and Semifermented Teas Analyzed by LC-MS-Based Nontargeted

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Effect of copper on fatty acid profiles in non- and semifermented tea analyzed by LCMS-based non-targeted screening Marc Pignitter, Klaus Stolze, Franz Jirsa, Lars Gille, Bernard A. Goodman, and Veronika Somoza J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02792 • Publication Date (Web): 03 Sep 2015 Downloaded from http://pubs.acs.org on September 10, 2015

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Journal of Agricultural and Food Chemistry

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Effect of copper on fatty acid profiles in non- and semi-fermented tea analyzed

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by LCMS-based non-targeted screening

3 4

Marc Pignitter1,*, Klaus Stolze2, Franz Jirsa3,4, Lars Gille5, Bernard A. Goodman6,

5

Veronika Somoza1

6 1

7

Department of Nutritional and Physiological Chemistry, Faculty of Chemistry, University of Vienna, 1090 Vienna, Austria

8 2

9

Veterinary Medicine Vienna, 1210 Vienna, Austria

10 3

11 12

4

Department of Inorganic Chemistry, University of Vienna, 1090 Vienna, Austria

Department of Zoology, University of Johannesburg, P. O. Box 524, Auckland Park, 2006 South Africa

13 14

5

Institute of Pharmacology and Toxicology, University of Veterinary Medicine Vienna, 1210 Vienna, Austria

15 16 17

Institute of Animal Nutrition and Functional Plant Compounds, University of

6

State Key Laboratory for Conservation and Utilization of Subtropical AgroBioresources, Guangxi University, 530004 Nanning, Guangxi, China

18 19 20

*Correspondence: Marc Pignitter, Althanstraße 14, 1090 Vienna, Austria. Tel: +43 1 4277 70621, fax: +43 1 4277 9706, e-mail: [email protected]

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Abstract

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Unsaturated fatty acids are well-known precursors of aroma compounds, which are

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considered important for green tea quality. Due to the known copper-induced

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oxidation of unsaturated fatty acids and the broad variability of the amount of copper

25

being present in tea infusion, this paper investigates the influence of copper, added

26

at a non-toxic concentration (300 µM) to non- and semi-fermented teas, on the

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degradation of fatty acids and fatty acid hydroperoxides thereof. The abundance of

28

fatty acids in green and oolong tea was determined by means of a non-targeted

29

approach applying high-resolution MS/MS. As a result, most of the fatty acids in

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green and oolong tea were already oxidized prior to copper addition. Addition of 300

31

µM CuSO4 to the oolong tea sample resulted in a decrease of 13-hydroperoxy-

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9Z,11E-octadecadienoic acid, an important flavor precursor, from 0.12 ± 0.02 µM to

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0.05 ± 0.01 µM (p=0.035), while other oxidized fatty acids decreased as well.

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However, copper-induced degradation of oxidized fatty acids was less pronounced in

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green tea compared to oolong tea most likely due to the formation of copper

36

complexes with low-molecular weight compounds as evidenced by electron

37

paramagnetic resonance spectroscopy.

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Key

39

epigallocatechin-3-gallate

words:

tea;

fatty

acid;

copper;

non-targeted

screening;

polyphenol;


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Introduction

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The tea infusion prepared from plant leaves of Camellia sinensis L. is, next to water,

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the most consumed beverage worldwide.1 The three major tea types, known as

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black, oolong and green tea, differ in their post-harvest processing and degree of

44

fermentation. Polyphenols, such as catechins, make up ~30% (w/w) of the solids of

45

tea leaves.1 They also, along with many minor components, such as fatty acids,

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contribute to the taste of tea.2 The proportion of lipids in fresh tea leaves is

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approximately 4-9% of the total dry weight.3,4 Lipids are also well known precursors

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of flavor-associated compounds of tea infusions.5 The degradation of unsaturated

49

fatty acids during tea production leads to the formation of volatile aroma compounds.

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One of the most abundant fatty acid found in tea infusions is linoleic acid. Oxidation

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of linoleic acid might lead to the formation of (Z)-3-hexenal, which is associated with

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a grassy odor.6 Thus, the fatty acids and their volatile oxidation products contribute to

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the aroma characteristics of tea infusions.

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In fermented teas, the susceptibility of tea constituents to oxidation is determined by

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the degree of fermentation during processing. While catechin-rich green tea does not

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undergo fermentation, oolong tea is partially fermented, yielding a mixture of

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monomeric polyphenols and high-molecular weight polymeric polyphenols, such as

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theaflavins.1 Also, lipoxygenase-catalyzed oxidation of unsaturated fatty acids has

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been reported to occur during the fermentation of teas.7

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Oxidation of fatty acids is catalyzed by trace amounts of transition metals, such as

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copper.8 Copper contents of tea infusions have been reported to be in the range of

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0.019-65.4 mg/kg, depending on the country of origin, the type of tea, the application

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of copper-containing fungicides, and the equipment used during processing.9,10,11,12



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Copper might promote the formation of advanced lipid oxidation end products in tea

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infusions, thereby influencing the flavor quality of tea.13

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To the best of our knowledge, there is no literature available on the effects of copper

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on the fatty acid profile in tea infusions. Thus, the aim of the current study was to

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investigate the effect of copper, as CuSO4, on the fatty acid profile of tea infusions

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prepared from non-fermented green tea or semi-fermented oolong tea, that are likely

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to differ in their composition. In this non-targeted screening approach, the abundance

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of the fatty acids and fatty acid oxidation products was identified by high-resolution

72

MS and MS/MS.

