Reagent Co., Ltd.,

135

Shanghai, China) using distilled water as eluent and Octadecylsilyl-A-HG (YMC Co.,

136

Ltd., Tokyo, Japan) using 2% methanol in water as eluent. The purity of the eluted

137

adduct was analyzed by HPLC, and those with purity over 98% were collected and

138

lyophilized using a Scientz-10N vacuum freeze-dryer (SCIENTZ Biotechnology Co.,

139

Ltd., Ningbo, China). The purified and lyophilized HMF–Lys Schiff base was

140

identified by high-resolution mass spectrometry (HRMS, X500R QTOF mass

141

spectrometer, AB Sciex, CA) and nuclear magnetic resonance (NMR, AVANCE III

142

600 MHz spectrometer, Bruker, Fällanden, Switzerland).

143

Cell Culture. The cytotoxicity of the HMF–Lys Schiff base was investigated in

144

the cell lines of GES-1, Caco-2, and EA.hy926. GES-1 and EA.hy926 cells were

145

cultivated in DMEM with 10% FBS, and Caco-2 cells were cultivated in DMEM with

146

20% FBS in a humidified incubator containing 5% CO2 and 95% air at 37 °C. The

147

densities of cells were maintained as recommended, and 0.25% trypsin was used for

148

detaching adherent cells from plates.

149

Measurement of Cell Viability. The cell counting kit-8 (CCK-8, Biosharp,

150

Hefei, China) was used to assess the growth inhibition of cells under different

151

treatments of HMF and HMF–Lys Schiff base for 24 and 48 h as previously described

152

by Zhao et al.22 Different levels of HMF and HMF–Lys Schiff base were set for the

153

treatments of different cells. In detail, HMF showed high cytotoxicity on GES-1 and

154

EA.hy926 cells. Therefore, concentrations of 4, 8, 12, 24, 28, and 32 mM were



155

selected for the HMF treatments of GES-1 and EA.hy926 cells. Meanwhile, the

156

HMF–Lys Schiff base showed comparably low cytotoxicity on these two cells, and

157

the concentrations of the HMF–Lys Schiff base were set as 8, 16, 32, 64, 90, and 128

158

mM. For the treatment of Caco-2 cells, the same concentration levels of 2, 4, 8, 16, 32,

159

64, 128, 192, and 256 mM were set for both HMF and HMF–Lys Schiff base.

160

Analysis of Apoptosis by Flow Cytometry. Caco-2 cells (3×104 cells/well)

161

were treated with different concentrations (2.5, 5.0, 10.0, and 15.0 mM) of HMF and

162

HMF–Lys Schiff base for 48 h in 6-well culture plates. The treated cells were

163

harvested by EDTA-free trypsinization and washed once with PBS. Cells at 1–5×105

164

were collected and then Annexin V-FITC (5 μL, BestBio, Shang Hai, China) was

165

added to the binding buffer (50 μL, BestBio, Shang Hai, China). After mixing to a

166

homogenous cell suspension, 450 μL of the binding buffer and 1 μL of PI were added.

167

Flow cytometric analysis using a NovoCyte Flow cytometer (ACEA Biosciences,

168

Hang Zhou, China) was then performed within 1 h.

169

Absorption of HMF and HMF–Lys Schiff Base by Caco-2 Cells. The

170

absorption of HMF and HMF–Lys Schiff base by Caco-2 cells was determined in

171

accordance with the method applied by Sampath et al. with some modifications.23

172

Cells (3×104 cells/well) were seeded in 24-well culture plates and then incubated in a

173

humidified incubator containing 5% CO2 and 95% air at 37 °C. The cells were treated

174

with 2 mM HMF and HMF–Lys Schiff base for 30, 60, 90, 120, and 150 min. At the

175

end of incubation, the medium was collected as the extracellular fluid. After washing

176

thrice with 100 μL of PBS, the cells were trypsinized and collected. RIPA cell lysis


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buffer (50 μL) was then added, and the cells were allowed to lyse in an ice bath for 30

178

min with vigorous mixing every 5 min. After cells were completely lysed, the

179

intracellular fluid was collected after centrifugation at 12,000×g for 15 min at 4 °C.

180

All samples were filtered through a 0.22 μm syringe filter into an autosampler vial

181

prior to the HPLC analysis of HMF and HML.

