Download Supplementary Material Experimental Patient selection and sample

Supplementary Material
Experimental
Patient selection and sample collection
Biological material was collected from patients qualified for a surgical procedure either for a radical
prostatectomy (RP) or transurethral resection of the prostate (TURP) in the Department of Urology. Jan Biziel
Hospital in Bydgoszcz. Poland. A total of 50 individuals were recruited into two main groups of patients - Cancer
group consisting of 25 patients diagnosed with prostate cancer qualifying for RP and a second benign prostatic
hyperplasia group (BPH) consisting of 25 patients qualifying for TURP with no evidence of malignancy but
diagnosed with benign prostate growth. All diagnoses were confirmed by histopathological examination of the
prostate gland removed from cancer patients. and from all prostatic tissue resected from patients with benign
prostatic hyperplasia. Due to a number of factors including ethical concerns patients health and potential
complications prostate glands of patients in the BPH group were not removed therefore histopathological
examination of the whole gland was not performed. Patients with a negative digital rectal exam, negative biopsy
results and no malignancy in resected tissue qualified for this group. Prostate volume in the cancer group was
measured by the pathologist and in the BPH group volume was determined by transrectal ultrasound. All
histopathological results were standardised according to WHO classification. Patients that had undergone prostate
biopsy or other urological procedure within 1 month prior to hospital admission were excluded from this study.
Two urine samples were collected from each patient. The first sample was collected in early morning
hours, into a sterile urine container. The second sample was collected after prostate massage performed by a
resident physician, employee of the Department of Urology, Jan Biziel Hospital in Bydgoszcz, Poland. Prostate
massage consisted of 3 sweeps per lobe, depressing the prostate gland (0.5–1 cm) in a milking action. All samples
were prepared for storage immediately after collection from patients. Sodium azide solution (approximately 1 mM
sodium azide final concentration in urine) was added to prevent bacterial contamination. Samples were divided in
to smaller aliquots and stored at -80°C until further analysis.
Materials and methods
Chemicals and solvents used throughout this study were purchased from Sigma Aldrich (Castle Hill.
Australia). All were either analytical or mass spectrometric grades. Deionized water (18.2 MΩ) was produced
using a Synergy UV Millipore System (Millipore).
Solutions of each amino acid or amine were prepared by dissolving individual amino acids or amines in
50% methanol containing 0.1% formic acid. The concentrated amino acid and amines solutions were then
combined to form a standard stock solution at a final concentration of 2.5 mM using volumetric glassware.
Supplementary Material
Calibration standards were generated by diluting the stock solutions to 100, 50, 25, 10, 5, 1, 0.5, 0.1, 0.05, and
0.01 μM in water containing 1 mM ascorbic acid and 10 mM tris(2-carboxyethyl)phosphine (TCEP).
Amino acids, amines and other low molecular weight compounds were extracted using 4°C methanol
solution containing internal standards which were used for extraction correction. 200 µL of the cooled internal
standard solution in methanol was transferred to an eppendorf tube with 100 µL of urine sample and vortexed for
30 seconds. Samples were cooled on ice for 5 minutes then centrifuged at 15.000 rpm for 10 minutes. 150 µL of
the supernatant was transferred to a fresh eppendorf tube.
Derivatization procedure was performed using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
(Aqc) according to Boughton B. et al. All information regarding method validation was presented in paper publish
by Boughton BA et al (2011) Comprehensive Profiling and Quantitation of Amine Group Containing Metabolites.
This paper is cited in manuscript. Within that publication Limit of Detections (LOD’s), Linear Calibration Ranges
(LCR’s) recovery and stability assays were validated for quantitation of amino acids extracted from human urine.
Amino acids were quantitated against an external calibration curve. Data from this paper is presented in table S1.
We have previously validated this approach (Boughton et al., Anal. Chem. 2011) and determined external
calibration curves generated in this manner are sufficient to provide relative quantification for metabolomics
comparative studies across a range of different biological matrices. We used 2-aminobutyric acid added to the
sample during the derivatization process to account for changes in instrument sensitivity between samples. We
added labelled valine during the extraction process to account for extraction efficiencies. To counter the common
effects of sample dilution we normalized between urine samples by normalizing to creatinine concentration. Pooled
Biological Quality Control was performed.
10 µL aliquot of each urine sample or standard solution was added to 70 µL of 200 mM borate buffer (pH
= 8.8 at 25°C) containing 25 µM 2-Aminobutyric acid (internal standard used for instrument/analyst error
correction). 1 mM ascorbic acid and 10 mM TCEP. The solution was vortexed and centrifuged then 20 µL of 10
mM Aqc reagent dissolved in 100% ACN was added. The solution was vortexed and centrifuged. then heated with
shaking at 55°C for 10 minutes. The derivatised solution was then centrifuged and transferred to HPLC vials
containing glass inserts.
