A sensitive HPLC method for the quantification of free and total p-cresol in patients with chronic renal failure

Clinica Chimica Acta 278 (1998) 1–21

A sensitive HPLC method for the quantification of free and total p-cresol in patients with chronic renal failure a, c b a Rita De Smet *, Frank David , Patrick Sandra , Jaqueline Van Kaer , a a a Gerrit Lesaffer , Annemieke Dhondt , Norbert Lameire , Raymond Vanholder a a

Renal Division, Department of Internal Medicine, University Hospital Gent, Gent, Belgium b Department of Organic Chemistry, University of Gent, Gent, Belgium c Research Institute for Chromatography, Kortrijk, Belgium

Received 9 March 1998; received in revised form 10 March 1998; accepted 7 August 1998

Abstract Para-cresol (4-methylphenol) is a volatile phenolic compound which is retained in chronic renal failure. Several recent studies suggest that p-cresol interferes with various biochemical and physiological functions at concentrations currently observed in uremia. Only a few methods are available for the determination of p-cresol concentration in serum. In addition, these methods have only been used for the determination of total p-cresol. In particular, the evolution of free (non-protein bound) p-cresol is of concern, because conceivably this is the biologically active fraction. The concentration of free p-cresol, is, however, markedly lower than that of total p-cresol, in view of its important protein binding. We report a method enabling the measurement of total and free p-cresol concentration in serum of healthy controls and uremic patients. Deproteinization, extraction and HPLC procedure are efficient, without interference of other protein bound ligands and / or precursors of p-cresol or phenol. By means of spiking experiments, the measurement of the UV absorbance over the 200–400 nm wavelength range, and capillary gas chromatography–mass spectrometry, the considered compound is identified as p-cresol. With a fluoresence detection at 284 / 310 nm as extinction / emission wavelengths the detection limit of p-cresol is 1.3 mmol / l (0.14 mg / ml). Recovery of added p-cresol to normal serum is 95.464.1%. For free p-cresol and total p-cresol determinations, intra-assay and day-to-day variation coefficients are 3.2%, 4.2%, 6.9% and 7.3%, respectively. Compared to healthy controls, the serum p-cresol levels are 7–10 times higher in continuous ambulatory peritoneal dialysis patients (CAPD), uremic outpatients, and hemodialysis patients: 8.663.0 vs. 62.0619.5, 87.8631.7 and 88.7649.3 mmol / l (0.9360.32 vs. 6.7062.11, 9.4963.43, and 9.6065.30 mg / ml) ( p , 0.05), *Corresponding author. Tel.: 1 32-9-2404518; fax: 1 32-9-2404599; e-mail: [email protected] 0009-8981 / 98 / $ – see front matter  1998 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 98 )00124-7

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respectively. The difference is even more important if free p-cresol is considered. This corresponds to a decreased protein binding in uremic patients. We conclude that the present method allows an accurate measurement of both total and free p-cresol, and that the measured concentrations in uremia are in the range which may cause biochemical alterations.  1998 Elsevier Science B.V. All rights reserved. Keywords: Uremia; Protein binding; Reversed-phase chromatography; Spectrophotometry; Gas chromatography–mass spectrometry

1. Introduction Para-cresol (4-methylphenol) is a metabolite of phenylalanine and tyrosine which is mainly produced by intestinal bacteria [1]. It is eliminated by the healthy kidneys [2] and the serum concentration is increased in renal failure [3]. We recently demonstrated that p-cresol exerts a dose-dependent inhibitory effect on the NADPH-oxidase enzyme system of polymorphonuclear granulocytes and on their capacity for glucose consumption and for production of free radical species in response to phagocytosis [4]. Hence, this compound might play a role in the enhanced susceptibility to infection of the uremic patient [5]. In addition, it was demonstrated that aluminum uptake and its toxicity on hepatocyte enzyme functions were enhanced in the presence of p-cresol [6]. Other biological and physiological functions are also affected by p-cresol: it increases the free active drug fraction of warfarin and diazepam after addition to normal serum [7]; it increases the permeability and composition of the cell membrane [8,9]; it inhibits the release by rat peritoneal macrophages of platelet activating factor [10], and it induces an increased LDH-leakage from rat liver slices into the incubation medium [11]. In spite of this suggested toxicity, the determination of the concentration of p-cresol remains cumbersome and labor-intensive, whereas recent comparative data between actual therapeutic strategies on serum levels in various subgroups of the uremic population is lacking. Various methods for the determination of p-cresol have been developed but most of them have been applied for the measurement of urinary levels [2,12,13], as p-cresol is a current marker for environmental contact with toluene [14,15]. However, these methods are in general not applicable to sera, since urinary estimations do not necessitate deproteinization, and since expected concentrations are markedly higher in urine than in normal and even uremic serum. To our knowledge, up to now only three reliable methods for the determination of p-cresol in serum have been described [16–18]. The latter methods have been used to measure total p-cresol. However, one of the major

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concerns, especially in renal failure, is the determination of free p-cresol, which can be considered as the fraction exerting toxicity. Free p-cresol concentrations can be expected to be extremely low, in view of the compound’s important protein binding. The present paper aims at the development and description of a method that is sensitive enough to measure these low concentrations of p-cresol, and at the analysis of its efficacy.

