Benzoxazinoids in Prostate Cancer Patients after a Rye-Intensive Diet

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Benzoxazinoids in Prostate Cancer Patients after a Rye-Intensive Diet: Methods and Initial Results Stine Krogh Steffensen, Hans Albert Pedersen, Khem B. Adhikari, Bente Birgitte Laursen, Elena-Claudia Jensen, Søren Høyer, Michael Borre, Helene Holm Pedersen, Mette Borre, David Edwards, and Inge S. Fomsgaard J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03765 • Publication Date (Web): 10 Oct 2016 Downloaded from http://pubs.acs.org on October 17, 2016

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

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

Page 1 of 45

Journal of Agricultural and Food Chemistry

54x130mm (300 x 300 DPI)

ACS Paragon Plus Environment


Page 2 of 45

Benzoxazinoids in Prostate Cancer Patients after a Rye-Intensive Diet: Methods and Initial Results Stine K. Steffensena*, Hans A. Pedersena, Khem B. Adhikaria, Bente B. Laursena, Claudia Jensena, Søren Høyerb, Michael Borrec, Helene H. Pedersenc, Mette Borred, David Edwardse, Inge S. Fomsgaarda. a

Department of Agroecology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark.

b

Department of Pathology, Aarhus University Hospital, Nørrebrogade 44, DK-8000 Aarhus

C, Denmark c

Department of Urology, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, DK-

8200 Aarhus N, Denmark d

Department of Medicine V (Hepatology and Gastroenterology), Aarhus University Hospital,

Nørrebrogade 44, DK-8000 Aarhus C, Denmark e

Department of Molecular Biology and Genetics, Aarhus University, Blichers Allé 20, DK-

8830 Tjele *Corresponding author: [email protected] Short title: Benzoxazinoids in Prostate Cancer Patients after a Rye-Intensive Diet

1 ACS Paragon Plus Environment

Page 3 of 45


1

Abstract

2

Rye bread contains high amounts of benzoxazinoids and in vitro studies have shown

3

suppressive effects of selected benzoxazinoids on prostate cancer cells. Thus research into

4

benzoxazinoids as possible suppressors of prostate cancer is demanded. A pilot study was

5

performed in which ten prostate cancer patients received a rye-enriched diet one week prior

6

to prostatectomy. Plasma and urine samples were collected pre- and post-intervention. Ten

7

prostate biopsies were obtained from each patient and histologically evaluated. The biopsies

8

exhibited concentrations above the detection limit of seven benzoxazinoids ranging from 0.15

9

to 10.59 ng/g tissue. An OPLS-DA analysis on histological and plasma concentrations of

10

benzoxazinoids classified the subjects into two clusters. A tendency of higher benzoxazinoid

11

concentrations towards the benign group encourages further investigations. Benzoxazinoids

12

were quantified by an optimized LC-MS/MS method and matrix effects were evaluated. At

13

low concentrations in biopsy and plasma matrices the matrix effect was concentration-

14

dependent and non-linear. For the urine samples the general matrix effects were small but

15

patient-dependent.

16

Keywords:

Benzoxazinoid, Prostate cancer, Matrix effect, Tissue, LC-MS/MS



Page 4 of 45

17

INTRODUCTION

18

Globally, prostate cancer accounts for about 15% of all new cancers diagnosed among

19

males.1 The age-standardized incidence of prostate cancer in Denmark is 138 per 100,000,

20

which is comparable to other European Union countries and the USA (106 and 129 per

21

100,000, respectively).2-4 However, as the elderly population increases, the prevalence of

22

prostate cancer is expected to increase dramatically in the coming decades. The dominant

23

type of prostate cancer is adenocarcinoma, an endocrine tumor. Despite treatment through

24

androgen deprivation, most patients eventually experience disease progression within a

25

median of 18-24 months.5

26

Rye wholegrain and bran intake has shown beneficial effects on prostate cancer progression

27

in animal models and humans, including lower tumor rates, smaller tumor volumes, and

28

reduced prostate-specific antigen (PSA) concentrations.6-8 The relationship to the ingredients,

29

however, was not investigated. The presence of benzoxazinoids, including the subgroups

30

benzoxazolinones, lactams, and hydroxamic acids (see Figure 1) in rye grains and pretreated

31

wheat and food products derived from these was reported recently.9-14 Benzoxazinoids have

32

various potential pharmacological and health-protecting properties, which have been

33

reviewed recently.15 The benzoxazinoid 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA)

34

inhibited the growth of the cancerous prostate cell line DU145.16, 17 Roberts et al.18 suggested

35

that this effect was due to the ability of DIBOA to induce cell death. The reported

36

suppressive effects of rye intake on prostate cancer, the newly discovered presence of

37

benzoxazinoids in rye grains and the in vitro inhibition by DIBOA of prostate cancer cell

38

growth, all provide compelling reasons for investigating the effect of a benzoxazinoid-

39

containing rye-based diet on human prostate cancer.


Page 5 of 45


40

We have reported earlier that dietary benzoxazinoids are bioavailable in pigs, rats, and

41

humans,12, 19, 20 but the extent to which benzoxazinoids are distributed to tissues in the human

42

body has not yet been investigated.

43

Analytical methods for the quantification of benzoxazinoids in plants and soil using LC-

44

MS/MS have been presented in several studies.21-23 In 2012, we first analyzed the

45

benzoxazinoid content of plasma and urine,12, 19 and several studies have since been

46

published on benzoxazinoids and their derivatives in either plasma or urine.15, 20, 24-27 The aim

47

of this initial study was to develop the analytical methodology for analysis of benzoxazinoids

48

in minute amounts of tissue obtained from prostate biopsies, to elucidate the role that matrix

49

effects play in the analysis of benzoxazinoids in biological samples, to present the first results

50

ever on the occurrence of benzoxazinoids in prostate tissue in men after a week on a high-rye

51

diet, and to examine the preliminary correlation between histological data and benzoxazinoid

52

concentrations in plasma, urine, and tissue. Based on the results of this study, a full cross-

53

over study will be planned.



Page 6 of 45

54

MATERIALS AND METHODS

55

Chemicals

56

HPLC-grade acetonitrile and methanol (Rathburn, Walkerburn, Scotland), glacial acetic acid

57

(Chromanorm, VWR, Fontenay-sous-Bois, France) and ultra-pure water (Milli-Q Advantage

58

A10 with LC-pack, Merck Millipore, Darmstadt, Germany) were used for all sample

59

preparations. LC-MS grade acetonitrile and Optima grade acetic acid (Fisher Scientific,

60

Denmark) were used for analyses. The benzoxazinoid and phenoxazinone standards

61

(systematic names and formulas in Figure 1) were obtained as described by Adhikari et al.20

62

Inclusion Criterion and Recruitment

63

Prostate cancer patients scheduled for radical prostatectomy were invited with a brief

64

explanation from the project nurse to participate in this study, and in total, 10 patients were

65

enrolled. The inclusion criterion was the presence of more than 10% cancerous tissue in at

66

least one diagnostic needle biopsy of the enlarged prostate.

