New biomarkers for monitoring the levels of isothiocyanates in humans

Article abstract of DOI:10.1021/tx900393t

Isothiocyanates (ITCs) found in cruciferous vegetables have demonstrated cancer preventive activity in animals, and increased dietary intake of ITCs has been shown to be associated with a reduced cancer risk in humans. ITCs exert their cancer chemopreventive action by multiple mechanisms, for example, by modulating the activities of phase I and phase II drug metabolism enzymes, by inhibiting the cell cycle and histone deacetylase, and by causing apoptotic cell death. In cells, protein adducts account for most of total cellular ITC uptake at 4 h after treatment. The time course of this protein binding correlates well with the inhibition of proliferation and the induction of apoptosis. Animal studies have shown that glutathione conjugates are the major products of ITCs. The major urinary excretion products of ITCs in human are N-acetyl cysteine conjugates. Urinary metabolites might provide the exposure history of the last 24 h, if the urine of the full next day is collected. However, this is not feasible in large epidemiological studies. Furthermore, the mercapturic acids of ITC are not stable. Therefore, stable biomarkers are needed that reflect a larger time span of the ITC exposure history. We developed a method to determine stable (not cysteine adducts) reaction products of ITCs with albumin and hemoglobin in humans and mice. We reacted albumin with the ITCs: benzyl isothiocyanate (BITC), phenylethyl isothiocyanate (PEITC), sulforaphane (SFN), and allyl isothiocyanate (AITC). After enzymatic digestion, we found one major product with lysine using LC-MS/MS. The identity of the adducts was confirmed by comparing the analyses with synthetic standards: N⁶-[(benzylamino) carbonothioyl]lysine (BITC-Lys), N⁶-{[(2-phenylethyl)amino] carbonothioyl}lysine (PEITC-Lys), N⁶-({[3-(methylsulfinyl)propyl] amino}carbonothioyl)lysine (SFN-Lys), and N⁶-[(allylamino] carbonothioyl]lysine (AITC-Lys). The adduct levels were quantified by isotope dilution mass spectrometry using the corresponding new ITC-[¹³C ₆¹⁵N₂]lysines as internal standards. The applicability of the method was tested for biological samples obtained from different experiments. In humans consuming garden cress, watercress, and broccoli and/or in mice exposed chronically to N-acetyl-S-{[(2-phenylethyl) amino]carbonothioyl}-l-cysteine, albumin and hemoglobin adducts were found. BITC-Lys, PEITC-Lys, and SFN-Lys released after enzymatic digestion of the proteins were quantified with LC-MS/MS. This new method will enable quantification of ITC adducts in blood proteins from large prospective studies about diet and cancer. Protein adducts are involved in the chemopreventive effects of ITCs. Therefore, blood protein adducts are a potential surrogate marker for the effects of ITCs at the cellular level. This new technique will improve the assessment of ITC exposure and the power of studies on the relationship between ITC intake and cancer.

Full text of DOI:10.1021/tx900393t

756
Chem. Res. Toxicol. 2010, 23, 756–765
New Biomarkers for Monitoring the Levels of Isothiocyanates in
Humans
Anoop Kumar and Gabriele Sabbioni*
Department of EnVironmental Health Sciences, School of Public Health and Tropical Medicine, Tulane
UniVersity, 1440 Canal Street, Suite 2100 (SL-29), New Orleans, Louisiana 70112
ReceiVed October 26, 2009
Isothiocyanates (ITCs) found in cruciferous vegetables have demonstrated cancer preventive activity
in animals, and increased dietary intake of ITCs has been shown to be associated with a reduced cancer
risk in humans. ITCs exert their cancer chemopreventive action by multiple mechanisms, for example,
by modulating the activities of phase I and phase II drug metabolism enzymes, by inhibiting the cell
cycle and histone deacetylase, and by causing apoptotic cell death. In cells, protein adducts account for
most of total cellular ITC uptake at 4 h after treatment. The time course of this protein binding correlates
well with the inhibition of proliferation and the induction of apoptosis. Animal studies have shown that
glutathione conjugates are the major products of ITCs. The major urinary excretion products of ITCs in
human are N-acetyl cysteine conjugates. Urinary metabolites might provide the exposure history of the
last 24 h, if the urine of the full next day is collected. However, this is not feasible in large epidemiological
studies. Furthermore, the mercapturic acids of ITC are not stable. Therefore, stable biomarkers are needed
that reflect a larger time span of the ITC exposure history. We developed a method to determine stable
(not cysteine adducts) reaction products of ITCs with albumin and hemoglobin in humans and mice. We
reacted albumin with the ITCs: benzyl isothiocyanate (BITC), phenylethyl isothiocyanate (PEITC),
sulforaphane (SFN), and allyl isothiocyanate (AITC). After enzymatic digestion, we found one major
product with lysine using LC-MS/MS. The identity of the adducts was confirmed by comparing the
analyses with synthetic standards: N6-[(benzylamino)carbonothioyl]lysine (BITC-Lys), N6-{[(2-
phenylethyl)amino]carbonothioyl}lysine (PEITC-Lys), N6-({[3-(methylsulfinyl)propyl]amino}carbonothioyl)-
lysine (SFN-Lys), and N6-[(allylamino]carbonothioyl]lysine (AITC-Lys). The adduct levels were quantified
by isotope dilution mass spectrometry using the corresponding new ITC-[13C615N2]lysines as internal
standards. The applicability of the method was tested for biological samples obtained from different
experiments. In humans consuming garden cress, watercress, and broccoli and/or in mice exposed
chronically to N-acetyl-S-{[(2-phenylethyl)amino]carbonothioyl}-L-cysteine, albumin and hemoglobin
adducts were found. BITC-Lys, PEITC-Lys, and SFN-Lys released after enzymatic digestion of the proteins
were quantified with LC-MS/MS. This new method will enable quantification of ITC adducts in blood
proteins from large prospective studies about diet and cancer. Protein adducts are involved in the
chemopreventive effects of ITCs. Therefore, blood protein adducts are a potential surrogate marker for
the effects of ITCs at the cellular level. This new technique will improve the assessment of ITC exposure
and the power of studies on the relationship between ITC intake and cancer.
cooking, the myrosinase is deactivated (4). The release of the
ITCs from GLs in the human can be performed by the gut
microflora (6). Naturally occurring ITCs found in cruciferous
vegetables have demonstrated cancer preventive activity in
animals, and increased dietary intake of ITCs has been shown
to be associated with a reduced cancer risk in humans (6, 7)
(Figure 1). ITCs exert their cancer chemopreventive action by
modulating the activities of phase I and phase II drug metabo-
lism enzymes (6, 8-11). ITCs and their thiol conjugates inhibit
the cell cycle and cause apoptotic cell death, possibly by
activation of vital signal transduction pathways (12, 13). ITCs
can induce cellular oxidative stress by rapidly conjugating and
thus depleting cells of glutathione (GSH) (14, 15). ITC
conjugates with thiols, including the thiols in GSH and cellular
proteins, are reversible (16). It has been shown with radiolabeled
phenethyl isothiocyanate (PEITC) and SFN that the initial
conjugation predominantly occurs with cellular GSH (12). With
increasing time, protein binding gradually becomes the major
reaction, at least in part because of dissociation of ITC from
Introduction
Isothiocyanates (ITCs)1 occur as glucosinolates (GLs) in
cruciferous vegetables (1, 2). They occur in all parts of these
plants but in different concentrations and profiles. ITCs are
released from GLs by the enzyme myrosinase (3), which occurs
in the same plants, separated cellularly from the GLs (4).
Myrosinase mixes with the GLs and hydrolyzes the GLs when
tissues of plants are damaged or chewed (5) (Figure 1). After
* To whom correspondence should be addressed. Tel/Fax: 504-988-2771.
E-mail: gabriele.sabbioni@bluewin.ch.
