1
Study of Urinary 2-{[2-(Acetylamino-2-carboxyethyl]sulfanyl}Butanedioic acid, a
2
Mercapturic Acid of Rats Treated with Maleic Acid
3
4
Yu-Syuan Luo† and Hsin-Yun Tsai†, Hsin-Chang Chen†, Charlene Wu†, Li-Ching
5
Shen‡, Wen-Sheng Chung‡, Su-Yin Chiang*§ and Kuen-Yuh Wu*†
6
7
†
Institute of Occupational Medicine and Industrial Hygiene, College of Public Health,
8
National Taiwan University, Taipei 10055, Taiwan
‡
9
Department of Applied Chemistry, College of Science, National Chiao Tung
10
11
University, Hsinchu 30010, Taiwan
§
School of Chinese Medicine, China Medical University, Taichung 404, Taiwan
12
13
Yu-Syuan Luo† and Hsin-Yun Tsai† contributed equally to this work.
14
15
*(S.-Y.
16
sychiang@mail.cmu.edu.tw; (K.-Y. W) Tel: +886-2-3366 8091, Fax +886-2-3366
17
8267; e-mail: kuenyuhwu@ntu.edu.tw; Address: Rm. 721, No.17, Xuzhou Rd.,
18
Zhongzheng Dist., Taipei City 10055, Taiwan.
C)
Tel:
+88-4-2205
3366-3305;
19
1
Fax:+886-4-2203
2295;
Email:
1
Table of Contents Graphic
2
3
4
5
6
7
2
1
Abstract
2
Maleic anhydride was reported illegally adulterated into starch to prepare
3
traditional foods for decades in Taiwan. Maleic acid (MA), hydrolyzed from maleic
4
anhydride, could cause kidney damages to animals. The potential health effects due to
5
long-term MA exposures through food consumption have been of great concerns.
6
Assessment of the dietary MA exposures could be very difficult and complicated. One
7
of the alternatives is to analyze an MA-specific biomarker to assess the daily total MA
8
intake. Therefore, this paper aimed to study the mercapturic acid of MA,
9
2-{[2-(acetylamino)-2-carboxyethyl]sulfanyl}butanedioic acid (MAMA), with our
10
newly-developed
11
chromatography tandem mass spectrometry (ID-SPE-LC-MS/MS) method. MAMA
12
was first synthesized, purified, and characterized with NMR to reveal two
13
diastereomers and used for developing the analytical method. The method was
14
validated to reveal excellent sensitivity with a LOD at 16.3 ng/mL and a LOQ at 20.6
15
ng/mL and used to analyze MAMA in urine samples collected from Sprague-Dawley
16
rats treated with a single dose of 0 mg/kg, 6 mg/kg, and 60 mg/kg (n=5) of MA
17
through gavage. Our results show dose-dependent increases in urinary MAMA
18
contents, and 70% MAMA was excreted within 12 h with no gender differences (p >
19
0.05). A half life of urinary MAMA was estimated at 6.8 h for rat. The formation of
isotope-dilution
online
3
solid-phase
extraction
liquid
1
urinary MAMA validates it as a chemically-specific biomarker for current MA
2
exposure. Future study of MA metabolism in vivo will elucidate mechanisms of
3
MAMA formation, and analysis of this marker in epidemiology studies could help to
4
shed light on the causal effects of MA on human.
5
6
Keywords: maleic acid; mercapturic acid; detoxication; LC-MS/MS
7
8
9
4
1
1. Introduction
2
Maleic anhydride, an illegal food additive, was intentionally added in starch to
3
prepare a wide variety of traditional foods (e.g. tapioca, rice noodles and oden), in
4
order to ameliorate viscosity, texture, and stability of the foods for decades in Taiwan.