182

HPLC-DAD Analysis of HMF and HMF–Lys Schiff Base in Reaction

183

Mixture, Cells, and Cell Cultures. For the determination of HMF and HMF–Lys

184

Schiff base, a 10 μL sample was injected into an LC-20AT HPLC equipped with an

185

SPD-M20AVP DAD linked to a CBM-20A controller (Shimadzu Corporation, Kyoto,

186

Japan). The sample was separated on an Agilent Zorbax SBAq C18 column (4.6 ×

187

250 mm, 5 μm) (Agilent Technologies, CA, USA) with the mobile phase of 5%

188

methanol in MQ-water at the column temperature of 40 °C. The flow rate was kept

189

constant at 0.6 mL/min. HMF and HMF–Lys Schiff base were detected at 284 and

190

253 nm, respectively. The contents of HMF and HMF–Lys Schiff base were

191

quantified using the calibration curves of the commercial standard and the standard (>

192

98%) prepared by our laboratory, respectively, as external standards.

193

Detection of the HMF–Lys Schiff Base in Foods. For determination of HMF–

194

Lys Schiff base content in foods, about 5 g of fully grinded sample was mixed with 50

195

mL of n-hexane and stirred occasionally for 24 h to remove fat-soluble components.

196

The supernatant was discarded after centrifugation at 10,000×g for 20 min at 4 °C,

197

and the residues were extracted twice with 30 mL of MQ-water. The supernatants

198

were collected and combined after centrifugation at 10,000×g for 20 min at 4 °C. The



199

sample was evaporated to dryness with a rotary evaporator under 40 °C, re-dissolved

200

with 4 mL of MQ-water, and then filtered through a 0.45 μm syringe filter. A sample

201

of 10 μL was injected into an Agilent 1100 Series LC (Agilent Technologies, CA,

202

USA) interfaced to an AB 4000 Q-Trap mass spectrometer (AB Sciex, MA, USA).

203

The sample was separated on an Agilent Zorbax SBAq C18 column (4.6 × 250 mm, 5

204

μm) (Agilent Technologies, CA, USA) with the same elution conditions described in

205

the HPLC-DAD analysis. The HMF–Lys Schiff base was detected with an

206

electrospray ionization mass spectrometer operated in the positive mode. The

207

capillary voltage was set at 5.5 kV, and the temperature was set at 500 °C. The

208

pressure of nebulizer gas and drying gas were 55 and 50 psi, respectively. The

209

qualification and quantification of the HMF–Lys Schiff base were achieved by

210

multiple reaction monitoring (MRM) with the transitions of m/z 255.1→130.0 and

211

m/z 255.1→84.1, respectively. The declustering potential and collision energy were

212

set at 50 and 19 V, respectively. The content of the HMF–Lys Schiff base was

213

quantified based on the calibration curve of the standard prepared by our laboratory.

214

pH Measurement of Foods. For pH determination, 5 mL of distilled water was

215

added to 5 g of food sample and then ground thoroughly. The pH of the slurry was

216

then measured using a STARTER300 pH meter incorporated with a ST 270 pH probe

217

(Ohaus Corporation, NJ, USA).

218

Statistical analysis. All experiments were conducted in triplicate. Statistical

219

analyses were performed using SPSS 16.0.1 (SPSS, Inc., Chicago, IL). Differences

220

between the effects of different concentrations and incubation times of HMF and


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HMF–Lys Schiff base on cell viability, and differences in values of pH, HMF and

222

HML between different food samples were investigated by one-way ANOVA.

223

Duncan’s multiple-range test (p < 0.05) was further applied to carry out the multiple

224

comparisons between different samples. Comparison between IC50 values of HMF

225

and HMF–Lys Schiff base treatments of various cell lines was conducted by an

226

independent-sample t test.

227

RESULTS AND DISCUSSION

228

Effect of Different Reaction Parameters on HMF Elimination. In previous

229

studies, HMF is largely eliminated by some amino acids through the Maillard reaction

230

and Michael addition reactions.11,12,17,24 In order to obtain sufficient amount of

231

HMF-Lys Schiff base standard, the effects of pH, temperature, reaction time, and

232

HMF-to-Lys concentration ratio on HMF elimination were investigated.

233

Results showed that the elimination of HMF by Lys was greatly influenced by

234

pH. At pH 1–5, HMF was only depleted by 6.14%–10.13%. As the pH was increased

235

to 9, HMF elimination sharply increased to 65.46% and then moderately increased

236

afterward (Figure 1A). Deprotonation of the amino group in amino acid is the

237

premise for the Maillard reaction. The pKa value of Lys is 10.4;25 as a result, higher

238

pH favors the Maillard reaction as a complete deprotonation of the amino group in

239

Lys would occur after pH 10.4. Although Lys could deplete 75.30% of HMF at

240

reaction pH of 13, pH 11 (70.21% HMF depletion) was chosen for the preparation of

241

the HMF–Lys Schiff base because of the least production of impurities.