67

Study Design and Sampling

68

All 10 patients received a high-benzoxazinoid diet and subsequently had a consultation (Visit

69

1) with the project dietician, who provided detailed information and instructions concerning

70

the diet (see below) and how to keep a diet diary. At Visit 1, the patients were asked to

71

provide blood and urine samples. Each patient would begin a high-benzoxazinoid diet one

72

week prior to the scheduled prostatectomy. At Visit 2 (the day of the prostatectomy), the

73

patient would bring a 24-hour urine sample and new blood samples (5 x 10 mL) would be

74

obtained prior to the prostatectomy. The study was approved through the Danish Ethical

75

Committee, Protocol no. 1-10-72-177-13.


Page 7 of 45


76

Diet

77

The diet was designed to have a high content of benzoxazinoids based on the chemical

78

analyses of bread and other cereal products presented in Steffensen et al. (in preparation)28 in

79

the same manner used in our previous clinical study.29 Two types of rye bread were provided,

80

as were rye flakes (“Rugflager”, Urtekram, Denmark) for the easy preparation of porridge.

81

The first type of rye bread (“Multikerne rugbrød”, Schulstad, Denmark) was a loaf baked

82

from rye kernels, whole-grain rye flour, sifted rye flour, barley malt, and wheat flour, and the

83

second (“Rugfler”, Hatting, Denmark) was a bun baked from whole-grain rye flour with flax,

84

sunflower, and pumpkin seeds. The patients were asked to consume a minimum amount of

85

75-100 g of rye flakes, 3 slices of rye bread and 2 buns per day and to register their intake in

86

a diet diary. Using a scoring system, the patients were offered the possibility of exchanging

87

products without lowering the desired minimum intake of benzoxazinoids. The patients were

88

allowed to consume any other food products according to their normal habits and tastes, with

89

the exception of wheat and oats.

90

Urine, Plasma and Tissue Samples

91

The urine samples were obtained in beakers at Visit 1 and in 3-L containers for 24 hours prior

92

to Visit 2. The samples were aliquoted and stored at -80°C until analysis. Furthermore, all

93

urine samples were analyzed for creatinine at the Department of Clinical Biochemistry at

94

Aarhus University Hospital in order to normalize the benzoxazinoid concentration across

95

varying urine volumes.30 Blood was drawn in heparinized tubes, incubated for 30 min and

96

subsequently centrifuged for 10 min at 2000 g (2.0 rcf) at room temperature to separate the

97

plasma. The plasma samples were aliquoted and stored at -80°C until analysis. Using 18 G

98

Bard Max-Core bioptomes, five random needle core biopsies were sampled from each lobe of

99

the prostatectomies for histological and chemical analysis. Each biopsy was immediately

100

placed in TissueTek (Sakura Finetek Europe B.V., Alphen aan den Rijn, The Netherlands) in 6 ACS Paragon Plus Environment


Page 8 of 45

101

a cryo vial, snap-frozen in liquid nitrogen and stored at -80˚C. An HE-stained 4-µm

102

cryosection was cut from each core for histological assessment prior to the chemical analysis.

103

The prostatectomies were processed for conventional histopathological analysis. An

104

experienced pathologist microscopically assessed each core, and malignant infiltrations were

105

assigned a Gleason score according to the ISUP 2005 guidelines.31

106

Chemical Analysis of Metabolites in Urine, Plasma and Tissue Samples

107

Preparation of plasma and urine samples:

108

Plasma and urine samples were purified prior to benzoxazinoid analysis using our previously

109

published methods.12 The SPE-cleaned urine and plasma extracts were diluted 1:3 with water

110

and filtered using a KX syringe filter from Kinesis (PTFE, 13 mm, 0.22 µm, Mikrolab,

111

Aarhus, Denmark) prior to injection into the LC-MS/MS.

112

Preparation of the prostate biopsy samples:

113

The tissue extraction method was developed using prostate tissue from mini-pigs (Ellegaard

114

Göttingen Minipigs, Dalmose, Denmark) due to the limited supply of human tissue. Upon

115

euthanasia the pig prostates were removed, bagged, frozen using dry ice and stored at -20°C

116

until further use. The pig prostate biopsies were taken from the frozen tissue using a 1.0-mm-

117

diameter biopsy punch with a plunger (Miltex GmbH, Reitheim-Weilheim, Germany). The

118

biopsies were submerged in TissueTek and left at -80°C overnight to mimic the storage of

119

human prostate tissue, thawed, transferred from the TissueTek to the extraction vials, and

120

spiked with 5 µL of 200 ng/mL standard solution, weighing every step for control. After the

121

solvent was evaporated, the samples were extracted. We tested several extraction methods

122

such as shaking, sonication, and accelerated solvent extraction (ASE) in combination with a

123

variety of extraction solvents containing either water, methanol, acetonitrile, or a mixture of

124

these, acidified or neutral. The optimum results as a compromise between recovery and 7 ACS Paragon Plus Environment

Page 9 of 45


125

matrix effect were obtained by 30 min sonication in 20% acetonitrile and 0.5% acetic acid in

126

water in a small glass vial using 500 µL for each biopsy. Prior to analysis, the extracts were

127

filtered using a Kinesis KX syringe filter (PTFE, 4 mm, 0.22 µm). The human tissue samples,

128

10 from each of 10 patients, having a mean weight of 5.6 mg, were extracted using this

129

method. The recovery, limit of detection (LOD) and limit of quantification (LOQ) were

130

determined in 6 replicate pig prostate samples following The International Council for

131

Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH)

132

harmonized tripartite guidelines for validation of analytical procedures.32 The determination

133

of recovery in spiked pig prostate samples was performed for three different standard

134

solutions containing standard compounds combined in groups that underwent no detectable

135

interconversion. The groups are indicated in Figure 1.

136

Instrumentation:

137

The chemical analysis of the benzoxazinoids was performed using an Agilent (Glostrup,

138

Denmark) 1260s HPLC system coupled to a Sciex (Copenhagen, Denmark) QTRAP 4500

139

mass spectrometer equipped with electrospray ionization source. The compound dependent

140

MS settings (declustering potential, collision energy, and collision cell exit potential) were

141

optimized through direct infusion for maximum signal intensity in multiple reaction

142

monitoring (MRM) mode. The resulting MRM transitions (Q1/Q3) and parameters are shown

143

in Figure 1. The analytical method was divided into periods for optimized intensity (Figure

144

1 and Table 1) and the general mass-spectrometric parameters (nebulizer gas, drying gas,

145

curtain gas, temperature, and ion spray voltage) were optimized individually using flow

146

injection analysis via the autosampler and HPLC flow. The analytes were separated using a

147

Phenomenex (Allerød, Denmark) Synergi Polar RP-80A column (250 × 2 mm, 4 µm particle

148

size), flow rate: 300 µL min-1; injection volume: 10 µL; column oven: 30.0°C; autosampler

149

tray: 10°C. The wash vial contained a 1:1 acetonitrile/water solution. Analyst 1.6.2 software 8 ACS Paragon Plus Environment


Page 10 of 45

150

from Sciex (Copenhagen, Denmark) was used for instrument control, data acquisition, and

151

subsequent quantifications. Data points of the standard curves were weighted according to x-

152

1

153

78% acetonitrile in water (v/v). Both solvents A and B contained 20 mM acetic acid. The

154

optimized chromatographic method and instrument settings are listed in Table 1. The

155

chromatographic method allowed sufficient separation of most of the compounds of interest

156

in a single acquisition method (Figure 2).

