Discovery of Dopamine Glucuronide in Rat and Mouse Brain

Anal. Chem. 2009, 81, 427–434

Discovery of Dopamine Glucuronide in Rat and Mouse Brain Microdialysis Samples Using Liquid Chromatography Tandem Mass Spectrometry Pa¨ivi Uutela,† Laura Karhu,† Petteri Piepponen,‡ Mikko Ka¨enma¨ki,‡ Raimo A. Ketola,§ and Risto Kostiainen*,† Division of Pharmaceutical Chemistry, Division of Pharmacology and Toxicology, and Centre for Drug Research (CDR), Faculty of Pharmacy, P.O. Box 56, FI-00014 University of Helsinki, Helsinki, Finland A liquid chromatographic-electrospray/tandem mass spectrometric (LC-ESI-MS/MS) method was developed for the analysis of dopamine and its phase I and phase II metabolites from brain microdialysis samples. The method provides for the first time the analysis of intact dopamine glucuronide and sulfate without hydrolysis. The paper describes also an enzymatic synthesis method using rat liver microsomes as biocatalysts and characterization of dopamine glucuronide as a reference compound. The method was validated for quantitative analysis by determining limits of detection and quantitation, linearity, repeatability, and specificity. Dopamine glucuronide was found for the first time in rat and mouse brain microdialysis samples. The concentrations of dopamine and its glucuronide in the microdialysates collected from the striatum of rat brains were approximately equal (2 nM). Dopamine sulfate was not detected in the microdialysates (limit of detection 0.8 nM). The main metabolites of dopamine were dihydroxyphenylacetic acid (DOPAC, 1200 nM) and homovanillic acid (HVA, 700 nM). Dopamine (DA) is an important neurotransmitter in the brain, and altered dopamine function has been linked to, for example, Parkinson’s disease and schizophrenia.1 The action of DA in the brain is terminated by uptake or metabolism. Enzyme-catalyzed metabolism can be divided into phase I and phase II reactions. A small polar functional group is added or exposed in phase I reactions, which are, for example, oxidation, reduction, hydrolysis, etc. A polar, hydrophilic moiety is added to the molecule in phase II metabolism reactions. DA in the brain is metabolized by phase I reactions to dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), the levels of which have been used as indices of the functional activity of dopaminergic neurons. However, in rat and human cerebrospinal fluid (CSF), which is assumed to reflect the metabolism of neurotransmitters in the brain, dopamine * Corresponding author. E-mail: [email protected]. Fax +358 9 191 59556. † Division of Pharmaceutical Chemistry. ‡ Division of Pharmacology and Toxicology. § Centre for Drug Research. (1) Cooper, J. R.; Bloom, F. E.; Roth, R. H. The Biochemical Basis of Neuropharmacology, 8th ed.; Oxford University Press: New York, 2003. 10.1021/ac801846w CCC: $40.75  2009 American Chemical Society Published on Web 11/26/2008

