Simultaneous Analysis of Seven Biomarkers of Oxidative Damage to

Subscriber access provided by Kaohsiung Medical University

Ecotoxicology and Human Environmental Health

Simultaneous analysis of seven biomarkers of oxidative damage to lipids, proteins, and DNA in urine Maria Martinez, and Kurunthachalam Kannan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00883 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on May 2, 2018

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

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

Page 1 of 31

Environmental Science & Technology

1 2

Simultaneous analysis of seven biomarkers of oxidative damage to

3

lipids, proteins, and DNA in urine

4 Maria P. Martinez a and Kurunthachalam Kannana,b*

5

6

7

a

Wadsworth Center, New York State Department of Health, and Department of Environmental

8

Health Sciences, School of Public Health, State University of New York at Albany, Empire

9

State Plaza, P.O. Box 509, Albany, New York 12201-0509, United States

10 11

b

Biochemistry Department, Faculty of Science and Experimental Biochemistry Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia

12

13

14

*Corresponding author: [email protected]

15

16

For submission to: Environmental Science and Technology

17 18 19 20 1 ACS Paragon Plus Environment


Page 2 of 31

21 22

Abstract

23

The determination of oxidative stress biomarkers (OSBs) is useful for the assessment of health

24

status and progress of diseases in humans. Whereas previous methods for the determination of

25

OSBs in urine were focused on a single marker, in this study, we present a method for the

26

simultaneous determination of biomarkers of oxidative damage to lipids, proteins, and DNA. 2,4-

27

Dinitrophenylhydrazine (DNPH) derivatization followed by solid phase extraction (SPE) and

28

high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) allowed

29

the

30

malondialdehyde (MDA), and four F2-isoprostane isomers: 8-iso-prostaglandinF2α (8-PGF2α),

31

11β-prostaglandinF2α

32

prostaglandinF2α (8,15-PGF2α) in urine. Derivatization with DNPH and SPE were optimized to

33

yield greater sensitivity and selectivity for the analysis of target chemicals. The limits of

34

detection of target analytes in urine were below 30 pg ml-1. The assay intra- and inter-day

35

variability was below 16% of the relative standard deviation, and the recoveries of target

36

chemicals spiked into synthetic urine were near 100%. The method was applied to the analysis of

37

21 real urine samples, and the analytes were found at a detection frequency of 85% for 8-PGF2α

38

and 15-PGF2α, 71% for 11-PGF2α, 81% for 8,15- PGF2α and 100% for diY, 8-OHdG, and MDA.

39

This method offers simultaneous determination of multiple OSBs of different molecular origin in

40

urine samples selectively with high accuracy and precision.

determination

of

8-hydroxy-2'-deoxyguanosine

(11-PGF2α),

(8-OHdG),

15(R)-prostaglandinF2α

41 42

2 ACS Paragon Plus Environment

o-o’-dityrosine

(15-PGF2α),

and

(diY),

8-iso,15(R)-

Page 3 of 31


43

Keywords: Biomarker, Oxidative Stress, Urine, Malondialdehyde, F2-isoprostane, o-o’-

44

dityrosine, 8-Hydroxy-2’-deoxyguanosine

45 46

Introduction

47 48

The imbalance between antioxidant and oxidant species in the body with radical oxygen species’

49

(ROS) exceeding the antioxidant capacity of the organism results in oxidative stress, which has

50

been claimed as an indicator for the prediction or progression of diseases, health status, or

51

exposure to external stressors. Oxidative stress implies that the excess ROS targets biomolecules,

52

resulting in their oxidation. Several compounds have been identified as the products of oxidation

53

of lipids, proteins, and DNA and were detected in biospecimens (e.g., urine, blood) as oxidative

54

stress biomarkers (OSBs) (Figure 1). The link between oxidative stress and breast cancer1 or

55

cardiovascular diseases2 has been reviewed previously. Studies that relate OSBs with Down

56

syndrome,3 Maple syrup disease,4 and exposure to external environmental factors, such as

57

asbestos,5 exercising,6 smoking,7 organic contaminants,8-10 poisons,11 anesthetics and alcohol

58

intake,12 have been reported. Under oxidative stress conditions, F2-isoprostanes (prostaglandin-

59

like compounds) are generated as oxidation products of arachidonic acid. Malondialdehyde

60

(MDA) is a product of the peroxidation of polyunsaturated fatty acids. These two are the most

61

common biomarkers of oxidation of lipids.13 DNA oxidation by ROS produces 8-hydroxy-2’-

62

deoxyguanosine (8-OHdG), and o,o’-diTyrosine (diY) is a biomarker of protein peroxidation.

63

The determination of these compounds in urine can provide an indication of the level of

64

oxidative stress.



Page 4 of 31

65

F2-isoprostanes, which are generated by the oxidation of arachidonic acid, can produce up

66

to 64 possible isomers. Bioactive F2-isoprostane isomers, such as 8-iso-prostaglandin F2α (8-

67

PGF2α), 15(R)-prostaglandin F2α, (15-PGF2α), 8-iso-15(R)-prostaglandin F2α (8,15-PGF2α), and

68

11β-prostaglandin F2α (11-PGF2α), have been used as OSBs.14 The most significant of the F2-

69

isoprostane isomers is 8-PGF2α, which has been the subject of over 200 publications that relate

70

human health with levels of PGF2α.7,10,15,16 The determination of F2-isoprostanes in biological

71

specimens by enzyme-linked immunosorbent assay (ELISA)3,4,10 or gas chromatography-mass

72

spectrometry (GC-MS) after a derivatization step17-20 has been described previously. The lack of

73

accuracy of ELISA and the extensive sample preparation steps needed for GC-MS analysis

74

highlight the need for alternative methods that include liquid chromatography-mass spectrometry

75

(LC-MS) for the analysis of F2-isoprostanes.5,14,21,22

76

Malondialdehyde (MDA) is the most important OSB due to its abundance in urine. MDA

