Identification and Quantification of Protein Adducts Formed by

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Identification and Quantification of Protein Adducts Formed by Metabolites of 1‑Methoxy-3-indolylmethyl Glucosinolate in Vitro and in Mouse Models Gitte Barknowitz,† Wolfram Engst,† Stephan Schmidt,† Mareike Bernau,† Bernhard H. Monien,† Markus Kramer,‡ Simone Florian,† and Hansruedi Glatt*,† †

German Institute of Human Nutrition (DIfE), Potsdam-Rehbrücke, Department of Nutritional Toxicology, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany ‡ Institute of Organic Chemistry, University of Tübingen, 72076 Tübingen, Germany S Supporting Information *

ABSTRACT: 1-Methoxy-3-indolylmethyl (1-MIM) glucosinolate (GLS) occurring in cabbage, broccoli, and other cruciferous plants is a potent mutagen requiring metabolic activation by myrosinase present in plant cells and intestinal bacteria. We previously reported that 1-MIM-GLS and its alcoholic breakdown product 1MIM-OH, which requires additional activation by sulfotransferases, form DNA adducts in mice. In the present study, the formation of protein adducts was investigated. First, two major adducts obtained after incubation of individual amino acids, serum albumin, or hemoglobin with 1-MIM-GLS in the presence of myrosinase were identified as τN-(1-MIM)-His and πN-(1-MIM)His using MS and NMR spectroscopy. After the development of a specific detection method using isotope-dilution UPLC-ESI-MS/ MS, adduct formation was confirmed in mice after oral treatment with 1-MIM-GLS. Adduct levels were highest in the cecum and colon, somewhat lower in serum albumin and the liver, and also readily detectable in the lung and hemoglobin. On the contrary, oral treatment with 1-MIM-OH produced the highest adduct levels in the liver. The higher ratio of albumin to hemoglobin adducts in 1-MIM-OH- compared to 1-MIM-GLS-treated animals (8.1 versus 3.5) suggests that in 1-MIM-OH-treated animals albumin adducts were produced mostly in the liver, the site of albumin synthesis. The formation of adducts was approximately linear over a range of single oral doses from 20 to 600 μmol/kg body mass. Repeated oral administration of 1-MIM-OH (up to 40 treatments, thrice per week) led to continuous accumulation of hemoglobin adducts, whereas the level of serum albumin adducts remained rather constant, which reflects the different turnover rates of these proteins (t1/2 nearly 1.9 d for serum albumin and 25 d for hemoglobin in the mouse). Accumulation of adducts was also noticed in the lung. Adduct levels were higher, but their accumulation was weaker in the liver and kidney. The method developed will be useful to assess the exposure of humans to reactive metabolites formed from 1-MIM-GLS present in many foods.

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INTRODUCTION Glucosinolates (GLS) are defense chemicals of cruciferous plants against microbial infections and herbivorous animals.1,2 Physical damage of plant tissue leads to the release of myrosinase from separate compartments. This β-thioglucosidase activates the GLS to chemically reactive intermediates, in particular isothiocyanates, and other toxic products to fight the enemy. As an alternative, this activation may be mediated by bacterial β-thioglucosidases, e.g., in the gut. Some GLS and their breakdown products have shown many different effects on mammalian organisms.2−7 They are major components of the smell and taste of Brassica vegetables such as broccoli, cabbage, mustard, cress, rocket salad, and radish.8,9 (2R)-2-Hydroxy-3butenyl-GLS (progoitrin) is goitrogenic at least in cattle when taken up in large quantities.10,11 Sulforaphane, an aliphatic isothiocyanate formed from 4-methylsulfinylbutyl-GLS (glucor© 2013 American Chemical Society

aphanin), activates the Nrf2 transcription factor, leading to the induction of various conjugating and antioxidative enzymes.4,12,13 Activation of Nrf2 has also been observed with allyl-, butyl-, and phenethyl isothiocyanate, breakdown products of other GLS.14 Indole-3-carbinol, formed from 3-indolylmethyl-GLS (glucobrassicin), and in particular its dimerization products, generated under the acidic conditions of the stomach, are activators of the arylhydrocarbon receptor.15 This activation leads to the induction of cytochromes P450 of the CYP1 family as well as various conjugating enzymes. Thus, activation of Nrf2 as well as the arylhydrocarbon receptor by breakdown products of some GLS can lead to altered biotransformation pathways for chemical carcinogens and other xenobiotics, often resulting Received: July 29, 2013 Published: December 31, 2013 188

dx.doi.org/10.1021/tx400277w | Chem. Res. Toxicol. 2014, 27, 188−199

Chemical Research in Toxicology

Article

Scheme 1. Structures of 1-MIM-GLS, Some Metabolites, and Potential Reaction Products with Cellular Nucleophiles (Nu)

in enhanced detoxification and chemoprotection.6 Allyl isothiocyanate, a breakdown product of allyl-GLS (sinigrin), was carcinogenic in rats, but not in mice.16,17 Yet, more than 120 different GLS have been identified, and it is evident that they strongly differ in their biological activities.1,18 Various GLS showed mutagenic or other genotoxic activities.7,19−22 In general, the observed activities were moderate. However, we recently detected that 1-methoxy-3-indolylmethyl glucosinolate (1-MIM-GLS, neoglucobrassicin; the structural formula is in Scheme 1) is a potent inducer of gene mutations in bacterial and mammalian cells in culture.22,23 This mutagenicity was associated with the formation of DNA adducts in the target cells of the mutagenicity tests.22,23 The same adducts were also found in tissues of animals exposed to 1-MIM-GLS or its breakdown product, 1-MIM alcohol (1-methoxyindole-3carbinol, 1-MIM-OH).24,25 However, the tissue distribution of DNA adducts differed between 1-MIM-GLS and 1-MIM-OH because these compounds require different enzymes for metabolic activation, i.e., myrosinase for 1-MIM-GLS and sulfotransferase for 1-MIM-OH, respectively.23 1-MIM-GLS primarily formed DNA adducts in the mucosa of the large bowel. 1-MIM-OH also produced DNA adducts in these tissues, but it formed even higher adduct levels in the liver, a tissue rich in SULT enzymes.26 Recently, Kumar and Sabbioni27,28 found that various GLS form serum albumin and hemoglobin adducts in experimental models and in humans. However, they did not study indole GLS, such as 1-MIM-GLS. In the present study, 1-MIM-GLS and 1-MIM-OH were shown to form protein adducts. Therefore, an isotope-dilution LC-MS/MS method was

developed and validated for the specific detection of amino acid adducts following digestion of the target proteins by Pronase E. The generation of protein adducts was examined in serum albumin and hemoglobin as well as in tissues in which DNA adducts from 1-MIM-GLS and 1-MIM-OH have been detected previously.

