Urinary excretion of the uraemic toxin p-cresol in the rat: contribution of glucuronidation to its metabolization

Gerrit Lesaffer1,, Rita De Smet1, Frans M. Belpaire2, Bruno Van Vlem1, Marijn Van Hulle3, Rita Cornelis3, Norbert Lameire1 and Raymond Vanholder1

1 Renal Division and 2 Department of Pharmacology, Heymans Institute, University Hospital and 3 Laboratory of Analytical Chemistry, University of Gent, Gent, Belgium



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Increasing evidence indicates that lipophilic and/or protein-bound substances such as p-cresol are responsible for adverse physiological alterations in uraemic patients. To better understand the evolution of p-cresol disposition in renal failure and dialysis patients, it is necessary to determine its kinetic characteristics and biotransformation pathways.

Methods. We studied the biotransformation of p-cresol after intravenous injection of the compound in eight rats with normal renal function. Urine was collected in four 1 h intervals. To evaluate the presence of p-cresol metabolites, ß-glucuronidase was added to urine samples and the isolated unidentified chromatographic peak observed in previous experiments was submitted to tandem mass spectrometry (MS/MS) analysis.

Results. Administration of p-cresol produced a p-cresol peak and an unknown peak, suggesting biotransformation of the compound. Addition of ß-glucuronidase to urine samples and incubation at 37°C resulted in a marked decrease in the unidentified peak height (P<0.001) together with an increase in p-cresol peak height (P<0.001), suggesting that the unidentified peak was composed, at least in part, of p-cresylglucuronide. Mass spectrometry (MS) and MS/MS analysis of the isolated unidentified peak confirmed the presence of p-cresylglucuronide. Linear regression between the peak height of p-cresylglucuronide before enzyme treatment and the increase in p-cresol peak height after enzyme treatment in samples incubated with ß-glucuronidase allowed us to calculate the amount of p-cresylglucuronide as its p-cresol equivalents. This revealed that 64% of the injected p-cresol was excreted as glucuronide. There was no change in peak heights when sulphatase was added to the urine. When p-cresol and p-cresylglucuronide levels were combined, ~85% of all administered p-cresol was recovered in the urine. In addition, the combined urinary excretion of p-cresol and p-cresylglucuronide was more than four times greater than excretion of p-cresol by itself (P<0.01).

Conclusions. In rats with normal renal function, intravenous administration of p-cresol results in immediate and extensive metabolization of the compound into p-cresylglucuronide. The elimination of p-cresol from the body depends largely on the urinary excretion of this metabolite.

Keywords: creatinine; glucuronidation; kinetics; metabolization; p-cresol; rats



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Numerous compounds having a broad range of molecular weights are normally removed by the healthy kidneys. However, these compounds progressively accumulate in the body in parallel with the deterioration of renal function [1] and several of these alter biochemical, biological and physiological processes [25].

p-Cresol is a small phenolic compound (MW=108.1 Da) with lipophilic properties [612]. It is strongly bound to serum proteins (>99% in healthy persons), but their binding is decreased in uraemic patients [9]. Furthermore, p-cresol excretion is probably impaired in these patients, resulting in higher unbound concentrations that are likely to be the biologically active fraction.

It has become clear over the past decade that lipophilic compounds, protein-bound substances or both are at the origin of functional alterations that contribute to the uraemic syndrome [25]. This suggests that p-cresol, together with other lipophilic compounds, should be removed as efficiently as possible, since these compounds can be considered as a prototype of protein-bound uraemic retention solutes. However, we recently demonstrated that serum concentrations of total (bound and unbound) p-cresol at the end of standard haemodialysis, both with low- and high-flux membranes, was only 30% below the starting value, compared with a 75% removal of urea [12].