77

has been used as a biomarker in studies that link oxidative stress to cancer,1,7 asbestos- or silica-

78

induced lung diseases,5 and exposure to polycyclic aromatic hydrocarbons (PAHs)23 and coke

79

oven dust,24 among others. Traditionally, MDA was quantified by a 2-thiobarbituric (TBA)

80

assay, and the MDA-TBA product was detected by fluorescence or spectrophotometry.25

81

However, the TBA assay lacks specificity, as the reaction can take place with other aldehydes in

82

the sample, yielding unknown TBA derivatives in addition to MDA-TBA, which leads to

83

overestimation of MDA.13,25,26 Analyses of MDA in urine after O-(2,3,4,5,6-pentafluorobenzyl)

84

hydroxylamine hydrochloride (O-PFB) derivatization followed by GC with electron capture

85

detection (GC-ECD),6 pentafluorobenzyl bromide (PFB-Br) derivatization followed by GC-MS

86

analysis,13

87

dinitrophenylhydrazine (DNPH) derivatization followed by liquid chromatography with

TBA

derivatization

followed

by

LC-fluorescence


detection,23,27

2,4-

Page 5 of 31


88

ultraviolet detection (LC-UV),28,29 and solid phase extraction (SPE)-LC-MS24 have been

89

reported.

90

Oxidative DNA damage is believed to be a trigger for diseases such as cancer. 8-oxo-

91

hydroxyguanosine (8-OHdG) is an OSB for DNA oxidation, a product of the reaction of ROS

92

with guanosine. 8-OHdG has been evaluated in relation to diseases such as cancer,1,7 Down

93

syndrome,3 and diabetes30 and in terms of linking oxidative stress with exposure to organic

94

contaminants.8,9,22,23 8-OHdG has been analyzed by ELISA3,6,30 or LC-MS8,23 methods.

95

When proteins are the targets of oxidation by ROS, o-o’-dityrosine (diY) and other

96

unnatural isomers of p-tyrosine are produced. diY is stable and resistant to enzymatic hydrolysis,

97

which makes it a good biomarker of protein oxidation.26 diY has been assessed as an OSB for

98

Down syndrome3 and diabetes.30 HPLC with fluorescence detection3 and LC-MS have been used

99

for the determination of diY in urine.6,26

100

Several studies have examined the association of oxidative stress with various diseases

101

and stressors.1,4,8-10,15,23 The majority of these studies have measured only a single OSB.1,15,9,10 A

102

few studies have developed analytical methods for simultaneous determination of 8-OHdG, 8-

103

PGF2α, 8-nitroguanine (biomarker of nitrative damage to DNA), and 4-hydroxy-2-nonenal-

104

mercapturic acid (HNE-MA)22 or the lipid peroxidation products, 8-PGF2α, MDA, and HNE.5

105

Limited studies have determined OSBs of lipids, proteins, and DNA simultaneously, but such

106

studies often used multiple analytical methods for the determination of each of the

107

biomarkers.3,6,11 Determination of OSBs produced by various cellular macromolecules would

108

provide a better understanding of the mechanisms and allow for a broader interpretation of the

109

results of toxicological studies, including the evaluation of correlations among various

110

biomarkers. Based on this background, this study was aimed at the development and validation 5 ACS Paragon Plus Environment


111

of an analytical method for simultaneous determination of major OSBs of lipids (viz., MDA and

112

PGF2α), proteins (viz., diY), and DNA (viz., 8-OHdG) in urine. The method can be applied in

113

epidemiological studies to examine oxidative stress patterns in cohorts inflicted with a disease or

114

an environmental condition as well as to evaluate antioxidant treatments. The method developed

115

and validated in this study, based on DNPH derivatization and SPE followed by HPLC with

116

tandem mass spectrometry (HPLC-MS/MS) detection and quantification, is accurate and precise

117

and allows for simultaneous determination of MDA, 8-OHdG, diY, and four F2-isoprostane

118

isomers, 8-PGF2α, 15-PGF2α, 8,15-PGF2α, and 11-PGF2α, in human urine.

119 120

Material and methods

121

Chemicals

122

Malondialdehyde tetrabutyl-ammonium salt (>97% purity), 50% glutaraldehyde solution, 8-

123

hydroxy-2’-deoxyguanosine (≥98%) (8-OHdG), acetic acid, hydrochloric acid (HCl), ethanol, n-

124

hexane, LC-MS grade methanol (MeOH), acetonitrile (ACN), HPLC grade water, ethyl acetate

125

(EtAc), synthetic urine, and 2,6-di tert-butyl-4-methylphenol (≥99%) (BHT) were purchased

126

from Sigma Aldrich (St. Louis, MO, USA). 2,4-Dinitrophenylhydrazine (DNPH) was purchased

127

from Spectrum (New Brunswick, NJ, USA). 1,1,3,3-Tetraethoxypropane-1,3-d2 (>98%) was

128

purchased from C/D/N isotopes (Point-Clair, Quebec, Canada). o,o’-Dityrosine (diY) (>99%)

129

was supplied by Toronto Research Chemicals (Toronto, Ontario, Canada). 13C12-o,o’-Dityrosine

130

(13C12-diY) (>98%) and

131

purchased from Cambridge Isotope Laboratories (Andover, MA, USA). 8-Iso-prostaglandin F2α

132

(>99%) (8-PGF2α), 15(R)-prostaglandin F2α (15-PGF2α) (>98%), 8-iso-15(R)-prostaglandin F2α

15

N5-8-hydroxy-2’-deoxyguanosine (15N5-8-OHdG) (>95%) were


Page 6 of 31

Page 7 of 31


133

(8,15-PGF2α) (>98%), 11β-prostaglandin F2α (11-PGF2α) (>98%), and D4-8-iso-prostaglandin F2α

134

(D4-8-PGF2α) (>99%) were obtained from Cayman Chemicals (Ann Arbor, MI, USA).