1 Abbreviations: AITC, allyl isothiocyanate; AcLys, NR-acetyl-L-lysine;
AITC-Lys, N6-[(allylamino]carbonothioyl]lysine; BITC, benzyl isothiocy-
anate; BITC-Cys, S-[(benzylamino)carbonothioyl]cysteine; BITC-Lys, N6-
[(benzylamino)carbonothioyl]lysine; BocLys, NR-(tert-butoxycarbonyl)-L-
lysine; BocCys, NR-(tert-butoxycarbonyl)-L-cysteine; CE, collision energy;
DP, declustering potential; GL, glucosinolate; GSH, glutathione; Hb,
hemoglobin; ITC, isothiocyanate; LOD, limit of detection; LOQ, limit of
quantitation; PEITC, phenylethyl isothiocyanate; PEITC-AcCys, N-acetyl-
S-{[(2-phenylethyl)amino]carbonothioyl}cysteine; PEITC-Lys, N6-{[(2-
phenylethyl)amino]carbonothioyl}lysine; SFN, D,L-sulforaphane; SFN-Lys,
N6-({[3-(methylsulfinyl)propyl]amino}carbonothioyl)lysine.
10.1021/tx900393t  2010 American Chemical Society
Published on Web 02/04/2010

New Biomarkers for Monitoring ITC LeVels in Humans
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 757
Figure 1. Release of BITC, PEITC, AITC, and SFN from the corresponding GLs.
tris(hydroxymethyl)amino-methane (#252859), sodium phosphate
monobasic monohydrate (#P0662), sodium thiocyanate (#251410),
[2H6]DMSO, dry 1,4-dioxane (#296309), L-Lys-[13C615N2]-
hydrochloride (#608041), NR-acetyl-L-lysine (Ac-Lys) (A2010),
L-valine, L-aspartic acid, copper(II) carbonate basic (#207896),
benzyl isothiocyanate (BITC) (#252492), S-[(benzylamino)car-
bonothioyl]cysteine (BITC-Cys) (#B7031), phenethyl isothiocyanate
(PEITC) (#53731), trifluoroacetic acid (TFA) (#T62200), human
serum albumin (#A1653), acylase I from porcine kidney (#A3010),
and water for LC-MS/MS (Chromasolv; #39253) were purchased
from Sigma-Aldrich (St. Louis, MO). A Chromabond C18ec
cartridge (500 mg/3 mL) was purchased from Macherey-Nagel. D,L-
Sulforaphane (S699115) (SFN) was purchased from Toronto
Research Chemicals, Inc. (North York, ON, Canada).
Instrumentation. An API 4000Q Trap (Applied Biosystems,
Foster City, CA) mass spectrometer interfaced to a HPLC (Shi-
madzu Prominance 20AD) was used for the LC-MS/MS analyses.
Chromatographic separation for the LC-MS/MS analyses was
achieved with a Luna 3 µ C18(2) (100 Å, 150 mm × 2 mm, 3 µm)
(Phenomenex Inc., Torrance, CA) attached to a C18 guard column
(AJO-4287; 4 mm L × 3 mm ID; Phenomenex Inc.) and a gradient
system with solvents A (10 mM ammonium formate) and B
(methanol) at a flow rate of 0.2 mL/min. HPLC analyses to
determine the purity of the compounds were performed on a
Lichrosphere RP18 (125 mm × 4.6 mm, 5 µm) column with a 20
min 30-80% methanol gradient in ammonium acetate (10 mM), a
flow rate of 1.0 mL/min, and λ ) 250 nm using a Hewlett-Packard
1100 system with a quaternary HPLC pump and a photodiode array
detector. A Beckman Coulter DU 800 spectrophotometer was used
for protein determination. Centrifugations were performed on a
Beckman Coulter Allegra X-22R centrifuge equipped with a
SX4250 swing out bucket rotor. NMR spectra were recorded on a
Bruker AC 500 instrument with [2H6]DMSO as the solvent and as
the internal standard (IS). The raw NMR data were processed with
the program MestRe-C (Magnetic Resonance Companion, J. C.
Cobas, J. Cruces, and F. J. Sordina, Departamente de Quimica
Organica, Universidad de Santiago de Compostela, 15706 Santjago
de Compostela, Spain).
the unstable adducts of ITC with the thiol group of GSH (12).
Eventually, proteins are the major binding sites of ITCs inside
cells. For example, PEITC-protein adducts account for 87%
of total cellular ITC uptake after 4 h of treatment. The time
course of this protein binding correlated well with the inhibition
of proliferation and the induction of apoptosis. This suggests
that cellular protein adducts of ITC may be an early event for
apoptosis induction (12). Therefore, biomarkers are needed that
show the presence of stable reaction products with proteins.
Animal studies have shown that following first pass metabo-
lism, GSH conjugates are the major products of ITCs (reviewed
in refs 17 and 18). The major urinary excretion products of GLs
and breakdown products in human are N-acetyl cysteine
conjugates, which have been used as urinary biomarkers of
exposure to dietary GLs (reviewed in ref 18). Reaction products
of thiols with ITCs are not stable (19-22), since an ITC
molecule may be regenerated and then react with other
nucleophiles.
Most epidemiological studies on the relation of diet and
cancer have relied on the information collected with question-
naires to monitor the food intake (7). Questionnaires suffer from
the disadvantage of information bias, especially recall bias,
which can lead to inaccurate exposure estimation. Urinary
metabolites provide the exposure history of the last 24 h, if the
urine of the full next day is collected. However, this is not very
practicable in large epidemiological studies. Furthermore, the
mercapturic acids of ITC in urine can react with other nucleo-
philes (21). Therefore, stable biomarkers are needed that reflect
a larger time span of the ITC exposure history. Thus, we propose
to determine stable (not cysteine adducts) reaction products of
ITCs with albumin and hemoglobin (Hb) in human. Stable Hb
adducts and albumin adducts have a lifetime of 120 days and a
half-life of 20-25 days, respectively (23). This new method
would enable one, for example, to quantify ITC adducts in blood
proteins from large prospective studies about diet and cancer.
Protein adducts have been discovered to be involved in the
chemopreventive effects of ITCs. Therefore, blood protein
adducts are a potential surrogate marker for the effects of ITCs
at the cellular level. This new technique will improve the
assessment of ITC exposure and of the power of studies on the
relationship between ITC intake and cancer.
Animals. Blood samples were obtained from Dr. Stephen Hecht
(University of Minnesota). The animals were part of a large
experiment. Five to six week old A/J mice received eight treatments
of a mixture of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(NNK) plus benzo(a)pyrene (BaP) (2 µmol of each) twice a week.
The mice of group 6 received N-acetyl-S-(N-2-phenethylthiocar-
bamoyl)-L-cysteine (PEITC-AcCys) (5 µmol/g diet) and indole-3-
carbinol (I3C) (5 µmol/g diet) 1 week after the last carcinogen dose.
The mice of group 11 were treated the same way, but myoinositol
(MI) (56 µmol/g diet) was given in addition to PEITC-AcCys and
I3C 1 week after the last carcinogen dose. The animals were
sacrificed after 19 weeks. Pooled frozen blood samples of group 6
and group 11 were sent to Tulane University for work-up and
chemical analyses. Fresh blood from control mice was obtained
from Dr. C. Miller (Tulane University, New Orleans).
Materials and Methods
Chemicals. Methanol (A454-4) for sample preparation, methanol
(Optima, A456-4) for LC-MS/MS, and Amicon Ultra Centrifuge
filters with 10 and 30 kDa (UFC803096) cutoffs were obtained
from Fisher Scientific (New Jersey). Pronase E from Streptomyces
griseus (#81748), formic acid (MS grade, #94318), ammonium
formate (#17843), NR-(tert-butoxycarbonyl)-L-lysine (BocLys)
(#15456), N-(tert-butoxycarbonyl)-L-cysteine (BocCys) (#15411),
sodium sulfide hydrate puriss p.a. (32-38%) (#71975), allyl
isothiocyanate (AITC) (#36682), and sodium hydroxide (#71687)
were purchased from Fluka (Buchs, Switzerland). The reagent grade
Isolation of Albumin and Hb from Frozen Whole Blood
Samples of Mice. The isolation procedure was used with a
modification of the method reported by Funk et al (24). Blood (200
µL) was diluted with water (370 µL) followed by dropwise addition
of cold ethanol (430 µL) while stirring for 2-3 min to precipitate

758 Chem. Res. Toxicol., Vol. 23, No. 4, 2010
Kumar and Sabbioni
the Hb. The supernatant was separated from Hb by centrifugation
at 4000 rpm for 5 min. The Hb pellet was washed twice with 10
mM Tris-HCl (pH 7.5, 2 mL), followed by sequential washing with
cold ethanol (2 mL), ethanol/ethyl ether (3:1, v/v, 1.5 mL), and
ethyl ether (2 mL). Hb was stored at -20 °C after it was dried at
room temperature in a desiccator. The purity of the Hb was checked
with HPLC. An HPLC-UV method was developed to check the
purity for Hb. Hb was dissolved in 50 mM ammonium bicarbonate
buffer (pH 9.0). HPLC-UV analysis was performed on a Phenom-
enex Jupitor (250 mm × 4.6 mm, 5 µm, 300 Å) column with a 70
min gradient run: 35 (0 min), 35 (10 min), 50 (60 min), 35 (65
min), and then 35% (70 min) acetonitrile in 0.1% TFA (B) and
0.1% TFA in water (A) [flow rate of 1 mL/min, tR (heme) ) 3.2
min, tR (ꢀa chain) ) 33.9 min, and tR (Ra chain) ) 39.5 min] (25).