242

Elevation of temperature increased the elimination of HMF. About 68.67% of



243

HMF was depleted when the temperature was increased to 160 ℃. A further increase

244

in the temperature showed limited impact on the elimination of HMF (Figure 1B),

245

whereas the production of impurities became apparent. Therefore, 160 ℃ was used

246

for the preparation of targeted HMF–Lys Schiff base.

247

Reaction time also influenced HMF elimination, but its impact was not as

248

remarkable as those of pH and temperature (Figure 1C). The elimination percentage

249

increased from 51.70% to 85.03% as the reaction time was prolonged from 10 min to

250

30 min. Thus, the reaction time of 25 min was finally chosen for the maximum

251

preparation of the HMF–Lys Schiff base and the minimum production of impurities.

252

Further prolongation of the reaction time (30 min) hardly influenced the formation of

253

the HMF–Lys Schiff base but increased the side-products, which might be attributed

254

to further reactions of the HMF–Lys Schiff base.

255

Molar ratio of HMF to Lys showed comparable impact on HMF elimination.

256

Higher ratio of Lys/HMF promoted the elimination of HMF. Figure 1D shows that

257

the elimination of HMF increased remarkably by two times as the molar ratio of HMF

258

to Lys changed from 2:1 to 1:2. The elimination of HMF increased but moderately as

259

the molar ratio further increased to 1:4. Although the highest elimination percentage

260

(70.60%) was reached at the molar ratio of 1:4 (HMF: Lys), the molar ratio of 1:2 was

261

chosen for the preparation of Schiff base to remove less Lys during the purification of

262

the HMF–Lys Schiff base.

263

On the basis of the results mentioned above, the preparation of the HMF–Lys

264

Schiff base was optimized as follows: 50 mM HMF and 100 mM Lys reacted at pH 11


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and 160 ℃ for 25 min. Under this condition, the elimination of HMF was 74.70%,

266

and the HMF–Lys Schiff base appeared as the predominant peak in the HPLC

267

chromatogram (Supplementary Figure S1).

268

Identification of HMF–Lys Schiff base. The HMF–Lys Schiff base was

269

produced from HMF and Lys under the optimized conditions obtained in the present

270

research. After successive purification using macroporous resin HP-20 and

271

Octadecylsilyl-A-HG, the target product was obtained as odorless white amorphous

272

powder with high hygroscopy, being soluble in water while insoluble in methanol.

273

The product with purity over 98% underwent identification analyses by HRMS and

274

NMR. The molecular weight of the product was 254.1 according to the molecular ion

275

detected at m/z 255.1331 in the positive mode of HRMS analysis (Supplementary

276

Figure S2), which was indicated as a Schiff base. The exact structure of the HMF–

277

Lys Schiff base was further elucidated by 1H and 13C NMR data and the correlations

278

from COSY and HMBC experiments (Figure 2). The downfield shift of the

279

corresponding chemical shifts for proton and carbon indicated that the Schiff base

280

formed between the ε-NH2 group in Lys and the aldehyde group of HMF. It was

281

assigned as (E)-N6-((5’-(hydroxymethyl)furan-2’-yl)methylene)lysine (HML). The

282

molecular formula is C12H18N2O4, with the theoretical molecular weight of 254.29,

283

which is consistent with the m/z value of 255.1331 [M + H]+ obtained by mass

284

spectrometric analysis.

285

Detection of HML in foods. In the current study, four types of bakery foods,

286

including biscuits, cookies, cakes, and breads, were analyzed and proved to contain



287

the HMF–Lys Schiff base, i.e., HML (Table 1). Among them, biscuits displayed the

288

highest amount of HML (p < 0.05), followed by cookies and breads, and cakes being

289

the lowest. The biscuit displayed the highest pH in all the samples, although the

290

difference between it and other samples was small but significant. A combined but

291

complex impact of rapid heat conduction and comparable alkaline condition of the

292

biscuit dough might contribute to the formation of high-level HML. Moreover, all the

293

samples tested are common bakery products. An enrichment of lysine as nutrient

294

supplements may further increase the formation of HML in some special nutritional

295

fortified foods.