135 136

Urine Samples

137

A pool of urine samples collected from healthy adult volunteers was used in method

138

development and validation. Concentration of the target analytes in the urine pool were: 1.29 ng

139

ml-1 diY, 3.08 ng ml-1 OHdG, 9.91 ng ml-1 MDA, 0.38 ng ml-1 8-PGF2α, 0.23 ng ml-1 11-PGF2α,

140

0.89 ng ml-1 15-PGF2α, and 0.19 ng ml-1 8,15-PGF2α. Urine samples collected from 11 adult

141

males and 10 adult females in 2017 in Albany, New York, USA, in 50-ml polypropylene tubes

142

were analyzed to demonstrate the feasibility of the developed method. The urine samples stored

143

at -20° C until analysis. No additional data other than gender were obtained from urine donors.

144 145

Standard Solutions, Reagents, and Derivatization

146

Stock solutions (1000 µg ml-1) of analytical standards were prepared for each OSB. An MDA

147

solution was prepared in MeOH and stored in glass vials filled with nitrogen gas. F2-isoprostane

148

standards were diluted with ethanol, diY solutions were prepared in MeOH, and 8-OHdG was

149

diluted in HPLC water. The working standard solution mixture, which contained all OSBs, was

150

prepared at 1000 ng ml-1 in MeOH. All stock and working solutions were stored at -20° C.

151

D2-malondialdehyde (d2-MDA) stock solution was prepared by the hydrolysis of 1,1,3,3-

152

tetraethoxypropane-1,3-D2 with 0.2 N HCl at room temperature for 2 h, as previously reported.24

153

An internal standard (IS) mixture, containing d2-MDA (IS for MDA),

154

15

155

concentration of 500 ng ml-1 for each compound.

13

C12-diY (IS for diY),

N5-8-OHdG (IS for 8-OHdG), and D4-8-PGF2α (IS for PGF2α isomers), was prepared at a



156

Derivatization reagent (i.e., DNPH) was prepared at a concentration 0.05M in a

157

water:ACN:acetic acid mixture (8:1:1 v/v). To reduce background contamination, DNPH

158

solution was extracted twice with 5 ml of n-hexane by shaking the mixture for 5 min. After

159

extraction, the organic layer was discarded, and DNPH solution was stored in darkness at 4° C

160

until use. For the method development, MDA-DNPH derivative was prepared at 10 µg ml-1 by

161

the addition of 200 µl of DNPH reagent to 500 µl of standard solution, incubated for 1 h at 60°

162

C, and extracted twice with 1 ml of hexane for 5 min. Extracts were collected, evaporated to

163

dryness, and reconstituted with MeOH. A BHT solution was prepared at a concentration of 2%

164

in ethanol.

165

The derivatization step was optimized by a 3 x 3 x 3 full-factorial design to examine the

166

effect of temperatures (25° C, 37° C, 60° C), acidic conditions of DNPH reagent (12M HCl, 1%

167

acetic acid, 2% formic acid) and concentrations of DNPH (0.005, 0.015, 0.05M), for a total of 27

168

experiments. To determine the optimal conditions, in addition to complete derivatization of

169

MDA, the stability of other non-aldehyde target analytes (i.e., 8-OHdG, diY, and four F2-

170

isoprostane isomers) was checked under the reaction conditions. For derivatization, 10 ng of

171

each IS were added to a 500-µl aliquot of sample. Under optimal conditions, 200 µl of

172

derivatization reagent were added to the sample and incubated at room temperature for 30 min.

173 174

Extraction of OSBs from Urine

175

Target analytes were extracted from urine after the derivatization step. ABS ElutNexus

176

cartridges (60mg, 3ml) (Agilent Technologies, Santa Clara, CA, USA) were used for SPE. For

177

the optimization of SPE, performances of water and 0.1% acetic acid (in water) as conditioning

178

solvents, water and 5% MeOH (in water) as washing solvents, and ACN, EtAc and MeOH 8 ACS Paragon Plus Environment

Page 8 of 31

Page 9 of 31


179

(MeOH) as elution solvents, were studied in a 2 x 2 x 3 full-factorial design of experiments

180

(DoEs).

181

After conditioning the cartridge with 2 ml of MeOH and equilibration with 2 ml of water,

182

samples (previously added with 2 ml of water) were loaded onto the cartridge and washed with 2

183

ml of 5% MeOH in water. Then, a vacuum was applied to dry the cartridges for 5 min. The target

184

analytes were eluted with 1 ml of MeOH followed by 1 ml of EtAc in the same tube. Eluate was

185

evaporated to near-dryness under a nitrogen stream, reconstituted with 150 µl of water:MeOH

186

(8:2 v/v), and transferred into vials with a glass insert for HPLC-MS/MS analysis.

187 188

HPLC-MS/MS Analysis

189

An HPLC-MS/MS system integrated by Agilent 1100 HPLC coupled with an ABSCIEX 4500

190

mass spectrometer (Applied Biosystems, Foster City, CA, USA) was used for identification and

191

quantification of target analytes. The chromatographic separation of target analytes was

192

accomplished with a Zorbax Aq 3.5 µm column (2.1 × 150 mm) (Agilent, Santa Clara, CA,

193

USA). The sample injection volume was 20 µl. The HPLC mobile phase comprised MeOH (A)

194

and 0.01% acetic acid (B). The initial mobile phase composition was 100% B, held for 2 min,

195

and then decreased to 45% B within 0.5 min. Then the composition was decreased to 25% B

196

within 19.5 min, decreased to 0% B in 0.5 min, and held for 1.5 min. Flushing of the HPLC

197

column and reverting to initial conditions were accomplished in the last 6 min, with a total run

198

time of 30 min. Carryover of target analytes was not detected under these conditions.