2. Material and methods

2.1. Patients Primary analyses were undertaken on pooled serum samples from six patients, collected before the start of a hemodialysis session. In addition, to analyze the evolution of p-cresol during various stages of renal failure, separate blood samples from nine healthy subjects, seven outpatients with end-stage renal disease, eleven patients on hemodialysis (blood collection before the start of the dialysis session) and nine patients on continuous ambulatory peritoneal dialysis (CAPD) were evaluated. The hemodialysis patients were treated 12 h weekly by conventional hemodialysis with low flux polysulfone membranes (BLS 643, Bellco-Sorin, Mirandola, Italy); blood flow was 280 ml / min and dialysate flow 500 ml / min. CAPD-treatment was applied with four exchanges of 2 l, with dialysate containing 1.36–3.86% glucose as osmotic agent. Glucose concentration was chosen as a function of the volume status of the patient. This investigation was approved by the local ethics committee.

2.2. Reagents All chemicals were obtained from Sigma Chemical (St. Louis, MO), except HPLC water, HPLC methanol and isopropyl ether which were purchased from Acros Organics (New Jersey) and ammonia solution which was purchased from BDH Laboratory Supplies (Poole, UK). The reagents for the determination of urea and of serum total protein were obtained from Sigma Diagnostics (St. Louis, MO), the creatinine reagent was from Analis (Namur, Belgium).

2.3. Sample preparation Blood was collected in tubes with gel and coagulation activator (Venoject II, Terumo Europe, Leuven, Belgium) and the blood samples were centrifuged at 3000 rpm. One ml serum was ultrafiltered at ambient temperature for 30 min at

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6000 rpm with a Centrifree ultrafiltration membrane (Centrifree Micropartition Devices, Amicon, Beverly, MA) with a cut-off of 30 000 Da to obtain serum filtrate. In the serum samples, total (protein bound 1 non-bound) p-cresol was determined; free (non-protein bound) p-cresol was determined in the serum filtrates. Standard solutions at various p-cresol concentrations [from 4.6 to 2311.8 mmol / l (0.50–250.00 mg / ml)] were made in HPLC water from a stock p-cresol solution containing 23.1 mmol / l (2.50 mg / ml). For comparison of chromatographic behavior with other structurally related solutes, a solution containing the phenolic compounds p-cresol [9.2 mmol / l (1.00 mg / ml)], o-cresol [46.2 mmol / l (5.00 mg / ml)] and phenol [26.6 mmol / l (2.50 mg / ml)] was prepared. In addition, another standard solution containing several protein bound uremic retention solutes as well as precursors of p-cresol and phenol was prepared: hippuric acid [22.3 mmol / l (4.00 mg / ml)], indole-3-acetic acid [22.8 mmol / l (4.00 mg / ml)], indoxyl sulfate [79.6 mmol / l (20.00 mg / ml)], phenylalanine [2.4 mmol / l (0.40 mg / ml)], tryptophan [19.6 mmol / l (4.00 mg / ml)], tyrosine [8.8 mmol / l (1.60 mg / ml)], 4-hydroxyphenylacetic acid [52.6 mmol / l (8.0 mg / ml)], 4-hydroxybenzoic acid [289.6 mmol / l (40.00 mg / ml)] and p-cresol [9.2 mmol / l (1.00 mg / ml)]. Since hippuric acid was not detectable at 22.32 mmol / l (4.00 mg / ml) with the fluorescence detection method (see HPLC analysis) applied for the present studies, a separate analysis was performed with a higher concentration of 11.2 mmol / l (2.00 mg / ml).