glucuronide (DA-Glu) and sulfate (DA-sulfate) have been found.2,3 For these analyzes, these phase II metabolites of DA were hydrolyzed by β-glucuronidase or arylsulfatase before they were detected as free DA. The concentrations of DA-Glu, DA-sulfate, and free DA in rat CSF were 70, 1, and 2 nM, respectively.2 In other studies, when acid hydrolysis was used, the conjugated form of DA was predominant (77-100%) in human and monkey CSF.4,5 In different regions of rat, monkey, and human brains, acid hydrolyzed conjugates of DA represented 0.5-14% of total DA (conjugated plus free DA).6-8 It has been suggested in the literature that the DA conjugates that were hydrolyzed by acid were sulfates, since it has been reported that 96% of DA-sulfate and only 10% of DA-Glu are hydrolyzed during acid boiling.3 However, the identity of the conjugate is still open to doubt. Buu et al.9 used arylsulfatase instead of acid for the hydrolysis of conjugates in brain samples, and they found that 1-30% of the total DA is in the sulfonated form, depending on the region of the rat brain. Sulfonation and glucuronidation are phase II metabolism reactions. Sulfonation is catalyzed by cytosolic sulfotransferase (ST) enzymes, which are divided, according to their amino acid sequence and enzyme function, into phenol (PST) and hydroxysteroid (HSST) sulfotransferase families.10 Sulfonation of catecholamines is catalyzed by monoamine-preferring PST, which has also been found in the human brain11,12 Glucuronidation is catalyzed by uridine diphosphoglucuronosyltransferases (UGTs), which are membrane-bound enzymes of the endoplastic reticu(2) Wang, P. C.; Kuchel, O.; Buu, N. T.; Genest, J. J. Neurochem. 1983, 40, 1435–1440. (3) Tyce, G. M.; Messick, J. M.; Yaksh, T. L.; Byer, D. E.; Danielson, D. R.; Rorie, D. K. Fed. Proc. 1986, 45, 2247–2253. (4) Tyce, G. M.; Rorie, D. K.; Byer, D. E.; Danielson, D. R. J. Neurochem. 1985, 44, 322–324. (5) Elchisak, M. A.; Powers, K. H.; Ebert, M. H. J. Neurochem. 1982, 39, 726– 728. (6) Karoum, F.; Chuang, L. W.; Wyatt, R. J. J. Neurochem. 1983, 40, 1735– 1741. (7) Elchisak, M. A.; Cosgrove, S. E.; Ebert, M. H.; Burns, R. S. Brain Res. 1983, 279, 171–176. (8) Elchisak, M. A. J. Neurochem. 1983, 41, 893–896. (9) Buu, N. T.; Duhaime, J.; Savard, C.; Truong, L.; Kuchel, O. J. Neurochem. 1981, 36, 769–772. (10) Weinshilboum, R. M.; Otterness, D. M.; Aksoy, I. A.; Wood, T. C.; Her, C.; Raftogianis, R. B. FASEB J. 1997, 11, 3–14. (11) Rivett, A. J.; Eddy, B. J.; Roth, J. A. J. Neurochem. 1982, 39, 1009–1016. (12) Young, W. F., Jr.; Okazaki, H.; Laws, E. R., Jr.; Weinshilboum, R. M. J. Neurochem. 1984, 43, 706–715.

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lum.13 UGTs exhibit tissue-specific expression and are mainly expressed in the liver, but isoforms UGT1A6, 2A1, and 2B7 have also been found in human brain.14 Even though UGT activity in rat brain microsomes has been reported, the glucuronidation of DA has not been observed thus far.15-17 Metabolism of neurotransmitters in brain can be studied by microdialysis, where physiological perfusion fluid is pumped through dialysis membrane which is surgically implanted into a region of interest in an animal’s brain.18-20 The extracellular fluid of brain contains synaptically released neurotransmitters and their metabolites, and also compounds from nonsynaptic sources.20 These low molecular weight compounds in the extracellular fluid are extracted to the perfusion fluid by passive diffusion. The recovery of compounds is dependent on many variables, for example, the temperature, molecular weight and charge, flow rate of the perfusion fluid, surface area of the dialysis membrane, etc.19 DA and its metabolites have commonly been analyzed using liquid chromatography (LC) with electrochemical (EC), fluorescence (FL), chemiluminescence, or mass spectrometric (MS) detection.21 The separation of analytes has commonly been achieved by reversed-phase liquid chromatography, utilizing an ion-pairing reagent. Derivatization of neurotransmitters has also been used to improve separation and ensure sensitive detection by LC-FL (limit of detection (LOD) 42-95 amol) because the concentrations of neurotransmitters in brain microdialysates are low (femtomole amounts).22 Higher specificity can be achieved by MS, though the sensitivity of FL detection is not reached (LODs by MS/MS analysis 2-10 fmol).23 Reversed-phase chromatography using volatile eluents has usually been used for the LC-MS/MS analysis of DA and its metabolites.23-25 However, these polar analytes are weakly retained on C18, and in this study, a pentafluorophenylpropyl stationary phase was used to enhance retention and achieve adequate separation of the analytes from the inorganic salts of the Ringer’s solution used in the microdialysis. So far, indirect analytical methods employing either acid or enzyme hydrolysis of the DA conjugates have been used, since DA-Glu and DA-sulfate standards are not commercially available. The indirect analysis methods, however, are prone to errors due to hydrolysis step that also complicates the analysis. The aim of (13) Mackenzie, P. I.; Bock, K. W.; Burchell, B.; Guillemette, C.; Ikushiro, S.; Iyanagi, T.; Miners, J. O.; Owens, I. S.; Nebert, D. W. Pharmacogenet. Genomics 2005, 15, 677–685. (14) Tukey, R. H.; Strassburg, C. P. Annu. Rev. Pharmacol. Toxicol. 2000, 40, 581–616, two plates. (15) Suleman, F. G.; Ghersi-Egea, J. F.; Leininger-Muller, B.; Minn, A. Neurosci. Lett. 1993, 161, 219–222. (16) El-Bacha, R. S.; Leclerc, S.; Netter, P.; Magdalou, J.; Minn, A. Life Sci. 2000, 67, 1735–1745. (17) Suleman, F. G.; Abid, A.; Gradinaru, D.; Daval, J. L.; Magdalou, J.; Minn, A. Arch. Biochem. Biophys. 1998, 358, 63–67. (18) Westerink, B. H. C. J. Chromatogr., B: Biomed. Sci. Appl. 2000, 747, 21– 32. (19) Plock, N.; Kloft, C. Eur. J. Pharm. Sci. 2005, 25, 1–24. (20) Bourne, J. A. Clin. Exp. Pharmacol. Physiol. 2003, 30, 16–24. (21) Tsunoda, M. Anal. Bioanal. Chem. 2006, 386, 506–514. (22) Yoshitake, T.; Yoshitake, S.; Fujino, K.; Nohta, H.; Yamaguchi, M.; Kehr, J. J. Neurosci. Methods 2004, 140, 163–168. (23) Hows, M. E. P.; Lacroix, L.; Heidbreder, C.; Organ, A. J.; Shah, A. J. J. Neurosci. Methods 2004, 138, 123–132. (24) Li, W.; Rossi, D. T.; Fountain, S. T. J. Pharm. Biomed. Anal. 2000, 24, 325–333. (25) Kushnir, M. M.; Urry, F. M.; Frank, E. L.; Roberts, W. L.; Shushan, B. Clin. Chem. (Washington, DC, U.S.) 2002, 48, 323–331.