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Materials and Methods

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Materials and chemicals

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Non-fermented leaves of C. sinensis, described as green tea, were obtained from a

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local supermarket in Guangxi province, China. The King’s oolong tea sample

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(originally from Gaoshan Xuefeng, Fujian, China) was obtained from Teahub,

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Germany. Both samples were stored under vacuum prior to analysis. All other

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chemicals were purchased from Sigma Aldrich, Vienna, Austria and Carl Roth,

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Karlsruhe, Germany.

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Sample preparation

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Green and king’s oolong tea infusions were prepared by first adding 50 mL double

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distilled, boiling water to one gram of dried leaves.14 After ten minutes, the infusion

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was re-filled with water to consider evaporated water and was passed through a

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Whatman cellulose filter with a pore size of 11 µm, and a polyvinylidene fluoride

86

syringe filter with a pore size of 0.45 µm. Additionally, samples were prepared to

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which 50 µL CuSO4 (300 mM) was added to freshly prepared tea infusions (50 mL) to 4 ACS Paragon Plus Environment

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yield a 300 µM surplus in copper concentration. After filtration, the samples were

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stored at -20°C prior to analysis.

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LCMS analyses

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Analyses of the fatty acid profiles of the tea samples were performed on a Nano-LC

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system (Dionex Ultimate 3000 RSLC, Thermo Fisher Scientific, Vienna, Austria)

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coupled to a Nano-ESI-LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific,

94

Vienna, Austria). The trap column (Acclaim PepMap 100, 100 µm x 2 cm nanoViper,

95

C18, 5 µm, Thermo Fisher Scientific, Vienna, Austria) was loaded with 5 µL of

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sample, which were separated on a nano LC column (Acclaim PepMap 100, 75 µm x

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15 cm nanoViper, C18, 3 µm, Thermo Fisher Scientific, Vienna, Austria).

98

columns were maintained at 35°C. The gradient program started with 3% acetonitrile

99

in water acidified with 0.1% formic acid (pH 2.69), and increased to 100% acidified

100

acetonitrile (pH 1.66) after 40 min at a flow rate of 300 nL/min. The ESI-MS was

101

operated in the negative mode, scanning a mass range of 100-1000 m/z. Ionization

102

of fatty acids as carboxylate anions might be facilitated in the ESI(-) mode.15 The

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capillary temperature and the capillary voltage were set at 300°C and 3.0 kV,

104

respectively. For structural identification, MS/MS experiments were performed by

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applying higher energy collisional dissociation with a normalized collision energy of

106

30%. To avoid cross-contamination, water was run as blank after each sample.

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Quantitation of 13-hydroperoxy-9Z,11E-octadecadienoic acid in tea samples was

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performed by external calibration using standard concentrations in the range of 0.01-

109

1 µM. The ion peak with a m/z of 457.0768 was identified as epigallocatechin-3-

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gallate, which eluted after 22.8 min, and showed characteristic fragments at m/z

111

305.067 (gallocatechin) and m/z 169.0143 (gallic acid). The ion peak at 457 m/z was

The



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used to quantitate epigallocatechin-3-gallate in tea by applying the external

113

calibration method.

114

Synthesis of 13-hydroperoxy-9Z,11E-octadecadienoic acid

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The synthesis of 13-hydroperoxy-9Z,11E-octadecadienoic acid was performed with

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slight modifications according to Schieberle et al.16 Briefly, 0.64 mmol linoleic acid

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were emulsified with 8 mL of 0.1% Tween-80 and approximately 2 mL of 1 M NaOH.

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The pH was adjusted to 9.0 after addition of 190 mL of 0.02 M sodium borate buffer

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(pH 9.0). After cooling on ice, the solution was saturated with oxygen and vigorously

120

stirred for 5 min. To start the enzymatic reaction, 4.2 mg soy lipoxygenase type I

121

dissolved in 1 mL of borate buffer were added to the substrate. The solution was then

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vigorously stirred and bubbled with oxygen for 2 hours. The reaction was stopped by

123

adding 2 M HCl to adjust the pH to 3.0. The reaction mixture was extracted three

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times with 200 mL diethyl ether, and the pooled extracts were washed with 200 mL

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double distilled water and dried with Na2SO4. The solvent was removed by rotating

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evaporation. A purity of 98% was determined by mass spectrometry. The

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synthesized product, 13-hydroperoxy-9Z,11E-octadecadienoic acid, was identified by