2.3.1. Extraction procedure To displace p-cresol from binding proteins, acidification with HCl and saturation with NaCl were used, followed by extraction. To a 100 ml sample, 15 ml HCl (6 mol / l) and 1.71 mmol (100 mg) NaCl were added, each time followed by thorough mixing. Then 4 ml isopropyl ether was transferred into the tube and p-cresol was extracted by vigorous vortex-mixing for 60 s followed by centrifugation at 3000 cycles / min for 5 min. Three ml of the organic layer was transferred into another test tube and 50 ml of a solution of 2,6-dimethylphenol [204.6 mmol / l (25.00 mg / ml)] in methanol was added as the internal standard. Then 100 ml (50 mmol / l) NaOH in methanol was added and the isopropyl ether and methanol were evaporated in a vacuum-centrifuge (RC 10.22, Jouan, Saint-Herblain, France). The dry residue was redissolved in 150 ml (50 mmol / l) HCl in 40% methanol and 50 ml was injected on the RP-HPLC column. To exclude methodological bias, the crude and the ultrafiltered serum samples as well as the standard solutions were all submitted to the same extraction procedure.

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2.4. HPLC analysis The chromatograph consisted of two high pressure HPLC pumps (2150), a gradient controller (2152) and a solvent degasser (Degasys DG-13 10) from Pharmacia (Bronima, Sweden). The HPLC apparatus was connected to a fluorescence detector (RF530, Shimadzu, Tokyo, Japan). Fifty ml of a sample was loaded on the column with a manual injector (Valco, Houston, TX). Analyses were performed on a 5 mm RP C18 column of 4.6 3 150 mm (Ultrasphere ODS, Beckman Instruments, Fullerton, CA). The column was kept at room temperature. The extracted samples were separated with a linear gradient of methanol and 50 mmol / l ammonium formate buffer (pH 5 3.3) from 40 to 75% methanol for 13 min with a flow-rate of 1 ml / min. In preliminary studies, various fluorescent excitation and emission spectra and different combinations of excitation and emission spectra reported in the literature for p-cresol and analogous compounds [13,18,19] were evaluated. For the fluorescence detection of p-cresol in the study, the excitation and emission wavelengths of 284 nm and 310 nm were applied. The isopropyl ether extracts of serum, serum filtrate and standard solutions were analyzed, the peak height of p-cresol and of the internal standard were detected by fluorescence analysis, registered with an integrator (2221, Pharmacia, Bromma, Sweden) and as an index of concentration the relative peak height was calculated.

2.5. Identification of p-cresol 2.5.1. Capillary gas chromatography–mass spectrometry ( CGC–MS) The presence of p-cresol in the isopropyl ether extract of uremic serum was confirmed by capillary gas chromatography–mass spectrometry (CGC–MS). An aliquot (1 ml) of the isopropyl ether extract was concentrated under nitrogen and derivatised by trimethylsilylation [3]. The analysis was performed on a HP 5890 GC coupled to a HP 5972 MSD (Hewlett-Packard, Palo Alto, CA). From the derivatised sample, 1 ml was splitlessly injected. The separation was done on a 30 m 3 0.25 mm I.D. 3 0.25 mm HP-SMS column. Helium was used as carrier gas at a constant flow of 0.8 ml / mn. The column was programmed from 508C to 3008C at 108C / min. The mass spectrometer was operated in full scan mode (40–500 amu). A standard was derivatised in the same way as the serum extract. The presence of p-cresol in uremic serum was evaluated by comparing the mass spectrum and retention time. The molecular ion with mass charge ratio 180 (m /z) and the most specific fragment ion 165 (m /z) originating from the loss of a methyl radical, were considered.

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2.5.2. Spiking experiment Uremic serum (1000 ml) was spiked with 40 ml of a 2311.8 mmol / l (250.00 mg / ml) standard solution of p-cresol. The native uremic serum and the spiked sample were submitted to the extraction procedure and analyzed by RP-HPLC (n 5 2). 2.5.3. UV spectra Extraction of a p-cresol standard solution of 138.7 mmol / l (15.00 mg / ml) was undertaken twenty times consecutively. The same procedure was applied to a pooled uremic serum sample. Each series of twenty samples were combined, and then separated with the same reversed phase column but with a 3 ml sample loop to obtain enough material. From the presumed p-cresol peaks a UV absorption spectrum was recorded from 200 to 400 nm (Uvikon Spectrophotometer 941, Kontron Instruments, Zurich, Switzerland). The two spectra were compared with each other and with a spectrum of the crude (non-extracted) p-cresol solution [138.7 mmol / l (15.00 mg / ml)] in water. The wavelengths at which UV absorption reached maxima were also registered. 2.5.4. Interference with other solutes The standard solution with the phenolic compounds and the standard solution containing precursors of p-cresol and other uremic retention solutes that are protein bound in serum, were submitted to RP-HPLC to compare their retention times with that of p-cresol (n 5 6). 2.6. Quantification 2.6.1. Linear response The linearity of the detector response for p-cresol was controlled over a wide concentration range of 0.0 to 462.4 mmol / l (50.00 mg / ml) (n 5 3) and in the lower concentration region from 0.0 to 9.2 mmol / l (1.00 mg / ml) (n 5 3). The lower detection limit of the p-cresol concentration measurement with this method was determined as the concentration corresponding to a signal 3 S.D. above the mean for a blank standard solution (n 5 6). 2.6.2. Calibration curves Calibration curves of standard solutions containing 0.0, 11.6, 23.1, 46.2, 57.8, 115.6 and 231.2 mmol / l (1.25, 2.50, 5.00, 6.25, 12.50 and 25.00 mg / ml) p-cresol were obtained for 30 consecutive days, by correlating the concentration of the standard solutions and the registered relative peak heights. The correlation coefficients were calculated (n 5 30).