Separation of derivatised compounds was performed on an Agilent Zorbax Eclipse XDB-C18 Rapid
Resolution HT 2.1 x 50 mm. 1.8 µm column. Mobile phase consisted of (A) 0.1% formic acid in water (v/v) and
(B) 0.1% formic acid in ACN (v/v). Flow rate was set to 300 µL.min-1. separation was performed at 30°C with
monitored pressure below 400 bar. Analysis time was 19 minutes. The gradient was run from 0-2 minutes using
Supplementary Material
1% solvent B, then linearly raised over 7 minutes from 1% to 15% solvent B. then raised to 30% solvent B over 5
minutes and dropped to 1% for re-equilibration which lasted 5 minutes.
Concentrations of derivatized alanine, arginine, aspartic acid, beta-alanine, citruline, cysteine, glutamine,
glutamic acid, glycine, homoserine, isoleucine, kynurenine, leucine, lysine, methionine, ornithine, phenylalanine,
proline, putrescine, sarcosine, serine, threonine, tryptophan, tyrosine, tyramine and valine were quantified by liquid
chromatography mass spectrometry using Agilent 1200 LC-system coupled to an Agilent 6410 ESI-QqQ-MS
(Santa Clara. CA). Injection volumes of 2 µL of samples or standards were used. Ions were monitored in the
positive ion mode using a dynamic MRM (DMRM) method optimized for each compound. The source, collision
energies and fragmentor voltages were optimized for each analyte by infusing a derivatised standard. Source
conditions were set to: sheath gas temperature 315°C. gas flow 10 L.min-1. nebulizer pressure 45 psi and capillary
voltage 3800 V.
Statistical Analysis
Statistical analysis was performed using Statsoft Statistica 10 and MetaboAnalyst 2.0. To compare groups
we performed T-test or U Mann-Whitney test analysis. To determine which statistical method should be used we
performed Shapiro-Wilk test. The diagnostic potential of selected amino acids was analysed by creating Receiver
Operating Characteristic (ROC) curves using ROCCET: ROC Curve Explorer & Tester (MetaboAnalyst 2.0.
www.metaboanalyst.ca). Statistical comparison of sample groups collected before prostate massage and after
prostate massage was not conducted. Urine collected before prostate massage differs significantly from urine
collected after prostate massage because of addition of prostatic fluid. This is why statistical analysis comparing
samples before and after prostate massage was not conducted. This might have generated false positive results.
Statistical analysis would be relevant only if samples collected from healthy patients were used for comparison.
A comparison of age between the two groups (t-test) showed no relevant differences, p>0.05 (mean age
in cancer group 61 and 63 in the benign prostate hyperplasia group) (Table S1). Four patients from the PCa group
had serum PSA levels <4 ng/ml, 17 patients had PSA levels between 4-10ng/ml and 4 patients presented with PSA
levels >10 ng/ml. In the benign group 15 patients had serum PSA levels <4 ng/ml, PSA of 9 patients was between
4 and 10 ng/ml, and 1 individual had PSA level higher than 10 ng/ml. Serum PSA levels were significantly higher
in the PCa group compared to the BPH group (p<0.001)
Extended Discution
Supplementary Material
N-methylglycine, also known as sarcosine is an amino acid that is being investigated as a potential
biomarker of prostate cancer. Opportunity to reduce the number of false positive diagnosis of prostate cancer
started an avalanche of studies that investigated the potential use of this molecule in the diagnostic process of
prostate cancer. In 2009 Sreekumar et al. [paper cited in mail text] reported that sarcosine concentrations vary in
benign prostate samples, clinically localized PCa and metastatic disease. Metabolic profiles performed by
gas/liquid chromatography and mass spectrometry in prostate tissue, plasma and urine suggested that sarcosine
metabolism may play a significant role in prostate cancer progression. Several follow up studies seeking to
translate these results including Jentzmik et al [S1], measuring the presence of sarcosine in urine after rectal exam
failed to detect prostate cancer and in 2010 found sarcosine levels in urine of PCa patients to not be significantly
different. In 2010 Struys et al. [S2] showed that serum sarcosine levels can’t be used as a marker for prostate
cancer. In our study we saw no differences in sarcosine concentrations when comparing PCa and BPH groups. We
observed no relevant changes in urine samples collected before and after prostate massage (p> 0.05), there were
also no differences in sarcosine concentration levels when PCa patients were divided into subgroups by Gleason
score and tumor stage. Our results agree with Jentzmik et al [S1] results providing further evidence that sarcosine
is not a suitable biomarker or indicator of the presence of prostate cancer.
