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Technical Briefs |
1 Laboratory of Digestion and Absorption,2 Laboratory of Nephrology, and3 Department of Medicine, Division of Nephrology, University Hospital Gasthuisberg K.U. Leuven, Leuven, Belgium;
aaddress correspondence to this author at: Laboratory of Digestion and Absorption, University Hospital Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium; fax 32-16-344399, e-mail Kristin.Verbeke{at}uz.kuleuven.ac.be
p-Cresol (4-methylphenol; 108 Da) is a protein-bound solute retained within the body in renal failure (1). p-Cresol is of interest because it has several toxic effects in vitro (2)(3)(4)(5)(6) and clinical correlates have been demonstrated (7)(8). In the absence of external exposure (9), p-cresol originates uniquely from bacterial tyrosine fermentation in the large intestine (10). During passage through the colonic mucosa and liver, it is detoxified by conjugation processes (sulfation and glucuronidation) (11)(12)(13). Thus, one might expect to find p-cresylsulfate and p-cresylglucuronide in the serum, but reports on conjugated p-cresol in renal failure patients are scarce (14)(15)(16). Most techniques to deproteinize serum samples (e.g., heat and acidification) may also partially hydrolyze sulfate esters and glucuronide bonds. Hence, the "total" (i.e., protein-bound and unbound) and "unbound" p-cresol reported in most studies probably reflect both unconjugated and (part of the) conjugated forms of the solute (17)(18)(19)(20)(21). We determined the extent of desulfation and deglucuronidation by deproteinization with heat and acid followed by gas chromatographic–mass spectrometric (GC-MS) analysis (19) with p-nitrophenylglucuronide and p-nitrophenylsulfate as model substrates. We also calculated exact amounts of unconjugated p-cresol, p-cresylsulfate, and p-cresylglucuronide in serum of hemodialysis patients and healthy controls.
Percentage desulfation and deglucuronidation by different methods (see below) was determined for random serum samples. Further analyses were performed on 9 serum pools from hemodialysis patients [n = 86; 49 male; mean (SD) age, 69.8 (1.5) years] and 5 serum pools from healthy controls [n = 29; 10 male; 31.0 (1.4) years; creatinine clearance, 87.1 (1.4) mL · min–1 · (1.73 m2)–1]. Serum pools were stored at –80 °C until analysis. The ethics committee of the University Hospital Leuven approved the study, and informed consent was obtained from all participants. p-Nitrophenylglucuronide, p-nitrophenylsulfate, and 2,6-dimethylphenol (all >98% purity) were from Sigma-Aldrich; p-cresol and p-nitrophenol were from Supelco; and ß-glucuronidase (Escherichia coli K12) was from Roche. Other materials were of analytical grade.
Several methods were used for sample preparation. Method A [acid and heat deproteinization (19)] consisted of dilution of 500 µL of serum with 350 µL of water; addition of 100 µL of p-nitrophenylglucuronide (1.2 g/L) or 100 µL of p-nitrophenylsulfate (1 g/L) to determine percentage deglucuronidation or desulfation, respectively; adjusting to pH 1 with H2SO4; heating to 90 °C for 30 min; cooling to room temperature; addition of 50 µL of 2,6-dimethylphenol (200 mg/L) as internal standard; extraction with 1 mL of ethyl acetate; mixing for 30 s; centrifugation at 1583g for 20 min; drying of 500 µL of supernatant over anhydrous sodium sulfate; and transfer of 100 µL to the GC-MS instrument.
Method B (enzymatic deglucuronidation + method A) included addition of 1 mL of phosphate buffer (0.1 mol/L; pH 7.0) to 500 µL of serum and 100 µL of p-nitrophenylglucuronide (1.2 g/L) or 100 µL of p-nitrophenylsulfate (1 g/L); addition of 20 µL of ß-glucuronidase; incubation at 37 °C for 30 min; and further acidification, heating, and extraction according to method A, except that 2 mL of ethyl acetate was used in the extraction step.