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EXPERIMENTAL PROCEDURES

Materials. 1-MIM-GLS was isolated from broccoli by extraction with boiling methanol followed by preparative HPLC as described previously.22 1-MIM-OH was synthesized from 1-hydroxyindole via 1methoxyindole and 1-methoxy-3-indolcarbaldehyde as reported elsewhere.23 Unlabeled L-histidine (His), other L-amino acids, [15N3]L-His, bovine serum albumin (BSA), bovine hemoglobin, glyceryl trioctanoate, Pronase E from Streptomyces griseus, myrosinase from Sinapis alba seeds, Drabkin’s reagent, Brij L23 solution (30%, w/v), and hydrazine monohydrate (64−65%) were purchased from SigmaAldrich (Steinheim, Germany). αN-acetyl-L-histidine (AcHis) was obtained from TCI Europe (Zwinjndrecht, Belgium). Human serum albumin (HSA) was provided by Fluka (Buchs, Switzerland). Chromabond C18ec columns (3 mL) were obtained from Macherey-Nagel (Dueren, Germany). Amicon Ultra Centrifugal Filters (30,000 molecular weight cutoff) were purchased from Millipore (Schwalbach/Ts, Germany). Instrumentation. Three LC systems were used. All components were obtained from Waters (Eschborn, Germany). System 1 was an Alliance 2695 HPLC System equipped with a Kromasil 100-5 C18 column (250 × 4.6 mm) interfaced with a 996 photodiode array (PDA) detector. System 2 was an Acquity ultraperformance liquid chromatography (UPLC) system equipped with an Acquity BEH Phenyl column (1.7 μm, 100 × 2.1 mm) and interfaced with a PDA detector and a Quattro Premier XE tandem quadrupole mass 189



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DMSO-d6). The spectra are presented in Figures S1 and S2 in the Supporting Information. H-3 was formed and isolated in smaller quantities, insufficient for NMR analysis. In a second approach, we reacted AcHis with activated 1-MIM-GLS, as described above for His. Two isomers of AcHis adducts were detected. They were designated as AcH-2 and AcH-3 since we later found that they represent the αN-acetylated derivatives of H-2 and H-3, respectively. They were produced in markedly larger quantities than H-2 and H-3 from His. Moreover, they differed strongly in their retention times compared to those of their nonacetylated congeners, which facilitated their chromatographic separation (performed as described above for H-2 and H-3). The 1 H NMR and H,H-COSY spectra of these compounds are presented in Figures S3−S6 in the Supporting Information. For their deacetylation, the separated AcH-2 and AcH-3 (ca. 1 mg of each isomer) were covered with 500 μL of 65% hydrazine monohydrate in a 1.5-mL reaction tube and kept at 100 °C overnight. After removing volatile components under reduced pressure, a pale brown residue was obtained, which was analyzed for the presence of H-2 and H-3 using LC system 1. Deacetylation of AcH-2 provided H-2 but no H-3, whereas deacetylation of AcH-3 provided H-3 but no H-2. This finding confirms that H-2 is τN-(1-MIM)-His, and implies that H-3 is πN-(1-MIM)-His. τN- and πN-(1-MIM)-[15N3]His were synthesized and separated as the unlabeled adducts, but using [15N3]L-His rather than unlabeled His as starting material. Since the quantities synthesized were not sufficient for weighing, they were quantified by LC (system 2) using defined levels of the unlabeled adducts for the calibration. Animal Experiments. All animal experiments were performed with permission (LUGV 23-2347-7-2010) of the Landesamt für Umwelt, Gesundheit, and Verbraucherschutz of the State of Brandenburg. Two sets of experiments were conducted. The first set of experiments involved single oral treatments of mice with 1-MIM-GLS or 1-MIM-OH. Eight-week old male FVB/N mice (Harlan, Rossdorf, Germany) received a GLS-free semisynthetic diet (C1000, Altromin, Lage, Germany) for one week before treatment. They were treated by gavage with 1-MIM-GLS (0, 20, 60, 200, and 600 μmol/kg body mass) using water as vehicle or equimolar doses of 1-MIM-OH in glyceryl trioctanoate (1.7 mL/kg body mass). Samples were collected 8 h after treatment (dose dependence experiment) or after 3, 8, 24, and 48 h (time dependence experiment, conducted with the dose of 600 μmol/kg). Animals were anesthetized with isoflurane, and blood was taken by puncture of the retrobulbar venous plexus into heparinized tubes. Then, the animals were killed, and the organs were immediately frozen in liquid nitrogen. In the second set of experiments, FVB/N-SULT1A1/2 mice received varying numbers (1−40) of oral treatments with 1-MIM-OH (150 μmol/kg body mass per treatment, 3 times per week, starting at the age of 5 weeks). These mice are transgenic for human SULT 1A1 and 1A2,29 enzymes that have demonstrated high efficiency in the activation 1-MIM-OH.23 This study was primarily designed to explore the subchronic toxicity of 1MIM-OH using various histological and biochemical end points (to be published separately). Blood was taken by cardiac puncture 24 h after last application and stored on ice in heparinized tubes. It was centrifuged (1500g, 10 min, 4 °C) to separate erythrocytes from plasma. Plasma samples were combined with one volume of saturated ammonium sulfate solution and centrifuged (10,000g, 10 min, 4 °C) to remove globulins. The supernatant was desalted by dilution with 15 mL of potassium phosphate buffer (50 mM, pH 7.4) and centrifugation (4000g, 20 min, 4 °C) in Amicon centrifugal filter tubes (30 kDa mass cutoff). The erythrocytes were washed three times by the addition of 2 volumes of saline, followed by centrifugation (1500g, 1 min, 4 °C) and removal of the supernatant. Following lysis by the addition of one volume of water, the samples were stored at −20 °C until further processing. The lysate was centrifuged (15,000g, 10 min, 4 °C) to remove cell debris. Hemoglobin was isolated with minor modification of published procedures.30,31 For determination of the hemoglobin content, a 20-μL aliquot was combined with 5 mL of Drabkin’s reagent

