Glucuronidation of 1-hydroxypyrene by human liver microsomes and human UDP-glucuronosyltransferases UGT1A6, UGT1A7, and UGT1A9: development of a high-sensitivity glucuronidation assay for human tissue.

Human UDP-glucuronosyltransferases (UGT, EC 2.4.1.17) involved in the biotransformation of pyrene were investigated by a sensitive fluorometric high-performance liquid chromatography (HPLC)method developed for determining activities toward 1-hydroxypyrene. The endpoint metabolite of pyrene, 1-pyrenylglucuronide, is a well-known urinary biomarker for the assessment of human exposure to polycyclic aromatic hydrocarbons. 1-Pyrenylglucuronide was synthesized using rat liver microsomes as biocatalyst. The yield was satisfactory, 22%. 1-Pyrenylglucuronide, identified by (1)H NMR and by electrospray mass spectrometry, was used for method validation and calibration. The HPLC assay was very sensitive with a quantitation limit of 3 pg (8 fmol) for 1-pyrenylglucuronide. The assay was precise, showing a relative standard deviation of 5% or less at 0.1 to 300 microM 1-hydroxypyrene. Only 2 microg of microsomal protein was required for the assay in human liver. The glucuronidation of 1-hydroxypyrene was catalyzed at high rates in microsomes from pooled or three individual liver samples, showing comparable apparent K(m) values. The formation of 1-pyrenylglucuronide was catalyzed by recombinant human UGT1A6, UGT1A7, and UGT1A9, the K(m) values being 45, 12, and 1 microM, respectively. The apparent K(m) values in human liver microsomes, ranging from 6.9 to 8.6 microM, agreed well with these results. The method provides a sensitive tool for measuring extremely low UGT activities and a specific means for assessing interindividual differences in 1-hydroxypyrene-metabolizing UGT activities in human liver and other tissues.

Abdominal radiography in monitoring the resolution of sand accumulations from the large colon of horses treated medically.

This retrospective study consisted of 14 horses (age 6 weeks-12 years) with radiographically evident sand accumulations cranioventrally in the abdomen and clinical signs suggestive of sand enteropathy. The horses were treated medically and resolution of sand was monitored radiographically. Routine treatment consisted of psyllium mucilloid, combined with magnesium sulphate and/or mineral oilif needed. Initially, the number, size and shape of the sand accumulations showed large variation and the response to therapy was not predictable based on the initial appearance of the accumulation. In 2 foals, some of the sand was passed and the rest was mixed with other intestinal contents within 2-4 days. Even large accumulations disappeared in 2-4 days with psyllium alone or combined with mineral oil in 4 horses. In another 4 horses, the size of the accumulations decreased but varying amounts remained approximately at the same site, despite treatment for 1-4 weeks, and all these horses also had either gastric or large colon impaction. Three horses had a limited response to psyllium treatment, but the accumulation resolved with repeated doses of magnesium sulphate, with or without mineral oil. One horse did not respond to prolonged laxative treatment but the accumulation resolved on pasture. Clinical improvement was not necessarily related to the resolution of sand. Radiography of the cranioventral abdomen was found to be a useful means for monitoring the resolution of sand and confirming the effect of medical treatment in removing sand from the large colon in the horse.

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.

Enzyme-assisted synthesis and structural characterization of nitrocatechol glucuronides.

Enzyme-assisted synthesis and characterization are described for 3-O-beta-D-glucuronides 1b-4b of the aglycons E- and Z-2-cyano-N, N-diethyl-3-(3,4-dihydroxy-5-nitrophenyl)propenamide (entacapone), 1a and 2a, respectively, 3-(3,4-dihydroxy-5-nitrobenzylidene)-2, 4-pentanedione (nitecapone) 3a and 4'-methyl-3, 4-dihydroxy-5-nitrobenzophenone (tolcapone) 4a, and 1-o- and 2-o-glucuronides 5b and 6b of the aglycon 1, 2-dihydroxy-4-nitrobenzene 5a. Liver microsomes from rats pretreated with Aroclor 1254 were used as catalyst in the synthesis. Glucuronidation was regio- and stereoselective in the case of 1a-4a; only one product was observed by HPLC, HPTLC, and NMR. The glucuronidation of 1,2-dihydroxy-4-nitrobenzene 5a resulted in equal amounts of 1-O-beta-D- and 2-O-beta-D-glucuronides. Purification of the crude products by C18 solid-phase extraction and/or flash chromatography gave compounds 1b-6b in 38-98% yields (50-84 mg). The structures of the glucuronides were characterized on the basis of UV and IR spectra and confirmed with FAB-MS and NMR spectroscopy.