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2.6.3. Deproteinization methods The deproteinization method, as applied in the present study with HCl / NaCl, was compared (n 5 6) for its yield with other deproteinization methods reported in the literature: deproteinization by heat or by the addition of trichloroacetic acid (TCA) [20]. The effect of the application of HCl or NaCl alone was also evaluated; all samples were then submitted to isopropyl ether extraction and analyzed for total p-cresol concentration, evaluating the yield of the total p-cresol after deproteinization. 2.6.4. Alternative extraction methods The efficiency of isopropyl ether to extract p-cresol from the samples was compared with two other organic solvents: ethyl acetate and acetonitrile; p-cresol was extracted from uremic serum and uremic serum filtrate. The same procedure was followed as described above, the composition of the extraction solvent being the only variable factor. 2.7. Validation experiments 2.7.1. Adsorption of p-cresol on Centrifree Micropartition Filters To exclude losses of solute by adsorption on the ultrafiltration filters during the preparation of ultrafiltrate, concentrations of p-cresol were checked before and after ultrafiltration (n 5 5) of p-cresol standards with a concentration from 4.6 to 231.2 mmol / l (0.50–25.00 mg / ml). 2.7.2. Variability of retention times The variability of the retention time of the p-cresol peak and of the internal standard in the HPLC elution procedure was evaluated daily in sixteen samples over a 5-day period. 2.7.3. Reproducibility Reproducibility was evaluated in three samples, a normal serum, a uremic serum and a uremic serum filtrate, covering the expected range of concentrations. Intra-assay variation coefficients were calculated from ten measurements of the sample on the same day. To determine the day-to-day variation, samples were kept deep frozen at 2 208C until their evaluation (six consecutive days). 2.7.4. Recovery To 1 ml uremic serum, in which p-cresol concentration had been determined, known quantities of p-cresol [20 ml of a solution containing 1849.5 mmol / l (200.00 mg / ml)], corresponding to a net quantity of 37 mmol (4 mg) were added; p-cresol concentration was measured again and the percentage recovery

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of the added amount was calculated (n 5 6). The same procedure was repeated for normal serum to which 24.8 mmol / l (2.68 mg / ml) or 103.7 mmol / l (11.22) mg / ml p-cresol was added.

2.7.5. Concentration measurement in a commercial standard solution of pcresol To check the accuracy of the method, a p-cresol solution of a commercial standard of 0.4% (v / v) p-cresol in HPLC-grade methanol (Sigma Chemicals, St. Louis, MO) was diluted 500 times in HPLC water. The concentration of p-cresol was determined as described above. 2.8. Assays of serum concentration Four groups were analyzed: healthy controls, non-dialyzed outpatients with chronic renal failure with a serum creatine . 530 mmol / l ( . 60 mg / ml), and patients treated by hemodialysis and continuous ambulatory peritoneal dialysis. The concentration of total and free p-cresol in the serum samples was determined from the registered relative peak heights ( p-cresol vs. internal standard) using daily standard curves. The percentage of protein bound p-cresol in serum was calculated from the total and free concentration. Serum creatinine (Screa), serum urea, serum total protein and residual endogenous creatinine clearance were determined by routine laboratory methods.

2.9. Statistical analysis The results were expressed as mean6S.D. The different patient groups and healthy controls were compared with the non-parametric Mann-Whitney U test.

3. Results

3.1. Determination of optimal excitation and emission wavelengths for fluorescence When combining five different excitation and emission wavelengths, maximum sensitivity of an isopropyl ether extract of a uremic serum pool or of a 138.7 mmol / l (15.00 mg / ml) p-cresol standard (not shown) was obtained at 284 nm excitation and at 310 nm emission wavelengths. Also when comparing various excitation / emission wavelengths reported in the literature for p-cresol and related compounds [18,19], registered peak height appeared to be maximal at 284 / 310 nm. Hence 284 / 310 nm excitation / emission wavelengths were used for all further analyses.