128

means of ESI(-)MS (m/z 311 and 293) and NMR. 1H-NMR (500 MHz, (CD3)2SO): δ

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(ppm) 4.40 [q, 1 H, CH, C(13)], 5.42 [m, 1 H, J = 11.1Hz; CH, C(9)], 5.59 [dd, 1 H, J =

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15.0Hz; CH, C(12)], 5.98 [t, 1 H, J = 11,1Hz; CH, C(10)], 6.46 [dd, 1 H, J = 15.0Hz;

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CH, C(11)], 13C-NMR (500 MHz; (CD3)2SO, HMBC): δ (ppm) 14.3 [CH, C(18)], 85.1

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[CH, C(13)], 128 [CH, C(11)], 129 [CH, C(10)], 133 [CH, C(9)], 134 [CH, C(12)], 175

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[COOH, C(1)].17

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Quantitation of copper in tea samples


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Total copper concentrations in tea samples were determined using a PinAAcle 900Z

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graphite furnace atomic absorption spectrometer (Perkin Elmer, Vienna Austria) as

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described previously.18 A volume of 8 mL of 34% HNO3 (TraceSELECT® Fluka) was

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added to 1 mL of each tea sample and heated up to 180°C in a microwave MARS

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XPRESS system (CEM Corporation, Kamp-Lintfort, Germany). After treatment in the

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microwave oven, double distilled water was added, leading to a final volume of 20 mL

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which was passed through a 0.2 µm polytetrafluoroethylene filter prior to

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measurement. The limit of detection for copper was determined to be 0.2 µg/L.

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EPR measurement of copper complex

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Tea extracts (1g/50ml H2O, 100°C, 10min) were incubated with Cu(II) (300 µM) at pH

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7.5 and recorded by a Bruker ESP300e EPR spectrometer at room temperature,

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using a TM110 resonator ER 4103 equipped with a quartz flat cell using the following

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parameters: center field 3203.5 G, field range 1507 G; microwave frequency 9.796

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GHz; microwave power 20 mW; modulation frequency 100 kHz; modulation

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amplitude 10.8 G; receiver gain 1 x 104 ; time constant 81.9 msec; conversion time

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81.9 msec; 1024 data points; 5 scans accumulated.

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Data processing

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The statistical analysis program XCMS online19 was applied to identify (i) the fatty

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acid profile in the tea samples by comparing tea infusions with the water blank, (ii)

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the difference with regard to fatty acids between green and oolong teas, and (iii) the

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differences of fatty acid patterns between untreated and copper-treated tea samples.

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Prior to data processing, the raw data files were converted to the mzXML format

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using MSConvert.20 The centWave algorithm was applied for highly sensitive feature

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detection.21 A maximally tolerated m/z deviation in consecutive runs of 2.5 ppm and a 7 ACS Paragon Plus Environment


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peak width range of 10-90 s were chosen for feature detection. Retention time

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correction was performed with the OBI-Warp method.22 Chromatographic alignment

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of the mass spectrometry data was defined by (i) a minimum fraction of samples of

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0.5 in at least one of the sample groups, (ii) a 5 s bandwidth of the Kernel smoother,

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and (iii) a 0.015 m/z width of overlapping m/z slices. To minimize the false discovery

164

rate for peak annotations of isotopes and adducts, the ppm error and the m/z

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absolute error were set to 5 ppm and 0.015 m/z, respectively. The METLIN

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metabolomics database23,24 was consulted for structural identification of [M-H]-

167

adducts. The tolerance for this database search was set to 5 ppm.

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Statistical analysis

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All experiments were performed three to four times independently with two technical

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replicates each. The principal component analysis (PCA) was performed to assess

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any effects of copper on the fatty acid profile. For identifying the fatty acid profiles,

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the copper-induced changes in the tea samples and the differences between tea

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samples in fatty acid profiles, the unpaired Welch t-test was applied. A difference was

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considered significant when the p-value was ≤ 0.01. The signal intensity was ≥ 105 or

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≥ 106, as indicated in the table and figure legends, and at least two-fold changes

176

were detected. Differences in copper concentrations between the oolong and green

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teas were determined by Kruskal-Wallis one way ANOVA on Ranks, where a p-value

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of < 0.05 was considered significant. Differences between non- and copper-treated

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tea samples with regard to 13-hydroperoxy-9Z,11E-octadecadienoic acid were

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calculated by one way ANOVA where also a p-value of < 0.05 was considered

181

significant.

182 183

Results and discussion 8 ACS Paragon Plus Environment

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Fatty acid profile in green and oolong tea

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Tables 1 and 2 show the most abundant fatty acids in the green and oolong tea

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samples, as identified by high-resolution MS and MS/MS fragmentation. A total of 13

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fatty acids were identified in the green tea, but only six fatty acids with a peak

189

intensity of at least 106 a.u. were detected in the oolong tea sample. In both, the

190

green and oolong tea samples, the vast majority of the fatty acids was already

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oxidized, and appeared as hydroxy- or hydroperoxy derivatives of medium- and long-

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chain saturated and unsaturated fatty acids. By comparing the fatty acid profiles of

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the green and oolong tea samples, five oxidized fatty acids were identified as having

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a more than two-fold higher abundance in green tea than in oolong tea, although

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more oxidized fatty acids were expected in the semi-fermented oolong tea. The most

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pronounced difference between the green and oolong tea with regard to the oxidized

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fatty acids was obtained for hydroperoxy octadecatrienoic acid and hydroxy

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octadecatrienoic acid. An at least two-fold higher abundance of hydroxy undecenoic

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acid (2.3 fold), hydroxy nonenoic acid (4.4 fold), hydroperoxy octadecadienoic acid

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(2.3 fold), trihydroxy octadecadienoic acid (3.4 fold), hydroperoxy octadecatrienoic

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acid (16 fold) and hydroxy octadecatrienoic acid (38 fold) could be shown in the

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green tea compared to the oolong tea sample.