5
Maleic anhydride can be completely hydrolyzed to maleic acid (MA) at room
6
temperature with a relative humidity of 96%.(Rosenfel.Jm and Murphy 1967)
7
Harrison et al., Al-Bander et al., and Eiam-ong et al. reported that MA could cause
8
kidney effects to rats and dogs.(Albander et al. 1982; Eiamong et al. 1995; Harrison
9
and Harrison 1954) The symptom is similar to the Fanconi syndrome in human, which
10
the tubular reabsorption is malfunctioned, and glucose, amino acids, phosphate and
11
bicarbonate could not be reabsorbed in proximal tubule.(Albander et al. 1982;
12
Harrison and Harrison 1954) The prevalence rates of end-stage renal disease in
13
Taiwan is ranked first worldwide.(USRDS 2013) Daily intakes of MA have been of
14
great concerns.1
15
The study of the potential adverse effects of MA requires information of MA
16
exposures through consumption of the MA-modified starch foods. The assessment of
17
MA exposures through food consumption could be very difficult and complicated due
1
Abbreviations: COSY, Correlation spectroscopy; DDW, double de-ionized water; GSH, glutathione;
HESI, heated electro spray ionization; HMBC, Heteronuclear multiple-bond correlation spectroscopy;
HMQC, Heteronuclear multiple-quantum correlation spectroscopy; LOD, limit of detection; LOQ,
limit of quantification; MA, maleic acid; MAMA,
2-{[2-(acetylamino)-2-carboxyethyl]sulfanyl}Butanedioic acid; PS1D, Pure shift 1D spectroscopy;
PBS, phosphate buffered saline.
5
1
to variation of daily food consumption rate and MA contents in the foods. One of the
2
alternatives is to analyze a MA-specific biomarker to assess daily total MA exposures.
3
In terms of biological monitoring, urine samples are non-invasive and easy to access.
4
If a urinary biomarker of MA can be detected, it can not only serve as a biomarker to
5
assess total MA exposures, but also imply MA metabolism and/or detoxication in vivo.
6
Although
the
metabolism
of
MA
is
not
well
understood,
7
glutathione-S-transferase (GST) usually plays an important role in detoxification of
8
industrial chemicals to form their corresponding glutathione conjugates, which are
9
further metabolized to N-acetyl cysteine adducts, also named as mercapturic
10
acids.(Habig et al. 1974; Hecht et al. 2008) These mercapturic acids have been well
11
studied to serve as biomarkers to assess current exposures for these chemicals in our
12
living environments and/or at workplaces.(Alary et al. 1995; Eckert et al. 2013;
13
Huang et al. 2007; Kotapati et al. 2014; Kuiper et al. 2010; Maestri et al. 1997;
14
Perbellini et al. 2002) If MA is also detoxified by GST, the resulting MA glutathione
15
conjugates
16
2-{[2-(acetylamino)-2-carboxyethyl]sulfanyl}butanedioic acid, the mercapturic acid
17
of MA (MAMA) excreted through urine. Therefore, the aims of this study were to
18
investigate urinary MAMA by using our newly-developed isotope-dilution online
19
solid-phase
can
extraction
be
liquid
further
chromatography
6
metabolized
tandem
mass
to
spectrometry
1
(ID-SPE-LC-MS/MS) method for rats treated with MA. Results from this study will
2
not only validate urinary MAMA as a biomarker for MA exposure, but also elucidate
3
in vivo metabolism of MA in part.
4
In order to accurately quantify urinary MAMA, MAMA and [13C2]-MAMA were
5
first synthesized, purified, and characterized to prepare their standard solutions for
6
developing the LC-MS/MS method. After validation of this method, MAMA in urine
7
samples collected from rats treated with single dose of 6 and 60 mg/kg (n=5) of MA
8
were analyzed, and compared with those of control rats. To the best of our knowledge,
9
MAMA is first quantified in urine of rats treated with MA. Further studies by
10
monitoring urinary MAMA as a biomarker for human exposed to MA will help to
11
shed light on the potential health effects caused by daily intakes of MA through food
12
consumption.
7
1
2. Materials and Methods
2
Caution
3
MA is irritative and corrosive to eye, skin and lung. Acetonitrile and formic acid
4
should be handled with proper personal protective equipment and operated in a
5
well-ventilated hood.
6
7
2.1 Chemicals
8
MA (99.0%, 500 g) was purchased from Showa (Tokyo, Japan). N-acetyl
9
cysteine (NAC) (≧ 99%, 25 g), phosphate buffered saline (PBS), formic acid (≧
10
98%, 500 mL), and [13C2]-MA were obtained from Sigma-Aldrich (St. Louis, MO).