199

The MS/MS method was split into two time periods, consisting of the first period with a

200

positive ionization mode for the detection of diY, 8-OHdG, and MDA, and, after 14 min,

201

switched to the negative ionization mode (the second period) for the detection of four F29 ACS Paragon Plus Environment


202

isoprostane isomers. Compound specific MS/MS parameters, m/z transitions of multiple reaction

203

monitoring, and ion source parameters were optimized by the injection of an individual analyte

204

standard solution at 1 µg ml-1 by flow injection analysis. Compound specific MS/MS parameters

205

as well as retention times of the target analytes are listed in Table 1. Ion source parameters were

206

optimized for improved ionization of the compounds eluted in that time window. Curtain gas

207

(CUR), collision activated dissociation gas (CAD), source temperature (TEM), nebulizer gas

208

(GS1), heater gas (GS2), and turbo ion spray voltage (IS) were set at 50 psi, 50 psi, 450° C, 50

209

psi, 50 psi, and 4500 V for Period 1 and 50 psi, 50 psi, 650° C, 45 psi, 40 psi, and -4500 V for

210

Period 2.

211

A six-port diverter valve integrated into the ABSCIEX 4500 mass spectrometer was used

212

to divert LC effluent to waste (position A) or to MS (position B) for the prevention of

213

accumulation of salts and impurities in the ion source. The diverter valve was kept in Position A

214

(waste) for up to 5.5 min of the analytical run, changed to B (MS) from 5.6 to 14.0 min (for the

215

detection of diY, 8-OHdG and MDA), diverted to waste from 14.1 to 19.4 min, and then to MS

216

from 19.5 to 29.0 min (for the detection of PGF2α isomers).

217 218

Quantification

219

Matrix-matched calibration solutions were prepared by the addition of increasing concentrations

220

from 0.05 to 50 ng ml-1 for each target analyte in urine and processed, following the method

221

described above. Linear regression of the response (area) of the ratio of the target compound to

222

the corresponding IS versus concentration was used for quantification. Target OSBs were not

223

detected in procedure blanks with the exception of MDA, which was found at 0.45 ng ml-1.

224 10 ACS Paragon Plus Environment

Page 10 of 31

Page 11 of 31


225

Results

226

Sample Preparation for LC-MS

227

Extraction

228

The extraction of OSBs from urine was first examined by liquid-liquid extraction using hexane,

229

EtAc, or the mixture ACN:EtAc (1:1 v/v). This approach was unsuccessful due to the poor

230

simultaneous extraction of target compounds in the solvents tested result of their different

231

polarities (Table S1). Whereas the optimal solvent for the extraction of MDA-DNPH was

232

hexane, F2-isoprostanes were preferably extracted in EtAc. diY was not effectively extracted by

233

hexane or EtAc, but the mixture ACN:EtAc was found effective for the extraction of diY (Figure

234

S1). Therefore, the SPE procedure was evaluated for the extraction and concentration of diY, 8-

235

OHdG, F2-isoprostane isomers, and MDA-DNPH in urine samples. To assess the recovery of

236

target analytes through the SPE procedure, urine samples were prepared by the addition of target

237

analytes, including MDA-DNPH derivative, at 40 ng ml-1. To simulate experimental conditions,

238

500 µl of samples were mixed with 200 µl of derivatization reagent, and 2 ml of water were

239

added to reduce the composition of the organic solvent in samples.

240

In the data analysis for the optimization of SPE, no statistical differences were found

241

between the two washing solvents, but 5% MeOH in water was selected to increase the strength

242

of the washing solution. Conditioning of cartridges with water yielded significantly higher

243

recoveries for 8-PGF2α (p-value = 0.023). Among the three elution solvents tested, acceptable

244

recoveries of OSBs (with minimal interferences) were observed with MeOH, which yielded good

245

recoveries of diY (p-value = 0.0001) and 8-OHdG (p-value = 0.017), while EtAc increased the

246

recovery of MDA (p-value = 0.005). The use of MeOH and ACN as elution solvents yielded

247

similar results, and these two solvents yielded better recoveries than did EtAc for 8-PGF2α (p11 ACS Paragon Plus Environment


248

value = 0.019). Mean graphs that show significant differences among various SPE parameters

249

are presented in Figure S2, and adjustment coefficients and p-values are listed in Table S2.

250

The elution step was further optimized for the combination/volume of elution solvents.

251

Five different solvent combinations were studied: 4 ml MeOH, 3 ml MeOH followed by 1 ml

252

EtAc, 2 ml MeOH followed by 2 ml EtAc, 1 ml MeOH followed by 3 ml EtAc, and 4 ml EtAc.

253

SPE cartridges were eluted with the 4 ml of solvent, which was collected in 1 ml fractions

254

(following elution). This test was performed in triplicate. In accordance with the DoEs, MeOH

255

increased the recovery of all target analytes except MDA, which showed a better recovery with

256

EtAc. The elution with 1 ml of MeOH followed by 1 ml of EtAc recovered more than 94% of the

257

target analytes from the cartridge (data not shown).

258

Under optimal conditions, the samples after derivatization were added with 2 ml of water

259

and extracted using an SPE cartridge, conditioned previously with 2 ml of MeOH and 2 ml of

260

water. After the sample addition, the cartridge was washed with 2 ml of 5% MeOH in water and

261

vacuum dried for 5 min. Elution was performed with 1 ml of MeOH followed by 1 ml of EtAc.

262

Both eluents were collected in the same tube and evaporated to near-dryness under a nitrogen

263

stream, reconstituted with 150 µl of water:MeOH (8:2 v/v), and transferred into a vial for HPLC-

264

MS/MS analysis.

265 266

Derivatization of MDA Using DNPH

267

Quantitative analysis of aldehydes is challenging due to their reactivity, volatility, and low

268

molecular weight (Table S1). A derivatization step would increase the stability, molecular

269

weight, and ionization of aldehydes. Previous methods of analysis of MDA were based on its

270

reaction with TBA, which required a high temperature for the formation of TBA derivatives.27 12 ACS Paragon Plus Environment

Page 12 of 31

Page 13 of 31


271

Derivatization, using DNPH followed by LC-MS analysis, has been reported for the

272

determination of MDA in urine.24 Nevertheless, the earlier method was not applied for the

273

simultaneous analysis of other OSBs in urine.