To facilitate the removal of a compound, it is necessary to understand how it is disposed in the body. To the best of our knowledge such investigations have not been undertaken for p-cresol. In a recent study, Ohmori et al. [13] demonstrated that dihydroethorphine, which contains a phenol group, is extensively conjugated with glucuronic acid. Because p-cresol is also a phenolic compound and since Ogata et al. [7] previously reported the presence of p-cresylglucuronide in the urine of healthy volunteers after oral administration of wood creosote [of which p-cresol comprises 13.7% (w/w)], we hypothesized that glucuronide or other metabolites may appear after administration of p-cresol to rats with normal renal function. In a study of this kind, assessing the kinetic behaviour of p-cresol in the body, it is indispensable to follow the evolution of its metabolites.

After intravenous bolus injection of p-cresol in normal rats, we had previously observed an unidentified peak of an unknown compound in addition to the p-cresol peak on the high-performance liquid chromatography (HPLC) chromatograms of serum and urine [14]. Given the possibility that p-cresol is glucuronidated, we began our investigations by adding ß-glucuronidase to the urine samples. In addition, when isolated fractions containing the unidentified peak were submitted to electrospray mass spectrometry (ES-MS) to evaluate the presence of one or more metabolites of p-cresol, this revealed the presence of p-cresylglucuronide. Thus, the total amount of p-cresol recovered in urine will be underestimated in a kinetic analysis that does not take into account formation of its metabolites. Therefore, the data in the present study collected for assessing urinary excretion of p-cresol were calculated twice, including one analysis for p-cresol and a second analysis for both p-cresol and p-cresylglucuronide.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Reagents and materials
OFA rats were purchased from Iffa Credo (Charles River, Wilmington, MA). All chemical reagents and liquids were of analytical or HPLC grade. p-Cresol was obtained from Supelco (Bellefonte, PA) and HPLC-water, methanol and isopropyl ether were from Acros Organics (Geel, Belgium). Acetate buffer, ß-glucuronidase (type H-3 from Helix pomatia), sulphatase (type VI from Aerobacter aeruginosa) and Tris were from Sigma Diagnostics (St Louis, MO) and isotonic saline solution was from Baxter (Lessines, Belgium). Polyethylene (PE) tubings were obtained from Medihac (St Michielsgestel, The Netherlands).

Animal instrumentation
Eight male OFA rats with an average body weight of 311±7 g were anaesthetized with an intraperitoneal injection of 60 mg/kg Na-pentobarbital (Nembutal®; Sanofi, Brussels, Belgium). The trachea was intubated with PE240 (ID, 1.67 mm; OD, 2.42 mm) and the left jugular vein was catheterized with PE50 tubing (ID, 0.58 mm; OD, 0.97 mm) for administration of the p-cresol solutions. The left carotid artery was catheterized with heparin-filled (250 IU/ml; B. Braun Pharma, Diegem, Belgium) PE50 tubing for blood collections. An abdominal incision was then made to expose the urinary bladder. All available urine was collected by puncturing the bladder with a syringe and then a PE160 tube (ID, 1.14 mm; OD, 1.57 mm) was inserted for periodical collection of all urine during the experiment. The animals were treated as recommended in ‘Use of Animals in Research’ [policy briefing 9 of the European Science Foundation (September 2000)]. The experiments were also in accordance with the guiding principles for the use of animals in toxicology [as stated in Toxicol Appl Pharmacol 2000; 162] and were approved by the Ethics Review Committee for Laboratory Animals at our hospital.

Injection of the compound and collection of the samples
A solution of p-cresol in isotonic saline (1.5 mg/ml) was prepared and kept at –20°C until its use. Immediately following the bolus intravenous injection of 2 ml (9.6 mg/kg) p-cresol, blood (0.7 ml) was collected from the carotid artery. Other collections were made at 5, 30, 60, 120, 180 and 240 min post-injection. Prior to each blood sampling, the cannula was flushed back and forth with the heparin solution and the first three drops of blood were discarded. Urine was collected in four 1 h fractions. Blood and urine samples were immediately centrifuged for 5 min at 3000 r.p.m. (500 g) and plasma or supernatant were collected and transferred into Eppendorf tubes, which were stored at -20°C until analysis. The urine fractions of all eight experimental rats were pooled per 1 h fraction.