Some effects of multiple administration of detomidine during the last trimester of equine pregnancy.

Detomidine was given to 11 pregnant mares at 3 week intervals during the last trimester of pregnancy. Maternal and fetal electrocardiographs were recorded and fetal activity studied by transabdominal ultrasonography, before and 2 h (2, 5, 10, 20, 30, 60, 90 and 120 min) after injection. After parturition, the foals were examined and weighed. Maternal and fetal heart rate showed an initial decline after detomidine administration. Maternal heart rate in the treatment group were lower already 2 min after injection, but a reduction in fetal heart was first seen 5 min after detomidine administration. Mean fetal heart rate at 2 min after detomidine injection was 109, 104, 95 and 90 beats/min, whereas at 5 min it was 80, 76, 72 and 66 beats/min in the 2nd, 3rd, 4th and 5th examination session, respectively. The heart rates did not revert to the control values during follow-up. Decline and recovery patterns were quite similar during all examination sessions. The mares exhibited conductive disturbances 2 min after detomidine administration, but fetal heart rhythm remained regular. Fetal activity was decreased at 5 min but had reverted to control values about 90 min after detomidine administration. Administration of detomidine (0.015 mg/kg) to healthy pregnant mares at 3 week intervals during the last trimester had no measurable detrimental effects on the outcome of pregnancy.

Characterization of 1-hydroxypyrene as a novel marker substrate of 3-methylcholanthrene-inducible phenol UDP-glucuronosyltransferase(s).

Rats were treated with acetone, pyrazole, phenobarbital, 4,4'-methylenebis-(2-chloroaniline) (MOCA), 3-methylcholanthrene, creosote oil, or a mixture of polychlorinated biphenyls (Aroclor 1254) to study the inducibility and enzyme kinetics of UDP-glucuronosyltransferases towards 1-hydroxypyrene, which is a human metabolite and a urinary biomarker of exposure to pyrene. The rate of 1-hydroxypyrene glucuronidation was analyzed in rat liver microsomes by a fluorometric HPLC assay of the formed glucuronide. The apparent K(m) and Vmax values in untreated controls (K(m) = 0.27 mM; Vmax = 31 nmol/min./mg protein) did not differ markedly from those in rats treated with acetone, pyrazole or phenobarbital, whereas the significantly decreased K(m) and increased Vmax values of the rats treated with the carcinogenic chemicals, MOCA (0.11; 51), creosote (0.06; 137), 3-methylcholanthrene (0.07; 141) or the Aroclor-1254 polychlorinated biphenyl (PCB) mixture (0.08; 226), implicated major changes in the hepatic expression of UDP-glucuronosyltransferases. 1-Hydroxypyrene proved to be a high affinity substrate and a sensitive marker of the polycyclic aromatic hydrocarbon (PAH) metabolizing UDP-glucuronosyltransferase(s). Catalytically, the most efficient isoforms were induced in creosote, 3-methylcholanthrene and PCB-treated rats showing Vmax/K(m) ratios which were 22-27 times greater than in untreated controls. Our findings suggest the existence of a 3-methylcholanthrene type inducible and a functionally efficient low-K(m)/ high-Vmax form(s) of UDP-glucuronosyltransferase(s) that detoxify 1-hydroxypyrene and probably other polycyclic aromatic hydrocarbons as well.

Oxidation of beta-blocking agents--VII. Periodate oxidation of labetalol.

The oxidation of labetalol with sodium metaperiodate is described. In spite of the bulky substituent on the amino group of labetalol, glycol cleavage of the molecule occurred. Spectrometric methods verified that the aromatic aldehyde formed was 2-hydroxy-5-formylbenzamide and that the amine was 1-methyl-3-phenylpropylamine. The polarographic behaviour of the aldehyde was examined as well. The half-wave potential in Britton-Robinson buffer pH 5.68 was -1.24 V. Direct linearity (r = 0.9999) was observed between the diffusion current and the concentration of the aldehyde. Quantitation of labetalol was carried out polarographically by measuring the concentration of 2-hydroxy-5-formylbenzamide formed in the oxidation.