However, our results showed higher concentrations of arginine in urine samples collected from PCa
patients both before and after prostate massage. The role of arginine metabolism in tumor growth has previously
been documented, Elgun et al. [S3] observed higher serum arginase II levels in patients with BPH compared to
PCa, also Mumenthaler et al. [S4] showed that arginase II in prostate cancer tissue samples and cell lines was
down regulated compared to BPH samples. Moreover, Mumenthaler et al [S4] showed a negative correlation
between the Gleason score and arginase II expression. This may explain higher concentrations of arginine in urine
samples from PCa patients. A more accurate investigation of arginine metabolism in tumor cells needs to be
performed to elucidate any changes to arginine metabolism that occur in prostate cancer compared to benign
growth.
Another amino acid, proline, also provided interesting results where higher levels of this amino acid were
found to be present in urine samples but only after pressing the gland by performing prostate massage. This may
indicate that higher concentrations of proline are related to prostate cancer and that this can only be observed by
analyzing urine collected after prostate massage. This is a good example of the fact that addition of prostatic fluid
in urine, when searching for prostate cancer biomarkers can be very helpful to identify small changes in metabolite
concentrations. Proline is connected to arginine metabolism (arginine-> ornithine-> proline). Proline is a major
Supplementary Material
product of arginine catabolism. Li et al [S5] have showed in animal models that there is a direct link between
arginase II expression and proline levels. They observed that greater expression of arginase II is correlated with
higher proline levels. It needs to be noted that this pathway requires arginase II to catalyze the synthesis of ornithine
from arginine thus making it a less likely explanation of higher concentrations of proline, when taking into
consideration results presented by Mumenthaler et al [S4]. At this time a definite link between arginine and proline
requires further research.
We also found higher concentrations of homoserine in urine samples of cancer patients both collected
before and after prostate massage. Homoserine is an amino acid that is not considered as a metabolite normally
present in tissues of mammals. It has been documented that small amounts of this amino acid are present in
metastasis of neuroblastoma or in urine of patients with homocystinuria. Homoserine was also present in urine of
patients with hepatitis B. [S6] Not a lot is known about pathways that lead to production of homoserine. It seems
that, in some pathological conditons homoserine is produced by splitting S-adenosylmethionine to
methylthioadenosine and alpha-amino-gamma-butyrolactone which is later converted to homoserine. This path is
linked to methionine metabolism. In our study we observed that patients with prostate cancer had significantly
higher concentrations of homoserine in urine when compared to patients with benign growth. Also when we
analyzed urine samples collected before prostate massage, cancer patients with higher Gleason score presented
with higher homoserine levels. More detailed analysis of methionine and homoserine metabolism in prostate
cancer needs to be performed.
References for supplementary data
S1. Jentzmik F, Stephan C, Miller K, Schrader M, Erbersdobler A, Kristiansen G et al. (2010) Sarcosine in urine
after digital rectal examination fails as a marker in prostate cancer detection and identification of aggressive
tumors. Eur Urol.58:12–18.
S2. Struys EA, Heijboer AC, van Moorselaar J, Jakobs C, Blankenstein MA (2010) Serum sarcosine is not a marker
for prostate cancer. Ann Clin Biochem.47:282
S3. Elgün S, Keskineğe A, Yilmaz E, Baltaci S, Bedük Y (1999) Evaluation of serum arginase activity in benign
prostatic hypertrophy and prostatic cancer. Int Urol Nephrol.31:95-99.
S4. Mumenthaler SM, Yu H, Tze S, Cederbaum SD, Pegg AE, Seligson DB et al. (2008) Expression of arginase
II in prostate cancer. Int J Oncol.32:357-365.
Supplementary Material
S5. Li H, Meininger CJ, Hawker JR Jr, Haynes TE, Kepka-Lenhart D, Mistry SK et al.(2001) Regulatory role of
arginase I and II in nitric oxide, polyamine, and proline syntheses in endothelial cells. Am J Physiol Endocrinol
Metab.280:75-82.
S6. Gazarian KG, Gening LV, Gazarian TG (2002) L-Homoserine: a novel excreted metabolic marker of hepatitis
B abnormally produced in liver from methionine. Med Hypotheses. 58:279-283.
Additional Tables and figures.
Fig S1. Concentrations of arginine (before and after prostate massage), homoserine (before and after
prostate massage), proline (after prostate massage) and tyramine (before prostate massage) in urine of cancer
patents (n=25) with different Gleason score, Gleason score 5 (n=6), Gleason score 6 (n=9), Gleason score 7 (n=10).