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3.2. Identification of p-cresol 3.2.1. Capillary gas chromatography–mass spectrometry ( CGC–MS) CGC–MS of the isopropyl ether extract of uremic serum revealed the presence of p-cresol: (1) the retention time of a component in the isopropylether extract during CGC was the same as a standard of p-cresol; (2) the extracted ion chromatogram at m /z 165 obtained for the derivatised isopropyl ether extract of uremic serum shows the presence of p-cresol (Fig. 1A). The spectrum taken at the peak corresponds to the spectrum of the trimethyl silylether of p-cresol (Fig. 1B). 3.2.2. Spiking experiments The result of a representative spiking experiment on an extract of uremic serum is illustrated in Fig. 2. The putative p-cresol peak eluted at 8.24 min and the internal standard at 10.83 min (Fig. 2A). The chromatogram of the same

Fig. 1. (A) Mass spectrum of trimethylsilylated p-cresol in an isopropyl ether extract of uremic serum, and (B) a reference mass spectrum of trimethylsilylated p-cresol. The molecular ion 180 (m /z) and fragment ion 165 (m /z) indicated the identity of the compounds.

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Fig. 2. (A) In a representative spiking experiment, an isopropyl ether extract of a uremic serum with 2,6-dimethylphenol as internal standard (IS), and (B) of the same serum to which p-cresol was added, analyzed by RP-HPLC. The presumed p-cresol peak became higher after the addition of extra p-cresol without the appearance of new peaks.

serum, to which p-cresol [40 ml of a 2311.8 mmol / l (250.00 mg / ml) standard] was added (Fig. 2B) showed that the peak eluting at 8.27 min had increased in height without the appearance of a new major peak. This suggested that the peak eluting at 8.24 min was indeed p-cresol.

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3.2.3. UV spectra The HPLC-peak collected after elution of combined isopropyl ether extracts of twenty uremic serum samples and the peak observed after a similar treatment of twenty p-cresol standard samples were submitted to UV spectrum analysis from 200 to 400 nm (Fig. 3A and Fig. 3B). The spectra obtained were compared to the UV spectrum of a crude p-cresol solution (Fig. 3C). For each sample the same UV spectrum was observed. There were two wavelengths at which UV absorption reached a maximum: 278 and 218 nm for the uremic serum extract, 278 and 220 nm for the extract of the p-cresol standard and 277 and 220 nm for the crude p-cresol. These data again suggest that the compound of interest was p-cresol.

3.2.4. Interference with other solutes Other protein bound uremic solutes or precursors of p-cresol did not co-elute with p-cresol. The retention times were for (1): tyrosine, 2.22 min; (2): indoxyl sulfate and phenylalanine, 2.82 min; (3): tryptophan, 3.13 min; (4): 4-hydroxyphenylacetic acid and 4-hydroxybenzoic acid, 3.93 min; (5): indole-3-acetic acid, 6.93 min, to be compared with (6): p-cresol, 8.24 min (Fig. 4A). Hippuric acid, only detectable at a high concentration of 11.2 mmol / l (2.00 mg / ml) eluted at 3.67 min. The retention time of the other phenolic compounds phenol and o-cresol did also not interfere with that of p-cresol. Phenol elutes at 5.82 min (a), p-cresol at 8.32 min (b) and o-cresol at 8.66 min (c) (Fig. 4B). Note that o-cresol is not a uremic retention solute.

3.3. Quantification 3.3.1. Linear response The fluorescence detector response with seven concentrations from 0.0 to 462.4 mmol / l (50.00 mg / ml) p-cresol was linear. Three regression analyses were performed resulting in a mean r 2 5 0.991260.0051. The detection was also linear in the low concentration ranges from 0.0 to 9.2 mmol / l (1.00 mg / ml) (five concentrations) with a mean r 2 5 0.994760.0046 for the three regression lines. The detection limit was 1.3 mmoI / l (0.14 mg / ml).

3.3.2. Calibration curves The daily calibration curves were drawn from 0.0 to 231.2 mmol / l (25.00 mg / ml) p-cresol (six data pairs). The mean slope of 30 calibration curves was 1.3060.13, with r 2 5 0.994260.0053.

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Fig. 3. (A) UV-absorption spectrum from 200–400 nm of the HPLC-peak obtained after isopropyl ether extraction of uremic serum, and (B) of a p-cresol solution submitted to the isopropyl ether extraction, compared to (C) the crude p-cresol solution. Identical spectra were obtained.