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this study was to develop a liquid chromatographic-electrospray/ tandem mass spectrometric (LC-ESI-MS/MS) method for the analysis of DA and its phase I (DOPAC, HVA) and phase II metabolites (DA-3-O- and DA-4-O-glucuronides and DA-3-O- and DA-4-O-sulfates), adrenaline (A), and noradrenaline (NA) from brain microdialysis samples. The method provides for the first time analysis of intact DA-Glu and DA-sulfate without hydrolysis. An enzymatic synthesis method was developed to produce DAGlu standard. The quantitative performance of the method was evaluated, and the method was used to study the conjugation metabolism of DA in brain by analyzing microdialysis samples collected from the rat or mouse brain. EXPERIMENTAL SECTION Reagents and Standards. 3-Hydroxytyramine hydrochloride (DA) and uridine-5′-diphosphoglucuronic acid (UDPGA, trisodium salt) were purchased from Sigma-Aldrich (Steinheim, Germany). Dihydroxyphenyl acetic acid (DOPAC), HVA, and saccharic acid 1,4-lactone were purchased from Sigma-Aldrich (St. Louis, MO), L-adrenaline and potassium dihydrogen phosphate were from Merck (Darmstadt, Germany), and DL-noradrenaline hydrochloride and disodium hydrogen phosphate dihydrate were from Fluka (Buchs, Switzerland). Dopamine-3-O-sulfate and dopamine-4-Osulfate were prepared in our laboratory.26 Acetonitrile (ACN) was purchased from Rathburn (Walkerburn, Scotland). Ringer’s solution, used for microdialysis and dilution of the standards, contained 147 mM NaCl, 1.2 mM CaCl2 · 2H2O (Merck, Darmstadt, Germany), 2.7 mM KCl (Riedel-deHae¨n, Seelze, Germany), 1.0 mM MgCl2 · 6H2O (Merck, Darmstadt, Germany), and 0.04 mM ascorbic acid (University Pharmacy, Helsinki, Finland). Synthesis of Dopamine Glucuronide. The enzymatic synthesis of DA-Glu was performed using microsomes prepared from rat, pig, bovine, and moose liver as described previously.27 Rat liver microsomes were prepared from male Sprague-Dawley rats induced by Aroclor 1254 (a mixture of polychlorinated biphenyls). The treatment of the rats was approved by the local Ethical Committee for Animal Studies. The pig and bovine livers were obtained from a commercial slaughterhouse (Paimion Teurastamo, Paimio, Finland). Protein concentrations of the microsomes were determined with the BCA protein assay kit (Pierce Chemical, Rockford, IL). The efficiencies of rat, moose, pig, and bovine liver microsomes toward dopamine glucuronidation were tested, and rat liver microsomes were selected as the biocatalyst for the synthesis of DA-Glu. In addition to rat liver microsomes (protein concentration 1 mg/mL), the incubation mixture contained 2 mM DA, 5 mM saccharic acid 1,4-lactone, 5 mM UDPGA, 5 mM MgCl2, 50 mM phosphate buffer (pH 7.4), and 2% ACN in a total volume of 30 mL. After 3 h of incubation (37 °C), 7 mg of DA and 56 mg of UDPGA were added, and the incubation was continued for an additional 2.5 h. The reaction mixture was then centrifuged for 14 min (18 500 rpm, 6 °C). The supernatant containing DA-Glu was acidified with 50 µL of formic acid (98-100%, Riedel-deHae¨n, Seelze, Germany) before filtration (0.45 µm). DA and DA-Glu were extracted from the supernatant by solid-phase extraction using mixed-mode strong (26) Ita¨aho, K.; Alakurtti, S.; Yli-Kauhaluoma, J.; Taskinen, J.; Coughtrie, M. W. H.; Kostiainen, R. Biochem. Pharmacol. 2007, 74, 504–510. (27) Luukkanen, L.; Elovaara, E.; Lautala, P.; Taskinen, J.; Vainio, H. Pharmacol. Toxicol. (Copenhagen) 1997, 80, 152–158.

