1 Renal Division and 2 Department of Pharmacology, Heymans Institute, University Hospital and 3 Laboratory of Analytical Chemistry, University of Gent, Gent, Belgium
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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:
| (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 MannWhitney 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 1 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.
|
|
|
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:
| (002) |
MS of the isolated unidentified peak. Figure 4 shows MS spectra of a 1:90 dilution of the isolated unidentified peak using ES-MS in the negative ion mode. In Figure 4A
, 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 4B
). 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 4A
corresponds to the [M-H]- fragment of p-cresylglucuronide (MW=284 Da). However, the presence of other peaks in Figure 4A
suggests that additional substances were present in the isolated unidentified peak fraction.
|
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 5 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.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 4). However, additional peaks in Figure 4A
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 |
---|
Conflict of interest statement. None declared.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|