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Departments of Nutrition (T.K., L.L.B., B.C., M.A.P., K.J., K.T., F.D., R.K., H.B.) and Pediatrics (R.K., W.D., D.K.), Case Western Reserve University, Cleveland, Ohio; and Children's Hospital (S.W.) and Department of Pediatrics (I.N.), University of Pennsylvania, Philadelphia, Pennsylvania
(Received May 14, 2003; accepted September 2, 2003)
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, 2001; Brusilow, 1991). These patients excrete nitrogen as phenylacetylglutamine (PAGN) (Batshaw et al., 1981). The latter is also formed when the patients are treated with phenylacetate (PA). However, PB is preferred as a prodrug of PA because it does not have the foul smell of the latter. In addition, PB shows promise for the treatment of cystic fibrosis because it increases trans-membrane chloride conductance (Rubenstein and Zeitlin, 2000; Zeitlin et al., 2002). Also, PB is used in clinical trials for the treatment of sickle-cell anemia and thalassemia because it induces the formation of fetal hemoglobin (Dover et al., 1994; Hoppe et al., 1999). Lastly, PB is used in clinical trials as a cytostatic antineoplastic agent, because it inhibits histone deacetylases and potentiates the effect of cytotoxic agents on tumors (Samid et al., 1997; Gilbert et al., 2001).
The clinical effectiveness of PB in some of these situations is limited by occasional incidences of toxicity at high doses (Carducci et al., 2001; Gore et al., 2002). Concerns have been raised by clinical investigators who treat patients with large doses of PB as a sodium salt. First, the total amount of PB and its known metabolites excreted in urine (PA, PAGN) is less than the administered PB dose, sometimes as low as 50%. Some of the unknown metabolites might contribute to PB toxicity at high doses. Second, the large sodium load of the treatment is potentially dangerous for patients with impaired cardiac and/or renal function. Third, the causticity of sodium PB can result in esophagal and/or gastric distress, even when it is administered as a powder suspended in water (this has been extensively debated on the Internet discussion group Metab-L at http://lists.franken.de/mailman/listinfo/metab-L). Neither the biochemical mechanism(s) of PB toxicity nor the identity of the missing metabolites of PB is known. Also, it is not clear whether the metabolism of PB (a modified fatty acid) interferes with, or is influenced by, the metabolism of fatty acids and carbohydrates present in foodstuffs. Lastly, it is not known whether stimulation of lipolysis under stress conditions interferes with PB metabolism.
Our original interest in PB metabolism was related to it being a precursor of PAGN, which can be used as a noninvasive probe of the 14C- or 13C-labeling pattern of citric acid cycle intermediates in human liver (Magnusson et al., 1991; Yang et al., 1996). As part of these investigations, we recently identified phenylbutyrylglutamine (PBGN) as a new metabolite of PB (Comte et al., 2002). PBGN is presumably formed from the reaction of PB-CoA with glutamine, by analogy with the formation of PAGN from the reaction between PA-CoA and glutamine (Webster et al., 1976). In normal adult subjects who ingested a fairly low dose of PB (5 g/75 kg), we found that the total excretion of PB + PA + PAGN + PBGN accounted for only half the ingested PB dose (Comte et al., 2002). The missing fraction of the dose may be disposed of either in urine as unknown metabolites, or in feces as unabsorbed PB and/or PB metabolites.
In the present study, we report the identification of additional metabolites of PB in humans, i.e., R- and S-3-hydroxy-4-phenylbutyrate (PHB), phenylacetone, and 1-phenyl-2-propanol, as well as PA and PB glucuronides. We studied the mechanism of PHB formation from PB in perfused rat livers and identified two additional metabolites, 4-phenyl-trans-crotonate (PtC) and 4-phenyl-3-ketobutyrate (PKB). Lastly, we prepared sodium-free esters of PB and investigated their bioavailability in rats. Glycerol-PB esters appear promising for the administration of large amounts of PB without the corresponding sodium load.