spectrometer. All analyses were conducted in the positive electrospray ionization (ESI+) mode. System 2 was used for the analyses of adducts formed in vitro. System 3 was equal to system 2, but the mass spectrometer was a Xevo TQ triple quadrupole. It was used for the analyses of adducts formed in the mouse models. The data were analyzed with MassLynx V4.1 software. All NMR measurements were performed on a Bruker AMX 600 MHz spectrometer (Ramrod Scientific, Gardner, Massachusetts, USA) equipped with a 3-mm dual probe head and operating at 600.13 MHz for 1H. Reaction of Amino Acids, HSA, and Hemoglobin with 1MIM-GLS and Analysis of the Products Formed. Individual Lamino acids (1 μmol) were incubated with 1-MIM-GLS (0.2 μmol) and myrosinase (1 mU) in 150 μL of sodium phosphate buffer (50 mM, pH 7.4) at 37 °C overnight. The samples were then centrifuged at 10,000g. The resulting supernatant was diluted 10-fold with water and directly injected into the UPLC. HSA (200 μg, 3 nmol) was incubated with 1-MIM-GLS (2.7 μmol) and myrosinase (6 mU) in 1 mL of sodium phosphate buffer (50 mM, pH 7.4) at 37 °C overnight. Human hemoglobin (200 μg, 3.1 nmol of the tetramer) was treated equally. The proteins were then digested with Pronase E, as described in a later section, but without the addition of internal standards. The resulting samples were sonicated for 10 s and centrifuged at 10,000g for 10 min. The supernatant was diluted 10-fold with water prior to analysis. Incubations of amino acids or HSA with myrosinase, but without 1-MIM-GLS, were used as negative controls. The analyses were conducted on LC system 2 in the single ion recording (SIR) and multiple reaction monitoring (MRM) modes. The chromatographic conditions were the same as those described in the last paragraph of the Experimental Procedures for the His adducts. The SIR analysis was conducted under the following settings: 1 kV capillary voltage, 10 V cone voltage, 400 °C desolvation temperature, and 850 L/h desolvation gas flow. The settings for the MRM analyses in system 2 were 0.65 kV capillary voltage, 20 V cone voltage, 450 °C desolvation temperature, 950 L/h desolvation gas flow, 0.11 mL/h collision gas flow (pressure 6.4 × 10−3 mbar), and 10 eV collision energy. System 3 involved a collision gas flow of 0.23 mL/h (pressure 3.3 × 10−3 mbar). All other parameters were equal to those in system 2. Synthesis, Purification, and Characterization of 1-MIM-His and 1-MIM-[15N3]His Adducts. L-Histidine (8 mg, 52 μmol) was incubated with 1-MIM-GLS (3 mg, 6.2 μmol) and myrosinase (33.5 mU) in 5 mL of potassium phosphate buffer (100 mM, pH 7.4) at 37 °C overnight. The product was desalted with a Chromabond C18ec column, evaporated to dryness, and dissolved in acetonitrile. After centrifugation (10,000g, 10 min), aliquots (20 μL) of the sample were injected for purification into LC system 1. A linear gradient of solvent A (water containing 0.9% formic acid) and solvent B (acetonitrile containing 0.9% formic acid) was used for the elution at a flow rate of 1 mL/min and a column temperature of 37 °C. The initial solvent composition (from 0 to 2 min) was 2% solvent B, followed by a linear gradient to 25% solvent B within 33 min resulting in the elution of the adducts. The column was washed with 90% solvent B for 4 min before re-equilibration. UV absorption was recorded at λ = 290 nm. Peaks were collected manually and analyzed using LC system 2 in the ESI+ full scan mode for m/z = 250−350. Three isomers of the His adducts (designated according to their retention times H-1, H-2, and H-3) were identified. H-1, probably αN-(1-MIM)-His, was of low interest, as it was only formed at appreciable levels from free His but not in HSA and hemoglobin. The collected peaks were applied on a Chromabond C18ec column, which had been preconditioned with methanol and equilibrated with water, in order to remove the formic acid, as the methoxy group of 1-MIM is acid-sensitive. After washing with 3 mL of water, the adduct was eluted with 3 mL of methanol. The samples were centrifuged at 10,000g for 1 min, and the resulting supernatants were freeze-dried and weighed. The purity of H-2 and H3 was ≥99%, as studied by HPLC analysis with UV detection. H-2 [τN-(1-MIM)-His] could be isolated in a quantity sufficient for 1H NMR and H,H-correlated spectroscopy (COSY) analysis (600 MHz, 190



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Figure 1. Mass spectrometric search for 1-MIM amino acid adducts in incubations of 1-MIM-GLS and individual amino acids (A1-F1), HSA (A2F2), and hemoglobin (A3-F3) in the presence of myrosinase. HSA and Hb were digested after the treatment. The reaction products were analyzed by UPLC-MS in MRM mode using the m/z transition of the protonated substitution product of the corresponding amino acid to 160 (1-MIM cation). The shaded peaks were absent in corresponding incubations without 1-MIM-GLS. Numbers for signal intensities refer to the largest peak.. the analytes m/z 315.0 (protonated molecule) → 160.0 and m/z 315.0 → 145.0. The signal intensity of the fragment m/z 160.0 (1-MIM residue) was used for quantification. The signal intensity of the fragment m/z 145.0 involving an additional scission of a methyl group from the 1-MIM-residue was recorded to confirm the identity of the substances. The settings for the MRM analyses were 0.5 kV capillary voltage, 20 V cone voltage, 450 °C desolvation temperature, 950 L/h desolvation gas flow, 0.23 mL/h collision gas flow, collision energy 13 eV (quantifier), and 29 eV (qualifier). The optimal cone voltage and collision energy for each MRM transition were determined using the IntelliStart tool of the MassLynx 4.1 software (Waters).