296

Impact of HMF and HML on the Cell Viability of GES-1, Caco-2, and

297

EA.hy926 Cells. Concerning the digestion and absorption of food components in

298

humans, epithelial cells in the gastrointestinal tract (GES-1 and Caco-2 cells) and

299

endothelial cells in the blood vascular system (EA.hy926 cells) were chosen to

300

investigate the effect of HML on cell viability, using its precursor HMF as a

301

control.22,26‒28

302

As shown in Figure 3, HML and HMF displayed distinguishing potent

303

cytotoxicity in the different cell lines. HMF caused significant injury on GES-1 and

304

EA.hy926 cells at low concentrations (below 32 mM). Over 95% of GES-1 cells died

305

after exposure to 28 and 24 mM HMF for 24 and 48 h, respectively; the same

306

situation occurred in EA.hy926 cells exposed to 24 and 12 mM HMF, respectively

307

(Figure 3). Based on the concentration test, the IC50 values of HMF were calculated

308

as 5.02 and 2.94 mM against GES-1 cells after incubation of 24 and 48 h, respectively,


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and 4.85 and 0.71 mM against EA.hy926 cells, respectively. By contrast, Caco-2 cells

310

showed higher resistance against the exposure to HMF than GES-1 and EA.hy926

311

cells. The IC50 values of HMF against Caco-2 cells were 36.84 and 18.51 mM after 24

312

and 48 h incubation, respectively, which were 7.2 and 6.1 times higher than those

313

against GES-1 cells and 7.6 and 26.1 times higher than those against EA.hy926 cells.

314

The formation of HML significantly decreased the toxicity of HMF toward all the

315

three cell lines. After HML incubation for 24 and 48 h, its IC50 value increased by

316

16.3- and 25.1-fold in GES-1 cells, 17.7- and 108.8-fold in EA.hy926 cells, and 4.2-

317

and 6.1-fold in Caco-2 cells, respectively, compared with those of HMF (Figure 3).

318

HML showed 1.5–2-fold lower toxicity toward Caco-2 cells than GES-1 and

319

EA.hy926 cells. Moreover, the concentration impact of HML on cell viability differed

320

between Caco-2 cells and the other two lines of cells. The viability of GES-1 and

321

EA.hy926 cells remained almost constant as that of the blank until the HML exposure

322

level reached 32 mM and then started to decrease sharply afterward in a

323

dose-dependent manner until almost total death of the cells occurred at the treatment

324

level of 128 mM. By contrast, only 40.62% and 60.85% of the Caco-2 cells died

325

under HML treatment at 128 mM for 24 and 48 h, respectively. The injury of Caco-2

326

cells occurred significantly at very low concentrations of HML and then increased

327

mildly as the treatment concentration was increased (Figure 3).

328

In our previous study,22 we reported the HMF–cysteine adduct DCH and

329

evaluated its cytotoxicity against Caco-2 cells. The IC50 value of DCH against Caco-2

330

cells was assessed to be 31.26 ± 1.50 mM, whereas that of HMF was 14.95 ± 1.09



331

mM, after 72 h of incubation. Therefore, lysine rather than cysteine might contribute

332

better in detoxification of HMF against Caco-2 cells.

333

Effects of HMF and HML on Apoptosis of Caco-2 Cells. In the current study,

334

flow cytometry was applied to evaluate cell apoptosis induced by HMF and HML.

335

Figure 4 shows that exposure to HML at 2.5-15.0 mM hardly induced the apoptosis

336

of Caco-2 cells, whereas exposure to HMF at 10-15 mM resulted in considerable cell

337

apoptosis. Approximately 8.54% of the normal cells were apoptotic; the population of

338

apoptotic cells remained almost unchanged below 9.68% after exposure to HML at

339

2.5-15.0 mM and HMF at 2.5-5.0 mM for 48 h; however, exposure to HMF at 10.0

340

and 15.0 mM for 48 h increased the percentage of apoptotic cells from 8.54% (control)

341

to 14.02% and 14.68%, respectively. These results further proved that the formation

342

of HML decreased the cytotoxicity of HMF against Caco-2 cells in terms of cell

343

apoptosis induction.