203 204

Copper content in green and oolong tea

205

To explain the higher abundance of oxidized fatty acids in non-fermented green tea,

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which was not subjected to oxidation after harvest, the amount of copper was

207

quantitated. The total copper concentration represents the maximum concentration of 9 ACS Paragon Plus Environment


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copper, which might, however, be partially bound to traces of tea proteins or amino

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acids, thereby restricting its participation in redox reactions. It could be shown that

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copper bound to proteins or amino acids led to a reduced formation of reactive

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oxygen species compared to free copper.25 In the current study, the total copper

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concentrations in the green tea and oolong tea infusions were determined by atomic

213

absorption spectroscopy to be 1.52 ± 0.38 µM and 0.25 ± 0.05 µM, respectively

214

(Figure 1). Thus, the approximately six-fold higher copper concentration in the green

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tea compared to the oolong tea (p < 0.05) may be an explanation for the higher

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abundance of oxidized fatty acids in this sample. Gallaher et al.26 reported a relatively

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narrow concentration range of 0.79 ± 0.05 µM copper in various teas, whereas other

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researchers quantified larger differences among different tea varieties. For example,

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Wong et al.11 quantitated a concentration of 0.96 ± 0.17 µM copper in a 1% aqueous

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extract green tea, but only 0.46 ± 0.39 µM in a similar extract of oolong tea.

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Similarly, Xie et al.12 also determined copper contents of 1.14 ± 0.38 µM and 0.49 ±

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0.04 µM, respectively, in 2% aqueous extracts of green and oolong teas. Thus, the

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higher concentration of copper in the green tea sample compared to the oolong tea

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sample of the present work was not unexpected. As reported previously, the

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concentration of copper in a tea infusion was shown to be more than 8 times higher

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than the concentration of iron.27 Other factors may also influence the copper contents

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of tea infusions. Soil pH influences the uptake of trace metals by tea plants,28 and

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elements including copper are more readily taken up from acidic soils.10,28

229 230

Effect of copper addition on the fatty acid profiles in green and oolong tea samples

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In addition to copper in tea infusions, copper in foods consumed together with tea

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might also affect the profiles of fatty acids and oxidized fatty acids in tea infusions. 10 ACS Paragon Plus Environment

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For example, 28.3 g of beef liver contains 4.13 mg copper.29 If this amount is

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consumed together with 250 mL tea, a total amount of approximately 16 mg (about

235

260 µM) of copper would result in the stomach. Thus, to model the effect of copper

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from other food or non-food sources on the fatty acid profiles of green and oolong

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tea, 300 µM CuSO4 were added to freshly prepared tea infusions. It was also aimed

238

to simulate a copper enrichment occurring, for example, as a result of the use of

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copper-containing pesticides, fertilizers or equipment during tea production.10,13 PCA

240

analysis was performed to determine any effects of copper on the fatty acid profile.

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Figure 2 demonstrates a distinct difference between copper-enriched tea and tea

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without copper addition. By applying non-targeted profiling, at least two-fold changes

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in the abundance of five fatty acids with a peak intensity of a minimum of 105 a.u.

244

were caused by the addition of copper to the green tea sample (Table 3). Compared

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to the untreated sample, copper supplementation of the green tea resulted in

246

significant

247

octadecatetraenoic acid (22-fold decrease) and hydroxy octadecatrienoic acid (3.3-

248

fold decrease), whereas methyl hydroperoxy bis-epidioxy eicosadienoate (3.0-fold

249

increase) and isopropyl hexanoic acid (2.2.-fold increase) were increased. In the

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oolong tea sample, twelve fatty acids with a peak intensity of ≥ 105 a.u. showed at

251

least a two-fold change as a result of copper addition (Table 4). Most of these were

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oxidized fatty acids such as hydroxy- and hydroperoxy derivatives. Ten of these were

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significantly reduced and only hydroxy nonanoic acid and hydroxy-decenoic acid

254

were increased. The types of fatty acids affected by the addition of copper to oolong

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tea were comparable to those in copper-supplemented green tea. The long-chain

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unsaturated fatty acids were decreased after addition of copper to oolong tea, the

257

most pronounced effect being observed with hydroxy octadecadienoic acid (49-fold

258

decrease). Overall, addition of copper resulted in more pronounced changes in the

decreases

of

methyl

tetradecadioic

acid

(5.8-fold

decrease),



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fatty acid profile in the oolong tea than in green tea. The different susceptibility to