157

Investigation of the instrument detection limit:

158

The instrument detection limit (IDL) was determined as a measure of the instrument

159

performance of the analytical system33 at the concentration where peak height ≥ 2*S/N (S/N

160

denotes the signal-to-noise ratio).32 The S/N ratio was calculated using the “Analyte signal to

161

noise” feature in the Analyst software and dividing this value by 4 to cover 95% of the noise,

162

assuming the noise is normally distributed. Low-concentration standard solutions of the

163

different standard mixtures at different dilutions were injected six times in random order in

164

order to measure the IDLs.

165

Investigation of matrix effects:

166

During optimization of the chromatographic method, the matrix effects of solvent and sample

167

blanks were investigated through the infusion of a mixed standard solution (16.0 ng/mL, 7.0

168

µL/min) into the HPLC stream to separate major matrix-effect contributors from target

169

analytes chromatographically.34, 35

170

The remaining matrix effect was individually determined for urine, plasma, and prostate

171

tissue. For each sample type, six mixed standard curves were prepared in parallel, three

172

curves in solvent and three curves in blank sample extracts of the same dilution as the

. The gradient was mixed from two eluent flasks: A, 7% acetonitrile in water (v/v), and B,


Page 11 of 45


173

corresponding sample matrices. Each of these standard curves had five dilution points,

174

covering the range of sample concentrations for the given sample type. The three standard

175

curves in sample extract were performed in blank samples from three different patients to

176

evaluate the presence of matrix effect differences between different patients within the same

177

matrix type (internal matrix effect) and the general matrix effect for each sample type

178

according to Matuszewski et al.36 The matrix effects were investigated at concentration

179

ranges from 100 to 0.391 ng/mL for urine and 1.60 to 0.00625 ng/mL for both prostate tissue

180

and plasma extracts. The dilution series was prepared by adding 30 µL of a higher

181

concentration to 90 µL of blank extract to form the next point in the series. Prior to analysis,

182

the serial dilutions were filtered.

183

Calculations and Statistics

184

Matrix effect evaluation:

185

The presence of an internal matrix effect within each matrix type was tested using Bartlett’s

186

test for homogeneity of variance. The variance in triplicate standard dilutions in the matrix

187

was tested against the triplicates in the solvent using a square-root transformation to

188

normalize the variance across dilution points.

189

The presence of a general matrix effect was investigated for each matrix type. The triplicate

190

standard curves were prepared in both solvent and sample matrices. Two additive models

191

were used to fit quadratic standard curves to the data points: The first model fitted one curve

192

to all six replicates while the second model fitted two curves: one for the three replicates in

193

solvent and one for the three replicates in sample matrix. The two models were compared

194

using ANOVA to determine whether the second model was a significantly better fit than the

195

first, thereby confirming the presence of a matrix effect. To determine a general numeric

196

value for the matrix effect of each compound in each matrix, the standard curves in the



197

matrix and in the solvent were integrated, and the matrix effect was calculated according to

198

Equation 1 over the range of the standard dilution in question:

199

=

(1)

Page 12 of 45

− 1.

200

Tests were performed using R version 3.2.1 statistical program.

201

An attempt was made to correct the quantification data for matrix effects using the two

202

triplicate sets of standard curves and Equation 2, where concquant is the quantified

203

concentration; concactual is the actual concentration; a, b, and c are coefficients of the terms of

204

the function in the absence of matrix; p, q, and r are correction factors to take into account the

205

matrix effect; and m = 1 or 0 depending on the presence or absence of matrix.

206 207

(2)

= ( + # × %) × ' + (( + ) × %) × + ( +

× %)

208

The 95% confidence intervals were obtained for both curves within each model using the

209

“predict” function of the stats package, and the root mean square prediction error (RMSPE)

210

was obtained using the “cvTools” package to perform a six-fold validation, leaving out one of

211

each of the six standard curve replicates (three with and three without matrix). For each

212

model, RMSPE, standard curves, parameter values, and confidence intervals for both curves

213

and parameters are given in Supporting Table 1.

214

Multivariate data analysis:

215

Histological scores of the human prostates were combined with the quantitative results of the

216

targeted benzoxazinoid analysis of the biopsies, plasma, and urine samples of the human

217

subjects, and then subjected to multivariate data analysis to differentiate the variables

218

between the benign and malignant cell groups of the prostate biopsies. The variables were


Page 13 of 45


219

mean-centered and scaled to unit variance prior to analysis using SIMCA 14 software

220

(Umetrics, Umeå, Sweden). Principal component analysis (PCA) was applied to evaluate the

221

overall structure of the data without considering any group information. After observing the

222

pattern of group differences between cell types in the PCA score plot, orthogonal partial least

223

squares discriminant analysis (OPLS-DA) was performed on data to identify discriminant

224

variables between the two cell groups. The quality of the models was evaluated through the

225

R2Y(cum) and Q2(cum) parameters. The OPLS-DA model was validated through the analysis

226

of the variance of cross-validated predictive residuals (CV-ANOVA), and the model was

227

considered valid when the p-value was lower than 0.05.37 The loading-line plots, variable

228

importance for projection (VIP), and S-plots generated from the model were used to visualize

229

the relative importance of different variables.



Page 14 of 45

230

RESULTS AND DISCUSSION

231

Histology

232

The results of the microscopic evaluation of the perioperative needle core biopsies and the

233

prostatectomies are shown in Table 2. A total of 10 biopsies were available from each of the

234

10 patients, with five biopsies being from each lobe. Acinar adenocarcima was observed in

235

three patients. The number of malignant cores was one core in patient 1, five cores in patient

236

9, and two cores in patient 10. The Gleason score in each core was 4+3=7, 3+5=8 and 3+4=7,

237

respectively. The Gleason scores matched the global Gleason score of each prostatectomy.

238

The estimated volume of carcinoma varied from 5% to 55% in the prostatectomies. The pT

239

stage38 was pT2a in two prostatectomies indicating less than 50% carcinoma in one lobe and

240

pT2c in eight prostatectomies indicating carcinoma in both prostate lobes.

241

Diet Adherence

242

Adherence was generally good for the short intervention period according to the dietary

243

records provided by the patients; however, some individuals complained about the large

244

quantities of rye consumed and the concomitant side effects, such as flatulence. These

245

findings should be considered as possible constraints on full adherence in future, longer

246

dietary interventions.

247

Method Validation of the Chemical Analysis

248

Instrument detection limit:

249

The instrument detection limit (IDL) is given as the mean concentrations, and S/N is

250

measured for each analyte at the dilution level, where all signals had S/N ≥ 2 (Table 3). Thus

251

the instrument was sensitive enough to measure picogram levels of compounds in 1 mL of

252

solvent.