344

Absorption of HMF and HML by Caco-2 cells. In the current study, we

345

incubated Caco-2 cells with HMF and HML at 2 mM for various periods from 30 min

346

to 150 min to determine the absorption of these compounds by Caco-2 cells. Table 2

347

shows that both HMF and HML could be absorbed by Caco-2 cells from 7.23% to

348

12.57%. Prolongation of incubation period hardly influenced the absorption of both

349

HMF and HML. Moreover, the absorption of the two compounds always remained at

350

the same statistical level (p > 0.05) after incubation of a same period, which indicated

351

that Caco-2 cells possess the same absorption affinity toward HMF and HML.

352

However, in our previous study, the HMF–cysteine adduct DCH22 showed less


Page 16 of 31

Page 17 of 31


353

absorption by Caco-2 cells compared with HMF after incubation for 4–24 h. About

354

4.51% DCH was absorbed by Caco-2 cells after 24 h treatment with 2 mM DCH,

355

whereas 36.5% HMF was absorbed after HMF treatment. Moreover, 91% of the

356

absorbed DCH disappeared upon absorption by Caco-2 cells and suggested to be

357

metabolized, of which 1% was transformed to HMF in intracellular fluids. Meanwhile,

358

HMF was detected neither in the extracellular fluid nor in the intracellular fluids in

359

the current study, indicating that HMF was not released from HML in Caco-2 cells

360

under the incubation conditions applied. However, the intracellular HML levels

361

(varied from undetectable level to 0.70 nmol/well) were considerably lower than the

362

absorbed amounts (180–250 nmol/well). Thus, we hypothesized that HML in Caco-2

363

cells was converted to other compounds. Further investigations of the in vitro and in

364

vivo metabolic pathways of these HMF–amino acid adducts should be conducted to

365

completely understand the metabolic route of these novel compounds and their safety.

366

HMF, a potential food-borne hazard, can easily form adducts with various amino

367

acids in food materials during the thermal processing or gastrointestinal digestion of

368

foods. Despite previous investigation on the HMF–cysteine adduct DCH, the safety of

369

the HMF–amino acid adducts and their absorption in the gastrointestinal tract was still

370

hardly evaluated. The present research evaluated the cytotoxicity of the HMF–lysine

371

Schiff base adduct HML against representative epithelial cells in the gastrointestinal

372

tract and endothelial cells in the blood vascular system. Results indicated that the

373

formation of HML remarkably lowered the toxicity of HMF against all the tested cell

374

lines. Moreover, HMF and HML were equally absorbed by Caoco-2 cells and largely



Page 18 of 31

375

metabolized to other compounds upon absorption. Further investigations on other

376

HMF–amino acid adducts and their absorption and metabolism in the digestion tract

377

and related target organs should be carried out to completely understand the potential

378

safety risks of these adducts.

379 380

ABBREVIATIONS USED

381

ANOVA, analysis of variance; ATCC, American type culture collection; CCK-8, cell

382

counting kit-8; Lys, cysteine; DCH, 1-dicysteinethioacetal-5-hydroxymehtylfurfural;

383

DMEM, Dulbecco’s modified Eagle’s medium; EDTA, ethylene diamine tetraacetic

384

acid;

385

(E)-N6-((5’-(hydroxymethyl)furan-2’-yl)methylene)lysine;

386

performance liquid chromatography-diode array detection; HRMS, high-resolution

387

mass spectrometry; IC50, half maximal inhibitory concentration; MRM, multiple

388

reaction monitoring; NMR, nuclear magnetic resonance; PBS, phosphate buffered

389

saline; PI, propidium iodide; RIPA, radio-immunoprecipitation assay; SMF,

390

5-sulfoxymethyfurfural.

391 392

AUTHOR INFORMATION

393

Corresponding Authors

394

*Tel.: +86 20 85226630; E-mail: [email protected] (J.Z.).

395

*Tel: +86 20 39366697; E-mail: [email protected] (X.Y.).

396

ORCID

397

Zheng Jie: 0000-0001-9755-5595

398

Shiyi Ou: 0000-0002-6779-0858

FBS,

fetal

bovine

serum;

HMF,

5-hydroxymethylfurfural;


HPLC-DAD,

HML, high

Page 19 of 31


399

Funding

400

This work was financially supported by the National Natural Science Foundation of

401

China (31671957; 81603165; 31972180), and Department of Science and Technology

402

of Guangdong Province (2018B050502008).

403

Notes

404 405

The authors declare no competing financial interest.

406

SUPPORTING INFORMATION

407

HPLC chromatogram detected at 205 nm of reaction mixture of 50 mM HMF and 100

408

mM lysine reacted at 160 ℃ for 25 min under pH 11 (Figure S1); High-resolution

409

full scan mass spectra of HMF–Lys Schiff base detected in positive mode (Figure

410

S2).