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Preparation of Unlabeled and Deuterated Standards. S-2-Phenylbutyryl chloride was prepared by reacting S-2-phenylbutyric acid with SOCl2, and was vacuum-distilled and stored at 4°C as a 0.5 M solution in benzene. [2H5]PB was prepared by aluminum chloride-catalyzed condensation of 
-butyrolactone with [2H6]benzene as previously described (Comte et al., 2002). -Phenyl-trans-crotonic acid was prepared by a Fridel-Crafts reaction of benzene with ethyl -bromo-trans-crotonate, followed by acid hydrolysis of ethyl -phenyl-trans-crotonate (Loffler et al., 1970). The trans configuration of the product was confirmed by 1H NMR. [2H7]Phenylacetylglycine was synthesized by reacting [2H7]phenylacetyl chloride with glycine, as described previously for the unlabeled analog (Ramsdell and Tanaka, 1977). [2H7]Phenylacetyl chloride was prepared by activation of [2H7]phenylacetic acid with freshly distilled dichloromethyl methyl ether and used immediately after evaporation of excess dichloromethyl methyl ether and of the methyl chloroformate byproduct. The synthetic protocol for preparation of PHB, PKB, [2H5]PHB, phenylacetone, and 1-phenyl-2-[2-2H]propanol is outlined in Fig. 1.
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Ethyl 4-Phenyl-3-ketobutyrate (2; Fig. 1) was synthesized by a method adapted from Capozzi et al. (1993). The commercial isopropylidene malonate (2,2-dimethyl-4,6-diketo-1,3-dioxane, also called Meldrum's acid; Aldrich Chemical Co., Milwaukee, WI) was reacted with phenylacetyl chloride in the presence of dry pyridine in anhydrous methylene chloride. The crude phenylacetylated Meldrum's acid (compound 1, Fig. 1) was refluxed in absolute ethanol until evolution of CO2 ceased (about 3 h). After evaporation of the solvent, ethyl 4-phenyl-3-ketobutyrate was purified on a silica gel column.
4-Phenyl-3-ketobutyric acid (PKB). A 10% molar excess of 1 N NaOH solution was added slowly to ice-cooled ethyl 4-phenyl-3-ketobutyrate and stirred at room temperature until the organic phase disappeared (approximately 12 h). The solution was acidified with 1 N HCl to pH 2 and extracted three times with 3 volumes of ethyl ether. After drying over Na2SO4, the solvent was evaporated to give the white solid product (yield 94%, m.p. 70°C). 1H NMR (300 MHz, 
, CDCl3): keto, 3.33 (s, 2H, CH2COO), 3.78 (s, 2H, CH2Ph), 7.17–7.33 (m, 5H, Ph); enol, 4.82 (s, 1H, CH), 12.13 (s, 1H, OH); keto/enol = 7.8:1. 13C NMR (75 MHz, , CDCl3): 48.36 (CH2COO), 49.87 (CH2Ph), 127.41 (C-4 Ph), 128.73 (C-3, C-5 Ph), 129.50 (C-2, C-6 Ph), 133.14 (C-ipso, Ph), 169.26 (COO), 201.57 (CO).
The identity of the product was also confirmed by 1) reduction of 4-phenyl-3-ketobutyrate with NaBH4, 2) extraction of R,S-PHB with ethyl acetate, and 3) TMS derivatization and NH3-positive chemical ionization GC-MS. The mass spectrum of the derivative was identical to that of PHB synthesized as described below. The 4-phenyl-3-ketobutyric acid was stored at –80°C to prevent decomposition. Just before use in liver perfusion experiments, the acid was dissolved in water and titrated to pH = 7.40.
R,S-3-Hydroxy-4-phenylbutyric acid (PHB). Ethyl 4-phenyl-3-ketobutyrate (2.06 g, 10 mmol) was mixed with 10 ml of H2O and a calculated amount of NaOH/H2O was added dropwise with cooling to give 0.5M solution of [OH-]. NaBH4 (0.37g, 10 mmol) was added and the mixture stirred for 24 h. After the reaction mixture was cooled to 0°C, the pH was brought to 7.0 with HCl (6N) and more than half of water removed by lyophilizer. The pH was brought to 2 by HCl (6 N) and the solution extracted 3 times with 3 volumes of ethyl ether. After drying the combined ether extract, the ether was evaporated giving a white solid product. Yield 79%. Purity of product was assayed by GC-MS after derivatization with TMS.