Identification of the metabolite(s)
Preparation of the samples. Acetate buffer (0.07 mol/l, pH 5.0) was added to 1 ml urine until a pH of 5.0 was reached. Similarly, Tris buffer (2.5 mol/l, pH 7.5) was gradually added to a second 1 ml urine sample until pH reached 7.5 [7]. Once the optimal pH for the enzyme activity was obtained, an additional 500 µl acetate or Tris buffer were added in excess to make certain that the pH was kept in the same range during the entire incubation period. At that time, dilution of the urine was 1:9 in both samples.

Time-response assessment. Additional samples of each (n=4) urine fraction were prepared as above in 1:9 proportions by adding 200 µl buffer (acetate or Tris) to 25 µl urine. Acetate-buffered urine samples were then supplemented with 1000 U (8.4 µl) ß-glucuronidase from a stock solution containing 119 000 U/ml. For the addition of sulphatase, a stock solution containing 19 U/ml was used and 0.019 U (1.0 µl) was added to Tris-buffered samples. An identical volume of an isotonic saline solution was added in parallel to another buffered urine sample as a control experiment. After mixing, the samples were incubated at 37°C for 0, 15, 30, 60 and 120 min. Incubations were performed in triplicate. After incubation, the samples were immediately analysed with HPLC as described below. Two peak heights (unidentified and p-cresol peaks) were plotted against the incubation period. The optimal incubation time was then deduced from the resulting graph. This optimal incubation time was then used for the following dose-response experiments.

Dose-response assessment. Only urine collected during the first hour of the experiment was used because this fraction contained the highest peaks of both p-cresol and the additional peak. ß-Glucuronidase was added to acetate-buffered urine samples in 500, 750, 1000, 1500, 2000 or 2500 U (4.2, 6.3, 8.4, 12.6, 16.8 and 21.0 µl, respectively) quantities. Similarly, Tris-buffered samples were supplemented with 0.0095, 0.0133, 0.0190, 0.0285 or 0.0380 U (0.5, 0.7, 1.0, 1.5 and 2.0 µl, respectively) of sulphatase. Again, identical volumes of isotonic saline were added to a parallel set of buffered urine samples as control experiments. After vortexing, the samples were incubated at 37°C. The selected incubation period was determined from the optimal incubation period assessed from the time-response experiments. After incubation, the samples were immediately analysed by HPLC, as described below. All experiments were performed in triplicate. Both peak heights of the additional peak and p-cresol were plotted against the added units of ß-glucuronidase or sulphatase.

Assay of p-cresol and the additional peak by HPLC. Samples were analysed by reverse phase (RP)-HPLC as previously described [9]. In brief, 15 µl HCl (6 mol/l) was added to 100 µl diluted urine to create optimal pH and to stop enzyme activity. After addition of 100 mg NaCl and vortexing, p-cresol and its metabolite were extracted with 4 ml isopropyl ether. After centrifugation, the organic phase was separated from the aqueous phase and 100 µl NaOH (0.05 mol/l in methanol) was added to 3 ml of the ether fraction. In experiments using acetate buffer, double the amount of NaOH was necessary. Subsequently, the internal standard (2,6-dimethylphenol) was added and then methanol and isopropyl ether were evaporated in a vacuum centrifuge (RC 10.22; Jouan, Saint-Herblain, France). The dry residue was then redissolved in 150 µl HCl (0.05 mol/l) and then 50 µl was analysed by RP-HPLC, as previously described [9]. A calibration curve was made from standard solutions that were prepared from a stock solution of p-cresol (0.032 g/l) and treated in the same way as explained above.

To evaluate whether the above procedure yielded a complete extraction of the additional peak, 250 µl urine was first submitted to extraction with 10 ml isopropyl ether followed by separation of the aqueous phase from the ether phase. Subsequently, 100 µl of the aqueous phase was again extracted with 4 ml isopropyl ether and analysed after redissolving the dried residue.