411 412



413

REFERENCE

414

(1) Ames, J. M. The Maillard reaction. In Biochemistry of Food Proteins; Hudson, B.

415

J. F., Ed.; Springer: Boston, MA, 1992; pp. 99−153.

416

(2) Capuano, E.; Fogliano, V. Acrylamide and 5-hydroxymethylfurfural (HMF): A

417

review on metabolism, toxicity, occurrence in food and mitigation strategies.

418

LWT-Food Sci. Technol. 2011, 44, 793−810.

419

(3) Murkovic, M.; Pichler, N. Analysis of 5-hydroxymethylfurfual in coffee, dried

420

fruits and urine. Mol. Nutr. Food Res. 2006, 50, 842−846.

421

(4) Ameur, L.; Mathieu, O.; Lalanne, V.; Trystram, G.; Birlouezaragon, I.

422

Comparison of the effects of sucrose and hexose on furfural formation and browning

423

in cookies baked at different temperatures. Food Chem. 2007, 101, 1407−1416.

424

(5) Shapla, U. M.; Solayman, M.; Alam, N.; Khalil, M. I.; Gan, S. H.

425

5-Hydroxymethylfurfural (HMF) levels in honey and other food products: effects on

426

bees and human health. Chem. Cent. J. 2018, 12, 35.

427

(6) Monien, B. H.; Frank, H.; Seidel, A.; Glatt, H. Conversion of the common food

428

constituent 5-hydroxymethylfurfural into a mutagenic and carcinogenic sulfuric acid

429

ester in the mouse in vivo. Chem. Res. Toxicol. 2009, 22, 1123−1128.

430

(7) De la Cueva, S. P.; Álvarez, J.; Végvári, Á.; Montilla-Gómez, J.; Cruz-López, O.;

431

Delgado-Andrade, C.; Rufián-Henares, J. A. Relationship between HMF intake and

432

SMF formation in vivo: An animal and human study. Mol. Nutr. Food Res. 2017, 61,

433

1600773.

434

(8) Kavousi, P.; Mirhosseini, H.; Ghazali, H.; Ariffin, A. A. Formation and reduction

435

of 5-hydroxymethylfurfural at frying temperature in model system as a function of


Page 20 of 31

Page 21 of 31


436

amino acid and sugar composition. Food Chem. 2015, 182, 164−170.

437

(9) EFSA (European Food Safety Authority). Opinion of the scientific panel on food

438

additives, flavourings, processing aids and materials in contact with food (AFC) on a

439

request from the commission related to Flavouring Group Evaluation 13 (FGE.13);

440

Furfuryl and furan derivatives with and without additional side-chain substituents and

441

heteroatoms from chemical group 14. EFSA J. 2005, 215, 1−73.

442

(10) Hamzalıoğlu, A.; Gökmen, V. Formation and elimination reactions of

443

5-hydroxymethylfurfural during in vitro digestion of biscuits. Food Res. Int. 2017, 99,

444

308−314.

445

(11) Hamzalıoğlu, A.; Gökmen, V. Investigation and kinetic evaluation of the reactions

446

of hydroxymethylfurfural with amino and thiol groups of amino acids. Food Chem.

447

2018, 240, 354−360.

448

(12) Zou, Y. Y.; Pei, K. H.; Peng, X. C.; Bai, W. B.; Huang, C. H.; Ou, S. Y. Possible

449

adducts formed between hydroxymethylfurfural and selected amino acids.; and their

450

release in simulated gastric model. Int. J. Food Sci. Technol. 2016, 51, 1002−1009.

451

(13) Qi, Y. J.; Zhang, H.; Zhang, H.; Wu, G. C.; Wang, L.; Qian H. F.; Qi, X. G.

452

Epicatechin adducting with 5‑hydroxymethylfurfural as an inhibitory mechanism

453

against acrylamide formation in Maillard reactions. J. Agric. Food Chem. 2018, 66,

454

12536−12543.

455

(14) Michalak, J.; Gujska, E.; Czarnowska, M.; Klepacka, J.; Nowak F. Effect of

456

storage on acrylamide and 5-hydroxymethylfurfural contents in selected processed

457

plant products with long shelf-life. Plant Food. Hum. Nutr. 2016, 71, 115−122.