Page 15 of 45


253

In general, the Analytical Methods Committee33 has recommended the investigation of the

254

limit of detection (LOD) in a blank sample; however, the complexity of the sample matrices

255

in this study and the possibility of internal matrix effects (see below) would provide an LOD

256

containing variation from the instrument performance, the matrix effects, and the sample

257

preparation procedures. This complexity would not contribute to the clarity of the overall

258

results, as the cause of systematic and random errors would remain unclear; hence, in this

259

case, IDL was preferred to classic LOD. Notably, the IDL is a measure used to describe the

260

performance of the analytical instrument: It is the lowest concentration at which an observed

261

peak can be taken as a true peak, rather than noise, with 95% certainty. It is not, however, the

262

concentration at which a true peak is first observed. Compounds can be detected at

263

concentrations well below the IDL, although the certainty of these measurements is lower

264

than that of the measurements above the IDL. The standard curves for quantifying the plasma

265

and biopsy extracts descend to concentrations around or below the IDL values reported in

266

Table 3. This finding is in accordance with the recommendations of the Analytical Methods

267

Committee,33 which state that measures lower than the detection limit should not be omitted

268

when performing multivariate statistical analysis of a dataset, as the low-concentration

269

samples might contain important information and omitting them might introduce bias. The

270

aim of our upcoming crossover study is to compare high and low concentrations to

271

investigate correlations between the benzoxazinoid content and prostate cancer scores for the

272

patients. Therefore, concentrations are measured as low as possible by visual inspection. To

273

minimize the uncertainty of these low-concentration measurements, standard curves are used

274

that approach the visual limit of detection. The random errors in these measurements are not a

275

problem, as the statistical models focus on systematic and not random variations.

276

Matrix effects:



Page 16 of 45

277

The matrix effects were treated in a two-step procedure, as suggested by Van Eeckhaut et

278

al.39 Firstly, during method development, matrix effects were detected by infusing an analyte

279

solution into a blank sample stream, resulting in negative peaks when a critical matrix effect

280

was present. Subsequently, the chromatographic method was adjusted to minimize co-elution

281

of analytes and matrix components. This was done for all three sample matrix types.

282

Secondly, after method development, the residual matrix effect was determined by

283

comparison of triplicate standard curves in solvent to triplicate standard curves in biopsy

284

extracts, plasma extracts, and urine extracts. Different patient sample lots were used for each

285

replicate to investigate both general as well as internal matrix effects.

286

Despite measures to reduce matrix effects during method development and sample

287

pretreatment, matrix effects were observed to some extent for most analytes in all three

288

matrices. The size and shape of the curves, however, differed between analytes, matrix types,

289

and concentration ranges. Figure 3 shows an example of a standard curve in solvent and in

290

matrix for a) biopsy extracts, b) plasma extracts, and c) urine extracts. The matrix effect was

291

dependent on concentration and was positive at low concentrations for most analytes but

292

gradually decreased and became negative at higher concentrations. The non-linear change in

293

the matrix effect indicated that the electrospray ionization depended on more than one

294

mechanism, with one mechanism being more important at low analyte concentrations, while

295

another mechanism was more important at high concentrations. To our knowledge this

296

concentration dependence has not previously been demonstrated. A detailed list of dilution

297

points for matrix and solvent curves and variations can be found in Supporting Table 2. A

298

summary of the matrix effects can be found in Table 3. The general matrix effects indicated

299

were calculated using Equation 1 and hence, do not reflect the concentration-dependent

300

variations. The presence of a general matrix effect was tested by ANOVA, and the level of

301

significance is marked by an asterisk (*) in the table. The ANOVA tests showed significant 15 ACS Paragon Plus Environment

Page 17 of 45


302

matrix effects for most analytes in the three matrices. Most matrix effects for the urine

303

extracts were negative; but, the values for most analytes were numerically small, indicating

304

that the matrix effect in these cases introduced only a minor inaccuracy to the result. The

305

matrix effects were generally positive for both biopsy and plasma extracts, although plasma

306

in particular exhibited a shift from a positive to a negative matrix effect with increasing

307

concentration, explaining why the mean-like values of the general matrix effect, shown in

308

Table 3, were low for plasma, while the ANOVA showed a significant matrix effect. These

309

results clearly demonstrate that elucidating matrix effects only through infusion of standard

310

compounds into a blank sample stream, or only determining the matrix effects at one

311

concentration is not sufficient to describe the accuracy of an analytical system in term of the

312

matrix effects.

313

The internal matrix effect was investigated by testing whether the variance of the dilution

314

points of the standard curve was larger in the matrix than in the solvent and the results are

315

listed in Table 3. For the urine extract, almost all analytes exhibited an internal matrix effect

316

in the examined concentration range, decreasing the overall precision of the quantification in

317

urine. The biopsy and plasma extracts exhibited few significant signs of internal matrix

318

effects, but at these low concentrations the normal variation of the method might disguise an

319

internal matrix effect when present.

320

As shown above, it is recommendable whenever possible to use matrix-matched standard

321

curves in the analysis of complex samples. However due to insufficient matrix material and

322

the presence of internal matrix effects, analytes like the benzoxazinoids examined in this

323

study had to be quantified against standard curves prepared only in solvent. We investigated

324

whether the triplicate standard curves in solvent and matrix could be used for correcting data

325

for matrix effect. For each of the combinations of 16 compounds × 3 matrices, a model giving

326

two quadratic curves was created to describe the quantified concentration (concquant) as a 16 ACS Paragon Plus Environment


Page 18 of 45

327

function of the actual concentration (concactual) in the solvent and in the matrix according to

328

Equation 2 (see Supporting Table 1). Estimated concentrations and their 95% confidence

329

intervals were found by solving the equations. The confidence intervals, however, were only

330

acceptable for some compounds and the approach was therefore abandoned.

331

Recovery of benzoxazinoids spiked to prostate tissue:

332

The recovery experiments were performed in pig prostate tissue as sufficient human prostate

333

tissue was unavailable. Biopsies were obtained from the pig tissue to mimic human samples,

334

then spiked and subsequently extracted as described above. Due to the small sample sizes,

335

extra efforts were undertaken to reduce variations due to lab procedures. Therefore, the

336

biopsies, spiking solutions, and the extraction solvent were weighed to correct for variations

337

in the pipetting procedure. The results of the recovery experiment are shown in Supporting

338

Table 3 both before and after correction for variance in the pipetting procedure. These results

339

showed that in most cases, as the recovery percentage increases, the variation decreases when

340

the weight of the pipetted solutions is accounted for. Pipetting thus introduces both

341

systematic and random errors to the analysis results. The accuracy expressed as recovery

342

percentage for most of the compounds was within or close to the range of 80-120% set by the

343

ICH harmonized tripartite guideline for analytical procedures validation.32 The precision of

344

this analytical method as measured by the coefficient of variation (CV) was in the range of 4-

345

13% (Supporting Table 3) demonstrating the high precision of the method.

346

To determine whether the recovery was dependent on the mass of the individual biopsies,

347

these data (not shown) were plotted against each other; however, no significant correlation

348

was observed.