The purity of Hb was measured at 210 nm.
In the supernatant, 600 µL of cold ethanol was added dropwise
while it was constantly stirred to precipitate albumin. Precipitated
albumin was dissolved in 10 mM Tris-HCl (pH 7.5, 2 mL) and
centrifuged at 4000 rpm for 5 min to remove further Hb. The
solution was loaded on a prewashed Amicon ultracentrifugal tube
(10 kDa molecular mass cutoff) and centrifuged with 4000 rpm
for 40 min at 4 °C. The procedure was repeated after 4 mL of
deionized water was added. The residue was dissolved in 10 mM
sodium phosphate buffer (1 mL, pH 7.0) for albumin concentration
determination.
(246 mg, 1 mmol) in sodium bicarbonate buffer (3 mL, 0.25 M,
pH 8.4) at room temperature. After 1 h at 80 °C, the reaction
mixture was collected and extracted with ethyl acetate (3 × 5 mL).
The organic phase was discarded, and the pH of the water phase
was adjusted to pH 4. The acidified water phase was extracted with
ethyl acetate (3 × 5 mL). The combined organic extracts were dried
over anhydrous sodium sulfate, filtered, and evaporated under
reduced pressure. The residue was covered with trifluoroacetic acid
(TFA). After 1 h, the TFA was evaporated under reduced pressure,
and the residue was dried using a high vacuum pump. After
recrystallization in ethyl acetate, a white powder was yielded (210.7
mg, 71.3%). The purity was 99% according to HPLC analysis (254
and 244 nm).
1H NMR ([2H6]DMSO) δ ppm: 8.02 (br s, 1H, NH), 7.78 (br s,
1H, NH), 7.29 (m, 4H, aromatic H), 7.24 (m, 1H, aromatic H in
para position), 4.69 (s, 2H, C6H5CH2), 3.42 [m, 5H, CH(R), CH2(ε),
NH2], 1.77 + 1.70 [m, 2H, CH2(δ)], 1.48 [m, 2H, CH2(ꢀ)], 1.39
[m, 2H, CH2(γ)]. ESI-MS (+) m/z: 613.1 [2 M + Na]+, 591.1 [2
M + H]+, 318.0 [M + Na]+, 296.0 [M + H]+. MS/MS of m/z
296.0: 189.0 (100%) [M - C7H9N]+, 130.0 (75%) [M -
C8H10N2S]+, 116.2 (75%) [M - C7H12N2O2S]+, 91.0 (57%) [M -
C7H13N3O2S]+. ESI-MS (-) m/z: 611.0 [2 M + Na]-, 589.1 [2 M
- H]-, 316.0 [M + Na]-, 294.0 [M - H]-. MS/MS of m/z 294.0:
145.0 (100%) [M - C8HNS]-, 130.8 (20%) [M - C8H7N2S]-
N6-[(Benzylamino)carbonothioyl]-[13C615N2]lysine
(BITC-
.
[13C615N2]Lys). One molequivalent of sodium hydroxide and then
one molequivalent of copper(II) carbonate were added to L-lysine-
[13C615N2] hydrochloride (10 mg, 0.06 mmol) dissolved in water
(3 mL). After 20 min at 100 °C, the reaction mixture was cooled
on an ice bath. The formed black precipitate was separated from
the blue supernatant by centrifugation. BITC (one molequivalent)
in 1,4-dioxane (0.5 mL) was added dropwise to the blue solution.
After 3 h at room temperature, sodium sulfide (16 mg, 0.2 mmol)
was added to the reaction mixture. Stirring for 10 min was followed
by centrifugation. The supernatant was washed with dichlo-
romethane (3 × 5 mL) and then acidified to pH 4. The acidic
solution was loaded in a preconditioned Chromabond C18ec
cartridge. After it was washed with water, the compound was eluted
with 0.1% formic acid/methanol (50:50 v/v, 6 mL). The eluate was
collected and evaporated under reduced pressure. The residue was
washed with n-hexane and dichloromethane and dried using a high
vacuum pump. A yellowish powder of BITC-[13C615N2]Lys (12.2
mg, 67.6%) was obtained.
The purity of the isolated albumin was controlled by HPLC on
a Phenomenex Jupitor (250 mm × 4.6 mm, 5 µm, 300 Å) column
with 0-80% methanol gradient in 0.1% TFA over 20 min and 5
min with 80% methanol in 0.1% TFA. Albumin eluted at 21 min
using a flow rate of 1 mL/min. The purity of albumin was >99.5%
at λ ) 220 nm. The retention time corresponded to commercially
available albumin (A3139, Sigma Aldrich) from mouse serum.
Human Sample. In a preliminary experiment, blood samples
were analyzed after cruciferous vegetable intake from one subject.
Garden cress (60 g) and water cress (100 g) were eaten as salad.
Blood samples were collected 1 day after the meal. Garden cress
and watercress were not part of the normal diet of the studied
subject. Broccoli (ca. 40 g in salads) was part of the regular diet of
the subject. The day before blood donation, 300 g of raw broccoli
was eaten. Plasma and erythrocytes were separated with centrifuga-
tion. The erythrocytes were washed three times with saline water
(0.9% NaCl).
Isolation of Albumin and Hb from Human Blood. Blood was
centrifuged to separate plasma from the erythrocytes. The eryth-
rocytes were washed three times with an equivalent volume of 0.9%
NaCl in water. Albumin was isolated from human plasma as
described earlier (26) using HiTrap Blue HP affinity column (1
mL volume, GE Life Sciences, Piscataway, NJ) (#17-0412-01). The
column was first equilibrated with 6.0 mL of binding buffer (20
mM NaH2PO4, pH 7.0). The plasma sample (0.5 mL) was diluted
with binding buffer (0.5 mL), applied on a pre-equilibrated column,
and washed with binding buffer (6 mL). The adsorbed albumin
was eluted with 6 mL of elution buffer (20 mM NaH2PO4 + 2 M
NaCl, pH 7.0). The HiTrap columns were regenerated and
equilibrated by washing with 10 mL of 50 mM Tris-HCl, pH 7.4,
+ 0.2 M NaSCN followed by 6.0 mL of binding buffer. Purified
fractions were concentrated in Amicon ultracentrifugal filter tube
(30 kDa molecular mass cutoff; 4 mL) by centrifuging with 4000
rpm at 4 °C for 10-15 min, followed by desalting with water (3
× 4 mL). Samples were redissolved in 10 mM sodium phosphate
buffer (pH 7.0).
ESI-MS (+) m/z: 326.1 [M + Na]+, 304.2 [M + H]+. MS/MS
of 304.2: 196.9 (100%) [M - C7H9N]+ 155.2 (30%) [M -
C8H7NS]+, 149.9 (35%) [M - C6H14N2S2]+, 137.0 (75%) [M -
C8H10N2S]+, 108.1 (75%) [M - C7H12N2O2S]+, 91.1 (57%) [M -
C7H13N3O2S]+. ESI-MS (-) m/z: 302.2 [M - H]+. MS/MS of
302.2: 194.9 (10%) [M - C7H9N], 153.0 (100%) [M - C8H7NS]-,
134.9 (14%) [M - C8H10N2S]-.
N6-{[(2-Phenylethyl)amino]carbonothioyl}lysine (PEITC-Lys).
The same procedure as described for the synthesis of BITC-Lys
was followed. Instead of BITC, PEITC was taken. A white powder
with 75% yield was obtained.
