High-performance liquid chromatographic quantification of 4-methylumbelliferyl-β-d-glucuronide as a probe for human β-glucuronidase activity in tissue homogenates

doi:10.1016/0378-4347(96)00139-9

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 685, Issue 1, 11 October 1996, Pages 181-184

https://doi.org/10.1016/0378-4347(96)00139-9 Get rights and content

Abstract

An internally standardized HPLC method to determine the concentration of 4-methylumbelliferone liberated from 4-methylumbelliferyl-β-d-glucuronide by human β-glucuronidase was developed. The assay allows the precise and rapid measurement of specific enzyme activity in human tissue homogenates. Without prior extraction the incubation mixture can be separated using a C₈ column followed by fluorescence detection. The assay showed good accuracy and precision with a detection limit of 20 nM and a limit of quantification of 167 nM. The suitability of the method was shown in enzyme kinetic experiments with human liver homogenates.

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Cited by (19)

Validation of a probe for assessing deconjugation of glucuronide and sulfate phase II metabolites assayed through LC–MS/MS in biological matrices
2017, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Citation Excerpt :
Some papers indicate the use of MUG and MUS as deconjugation markers, but without providing any reference regarding a validated assay method for MU, MUG and MUS. The papers describing MU, MUG and/or MUS analysis used liquid chromatographic methods coupled to a UV or fluorimetric detector [21,26–28], which do not correspond to the current gold standard in bioanalysis, i.e. LC–MS/MS. Sample preparation is very rapid and easy to perform.
LC–MS/MS has been proposed in various areas such as Therapeutic Drug Monitoring (TDM), Human Biomonitoring (HBM), disease diagnosis, clinical toxicology and doping control to identify and quantify chemical parents and their metabolites in biological matrices. To determine the total content of a xenobiotic (unconjugated + conjugated forms), an enzymatic hydrolysis step is required. Most studies in the literature have not controlled the effectiveness of the deconjugation process because no method has been described for that purpose. Therefore the aim of this study was to develop and validate a deconjugation probe using a LC–MS/MS method. In order to estimate deconjugation using β-glucuronidase and/or sulfatase, 4-methyl-umbelliferone (MU) and its conjugates were used as markers.
Glucuronidase/sulfatase was added to plasma or urine spiked with 4-methylumbelliferyl-β-d-glucuronide (MUG) and 4-methylumbelliferyl sulfate (MUS) and umbelliferone, which was used as the internal standard. After incubation at 37 °C during 90 min, MU appears as a result of the deconjugation of MUG and MUS. The concentrations of the 3 markers were determined using LC–MS/MS. Trueness and precision of the LC–MS/MS method were determined by quality control analysis at three different levels of concentration covering the whole range of calibration.
In both matrices, the analytical method allows quantification of the different compounds, with good linearity, trueness and precision and negligible matrix effects. The method was applied with success to deconjugation assay using active glucuronidase/sulfatase in plasma and urine. The probe developed in this study allows to ensure that enzymatic preparation is working properly in the frame of a quality system.
The nucleotide transporter MRP4 (ABCC4) is highly expressed in human platelets and present in dense granules, indicating a role in mediator storage
2004, Blood
Citation Excerpt :
Mepacrine accumulation in the obtained subcellular fractions was analyzed by fluorescence detection (excitation at 485 nm, emission at 535 nm). Activity of β-glucuronidase was measured as described previously.33 Membrane fractions were loaded onto a 7.5% sodium dodecylsulfate–polyacrylamid gel after incubation in sample buffer at 37°C for 30 minutes.
Platelet aggregation is initiated by the release of mediators as adenosine diphosphate (ADP) stored in platelet granules. Possible candidates for transport proteins mediating accumulation of these mediators in granules include multidrug resistance protein 4 (MRP4, ABCC4), a transport pump for cyclic nucleotides and nucleotide analogs. We investigated the expression of MRP4 in human platelets by immunoblotting, detecting a strong signal at 170 kDa. Immunofluorescence microscopy using 2 MRP4-specific antibodies revealed staining mainly in intracellular structures, which largely colocalized with the accumulation of mepacrine as marker for delta-granules and to a lower extent at the plasma membrane. Furthermore, an altered distribution of MRP4 was observed in platelets from a patient with Hermansky-Pudlak syndrome with defective delta-granules. Adenosine triphosphate (ATP)–dependent cyclic guanosine monophosphate (cGMP) transport codistributed with MRP4 detection in subcellular fractions, with highest activities in the dense granule and plasma membrane fractions. This transport was inhibited by dipyramidole, indomethacin, and MK571 with median inhibitory concentration (IC₅₀) values of 12, 22, and 43 μM, and by ibuprofen. Transport studies with [³H]ADP indicated the presence of an orthovanadate-sensitive ADP transporting system, inhibited by dipyramidole, MK571, and cyclic nucleotides. The results indicate a function of MRP4 in platelet mediator storage and inhibition of MRP4 may represent a novel mechanism for inhibition of platelet function by some anti-inflammatory drugs.