260

oxidation of the fatty acids in green and oolong tea as a result of the copper addition

261

was

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octadecadienoic acid (Figure 3). Whereas copper addition did not induce significant

263

changes in the concentration of 13-hydroperoxy-9Z,11E-octadecadienoic acid in

264

green tea, it resulted in a clear reduction from 0.12 ± 0.02 µM to 0.05 ± 0.01 µM in

265

the oolong tea. This result suggests that green tea, but not oolong tea, contains

266

substances which might interact with copper to inhibit the copper-induced

267

degradation of the oxidized fatty acids.

also

observed

by

quantitative

analysis

of

13-hydroperoxy-9Z,11E-

268 269

Effect of addition of copper on the concentration of epigallocatechin-3-gallate in

270

green and oolong tea samples

271

To investigate a possible cause for the different susceptibilities of the green and

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oolong tea samples to copper-induced fatty acid oxidation, changes in the

273

abundance of epigallocatechin-3-gallate, the major polyphenol in untreated tea

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leaves, were also investigated for the green and oolong tea samples in the presence

275

and absence of 300 µM CuSO4. The green tea polyphenols, such as epicatechin,

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epicatechin-3-gallate, epigallocatechin and epigallocatechin-3-gallate, constitute 25 -

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35% of the dry weight of green tea leaves30,31,32,33 and represent approximately 50

278

mg/g tea infusion,34 whereas in black tea, the more highly condensed thearubigin and

279

theaflavin represent the main polyphenolic compounds.35 In the current study,

280

extracted

281

epigallocatechin-3-gallate, are illustrated in Figure 4. Epigallocatechin-3-gallate was

282

approximately two-fold higher in the green tea sample than in the oolong tea sample,

ion

chromatograms

for

m/z

457.0765-457.0780,

representing


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and is, thus, a candidate for mitigating the copper-induced degradation of oxidized

284

fatty acids in green tea.

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The antioxidative activity of tea flavanols has been demonstrated in several in vitro

286

and in vivo studies.36,37,38 Higdon and Frei39 reported a 2 - 15% increase in plasma

287

antioxidant activity after consumption of tea. In vitro experiments revealed that

288

epigallocatechin-3-gallate is a more effective radical scavenger than other major

289

green tea polyphenols.38 It has been proposed that catechin polyphenols exert their

290

antioxidant activities by chelating redox-active transition metals, such as copper,

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thereby preventing the formation of free radical and non-radical oxidation

292

products.40,41 Mira et al.41 showed that catechins chelate Cu(II) ions at the ortho-

293

catechol group at pH 7.4. Similar results were obtained by Yoshioka et al.42 who

294

confirmed the copper-coordinating activity of catechins by showing that 125 µM

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epigallocatechin-3-gallate formed a complex with 63 µM Cu(II) ions in phosphate

296

buffer. Furthermore, the green tea polyphenols gallic acid and epigallocatechin-3-

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gallate tend to form polymeric complexes with Cu(II) at acidic and neutral pH values,

298

and precipitate from pure solutions.43 However, in the present study, the

299

concentration of epigallocatechin-3-gallate was not only substantially decreased by a

300

factor of two in the green tea sample (252 ± 7.97 µM to 128 ± 4.00 µM) but also in

301

oolong tea sample (129 ± 4.07 µM to 59.3 ± 1.87 µM) after addition of 300 µM

302

CuSO4, suggesting that epigallocatechin-3-gallate was not the predominant

303

“antioxidant” source in the green tea sample.

304 305

EPR measurements of green and oolong tea with Cu(II) at neutral pH



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In the present study, the LCMS experiments did not reveal any copper-derived

307

compounds in the copper-treated teas, possibly because of instability of the

308

complexes during the ionization process in the mass spectrometer. However, EPR

309

measurements by Goodman et al.44 indicated the formation of a number of Cu(II)

310

complexes in copper supplemented teas at neutral and weakly acidic pH values.

311

Goodman et al.44 proposed involvement of tea-containing amino acids, such as

312

theanine, since the EPR results were inconsistent with the formation of soluble Cu(II)

313

chelates of epigallocatechin-3-gallate. Goodman et al.44 also observed a substantial

314

reduction in the EPR detectable Cu(II) in tea solutions at these pH values, a result

315

which is consistent with the formation of polymeric species or precipitates as

316

observed in reactions of Cu(II) with solutions of the green tea polyphenols gallic acid

317

and epigallocatechin-3-gallate.43,45 In the current study, the EPR spectra of all tea

318

extracts were dominated by a 6-line signal from solvated Mn(II) (not shown), which

319

has been discussed previously,44,46 and were subtracted from all spectra in order to

320

record the Cu(II) signals resulting from the addition of 300 µM of CuSO4 (Figure 5).