Page 19 of 45


349

Benzoxazinoid Content of Prostate Tissue

350

The quantification data for the prostate tissue biopsies from the patients after one week on an

351

intensive rye diet is shown in Figure 4a. The prostate tissue of patients 6 and 8 had

352

significantly higher benzoxazinoid content (>2.6 ng/g tissue) than that of the other patients

353

(2. In urine, several methoxylated benzoxazinoids

422

were observed at low concentrations, indicating that these compounds were both absorbed

423

and excreted, although they were not detected in plasma and prostate tissue samples. Small 20 ACS Paragon Plus Environment


Page 22 of 45

424

amounts of the phenoxazinones 2-aminophenoxazin-3-one (APO) and 2-

425

acetylaminophenoxazin-3-one (AAPO), which are degradation products of benzoxazinoids,42

426

were primarily detected in the urine samples from post-intervention samples. It was not

427

possible, however, to establish whether these transformation products were formed in the

428

body or after delivery of the urine sample. APO forms from 2-aminophenol, a hydrolysis

429

product of BOA or HBOA, and this reaction spontaneously occurs in the presence of oxygen

430

and is catalyzed by microbial enzymes.42 The transformation of APO into AAPO is unlikely

431

to occur spontaneously in urine and is therefore an indicator of biological transformation.

432

This reaction could, however, reflect microorganisms deposited in the urine sample during

433

the 24-h sampling period in the patient’s home.

434

OPLS-DA analysis resulted in a 1+1 OPLS model with R2Y(cum) and Q2(cum) values of

435

0.71 and 0.27, respectively, illustrating the poor predictivity of the model (Figure 8a), which

436

was not unexpected in this short pilot study. This may have been due to the short intervention

437

period and few patients in the malignant cell group. Thus, despite the poor predictivity of the

438

discriminant analysis of the variables between the two cell types in this pilot study, a longer

439

intervention study with more patients could be relevant. The observations spread over the

440

vertical direction and separated along the orthogonal component t0 [1] showed variation

441

before and after the intervention. Histological data were the most discriminant variables

442

contributing to the differentiation of the malignant carcinoma group from the benign cell

443

group (Figure 8b). As in plasma, most benzoxazinoids were correlated with the benign cell

444

group. The negative correlation of benzoxazinoids and histological data suggested that

445

benzoxazinoids might play a role in carcinoma progression, thereby requiring a longer-

446

duration intervention study.

447

The inhibiting effect of consumption of wholegrain rye on prostate cancer is of great potential

448

in public health management. However, causality must first be established, in order to take 21 ACS Paragon Plus Environment

Page 23 of 45


449

full advantage of the potential of rye-based food products. Rye phytochemicals are likely

450

candidates, and the benzoxazinoids must be considered prime suspects, as they have

451

previously shown anti-prostate cancer activity in in vitro experiments. We have established a

452

comprehensive methodology for testing this hypothesis by analyzing the benzoxazinoids in

453

prostate biopsies in addition to urine and plasma samples. The benzoxazinoids were

454

detectable in urine and plasma as a picture of the dynamic metabolic processes in the body.

455

Most interestingly, the benzoxazinoids, which were also detectable in prostate tissue after

456

prostate cancer patients had spent just one week on a rye-enriched diet, could cause a direct

457

effect on the prostate tissue following long-term dietary exposure. Our preliminary statistical

458

results indicated an inverse correlation between the concentrations of benzoxazinoids and

459

histological data of malignant tissue in the prostatectomies, but more research is still needed

460

to confirm these indications. Causality testing requires robust analytical methods. Elaborate

461

matrix effect studies is a way to test both accuracy and precision of such analytical methods.

462

Furthermore, the elucidation of inter-patient matrix effects may also reveal a source of

463

causalities indicated by statistics and should always be considered before final conclusions

464

are made. We show that matrix effects exhibit complicated patterns and that simplistic matrix

465

effect testing will not give a good indication of the true influence of the sample matrix.

466

ASSOCIATED CONTENT

467

Supporting Information

468

Detailed list of dilution points for matrix and solvent standard curves and variations

469

(Supplementary Table 1), model parameters of matrix effect analysis (Supplementary Table

470

2), and recovery analysis (Supplementary Table 3) (PDF).



Page 24 of 45

471

AUTHOR INFORMATION

472

Corresponding Author

473

Email: [email protected]. Phone: (45) 87158178. Fax: (45) 87156082

474

Funding

475

This study was conducted as part of the project “Whole grain rye as a functional food for

476

suppression of prostate cancer - elucidating the role of benzoxazinoids and other bioactive

477

constituents” (RyeproC) and generously funded through the grant 0602-02416B from the

478

Danish Council for Independent Research, Technology and Production (FTP).

479

Notes

480

Inge S. Fomsgaard is listed as co-inventor on the patent application, PA 84245 "Use of

481

benzoxazinoids-containing cereal grain products for health-improving purposes". The

482

remaining authors have no conflicts of interest.

483

The authors declare no competing financial interest.

484

ACKNOWLEDGMENTS

485

The authors would like to thank Ellegaard Göttingen Minipigs A/S (Dalmose, Denmark) for

486

supplying pig tissue for method development, CytoTrack ApS (Lyngby, Denmark) for

487

running the circulating tumor cell screenings, and Lantmännen (Stockholm, Sweden) for

488

supplying bread for the diets.

489

ABBREVIATIONS USED

490

AAPO, 2-acetylaminophenoxazin-3-one; APO, 2-aminophenoxazin-3-one; BOA,

491

benzoxazolin-2-one; DIBOA, 2,4-dihydroxy-1,4-benzoxazin-3-one; DIBOA-glc, 2-β-D-

492

glucopyronosyloxy-4-hydroxy-1,4-benzoxazin-3-one; DIMBOA-glc, 2-β-D-

493

glucopyranosyloxy-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one; HBOA, 2-hydroxy-1,423 ACS Paragon Plus Environment

Page 25 of 45


494

benzoxazin-3-one; HBOA-glc, 2-β-D-glucopyronosyloxy -1,4-benzoxazin-3-one; HMBOA,

495

2-hydroxy-7-methoxy-1,4-benzoxazin-3- one; HMBOA-glc, 2-β-D-glucopyranosyloxy-7-

496

methoxy-1,4-benzoxazin-3-one; CV-ANOVA, analysis of variance of cross-validated

497

predictive residuals; IDL, instrument detection limit; MRM, multiple reaction monitoring;

498

OPLS-DA, orthogonal partial least squares discriminant analysis; PCA, principal component

499

analysis; RMSPE, root mean square prediction error.



Page 26 of 45

500

References

501

1.

502

Parkin, D. M.; Forman, D.; Bray, F. GLOBOCAN 2012 v1.0, Cancer incidence and mortality

503

worldwide: IARC CancerBase No. 11 [Internet]. Lyon, France: International Agency for

504

Research on Cancer; 2013. http://globocan.iarc.fr, accessed on 11/07/2016

505

2.

506

accessed on 11/07/2016

507

3.

508

accessed on 11/07/2016

509

4.

510

2009 (Tal og Analyse:Cancerregistret 2009) (In Danish). 2009.

511

5.