R-3-Hydroxy-4-phenylbutyrate (R-PHB) was prepared by reducing the corresponding 4-phenyl-3-ketobutyric acid with B-chlorodiisopinocampheylbo rane [(–)-DIP-Cl] (Wang et al., 1999). The yield was 89% from PKB; enantiomeric excess was 97% [GC-MS of the methyl S-2-phenylbutyryl derivative (see below), and purity was confirmed by NMR].
R,S-3-Hydroxy-4-phenyl[2,2,3,4,4-2H5]butyrate (R,S-[2H5]PHB). 4-Phenyl-3-ketobutyric acid (0.267 g, 1.5 mmol) was suspended in 3 ml of 2H2O (99.9%) to which 0.36 ml of 40% of NaO2H (3.6 mmol) in 2H2O was slowly added at 0–5°C. This procedure exchanges 1H for 2H atoms on the methylene groups adjacent to the carbonyl. The solution was stirred at room temperature overnight and then lyophilized. The residue was dissolved in 3 ml of 2H2O and stirred for another 5 h at room temperature. The solution was cooled on ice, treated with NaB2H4 (63 mg, 1.5 mmol), and stirred overnight at room temperature (Des Rosiers et al., 1988). After acidification to pH 1 to 2 with HCl (6 N), the solution was saturated with NaCl and extracted three times with 3 volumes of diethyl ether. Solvent evaporation yielded R,S-[2H5]PHB (yield 74%, purity 99% by NMR, M5 isotopic enrichment 95% by GC-MS of the TMS derivative). The free acid was titrated to pH 8 with NaOH and stored frozen as a 0.5 M solution until use.
Phenylacetone was prepared by decarboxylating phenylketobutyrate in acid at 100°C. It was reduced to 1-phenyl-2-[2-2H]propanol with NaB2H4.
Esters of Phenylbutyrate. Dihydroxyacetone-di-PB, glycerol-tri-PB, ribose-tetra-PB, glucose-penta-PB, and sorbitol-hexa-PB were prepared by reacting the polyol/sugar with excess phenylbutyryl chloride in the presence of pyridine and catalytic amounts of N,N-dimethylaminopyridine. Products were purified by flash column chromatography on silica. To prepare glycerol-mono-PB, isopropylidene glycerol was reacted with phenylbutyryl chloride as above, and the isopropylidene group was removed by mild acidic hydrolysis in water. The structure and purity of all products were confirmed by 1H and 13C NMR. The structure of glycerol-mono-PB was confirmed by acetylation with acetic anhydride, followed by GC-MS analysis of the derivative.
Sample Preparation. For the determination of the free acids concentration (PA, PHB, PKB, PtC, and PB) in perfusate, samples (0.1 ml) were spiked with 0.17 µmol of [2H7]PA, [2H5]PB, and R,S-[2H5]PHB before deproteinization with 20 µl of saturated sulfosalicylic acid. The slurries were saturated with NaCl, acidified with one drop of 6 M HCl, and extracted three times with 5 ml of diethyl ether. For the assay of conjugates of PB and PA, 0.1-ml aliquots of final liver perfusate or of human urine were spiked with internal standards and treated with 1.0 ml HCl (6 N) at 90°C overnight to hydrolyze the conjugates. Glucuronides of PB and PA were identified by the amount of these compounds released after incubation of perfusate and urine samples with ß-glucuronidase in 0.2 M ammonium acetate buffer, pH 5.0, overnight at 37°C.
Bile samples were analyzed for free and total (conjugated + free) PB and its metabolites. In the first series of assays, 0.05-ml samples of bile were spiked with internal standards, acidified to pH 2 to 2.5, and extracted three times with diethyl ether. In the second series of assays, samples spiked with internal standards were hydrolyzed with 0.3 ml of NaOH (6 N) at 90°C for 3 h before acidification and extraction.
Phenylacetylglycine was extracted in acid and derivatized with methanol/HCl. For the assay of phenylacetone and 1-phenyl-2-propanol, urine and perfusate samples were spiked with the structural analog 1-phenyl-[2H5]ethanol and then treated with NaB2H4 to reduce phenylacetone to monodeuterated 1-pheny-2-propanol. The labeled and unlabeled 1-phenyl-2-propanol were assayed as TMS derivatives.