Linear regression. To evaluate whether the additional peak represented p-cresylglucuronide, in whole or in part, and whether its decrease after enzyme treatment resulted in the generation of an equivalent amount of p-cresol, a linear regression analysis was made between the peak heights of the additional peak both before addition of the enzyme and the increase in p-cresol peak height after the enzyme treatment. All data from the plateau of the dose response experiment were used for the analysis.

Assay of p-cresylglucuronide by ES-MS. ES-MS was performed using a Quattro LC (Micromass, Manchester, UK) electrospray triple quadrupole mass spectrometer equipped with a Z-sprayTM interface. The samples were infused using a syringe pump (Harvard Apparatus, South Natick, MA) set at a flow rate of 10 µl/min. The electrospray needle was held at 2.8 kV and was operated in the negative ion mode. Standards of p-cresol, p-nitrophenylglucuronide and the isolated fraction from the RP-HPLC separation were diluted in methanol/water (1:1), to which 1% NH3 was added to improve ionization. The product ion scans were performed using a collision energy of 10 eV.

Calculation of the urinary excretion of p-cresol
The percentage of the administered dose that was excreted in the urine was calculated at time points 60, 120, 180 and 240 min according to: Go


(001)

Urinary excretion was expressed as the amount of p-cresol in mg (before and after addition of ß-glucuronidase to the urine) that had been recovered in the urine from the start of the experiment up to the time-point t of measurement (At,urine).

Statistics
Results are expressed as means±SD. Control and enzyme-treated samples were compared using Mann–Whitney U-tests. A one-way analysis of variance was used to detect differences in peak heights during the time-response and dose-response experiments. Bonferroni tests for multiple comparisons were used for post-test comparisons. P-values of<0.05 were considered significant.



   Results
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 Abstract
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 Subjects and methods
 Results
 Discussion
 References
 
Validation of the extraction method for the assessment of the unidentified peak
Neither p-cresol nor the unidentified peak were found in the aqueous phase of samples submitted to the extraction procedure (data not shown), indicating that extraction was nearly complete for both compounds.

Addition of ß-glucuronidase
Peak heights of both p-cresol and the unidentified peak in urine obtained after intravenous administration of p-cresol were not influenced by addition of acetate buffer by itself, isotonic saline by itself or by a combination of both (data not shown). Addition of 500, 750, 1000, 1500, 2000 or 2500 U ß-glucuronidase in non-buffered samples resulted in smaller peak height changes than when buffer was added (data not shown).

Time response. Figure 1Go depicts HPLC chromatograms of a buffered urine sample before and after addition of 1000 U ß-glucuronidase with 60 min of incubation. After addition of the enzyme, there was a decrease in the unidentified peak (metabolite) and a substantial increase in the p-cresol peak.



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Fig. 1.  HPLC chromatograms of a urine sample before (A) and after (B) addition of 1000 U ß-glucuronidase and 60 min of incubation at 37°C. IS, internal standard.

 
Figure 2Go shows the evolution of both peaks in the first 1 h urine collection (F0–60) during the incubation period, after the samples had been buffered and supplemented with 1000 U ß-glucuronidase. Although the evolution of both peaks after treatment was similar in collections after the first hour, they showed progressively lower peak heights. Therefore, we described only the evolution of F0–60. In control experiments, incubation did not affect peak heights. In enzyme-treated samples, a progressive decrease in the unidentified peak and a parallel increase in the p-cresol peak were observed up to 30 min, followed by a plateau for both peaks. Although the unidentified peak did not completely disappear, a decrease of at least 85% was registered during the plateau phase. Because the 60 min incubation period produced peak heights located in the middle of the plateau, this period length was used in the dose-response experiments.



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Fig. 2.  Time course of urinary p-cresol (dashed lines) and the unidentified peak (solid lines) in F0–60 after addition of 1000 U ß-glucuronidase (circles) or an equivalent amount of isotonic saline (control, open squares), followed by incubation at 37°C. Filled circles, P<0.001 vs 0 min; *P<0.01 vs 15 min; filled squares, P<0.05 vs control.