1H NMR ([2H6]DMSO) δ ppm: 7.55 (br s, NH), 7.51 (br s, NH),
7.29 (“t”, J ) 7.4, 7.3 Hz 2H, aromatic H in meta position), 7.23
(“d”, J ) 7.3 Hz, 2H, aromatic H in ortho position), 7.19 (“t”, J )
7.4 Hz 1H, aromatic H in para position), 3.59 (m, 2H,
C6H5CH2CH2), 3.53 (m, 1H, CHCOO), 3.34 (m, 2H, CH2NH), 2.80
(t, J ) 7.5 Hz, 2H, C6H5CH2), 1.77 + 1.70 [m, 2H, CH2(δ)], 1.47
[m, 2H, CH2(ꢀ)], 1.38 [m, 2H, CH2(γ)]. ESI-MS (+) m/z: 641.2 [2
M + Na]+, 619.2 [2 M + H]+, 332.1 [M + Na]+, 309.9 [M +
H]+. MS/MS of m/z 309.9: 189.1 (85%) [M - C8H11 N]+, 147.0
(35%) [M - C9H9NS]+, 130.1 (55%) [M - C9H12N2S]+, 122.1
(100%) [M - C7H14N2O2S]+, 105.1 (30%) [M - C7H15N3O2S]+.
ESI-MS (-) m/z: 639.2 [2 M + Na]-, 617.0 [2 M - H]-, 329.9
[M + Na]-, 308.1 [M - H]-. MS/MS of 308.1: 187.0 (9%) [M -
C8H11N]-, 145.0 (100%) [M - C9H9NS]-.
Hb was isolated from erythrocyte (200 µL) as described earlier
(27). Erythrocytes were lysed with 4 volumes of water on ice for
30 min. The cell debris was separated with centrifugation. Cold
ethanol was added (2 mL) dropwise to the supernatant to precipitate
the Hb. Precipitated Hb was sequentially washed with cold ethanol
(2 × 3 mL), 1.5 mL (3:1, ethanol/ethyl ether v/v), and finally with
2 mL of ethyl ether. Hb was stored at -20 °C after it was dried at
room temperature for 1 h.
PEITC-[13C615N2]Lys. The same procedure used to synthesize
BITC-[13C615N2]Lys was followed and yielded PEITC-[13C615N2]Lys
(white solid, 9.4 mg, 45.6% yield, purity 99% at 250 nm). ESI-
N6-[(Benzylamino)carbonothioyl]lysine (BITC-Lys). BITC
(149 µL, 1 mmol) in 1,4-dioxane was added dropwise to BocLys

New Biomarkers for Monitoring ITC LeVels in Humans
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 759
MS (+) m/z: 340.2 [M + Na]+, 318.2 [M + H]+. MS/MS of m/z
318.2: 197.1 (82%) [M - C8H11N]-, 137.0 (55%) [M -
C9H12N2S]+, 122.1 (100%) [M - C7H14N2O2S]+, 104.8 (30%) [M
- C7H15N3O2S]+. ESI-MS (-) m/z: 632.8 [2 M - H]-, 316.1 [M
- H]-. MS/MS of 316.1: 185.0 (9%) [M - C8H11N]-, 153.0
(100%) [M - C9H9NS]-, 135.0 (8%) [M - C9H12N2S]-.
N6-({[3-(Methylsulfinyl)propyl]amino}carbonothioyl)lysine (SFN-
Lys). AcLys (60 mg, 0.32 mmol) was dissolved in sodium
bicarbonate (0.25 M, pH 8.4, 5 mL) buffer followed by dropwise
addition of SFN (35 µl, 0.20 mM) in 1,4-dioxane (0.5 mL) at 80
°C with continuous stirring for 1 h. After 1 h, the reaction mixture
was cooled down on a ice bath, extracted with ethyl acetate (3 ×
5 mL), and then acidified with 2 M HCl to pH 4. SFN-AcLys was
purified using a Chromabond C18ec cartridge. Before the sample
was loaded, the cartridge was conditioned and equilibrated with 3
mL of methanol and 0.1% formic acid (pH 4), respectively. SFN-
AcLys was eluted with 6 mL of methanol and concentrated to
dryness. The residue was dissolved in 50 mM sodium phosphate
buffer (pH 7.4, 3 mL) and incubated (37 °C, 60 h) with 50 mg of
acylase I from porcine kidney (#A3010 Sigma). The reaction
mixture was centrifuged and acidified with 2 M HCl to pH 4.0.
AITC-Lys was isolated using solid-phase extraction as described
above. Precipitated acylase I was removed by centrifugation. The
supernatant was dried using at reduced pressure and yielded SFN-
Lys (72 mg, 69.9%) as a white solid. HPLC-UV analysis was
performed on a LiChrosphere RP18 (125 mm × 4 mm, 5 µm)
column with a 14 min: gradient run 5% (1 min), 10% (7 min),
then hold 10% (11 min), 5% (12 min) methanol in 10 mM
ammonium acetate [flow rate of 1 mL/min, tR (SFN-NR-AcLys) )
9.2 min, tR (SFN-Lys) ) 6.3 min]. The purity for SFN-Lys was
99% at λ ) 220 nm.
N6-[(Allylamino)carbonothioyl]lysine (AITC-Lys). AITC (1
mmol) in 1,4-dioxane (0.5 mL) was added dropwise to AcLys (188
mg, 1 mmol) dissolved in sodium bicarbonate (0.25 M, pH 8.4, 5
mL) at 80 °C. After 1 h, the reaction mixture was cooled down on
a ice bath, extracted with ethyl acetate (3 × 5 mL), and then
acidified with 2 M HCl to pH 4. AITC-AcLys was purified using
a Chromabond C18ec cartridge. Before the sample was loaded, the
cartridge was conditioned with methanol (3 mL) and equilibrated
with 0.1% formic acid (3 mL, pH 4). AITC-AcLys was eluted with
methanol (6 mL) and concentrated to dryness. The residue was
dissolved in 50 mM sodium phosphate buffer (pH 7.4, 3 mL) and
incubated (37 °C, 24 h) with 25 mg of acylase I from porcine kidney
(#A3010 Sigma). The reaction mixture was centrifuged and acidified
with 2 M HCl to pH 4.0 before solid-phase purification as described
above. AITC-Lys was eluted with methanol. Precipitated acylase I
was removed by centrifugation, and the supernatant was evaporated
at reduced pressure and yielded AITC-Lys as a white solid product
(112.7 mg, 46.0%). HPLC-UV analysis was performed on a
Lichrosphere RP18 (125 mm × 4 mm, 5 µm) column with a 15
min, isocratic run: 20% methanol in 0.1% formic acid [flow rate
of 1 mL/min, tR (AITC-AcLys) ) 5.9 min, and tR (AITC-Lys) )
3.1 min]. The purity for AITC-Lys was found to be 99% at λ )
220 nm.
1H NMR (COSY) ([2H6]DMSO) δ ppm: 8.3 (br s, 3H, NH, NH,
COOH), 3.39 (m, CH2N of SFN), 3.31 (m, CH2N of Lys, and
CHCOO of Lys), 2.76 (m, 1H, CH2SO), 2.66 (m, 1H, CH2SO),
2.6 (s, 3H, CH3SO), 1.72 [m, 1H, CH2(δ)], 1.60 [m, 1H of CH2(δ)
+ 4H of SOCH2CH2CH2CH2], 1.44 [m, 2H, CH2(ꢀ)], 1.35 [m, 2H,
CH2(γ)]. 13C NMR ([2H6]DMSO) δ ppm: 173.7 and 171.2 (COOH,
CS), 54.06 (CHCOOH), 52.90 (CH2SO), 43.2 and 42.8
(CH2NHCSNHCH2), 37.95 (CH3SO), 30.84 [CH2(ꢀ)], 28.29 and
28.05 [CH2(δ) and CH2CH2NHSO], 22.42 [CH2(γ)], 19.51
(CH2CH2SO). ESI-MS (+) m/z: 669.2 [2 M + Na]+, 646.0 [2 M
- H]+, 346.3 [M + Na]+, 324.1 [M + H]+. MS/MS of 324.1:
1H NMR ([2H6]DMSO) δ ppm: 7.93 (br s, 3H, NH, NH, COOH),
5.84 (m, 1H, CH2CHCH2N), 5.14 (d, J ) 17.2 Hz, 1H, trans
CH2dCHCH2N), 5.04 (dd,
J ) 10.4 Hz, 1.5, 1H, cis
CH2dCHCH2N), 4.04 (s, 2H, CHCH2N), 3.42 [m, 2H, CH2(ε)],
3.35 (m, 1H, CHCOO), 1.77 + 1.70 [m, 2H, CH2(δ)], 1.46 [m,
2H, CH2(ꢀ)], 1.37 [m, 2H, CH2(γ)]. ESI-MS (+) m/z: 736.3 [3 M
+ H]+, 513.3 [2 M + Na]+, 491.1 [2 M + H]+, 268.1 [M + Na]+,
246.0 [M + H]+. MS/MS of m/z 246.0: 201.1 (100%) [M -
CHO2]+, 189.3 (10%) [M - C3H7N]+, 147.2 (5%) [M - C4H5NS]+,
143.2 (22%) [M - C4H9NO2]+, 129.9 (32%) [M - C4H8N2S]+,
116.8 (30%) [M - C6H11NO2]+, 58.2 (17%) [M - C7H12N2O2S]+.