321

Both tea samples showed EPR signals at pH 7.5 that are similar to those reported

322

previously for green and black teas (aCu = 70 G; g = 2.138).44 The line width variation

323

in these spectra is the result of incomplete averaging of the anisotropy through

324

molecular motion, and is a normal phenomenon in the EPR solution spectra of Cu(II)

325

complexes. In addition, the better resolution of the spectrum from the green tea

326

sample compared to that from the oolong tea is indicative of more rapid molecular

327

motion, and hence a lower molecular mass complex. Since the EPR signals of the

328

individual Cu complexes could only be obtained after a series of spectral

329

subtractions, the reported values for the spectral parameters must be considered to

330

be approximate, since unequivocal assignment to a specific complex was not

331

possible. However, the parameters obtained from the green tea spectrum are 14 ACS Paragon Plus Environment

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consistent with Cu(II) bis(amino acid) complexes,47 and the bis complex of Cu(II) with

333

theanine showing solution spectral parameters of aCu = 70 G, g = 2.135.44

334

Polyphenols, on the other hand, tend to form EPR-silent complexes and precipitate in

335

solutions around neutral pH, and although a spectrum was observed with

336

Cu(II)/epigallocatechin-3-gallate solutions,43 it has greater line width anisotropy than

337

that observed with the green tea solution. Therefore, the spectrum in Figure 5A does

338

not correspond to a bis complex of Cu(II) with epigallocatechin-3-gallate, although

339

the involvement of catechins in mixed complexes with amino acids cannot be

340

discounted. This tentative assignment to mixed complexes is consistent with the

341

differences between the Cu(II) spectra from the green and oolong teas (Figure 5A

342

and 5B), because of the overall higher molecular mass of the polyphenolic

343

components in fermented teas.

344

Thus, green tea polyphenols and other low-molecular weight compounds, such as

345

amino acids, might help to protect the oxidized fatty acids from copper-induced

346

degradation (Figure 6). A redox reaction of oxidized fatty acids or polyphenols with

347

Cu(II) resulting in the formation of Cu(I) and peroxy radicals17,48,49 is illustrated in

348

Figure 6. Re-oxidation of Cu(I) by hydroperoxides produces alkoxy radicals, which

349

might further decompose into aldehydes.50 Formation of polymeric Cu(II) species by

350

reaction with green tea polyphenols43,45 or amino acids47 could remove Cu(II) from

351

the reaction cycle and, therefore, inhibit the degradation of oxidized fatty acids.

352

However, the higher copper concentration of the green tea sample and the higher

353

abundance of fatty acid oxidation products prior to copper addition might have

354

contributed to the less pronounced effect of copper administration compared to

355

oolong tea.



Page 16 of 31

356

Overall, the present study suggests that low concentrations of copper which occur

357

naturally in teas might contribute to the oxidation of tea fatty acids, whereas higher

358

concentrations of copper may lead to degradation of these oxidized fatty acids.

359

Future mechanistic studies should verify whether copper-induced decomposition of

360

oxidized fatty acids might be hampered by green tea polyphenols and amino acids.

361

Since fatty acids are among the precursors of aroma molecules, such reactions may

362

contribute to the aroma characteristics of tea infusions.

363 364

Acknowledgments

365

The authors want to thank Prof. Dr. Peter Schieberle, Head of the German Research

366

Centre for Food Chemistry in Freising, Germany, for carefully reading the manuscript

367

and providing constructive comments. We also gratefully acknowledge Prof. Dr.

368

Galanski for recording the NMR spectra.

369 370

Conflicts of interest

371

The authors declare that there are no conflicts of interest.

372


Page 17 of 31


373

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(41) Mira, L.; Fernandez, M. T.; Santos, M.; Rocha, R.; Florencio, M. H.; Jennings, K. R. Interactions of flavonoids with iron and copper ions: A mechanism for their antioxidant activity. Free Radic Res 2002, 36, 1199-1208. (42) Yoshioka, H.; Senba, Y.; Saito, K.; Kimura, T.; Hayakawa, F. Spin-trapping study on the hydroxyl radical formed from a tea catechin-Cu(II) system. Biosci Biotech Bioch 2001, 65, 1697-1706. (43) Pirker, K. F.; Baratto, M. C.; Basosi, R.; Goodman, B. A. Influence of pH on the speciation of copper(II) in reactions with the green tea polyphenols, epigallocatechin gallate and gallic acid. J Inorg Biochem 2012, 112, 10-6. (44) Goodman, B. A.; Severino, J. F.; Pirker, K. F. Reactions of green and black teas with Cu(II). Food Funct 2012, 3, 399-409. (45) Severino, J. F.; Goodman, B. A.; Reichenauer, T. G.; Pirker, K. F. Is there a redox reaction between Cu(II) and gallic acid? Free Radic Res 2011, 45, 115-24. (46) Pirker, K. F.; Severino, J. F.; Reichenauer, T. G.; Goodman, B. A. Free radical processes in green tea polyphenols (GTP) investigated by electron paramagnetic resonance (EPR) spectroscopy. Biotechnol Annu Rev 2008, 14, 349-401. (47) Goodman, B. A.; McPhail, D. B.; Powell, H. K. J. Electron spin resonance study of copper(II)–amino-acid complexes: evidence for cis and trans isomers and the structures of copper(II)–histidinate complexes in aqueous solution J. Chem. Soc., Dalton Trans 1981, 822-827. (48) Waters, W. A. Kinetics and Mechanism of Metal-Catalyzed Autoxidation. J Am Oil Chem Soc 1971, 48, 427-433. (49) Marino, D. C.; Sabino, L. Z.; Armando, J., Jr.; De Andrade Ruggiero, A.; Moya, H. D. Analysis of the polyphenols content in medicinal plants based on the reduction of Cu(II)/bicinchoninic complexes. J Agric Food Chem 2009, 57, 11061-6. (50) Patel, R. P.; Svistunenko, D.; Wilson, M. T.; Darley-Usmar, V. M. Reduction of Cu(II) by lipid hydroperoxides: Implications for the copper-dependent oxidation of low-density lipoprotein. Biochem J 1997, 322, 425-433.

