Paracetamol glucuronidation

FIGURE 7.15 Continuous-flow CEC-NMR spectra (a) Paracetamol glucuronide (b) paracetamol sulfate and (c) endogenous hippurate. (Reproduced from Pusecker, K. et al. Anal. Commun. 1998, 35, 213-215. With permission from RSC publishers.)... 

The ability to identify metabolites and biotransformed products in biological fluids during separation is a considerable challenge. The potential application of CE-NMR and CEC-NMR for analysis of metabolites in biofluids has been demonstrated [51,52], For example, CE-NMR has successfully analyzed the major metabolites of paracetamol in human urine [51]. Two major metabolites, paracetamol glucuronide and paracetamol sulfate conjugates, as well as endogenous material (hippurate) have been characterized. Comparison of chemical shifts has confirmed the presence of these compounds. The estimated amount that can be detected in this study with a S/N of 3 is —10 ng. 

Figure 14 Stopped-flow TOCSY CEC-NMR spectra of paracetamol glucuronide. Acquisition parameters number of 144 scans for each of 256 increments. Spectral width of 4716 Hz, number of points 4096. Processing parameters zero filled and multiplied by apodization function of 3 Hz in both dimensions. (From Ref. 52 reproduced with permission from The Royal Society of Chemistry.)...

Jensen, L.S., Valentine, J., Milne, R.W., Evans, A.M. The quantification of paracetamol, paracetamol glucuronide and paracetamol sulphate in plasma and urine using a single high-performance liquid chromatography assay. J. Pharm. Biomed. Anal. 24, 585-593 (2004)... 

Figure 2.24 Representative pre-dose (a) and post-dose (b) proton NMR spectra of urine samples from a rat dosed with paracetamol (600 mpk). Inset in (a) is an expansion. 2-OG, 2-oxoglutarate G, paracetamol glucuronide. (Reprinted by permission from Clayton, T.A., et al. Pharmaco-metabonomic phenotyping and personalized drug treatment. Nature 2006, 440, 1073-1077, copyright 2006, Macmillan Publishers Ltd.)...

Nizatidine 300 mg given to 5 healthy subjects with paracetamol 1 g modestly increased the paracetamol AUC in the first 3 hours by 25%. Over this time period, there was also a nonsignificant 4% reduction in formation of paracetamol glucuronide, but this did reach statistical significance at 30 and 45 minutes. Nizatidine 150 mg had a similar, but smaller, effect. ... 

A patient taking zidovudine and co-trimoxazole took 3.3 g of paracetamol over 36 hours. Within 2 days he developed severe hepatotoxicity, and as other causes were excluded, the reaction was attributed to the paracetamol. The authors suggested that zidovudine may have augmented the paracetamol toxicity. However, in a single-dose study, reduced paracetamol glucuronidation and increased formation of hepatotoxic metabolites was seen in patients with advanced HIV infection compared with healthy HIVpositive subjects and those without HIV, and this effect was independent of zidovudine use.In contrast, in another study, disease state (AIDS versus healthy HIV-positive subjects) was not found to alter paracetamol metabolism, and zidovudine was found to increase paracetamol glucuronidation in some patients."... 

Drugs must also be considered as foreign compounds, and an essential part of drug treatment is to understand how they are removed from the body after their work is completed. Glucuronide formation is the most important of so-called phase II metabolism reactions. Aspirin, paracetamol, morphine, and chloramphenicol are examples of drugs excreted as glucuronides. 

Figure 6.48 Two-dimensional NOESY spectrum (mixing time = 2 s) of the ether glucuronide of 3-methoxy-paracetamol at 600 MHz in a 3-mm cryogenic probe head (total experiment time 20 h). The sample was recovered from a conventional 3-mm LC probe head after a triple trapping SPE-NMR run (result shown in Eigure 6.41). Reproduced from [59] with permission from Elsevier.

The analgesic paracetamol is largely excreted in the urine of adults as the glucuronide, only around 30% appearing as the sulphate. When human foetal liver cells were incubated with paracetamol, however, they produced the sulphate conjugate but no glucuronide. 

Figure 7.10 Metabolism of paracetamol. With therapeutic doses, paracetamol is metabolised to the glucuronide and sulphate conjugates. With higher doses these pathways become saturated and metabolism proceeds via die P-450-mediated route, with the formation of the toxic metabolite benzoquinone. This is normally metabolised by conjugation with glutathione. When glutathione is depleted benzoquinone is free to interact with cellular macromolecules, leading to cellular damage.

Paracetamol reaches peak plasma concentrations within the first hour after oral administration and shows only a low tendency for plasma protein binding at therapeutic concentrations. The elimination half-life is between 1 and 3 h. Paracetamol is metabolized mainly in the liver and excreted in the urine as glucuronide and sulphate conjugates. The metabolic pathway of paracetamol is shown in Schemes 66 and 67 ... 

Scheme 66 Formation of the glucuronide and sulphate conjugates of paracetamol.

The amount of futile acetylation observed for this compound in the rat, at ca. 7-10% for parent compound and metabolites, was less than that seen for phenacetin and similar to that found for paracetamol itself. The bulk of the radio-label was rapidly excreted in urine as practolol itself (albeit with 7-10% reacetylation) and the remainder as either the ring-hydroxylated metabolite or its glucuronide conjugate. As in previous examples, deuterated methanol was used in the mobile phase rather than methanol in order to be able to more easily observe the acetyl resonances of practolol and related compounds. 

Figure 4.1 Pseudo-2D plot of continuous-flow 1 H LC-NMR data obtained on human urine after dosing with paracetamol (1). The resonances from the glucuronide (2) and sulfate (3) conjugate metabolites of paracetamol and their ID slices are shown...

Ullrich D, Sieg A, Blume R, et al. Normal pathways for glucuronidation, sulphation and oxidation of paracetamol in Gilbert s syndrome. Eur J Clin Invest 1987 17(3) 237-240. 

Bock KW, Wiltfang J, Blume R, et al. Paracetamol as a test drug to determine glucuronide formation in man. Effects of inducers and of smoking. Eur J Clin Pharmacol 1987 31(6) 677-683. 

The metabolism of paracetamol (Figure 6.6) is an example of potential toxication. Paracetamol is metabolised primarily in the liver, via phase II metabolism, where its major metabolites include inactive sulphate and glucuronide conjugates, which are excreted by the kidneys. 

---