ESI-MS (-) m/z: 511.0 [2 M + Na]-, 489.0 [3 M - H]-, 489.0 [2
M - H]-, 266.1 [M + Na]-, 244.0 [M - H]-. MS/MS of 244.0:
186.6 (5%) [M - C3H7N]+, 145.0 (100%) [M - C4H5NS]-, 56.0
(7%) [M - C7H12N2O2S]-.
136.0 (100%) [M
- -
C7H12N2O2S]+, 147.2 (53%) [M
C6H11NOS2]+, 178.2 (13%) [M - C6H14N2O2]+, 260.3 (14%) [M
- CH3OS]+. ESI-MS (-) m/z: 645.1 [2 M - H]-, 344.1 [M +
Na]-, 322.0 [M - H]-. MS/MS of 322.2; 145.0 (100%) [M -
C6H11NOS2]-, 186.9 (13%) [M - C5H13NOS]-
.
SFN-[13C615N2]Lys. L-Lys [13C615N2]hydrochloride (10 mg, 0.06
mmol) was dissolved in water (3 mL), and one molequivalent of
sodium hydroxide was added followed by one molequivalent of
cupric(II) carbonate. After 20 min at 100 °C, the reaction was cooled
down on an ice bath, and the black precipitate was eliminated by
centrifugation. SFN (one molequivalent in 1,4-dioxane) was added
dropwise to the blue supernatant. The reaction was carried out at
room temperature for 4 h. Sodium sulfide (16 mg, 0.2 mmol) was
added to the reaction mixture and stirred for 10 min at room
temperature. After centrifugation, the supernatant was washed with
dichloromethane (3 × 3 mL), acidified with 2 M HCl to pH 4, and
loaded on a preconditioned Chromabond C18ec cartridge (500 mg/3
mL). The compound was eluted with 0.1% formic acid/methanol
(50:50 v/v, 6 mL). After evaporation under reduced pressure, the
residue was washed with n-hexane and dichloromethane. After this
was dried, SFN-[13C615N2]Lys (16.1 mg, 81%) was obtained.
AITC-[13C615N2]Lys. L-Lysine [13C615N2]hydrochloride (10 mg,
0.06 mmol) was dissolved in water (3 mL), and one molequivalent
of sodium hydroxide was added followed by one molequivalent of
cupric(II) carbonate and heated to 100 °C. After 20 min, the reaction
was cooled down on an ice bath, and a black precipitate was
eliminated by centrifugation. AITC (one molequivalent in 1,4-
dioxane) was added dropwise to the blue supernatant. After 4 h at
room temperature, sodium sulfide (16 mg, 0.2 mmol) was added
to the reaction mixture and stirred for 10 min followed by the
centrifugation. The supernatant was washed with dichloromethane
(3 × 3 mL), and then, the aqueous phase was acidified with 2 M
HCl up to pH 4. The acidified aqueous phase was loaded on a
preconditioned Chromabond C18ec cartridge, and the retained
compound was eluted with 0.1% formic acid/methanol (50:50 v/v,
6 mL). Fractions were collected and evaporated under reduced
pressure. The final compound was washed with n-hexane and
dichloromethane and yielded AITC-[13C615N2]Lys (18.9 mg, 93.2%).
HPLC analysis showed a purity of 99% at λ ) 244 nm.
ESI-MS (+) m/z: 354.2 [M + Na]+, 332.0 [M + H]+. MS/MS
of 332.0: 136.0 (100%) [M - C7H12N2O2S]+, 155.0 (46%) [M -
C6H11NOS2]+, 178.3 (13%) [M - C6H14N2O2]+, 268.1 (14%) [M
- CH3OS]+. ESI-MS (-) m/z: 330.2 [M - H]-. MS/MS of 330.2:
153.0 (100%) [M - C6H11NOS2]-, 194.9 (10%) [M - C5H13NOS]-.
N-[(Benzylamino)carbonothioyl]valine (BITC-Val). BITC (149
µL, 1 mmol) in 1,4-dioxane (0.5 mL) was added dropwise to
L-valine (116 mg, 1 mmol) in sodium bicarbonate (3 mL, 0.25 M,
pH 8.4). After 30 min at 80 °C, the cooled reaction mixture was
extracted with ethyl acetate (three times). The organic phase was
discarded, and the pH of the aqueous phase was adjusted to pH 4.
The acidified water phase was extracted with ethyl acetate. The
organic extract was dried over anhydrous sodium sulfate, filtered,
and evaporated under reduced pressure. The residue was dried using
ESI-MS (+) m/z: 529.5 [2 M + Na]+, 507.4 [2 M + H]+, 254.0
[M + H]+. MS/MS of m/z 254.0: 58.1 (66%) [M - C7H12N2O2S]+,
89.9 (90%) [M - C7H12N2S]+, 137.0 (100%) [M - C4H8N2S]+,
149.9 (75%) [M - C4H7NO2]+, 197.1 (90%) [M - C3H7N]+. ESI-
MS (-) m/z: 527.3 [2 M + Na]-, 504.8 [2 M - H]-, 252.1 [M -
H]-. MS/MS of 252.1; 59.0 (17%) [M - C7H12N2O2S]-, 134.7
(7%) [M - C4H8N2S]-, 153.0 (100%) [M - C4H5NS]-
.

760 Chem. Res. Toxicol., Vol. 23, No. 4, 2010
Kumar and Sabbioni
a high vacuum pump. A yellowish oily product of BITC-Val (43
mg, 16%) was obtained after recrystallization with ethyl acetate.
The purity was 99% according to HPLC analysis (200 and 254
nm). The compound was not stable at -20 °C. Gradually, the
benzylthiohydantoin-valine (BITC-Hyd-Val) formed.
carried out with 100 pg/µL solution of analyte with a flow rate of
10 µL/min in negative ionization mode for BITC-Lys, PEITC-Lys,
and AITC-Lys and positive ionization mode for SFN-Lys, showing
corresponding peaks at m/z 294.0, 308.1, 244.1 [M - H]-, and
324.1 [M + H]+. Quantitative optimization mode was used to
maximize the signal and set the maximum suitable MS parameters
for the compounds. For better resolution and sensitivity of the
analyte, quadrupole mass analyzers (Q1 and Q3) were set on 0.7
( 0.1 amu resolution window.
The mass spectrometer was operated in negative ionization mode
with a electrospray voltage at -4500 V for negative ionization and
+5500 V for positive ionization mode and a source temperature of
500 °C. Nitrogen was used as ion spray (GS1), drying (GS2), and
curtain gas at 40, 45, and 10 arbitrary units, respectively. The
declustering potential (DP) and collision energy (CE) for BITC-
Lys, PEITC-Lys, AITC-Lys, and SFN-Lys were -45, -50, -40,
+61 and -24, -24, -25, +23 V, respectively. The entrance
potential (EP) for all compounds was -10 V. All data were
processed using Analyst software 1.4.2 (Applied Biosystems/MDS
Sciex).
The BITC-Lys, PEITC-Lys, AITC-Lys, and SFN-Lys were
detected with MRM transition of 294.0/145.0, 308.1/145.0, 244.1/
145.0 [M - H]- and 324.1/136.0 [M + H]+, respectively, along
with their corresponding stable isotope-labeled compounds as ISs
PEITC-[13C615N2]Lys {316.1/153.0 [M - H]-}, BITC-[13C615N2]-
Lys {302.1/153.0 [M - H]-}, AITC-[13C615N2]Lys {252.1/153.0
[M - H]-}, and SFN-[13C615N2]Lys {332.1/136.0 [M + H]+}.
