Leena Luukkanen

Expression and Characterization of Recombinant Human UDP-glucuronosyltransferases (UGTs) UGT1A9 IS MORE RESISTANT TO DETERGENT INHIBITION THAN THE OTHER UGTs AND WAS PURIFIED AS AN ACTIVE DIMERIC ENZYME

Eight human liver UDP-glucuronosyltransferases (UGTs) were expressed in baculovirus-infected insect cells as fusion proteins carrying a short C-terminal extension that ends with 6 histidine residues (His tag). The activity of recombinant UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B7, and UGT2B15 was almost fully inhibited by 0.2% Triton X-100. In the case of UGT1A9, however, glucuronidation of α-naphthol and scopoletin was resistant to such inhibition, whereas glucuronidation of entacapone and several other aglycones was sensitive. His-tagged UGT1A9 was purified by immobilized metal-chelating chromatography (IMAC). Purified UGT1A9 glucuronidated scopoletin at a high rate, whereas its glucuronidation activity toward entacapone was low and largely dependent on phospholipid addition. Recombinant UGT1A9 in which the His tag was replaced by hemagglutinin antigenic peptide (HA tag) was also prepared. Insect cells were co-infected with baculoviruses encoding both HA-tagged and His-tagged UGT1A9. Membranes from the co-infected cells, or a mixture of membranes from separately infected cells, were subjected to detergent extraction and IMAC, and the resulting fractions were analyzed for the presence of each type of UGT1A9 using tag-specific antibodies. In the case of separate infection, the HA-tagged UGT1A9 did not bind to the column. When co-infected with His-tagged UGT1A9, however, part of the HA-tagged enzyme was bound to the column and was eluted by imidazole concentration gradient together with the His-tagged UGT1A9, suggesting the formation of stable dimers that contain one His-tagged and one HA-tagged UGT1A9 monomers.

Prominent but Reverse Stereoselectivity in Propranolol Glucuronidation by Human UDP-Glucuronosyltransferases 1A9 and 1A10

Propranolol is a nonselective β-adrenergic blocker used as a racemic mixture in the treatment of hypertension, cardiac arrhythmias, and angina pectoris. For study of the stereoselective glucuronidation of this drug, the two propranolol glucuronide diastereomers were biosynthesized, purified, and characterized. A screen of 15 recombinant human UDP-glucuronosyltransferases (UGTs) indicated that only a few isoforms catalyze propranolol glucuronidation. Analysis of UGT2B4 and UGT2B7 revealed no significant stereoselectivity, but these two enzymes differed in glucuronidation kinetics. The glucuronidation kinetics of R-propranolol by UGT2B4 exhibited a sigmoid curve, whereas the glucuronidation of the same substrate by UGT2B7 was inhibited by substrate concentrations above 1 mM. Among the UGTs of subfamily 1A, UGT1A9 and UGT1A10 displayed high and, surprisingly, opposite stereoselectivity in the glucuronidation of propranolol enantiomers. UGT1A9 glucuronidated S-propranolol much faster than R-propranolol, whereas UGT1A10 exhibited the opposite enantiomer preference. Nonetheless, the Km values for the two enantiomers, both for UGT1A9 and for UGT1A10, were in the same range, suggesting similar affinities for the two enantiomers. Unlike UGT1A9, the expression of UGT1A10 is extrahepatic. Hence, the reverse stereoselectivity of these two UGTs may signify specific differences in the glucuronidation of propranolol enantiomers between intestine and liver microsomes. Subsequent experiments confirmed this hypothesis: human liver microsomes glucuronidated S-propranolol faster than R-propranolol, whereas human intestine microsomes glucuronidated S-propranolol faster. These findings suggest a contribution of intestinal UGTs to drug metabolism, at least for UGT1A10 substrates.

Analysis of catechol-type glucuronides in urine samples by liquid chromatography–electrospray ionization-tandem mass spectrometry

A direct and fast liquid chromatographic–electrospray ionization-tandem mass spectrometric (LC–ESI-MS–MS) method is described for the determination of 3-O-glucuronides of E- and Z-entacapone, nitecapone and tolcapone and 1-O- and 2-O-glucuronides of 4-nitrocatechol in urine. p-Nitrophenyl β-d-glucuronide was used as internal standard. Spiked urine samples were prepared by solid-phase extraction and analysed by isocratic LC–ESI-MS–MS in negative ion mode. The ESI mass spectra showed an abundant deprotonated molecule [M–H]−, which was chosen as precursor ion. Collisionally induced dissociation of [M–H]− in MS–MS resulted in the loss of neutral glucuronide moiety and in the appearance of an intense negatively charged drug molecule, which was chosen as the product ion to be monitored in the LC–MS–MS analysis. The new method showed good linearity (r2>0.997) and repeatability of the method (relative standard deviation <2.56%). The limits of detection were determined to be 0.1–0.2 μg/ml when 5 μl of the spiked urine was used for the analysis (5–10 pg of glucuronide introduced to ESI-MS–MS).