 
Dose responses. The influence of increasing amounts of ß-glucuronidase on peak heights in F0–60 after 60 min of incubation is shown in Figure 3Go. In control experiments, equivalent volumes of isotonic saline did not influence peak heights. Increasing amounts of ß-glucuronidase caused progressive decreases in the unidentified peak and parallel increases in the p-cresol peak up to 750 U of the enzyme, followed by a plateau phase for both peaks. The enzyme failed to produce complete disappearance of the unidentified peak. Nevertheless, the unidentified peak decreased by at least 86% during the plateau phase.



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Fig. 3.  Influence of ß-glucuronidase (circles) or equivalent amount of isotonic saline (control, squares) on the evolution of urinary p-cresol (dashed lines) and the unidentified peak (solid lines) in F0–60 after incubation at 37°C for 60 min. *P<0.001 vs 0 U; filled squares, P<0.001 vs 500 U; filled squares, P<0.01 vs control.

 
Linear regression analyses. Absolute differences between peak heights obtained before and after enzyme treatment were calculated both for the p-cresol and unidentified peaks.

For each of the incubation periods (30, 60 and 120 min), a very strong linear relationship was reached between both peaks (r=0.997, 0.998 and 0.997, respectively). The 60 min incubation with ß-glucuronidase, given as a representative experiment for all time-response studies, yielded a regression curve with the equation: Go


(002)
(n=12, r2=0.996, P<0.0001), where {Delta}pC represents the increase in p-cresol peak height after the addition of ß-glucuronidase and M represents the peak height of the unidentified peak before enzyme treatment. The regression equations for 30 and 120 min yielded {Delta}pC-values that were not significantly different from the values at 60 min.

MS of the isolated unidentified peak. Figure 4Go shows MS spectra of a 1:90 dilution of the isolated unidentified peak using ES-MS in the negative ion mode. In Figure 4AGo, a fragment with m/z=283.2 is distinguished. The product ion spectrum of the isolated fragment with m/z 283.2 shows daughter ions with m/z at 174.8, 112.9 and 107.0 (Figure 4BGo). The fragments with m/z 112.9 and 174.8 can also be found in the product ion spectrum of a standard of p-nitrophenylglucuronide (data not shown), suggesting that these peaks represent fragments of the glucuronide ion. A separate MS analysis of a p-cresol standard solution produced a fragment with m/z 107, representing the [M-H]- fragment of p-cresol (data not shown). Altogether, these data indicate that the peak with m/z 283.2 in Figure 4AGo corresponds to the [M-H]- fragment of p-cresylglucuronide (MW=284 Da). However, the presence of other peaks in Figure 4AGo suggests that additional substances were present in the isolated unidentified peak fraction.



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Fig. 4.  MS spectrum (A) of the isolated unidentified peak and MS/MS spectrum (B) of the peak with m/z=283.2.

 

Addition of sulphatase
Peak heights of both p-cresol and the unidentified peak in urine obtained after administration of p-cresol to rats were not influenced by adding Tris buffer alone, isotonic saline alone or a combination of both (data not shown). Addition of 0.0095, 0.0133, 0.0190, 0.0285 and 0.0380 U sulphatase to Tris-buffered urine and subsequent incubation for 60 min did not alter the p-cresol and unidentified peak heights (data not shown). Neither the highest sulphatase dose (0.0380 U) followed by 3 h incubation nor addition of five times higher amounts of sulphatase (0.1900 U, 10 µl) resulted in altered peak heights (data not shown).

Calculation of urinary excretion and renal clearance
In a previous study that examined only p-cresol, administration of the compound resulted in a urinary excretion of 0.61±0.28 mg of p-cresol (20.3±9.3% of the administered dose) in rats with normal renal function [13]. Because our findings indicate that the unidentified peak mainly represents p-cresylglucuronide, excretion of both p-cresol and p-cresylglucuronide are urinary removal pathways for p-cresol. By extrapolation of Equation 2 as applied to the unidentified peak heights in the urine from a previous study [13], we calculated that addition of 1000 U ß-glucuronidase to each of the four urine fractions would liberate 1.92±0.35 mg p-cresol from the four urine fractions. Thus, 64.0±11.7% of the initially injected p-cresol had been glucuronated and excreted as a glucuronide in the urine during the experiment. The total amount of p-cresol (unconjugated and conjugated) excreted in the urine was therefore 2.53±0.29 mg (84.3±9.7% of the administered dose). This value was substantially higher than values obtained when p-cresol was tested by itself (20.1±5.4%; P<0.01).