Group comparison performed using U Mann-Whitney test. Graphic presentation: square- median, box- 25/75
percentile, whiskers: min/max below upper fence, circle- outlier, asterisk- far outlier.
Arginine concentrations in urine
collected before prostate massage
Arginine concentrations in urine
collected after prostate massage
220
240
Gleason score
Homoserine concentrations in urine
collected before prostate massage
Gleason score
Gleason score
Homoserine concentrations in urine
collected after prostate massage
Gleason score
Supplementary Material
Proline concentrations in urine
collected after prostate massage
Tyramine concentrations in urine
collected before prostate massage
Gleason score
Gleason score
Tab S1. Group Characteristics
Prostate Cancer
Number of patients
Age Mean
(Range)
pT2a
pT2b
pT2c
Gleason score 5
Gleason score 6
Gleason score 7
25
61
(55-74)
8
5
12
6
9
10
Benign Prostate
Growth
25
63
(52-80)
-
Tab S2: Calibration parameters for each metabolite measured. including: Limit of Detection (LOD. µM).
Linear Calibration Range (LCR. µM). where concentrations reported are of the original standard solution not the
injected derivatised solution which has a concentration of 10 fold less. correlation coefficient for calibration curve
(R2); and recoveries of spiked amino acids and biogenic amines from protein precipitated urine. Experiment
performed on an Agilent 6410 LC-ESI-QqQ.
Amino Acid / Biogenic
Amine
Calibration Paramaters
Matrix
LOD
(µM)
LCR
(µM)
R2
Arginine
0.5
1-100
Serine
0.1
Glutamine
0.1
Urine
%RSD
%
Recovery
(n = 3)
0.99
138.8%
5.4%
0.5-100
0.99
114.1%
2.3%
0.5-100
0.99
146.6%
1.9%
Supplementary Material
Homoserine
0.1
0.5-100
0.99
74.6%
3.5%
Glycine
0.1
1-100
0.99
105.0%
2.8%
Aspartic acid
0.05
0.1-100
0.99
70.1%
2.1%
Alanine
0.1
0.5-100
0.99
107.4%
2.2%
Citrulline
0.1
0.5-100
0.99
91.8%
2.0%
Glutamic acid
0.05
0.1-100
0.99
-
-
Threonine
0.05
0.1-100
0.99
101.7%
1.9%
β-Alanine
0.5
1-100
0.99
-
-
Cysteine
0.1
0.5-100
0.95
-
-
Proline
0.1
0.5-100
0.99
91.7%
1.8%
Ornithine
0.05
0.1-100
0.99
89.0%
4.0%
Lysine
0.05
0.1-100
0.99
123.5%
5.0%
Putrescine
0.1
0.5-100
0.99
93.1%
1.5%
Tyrosine
0.05
0.1-100
0.99
103.6%
3.7%
Methionine
0.05
0.1-100
0.99
83.4%
5.1%
Valine
0.05
0.1-100
0.99
101.1%
2.6%
Tyramine
0.1
0.5-100
0.99
108.1%
3.2%
Leucine
0.05
0.1-100
0.99
96.2%
2.7%
Isoleucine
0.05
0.1-100
0.99
98.9%
3.2%
Kynurenine
0.1
0.5-100
0.99
-
-
Phenylalanine
0.05
0.1-100
0.99
98.9%
3.2%
Tryptophan
0.05
0.1-100
0.99
95.4%
3.8%
Sarcosine
0.05
0.1-100
0.99
-
-
Table S2: List of MRM transitions for targeted amino acids and biogenic amines. In all cases the dominant adduct was the
protonated form present as the [M+H]+ form. unless otherwise stated [M+2H]2+ forms.
Amine
MRM
Fragment
(m/z)
ion (m/z)
2-Aminobutyric acid
274
171
Alanine
260
171
Arginine
345
171
Aspartic acid
304
171
Citrulline
346
171
Cysteine
292
171
Glutamic acid
318
171
Glutamine
317
171
Glycine
246
171
Homoserine
290
171
Isoleucine
302
171
Kynurenine
379
171
Lysine 2xAqc
[M+2H]2+
244
171
Lysine 2xAqc
[M+H]+
487
171
Methionine
320
171
Ornithine 2xAqc [M+2H]2+
237
171
Supplementary Material
Phenylalanine
336
171
286
171
215
171
Serine
276
171
Threonine
290
171
Tryptophan
375
171
Tyramine
308
171
Tyrosine
352
171
Valine
288
171
Proline
Putrescine 2xAqc
[M+2H]2+