and incubated at room temperature for 15 min. Absorption was measured at 540 nm on a Beckmann DU 7400 spectrophotometer from Beckmann Coulter (Krefeld, Germany). Solutions of bovine hemoglobin (60−180 mg per mL Drabkin’s solution) served as standards for the quantification. Deep-frozen (−80 °C) organs were ground in a Tissuelyser ball mill (Qiagen, Hilden, Germany) under cooling with liquid nitrogen. Aliquots (5 mg) of the ground powder were taken up in 1 mL of potassium phosphate buffer (50 mM, pH 7.4) and further homogenized with an Ultra Turrax T 25 (Rose Scientific Inc., Cincinnati, Ohio, USA). Total tissue homogenate was used for the analysis of protein adducts. The protein contents were quantified with the BCA Protein Assay Kit (Thermo Fisher Scientific, Bonn, Germany). Digestion of Proteins and Quantification of the Released Analytes [τN-(1-MIM)-His and πN-(1-MIM)-His]. The target protein (1 mg), spiked with the isotope-labeled internal standards (4 and 21 pmol of τN- and πN-(1-MIM)-[15N3]His), was digested with Pronase E (0.33 mg) in 1 mL of potassium phosphate buffer (50 mM, pH 7.4) at 37 °C for 18 h. The samples were applied on Chromabond C18ec columns activated with 3 mL of methanol and preconditioned with 3 mL of water. After washing with 3 mL of water, the analytes were eluted with 5 mL of methanol/water (4:1, v/v). After concentration under reduced pressure, the samples were taken up in 50 μL of methanol/water (1:1, v/v) containing 0.1% formic acid. Aliquots of 7.5 μL were subjected to UPLC-ESI(+)-MS/MS analysis on LC systems 2 or 3. Acidified (0.25% acetic acid and 0.25% formic acid) water (solvent C) and acidified acetonitrile (solvent D) were used as eluents at a flow rate of 0.35 mL/min. The column temperature was 37 °C. The initial solvent composition (from 0 to 1 min) was 2% solvent D, followed by a linear gradient to 15% solvent D within 5 min resulting in the elution of the analytes. The total running time per analysis was 11 min. The following MS/MS mass transitions were recorded (for the fragmentation pattern, see Figure 2): for the internal standards, τN- and πN-(1-MIM)-[15N3]His, m/z 318.0 (protonated molecule) → 160.0 and m/z 318.0 → 145.0, and for

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RESULTS AND DISCUSSION Reactions of Activated 1-MIM-GLS with Free Amino Acids, HSA, and Human Hemoglobin. We previously found that incubation of 1-MIM-GLS with 2′-deoxyguanosine, 2′deoxyadenosine, or DNA in the presence of myrosinase leads to the adduction of the 1-MIM moiety to the exocyclic amino group of the purine bases.24 We investigated now whether analogous substitution reactions also occur in proteins. Six amino acids that contain nucleophilic moieties in their side chain (Cys, His, Glu, Lys, Ser, and Trp) were individually incubated with 1-MIM-GLS and myrosinase. Substitution reactions with 1-MIM (Scheme 1) would increase the mass of the amino acid by 160 units. However, it is likely that the primary reactive intermediate of 1-MIM-GLS is an isothiocyanate, and it is known that some isothiocyanates derived from other GLS, in particular aliphatic isothiocyanates, undergo addition, rather than substitution, reactions with amino acids and proteins.28 Addition reactions of amino acids with 1-MIM isothiocyanate (Scheme 1) would increase the mass by 219 units. Therefore, we analyzed the samples for both types of 191



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Figure 2. Daughter ion spectra of products H-2 [τN-(1-MIM)-His] and H-3 [πN-(1-MIM)-His]. The m/z ratio of both parent ions was 315. The spectra were recorded using collision energies of 8 eV (upper panels) and 30 eV (lower panels). The assignment to structures was verified by 1H NMR analysis. Possible fragmentations explaining the daughter ions are indicated.

hemoglobin (α2β2) contains 6 Cys, 38 His, and 6 Trp residues (578 amino acids). Furthermore, we detected two putative His adducts that were well separated and produced strong signals under the experimental conditions used (H-2 and H-3) and two minor products in HSA as well as hemoglobin (Figure 1, H-1 and H-4 in panels D2 and D3). Since the presence of two well detectable adducts may be useful for the corroboration of findings in epidemiological studies, we focused the subsequent investigations on these His adducts. Characterization of the 1-MIM-His Adducts Formed in Proteins. Incubation of free His with activated 1-MIM-GLS resulted in the formation of three products with mass spectrometric properties of substitutional adducts. They were termed H-1, H-2, and H-3 in the sequence of their elution in the UPLC (Figure 1, panel D1). Only two of these products (H-2 and H-3) were observed at appreciable levels in incubations with HSA (Figure 1, panel D2). We suspect that the other product (H-1) formed from free His involved binding at the α-amino group. Daughter ion spectra of H-2 and H-3 (the products of interest, as occurring in HSA) were similar (Figure 2). Larger quantities were required for the elucidation of their exact structure by NMR spectroscopy. Baseline separation of H-1, H-2, and H-3, as achieved by UPLC, was not successful with preparative columns. However, using multiple runs on the analytical column, sufficient quantities of H-2 could be collected for 1H NMR and H,H-COSY analyses. The spectra (Figures S1 and S2 of Supporting Information) indicate that the 1-MIM group is attached to an imidazo nitrogen. However, it could not be decided whether it was the proximal (π) or remote (tele, τ) ring-nitrogen of His. Moreover, H-3, which probably represents the other positional isomer, was not available in sufficient quantities for NMR analyses. As an alternative, we reacted AcHis with activated 1MIM-GLS. Because of the protection of the α-amino group, only two products (termed AcH-2 and AcH-3) were formed.

potential products by LC-MS, using incubations without 1MIM-GLS and incubations without amino acid as negative controls. In the first round, we screened by SIR in the positive ionization mode. With all six amino acids, products with the mass of substitutional adducts were detected; in contrast, we did not find any appreciable signals matching the mass of addition products (data not shown). This finding agrees with the results of our previous studies using 2′-deoxynucleosides as reactants.24 The daughter ions spectra of all 1-MIM 2′deoxynucleoside adducts contained a characteristic signal at m/ z = 160, indicating fragmentation to the 1-MIM cation.24 On this basis, we repeated the analysis of the products formed from the amino acids using the more sensitive and more specific MRM mode (parent → 160). Again, appropriate signals that might reflect substitutional adducts were observed with all six amino acids studied (Figure 1, panels A1−F1). Of course, some adducts may involve reaction with the α-amino group. Such adducts are of minor interest, as they could be formed only with N-terminal amino acids in proteins. Subsequently, we incubated HSA and human hemoglobin, rather than the individual amino acids, with 1-MIM-GLS and myrosinase. Analyses of the resulting digests by LC-MS in the MRM mode yielded several signals that matched products formed by bioactivated 1-MIM-GLS with free amino acids (Figure 1, panels A2−F2 and A3−F3 versus A1−F1). The strongest signals in the HSA digest were observed for putative adducts with Cys, Trp, and His (Figure 1, panels A2−F2). These amino acids were also the major binding sites in hemoglobin (Figure 1, panels A3−F3), but the order of the signal intensities differed. In particular, the Cys adduct generated the strongest signal with HSA, but its signals were weaker in hemoglobin than those of the Trp and His adducts. The differences were not surprising since the relative abundance of these amino acids is very different in the proteins studied. HSA comprises 35 Cys, 16 His, and 2 Trp residues out of a total of 609. Human 192