Method C (acetone deproteinization) consisted of the addition of 100 µL of p-nitrophenylglucuronide (7 g/L) or 100 µL of p-nitrophenylsulfate (6 g/L) to 500 µL of serum; addition of 3 mL of cold acetone; centrifugation at 1583g for 10 min; separation of supernatant from pellet by decantation; adjusting to pH 5 with HCl (0.6 mol/L); addition of 100 µL of 2,6-dimethylphenol (200 mg/L) as internal standard; extraction with 3 mL of chloroform; mixing for 30 s; centrifugation at 1583g for 20 min; drying of 500 µL of the lower layer over anhydrous sodium sulfate; and transfer of 100 µL to the GC-MS instrument.
Method D (ultrafiltration + method A) for determination of the unbound fraction of p-cresol included ultrafiltration of 1 mL of serum at 2000g for 60 min at room temperature with ultrafiltration membrane (cutoff, 30 000 Da; Centrifree and MPS Micropartition UF Devices; Amicon); dilution of 500 µL of filtrate with 450 µL of water; and further acidification, heating, and extraction according to method A (19).
Sample preparation was followed by separation and identification by GC-MS (Trace GC-MS; Thermofinnigan) as follows: splitless injection of 0.5 µL of sample on an AT5-ms analytical column [30 m x 0.32 mm (i.d.); 1-µm film thickness; Alltech Associates] with GC-grade helium (1.3 mL/min) as the carrier gas; oven programming from 75 °C (isotherm for 5 min) to 280 °C (15 °C/min); and identification by MS (electron impact full scan mode from m/z 59 to m/z 590 at 2 scans/s). Quantitative results were obtained by the internal standard method. The detection limit (LOD) for p-cresol was 0.15 mg/L and the quantification limit (LOQ) was 0.30 mg/L. The LOD and LOQ for p-nitrophenol were 0.5 and 2 mg/L, respectively. The LOD and LOQ were defined empirically (22). Values below the LOQ were considered zero for calculations. Three aliquots of each sample were measured, and mean values are reported. Interassay CVs were calculated from these triple measurements. The intraassay CV was determined by injecting a single sample 3 times.
The mean (SD) yield of hydrolysis of p-nitrophenylglucuronide by method A was 16.0 (0.2)% and that for p-nitrophenylsulfate was 97.9 (6.5)%. Thus, the amount of p-cresol measured by method A comprises the unconjugated p-cresol, 16% of glucuronidated p-cresol, and almost all p-cresylsulfate present in the serum (Eq. 1
).
 | (1) |
The enzymatic reaction used in method B led to complete deconjugation of p-nitrophenylglucuronide [yield, 101.3 (3.0)%]. Hydrolysis of p-nitrophenylsulfate was 97.8 (2.2)%. Thus, the amount of p-cresol measured by method B comprises almost all forms of p-cresol in the serum (Eq. 2 ).
 | (2) |
Method C was intended to avoid deconjugation and hence to measure only unconjugated p-cresol. Although there was no measurable deconjugation of the glucuronidated substrate (below the LOD), limited hydrolysis [7.2 (5.1)%] of p-nitrophenylsulfate was observed (Eq. 3 ).
