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
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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
2 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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.
3 ACS Paragon Plus Environment
Page 5 of 45
Journal of Agricultural and Food Chemistry
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.
4 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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.
5 ACS Paragon Plus Environment
Page 7 of 45
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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,
9 ACS Paragon Plus Environment
Page 11 of 45
Journal of Agricultural and Food Chemistry
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
10 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
11 ACS Paragon Plus Environment
Page 13 of 45
Journal of Agricultural and Food Chemistry
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.
12 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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.
13 ACS Paragon Plus Environment
Page 15 of 45
Journal of Agricultural and Food Chemistry
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:
14 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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.
17 ACS Paragon Plus Environment
Page 19 of 45
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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).
22 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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.
24 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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.;
25 ACS Paragon Plus Environment
Page 27 of 45
Journal of Agricultural and Food Chemistry
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
26 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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,
27 ACS Paragon Plus Environment
Page 29 of 45
Journal of Agricultural and Food Chemistry
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
28 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
29 ACS Paragon Plus Environment
Page 31 of 45
Journal of Agricultural and Food Chemistry
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
30 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
31 ACS Paragon Plus Environment
Page 33 of 45
Journal of Agricultural and Food Chemistry
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.
32 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
Journal of Agricultural and Food Chemistry
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
34 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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.
35 ACS Paragon Plus Environment
Page 37 of 45
Journal of Agricultural and Food Chemistry
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.
36 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 38 of 45
Figures Figure 1
37 ACS Paragon Plus Environment
Page 39 of 45
Journal of Agricultural and Food Chemistry
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
38 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 40 of 45
Figure 3
39 ACS Paragon Plus Environment
Page 41 of 45
Journal of Agricultural and Food Chemistry
Figure 4
40 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 42 of 45
Figure 5
41 ACS Paragon Plus Environment
Page 43 of 45
Journal of Agricultural and Food Chemistry
Figure 6
42 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 44 of 45
Figure 7
43 ACS Paragon Plus Environment
Page 45 of 45
Journal of Agricultural and Food Chemistry
Figure 8
44 ACS Paragon Plus Environment