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Figure 3. Conformers of τN-(1-MIM)-His (A) and πN-(1-MIM)-His (B) possessing the lowest energy and aromatic part of the NOE spectra of AcH-2 (C) and AcH-3 (D). The presence of a NOE H15-H12 resonance signal in AcH-2 and its absence in AcH-3 indicate that AcH-2 is τN-(1MIM)-His.

Their 1H NMR and H,H-COSY spectra (Figures S3−S6 and S2 of Supporting Information) showed the binding of the 1-MIM group to ring nitrogens of His. However, it was not possible to assign the isolated compounds to both possible positional isomers. In addition, nuclear Overhauser effect (NOE) spectroscopic data were available from the NMR analysis. Therefore, we executed a grid search for πN- and τN-(1-MIM)AcHis and compared the theoretical NOEs for the conformers possessing the lowest calculated energy with the NOEs observed with AcH-2 and AcH-3. The grid search indicated that H12 and H15 should be close (4 Å) in πN-(1-MIM)AcHis (Figure 3B). A corresponding NOE signal was observed in AcH-2 (Figure 3C) but not AcH-3 (Figure 3D). Therefore, we assigned AcH-2 to τN-(1-MIM)-AcHis and AcH-3 to πN(1-MIM)-AcHis. Removal of the protection (acetyl) group in AcH-2 and AcH3 posed some problems due to the instability of the 1-methoxy group in the indole ring, in particular its acid sensitivity. Eventually, we succeeded in removing the acetyl group with hydrazine. AcH-2 yielded H-2 but no H-3, whereas AcH-3 yielded H-3 but no H-2 (data not shown). This finding implies that H-2 is τN-(1-MIM)-His and H-3 is πN-(1-MIM)-His.

The imidazo nitrogens are common sites of alkylation or arylation of proteins and other suitably protected His and histamines. The reaction results almost always in a mixture of τN- and πN-adducts, often with a certain preference of the former.32,33 Recovery, Interday and Intraday Variation, Limit of Detection, and Linear Detection Range. The analytical method was validated using BSA reacted with 1-MIM-GLS and myrosinase or, for recovery and determination of linear range, untreated BSA spiked with unlabeled adduct standards (Table 1). When BSA was incubated with bioactivated 1-MIM-GLS, τN- and πN-(1-MIM)-His were formed at similar levels. However, recovery (80%), intraday variation (1.3%), and interday variation (6.8%) were somewhat better for τN-(1MIM)-His. Moreover, background signals were markedly stronger at the retention time of πN-(1-MIM)-His, implying higher values for the limits of detection (LOD) and limits of quantification (LOQ) for πN-(1-MIM)-His than for τN-(1MIM)-His. For these various reasons, the level of τN-(1-MIM)His adducts formed in BSA was nearly 60-fold above the LOQ, whereas the corresponding margin was only 10 for πN-(1MIM)-His. 193



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this article, but all corresponding data for πN-(1-MIM)-His is available in the Supporting Information. Furthermore, we analyzed serum albumin of a 1-MIM-OHtreated mouse for the presence of adducts with other amino acids (Figure 4), using the same methods as those for the

Table 1. Validation Parameters for the Analysis of (1-MIM)His Adducts in BSA parameter recovery [%]a intraday variation [%]b interday variation [%]b LOD on column [fmol]c LOD [fmol/mg BSA]c LOQ [fmol/mg BSA]c level of adducts formed in vitro [fmol/mg BSA]b linearity in range 1−10000 × LOQ, r2d

τN-(1-MIM)His

πN-(1-MIM)His

79.8 ± 1.1 1.3 ± 0.2 6.8 ± 1.4 3 32 67 4060 ± 100

68.1 ± 4.1 2.7 ± 0.6 25.7 ± 2.6 10 120 280 3830 ± 320

0.9996

0.9995

Untreated BSA (1 mg per sample) was spiked with τN- and πN-(1MIM)-His (4 and 21 pmol, respectively) before digestion. Means ± SE of 10 separate samples. bBSA (40 mg) was treated with 1-MIMGLS (400 fmol) and myrosinase (0.2 mU) in a total volume of 40 mL of potassium phosphate buffer (50 mM, pH 7.4) at 37 °C overnight and then stored at −80 °C until analysis. For the analysis, aliquots (1 mL) were spiked with τN- and πN-1-MIM)-[15N3]His (4 and 21 pmol, respectively) and digested. Values are the means ± SE for 10 aliquots analyzed on a single day (intraday variation) or on separate occasions over a period of 151 days (interday variation and adduct levels). The individual values are given in Tables S1 and S2 in the Supporting Information. cLOD and LOQ were defined as (xB + 3 σB) and (xB + 9 σB), respectively. xB is the mean value, and σB is the standard deviation of background signals of the quantifier transition for 10 samples of unexposed BSA (1 mg per sample). dSamples of untreated BSA were spiked with fixed levels of τN-(1-MIM)-[15N3]His (4 pmol) and πN(1-MIM)-[15N3]His (21 pmol) and varying levels of the unlabeled analytes (geometric row from 1 to 10000 × LOQ). The decadic logarithms of the peak areas of the adducts were plotted against the decadic logarithms of the concentrations of the analytes (Supporting Information, Figure S7). These plots were analyzed by linear regression analysis. a