 | (3) |
The p-cresol concentrations measured in serum pools of HD patients (pools I to IX) and healthy controls (pools X to XIV) when we used methods A, B, and C are shown in Table 1
. Combining of Eqs. 1–3
allowed calculation of the exact amounts of unconjugated p-cresol, p-cresylglucuronide, and p-cresylsulfate in each pool (Table 1
, middle portion). Total p-cresol was significantly higher in patients than in controls [24.25 (4.98) mg/L vs 1.86 (0.31) mg/L; P <0.0001]. In both groups, most p-cresol was in its sulfated form [95.5 (3.3)% vs 98.2 (1.6)%; difference not significant], and a small proportion was glucuronidated [3.8 (2.4)% vs 1.8 (1.6)%; difference not significant]. Unconjugated p-cresol was undetectably low in most of the participants. p-Cresol concentrations measured with method A constituted 90.2 (3.0)% of the total p-cresol [patients, 90.6 (3.6)%; controls 89.5 (1.1)%; difference not significant]. The median (range) concentration of unbound p-cresol (method D) was 3.22 (2.01–5.65) mg/L in the patients (nongaussian distribution). This concentration was 16.8 (5.4)% of the concentration found when method A was applied to unfiltered samples. Unbound p-cresol was below the LOD in the controls (Table 1
, last rows). The interassay variability was low for all methods (Table 1
, last column). The intraassay CV for measurement of p-cresol was 0.68%.
|
Three important conclusions can be made from our study. First, most p-cresol in human serum is present as its sulfated metabolite, and a small proportion is glucuronidated. Unconjugated p-cresol is undetectably low in most cases. p-Cresylsulfate has previously been reported to be the major metabolite of p-cresol (14)(15). This finding is not surprising because colonic mucosal cells, considered to be more important than the liver for detoxification of phenols originating from the intestinal lumen (12), conjugate phenols with a preponderance of sulfation over glucuronidation (12)(13). By contrast, the major p-cresol metabolite after intravenous administration in rats was found to be p-cresylglucuronide (16), possibly resulting from shortcutting of the intestinal mucosa in addition to reflecting species differences between rats and humans (23) (see Supplement 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue8/).
The second conclusion is that non–protein-bound p-cresol (method A on ultrafiltered serum) accounts for 16.8 (5.4)% of total p-cresol in uremic serum and is below the LOD in healthy controls, which is in agreement with data in the literature (18) (see Supplement 2 in the online Data Supplement).
Finally, our combined acid and heat deproteinization method (19) hydrolyzed 
16% of the p-cresylglucuronide and 98% of p-cresylsulfate. p-Nitrophenol conjugates were used as model substrates for determination of the percentage of deconjugation because of their structural similarities with conjugated p-cresol metabolites and because no background p-nitrophenol was detected in native serum (data not shown). For calculation of the exact concentrations of p-cresol and its metabolites in serum, 2 other sample preparation methods were developed, each measuring clearly different portions of total p-cresol and, as such, allowing mathematical solution of 3 equations with 3 unknowns. The data clearly indicated that the measured concentrations of p-cresol may be different when different analytical procedures are used. This may explain the higher serum concentrations of p-cresol that we found compared with other values in the literature (14)(17)(18). Our acid and heat deproteinization method measured 90% of total serum p-cresol [median (range) reference values, 1.58 (0.39–6.23) mg/L for a control population (n = 24) and 20.10 (0.30–60.45) mg/L for hemodialysis patients (n = 107)]. Ideally, by combining complete enzymatic deconjugation and deproteinization, one would be able to measure 100%. This approach would be cumbersome, time-consuming, and more expensive. It appears reasonable to adopt an analytical protocol that enables measurement of a well-defined portion of the total concentration. Nevertheless, the applied sample preparation should be taken into account when comparing the results of different analytical methods.
Acknowledgments
We thank M. Dekens for excellent technical assistance. D. Kuypers is acknowledged for providing scientific comments and reviewing the manuscript. We also wish to thank all participants as well as the renal nurses and physicians taking care of them. The study was supported by governmental grants from the Fonds voor Wetenschappelijk Onderzoek (Grants 1127602N and G.0230.04).
References
The following articles in journals at HighWire Press have cited this article:
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  T. A. Clayton, D. Baker, J. C. Lindon, J. R. Everett, and J. K. Nicholson From the Cover: Pharmacometabonomic identification of a significant host-microbiome metabolic interaction affecting human drug metabolism PNAS, August 25, 2009; 106(34): 14728 - 14733. [Abstract] [Full Text] [PDF]
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