3.3.3. Deproteinization methods The highest yield (100%) of total p-cresol [80.163.6 mmol / l (8.6660.39 mg / ml)] after deproteinization was obtained with the combination of HCl / NaCl compared to 22.4% [17.961.0 mmol / l (1.9460.11 mg / ml)] for HCl alone and 0.56% [1.460.5 mmol / l (0.1560.06 mg / ml)] for TCA. No p-cresol could be detected in uremic serum deproteinized by heat and with NaCl alone.

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Fig. 4. (A) Comparison of the elution of p-cresol with its precursors and other protein bound uremic retention solutes; 1: tyrosine; 2: indoxyl sulfate and phenylalanine; 3: tryptophan; 4: 4-hydroxyphenylacetic acid and 4-hydroxybenzoic acid; 5: indole-3- acetic acid; and 6: p-cresol. Hippuric acid was not detectable at 22.3 mmol / l (4.00 mg / ml), but was demonstrated to elute at 3.67 min when a separate analysis with 11.2 mmol / l (2.00 mg / ml) was performed. (B) Comparison with other phenolic compounds; a: phenol; b: p-cresol; and c: o-cresol.

3.3.4. Alternative extraction methods Para-cresol was extracted from uremic serum and uremic serum filtrate with three organic solvents: isopropyl ether, acetonitrile and ethyl acetate. In uremic serum, a yield of only 27.164.6% and 5.661.0% of total p-cresol (bound 1 unbound) was obtained with acetonitrile and ethyl acetate extraction compared

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to isopropyl ether (100%). The yield for free p-cresol in the filtrate was 55.968.3% with acetonitrile and 0.0% with ethyl acetate in comparison with isopropyl ether.

3.4. Validation experiments 3.4.1. Adsorption of p-cresol on Centrifree Micropartition Filters Aqueous solutions of 4.6, 11.6, 23.1, 57.8, 115.6 and 231.2 mmol / l (0.50, 1.25, 2.50, 6.25, 12.50 and 25.00 mg / ml) p-cresol showed recoveries of 101.2%, 95.7%, 94.7%, 95.9%, 94.5% and 92.4%, respectively. 3.4.2. Variability of retention times The mean retention time of p-cresol and the internal standard in sixteen samples evaluated daily over a 5-day period varied between 8.1360.02 min and 8.3360.04 min for p-cresol and between 10.7360.02 min and 10.9360.05 min for the internal standard. 3.4.3. Reproducibility The intra-assay variance and the day-to-day variation coefficient of free p-cresol in a uremic serum pool were 3.2% and 4.2%, respectively. For determination of total p-cresol intra-assay variance and day-to-day variation coefficient were 6.9% and 7.3%, respectively. The intra-assay and the day-today precision in normal serum were 4.7% and 6.3%, respectively. 3.4.4. Recovery The recovery of an added amount of p-cresol of 37.0 mmol / l (4.00 mg / ml) to uremic serum was 95.464.1% (n 5 6). The addition of low 24.8 mmol / l (2.68 mg / ml) and high 103.7 mmol / l (11.22 mg / ml) concentrations of p-cresol to normal serum yielded recoveries of 98.665.0 and 97.763.6%. 3.4.5. Concentration measurement in a commercial standard solution of pcresol The determined p-cresol in the commercial standard solution of 37.0 mmol / l (4.00 mg / ml), diluted to 74.0 mmol / l (8.00 mg / ml) was 72.160.09 mmol / l (7.8060.01 mg / ml) (mean6S.D. of three determinations). 3.5. In vivo p-cresol serum concentrations and protein binding The characteristics of healthy controls and the different patient groups are illustrated in Table 1. Their p-cresol concentrations are represented in Fig. 5. Total p-cresol is the sum of free and bound p-cresol. The total p-cresol concentration was 8.663.0 mmol / l (0.9360.32 mg / ml) for the controls [range:

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Table 1 Characteristics of the healthy controls and the different patient groups

Age (years) Sex (F / M) Time since the start of dialysis (months) Screa (mmol / l) Urea (mmol / l) Serum total protein (g / 100 ml) Ccrea (ml / min)

Healthy controls n59

Outpatients n57

CAPDpatients n59

HDpatients n 5 11

50618 4/5 –

56611 2/5 –

60610 3/6 35650

63613 4/7 48657

8869 561 6.960.5

6986150 b 3469 b 6.761.1

6896212 b 1867 b,c 6.360.4

7696150 b 2666 b,d 6.660.5 d

96.4630.9

14.667.4 b

3.364.1 b,c

0.560.8 b,c

Screa: serum creatinine; Ccrea: creatinine clearance; CAPD: chronic ambulatory peritoneal dialysis; HD: hemadialysis. a p , 0.05; b p , 0.01 vs. healthy controls; c p , 0.01 vs. outpatients; d p , 0.05 vs. CAPDpatients.