In order to determine the size of the MS/MS signal of the quantifier as a function of the analyte level, unexposed BSA was spiked with fixed amounts of isotope-labeled internal standards as well as varying amounts of the analytes (1, 3, 10, 30, 100, 300, 1000, 3000, and 10000 × LOQ). The decadic logarithm of the peak areas of the analytes, standardized for the responsiveness of the internal standard, was plotted against the decadic logarithm of the concentration of the analytes (Figure S7, Supporting Information). The linear correlation coefficients of these curves were excellent (r2 > 0.9995). However, the slopes of the regression lines were slightly below 1. They amounted to 0.97 for τN-(1-MIM)-His and 0.96 for πN-(1-MIM)-His implying that the corrected peak areas per concentration unit were decreased by 21 and 34%, respectively, at the highest concentration (10000 × LOQ) compared to the lowest concentration (1 × LOQ) of the analyte used. We recommend the adjustment of the level of the internal standards or mathematical correction if samples with extreme adduct levels are to be investigated. Relative Levels of τN- and πN-(1-MIM)-His and 1-MIMAmino Acid Adducts Formed in Mouse Proteins in Vivo. As reported in the preceding section, τN- and πN-(1-MIM)-His adducts were formed at similar levels in BSA in vitro, but τN-(1MIM)-His was detected with higher sensitivity due to its lower LOQ. This was also true for the adduct formation and detection in blood and tissue proteins in vivo. For these reasons, we only show the results for τN-(1-MIM)-His in the figures of

Figure 4. Mass spectrometric search for 1-MIM amino acid adducts in serum albumin of a mouse treated with 1-MIM-OH. A male FVB/N mouse received a single oral treatment with 1-MIM-OH (600 μmol/ kg) and was killed 8 h later. Serum albumin was isolated, digested, and purified with solid phase extraction. The purified digests were analyzed by UPLC-MS in the MRM mode using the m/z transition of the protonated substitution product of the corresponding amino acid to 160 (1-MIM cation). The shaded peaks were absent in negative control animals that only received the vehicle. Numbers for signal intensities refer to the largest peak.

adducts formed in cell-free systems (Figure 1). The strongest signals were observed for τN-(1-MIM)-His in serum albumin adducted in vivo, confirming that the selection of this adduct for further analyses was favorable. Levels of 1-MIM-His Adducts in Serum Albumin of Mice Treated with Varying Doses of 1-MIM-GLS or 1MIM-OH. We previously showed that two metabolites of 1MIM-GLS can form DNA adducts.23,24,26 Generation of the first active metabolite, probably 1-MIM isothiocyanate, only requires the presence of myrosinase (Scheme 1). This intermediate is unstable and is rapidly hydrolyzed to 1-MIMOH,34,35 which can be reactivated by the action of mammalian SULT,23 as indicated in Scheme 1. In the first animal 194



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1-MIM-OH. In the main experiment, mice were sacrificed 8 h after a single oral treatment with 1-MIM-OH or 1-MIM-GLS (600 μmol per kg body mass). The local distribution of the adducts formed varied substantially between both treatments (Table 2). Treatment with 1-MIM-GLS led to the highest adduct levels in the colon and cecum, probably due to the activation of 1-MIM-GLS by bacterial myrosinases. Only marginally lower adduct levels were detected in the liver and serum albumin, which may be reached rapidly by the reactive metabolite formed and absorbed in the gut. After treatment with 1-MIM-OH, exceptionally high adduct levels were detected in the liver, a tissue showing high expression of SULT, enzymes required for activation of 1-MIM-OH. The second highest level of adducts in 1-MIM-OH-treated mice was detected in serum albumin. These adducts could have been formed either in the blood or in the liver, the site of albumin synthesis. The half-life time of serum albumin in mice is 1.9 d,36 implying that nearly 8% of the albumin in the serum was renewed within the treatment period (8 h). Supposing that the adduction level of hepatic albumin is similar to that of other hepatic proteins (11 times higher than the adduct level recorded in blood serum albumin, Table 2), a substantial portion of the adducts in blood serum albumin might have been formed in the liver in 1-MIM-OH-treated mice. The situation was different after treatment with 1-MIM-GLS, with a quotient between liver protein and blood serum albumin adducts of 0.9 (rather than 11 after treatment with 1-MIM-OH) suggesting that the major portion of these adducts was formed in the blood. Serum albumin was not the only blood protein in which we studied the formation of 1-MIM-His adducts. We also analyzed hemoglobin. This protein is formed during the hematopoiesis in the bone marrow. The life span of erythrocytes (and hemoglobin) in mice is 20−30 d.37 Therefore, the adducts found under our experimental conditions had to be formed in the peripheral erythrocytes. Both, 1-MIM-GLS and 1-MIM-OH, treatments led to the formation of 1-MIM-His adducts in hemoglobin, but the levels were much lower than in serum albumin. It is probable that the erythrocyte membrane reduced the access of the reactive metabolites to the hemoglobin. Moreover, the ratio of serum albumin to hemoglobin adducts was larger in 1-MIM-OHcompared to 1-MIM-GLS-treated animals (8.1 versus 3.5), which supports the idea that some serum albumin adducts may have been formed in the liver of the 1-MIM-OH-treated animals. To further explore the relationship between hepatic protein and the serum albumin adduct, we studied the time course of the levels of these adducts in 1-MIM-OH-treated animals. Groups of animals were sacrificed 3, 8, 24, and 48 h after treatment. Maximal adduct levels were observed in both liver proteins and serum albumin after 8 h (1.5−2.8 higher than at any other time point studied, Figure S8 in the Supporting

experiment of the present study, mice received a single oral treatment with varying doses of 1-MIM-GLS or 1-MIM-OH. They were killed 8 h later for the determination of 1-MIM-His adducts in serum albumin. Adducts were detected at the highest three doses of 1-MIM-GLS (60−600 μmol per kg body mass) and at all four doses of 1-MIM-OH (20−600 μmol per kg body mass) (Figure 5). Adduct formation increased nearly linearly

Figure 5. Dose−response relationship of τN-(1-MIM)-His adducts formed in the serum albumin of male FVB/N mice 8 h after single oral doses of 1-MIM-GLS (upper panel) and 1-MIM-OH (lower panel), respectively. Values are the means ± SE of 5 animals. Bars (within a panel) that do not share a letter are statistically significantly different (p < 0.05, one-way ANOVA with Bonferroni’s correction for multiple comparisons). Numbers indicated above the bars represent adduct levels (in fmol per mg serum albumin) divided by the dose used (in μmol per kg body mass). *, adduct levels were below LOD (30 fmol/ mg protein). Similar results were obtained with the isomeric adduct, πN-(1-MIM)-His (Supporting Information, Figure S9).