For the assays in urine, 0.1-ml samples were spiked with 0.15 µmol [2H5]PHB, acidified to pH 1 to 2 with HCl, saturated with NaCl, and extracted three times with 3 ml of diethyl ether. The combined extracts were dried with Na2SO4 and evaporated before reacting the residues with 70 µl of TMS at 60°C for 20 min.
For the chiral assay of PHB enantiomers (Powers et al., 1994), 0.1-ml samples were spiked with R,S-[2H5]PHB, and either deproteinized with 50 µl of saturated sulfosalicylic acid (if containing proteins) or acidified to pH 1 to 2 with HCl (for urine). Then, the slurries or solutions were saturated with NaCl and extracted three times with 3 ml of diethyl ether. The combined extracts were dried with Na2SO4 and evaporated before reacting the residues with 0.15 ml of methanol/HCl for 1 h at 65°C, to derivatize the carboxyl groups of the PHB enantiomers. After cooling, 1 ml of water was added to the mixture and the hydroxyacid methyl ester was extracted with diethyl ether (three times in 3 ml). After complete evaporation of the combined ether extract, S-2-phenylbutyryl chloride benzene solution (0.1 ml, 0.5 M) and 0.05 ml of aqueous 12 N NaOH were added. After vortexing, the mixture was incubated for 1 h on a slow shaker at room temperature. The derivatives were extracted with ether (three times in 3 ml) and 1 ml of water. The combined ether phase was dried with Na2SO4 and evaporated completely. The residue was dissolved in 0.1 ml of ethyl acetate, and 1 µl was injected into the GC-MS.
GC-MS Methods. All of the metabolites, except phenylacetylglycine, were analyzed as their TMS derivatives on a Hewlett-Packard 5890 gas chromatograph equipped with a ZB-5 capillary column (60 m x 0.25 mm i.d., 0.5 mm film thickness; Hewlett Packard, Palo Alto, CA) and coupled to a 5989A mass selective detector. Samples (0.2–1 µl) were injected with a split ratio 20 to 50:1. The carrier gas was helium (1 ml/min) and nominal initial pressure was 20.61 psi. The injector port temperature was at 270°C, the transfer line at 305°C, the source temperature at 200°C and quadrupole at 150°C. The column temperature program was: start at 100°C, hold for 1 min, increase by 8°C/min to 236°C, increase by 35°C/min to 310°C, 6 min at 310°C. After automatic calibration, the mass spectrometer was operated under ammonia-positive ionization mode. Appropriate ion sets were monitored with a dwell time of 25 to 35 ms/ion, at m/z 226/233 (PA/[2H7]PA), 254/259 (PB/[2H5]PB), 325/330 (PHB/[2H5]PHB), and 252 (PtC). Note that PKB and phenylacetone had been reduced with NaB2H4 to monodeuterated R,S-3-hydroxy-4-phenylbutyrate (monitored at m/z 326/330) and 1-phenyl-2-propanol (monitored at m/z 200/210 and/or 217/227). Also, since 1-phenyl-2-propanol was assayed with a standard of 1-phenyl[2H5]ethanol, ions monitored for this assay were 200/209 or 217/226.
For the analysis of chiral PHB derivatives, the GC injector temperature was set at 280°C. The column (DB-5, 60 m x 0.25 mm i.d., 0.5 mm film thickness; Hewlett Packard) program was modified to the initial 150°C for 2 min, increased by 15°C/min to 230°C, 25 min at 230°C, increased by 35°C to 290°C, and held 10 min. Ions monitored were 1) 358 (M + 18, i.e., M + NH4+) for analytes and 2) 363 (M + 18 + 5, i.e., M + 5+NH4+) for [2H5]PHB. The mass spectrometer was operated under ammonia-positive chemical ionization and was tuned automatically.