Figure 1. Structures of NA, A, DA, and its phase I and II metabolites used in the study.

cation-exchange and reversed-phase cartridges (Oasis MCX 150 mg, 6 cc, Waters, MA). The cartridge was conditioned with 3 mL of methanol and 3 mL of 0.1% aqueous formic acid. After addition of 5 mL of supernatant, the cartridge was washed with 4 mL of 2% aqueous formic acid and 4 mL of methanol, and the compounds were eluted with 10 mL of methanol containing 2% ammonia. To the collection tubes, 2 mL of methanol and 750 µL of formic acid were added to neutralize the ammonia used in the elution. Methanol was evaporated with a rotary evaporator, and DA-Glu was fractionated from the residue with an Agilent HP 1100 liquid chromatograph (Hewlett-Packard GmbH, Waldbronn, Germany) equipped with a binary pump, an autosampler, a column compartment, UV diode array detector, and fraction collector. The separation of DA and DA-Glu was performed on a Discovery HS F5 column (150 mm × 4 mm, 3 µm, Sigma-Aldrich, Bellefonte, PA) using ACN and aqueous 0.1% formic acid as eluents. A linear gradient of 5-25% ACN for 0-7 min, 25-80% ACN for 7-7.1 min, 80% ACN for 7.1-9 min, 80-5% ACN for 9.0-9.1 min, and 5% ACN for 9.1-25 min was used. The flow rate was 0.9 mL/min, and injection volume was 100 µL. A wavelength of 266 nm was used for peak detection. The DA-Glu fraction was evaporated to dryness with nitrogen and lyophilized. The purity of DA-Glu was studied using an LC-UV diode array detector. A linear gradient of 5-95% ACN for 30 min was used. From the spectra, it was observed that the absorption maximum of the impurities was 210 nm, which was used for the determination of purity of DA-Glu. NMR Spectroscopy. The DA and lyophilized DA-Glu samples were dissolved in 600 µL of D2O for nuclear magnetic resonance spectroscopy (NMR) analysis. The 1H and 13C chemical shifts were referenced to acetone added to the sample. The NMR experiments were carried out on a Varian Mercury Plus 300 spectrometer (Varian, Palo Alto, CA) at 23 °C. Liquid Chromatography-Mass Spectrometry. The HPLC method used for the fractionation of DA-Glu was modified for the analysis of microdialysis samples and standards diluted in Ringer’s solution. The separation of DA-3- and 4-O-sulfates, DA-Glu, DA, NA, A, DOPAC, and HVA (Figure 1) was achieved with a linear gradient of 5-35% ACN for 0-10 min using a Discovery HS F5 column. Eluent A was aqueous 0.1% formic acid. The column was washed with 35-80% ACN for 10-10.1 min, 80% ACN for 10.1-13 min, 80-5% ACN for 13-13.1 min followed by the equilibration