Fluorogenic assay for β-glucuronidase using microchip-based capillary electrophoresis
2001, Journal of Chromatography B: Biomedical Sciences and Applications
Microchip capillary electrophoresis (CE) was used with a model enzyme assay to demonstrate its potential application to combinatorial drug screening. Hydrolysis with β-glucuronidase of the conjugated glucuronide, fluorescein mono-β-d-glucuronide (FMG), liberated the fluorescent product, fluorescein. FMG and fluorescein were detected by fluorescence, with excitation and emission at 480 and 520 nm, respectively. Microchip CE was used to separate FMG and fluorescein. Fluorescein production was monitored to assess β-glucuronidase activity. Michaelis–Menten enzyme kinetics analysis yielded the K_m value. The results were compared with those from experiments done by conventional CE. The K_m value for β-glucuronidase with FMG is being reported for the first time as 18 μM. The inhibition of β-glucuronidase by the competitive inhibitor d-saccharic acid-1,4-lactone (SL) was also determined using microchip CE. Reactions were done with various concentrations of inhibitor and constant β-glucuronidase and FMG concentrations. A dose–response plot was acquired and the IC₅₀ value for SL was determined to be 3 μM.
Does pH 6 β-galactosidase activity indicate cell senescence?
1999, Mechanisms of Ageing and Development
Apparently two forms of β-galactosidase (β-GAL) in cells or tissue sections can be detected by enzyme histochemical staining (X-GAL). Using a sensitive and specific HPLC method we have determined the pH dependent activity of β-GAL in cell lines of lung carcinoma (A549), colon carcinoma (Caco2-TC7), promyelocytic leukemia (HL60), hepatoma (HepG2) and human liver homogenates. The HPLC method has been validated and the influence of pH and substrate concentration was studied. There was a good linear correlation between HPLC and quantitative enzyme histochemistry (pH 4.5: r=0.985; pH 6.0: r=0.967). Both, pH 4.5 β-GAL and pH 6 β-GAL could be demonstrated in all biological material tested and pH 6 β-GAL activity was always lower (25–50%) than pH 4.5 activity. In Caco2-TC7 cells both activities increased by a factor of 10 from day 3 to day 17 after seeding. In addition, since the β-GAL activity decreased with increase in pH both in human liver homogenates (independent of the age of the donor) as well as in tumor cell lysates in a similar fashion we believe that the activity at pH 6 can hardly be considered as an exclusive ‘senescence marker’. In addition, the more sensitive HPLC method could demonstrate activity in cells that showed negative reaction with X-GAL.
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1999, Journal of Chromatography B: Biomedical Sciences and Applications
A capillary electrophoresis (CE) method was developed using paracetamol glucuronide as a novel probe for human β-glucuronidase activity. Using UV detection without prior sample clean-up procedures, fast and reliable quantitation of the released paracetamol was possible. The method showed good precision, accuracy and sensitivity with a limit of detection of 0.25 μM (38 ng/ml) and a limit of quantitation of 1 μM (151 ng/ml). The suitability of the method has been shown for enzyme kinetic studies using different liver and kidney homogenates, respectively. Our data clearly demonstrate that paracetamol glucuronide is cleaved by human β-glucuronidase thereby releasing paracetamol. The CE method presented is not only a valuable tool for measuring human β-glucuronidase activity, but also allows investigation of the contribution of deglucuronidation of paracetamol glucuronide to the disposition of paracetamol.
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Short communicationHigh-performance liquid chromatographic quantification of 4-methylumbelliferyl-β-d-glucuronide as a probe for human β-glucuronidase activity in tissue homogenates

Abstract

J. Pediatr.

Clin. Chim. Acta

Clin. Chim. Acta

Biochem. Pharmacol.

Clin. Chim. Acta

J. Biol. Chem.

Pediatr. Res.

Short communication
High-performance liquid chromatographic quantification of 4-methylumbelliferyl-β-d-glucuronide as a probe for human β-glucuronidase activity in tissue homogenates