274

For the DoE study, 500 µl of urine (pooled sample) were spiked at 40 ng ml-1 of each

275

target analyte. An IS mixture and 200 µl of DNPH were added, vortexed, and incubated for 1 h

276

at the temperatures listed above. An incubator was used for the incubation of test solutions at 25°

277

C, 37° C, and 60° C. Samples were then extracted by SPE and analyzed by LC-MS/MS. No

278

differences in the recoveries of target chemicals were found between various concentrations of

279

DNPH. With regard to the use of different acids during derivatization, acetic acid and formic

280

acid yielded similar results, while HCl affected the concentration of non-aldehyde OSBs by

281

decreasing the levels of F2-isoprostanes (p-value < 0.0001) and 8-OHdG (p-value < 0.0001) and

282

increasing diY levels (p-value < 0.0001). Therefore, acetic acid was selected for further

283

experiments. With the increase in temperature, the concentration of MDA-DNPH increased (p-

284

value = 0.018). 8-OHdG levels also increased (p-value = 0.019), however, while 8-PGF2α, 11-

285

PGF2α, 15-PGF2α, and 8,15-PGF2α (p-values = 0.004, 70% of the samples.


Page 18 of 31

Page 19 of 31


406

The proposed method represents a major improvement over the previously reported methods

407

for the individual OSB determination in terms of specificity and efficiency. ELISA methods

408

were used for the analysis of 8-OHdG or 8-PGF2a and TBA assay was used for MDA analysis,

409

but they lacked specificity. Other methods for the analysis of F2-isopostanes by GC-MS require

410

extended sample preparation. A major strength of this study lies in the measurement of several

411

OSBs in a single extraction and detection method. A limited number of studies have reported a

412

method for simultaneous determination of more than one OSB in urine. A method for the

413

analysis of MDA, 8-PGF2α, and hydroxynonenal (HNE; biomarker of lipid peroxidation) in

414

exhalated breath condensate, plasma, and urine has been reported.5 Simultaneous analysis of 8-

415

OHdG, HNE-MA (hydroxynonenal-mercapturic acid), and 8-PGF2α in urine also has been

416

reported.22 Our goal was to optimize and validate a method for the determination of

417

concentrations of major OSBs produced by the oxidation of lipids, proteins, and DNA in urine.

418

Apart from simultaneous determination of seven OSBs in urine, the LODs of this method are

419

below the actual concentrations found in urine from healthy individuals. As the levels of OSBs

420

are expected to be higher in individuals with disease conditions or exposed to stressors, this

421

method can be applied to a range of population-based studies. Our method allows for

422

simultaneous analysis of degradation products of lipids, proteins, and DNA, which implies that

423

mechanisms of toxicity or disease progression can be evaluated by measuring effects on major

424

biomolecules present in human cells.

425

The proposed method, based on DNPH derivatization followed by SPE-LC-MS/MS, has been

426

optimized and validated for the analysis of seven OSBs in urine. Optimization of DNPH

427

derivatization ensured maximum yield for MDA-DNPH derivative without affecting the

428

quantification of other OSBs in the same sample. The optimization of SPE for the extraction of 19 ACS Paragon Plus Environment


429

target OSBs was accomplished, and the need for a suitable elution solvent for quantitative

430

recovery of all seven OSBs from the SPE cartridge was enabled. The applicability of the method

431

was tested by the analysis of 21 real urine samples. diY, 8-OHdG, and MDA were found in all

432

samples, and F2-isoprostanes were found in >70% of the urine samples. The proposed method

433

can be applied in epidemiological and toxicological studies that link OSBs with health outcomes

434

from exposure to environmental stressors in human populations.

435

Supporting Information

436

Additional information is provided in the supporting information, as listed above (Table S1–S6,

437

Fig. S1–S6). Tables showing physicochemical properties of target chemicals, SPE method

438

development and optimization results, optimization of derivatization steps, slopes of calibration

439

curves prepared in urine, concentrations of OSBs in individual urine samples, and reported

440

LOQs for target analytes; figures showing recoveries from liquid-liquid extraction, mean plots of

441

SPE optimization and derivatization results, derivatization time on OSB recoveries,

442

derivatization reaction, Box and Whisker plots of ODB distribution in males and females. The

443

Supporting Information is available free of charge on the ACS Publications website.

444

Author Contributions

445

The manuscript was written with contributions from all authors. All authors have given approval

446

of the final version of the manuscript.

447

Notes

448

The authors declare no competing financial interest.

449

Acknowledgments 20 ACS Paragon Plus Environment

Page 20 of 31

Page 21 of 31


450

Research reported in this publication was supported in part by the National Institute of

451

Environmental Health Sciences of the National Institutes of Health under Award Number

452

U2CES026542-01. The content is solely the responsibility of the authors and does not

453

necessarily represent the official views of the National Institutes of Health.

454 455

References

456 457

(1) Lee, J.D.; Cai, Q.; Shu, X.O; Nechuta S.J. The role of Biomarkers of Oxidative Stress in

458

Breast Cancer Risk and Prognosis: A Systematic Review of the Epidemiologic Literature. J.

459

Womens Health 2017, 26, 467-482.

460

(2) Ho, E.; Galougahi, K.K.; Liu, C-C.; Bhindi, R.; Figtree, G.A. Biological markers of

461

oxidative stress: Applications to cardiovascular research and practice. Redox Biol. 2013, 1,

462

483-491.

463

(3) Campos, C.; Guzman, R.; Lopez-Fernandez, E.; Casado, A. Evaluation of urinary

464

biomarkers of oxidative/nitrosative stress in children with Down syndrome. Life Sci. 2011,

465

89, 655-661.

466

(4) Guerreiro, G.; Mescka, C.P.; Sitta, A.; Donida, B.; Marchetti, D.; Hammerschmidt T.;

467

Faverzani, J.; de Moura Coelho, D.; Wajner, M.; Severo, C.; Dutra-Filho, Vargas; C.R.