512



Page 20 of 31

513

Figure captions

514

Figure 1. Concentration [µM] of copper in green and oolong tea infusion. Data are

515

expressed as mean ± SD (n=4). Different letters indicate statistically significant

516

differences between the tea samples (p < 0.05).

517

Figure 2. PCA scores plot obtained from green tea (A) and oolong tea (B) without or

518

with addition of 300 µM CuSO4.

519

Figure 3. Concentration of 13-hydroperoxy-9Z,11E-octadecadienoic acid in green

520

and oolong tea infusion with or without addition of 300 µM CuSO4. Data are

521

expressed as mean ± SD (n=4). The asterisk indicates statistically significant

522

difference versus control (p < 0.05).

523

Figure 4. Extracted ion chromatogram of epigallocatechin-3-gallate in green (A) and

524

oolong tea (B) samples in the presence or absence of 300 µM CuSO4 at a mass

525

range of m/z 457.0765-457.0780. Representative chromatogram of four experimental

526

and two technical replicates.

527

Figure 5. EPR measurements of copper complexes formed at pH 7.5 by adding 300

528

µM CuSO4 to green (A) and oolong (B) tea infusion.

529

Figure 6. Proposed reaction explaining the fate of oxidized fatty acids in the

530

presence of cupric sulfate in non- and semi-fermented tea.


Page 21 of 31


Table 1. Most abundant fatty acids with a peak intensity of ≥ 106 in a green tea infusion as identified by means of LCMS in a range of 100-1000 m/z. fatty acid

rt [min]

[M-H][m/z]

error [ppm]

nonanedioic acid (C9H16O4)

25.5

187.0971

2

125.0973 (C8H13O; octanone)

dodecenedioic acid (C12H20O4)

29.8

227.1285

1

183.1393 (C11H19O2; -CO2; undecenoic acid), 165.1287 (C11H17O; -H2O)

hydroxy nonenoic acid (C9H16O3)

28.9

171.1023

2

127.1130 (C8H15O; -CO2), 153.0924 (C9H13O2; -H2O)

hydroxy nonenediynoic acid (C9H8O3)

25.9

163.0396

3

119.0503 (C8H7O; -CO2)

hydroxy undecenoic acid (C11H20O3)

28.7

199.1335

2

155.1079 (C9H15O2; nonenoic acid)

hydroxy dodecanedioic acid (C12H22O5)

26.5

245.1389

2

185.1187 (C10H17O3; hydroxy decenoic acid)

hydroxy octadecatrienoic acid (C18H30O3)

36.8

293.2118

2

275.2025 (C18H27O2; -H2O), 235.1710 (C15H23O2)

dihydroxy palmitic acid (C16H32O4)

30.3

287.2224

1

187.1345 (C10H19O3; hydroxy decanoic acid)

trihydroxy stearic acid (C18H36O5)

29.5

331.2484

2

313.2407 (C18H33O4; -H2O), 287.0204 (C17H35O3; -CO2)

trihydroxy octadecenoic acid (C18H34O5)

29.7

329.2329

1

311.2245 (C18H31O4; -H2O), 171.1036 (C9H15O3; hydroxy nonenoic acid), 229.1445 (C12H21O4; dodecanedioic acid)

trihydroxy octadecadienoic acid (C18H32O5)

28.9

327.2172

2

309.2099 (C18H29O4; -H2O), 171.1033 (C9H15O3; oxononanoic acid)

hydroperoxy octadecadienoic acid (C18H32O4)

35.1

311.2222

2

293.2128 (C18H29O3; -H2O; epoxy octadecadienoic acid), 171.1028 (C9H15O3; hydroxy nonenoic acid)

hydroperoxy octadecatrienoic acid (C18H30O4)

31.3

309.2066

1

291.1972 (C18H27O3; -H2O; hydroxy octadecadienynoic acid), 171.1029 (C9H15O3; hydroxyl nonenoic acid)

fragments [m/z]

21

ACS Paragon Plus Environment


Page 22 of 31

Table 2. Most abundant fatty acids with a peak intensity of ≥ 106 in oolong tea infusion as identified by means of LCMS in a range of 100-1000 m/z. fatty acid

rt [min]