11
Acetonitrile (≧ 99.9%, 4 L) was supplied by Merck (Darmstadt, Germany). Double
12
de-ionized water (DDW) was prepared by a Milli-Q system (Billerica, MA).
13
14
2.1.1 Synthesis, Purification and Characterization of MAMA Conjugate
15
MA (0.10 mmol, 11.67 mg) and NAC (0.11 mmol, 18.01 mg) were added into 10
16
mL of PBS buffer (2 g of PBS tablet dissolved in 200 mL of DDW) and stirred at 37
17
o
18
semi-preparative C18 column (5 μm, 10 × 250 mm, Phenomenex Co., Torrance, CA),
19
a binary pump (Jasco PU-980) and an UV/Vis detector (205 nm, Jasco UV-975,
C for 96 h. MAMA was purified with an LC-UV system consisted of a
8
1
Easten, MD). Isocratic elution with mobile phase A (0.1% FA in H2O) and B
2
(acetonitrile) at a ratio of 88:12 and a flow rate of 1.2 mL/min was adopted for
3
chromatographic separation. MAMA was collected at retention time between 16.5 and
4
17.5 min by using a fraction collector (CHF121SA, Toyo Seisakusho Kaisha, Ltd,
5
Japan), and then the collected solutions were combined and dried under vacuum.
6
MAMA was characterized by using a triple quadruple tandem mass spectrometer
7
(MS/MS) with a heated electrospray ionization (HESI) source in negative ion mode.
8
MAMA was prepared in DDW and analyzed through direct infusion at a flow rate of
9
10 μL/min. A spray voltage was set at 2000 V, and sheath gas and auxiliary gas flow
10
rate were at 35 and 10 psi; capillary and HESI temperature were set at 150 oC and 200
11
o
12
pressure of 1.5 mtorr and a scan period of 1 min. The precursor ion was m/z 278
13
([M-H]-) and the product ions were m/z 149 ([M-C5H8NO3-H]-), m/z 162
14
([M-C4H5O4-H]-), and m/z 115 ([M-C5H8NO3S-H]-). MAMA was dissolved in
15
deuterium oxide and further characterized with NMR (Varian VNMRS-600 NMR
16
Spectrometer). Results from 1H and 13C NMR analysis were described as following:
C, respectively. Product ion spectrum of MAMA was obtained with a collision gas
1H
17
NMR (600 MHz, D2O). MAMA (Diastereomer A): δ [ppm] = 4.51 (dd, J3, a’
18
= 7.8 Hz, J3, a = 4.8 Hz, 1H, H-3), 3.65
19
3
3.68 (m, 1H, H-c), 3.11 (dd, Ja, a’ = 13.8, Ja,
= 4.8 Hz, 1H, H-a), 2.99 (dd, Ja’, a = 13.8, Ja’, 3 = 7.8 Hz, 1H, H-a’), 2.84 (dd, Jd, d’ =
9
1
17.4, Jd, c = 9.6 Hz, 1H, H-d), 2.70 (dd, Jd’, d = 15.0, Jd’, c = 6.0 Hz, 1H, H-d‘), 1.91 (s,
2
3H, H-1); MAMA (Diastereomer B): δ [ppm] = 4.52 (dd, J3, a’ = 8.4, J3, a = 4.8 Hz,
3
1H, H-3), 3.65
4
2.91 (dd, Ja’, a = 13.8, Ja’, 3 = 8.4 Hz, 1H, H-a’), 2.84 (dd, Jd, d’ = 17.4, Jd, c = 9.6 Hz,
5
1H, H-d), 2.68 (dd, Jd’, d = 17.4, Jd’, c = 6.0 Hz, 1H, H-d’ ), 1.92 (s, 3H, H-1).
6
13C
3.68 (m, 1H, H-c), 3.13 (dd, Ja, a’ = 14.4, Ja, 3 = 4.2 Hz, 1H, H-a),
NMR (150 MHz, D2O). MAMA (A form): δ [ppm] = 178.1 (s, C-b), 177.2
7
(s, C-e), 176.8 (s, C-2), 176.3 (s, C-4), 55.3 (s, CH-3), 45.1 (s, CH-c), 38.8 (s, CH2-d),
8
35.0 (s, CH2-a), 24.2 (s, CH3-1); MAMA (B form): δ [ppm] = 178.0 (s, C-b), 177.2 (s,
9
C-e), 176.8 (s, C-2), 176.3 (s, C-4), 54.8 (s, CH-3), 44.3 (s, CH-c), 38.6 (s, CH2-d),
10
34.6 (s, CH2-a), 24.2 (s, CH3-1).