with the dose (r2 = 0.9989 and 0.9322 for linear correlation with 1-MIM-OH and 1-MIM-GLS, respectively, see also Figure S11 in the Supporting Information), as indicated by rather constant ratios of adduct and dose levels (values above bars in Figure 5). The number of adducts formed per dose unit was 3 times higher for 1-MIM-OH than for 1-MIM-GLS. 1-MIM-His Protein Adducts in Various Tissues and Blood Components in Mice Treated with 1-MIM-GLS or

Table 2. Levels of τN-(1-MIM)-His Protein Adducts in Blood Components and Various Tissues of Mice Treated with 1-MIMGLS or 1-MIM-OHa

adducts in 1-MIM-OH-treated mice (fmol/mg protein) adducts in 1-MIM-GLS-treated mice (fmol/mg protein) ratio of adducts in 1-MIM-OH- and 1-MIM-GLS-treated mice

serum albumin

hemoglobin

cecum

colon

liver

lung

5890 ± 960 1630 ± 470 3.6

723 ± 69 463 ± 112 1.6

2340 ± 210 2260 ± 570 1.0

4590 ± 250 2260 ± 420 2.3

66500 ± 9100 1520 ± 440 44

1340 ± 190 380 ± 97 * 3.5

Male FVB/N mice received a single oral treatment with the compound (600 μmol per kg body mass) and were killed 48 h later. Adduct levels are the means ± SE of 5 (or *4) animals. Similar results were obtained with the isomeric adduct, πN-(1-MIM)-His (Supporting Information, Table S3). a

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albumin in the mouse. On the contrary, adduct levels in hemoglobin continuously increased. After 40 treatments (90 d), they were 7 times higher than that after a single treatment. Thus, the adducts accumulated, in agreement with the longer life span of hemoglobin/erythrocytes (25 d in the mouse) compared to serum albumin. Continuous accumulation of protein adducts was also observed in the lung, whereas adduct levels in the liver and kidney showed negligible variation over large periods of the experiment with some increase toward the end (Figure S12 in the Supporting Information). Adduct levels at the end of this chronic study followed the order liver > kidney > serum albumin > lung > hemoglobin. This data has to be taken with some reservation for various reasons: (i) We only determined adduction to His residues. As illustrated in a preceding section for serum albumin and hemoglobin, the abundance of His and other nucleophilic amino acids as well as the preferred sites of adduction may substantially vary between different proteins. Therefore, the level of the His adducts may not accurately correlate with the total level of adducts in different proteins. (ii) We have not identified the proteins that were modified in the various tissues. It is probable that many different proteins were adducted, but to varying extents. Likewise, the efficiency of the digestion procedure used may vary between different proteins. (iii) Turnover rates may drastically vary between different tissue proteins, for example, due to degradation or export into the blood (as is the case for serum albumin synthesized in hepatocytes). Therefore, the protein adduct level determined after repeated treatments is not suited for comparing the cumulative exposure to the active metabolites between different tissues. General Considerations on the Levels of 1-MIM-His Protein Adducts Formed in Vivo. 1-MIM-OH formed higher adduct levels than 1-MIM-GLS in the mouse models. After a single treatment at a dose of 600 μmol (106 mg) per kg body mass, a total of 1.9, 8.9, and 130 pmol 1-MIM-His adducts (both isomers combined) were detected per mg protein in hemoglobin, serum albumin, and liver tissue, respectively. Since the molecular mass of hemoglobin and serum albumin is known, one can calculate the modification level. It amounted to 1.2 and 5.9 His adducts per 104 molecules of hemoglobin (tetramer) and albumin, respectively, supposing that recovery was complete. Preliminary analysis indicated that release of His from hemoglobin in the digest was in a range of 50−100%, depending on the unidentified contribution of His resulting from autodigestion of the Pronase (Bernau, M., Barknowitz, G., and Glatt, H. R., unpublished results). Even if the estimated values are multiplied by a factor of 5−10 to take into account incomplete recovery of the His adducts as well as adduction with other amino acids, the putative modification levels in hemoglobin and albumin remain low, probably without any appreciable impact on the function of these proteins. Hazard assessment is more difficult for the liver due to higher adduction and the possibility that adduct formation may be concentrated to certain proteins. However, it may be helpful to make a comparison with other compounds that form protein adducts. Thus, it was reported that acetaminophen (400 mg/kg body mass) formed 2.3 and 0.177 nmol adducts per mg protein in liver and hemoglobin, respectively, in phenobarbital-treated mice.38 Waidyanatha et al.39 treated rats with varying doses of naphthalene and determined four different Cys adducts in blood proteins. At a dose of 400 mg/kg body mass, they recorded a total of 360 and 300 nmol adducts per mg protein in

Information). This time course may reflect the turnover of the proteins rather than providing additional information on the site of formation of serum albumin adducts. Levels of 1-MIM-His Adducts in Blood and Tissue and in Mice after Repeated Administration of 1-MIM-OH. We recently investigated the subchronic toxicity of 1-MIM-OH in male FVB/N-hSULT1A1/2 mice (manuscript in preparation). These transgenic mice express human SULT1A1/2, able to activate 1-MIM-OH, in many different tissues like those in humans, whereas expression of the endogenous SULT1A enzyme of mice is largely restricted to the liver and the gut. 1MIM-OH was administered by gavage, 150 μmol/kg body mass and treatment, three times per week, in total up to 40 times. Subgroups of animals were killed after varying numbers of treatments for histological and biochemical analyses. Organ homogenates and blood collected at sacrifice were available for the present study. Figure 6 shows the time courses of the 1-

Figure 6. τN-(1-MIM)-His adducts in serum albumin (upper panel) and hemoglobin (lower panel) of mice subchronically treated with 1MIM-OH. Male FVB/N-hSULT1A1/2 mice were treated with 1MIM-OH by gavage, 150 μmol per kg body mass and treatment, three times per week. Subgroups were sacrificed 24 h after varying numbers of treatments. Values are the means ± SE of at least 3 animals. No adducts were detected in negative control animals treated with the vehicle. Bars (within a panel) that do not share a letter are statistically significantly different (p < 0.05, one-way ANOVA with Bonferroni’s correction for multiple comparisons). Similar results were obtained with the isomeric adduct, πN-(1-MIM)-His (Supporting Information, Figure S4).