[M-H][m/z]

error [ppm]

nonanedioic acid (C9H16O4)

25.6

187.0971

2

125.0972 (C8H13O; octanone)

hydroxy dodecenoic acid (C12H22O3)

30.3

213.1493

1

195.1394 (C12H19O2; -H2O; dodecadienoic acid), 183.1396 (C11H19O2; undecenoic acid)

dihydroxy palmitic acid (C16H32O4)

30.3

287.2224

1

187.1345 (C10H19O3; hydroxydecanoic acid)

dihydroxy octadecenoic acid (C18H34O4)

33.9

313.2378

1

269.2156 (C16H29O3; hydroxy hexadecenoic acid)

trihydroxystearic acid (C18H36O5)

29.4

331.2485

1

313.2407 (C18H33O4; -H2O), 287.0204 (C17H35O3; -CO2)


29.7

329.2329

1


fragments [m/z]

22


Page 23 of 31


Table 3. A more than 2-fold higher or lower abundance (p ≤ 0.01) of oxidized fatty acids with a peak intensity of ≥ 105 in green tea compared to green tea infusion supplemented with 300 µM CuSO4 as identified by means of LCMS in a range of 100-1000 m/z. fatty acid

rt [min]

[M-H][m/z]

isopropyl hexanoic acid (C9H18O2)

35.0

157.1232

1

113.0612 (C6H9O2; hexenoic acid)

2.2

methyl tetradecanedioic acid (C15H28O4)

34.9

271.1913

1

253.1814 (C15H25O3; -H2O), 209.1912 (C14H25O)

5.8

octadecatetraenoic acid (C18H28O2)

36.6

275.2015

1

231.2118 (C17H27; -CO2), 177.1647 (C13H21)

22

hydroxy octadecatrienoic acid (C18H30O3)

36.8

293.2118

2

275.2022 (C18H27O2; -H2O; octadecatetraenoic acid), 235.1708 (C15H23O2)

3.3

methyl

31.8

413.2178

0

221.1549 (C14H21O2; dodecatrienyl acetate)

3.0

hydroperoxy

bisepidioxy

error fragments [m/z] [ppm]

effect of Cu2+ addition on the relative abundance of fatty acid

eicosadienoate (C21H34O8)

23



Page 24 of 31

Table 4. A more than 2-fold higher or lower abundance (p ≤ 0.01) of oxidized fatty acids with a peak intensity of ≥ 105 in oolong tea compared to oolong tea infusion supplemented with 300 µM CuSO4 as identified by means of LCMS in a range of 100-1000 m/z. fatty acid

rt [min]

[M-H][m/z]

error [ppm]

hydroxy nonanoic acid (C9H18O3)

31.0

173.1181

1

hydroxy-decenoic acid (C10H18O3)

31.6

185.1180

2

167.1079 (C10H15O2; -H2O), 115.0400 (C5H7O3)

7.3

hydroxy undecenoic acid (C11H20O3)

28.7

199.1335

2

155.1079 (C9H15O2; nonenoic acid)

3.5

undecenoic acid (C11H20O2)

30.0

183.1387

2

139.1128 (C9H15O)

6.4

dodecadienoic acid (C12H20O2)

34.7

195.1389

1

methyl dodecanedioic acid (C13H24O4)

32.1

243.1599

1

225.1499 (C13H21O3; -H2O), 163.0406 (C9H7O3)

11

hydroxy octadecadienoic acid (C18H32O3)

38.3

295.2276

0

277.2186 (C18H29O2; -H2O) 230.9827

49


29.7

329.2329

1


15

trihydroxy octadecadienoic acid (C18H32O5)

28.9

327.2172

2

309.2099 (C18H29O4;-H2O), 171.1033 (C9H15O3; oxo nonanoic acid)

3.7

hydroperoxy octadecadienoic acid (C18H32O4)

35.4

311.2225

1

293.2128 (C18H29O3; -H2O; epoxy octadecadienoic acid), 171.1028 (C9H15O3; hydroxy nonenoic acid)

2.2

hydroperoxy hydroxy octadecatrienoic acid (C18H30O5)

29.2

325.2015

1

hydroperoxy dihydroxy octadecenoic acid (C18H34O6)

29.2

345.2275

2

fragments [m/z]

effect of Cu2+ addition on the relative abundance of fatty acid [xfold change] 12

4.3

2.4

171.1025 (C9H15O3; hydroxy nonenoic acid), (C12H19O4; dioxo dodecanoic acid)

5.6

24


Page 25 of 31


Figure 1



Page 26 of 31

Figure 2


Page 27 of 31


Figure 3



Page 28 of 31

Figure 4


Page 29 of 31


Figure 5

g ~ 2.05 green tea

A

pH 7.5; 300 µM Cu2+

2400

2600

2800

3000

3200

3400

3600

3800

4000

[G]

oolong tea

B

pH 7.5; 300 µM Cu2+



Page 30 of 31

Figure 6


Page 31 of 31



and Semifermented Teas Analyzed by LC-MS-Based Nontargeted

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