4.0–12.7 mmol / l (0.43–1.37 mg / ml)]; 87.8631.7 mmol / l (9.4963.43 mg / ml) for the outpatients [range: 53.5–148.0 mmol / l (5.79–16.00 mg / ml)]; 62.0619.5 mmol / l (6.7062.11 mg / ml) for the CAPD-patients [range: 31.2–84.1 mmol / l (3.37–9.10 mg / ml)]; and, 88.8649.3 mmol / l (9.6065.33 mg / ml) for hemodialysis patients [range: 96.1–176.6 mmol / l (1.04–19.10 mg / ml)]. No free p-cresol could be detected in the healthy subjects. The free p-cresol was the highest in the hemodialysis patients 11.068.9 mmol / l (1.1960.96 mg / ml) [range: 1.8–23.8 mmol / l (0.19–2.57 mg / ml)]. In the outpatients and CAPDpatients the free concentration was 7.163.0 mmol / l (0.7760.33 mg / ml) [range: 4.7–11.6 mmol / l (0.51–1.26 mg / ml)]; and, 6.062.0 mmol / l [range: 3.2–9.0 mmol / l (0.35–0.97 mg / ml)]. The calculated protein binding for controls, outpatients, CAPD-patients and hemodialysis patients was 100%, 91.762.8%, 90.263.2% and 87.766.0%, respectively, and varied within the ranges 86.6– 95.3%, 82.7–93.3% and 74.9–95.2%. Statistically significant differences were found in the three patient groups vs. control for total and free p-cresol and for percentage protein binding.

4. Discussion In this paper, a method for the quantification of total and free serum p-cresol in healthy controls and in patients in various stages of uremia is reported. After an extraction procedure with isopropyl ether, p-cresol was analyzed by RPHPLC with fluorescence detection. The method appeared to be accurate, the

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Fig. 5. Serum p-cresol concentrations in healthy controls (C, n 5 9), uremic outpatients (OP, n 5 7), Chronic ambulatory peritoneal dialysis patients (CAPD, n 5 9) and hemodialysis patients (HID, n 5 11). The characteristics are given in Table 1. (A) Total concentration (hatched: free fraction; open: protein bound fraction). (B) Free concentration. (C) Percentage protein binding. *: p , 0.05 vs. healthy controls.

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deproteinization and extraction procedure were efficient, there was no interference with other solutes, and the recovery was . 95%. The detection limit was low due to an optimal extraction procedure and the well considered choice of excitation and emission wavelengths; this allowed us to determine even free p-cresol in an appropriate way. The identity of p-cresol was proven by spiking experiments, registration of the UV-spectrum and mass spectrometry. Concentrations were increased in uremic patients. Concentrations in uremia were in a range causing biochemical alterations [5–7,11]. The biological fluid most frequently studied up to now for p-cresol concentration was urine, where the substance was used as an index of exposure to toluene. For the determination of p-cresol in serum of healthy controls and uremic patients different methods have been used. Paper chromatography was the first technique reported [21]. This method was quantitative but labor-intensive. Colorimetric determinations were non-specific as a separation from other phenolic compounds was impossible [22,23]. Gas chromatography by itself was accurate [16]; however, p-cresol loss was observed during the evaporation and dehydration procedure preceding the chromatography [3], unless labor-intensive preparative measures were taken [16]. RP-HPLC was up to now applied in two studies, only for the determination of serum total p-cresol and not for the much lower free p-cresol [17,18]. With the present method, it became possible to measure total as well as free p-cresol concentration as a consequence of a number of specific measures. The determination of total p-cresol was strongly dependent upon the means of deproteinization. The highest yield in the present study was obtained by the combined use of HCl / NaCl. All other methods were less efficient. Although heat denaturation was a reliable method for the release of many other uremic retention compounds from their protein binding sites [20], this was not the case for p-cresol. Being a volatile compound, p-cresol could be lost due to the heating of the serum. With the present deproteinization method, which applied acidification with HCl and saturation with NaCl, no loss of p-cresol occurred. The volatilization of the extracted p-cresol was prevented by the addition of NaGH before the evaporation of the solvent. In addition, isopropyl ether extraction, as applied in the present study, resulted in a maximum yield of p-cresol. In our hands, alternative extraction solutes such as ethylacetate and acetonitrile extraction resulted in a markedly lower yield in our method. The better yield with isopropyl ether indirectly resulted in a low detection limit; the latter made it possible to estimate reliable concentrations even in the low range, e.g. for the determination of free p-cresol. In the HPLC-methods for determination of p-cresol in serum that were published earlier, either UV-detection [17], or fluorescence detection at different wavelengths were used [18]. In our experience these approaches were associated with difficulties for the determination of low concentrations. For RP-HPLC