512

Gulley, J. L. A retrospective study of the time to clinical endpoints for advanced prostate

513

cancer. BJU Int. 2005, 96, 985-989.

514

6.

515

P.; Adlercreutz, H.; Nilsson, T. K.; Hallmans, G.; Bergh, A.; Stattin, P. Randomised

516

controlled short-term intervention pilot study on rye bran bread in prostate cancer. Eur. J.

517

Cancer Prev. 2003, 12, 407-415.

518

7.

519

diet increases epithelial cell apoptosis and decreases epithelial cell volume in TRAMP

520

(transgenic adenocarcinoma of the mouse prostate) tumors. Nutr. Cancer 2005, 53, 111-116.

521

8.

522

Adlercreutz, H.; Kamal-Eldin, A.; Åman, P.; Hallmans, G. Rye whole grain and bran intake

523

compared with refined wheat decreases urinary c-peptide, plasma insulin, and prostate

524

specific antigen in men with prostate cancer. J. Nutr. 2010, 140, 2180-2186.

Ferlay, J.; Soerjomataram, I.; Ervik M.; Dikshit, R.; Eser, S.; Mathers, C.; Rebelo, M.;

EUCAN statistics. http://eco.iarc.fr/eucan/CancerOne.aspx?Cancer=29&Gender=1,

SEER stat fact sheets: Prostate cancer. http://seer.cancer.gov/statfacts/html/prost.html,

The National Board of Health (Sundhedsstyrrelsen). Cancer incidence in Denmark

Sharifi, N.; Dahut, W. L.; Steinberg, S. M.; Figg, W. D.; Tarassoff, C.; Arlen, P.;

Bylund, A.; Lundin, E.; Zhang, J. X.; Nordin, A.; Kaaks, R.; Stenman, U.-H.; Åman,

Wikstrom, P.; Bylund, A.; Zhang, J.-X.; Hallmans, G.; Stattin, P.; Bergh, A. Rye bran

Landberg, R.; Andersson, S.-O.; Zhang, J.-X.; Johansson, J.-E.; Stenman, U.-H.;


Page 27 of 45


525

9.

Fomsgaard, I. S.; Mortensen, A. G.; Holm, P. B.; Gregersen, P. L. Use of

526

benzoxazinoids-containing cereal grain products for health-improving purposes. EP 2 265

527

133 A1, 2010, 2010.

528

10.

529

cereal cultivars contains an important array of neglected bioactive benzoxazinoids. Food

530

Chem. 2011, 127, 1814-1820.

531

11.

532

Qualitative characterization of benzoxazinoid derivatives in whole grain rye and wheat by

533

LC-MS metabolite profiling. J. Agric. Food Chem. 2011, 59, 921-927.

534

12.

535

benzoxazinoids in rye bread are absorbed and metabolized in pigs. J. Agric. Food Chem.

536

2012, 60, 2497-2506.

537

13.

538

certain bioactive components in whole grain wheat and rye. J. Cereal Sci. 2014, 59, 294-311.

539

14.

540

by HPLC-DAD and UPLC-QTOF MS. Food Chem. 2016, 204, 400-408.

541

15.

542

Poulsen, L. K.; Nielsen, C. H.; Høyer, S.; Borre, M.; Fomsgaard, I. S. Benzoxazinoids:

543

Cereal phytochemicals with putative therapeutic and health-protecting properties. Mol. Nutr.

544

Food Res. 2015, 59, 1324-1338.

545

16.

546

Isolation and characterization of a cyclic hydroxamic acid from a pollen extract, which

547

inhibits cancerous cell wrowth in vitro. J. Med. Chem. 1995, 38, 735-738.

548

17.

549

prostate inhibitory substance in a pollen extract. Prostate 1995, 26, 133-139.

Pedersen, H. A.; Laursen, B.; Mortensen, A.; Fomsgaard, I. S. Bread from common

Hanhineva, K.; Rogachev, I.; Aura, A.-M.; Aharoni, A.; Poutanen, K.; Mykkänen, H.

Adhikari, K. B.; Laursen, B. B.; Laerke, H. N.; Fomsgaard, I. S. Bioactive

Andersson, A. A. M.; Dimberg, L.; Åman, P.; Landberg, R. Recent findings on

Pihlava, J. M.; Kurtelius, T. Determination of benzoxazinoids in wheat and rye beers

Adhikari, K. B.; Tanwir, F.; Gregersen, P. L.; Steffensen, S. K.; Jensen, B. M.;

Zhang, X.; Habib, F. K.; Ross, M.; Burger, U.; Lewenstein, A.; Rose, K.; Jaton, J.-C.

Habib, F. K.; Ross, M.; Lewenstein, A.; Zhang, X.; Jaton, J. C. Identification of a



Page 28 of 45

550

18.

Roberts, K. P.; Iyer, R. A.; Prasad, G.; Liu, L. T.; Lind, R. E.; Hanna, P. E. Cyclic

551

hydroxamic acid inhibitors of prostate cancer cell growth: Selectivity and structure activity

552

relationships. Prostate 1998, 34, 92-99.

553

19.

554

concentrations of bioactive dietary benzoxazinoids and their glucuronidated conjugates in rats

555

fed a rye bread-based diet. J. Agric. Food Chem. 2012, 60, 11518-11524.

556

20.

557

Poulsen, L. K.; Jensen, B. M.; Fomsgaard, I. S. Absorption and metabolic fate of bioactive

558

dietary benzoxazinoids in humans. Mol. Nutr. Food Res. 2013, 57, 1847-1858.

559

21.

560

Fomsgaard, I. S. Allelochemicals in rye (Secale cereale L.): cultivar and tissue differences in

561

the production of benzoxazinoids and phenolic acids. Nat. Prod. Commun. 2009, 4, 199-208.

562

22.

563

Bastidas, A.; Macías, F. A.; Stochmal, A.; Oleszek, W.; Shakaliene, O.; Barceló, D. First

564

European interlaboratory study of the analysis of benzoxazinone derivatives in plants by

565

liquid chromatography. J. Chromatogr. A 2004, 1047, 69-76.

566

23.

567

chromatography–electrospray ionization mass spectrometry analysis of benzoxazinoid

568

derivatives in plant material. J. Chromatogr. A 2007, 1157, 108-114.

569

24.

570

H.; Poutanen, K. UPLC-QTOF/MS metabolic profiling unveils urinary changes in humans

571

after a whole grain rye versus refined wheat bread intervention. Mol. Nutr. Food Res. 2013,

572

57, 412-422.

Adhikari, K. B.; Laerke, H. N.; Mortensen, A. G.; Fomsgaard, I. S. Plasma and urine

Adhikari, K. B.; Laursen, B. B.; Gregersen, P. L.; Schnoor, H. J.; Witten, M.;

Carlsen, S. C. K.; Kudsk, P.; Laursen, B.; Mathiassen, S. K.; Mortensen, A. G.;

Eljarrat, E.; Guillamón, M.; Seuma, J.; Mogensen, B. B.; Fomsgaard, I. S.; Olivero-

Villagrasa, M.; Guillamón, M.; Eljarrat, E.; Barceló, D. Matrix effect in liquid

Bondia-Pons, I.; Barri, T.; Hanhineva, K.; Juntunen, K.; Dragsted, L. O.; Mykkänen,


Page 29 of 45


573

25.