458

(15) Zhao, Q. Z.; Ou, J. Y.; Huang, C. H.; Qiu, R. X.; Wang, Y.; Liu, F.; Zheng, J.; Ou,

459

S. Y. Absorption of 1-dicysteinethioacetal-5-hydroxymehthylfurfural (DCH) in rats,

460

and its effect on oxidative stress and gut microbiota. J. Agric. Food Chem. 2018, 66,

461

11451−11458.

462

(16) Cai, Y.; Zhang, Z. H.; Jiang, S. S.; Yu, M.; Huang, C. H.; Qiu, R. X.; Zou, Y. Y.;

463

Zhang, Q. R.; Ou, S. Y.; Zhou, H.; Wang, Y.; Bai, W. B.; Li, Y. Q. Chlorogenic acid

464

increased acrylamide formation through promotion of HMF formation and

465

3-aminopropionamide deamination. J. Hazard. Mater. 2014, 268, 1−5.

466

(17) Gökmen, V.; Kocadağlı, T.; Göncüoğlu, N.; Mogol, B. A. Model studies on the

467

role of 5-hydroxymethyl-2-furfural in acrylamide formation from asparagine. Food

468

Chem. 2012, 132, 168−174.

469

(18) Elmore, J. S.; Koutsidis, G.; Dodson, A. T.; Mottram, D. S.; Wedzicha, B. L.

470

Measurement of acrylamide and its precursors in potato, wheat, and rye model

471

systems. J. Agric. Food Chem. 2005, 53, 1286–1293.

472

(19) Mustafa, A.; Aman, P.; Andersson, R.; Kamal-Eldin, A. Analysis of free amino

473

acids in cereal products. Food Chem. 2007, 105, 317–324.

474

(20) Flodin, N. W. Lysine supplementation of cereal foods: a retrospective. J. Am. Coll.

475

Nutr. 1993, 12, 486−500.

476

(21) Friedman, M.; Levin, C. E. Review of Methods for the Reduction of Dietary

477

Content and Toxicity of Acrylamide. J. Agric. Food Chem. 2008, 56, 6113–6140.

478

(22) Zhao, Q. Z.; Zou, Y. Y.; Huang, C. H.; Lan, P.; Zheng, J.; Ou, S. Y. Formation of a

479

hydroxymethylfurfural−cysteine adduct and its absorption and cytotoxicity in Caco‑2


Page 22 of 31

Page 23 of 31


480

cells. J. Agric. Food Chem. 2017, 65, 9902–9908.

481

(23) Sampath, C.; Zhu, Y.; Sang, S.; Ahmedna, M. Bioactive compounds isolated from

482

apple, tea, and ginger protect against dicarbonyl induced stress in cultured human

483

retinal epithelial cells. Phytomedicine 2016, 23, 200−213.

484

(24) Nikolov,

485

5-hydroxtmethyl-2-furaldehyde (HMF) and its further transformations in the presence

486

of glycine. J. Agric. Food Chem. 2011, 59, 10104–10113.

487

(25) André, I.; Linse, S.; Mulder, F. A. A. Residue-specific pKa determination of

488

lysine and arginine side chains by indirect

489

Application to apo calmodulin. J. Am. Chem. Soc. 2007, 129, 15805−15813

490

(26) Carrillo, E.; Ramírez-Rivera, S.; Bernal, G.; Aquea, G.; Tessini, C.; Thomet, F. A.

491

Water-soluble Ru(II)-anethole compounds with promising cytotoxicity toward the

492

human gastric cancer cell line AGS. Life Sci. 2019, 217, 193−201.

493

(27) Moyes, S. M.; Morris, J. F.; Carr, K. E. Culture conditions and treatments affect

494

Caco-2 characteristics and particle uptake. Int. J. Pharmacol. 2010, 387, 7−18.

495

(28) Wang,

496

TNF-alpha-induced oxidative stress and endothelial dysfunction in EA.hy926 cells is

497

prevented by mate and green coffee extracts, 5-caffeoylquinic acid and its microbial

498

metabolite, dihydrocaffeic acid. Int. J. Food Sci. Nutr. 2019, 70, 267-284.

P.

S.;

Y.;

Sarria

Yaylayan,

B.;

Mateos

V.

R.;

A.

15

Thermal

N and

Goya,

499


13

L.;

decomposition

of

C NMR spectroscopy:

Bravo-Clemente,

L.


500

Figure captions

501

Figure 1. Effects of pH (A), temperature (B), reaction time (C), and concentration

502

ratio of HMF:Lys (D) on the elimination of HMF. Error bars represent the standard

503

deviations of the mean of three replicates (n = 3). For the concentration ratio,

504

equivalent of 1 refers to 50 mM in concentration.