11
[13C2]-MAMA was obtained from the reaction of [13C2]-MA and NAC by
12
following with the identical procedures of MAMA synthesis. The MS fragmentation
13
of [13C2]-MAMA shows the similar pattern with MAMA. The precursor ion was m/z
14
280 ([M-H]-) and the product ions were m/z 151 ([M-C5H8NO3-H]-), m/z 162
15
([M-C213C2H5O4-H]-), and m/z 117 ([M-C5H8NO3S-H]-) (Figure S1).
16
17
2.2 Quantitative Analysis of MAMA Conjugate
18
2.2.1 Online Solid-Phase Extraction Coupled with LC-ESI-MS/MS
19
The online SPE system included a trapping column (Intersil ODS-3, 3 μm, 2.1 ×
10
1
33 mm, GL Sciences Inc., Japan), an analytical column (Hypurity C18, 3 μm, 2.1 ×
2
150 mm, Thermo), a quaternary LC pump (Accela micropump, Thermo Fisher
3
Scientific), a L-2100 pump (Hitachi High-Technologies, Japan) and an auto-sampler
4
(Accela, Thermo Fisher Scientific). The gradient mobile phases consisted of solution
5
A (0.1 % formic acid in methanol) and solution B (0.1 % formic acid in H2O). The
6
composition of mobile phase for the online SPE program was described in Table S1.
7
A triple quadruple mass spectrometry (TSQ Quantum Access, Thermo Fisher
8
Scientific, Ringoes, NJ) with an HESI was used for MS analysis. A spray voltage was
9
set at 2600 V, and sheath gas and auxiliary gas flow rate were at 30 and 25 psi;
10
capillary and HESI temperature were set at 200 oC and 250 oC, respectively. The
11
collision energy, tube lens, scan width ,and scan time were set at 15 V, 47 unit, m/z 2.0
12
and 0.05 s correspondingly. The mass spectrometer was operated under multiple
13
reaction monitoring (MRM) for quantitation of MAMA. The ion transitions
14
monitored were m/z 277.9 → 149.0 (qualifier) and m/z 277.9 → 115.1 (quantifier)
15
for MAMA, and m/z 280.0 → 151.1 (qualifier) and m/z 280.0 →117.3 (quantifier)
16
for [13C2]-MAMA.
17
18
19
2.2.2 Sample Preparation
Two hundred micro liter of urine sample was spiked with 0.05 μg of
11
1
[13C2]-MAMA, mixed vigorously, and precipitated with 400 μL of methanol. The
2
mixture was centrifuged at 5,000 rpm for 5 min, and then 250 μL of the supernatant
3
was diluted with 250 μL of ammonium formate buffer (10 mM, pH = 3). The
4
processed sample was then filtrated with a polyvinylidene fluoride (PVDF) filter (0.22
5
μm) prior to LC-MS/MS analysis.
6
7
2.2.3 Method Validation
8
The method was validated according to the guidance for industry bioanalytical
9
method validation, published by US food and drug administration.(Center for Drug
10
Evaluation and Research (U.S.) and Center for Veterinary Medicine (U.S.) 2001)
11
MAMA standard solutions at 25, 50
12
ng/mL were prepared in mobile phase solutions or a
13
mixture of urine collected from six control rats (3 female and 3 male rats). Calibration
14
curves were established by repeated analysis of these standard solutions with triplicate
15
on 3 different days. Limit of detection (LOD) and limit of quantification (LOQ) were
16
determined as 3 and 10 fold of the standard deviation of urine blank spiked with
17
internal standard. Linearity of the calibration curves was determined with simple
18
linear regression, with the ratios of peak area of standard solution versus that of the
19
spiked internal standard (STD/IS). Quality control samples (QCs) were prepared at 30,
12
1
200, and 800 ng/mL to evaluate the inter- and intra-day accuracy and precision of this
2
method. These standard solutions were analyzed with triplicate for 4 consecutive days.