MIM-His adducts in serum albumin and hemoglobin. The level of adducts in serum albumin showed a dip after 4 to 10 treatments and reached its maximum after the last treatment, but the deviation from the level observed after the first treatment was not statistically significant at any time point. If real, the observed variation might have resulted from treatmentor age-dependent changes in the biotransformation of 1-MIMOH. The low temporal variation of the adduct level was plausible, as the mean interval between the treatments was 2 1/ 3 d, which is slightly more than the half-life time of serum 196



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subjects with high consumption of green cabbage or Chinese cabbage. Similarly, complex situations were noticed for other cancers.41,42,44,45 Thus, it might be important to focus on individual cruciferous vegetables, which can differ in their GLS profile or, even better, on the intake of individual GLS. As indicated in the Introduction, they differ in their biological activities. It is not likely that the formation of DNA adducts and the induction of mutations, as observed with 1-MIM-GLS, is beneficial to health. This does not rule out that 1-MIM-GLS may promote health via other ways. In any case, blood protein adducts, representing biomarkers for the individual exposure to its active metabolites, should be promising to clarify the situation, ideally in combination with similar adducts formed by other GLS.

serum albumin and hemoglobin, respectively. It is evident that these adduct levels observed at toxic doses of the test compounds are higher by several orders of magnitude than formed in our study by 1-MIM-GLS or 1-MIM-OH, although we used these compounds at high doses, as compared to human dietary exposure. Steinbrecher and Linseisen40 estimated the mean daily intake of individual GLS from 24-h diet recall in nearly 2000 subjects of the EPIC-Heidelberg cohort. The values for 1-MIM-GLS were 0.68 mg for men and 0.60 mg for women. This is approximately 1/30000 of the highest dose of 1-MIM-GLS used. Therefore, it is unlikely that 1-MIM protein adducts formed in humans pose a health hazard as such, but 1-MIM hemoglobin adducts may be used as a biomarker for estimating the systemic exposure to active metabolites of the mutagen, 1-MIM-GLS.

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ASSOCIATED CONTENT

S Supporting Information *

CONCLUSIONS In the present study, we demonstrate that 1-MIM-GLS can form protein adducts after metabolic activation. The same two pathways that lead to the formation of DNA adducts23,24,26 are involved in protein adduction. The first pathway only requires 1-MIM-GLS, myrosinase, and the nucleophile (DNA or protein). It is probable that 1-MIM isothiocyanate is the active intermediate in this pathway (Scheme 1), although this putative metabolite is not yet available as a synthetic standard for verification of the hypothesis. 1-MIM isothiocyanate is rapidly hydrolyzed to 1-MIM-OH,34,35 the substrate for the second activation pathway mediated by SULT. The observations that 1-MIM-OH formed protein adducts in mice and that the adduct levels were particularly high in the liver, a tissue with high SULT expression, demonstrates that this pathway was operative in our in vivo model. The operativity of the other pathway was demonstrated unambiguously in the SULT-free in vitro model used. We showed that activated 1-MIM-GLS reacts with several different sites of proteins. We identified the structure of two adducts, τN-(1-MIM)-His and πN-(1-MIM)-His, and developed methods for their quantification in proteins. The presence of these adducts was demonstrated in various tissues as well as in serum albumin and hemoglobin in mouse models. Pilot studies indicate that 1-MIM His adducts are also detectable in serum albumin and hemoglobin from humans, even without intervention with Brassica-rich diet (Barknowitz, G., Bernau, M., and Glatt, H. R., unpublished findings). Various epidemiological studies found a reduced risk of cancer in subjects with a high consumption of cruciferous vegetables.41−45 Good data sets are available for lung cancer in women, as recently reported by Wu et al.43 In a prospective study on 74,914 Chinese women, they observed a hazard ratio of 0.73 for the highest compared with the lowest quartile regarding the amount of cruciferous vegetables consumed. This result was of borderline statistical significance (95% confidence interval 0.54−1.00, p for trend 0.1607), as was the case in several similar studies. In a meta-analysis, involving nine additional studies, clear statistical significance was reached (hazard ratio 0.75, 95% confidence interval 0.63−0.89). In the study of Wu et al.,43 the inverse association between lung cancer risk and intake of cruciferous vegetables was strengthened if the analysis was restricted to never smokers. The association in this group was also statistically significant when consumption of Chinese greens rather than the total of cruciferous vegetables was considered. In contrast, the authors found no clear indication for a reduced risk of lung cancer in

1 H NMR and H,H-COSY spectra of τN-(1-MIM)-His, τN-(1MIM)-AcHis, and πN-(1-MIM)-AcHis; the individual values of τN- and πN-(1-MIM)-His adducts determined in aliquots of modified BSA determined on a single day (intraday variation) or over a time period of approximately five months (interday variation); data on the linearity of result parameters for BSA spiked with fixed levels of τN- and πN-(1-MIM)-[15N3]His and varying levels of unlabeled τN- and πN-(1-MIM)-His (geometric row from 1 to 10000 × LOQ); the time course of the levels of τN-(1-MIM)-His adducts in serum albumin and liver proteins of mice treated with a single oral dose of 1-MIM-OH; figures and tables equivalent to Figure 5, Figure 6 and Table 2 but showing the levels of πN-(1-MIM)-His rather than those of τN-(1-MIM)-His; a plot of the linear correlation analysis for the levels of τN-(1-MIM)-His adducts formed in serum albumin of mice treated with varying doses of 1-MIM-GLS and 1-MIM-OH; and time course of τN-(1-MIM)-His and πN(1-MIM)-His adducts formed in the liver, kidney, and lung proteins of mice subchronically treated with 1-MIM-OH for varying periods. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49-30-6916846. Fax: +49-33200-882426. E-mail: [email protected]. Funding

This work was financially supported by the German Federal Ministry of Education and Research (grant 0315370D) and German Federal Institute for Risk Assessment (grant FZ 1329437.2). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Brigitte Knuth for excellent technical assistance. ABBREVIATIONS AcHis, αN-acetyl-L-histidine; BSA, bovine serum albumin; COSY, correlated spectroscopy; ESI+, positive electrospray ionization; GLS, glucosinolate(s); HSA, human serum albumin; LOD, limit of detection; LOQ, limit of quantification; 1-MIM, 1-methoxy-3-indolylmethyl; MRM, multiple reaction monitoring; MS, mass spectrometry; NOE, nuclear Overhauser effect; PDA, photodiode array; SIR, single ion recording; SULT, 197



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sulfotransferase(s); UPLC, ultra performance liquid chromatography

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