505

Figure 2. Chemical structure and 1H (500 MHz) and 13C (125 MHz) NMR data of the

506

HMF–Lys Schiff base in D2O.

507

Figure 3. Effect of different concentrations of HMF and HML on the viability of

508

GES-1, Caco-2, and EA.hy926 cells, respectively, after incubation for 24 and 48 h.

509

Different letters indicate significant differences (p < 0.05) between different

510

treatments. * indicates significant differences (p < 0.05) between IC50 values of HMF

511

and HML treatments at 24 and 48 h, respectively.

512

Figure 4. Apoptosis in Caco-2 cells after 48 h treatment with various concentrations

513

of HMF and HML.

514 515


Page 24 of 31

Page 25 of 31


516 517

518 519

Table 1. Contents of HMF and HML in different bakery foods Sample

pH

HMF (mg/kg)

HML (μg/kg)

Biscuit

7.24 ± 0.14d

8.98 ± 0.02a

1.9 ± 0.1c

Bread

5.66 ± 0.03a

9.94 ± 0.90ab

0.3 ± 0.1ab

Cookie 1

6.99 ± 0.07c

10.76 ± 0.21b

0.7 ± 0.2b

Cookie 2

6.29 ± 0.03b

12.54 ± 1.10c

0.2 ± 0.0ab

Cake

7.13 ± 0.09cd

9.54 ± 0.19ab

0.1 ± 0.0a

Different letters in upper cases indicate significant differences (p < 0.05) between different samples.



Page 26 of 31

Table 2. Extracellular and intracellular levels of HMF and HML in Caco-2 cells after different incubation time Treatment

HMFcontent (μmoL/well)

HML content (μmoL/well)

Incubation time (min)

Extracellular

Intracellular (×10 )

Extracellular

Intracellular (×10-3)

2 mM HMF

30 60 90 120 150

1.84 ± 0.02bc 1.86 ± 0.04c 1.78 ± 0.08ab 1.79 ± 0.01abc 1.75 ± 0.06a

ND ND 0.06 ± 0.05 0.07 ± 0.05 0.11 ± 0.07

— — — — —

— — — — —

2 mM HML

30 60 90 120 150

— — — — —

— — — — —

1.82 ± 0.05abc 1.77 ± 0.04ab 1.75 ± 0.01a 1.75 ± 0.04a 1.75 ± 0.02a

ND ND 0.40 ± 0.00 0.70 ± 0.00 0.50 ± 0.10

-3

Different letters in upper cases indicate significant differences (p < 0.05) between different treatments.



60 40 20 0 2

4

6 pH

8

10

12

80 60 40 20 0 70 80 90 100 110 120 130 140 150 160 170 180 190 Temperature (ºC）

14

C

100 Eliminaton of HMF (%)

Elimination of HMF (%)

80

0

B

100

A

100

Elimination of HMF (%)

Elimination of HMF (%）

Page 27 of 31

80 60 40 20

100

D

80 60 40 20 0

0 5

10

15

20 Time (min)

25

30

35

Figure 1.


2:1

1:1 1:2 1:3 1:4 Concentration ratio of HMF: Lys


Position 1 2 3 4 5 6 1’ 2’ 3’ 4’ 5’ 5’-CH2

δC (ppm) 175.21 s 54.59 d 30.24 t 21.52 t 30.26 t 56.29 t 135.31 d 137.67 s 134.53 d 128.70 d 165.76 s 58.85 t

δH (ppm)

1

H-1HCOSY

3.60 (t, J = 6.36 Hz) 1.79 (m) 1.39 (m) 1.90 (m) 4.30 (t, J = 7.74 Hz) 7.67 (d, J = 2.76 Hz)

H-3 H-2, 4 H-3, 5 H-4, 6 H-5

7.32 (dd, J = 8.94, 2.76 Hz) 7.47 (d, J = 8.94 Hz)

H-4’ H-3’

4.70 (m)

Figure 2.


Page 28 of 31

HMBC H-2 H-3 H-2, 5 H-2, 6 H-6 H-1’ H-6 H-6, 1’, 3’, 4’, 5’-CH2 H-1’ H-3’, 5’-CH2 H-4’ H-4’

Page 29 of 31


Figu ure 3.



F Figure 4.


Page 30 of 31

Page 31 of 31


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