Chromatographic separation of BITC-Lys and PEITC-Lys was
achieved on a Luna 3 µ C18(2) column with a gradient system
using solvent A (10 mM ammonium formate) and B (methanol) at
a flow rate of 0.2 mL/min. The gradient program was as follows:
0.10 (B 20%), 3 (B 20%), 16 (B 90%), 20 (B 90%), and 21-26
min (B 20%). The retention times (tR) of BITC-Lys and PEITC-
Lys were 13.7 and 15.2 min, respectively. The column flow was
diverted away from the ESI ion source except for the time period
from 8 to 18 min. The limits of detection (LOD) for BITC-Lys
and PEITC-Lys were 1.69 and 1.61 fmol (on column), while the
limits of quantitation (LOQ) were 18.8 and 17.9 fmol/mg albumin,
respectively.
SFN-Lys and AITC-Lys separation was achieved on a Luna 3 µ
C18(2) with a gradient system using solvents A (10 mM ammonium
formate) and B (methanol) at a flow rate of 0.2 mL/min. The
gradient program was as follows: 0.10 (B 5%), 1 (B 5%), 8 (B
80%), 9 (B 80%), and 11-15 min (B 5%). The retention times
(tR) of SFN-Lys and AITC-Lys were 7.71 and 8.30 min, respec-
tively. The column flow was diverted away from the ESI ion source
except for the time period from 4 to 10 min. The LODs for SFN-
Lys and AITC-Lys were 1.54 and 4.07 fmol (on column), while
the LOQs were 34.3 and 113.7 fmol/mg albumin, respectively.
Calibration Line. To generate the calibration line, 9 mg of
human serum albumin (Sigma) was spiked with different amounts
of BITC-Lys (0.00, 0.17, 0.34, 3.34, 33.88, and 169.40 pmol),
PEITC-Lys (0.00, 0.16, 0.32, 3.23, 32.35, and 161.75 pmol), SFN-
Lys (0.00, 0.31, 0.77, 1.54, 3.09, 15.47, and 30.95 pmol), and AITC-
Lys (0.00, 0.40, 1.01, 2.03, 4.07, 20.39, and 40.79 pmol) along
with the corresponding BITC-[13C615N2]Lys (1.61 pmol), PEITC-
[13C615N2]Lys (1.57 pmol), SFN-[13C615N2]Lys (15.1 pmol), and
AITC- [13C615N2]Lys (19.7 pmol). Samples were incubated with 3
mg of Pronase E in 50 mM sodium bicarbonate (2 mL, pH 8.9) at
37 °C overnight. Digested samples were acidified (up to pH, 4.0)
using 2 M HCl. Chromabond C18ec cartridges were used for the
solid-phase extraction cleanup. The column was first activated with
3 mL of methanol and then equilibrated with 3 mL of 0.1% formic
acid (pH 4.0). The sample was applied on the column and
subsequently washed with 0.1% formic acid (3 mL, pH 4.0). BITC-
Lys, PEITC-Lys, SFN-Lys, and AITC-Lys were eluted with a 6
mL fraction of 80% methanol in 0.1% formic acid (pH 4.0). The
fraction was evaporated up to 1 mL and injected in the LC-MS/
MS system for further analysis.
1H NMR ([2H6]DMSO) δ ppm: 12.5 (br s, COOH), 8.02 (t, J )
5.1 Hz, CH2NH), 7.51 (“d”, J ) 5.7 Hz, CHNH), 7.32 (m, 4H,
aromatic H), 7.25 (m, 1H, aromatic H in para position), 4.86 (m,
1H, CHNH), 4.68 (dd, J ) 2.2, 5.1 Hz, 2H, CH2NH), 2.13 [m, 1H,
CH(CH3)2], 0.91 (d, J ) 6.8 Hz, 3H, CH3), 0.90 (d, J ) 6.9 Hz,
3H, CH3), 15% BITC-Hyd-Val was present. ESI-MS (+) m/z: 598.9
[2 M + 3Na]+, 576.9 [2 M + 2Na]+, 555.1 [2 M + Na]+, 289.1
[M + Na]+, 267.1 [M + H]+. MS/MS of 267.1; m/z: 71.8 [M -
C13H16N2O2S]+, 90.8 [M - C6H12N2O2S]+, 118.2 [M - C8H7NS]+.
ESI-MS (-) m/z: 530.8 [2 M + H]+, 265.1 [M + H]+. MS/MS of
265.1; m/z: 115.8 (100%) [M - C8H7NS]-
.
BITC-Cys. BITC-Cys can be obtained commercially or from
the reaction of BITC with BocCys for 1 h at room temperature in
sodium bicarbonate (3 mL, 0.25 M, pH 8.4). LC-MS/MS chro-
matographic separation of BITC-Cys was achieved on a Luna 3 µ
C18(2) with gradient system using solvents A (10 mM ammonium
formate) and B (methanol) at a flow rate of 0.2 mL/min. The
gradient program was as follows: 0.1 (B 20%), 3 (B 20%), 16 (B
90%), 20 (B 90%), and 21-26 min (B 20%). The retention time
(tR) of BITC-Cyst was 14.1 min. The column flow was diverted
away from the ESI ion source except for the time period from 10
to 17 min. The BITC-Cys was detected with MRM transition of
268.9/119.9 [M - H]-
.
ESI-MS (+) m/z: 293.1 [M + Na]+, 271.1 [M + H]+. MS/MS
of m/z 271.1; 91.0 (100%) [M - C4H6N2O2S2]+, 122.0 (25%) [M
- C8H8NS]+, 245.0 (15%) [M - OH]+. ESI-MS (-) m/z: 268.9
[M - H]-. MS/MS of m/z 268.9; 119.9 (100%) [M - C8H8NS]-.
N-[(Benzylamino)carbonothioyl]aspartic Acid (BITC-Asp).
BITC (149 µL, 1 mmol) in 1,4-dioxane (0.5 mL) was added
dropwise to L-aspartic acid (133 mg, 1 mmol) in sodium bicarbonate
(3 mL, 0.25 M, pH 8.4). After 15 min at 80 °C, the cooled reaction
mixture was extracted with ethyl acetate (3 × 5 mL). The organic
phase was discarded, and the pH of the aqueous phase was adjusted
to pH 5. The acidified water phase was loaded on a preconditioned
Chromabond C18ec cartridge and eluted with 0.1% formic acid/
methanol (50:50 v/v, 6 mL). The eluate was collected and
evaporated under reduced pressure. The residue was washed with
n-hexane:methyl t-butyl ether (1:5) and dried using a high vacuum
pump. A whitish thick oil of BITC-Asp (210 mg, 74.4%) was
obtained. The purity was 53% according to HPLC analysis (244
nm).
HPLC-UV analysis was performed on a Lichrosphere RP18 (125
mm × 4 mm, 5 µm) column, with a linear methanol gradient in
0.1% formic acid: 30% (0-2 min), 30-90% (15 min), and BITC-
Asp and benzylthiohydantoin-aspartic acid [)1-benzyl-5-oxo-2-
thioxoimidazolidin-4-yl)acetic acid] (BITC-Asp-Hyd) were found
to be 53 and 47% at λ ) 244 nm, respectively. LC-MS/MS
chromatographic separation of BITC-Asp was achieved with a Luna
3 µ C18(2) with a gradient system using solvent A (10 mM
ammonium formate) and B (methanol) at a flow rate of 0.2 mL/
min. The gradient program was as follows: 0.1 (B 20%), 3 (B 20%),
16 (B 90%), and 23 min (B 90%). The retention times (tR) of BITC-
Asp and BITC-Asp-Hyd were 11.9 and 15.8 min, respectively. ESI-
MS for BITC-Asp and BITC-Asp-Hyd showed molecular ions of
m/z 281.0 and 263.0, respectively.
ESI-MS (+) m/z: 565.2 [2 M + H]+, 305.0 [M + Na]+, 283.0
[M + H]+ MS/MS of m/z 283.0; 116.0 (55%) [M - C8H8N2S]+,
.
133.9 (100%) [M - C8H7NS]+. ESI-MS (-) m/z: 563.2 [2 M -
H]-, 281.0 [M - H]-, MS/MS of m/z 281.0; 132.0 (100%) [M -
C8H7NS]-.