Figure 5Go shows the cumulative urinary excretion of p-cresol compared with the sum of p-cresol and p-cresylglucuronide (expressed as equivalent amounts of p-cresol) as a function of time after administration of p-cresol. Significant differences were found at the 120th min and thereafter.



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Fig. 5.  Cumulative excretion of p-cresol compared with the sum of p-cresol and p-cresylglucuronide (expressed as equivalent amounts of p-cresol) in the urine. *P<0.05 vs p-cresol alone; **P<0.01 vs p-cresol alone; filled squares, P<0.01 vs min 60.

 



   Discussion
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 Subjects and methods
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 References
 
Administrated compounds are eliminated from the blood either by excretion or by biotransformation. p-Cresol is a compound that accumulates in uraemic patients and is not adequately removed by haemodialysis [12]. Adequate removal of such compounds depends upon knowledge of their kinetic behaviour and metabolic pathways. The present study was undertaken to investigate the biotransformation of p-cresol. We found that intravenous p-cresol injection in rats produced an immediate p-cresol peak and an unidentified peak on chromatograms from serum and urine. The main findings of the present experiments were:

  1. Addition of ß-glucuronidase to urine samples resulted in a decrease in peak height of the unidentified peak and a parallel increase in the p-cresol peak.
  2. MS and tandem MS (MS/MS) analysis of the isolated unidentified peak showed the presence of p-cresylglucuronide.
  3. The unidentified peak was not entirely hydrolysed by treatment with ß-glucuronidase.
  4. When urinary p-cresylglucuronide was combined with p-cresol, ~85% of administered p-cresol was recovered in the urine.
Decreases in the unidentified peak and parallel increases in the p-cresol peak following addition of ß-glucuronidase to the urine indicated the presence of p-cresol. Time- and dose-response experiments (Figures 2Go and 3Go) showed plateau phases for both peaks, but enzyme treatment never produced the complete disappearance of the unidentified peak.

The presence of p-cresylglucuronide was also confirmed by MS analysis of the isolated unidentified HPLC-peak, revealing a peak with m/z 283 and, by MS/MS analysis, showing two daughter peaks with respective m/z ratios of 107 (p-cresol) and 175 (glucuronide ion) (Figure 4Go). However, additional peaks in Figure 4AGo indicated that other substances were also present in the isolated fraction of the unidentified peak, which explains why this peak did not entirely disappear after enzyme treatment. We were unable to identify these compounds in the present study. Nevertheless, our results indicate that p-cresylglucuronide represents ~85% of the unknown peak.

If a substantial portion of administered p-cresol is conjugated, then interpretation of the urinary excretion using only p-cresol (as was performed in our previous study [14]) may lead to the erroneous conclusion that large amounts of p-cresol, in whatever form, still remain in the body or are eliminated via other routes. We performed a linear regression analysis to determine the amount of p-cresol represented in the unidentified peak. From this regression line and without taking this metabolite into account, we attempted to obtain kinetic data values similar to those previously reported [14]. Such a calculation produces only approximate figures, since data obtained in the present study were extrapolated from results obtained from previous experiments. Nevertheless, it is possible that these calculations provided accurate results since these experiments were performed with the same rat strains having normal renal function, similar body weight and urinary production and under similar conditions.