468

Urinary biomarkers of oxidative damage in Maple syrup urine disease: The L-carnitine role.

469

Int. J Dev. Neurosci. 2015, 42, 10-14.

470

(5) Syslova, K.; Kacer, P.; Kuzma, M.; Najmanova, V.; Fnclova, Z.; Vlckova, S.; Lebedova J.;

471

Pelclova, D. Rapid and easy method for monitoring oxidative stress markers in body fluids

472

of patients with asbestos or silica-induced lung diseases. J. Chromatogr. B 2009, 877,

473

2477-2486.

474

(6) Ohran, H.; Van Holland, B. Krab, B.; Moeken, J.; Vermeulen, N.P.E.; Hollander, P.;

475

Meerman, J.H.N. Evaluation of a Multi-parameter Biomarker Set for Oxidative Damage in

476

Man: Increased Urinary Excretion of Lipid, Protein and DNA Oxidation Products after One

477

Hour of Exercise. Free Radic. Res. 2004, 38, 1269-1279.



478

(7) Lowe, F.J.; Luettich, K.; Gregg, E.O. Lung cancer biomarkers for the assessment of

479

modified risk tobacco products: an oxidative stress perspective. Biomarkers 2013, 18, 183-

480

195.

481

(8) Asimakopoulos, A.G.; Xue, J.; Pereira De Carvalho, B. Iyer, A.; Abualnaja, K.O.;

482

Yaghmoor, S.S.; Kumosani, T.A.; Kannan, K. Urinary biomarkers of exposure to 57

483

xenobiotics and its association with oxidative stress in a population in Jeddah, Saudi Arabia.

484

Environ. Res. 2016, 150, 573-581.

485

(9) Iyer, A. P.; Xue, J.; Honda, M.; Robinson, M.; Kumosani, T. A.; Abulnaja K.; Kannan K.

486

Urinary levels of triclosan and triclocarban in several Asian countries, Greece and the USA:

487

Association with oxidative stress. Environ. Res. 2018, 160, 91-96.

488

(10) Wu, H.; Olmsted, A.; Cantonwine, D.E.; Shahsavari, S.; Rahil, T.; Sites, C.; Pilsner, J.R.

489

Urinary phthalate and phthalate alternative metabolites and isoprostane among couples

490

undergoing fertility treatment. Environ. Res. 2017, 153, 1-7.

491

(11) Kadiiska, M.B.; Gladen, B.C.; Baird, D.D.; Germolec, D.; Graham, L.B.; Parker, C.E.;

492

Nyska, A.; Wachsman, J.T.; Ames, B.N.; Basu, S.; Brot, N.; FitzGerald, G.A.; Floyd, R.A.;

493

George, M.; Heinecke, J.W.; Hatch, G.E.; Hensley, K.; Lawson, J.A.; Marnett, L.J.;

494

Morrow, J.D.; Murray, D.M.; Plastaras, J.; Roberts II, L.J.; Rokach, J.; Shigenaga, M.K.;

495

Sohal, R.S.; Sun, J.; Tice, R.R.; Van Thiel, D.H.; Wellner, D.; Walter, P.B.; Tomer, K.B.;

496

Mason, R.P.; Barrett, J.C. Biomarkers of Oxidative Stress Study II. Are oxidation products

497

of lipids, proteins, and DNA markers of CCl4 poisoning? Free Radic. Biol. Med. 2005, 38,

498

698-710.

499

(12) Kim, H.; Oh, E.; Im, H.; Mun, J.; Yang, M.; Khim, J-Y.; Lee, E.; Lim, S.H.; Kong, M.H.;

500

Lee M.; Sul, D. Oxidative damages in the DNA, lipids, and proteins of rats exposed to

501

isofluranes and alcohols. Toxicol. 2006, 220, 169-178.

502

(13) Tsikas, D. Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and

503

relatives in biological samples: Analytical and biological challenges. Anal. Biochem. 2017,

504

524, 13-30.

505

(14) Aszyk, J.; Kot, J.; Tkachenko, Y.; Wozniak, M.; Bogucka-Kocka, A.; Kot-Wasik, A. Novel

506

liquid chromatography method based on linear weighted regression for the fast

507

determination of isoprostane isomers in plasma samples using sensitive tandem mass

508

spectrometry detection. J. Chromatogr. B 2017, 1051, 17-23. 22 ACS Paragon Plus Environment

Page 22 of 31

Page 23 of 31

509


(15) Van’tErve, T.J.; Kadiiska, M.B.; London, S.J.; Mason, R.P. Classifying oxidative stress by

510

F2-isoprostane levels across human diseases: A meta-analysis. Redox Biol. 2017, 12, 582-

511

599.

512

(16) Kataria, A.; Levine, D.; Wertenteil, S.; Vento, S.; Xue, J.; Rajendiran, K.; Kannan, K.;

513

Thurman, J.M.; Morrison, D.; Brody, R.; Urbina, E.; Attina, T.; Trasande L.; Trachtman, H.

514

Exposure to bisphenols and phthalates and association with oxidant stress, insulin

515

resistance, and endothelial dysfunction in children. Pediatr. Res. 2017, 81, 857-864.

516

(17) Liu, W.; Morrow, J.D.; Yin, H. Quantification of F2-isoprostanes as a reliable index of

517

oxidative stress in vivo using gas chromatography-mass spectrometry (GC-MS) method.

518

Free Radic. Biol. Med. 2009, 47, 1101-1107.

519 520 521

(18) Milne, G.L.; Sanchez, S.C.; Musiek, E.S.; Morrow J.D. Quantification of F2-isoprostanes as a biomarker of oxidative stress. Nat. Protoc. 2007, 2, 221-226. (19) Tsikas, D.; Suchy. M-T. Protocols for the measurement of the F2-isoprostane, 15(S)-8-iso-

522

prostaglandin F2α, in biological samples by GC-MS or GC-MS/MS coupled with

523

immunoaffinity column chromatography. J. Chromatogr. B 2016, 1019, 191-201.