Hanhineva, K.; Keski-Rahkonen, P.; Lappi, J.; Katina, K.; Pekkinen, J.; Savolainen,

574

O.; Timonen, O.; Paananen, J.; Mykkänen, H.; Poutanen, K. The Postprandial Plasma Rye

575

Fingerprint Includes Benzoxazinoid-Derived Phenylacetamide Sulfates. J. Nutr. 2014.

576

26.

577

phenylacetamides derived from bioactive benzoxazinoids are bioavailable in humans after

578

habitual consumption of whole grain sourdough rye bread. Mol. Nutr. Food Res. 2013, 57,

579

1859-1873.

580

27.

581

Martínez-González, M.; Corella, D.; Fitó, M.; Estruch, R.; Serra-Majem, L.; Andres-

582

Lacueva, C. Nutrimetabolomics fingerprinting to identify biomarkers of bread exposure in a

583

free-living population from the PREDIMED study cohort. Metabolomics 2015, 11, 155-165.

584

28.

585

Jensen, C.; Fomsgaard, I. S. Bioactive small molecules in commercially available cereal

586

products from Danish supermarkets (I): Benzoxazinoids. In preparation 2016.

587

29.

588

Poulsen, L. K. Quantitative analysis of absorption, metabolism, and excretion of

589

benzoxazinoids in humans after the consumption of high- and low-benzoxazinoid diets with

590

similar contents of cereal dietary fibres: a crossover study. Eur. J. Nutr. 2015, 1-11.

591

30.

592

L. Urinary creatinine concentrations in the U.S. Population: implications for urinary biologic

593

monitoring measurements. Environ. Health Perspect. 2005, 113, 192-200.

594

31.

595

international society of urological pathology (ISUP) consensus conference on gleason

596

grading of prostatic carcinoma. Am. J. Surg. Pathol. 2005, 29, 1228-42.

Beckmann, M.; Lloyd, A. J.; Haldar, S.; Seal, C.; Brandt, K.; Draper, J. Hydroxylated

Garcia-Aloy, M.; Llorach, R.; Urpi-Sarda, M.; Tulipani, S.; Salas-Salvadó, J.;

Steffensen, S. K.; Adhikari, K. B.; Borre, M.; Borre, M.; Høyer, S.; Laursen, B.;

Jensen, B. M.; Adhikari, K. B.; Schnoor, H. J.; Juel-Berg, N.; Fomsgaard, I. S.;

Barr, D. B.; Wilder, L. C.; Caudill, S. P.; Gonzalez, A. J.; Needham, L. L.; Pirkle, J.

Epstein, J. I.; Allsbrook, W. C., Jr.; Amin, M. B.; Egevad, L. L. The 2005



Page 30 of 45

597

32.

The International Council for Harmonisation of Technical Requirements for

598

Pharmaceuticals for Human Use (ICH). ICH harmonised tripartite guideline. Validation of

599

analytical procedures: Text and methodology Q2(R1). International Conference on

600

Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human

601

Use. In 1994/1996; p 13.

602

33.

603

estimation and use of the detection limit. Analyst 1987, 112, 199-204.

604

34.

605

of matrix effects, its removal and estimation in ESI-LC-MS/MS bio-analysis. J. Anal.

606

Bioanal. Tech. 2010, 1, 7.

607

35.

608

matrix effect of different sample matrices for 33 pharmaceuticals by post-column infusion. J.

609

Chromatogr. B 2015, 1000, 84-94.

610

36.

611

assessment of matrix effect in quantitative bioanalytical methods based on HPLC−MS/MS.

612

Anal. Chem. 2003, 75, 3019-3030.

613

37.

614

OPLS® models. J. Chemom. 2008, 22, 594-600.

615

38.

616

seventh edition; Sobin, L. H., Gospodarowicz, M. K., Wittekind, Ch., Eds.; Wiley-Blackwell:

617

West Sussex, UK, 2009; pp. 243-248.

618

39.

619

bioanalytical LC–MS/MS assays: Evaluation of matrix effects. J. Chromatogr. B 2009, 877,

620

2198-2207.

Analytical Methods Committee. Recommendationdations for the definition,

Ghosh, C. S., Chandrakant P.; Chakraborty, Bhaswat S. Ionization polarity as a cause

Rossmann, J.; Gurke, R.; Renner, L. D.; Oertel, R.; Kirch, W. Evaluation of the

Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M. Strategies for the

Eriksson, L.; Trygg, J.; Wold, S. CV-ANOVA for significance testing of PLS and

UICC International Union Against Cancer. TNM classification of malignant tumours,

Van Eeckhaut, A.; Lanckmans, K.; Sarre, S.; Smolders, I.; Michotte, Y. Validation of


Page 31 of 45


621

40.

Azorin-Ortuno, M.; Yanez-Gascon, M. J.; Vallejo, F.; Pallares, F. J.; Larrosa, M.;

622

Lucas, R.; Morales, J. C.; Tomas-Barberan, F. A.; Garcia-Conesa, M. T.; Espin, J. C.

623

Metabolites and tissue distribution of resveratrol in the pig. Mol. Nutr. Food Res. 2011, 55,

624

1154-1168.

625

41.

626

radical–scavenging, and cytotoxic activities of Acanthus hirsutus boiss. J. Med. Food 2011,

627

14, 767-774.

628

42.

629

products of benzoxazolinone and benzoxazinone allelochemicals––a review. Chemosphere

630

2004, 54, 1025-1038.

Harput, U. S.; Arihan, O.; Iskit, A. B.; Nagatsu, A.; Saracoglu, I. Antinociceptive, free

Fomsgaard, I. S.; Mortensen, A. G.; Carlsen, S. C. K. Microbial transformation



Page 32 of 45

631

Figure caption

632

Figure 1

633

The benzoxazinoids and phenoxazinones. Compound abbreviations, structural information, chemical

634

names, mass spectrometric parameters, and analytical groups. Q1/Q3: Mother/daughter ion mass

635

transition; DP: Declustering potential; CE: Collision energy; CXP: Collision cell exit potential.

636

Figure 2

637

MRM chromatograms of benzoxazinoid and phenoxazinone standards at a concentration of 50 ng/mL.

638

The MBOA peak is cut off due to scaling up or low-intensity peaks such as DIBOA. Retention times

639

are given in parentheses. Signal intensity is given in counts per second (cps).

640

Figure 3

641

Standard curves in matrix versus standard curves in solvent for DIBOA-glc in the three different

642

sample matrices. This example is representative of the general tendencies for all analytes in the three

643

matrices. Solid line and black points: standard curve in matrix. Dashed line and white points: standard

644

curve in solvent.

645

Figure 4

646

Benzoxazinoid content of prostate tissue. a) Mean content of each benzoxazinoid in prostate tissue

647

from each patient. Data from peaks where S/N > 2 or where compounds were present in only one

648

biopsy were excluded. b) Relative contributions of individual biopsies to the total benzoxazinoid

649

content for each patient. Ten biopsies were taken from each patient. Asterisks indicate biopsies

650

containing malignant tissue.