3
Online SPE recoveries were calculated according to a report by Ye et al. Briefly, two
4
subsequent analysis were conducted to evaluate the recoveries: (A) Twenty microliter
5
of MAMA standard solutions prepared in urine at 30, 200, and 800 ng/mL were
6
injected onto the trapping column. Then, 5 ng of [13C2]-MAMA prepared in the
7
mobile phase was automatically injected into the LC column by using a six port
8
electronic valve. Despite the fact that MAMA and [13C2]-MAMA were injected
9
separately, they were co-eluted. The measured MAMA and [13C2]-MAMA
10
concentrations were defined as STD(A) and IS(A). (B) Twenty microliter of the
11
standard solutions spiked with 5 ng of [13C2]-MAMA were injected, subjected to SPE
12
and chromatography with analytical column, and analyzed with the tandem mass
13
spectrometry. The measured MAMA and [13C2]-MAMA were defined as STD(B) and
14
IS(B), respectively. The online SPE recoveries were calculated from (STD(A) / IS(A)) /
15
(STD(B) / IS(B)) .
16
17
Stability was determined by analysis of the QC samples stored at -20 oC for 7, 21
and 30 days.
18
19
2.3 Animal Study
13
1
All procedures involved in the animal study were approved by the Institutional
2
Animal Care and Use Committee of Chinese Medical University (No. 102-258-N).
3
Male and female Sprague-Dawley rats of 7 weeks were obtained from BioLasco
4
(Taiwan), and maintained on 12:12 h light / dark cycle with food and water ad libitum.
5
Animals were divided into control (body weight: 263.2 ± 9.6 g for male; 206.2 ±
6
8.0 g for female, n = 5 per sex), low dosage (6 mg/kg, body weight: 262.2 ± 19.6 g
7
for male; 215.0 ± 19.2 g for female, n = 5 per sex), and high dosage (60 mg/kg, body
8
weight: 269.0 ± 13.3 g for male; 199.0 ± 2.79 g for female, n = 5 per sex) groups,
9
and acclimatized for 7 days prior to the animal study. MA solutions were freshly
10
prepared in DDW on the day of treatment. Each rat was treated with a single dose of
11
MA through gavage, and then individual rat was placed in a metabolic cage for urine
12
collection. Urine samples were collected at 12, 24, 48, 72, 96, 120, 144, 168 h after
13
MA treatment. The collected samples were stored at -20oC until used for analysis.
14
15
2.4 Kinetic Parameters
16
Kinetic parameters of MAMA were estimated by using the WinNonlin software
17
version 5.3 (Pharsight Corp., Mountain View, CA). Non compartmental analysis
18
(urine, model 210) was applied to fit our experimental data. The elimination rate
19
constant (Ke) and half life (T1/2) were estimated by fitting the urinary MAMA
14
1
concentrations obtained at 12, 24 and 48 h. The maximum excretion rate (ER max) was
2
calculated from the maximum concentration, urine volume, and the period between
3
urine collection with the following equation:
4
5
MAMA concentration × urine volume / the period between urine collection.
Area under the curve (AUC) were calculated by using the trapezoidal rule.
6
3. Results and Discussion
7
3.1 Synthesis, Purification, and Characterization of MAMA
8
To purify MAMA by using the HPLC system, the fraction between retention
9
time from 16.5 min to 17.5 min was collected by setting the UV absorbance
10
wavelength at 205 nm. The yield was estimated to be 42 %. Purified MAMA was
11
characterized with MS and NMR. Characterization of MAMA with LC-MS/MS by
12
infusion under negative ion mode, the most abundant fragment ion of MAMA were
13
m/z 149 by loss of acetyl alanine moiety, according to their chemical structures (see
14
Figure S1). Characterized with 1H NMR and
15
characterized with HMQC and HMBC to investigate the direct and multi
16
carbon-proton relationships. Proton-proton relationships were revealed by using
17
COSY. Pure shift 1D (PS1D) NMR was further used to characterize the diastereomers
18
of MAMA.