of the column for 6 min with 5% ACN. The flow was directed to waste for the first 3.8 min to prevent the inorganic ions of the Ringer’s solution from entering the mass spectrometer. After the elution of HVA (at 9.2 min) the flow was directed back to the waste. The flow was split in a ratio of 1:10 before the mass spectrometer. A PE Sciex API3000 triple-quadrupole mass spectrometer (Applied Biosystems/MDS Sciex, Concord, Canada) with a turbo ion spray source was used for detection. Purified air (Atlas Copco CD 2, Wilrijk, Belgium) was used as a nebulizing gas, and nitrogen generated with a Whatman 75-72 generator (Haverhill, MA) was used as turbo, curtain, and collision gas. The turbo gas flow rate was 6 L/min, and it was heated to 280 °C. Analyzes were carried out in the positive ion mode for DA-Glu, DA, NA, and A. The negative ion mode was used in the analysis of DA-sulfates, DOPAC, and HVA. The selected reaction monitoring (SRM) pairs for each compound are shown in Table 1. The ion spray and orifice (declustering) voltages, the collision energy, and the collision cell exit potentials for different SRMs were individually optimized for each compound. The data were collected and processed by Analyst 1.4.2 software. The stock solutions (10 mM) were prepared by dissolving the compounds in Ringer’s solution. External calibration was used for the quantification of the analytes in brain microdialysates. The calibration standards were diluted with Ringer’s solution from the stock solutions to the concentration levels of 0.8, 2, 5, 10, 30, 50, 70, 100, 180, 260, 360, 500, 600, 800, 1000 nM. Six or more concentration levels were used in the calibration (Table 1). Animals. Male and female mice of mixed 129 Sv/C57BL/6J background or male Wistar rats were used at 8-16 weeks of age. All procedures with animals were performed according to European Community Guidelines for the use of experimental animals (European Communities Council Directive 86/609/EEC) and reviewed by State Provincial Office of Southern Finland and approved by the Animal Experiment Board in conformance with current legislation. The rats and mice were housed in groups of 2-8 to a cage and had free access to chow and water. They were maintained under a 12:12 h light/dark cycle with lights on from 06:00 to 18:00 at an ambient temperature of 20-22 °C. Microdialysis. The animals were implanted with guide cannulae (rats, BAS MD-2250, Bioanalytical Systems Inc., IN, for striatum and MAB 4.10 IC, AgnTho’s, Lidingo¨, Sweden for nucleus Analytical Chemistry, Vol. 81, No. 1, January 1, 2009

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Table 1. Results of Validation Tests of the LC-MS/MS Method for the Analysis of DA and Its Phase I and II Metabolitesa repeatability, % RSD, n ) 6 compd DA-4-SO4 DA-3-SO4 DA-Glu NA A DOPAC DA HVA

SRM pairs 232 232 330 170 184 167 154 181

f f f f f f f f

152 152 137 135 107 123 137 122

(122, 80) (122, 80) (154, 91) (107) (77) (95) (91, 65)

LOD S/N > 3 LOQ S/N > 10 linearity nM (pmol) nM (pmol) range (nM) 2 (0.2) 0.8 (0.08) 0.25 (0.025) 5 (0.5) 5 (0.5) 10 (1) 0.25 (0.025) 30 (3)

5 (0.5) 2 (0.2) 0.8 (0.08) 10 (1) 10 (1) 30 (3) 0.8 (0.08) 70 (7)

5-600 2-600 0.8-100 10-600 10-600 260-1000 0.8-100 70-1000

r

accuracy %

0.999 0.998 0.998 0.998 0.998 0.998 1.0 0.999

87-109 78-116 93-139 89-106 72-107 90-104 98-105 85-109

10 nM std in 100 nM std microdialysis Ringer in Ringer % RSD (concn) 7.3 6.8 7.0 8.8 20.5 8.0 3.3