651

Figure 5

652

Pre- and post-intervention content of benzoxazinoids and phenoxazinones in plasma.

653

Figure 6


Page 33 of 45


654

Tentative correlations between plasma benzoxazinoids and histological data for prostate cancer

655

patients. a) OPLS-DA score plot for plasma samples showing a benign (green circle) and malignant

656

(blue circle) group. Statistical parameters for the 1+2 OPLS-DA model were R2X = 0.78, R2Y = 0.83,

657

Q2 = 0.65, p[CV-ANOVA] = 0.01 b) OPLS-DA loading plot for plasma samples.

658

Figure 7

659

Pre- and post-intervention content of benzoxazinoids and phenoxazinones in urine. The 24h post-

660

intervention urine sample for patient 4 was not collected, and is therefore absent.

661

Figure 8

662

Tentative correlations between urine benzoxazinoids and histological data for prostate cancer patients.

663

a) OPLS-DA score plot for urine samples showing separation of a benign (green circle) and malignant

664

(blue circle) group. Statistical parameters for the 1+1 OPLS-DA model were R2X = 0.53, R2Y = 0.71,

665

Q2 = 0.27, p[CV-ANOVA] = 0.31 b) OPLS-DA loading plot for urine.



Page 34 of 45

Tables Table 1. Time table for the chromatographic and instrument settings for the LC-MSMS method. Time (min) Eluent B (%) LC stream Acquisition period (min) Ionization mode Curtain gas (psi) Ion spray voltage (V) Entrance potential (V) Temperature (°C) Drying gas (psi) Nebulizer gas (psi)

0 0

1 8

2

3 4 5 10 to waste 0.0-8.4

6

7

8

9

10

11

8.4-11.3

12

13 14 70 90 to MS 11.3-15.0

negative 20 -4500 -2 550 85

450 50

15

16 90

17 0

18

19

20

21

22

23 0

to waste 15.0-23.0 positive 40 4500 5 500 60

90

33

ACS Paragon Plus Environment

Page 35 of 45


Table 2. Histological data of the prostate tissues of the patients involved in the study Needle core biopsies a

Prostatectomy Histological Data

Biopsy No. 1 2 3 4 5 6 7 8 9 10 % Carcinoma Gleason score pT

a

Patient 1

BM B B B B B B B B

30

4+3

pT2c

Patient 2

BB B B BBB BB B

5

3+4

pT2a

Patient 3

BB B B BBB BB B

30

3+4

pT2c

Patient 4

BB B B BBB BB B

15

4+3

pT2c

Patient 6

BB B B BBB BB B

25

4+3

pT2c

Patient 7

BB B B BBB BB B

15

3+3

pT2c

Patient 8

BB B B BBB BB B

20

4+3

pT2c

Patient 9

B B M B MBMMBM

30

3+5

pT2c

Patient 10 B B B M B B M B B B

55

3+4

pT2c

Patient 11 B B B B B B B B B B

5

3+3

pT2a

B denotes benign prostate cell type, M denotes malignant prostate cell type



Page 36 of 45

Table 3: Instrument detection limit and matrix effect. Biopsy and plasma matrices were investigated at analyte concentrations of 1.60, 0.400, 0.100, 0.0250, and 0.00625 ng/mL, and urine at 100, 25.0, 6.25, 1.56, and 0.391 ng/mL.

Compound

Solvent

Biopsy

Plasma

Urine

IDLa mean concentration

General

Internal

General

Internal

General

Internal

(ng/mL), [mean S/N]

matrix

matrix

matrix

matrix

matrix

matrix

effect b

effect c

effect

effect c

effect

effect c

HBOA

0.00171 [2.7]

0.08**

0.05***

-0.15***

#

BOA

0.00654 [3.1]

-0.23***

0.03***

-0.25***

###

MBOA

0.00128 [2.4]

-0.39***

-0.02***

-0.18***

#

HMBOA

0.000960 [2.5]

0.02

0.03***

-0.24***

###

DIBOA

0.326 [5.6]

0.16*d

-0.08*** d

-0.10**

DIMBOA

0.311 [2.4]

0.09 d

-0.17*** d

-0.23***

###

HBOA-glc

0.00571 [3.0]

0.19***

0.08***

-0.08**

##

HBOA-glc-hex

0.0117 [2.5]

0.22***

0.12***

-0.04*

##

HMBOA-glc

0.00324 [2.5]

0.22***

0.08***

-0.12***

###

DIBOA-glc

0.00645 [2.9]

0.20***

0.07***

-0.09***

#

DIMBOA-glc

0.0219 [2.9]

0.20***

-0.00***

-0.08*

#

DIBOA-glc-

0.00237 [2.7]

0.26***

0.05***

-0.03*

#

APO

0.00292 [2.7]

0.64***

0.38***

0.00

AMPO

0.00225 [2.2]

0.88***

0.78***

0.02*

AAPO

0.000699 [2.3]

0.28***

0.14**

-0.03

AAMPO

0.000713 [2.6]

0.15*

0.06***

-0.03*

##

#

hex

a

##

IDL = Instrument detection limit. The concentration measured for six injections of a low-concentration

standard solution where all injections had a signal-to-noise ratio (S/N) >2. b

The general matrix effect was calculated as the integrated matrix standard curve divided by the integrated

solvent standard curve minus 1. This value, however, does not reflect intersecting standard curves (Figure 3). Asterisks indicate statistically significant differences between standard curves in matrix and solvent with pvalues denotaed as follows: p < 0.001: ***, 0.001 < p < 0.01: **, 0.01 < p < 0.05: *. c

The presence of an internal matrix effect was confirmed when Bartlett’s test showed that the variance of the

dilution points of the matrix standard curve was significantly greater than that of the solvent standard curve.


Page 37 of 45


Hashtags indicate statistical significance, with p-values denoted as follows: p < 0.001: ###, 0.001 < p < 0.01: ##, 0.01 < p < 0.05: #. d

Reduced sample set reflecting a higher detection limit.



Page 38 of 45

Figures Figure 1


Page 39 of 45


Figure 2 3E+06

HBOA-Glc-Hex (7.49) MBOA (13.83)

DIMBOA-Glc (10.24)

DIBOA-Glc-Hex (7.53)

AAPO (17.51)

3E+06 AAMPO (17.86)

Intensity, cps

DIBOA-Glc (8.81)

HBOA (10.67)

2E+06 HMBOA-Glc (10.09)

2E+06

APO (16.45)

HMBOA (11.79)

HBOA-Glc (8.72)

1E+06

AMPO (16.92) BOA (13.00)

DIBOA (10.58)

5E+05 0E+00 6

8

10

12

14

16

18

20

Time, min



Page 40 of 45

Figure 3


Page 41 of 45


Figure 4



Page 42 of 45

Figure 5


Page 43 of 45


Figure 6



Page 44 of 45

Figure 7


Page 45 of 45


Figure 8


Benzoxazinoids in Prostate Cancer Patients after a Rye-Intensive Diet

Recommend Documents