19
13
C NMR, MAMA was further
Although the alkenyl group of MA has two prochiral centers, the symmetrical
15
1
structure of MA and the identical steric hindrance of the re and si faces result in two
2
diastereomers of MAMA. The diastereomers are co-eluted at 17 min with HPLC
3
chromatography. The structures of diastereomers were further resolved with NMR.
4
The 1H NMR spectrum of MAMA (Figure 1) reveals that the singlet signals
5
located at 1.91 (A form) and 1.92 (B form) ppm were assigned to the acetyl protons
6
H-1, and the 13C NMR signal at 24.2 ppm (A and B form) was assigned to C-1 (A and
7
B form) according to the HMQC spectrum. The integration of peak area of proton
8
decoupled PS1D spectra assisted to confirm the signals of A (less abundant) and B
9
(more abundant) form (Figure S3). The downfield chemical shifts at 4.51 (A form)
10
and 4.52 (B form) ppm of the 1H NMR spectrum were assigned to
11
H-3; accordingly, the 13C signals at 55.3 (A form) and 54.8 (B form) ppm were
12
assigned to C-3 based on the HMQC spectrum (Figure S4). We assigned the chemical
13
shifts around 2.91
14
spectrum (Figure S5). Subsequently, the chemical shifts at 3.11 and 2.99 ppm were
15
assigned to H-a and H-a’ as one diastereomer of MAMA (A form), while 3.13 and
16
2.91 ppm were considered as H-a and H-a’ for B form. The signal at 3.66
17
further assigned to H-c for its proximity with the sulfur atom; correspondingly, the 13C
18
signals at 45.1 and 44.3 ppm were assigned to C-c based on the HMQC spectrum. On
19
the basis of correlation with H-c in COSY, the signals around 2.68
H of amino acid,
3.13 ppm to H-a for its correlation with H-3 of the COSY
16
3.68 was
2.84 ppm were
1
assigned as H-d and H-d’. The COSY revealed that signals at 2.84 and 2.70 ppm were
2
H-d and H-d’ for A form, and 2.84 and 2.68 ppm were for B form (Table 1). The
3
carbon signal at 176.8 ppm was assigned to C-2 based on its coupling with H-1 (2JCH)
4
in the HMBC spectrum (Figure S6), and the carbon signal at 176.3 ppm was assigned
5
to C-4 for its correlation with H-3 (2JCH) and Ha in the HMBC spectrum. Likewise,
6
the carbon signals at 178.1 and 178.0 ppm were assigned to C-b based on its coupling
7
with H-c (2JCH) in the HMBC spectrum. Finally, the carbon signal at 177.2 ppm was
8
assigned to C-e according to its correlation with H-d (2JCH) in the HMBC spectrum
9
(Table 1).
10
The correlation of H-3 and H-a / H-a’ was used to distinguish diastereomers from
11
A and B forms in the COSY spectrum. The J3, a and J3, a’ of A form (H-3, 4.51 ppm)
12
are 4.8 and 7.8 Hz, which are the same as Ja, 3 (H-a, 3.11 ppm) and Ja’, 3 (H-a’, 2.99
13
ppm). Accordingly, the assignments of A and B form were further confirmed.
14
17
1
2
Figure 1. 1H Spectrum of MAMA (600 MHz). Diastereomers of MAMA are
3
represented as A and B form.
4
3.2 Quantitative Analysis of MAMA Conjugate
5
MAMA was successfully quantified with our newly-developed LC-MS/MS
6
method. A retention time of MAMA is at 7.9 min, and [13C2]-MAMA is co-eluted
7
(Figure 2).
18
1
2
Figure 2. Representative LC-MS/MS Chromatogram of MAMA and [13C2]-MAMA
3
generated from analysis of a urine sample collected from a female rat treated with 6
4
mg/kg MA.
5
6
The results of our method validation were summarized in Table 2. The LOD and
7
LOQ of urinary MAMA were 16.3 and 20.6 ng/mL, respectively. The calibration
8
curve in urine showed excellent linearity (R2 = 0.9989), and the inter-day and
9
intra-day accuracy, and precision are complied with the guidance of US FDA.(Center
10
for Drug Evaluation and Research (U.S.) and Center for Veterinary Medicine (U.S.)