LC-MS/MS Method and Calibration Line for BITC-Lys,
PEITC-Lys, AITC-Lys, and SFN-Lys. A Shimadzu Prominance
20AD interfaced to a API 4000Q Trap LC-MS/MS (Applied
Biosystems) mass spectrometer system was used for all of the
quantitative analyses. The MS parameters were optimized in the
electrospray ionization mode (ESI). Parameter optimization was
The calibration lines for BITC-Lys, PEITC-Lys, SFN-Lys, and
AITC-Lys were generated over the ranges of 0.00-169.40,

New Biomarkers for Monitoring ITC LeVels in Humans
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 761
Figure 2. Synthesized adducts of BITC, PEITC, AITC, and SFN with
lysine.
0.00-161.75, 0.00-30.95, and 0.00-40.79 pmol/9 mg albumin,
respectively. The concentration levels were plotted against the peak
area ratio of the analyte against the peak area of the IS (e.g., peak
area ratio BITC-Lys/BITC-[13C615N2]Lys). The regression coef-
ficients r2 ) 0.999 (BITC-Lys), 0.998 (PEITC-Lys), 0.999 (SFN-
Lys), and 0.998 (AITC-Lys) were found using linear regression
and a weighting factor of 1/x.
Reaction of ITC with Albumin. The following procedure was
applied for the in vitro modification of albumin with ITCs (BITC,
PEITC, AITC, and SFN). Below the method is described in detail
for BITC. Human serum albumin (20 mg) was dissolved in 50 mM
sodium hydrogen phosphate buffer (3 mL, pH 7.4). A different
molar ration of BITC (in 1,4-dioxane) (1:10, 1:1, 1:0.1, and 1:0.01)
was added in the dissolved albumin solution. The reaction was kept
at 37 °C overnight with continuous stirring. After it was stirred
overnight at 37 °C, the reaction mixture was centrifuged to eliminate
the formed precipitate. The supernatant was washed with ethyl
acetate (three times), and the organic phase was discarded. The
aqueous solution was dialyzed against 4 L for 4 h at 4 °C. Albumin
was precipitated after cold acetone (4:1) and 25 µL of acetic acid
were added. After filtration, the precipitate was reconstituted in 2
mL of water for protein concentration determination. The protein
determination was done using a Coomassie protein Assay kit
(23236) with the standards protocol. The protein recovery was
50-70%. The samples were digested with Pronase E and worked
as described above. Human serum albumin (0.5 mg) was spiked
with BITC-[13C615N2]Lys (82.4 pmol). Samples were digested and
incubated with 166.6 µL of fresh Pronase E solution (1 mg/mL;
50 mM ammonium bicarbonate, pH 8.9) at 37 °C overnight.
Digested samples were acidified (up to pH 4.0) with 2 M HCl, and
Chromabond ec C18 was used for the solid-phase extraction
procedure. The column was first activated with 3 mL of methanol
and then equilibrated with 3 mL of 0.1% formic acid (pH 4.0).
The sample was applied on the column and subsequently washed
with a 3 mL fraction of 0, 10% methanol in 0.1% formic acid.
BITC-Lys eluted with a 6 mL fraction of 80% methanol in 0.1%
formic acid. After evaporation at the speed-vac to approximately 1
mL, 2 µL was taken for LC-MS/MS analysis.
Figure 3. Work-up scheme for the analysis of albumin adducts of
BEITC, PEITC, AITC, and SFN. The ISssBITC-[13C615N2]Lys, PEITC-
[13C615N2]Lys, AITC-[13C615N2]Lys, and SFN-[13C615N2]Lysswere added
prior to albumin digestion.
reaction was performed in sodium carbonate at 80 °C. The Boc
group was cleaved in neat TFA. BITC-Cys can be obtained
commercially or from the reaction of BITC with BocCys at room
temperature in sodium bicarbonate. Valine and aspartic acid
were reacted at room temperature with BITC. The loss of water
and subsequent ring closure to the hydantoin could not be
avoided. For PEITC, AITC, and SFN, only the adducts with
lysine were synthesized (Figure 2). The same procedure used
for BITC-Lys was applied for PEITC-Lys. In the case of SFN
and AITC, AcLys was taken since the cleavage procedure of
Boc with TFA yielded side products. The acetyl group from
AcLys was cleaved with acylase I as shown previously for
aflatoxin adducts of AcLys (28). AITC-Lys was synthesized
recently from BocLys and AITC (21). No NMRs were reported.
The corresponding isotope-labeled standards of BITC-Lys,
PEITC-Lys, AITC-Lys, and SFN-Lys were synthesized with
[13C615N2]Lys. The R amino group was protected as cupper
complex (29). After reaction with the corresponding isothio-
cyanates, the copper complex was released using sodium sulfide
as the reagent (30). The enantiomeric purity of all final products
was not investigated.
BITC-Cys Incubation with Albumin. Albumin (20 mg) was
incubated with BITC-Cys (74.07 pmol) in 50 mM sodium phosphate
buffer (1 mL, pH 7.4) and 50 mM sodium bicarbonate buffer (1
mL, pH 9) at 37 °C overnight. Pronase E (2 mg) was added and
incubated overnight in a shaking bath at 37 °C. The samples were
purified with Chromabond C18ec and analyzed with LC-MS/MS.
BITC was reacted in vitro with albumin overnight at
physiological conditions. The proteins were digested with
Pronase E following an optimized procedure developed for 4,4′-
methylenediphenyl diisocyanate adducts with albumin found in
vivo (31). The digests were prepurified with solid-phase
extraction (Figure 3). The extracts were analyzed with LC-MS/
MS (Figure 4). The major reaction product appears to be with
lysine. No cysteine adducts were found. Other potential amino
acid adducts with tryptophan, tyrosine, and serine were moni-
tored, although no synthetic standard was available. The
N-terminal adduct with aspartic acid appears not to be released
after hydrolysis with Pronase E.
Results
The potential reaction products of BITC with blood proteins
present in vivo were synthesized from the single amino acids.
Cysteine, the N-terminal amino acids, and lysine (Figure 2) are
the most reactive amino acids. The N-terminal amino acids in
human serum albumin and Hb are aspartic acid and valine,
respectively. For the synthesis of adducts with the ε amino group
of lysine, NR-Boc-protected lysine was used as reagent. The

762 Chem. Res. Toxicol., Vol. 23, No. 4, 2010
Kumar and Sabbioni
Figure 4. LC-MS/MS analysis of albumin digested with Pronase E. The retention times of synthesized standards BITC-Lys, BITC-Asp, and BITC-
Cys synthesized from BITC and the single amino acids were 13.1, 11.9, and 14.1, respectively. BITC-Ser, BITC-His, BITC-Trp, and BITC-Tyr
standards were not available. The chosen mass fragmentations were deduced from the MS properties of the ITC adducts with other amino acids.
MS/MS of the molecular ion of amino acid-ITC adducts yields the corresponding amino acid as a daughter ion.
For the analysis of biological samples, a LC-MS/MS method
was developed to analyze the major adducts. First, in vitro
experiments with albumin were performed to develop a method
to quantify ITC adducts with amino acids. To detect single
amino acid adducts, Pronase E was used to digest the proteins.
The digests were purified using solid-phase extraction (Figure
3). The eluates were analyzed with LC-MS/MS. MS/MS of the
molecular ion of amino acid-ITC adducts yields the corre-
sponding amino acid as daughter ion. The sensitivity of the assay
was tested by spiking albumin samples with synthesized standard
compounds. For BITC-Lys, PEITC-Lys, and AITC-Lys, the best
results were obtained with negative ESI. For SFN-Lys, the best
results were obtained with positive ESI. Starting from 9 mg,
the LOQs were established as follows: BITC-Lys, PEITC-Lys,
AITC-Lys, and SFN-Lys as 18.8, 17.9, 113.7, and 34.3 fmol/
mg albumin.
Albumin was modified in vitro with four different doses of
BITC. The adduct levels of the formed BITC-Lys increased
linearly with the dose (Figure 5).
Albumin was modified in vitro with BITC, PEITC, AITC,
and SFN. The reactions were performed with a molar ratio 1:1
of ITC to albumin. Albumin was digested with Pronase E and
analyzed with LC-MS/MS. The adduct levels decreased in the
following way: BITC-Lys > PEITC-Lys > AITC-Lys > SFN-
Lys.
Figure 5. Albumin was modified in vitro with four different ratios of
BITC/albumin ) 0.01/1, 0.1/1, 1/1, and 10/1. Albumin was digested
and analyzed with LC-MS/MS.