Accurate Mass Measurements of Some Glucuronide Derivatives by Electrospray Low Resolution Quadrupole Mass Spectrometry

The measurement of exact masses using negative-ion quadrupole low resolution electrospray mass spectrometry (ESMS) is demonstrated with p-nitrophenol, entacapone, mitecapone and 1-hydroxypyrene glucuronides. The results are compared with those obtained by high resolution fast-atom bombardment mass spectrometry (FAB-MS). The errors with ESMS between measured and calculated values ranged from −1.7 to 3.1 ppm (−0.8 to 1.2 milli-mass units (mmu). The standard deviations of the measurements were between ±1.1 and 4.8 ppm (±0.5 and 2.3 mmu). The errors with high resolution FAB-MS were between −2.5 and 3.2 ppm (−1.1 and 1.0 mmu). These results show that accurate mass measurements using low resolution quadrupole ESMS are comparable to those achievable in practice using high resolution instruments. A precondition for a successful measurement with low resolution mass spectrometry is that the sample ion be completely separated from background ions, since even very small levels of unresolved ions can interfere with the accuracy of mass analysis.

ASSESSMENT OF CATECHOL INDUCTION AND GLUCURONIDATION IN RAT LIVER MICROSOMES

Catechols are substances with a 1,2-dihydroxybenzene group from natural or synthetic origin. The aim of this study was to determine whether catechols (4-methylcatechol, 4-nitrocatechol, 2,3-dihydroxynaphthalene) and the antiparkinsonian drugs, entacapone and tolcapone, at doses 150 to 300 mg/kg/day, for 3 days, are able to enhance their own glucuronidation. The induction potency of catechols on rat liver UDP-glucuronosyltransferases (UGTs) was compared with that of a standard polychlorinated biphenyl (PCB) inducer, Aroclor 1254. The glucuronidation rate of these catechols was enhanced up to 15-fold in the liver microsomes of PCB-treated rats, whereas treatment with catechols had little effect. Entacapone, tolcapone, 4-methylcatechol, catechol, 2,3-dihydroxynaphthalene, and 4-nitrocatechol were glucuronidated in control microsomes at rates ranging from 0.12 for entacapone to 22.0 nmol/min/mg for 4-nitrocatechol. Using 1-naphthol, entacapone, and 1-hydroxypyrene as substrates, a 5-, 8-, and 16-fold induction was detected in the PCB rats, respectively, whereas the catechol-induced activities were 1.1- to 1.5-fold only. Entacapone was glucuronidated more efficiently by PCB microsomes than by control microsomes (Vmax/Km, 0.0125 and 0.0016 ml/min/mg protein, respectively). Similar kinetic results were obtained for 1-hydroxypyrene. The Eadie-Hofstee plots suggested the contribution of multiple UGTs for the glucuronidation of 1-hydroxypyrene (Km1, Km2, Km3 = 0.8, 9.7, and 63 μM, and Vmax1, Vmax2, Vmax3 = 11, 24, and 55 nmol/min/mg, respectively), whereas only one UGT could be implicated in the glucuronidation of entacapone (Km = 130 μM, Vmax = 1.6 nmol/min/mg). In conclusion, catechols are poor inducers of their own glucuronidation supported by several UGT isoforms. Their administration is unlikely to affect the glucuronidation of other drugs administered concomitantly.

Mass Spectrometric and Tandem Mass Spectrometric Behavior of Nitrocatechol Glucuronides: A Comparison of Atmospheric Pressure Chemical Ionization and Electrospray Ionization

The mass spectrometric (MS) and tandem mass spectrometric (MS/MS) behavior of six nitrocatechol-type glucuronides using atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) was systematically studied, and the effect of operation parameters on the fragmentations are presented. The positive ion APCI- and ESI-MS spectra showed an intense protonated molecule and the respective negative ion spectra a deprotonated molecule with minimal fragmentation. The main fragment ions in the MS/MS spectra of the protonated and deprotonated molecules were [M+H−Glu]+ and [M−H−Glu]−, respectively, formed by the loss of the glucuronide moiety. The measured limits of detection indicated that ESI is a significantly more efficient ionization method than APCI in the negative and positive ion modes for the compounds studied. MS/MS was found to be less sensitive, but more reliable and simple than MS due to the absence of chemical noise.