The combination of p-cresol with its glucuronide produced ~85% urinary recovery of administered p-cresol: 20% as p-cresol and ~65% as p-cresylglucuronide. Thus, the elimination of p-cresol under normal conditions in the rat can be largely attributed to the urinary excretion of p-cresylglucuronide. It is generally accepted that anaesthesia may have a negative impact on glomerular filtration and renal blood flow. However, it is not presently known whether Na-pentobarbital influences the urinary excretion of p-cresol, p-cresylglucuronide or similar compounds or whether other anaesthetics would have produced the same results. However, it would have been difficult, if not impossible, to perform similar experiments in unanaesthetized rats.

Conjugation by glucuronidation is a major route of biotransformation for compounds with functional hydroxyl, thiol, amine or carbonyl groups [15]. As a result, substances become more hydrophilic, which enhances their renal elimination [16]. This also explains why patients with renal insufficiency accumulate glucuronides [16].

There is evidence that glucuronides of certain drugs, such as morphine, have a direct pharmacological activity (for review see [16]). However, the endogenous presence of ß-glucuronidase increases the risk that these metabolites become retransformed into the often more toxic parent compound. Nevertheless, interindividual variability in ß-glucuronidase activity has been shown in human liver and kidney [17]. These considerations indicate that glucuronides, although they are metabolites, might play a role in biochemical and biological side-effects. However, to our knowledge, the biochemical impact and toxicity of p-cresylglucuronide have never been investigated.

Ogata et al. [7] reported the presence of a sulphate conjugate of p-cresol after administration of wood creosote to healthy volunteers and recently, urinary p-cresylsulphate was linked with clinical changes in multiple sclerosis [18]. We previously demonstrated that the chemically prepared conjugate p-cresylsulphate had no capacity to inhibit leukocyte responses to phagocytosis, which is in contrast to p-cresol [4]. Based on these observations, we performed additional experiments in order to add sulphatase to the urine samples. We did not observe a decrease in the unidentified peak or an increase in the p-cresol peak. This finding indicates that p-cresylsulphate was not produced by the metabolization of p-cresol and was therefore not present in the fraction represented by the unidentified peak. This is in agreement with the recent suggestion that p-cresylsulphate is probably generated from tyrosine sulphate [19]. Nevertheless, it is possible that p-cresylsulphate may have been cleaved, at least in part, as a result of the deproteinization methods. This latter effect may have been kinetically less important, at least for urinary excretion, because the metabolite would then have been recovered as p-cresol in the urine. In addition, it has been demonstrated that large doses of phenolic substances, as were present in the current experiments, are glucuronidated rather than sulphated [20]. The amount of p-cresol injected in the present study had been previously determined to induce a serum concentration, 5 min after the injection, that corresponded to the concentration currently observed in end-stage renal disease patients (1 mg/dl).

Even after enzyme treatment, ~15% of administered p-cresol was still not recovered. Further experiments are warranted to determine the distribution of the remaining portion of p-cresol in the body (e.g. fat tissue). It is likely that at least part of the missing 15% is excreted with bile. It is additionally possible that the conjugation of p-cresol to p-cresylglucuronide, its renal excretion or both are hampered in renal failure. Therefore, we will perform future experiments with uraemic rats.

In conclusion, the majority of the unidentified peak that appeared in urine chromatograms from p-cresol-treated rats was identified as p-cresylglucuronide. This finding was corroborated both by incubation of the urine with ß-glucuronidase and by MS and MS/MS analysis of the unidentified peak. We further established a regression equation that enables calculation of the underlying amount of p-cresol in rats with normal renal function.



   Acknowledgments
 
This study was supported by a grant (51U00198) from the Baxter Healthcare Extramural Grant Program and a grant (993212) from the fund for Scientific Research (Fonds voor Wetenschappelijk Onderzoek, FWO-Vlaanderen). G.L. was supported by a scholarship from the Else Kröner Fresenius Stiftung.

Conflict of interest statement. None declared.



   Notes
 
Correspondence and offprint requests to: Gerrit Lesaffer, Analytical Chemistry, KAHO Sint-Lieven, Gildestraat 17, Gent, Belgium. Email: gerrit.lesaffer{at}kahosl.be Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 23. 6.02
Accepted in revised form: 19.11.02