524

(20) Briskey, D.R.; Wilson, G.R.; Fassett, R.G.; Coombes, J.S. Optimized method for

525

quantification of total F2-isoprostanes using gas chromatography-tandem mass

526

spectrometry. J. Pharm. Biomed. Anal. 2014, 90, 161-166.

527

(21) Xiao, Y.; Fu, X.; Pattengale, P.; Dien Bard, J.; Xu, Y-K.; O’Gorman, M.R. A sensitive LC -

528

MS/MS method for the quantification of urinary 8-iso-prostaglandin F2α (8-iso-PGF2α)

529

including pediatric reference interval. Clin. Chim. Acta 2016, 460, 128-134.

530

(22) Wu, C.; Chen, S-T.; Peng, K-H.; Cheng T-J.; Wu, K-Y. Concurrent quantification of

531

multiple biomarkers indicative of oxidative stress status using liquid chromatography-

532

tandem mass spectrometry. Anal. Biochem. 2016, 512, 26-35.

533

(23) Lu, S-y.; Li, Y-x; Zhang, J-q; Zhang, T.; Liu, G-h.; Huang, M-z.; Li, X.; Ruan, J-j.; Kannan

534

K.; Qiu, R-l. Associations between polycyclic aromatic hydrocarbon (PAH) exposure and

535

oxidative stress in people living near e-waste recycling facilities in China. Environ. Int.

536

2016, 94, 161-169.

537 538

(24) Chen, J-L.; Huang, Y-J.; Pan, C-H.; Hu, C-W.; Chao, M-R. Determination of urinary malondialdehyde by isotope dilution LC-MS/MS with automated solid-phase extraction: A



539

cautionary note on derivatization optimization. Free Radic. Biol. Med. 2011, 51, 1823-

540

1829.

541

(25) Giera, M.; Lingeman, H.; Niessen ,W.M.A. Recent Advancements in the LC- and GC-Based

542

Analysis of Malondialdehyde (MDA): A Brief Overview. Chromatographia 2012, 75, 433-

543

440.

544

(26) Orhan, H. Analyses of representative biomarkers of exposure and effect by

545

chromatographic, mass spectrometric, and nuclear magnetic resonance techniques: Method

546

development and application in life sciences. J. Sep. Sci. 2007, 30, 149-174.

547 548 549

(27) Agarwal, R.; Chase, S.D. Rapid, fluorometric-liquid chromatographic determination of malondialdehyde in biological samples. J. Chromatogr. B 2002, 775, 121-126. (28) Kim, B.; Jung, W.; Kho, Y. Quantifiation of Malondialdehyde in Human Urine by HPLC-

550

DAD and Derivatization with 2,4-Dinitrophenylydrazine. Bull. Korean Chem. Soc. 2017,

551

38, 642-645.

552

(29) Pilz, J.; Meineke, I.; Gleiter, C.H. Measurement of free and bound malondialdehyde in

553

plasma by high-performance liquid chromatography as the 2,4-dinitrophenylhydrazine

554

derivative. J. Chromatogr. B 2000, 742, 315-325.

555

(30) Kato, Y.; Dozaki, N.; Nakamura, T.; Kitamoto, N.; Yoshida, A.; Naito, M.; Kitamura, M.;

556

Osawa, T. Quantification of Modified Tyrosines in Healthy and Diabetic Human Urine

557

using Liquid Chromatography/Tandem Mass Spectrometry. J. Clin. Biochem. Nutr. 2009,

558

44, 67-78.

559

(31) Czauderna, M.; Kowalcyk, J.; Marounek, M. The simple and sensitive measurement of

560

malondialdehyde in selected specimens of biological origin and some feed by reversed

561

phase high performance liquid chromatography. J. Chromatogr. B 2011, 879, 2251-2258.


Page 24 of 31

Page 25 of 31


Figure 1. Schematic of oxidative stress biomarkers formation from proteins, deoxyribonucleic acid (DNA), arachidonic acid and polyunsaturated fatty acids (PUFA).



Figure 2. Total ion chromatogram (TIC) and extracted ion chromatograms of A. Real urine sample from a healthy adult (F8: 0.76 ng ml-1 diY, 5.41 ng ml-1 8-OHdG, 14.1 ng ml-1 MDA, 0.46 ng ml-1 8-PGF2α, 0.33 ng ml-1 11-PGF2α, 0.54 ng ml-1 15-PGF2α, 0.65 ng ml-1 8,15-PGF2α); B. Spiked urine at 0.6 ng ml-1 of target chemicals. Peak identification: 1. diY and 13C12-diY; 2. 8OHdG and 15N5-8-OHdG; 3. MDA and D2-MDA; 4. 8,15-PGF2α, 5. 8-PGF2α and D4-8-PGF2α, 6. 11-PGF2α and 7. 15-PGF2α.


Page 26 of 31

Page 27 of 31


Table 1. Retention time (RT) and compound dependent tandem mass spectrometric parameters ionization mode, multiple reaction monitoring (MRM) period, MRM transition, declustering potential (DP), entrance potential (EP), collision energy (CE) and collision cell exit potential (CXP) used in the analysis of oxidative stress biomarkers in urine.