11
2001) There was no carry-over between runs. The matrix effect was compensated by
12
using isotope-dilution quantification (Figure 3). The online SPE recoveries of the QCs
19
1
were all higher than 90% and showed precision ranging from 3.4% to 7.2%. MAMA
2
was stable for one month under -20 oC and after three freeze and thaw cycles (Table
3
2).
4
3.5
Ratio in urine
Ratio in 0.1% FA
y  0.0037x - 0.0596
R 2  0.9989
MAMA/[13C2]-MAMA Ratio
3.0
y  0.0037x - 0.1387
R 2  0.9944
2.5
2.0
1.5
1.0
0.5
0.0
0
200
400
600
800
1000
1200
MAMA concentration (ng/mL)
5
6
Figure 3. Calibration Curves in Urine and 0.1% FA(aq). The matrix effect was
7
compensated
8
MAMA/[13C2]-MAMA ratio in urine; ○ MAMA/[13C2]-MAMA ratio in 0.1% FA
9
3.3 Animal Study
by
applying
the
isotope-dilution
method.
Labels:
●
10
Urinary MAMA was successfully quantified, and shows a dose-dependent
11
increase (Figure 4). MAMA was detected in urine of control female rats at 62.7 ±
12
12.3 μg/g creatinine (n = 5), but below detection limit in urine of control male rats.
13
However, no gender difference was observed in exposed groups. The dose-dependent
20
1
increase in urinary MAMA contents suggests that MAMA could serve as a biomarker
2
for current MA exposure.
3500
g/ g creatinine
3000
2500
Male
Female
2000
1500
1000
500
0
Control
3
6 mg/kg
60 mg/kg
Exposed MA (gavage, n=5)
4
Figure 4. Urinary MAMA increased in a dose-dependent manner in both genders
5
(urine samples were collected at 12 h).
6
7
Analysis of MAMA in urine collected from rats treated with MA reveals it
8
could be rapidly generated and excreted through urine. Up to 70% of MAMA was
9
excreted within 12 h (Figure 5). Fitted with non compartmental model, urinary
10
MAMA manifested a half life of 6.8 h for rats treated with 60 mg/kg MA (Table 3).
11
The half life of urinary MAMA is longer than that of urinary MA, suggesting that
12
MAMA is suitable to serve as a biomarker for current MA exposure. Previous studies
13
in styrene (Seutter-Berlage et al. 1978), 1,2-diethylbenzene (Linhart 1996),
21
1
1,3-Butadiene (Kotapati et al. 2011; Sterz et al. 2012), 1,2,4-trimethylbeneze
2
(Tsujimoto 2000), 2-chloroprene , allyl chloride (De Rooij 1996), acrylonitrile
3
(Kedderis 1993), and acrylamide (Sumner et al. 1992) have demonstrated that
4
mercapturic acids are the major detoxification products and the biomarkers of
5
exposure. Our results also reveal that the MA exposure can lead to the formation of
6
MAMA in rat urine, which suggests that MA could be potentially detoxified through
7
GSH conjugation in vivo and excreted through urine. Although MA was reported to be
8
metabolized to carbon dioxide (Sacks 1958), the metabolism of MA was rarely
9
studied. In this paper, the biotransformation of MA to MAMA was first demonstrated
10
in rat. Further studies are needed to elucidate the metabolism of MA in vivo.
11
12
13
Figure 5. Cumulative Excretion of MAMA in Rat Urine (60 mg/kg, n = 5). Urinary
22
1
MAMA mostly excreted within 12 h.
2
3
In conclusion, we successfully synthesized, purified, and characterized MAMA,
4
and developed an online ID-SPE-LC-MS/MS method to quantify MAMA in urine.
5
Although the metabolism of MA in vivo has not well understood, analysis of urinary
6
MAMA revealed one potential metabolic pathway of MA in vivo. The dose-dependent
7
increase in urinary MAMA validated itself to serve as a biomarker for current MA
8
exposure. Analysis of this biomarker to assess MA exposure may shed light on the
9
association between MA and potential health effects. However, further studies may be
10
needed to elucidate metabolic pathways and mechanisms associated with the effects
11
on kidneys in animal studies.