The stability of cysteine adducts was tested. BITC-Cys was
incubated at pH 7.4 with albumin overnight. After the digestion
of albumin, no BITC-Cys could be found but newly formed
BITC-Lys.
found in the mice (Figure 6). In the pooled blood samples of
the mice, 6ABCD and 11CD, 1.94 and 1.09 pmol PEITC-Lys
per mg albumin were found. In the control mice, no such adducts
were found.
The adduct formation in vivo and the applicability of the
method was investigated from biological material obtained with
different precursors of ITC: mercapturic acid adduct of PEITC
and/or diet. Mice were chronically exposed to PEITC-AcCys.
Hb and albumin were isolated from frozen whole blood samples.
Hb could be not isolated pure from whole frozen blood.
Impurities were present according to the HPLC analyses of Hb.
Hb of the control mice obtained from washed erythrocytes
showed the typical signals for the heme group and for the R
and ꢀ chain of Hb (25). Therefore, Hb of the exposed mice
was not analyzed. Albumin from exposed mice was obtained
from ethanol precipitation. Albumin adducts of PEITC were
Human blood samples were obtained from one subject on
three different occasions: (1) 1 day after eating 60 g of garden
cress and 100 g of watercress, (2) 30 days after eating garden
cress and watercress and 1 day after eating 300 g of broccoli,
and (3) 4 months after eating garden cress and watercress.
Watercress and garden cress were never eaten before and after
the experiment. Broccoli was part of the daily diet. Albumin
was purified with affinity chromatography, digested with Pronase
E, and analyzed with LC-MS/MS (Figures 6 and 7). The results
are summarized in Table 1. PEITC-Lys and BITC-Lys were
present in large amounts in albumin 1 day after the meal. After
4 months, the PEITC-Lys and BITC-Lys levels were close to

New Biomarkers for Monitoring ITC LeVels in Humans
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 763
Figure 6. LC-MS/MS analyses of albumin digests obtained from (A) mouse control, (B) mouse exposed to PEITC-AcCys, (C) human exposed to
watercress, and (D) PEITC-Lys standard compound. The right panels show the chromatogram of the IS, PEITC-[13C615N2]Lys, which was added
at the beginning of digestion procedure (Figure 3). PEITC-Lys and PEITC-[13C615N2]Lys were detected with the MRM transitions m/z 308.1/145.0
and 316.1/153.0, respectively.
Figure 7. LC-MS/MS analyses of albumin digests of a human subject regularly eating broccoli. The panels on the left represent the chromatograms
of albumin digests from (A) commercially available human serum albumin, (B) human sample collected at day 1, (C) human sample collected at
day 30, and (D) SFN-Lys standard compound. The right panels show the chromatogram of the IS, SFN-[13C615N2]Lys, which was added at the
beginning of digestion procedure (Figure 3). SFN-Lys and the IS SFN-[13C615N2]Lys were detected with the MRM transitions m/z 324.1/136.0 and
332.1/136.0, respectively.

764 Chem. Res. Toxicol., Vol. 23, No. 4, 2010
Kumar and Sabbioni
Table 1. Albumin (Alb) and Hb in One Human Subject
Eating Garden Cress and Watercress on Day 0 and
Regularly Eating Broccoli (ca. 40 g Per Day), except for a
Large Meal on Day 29 (300 g)
measured in the past in urine and in serum (see Introduction).
For example, recently, mercapturic acids of ITCs have been
quantified in urine (33) after the consumption of certain
vegetables. Biologically free available ITCs have been found
in urine and serum of humans (34). Protein adducts after in
vitro experiments with cells have been published recently
(35). In this case, cysteine adducts were postulated (13). Our
experiments and the data from the literature (21) indicate
that such adducts will not be stable.
pmol/mg Alb
pmol/mg Hb
days PEITC-Lys BITC-Lys SFN-Lys PEITC-Lys BITC-Lys SFN-Lys
1
2.365
0.823
0.046
1.011
0.480
0.029
2.203
3.306
1.372
0.050
0.020
a
0.023
0.021
a
0.140
0.203
a
30
121
a Below the LOQ.
For this study, human albumin was isolated with Cibacron
blue affinity columns (HiTrap Blue), which yields purer
albumin than ammonium sulfate precipitation (36). In larger
studies, we suggest using the precipitation method since many
more samples could be processed per day (8 vs 30). The
developed method was sensitive enough to analyze small
samples of albumin (9 mg). Increasing the amount of albumin
also increases the ion suppression effects for the detection
of the adducts in LC-MS/MS. Therefore, to increase the
sensitivity of the assay, the digests would have to be purified
with specific ITC immunoaffinity columns.
In a 75 kg man, approximately 100 g of albumin is present.
It has been reported that from 30 g of watercress 46.6 µmol
of PEITC can be released (37). Therefore, consuming 100 g
of watercress could yield approximately 155 µmol of PEITC.
We found 2.365 nmol PEITC-Lys per g of albumin. Thus,
approximately 0.15% of the PEITC dose was bound to
albumin. In humans eating 30 g of watercress, 30-67% of
the PEITC conjugate (PEITC-AcCys) was found in 24 h urine
(37). In humans eating 100 g of watercress, up to 1 µmol of
free PEITC per L plasma could be found (34). Comparing
these values from different laboratories, the levels of albumin
adducts (present data) were 10 and 300 times lower than
PEITC in plasma (34) and PEITC metabolites in urine (37),
respectively. After 48 h, usually no urinary metabolites were
found (37). The half-life of PEITC in plasma was 4.9 ( 1.1 h
(34). After 48 h, approximately 10 half-lives, only 0.09%
(0.9 nmol/L plasma) of the original PEITC levels (1 µmol/L
plasma), will be available in plasma. Therefore, only blood
protein adducts will be measurable 48 h after the last exposure
to ITCs, since the half-life of the adducts is approximately
21-23 days. For chronically exposed people, we expect a
steady state albumin adduct level that is 29 times higher
adduct level after chronic dosing in comparison to a single
dose (28). Therefore, in chronically exposed people, higher
adduct levels will be found than after a single dose.
Consequently, the concentration difference between the short-
term markers (urine and plasma metabolites) and the long-
term markers (albumin adducts) will be smaller under chronic
exposure.
Figure 8. Kinetics of albumin adducts of a human subject eating 100 g
of watercress and 60 g of garden cress on day 0.
the quantitation limit of the assay. SFN-Lys was present at all
three time points since the subject was consuming broccoli
regularly. The highest levels were found 1 day after the
consumption of a larger amount of broccoli (300 g). Adducts
with lysine were found in human Hb. The adduct with the
N-terminal valine, BITC-Val, was not found. Probably BITC-
Val was not released with Pronase E. The levels of BITC-Lys
and PEITC-Lys were approximately 50 times lower in Hb than
in albumin (Table 1). Therefore, albumin adducts are a more
sensitive marker to monitor ITC exposure.
Plotting the adduct levels of PITC-Lys and or BITC-Lys
against the days postexposure on a half-logarithmic scale (Figure
8) yielded a half-life of the albumin adducts of 21.3 and 23.2
days, which was close to the values of the half-life of albumin,
20-25 days (32).
Conclusions
ITCs (PEITC, BITC, SFN, and AITC) react with lysine
present in proteins such as albumin. Using LC-MS/MS, ITC-
Lys can be determined as low as 18 fmol/mg albumin. Because
cell protein adducts are involved in the chemopreventive effects
of ITCs, blood protein adducts are probably not only a bio-
marker of exposure but also a potential surrogate marker for
the effects of ITCs at the cellular level. This new method will
enable one to quantify ITC adducts in blood proteins from large
prospective studies about diet and cancer. This new technique
will improve the assessment of ITC exposure and of the power
of studies on the relationship between ITC intake and cancer.
The adduct levels in albumin-depleted plasma were lower
than in albumin. In the samples 1 day after watercress and
garden cress, the adduct levels for albumin-depleted plasma
proteins were 1.17 pmol/mg PEITC-Lys and 0.60 pmol/mg
BITC-Lys. This is a factor two lower than the binding levels in
albumin (Table 1).
Discussion
This is the first report of albumin and Hb adducts of ITCs
present in vivo. Albumin adducts were found in mice after
chronic exposure to PEITC-AcCys, and albumin adducts were
found in a human subject after eating broccoli, garden cress,
and watercress. Biomarkers of ITC exposure have been
Acknowledgment. This research was supported by the Tulane
Cancer Center. The NMR spectra were run by Dr. Qi Zhao at

New Biomarkers for Monitoring ITC LeVels in Humans
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 765
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