RT

Ionization

MRM

(min)

mode

Period

diY

7.6

+

1

13

7.6

+

8-OHdG

8.0

15

N5-8OHdG

MRM

DP

EP

CE

CXP

(V)

(V)

(V)

(V)

353.0 > 315.0

45

10

23

10

1

373.0 > 327.0

56

10

23

10

+

1

284.0 > 168.0

25

3

18

4

8.0

+

1

289.0 > 173.0

25

3

18

4

MDA-DNPH

13.0

+

1

235.0 > 159.0

30

8

30

3

D2-MDA-DNPH

13.0

+

1

237.0 > 159.0

30

8

30

3

8,15-PGF2α

20.4

-

2

353.0 > 193.0

-40

-6

-40

-2

8-PGF2α

21.2

-

2

353.0 > 193.0

-45

-8

-35

-2

D4-8-PGF2α

21.2

-

2

357.0 > 197.0

-45

-8

-35

-2

11-PGF2α

21.7

-

2

353.0 > 193.0

-30

-8

-35

-2

15-PGF2α

22.7

-

2

353.0 > 193.0

-35

-8

-35

-2

Compound

C12-diY

Transition (m/z)



Page 28 of 31

Table 2. Limits of detection (LOD) and quantification (LOQ), repeatability, intra-day precision and accuracy of the method developed for the analysis of oxidative stress biomarkers in urine. Repeatability, intra-day repeatability and recovery were measured at three concentrations: A. 0.1 ng ml-1, B. 1.0 ng ml-1, C. 10 ng ml-1

Compound

LOD,

LOQ,

Repeatability,

Intra-day

ng ml-1

ng ml-1

RSD %

precision RSD %

Recovery, % ± 95%CI

A

B

C

A

B

C

A

B

C

diY

0.030

0.10

9

9

9

10

9

9

109 ± 20

103 ± 20

98 ± 9

8-OHdG

0.030

0.10

7

7

8

9

5

5

92 ± 17

101 ± 22

97 ± 10

MDA

0.024

0.08

10

4

6

10

9

16

109 ± 24

112 ± 16

91 ± 18

8,15-PGF2α

0.013

0.04

8

8

6

12

7

14

105 ± 15

102 ± 5

95 ± 26

8-PGF2α

0.010

0.03

7

7

4

8

9

8

103 ± 23

96 ± 14

93 ± 18

11-PGF2α

0.016

0.05

8

6

4

2

13

9

107 ± 16

96 ± 8

93 ± 11

15-PGF2α

0.012

0.04

7

10

6

13

10

9

94 ± 26

92 ± 23

103 ± 18


Page 29 of 31


Table 3. Stability of oxidative stress biomarkers in urine samples after storage at -20°C, three freeze-thaw cycles or light exposure during derivatization and analysis (expressed as % recovery ± 95% confidence interval). A. No BHT addition. B. BHT addition at urine collection. C. BHT addition at the beginning of analysis.

diY

8-OHdG

MDA

8,15-PGF2α

8-PGF2α

11-PGF2α 15-PGF2α

A

100 ± 12

100 ± 5

100 ± 14

100 ± 11

100 ± 8

100 ± 6

100 ± 13

B

105 ± 20

105 ± 8

105 ± 13

102 ± 1

102 ± 2

104 ± 20

100 ± 2

C

112 ± 14

96 ± 2

98 ± 2

98 ± 11

95 ± 4

88 ± 6

100 ± 2

A

112 ± 10

88 ± 7

90 ± 14

94 ± 13

88 ± 10

106 ± 8

89 ± 12

B

102 ± 15

91 ± 6

87 ± 8

98 ± 21

89 ± 15

112 ± 25

91 ± 18

C

116 ± 13

98 ± 5

84 ± 2

98 ± 10

96 ± 2

116 ± 3

95 ± 5

A

104 ± 13

98 ± 4

96 ± 8

86 ± 10

95 ± 2

116 ± 12

98 ± 14

B

97 ± 12

99 ± 4

92 ± 11

89 ± 10

96 ± 7

112 ± 16

100 ± 5

C

116 ± 9

98 ± 4

97 ± 9

90 ± 10

98 ± 14

106 ± 3

105 ± 2

A

109 ± 14

93 ± 8

43 ± 1

80 ± 13

95 ± 9

138 ± 24

97 ± 22

B

112 ± 10

88 ± 3

42 ± 4

79 ± 12

96 ± 6

146 ± 5

98 ± 4

C

109 ± 24

93 ± 3

43 ± 5

81 ± 13

95 ± 4

138 ± 4

108 ± 4

A

107 ± 16

111 ± 16 130 ± 25

79 ± 1

97 ± 1

108 ± 12

122 ± 12

B

117 ± 15

112 ± 10

120 ± 7

77 ± 6

97 ± 7

120 ± 21

135 ± 12

C

103 ± 27

105 ± 25

130 ± 22

79 ± 19

93 ± 8

102 ± 17

143 ± 10

A

98 ± 15

101 ± 6

104 ± 5

91 ± 15

101 ± 10

93 ± 1

89 ± 2

B

115 ± 5

98 ± 5

103 ± 3

108 ± 18

99 ± 14

96 ± 13

99 ± 10

C

95 ± 5

102 ± 19

116 ± 5

112 ± 18

108 ± 4

105 ± 12

105 ± 9

Storage time Collection (0 day)

1 day

7 days

30 days

Freeze-thaw cycles

Light exposure



Page 30 of 31

Table 4. Concentrations of seven oxidative stress markers (ng ml-1) in urine sample collected from healthy adults, 11 males (M) and 10 females (F) from Albany, New York, USA.

diY

8-OHdG

MDA

8-PGF2α

11-PGF2α

15-PGF2α

8,15-PGF2α

M Median

1.64

12.7

15.1

0.81

0.34

1.34

1.11

M Min

0.54

6.64

8.14

0.26

0.14

0.19

0.30

M Max

4.34

21.8

33.3

2.15

1.06

2.17

1.41

F Median

0.68

5.91

13.18

0.34

0.13

0.36

0.53

F Min

0.20

1.45

1.98

0.28

0.10

0.051

0.048

F Max

1.10

16.93

31.6

0.69

0.33

0.70

0.77

Median

0.99

9.18

14.07

0.55

0.28

0.56

0.76

Min

0.20

1.45

1.98

0.03

0.10

0.051

0.048

Max

4.34

21.8

31.6

2.15

1.06

2.17

1.41

100

100

100

85

71

85

81

Detection frequency, %


Page 31 of 31


TOC_ es-2018-00883t.R1 Simultaneous analysis of seven biomarkers of oxidative damage to lipids, proteins, and DNA in urine

ACS Paragon Plus Environment

Simultaneous Analysis of Seven Biomarkers of Oxidative Damage to

Recommend Documents