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determinations of p-cresol in urine, UV detection at wavelengths of 210 nm [2] or 270 nm [13] has been used, which is close to the registered absorption maxima of 218 and 278 nm of the UV spectrum of p-cresol, reported in the present study (Fig. 3). However, to measure p-cresol accurately at low concentrations in serum and serum filtrate, we opted here for the more specific and more sensitive fluorescence detection method. The optimal choice of the excitation and emission wavelengths at 284 / 310 nm Ex / Em made it possible to detect free p-cresol in uremic serum with a detection limit as low as only 1.3 mmol / l (0.14 mg / ml) p-cresol. To our knowledge, this combination of wavelengths had up to now only been used to detect p-cresol in tobacco smoke [19]. The total p-cresol in hemodialysis patients were higher than the concentrations found by Niwa et al. [18]; this may be attributable to the differences in nutritional habits and / or in dialysis methodology or adequacy. Determination of free p-cresol concentration necessitates the use of ultrafiltration. The ultrafiltration devices were shown to adsorb some of the p-cresol (0–7.6%), but this effect was most pronounced at concentrations higher than those currently observed in end-stage renal disease patients (ESRD). For the currently observed mean free concentrations in hemodialysis patients of 11.0 mmol / l (1.19 mg / ml) (Fig. 5), this adsorption would be in the range of 4.3%, resulting in a reciprocal underestimation of free p-cresol and an overestimation of protein binding by 60.5%. Thus, the combination of an effective deproteinization or ultrafiltration, and an adequate extraction and detection method allowed a reproducible determination of total or free p-cresol in normal and uremic serum or uremic serum filtrate. The gradient profile made our method specific for the elution of p-cresol; no interferences with other compounds chemically related to p-cresol were observed (Fig. 4). The spiking with p-cresol showed no additional peak in the HPLC of uremic serum extract (Fig. 2); the UV spectra of the presumed and native p-cresol were similar (Fig. 3). The GC–MS procedure using the silylation technique confirmed the presence of p-cresol in the isopropyl ether extract of uremic serum (Fig. 1). A marked increase in both total and free concentration of p-cresol and a decrease in protein binding were observed in uremic patients vs. healthy controls. Among the patient groups, total and free p-cresol concentrations were highest in hemodialysis patients (Fig. 5). It should be noted that retention in renal failure was associated with a more pronounced increase in free than in total concentration (Fig. 5). It is of note that, although no statistical significance was reached, free and total p-cresol were lower in CAPD-patients compared to hemodialysis patients. In analogy to the findings by Niwa et al [24] regarding 3-carboxy-4-methyl-5propyl-2-furanpropionic acid, another strongly protein bound compound, remov-

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al of p-cresol by CAPD might be more important than with hemodialysis due to the gradual solute exchange and the protein losses in the peritoneal dialysis procedure. Alternatively, residual renal function might persist longer in CAPDpatients. It may be interesting to evaluate this phenomenon during further studies, as well as other aspects, such as the progress of p-cresol concentration in non-dialyzed renal failure outpatients. The study and measurement of free and total concentration of p-cresol in various conditions of the uremic status may be of interest, because of its specific chemical and physical characteristics. The hydroxyl and the methyl group on the benzene ring makes this aromatic compound partially hydrophilic and partially lipophilic. It has a high affinity for serum proteins. In analogy with protein bound drugs, metabolic or toxic effects might to be mediated by free unbound concentration [25]. Because of these properties, p-cresol may be representative for a large group of partially lipophilic compounds with strong protein binding. Those compounds with proven toxicity and similar physical characteristics are phenol, 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid [26] and several of the indoles (tryptophan, indoxyl sulfate and indole-3-acetic acid) [27]. Their toxicity may at least in part be attributable to interference with cell membrane structure [8,9] and to competition at the binding sites of proteins with other retention solutes [28]. It should be reminded that until now little attention has been paid to these partially lipophilic compounds in relation to dialysis treatment, both in the conceptualization of solute removal and in the choice of markers for that removal; up to now hydrophilic and non-protein bound compounds have been emphasized. In conclusion, in the present paper a method for the determination of total and free p-cresol concentration in serum is reported. The method is appropriate, and allows the monitoring of the concentration of p-cresol in various uremic conditions. The concentration tends to rise during the progress of renal failure. Increases in free active concentrations are even more substantial than for total (protein bound 1 non-bound) concentration.

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