12
13
Conflict of interest
14
The authors declare that there are no conflicts of interest.
15
16
Funding Sources
17
This study was supported by a grant from National Health Research Institute (grant
18
number. NHRI-EX104-10433PI )
19
23
1
Acknowledgement
2
The NMR analysis was supported by the Instrumentation center of National Chiao
3
Tung University. We also appreciate National Taiwan University for instrumental
4
support and China Medical University for animal experiments.
5
6
Supplementary Data
7
The product ion spectrum of MAMA and [13C2]-MAMA, 2D NMR spectra of MAMA
8
and the online SPE program for MAMA Analysis are available free of charge via the
9
internet at http://www.sciencedirect.com.
10
Reference
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3
29
Tables
Table 1. 1H and 13C NMR Interpretation of MAMA.
A form
Position
B form
C
mult.
H
mult.
J (Hz)
C
mult.
H
mult.
J (Hz)
1
24.2
s
1.91†
s
-
24.2
s
1.92†
s
-
2
176.8
s
-
-
-
176.8
s
-
-
-
3
55.3
s
4.51
dd
4.8, 7.8
54.8
s
4.52
dd
4.8, 8.4
4
176.3
s
-
-
-
176.3
s
-
-
-
a
35.0
s
3.11
dd
4.8, 13.8
34.6
s
3.13
dd
4.2, 14.4
2.99
dd
7.8, 13.8
2.91
dd
8.4, 13.8
a’
b
178.1†
s
-
-
-
178.0†
s
-
-
-
c
45.1†
s
3.65-3.68‡
m
-
44.3†
s
3.65-3.68‡
m
-
d
38.8†
s
2.84
dd
9.6, 17.4
38.6†
s
2.84
dd
9.6, 17.4
2.70
dd
6.0, 15.0
2.68
dd
6.0, 17.4
-
-
-
-
-
-
d’
e
177.2
s
177.2
†
Assignments are interchangeable between diastereomer A and B.
‡
Signals are overlapped.
30
s
Table 2. Results of Method Validation for Analysis of Urinary MAMA
MAMA
QCs (ng/mL)†
Inter- and intra-day accuracy (n = 9 × 4)
30
109 ± 1%
105 ± 5%
108 ± 5%
96 ± 4%
200
101 ± 7%
97 ± 7%
96 ± 7%
91 ± 7%
800
105 ± 6%
105 ± 6%
95 ± 5%
94 ± 1%
Inter- and intra-day precision (n = 9 × 4)
30
1.3%
4.9%
4.9%
4.7%
200
7.3%
7.4%
6.9%
7.2%
800
5.5%
6.1%
5.4%
1.4%
Recovery (n=5)
30
98.2 ± 7.2 %
200
109.0 ± 6.9 %
800
104.9 ± 3.4 %
Stability (n=3)
†
7 day
21 day
30 day
30
99.9 ± 15.7 %
104.2 ± 6.4 %
104.7 ± 12.7 %
200
103.8 ± 6.3 %
104.1 ± 1.9 %
94.3 ± 1.7 %
800
99.7 ± 0.7 %
97.7 ± 4.3 %
100.0 ± 7.3 %
QCs, Quality Control Samples
31
Table 3. Kinetic Parameters of MAMA in Rat Urine†
Unit
Male
Female
Ke
1/hr
0.845 ~ 0.999
0.11 ± 0.02
0.73 ~ 0.91
0.11 ± 0.03
T1/2
hr
6.8 ± 1.2
6.9 ± 2.5
ng/hr
249.6 ± 155.4
354.8 ± 245.4
Urine volume
mL
75.0 ± 24.9
65.8 ± 20.9
Amount_Recovered
ng
4513.9 ± 1778.1
5540.7 ± 3250.1
R‡
ERmax
Data were expressed as mean ± standard deviation (n=5 for rats treated with 60 mg/kg MA)
†
Kinetic parameters of MAMA were calculated with non compartmental model by WinNonlin ver 5.3 (Model 210, Pharsight Corp., Mountain
View, CA).
‡
R, goodness of fit statistic for the estimate of elimination rate constant; Ke, elimination rate constant; T1/2, elimination half life; ERmax,
maximum observed excretion rate.
32