Phenylacetylglycine was analyzed as its methyl ester derivative using an OV-225 column (29 m x 0.32 mm i.d., 1 µm film thickness; Quadrex Corporation, Woodbridge, CT). This column yielded better resolution of N-phenylacetylglycine methyl ester with no peak tailing. Samples (0.2–1 µl) were injected with a split ratio 20:1. The carrier gas was helium (constant flow: 1.2 ml/min). The injector port temperature was at 220°C, the transfer line at 240°C, the source temperature at 200°C, and quadrupole at 106°C. The column temperature program was: start at 90°C, hold for 1 min, increase by 10°C/min to 240°C, 15 min at 240°C. After automatic calibration, the mass spectrometer was operated under ammonia-positive ionization mode (pressure adjusted to optimize peak areas). Ions monitored were 1) 208 (M + 1, i.e., M + H+) and 225 (M + 18, i.e., M + NH4+) for the analyte and 2) 215 (M + 7 + 1, M + 7 + H+) and 232 (M + 7 + 18, M + 7 + NH4+) for N-[2H7]PA-glycine with a dwell time of 25 ms/ion.
Areas under each chromatogram were determined by interactive computer integration, and corrected for naturally occurring heavy isotopes and light isotopic impurities in the synthesized labeled internal standards.
NMR Spectroscopy. Proton NMR spectroscopy was performed at 400 MHz on a Bruker Avance (Bruker, Newark, DE) DMX 400 wide-bore spectrometer using a 5-mm inverse probe. Full-strength urine samples were obtained by lyophilizing 5 ml of urine to dryness. The residue was dissolved in 0.5 ml of 2H2O, and the solution was introduced into a 5-mm NMR tube. An external standard made of a sealed capillary containing a solution of trimethylsilylpropionic acid in 2H2O was introduced into the NMR tube and used as chemical shift reference. Standard acquisition conditions were as follows for one-dimensional spectra: 45° pulse, 8-s repetition time, water saturation during the relaxation delay, sweep width (SW) 6775 Hz, 64K data points (TD), and 32 scans of data collection. Two-dimensional correlation spectroscopy (COSY) spectra were obtained with the following conditions for the second dimension: SW 3500 Hz, TD 2K, 16 scans, and for the first dimension, 512 increments of 278 µs zerofilled to 1K. A nonshifted sinebell window was applied in both dimensions, and magnitude spectra were calculated. Two-dimensional 1H/13C correlations via double insensitive nuclei enhanced by polarization transfer (HSQC) were performed in the phase-sensitive mode (TPPI; time-proportional receiver phase incrementation) using gradients for coherence selection and carbon decoupling during acquisition. The following conditions were used in the second dimension: SW 3200 Hz, TD 2K, 128 scans, and in the first dimension: SW 12 kHz, 256 increments of 20.7 µs zerofilled to 512. A shifted sinebell window was applied in both dimensions.
Proton-decoupled carbon spectra of the concentrated urine samples were obtained at 100.62 MHz in a 5-mm dual probe. Acquisition conditions were as follows: 20° pulse, repetition time 1.3 s, SW 25 kHz, TD 64K, 40,000 scans. The free induction decays were zerofilled to 128K, and a Lorentz to Gauss transformation (LB = –1 Hz, GB = 0.1) was applied before Fourier transformation.
Clinical Investigation. The protocol was reviewed and approved by the Institutional Review Board of University Hospitals of Cleveland. All subjects were free of any chronic or acute illness. Women had a negative pregnancy test and were not breastfeeding. Seven subjects (three men, four women; 31.7 ± 5.0 years; 171.3 ± 3.4 cm; 79.5 ± 5.9 kg) received detailed information on the purpose of the investigation and signed an informed consent form. After an overnight fast, the subjects were admitted to the Clinical Research Center at 7:30 AM. They remained fasting until the completion of the study. An intravenous line was installed in the forearm with a saline infusion (20 ml/h) and a short blood sampling catheter was inserted into a superficial vein of the contralateral hand. The hand was placed in a heating box at 60°C for sampling of arterialized venous blood. At 8:00 AM, after baseline blood and urine sampling, each subject ingested 0.36 mmol/kg (5 g/75 kg) Na-PB. This dose corresponds to 11 to 17% of the doses commonly used in the treatment of patients with inborn errors of urea synthesis (0.4–0.6 g · kg–1 · day–1). Water intake was adjusted to induce a diuresis of at least 100 ml/30 min. Urine samples were collected at 30-min intervals for the first 3 h after PB ingestion, and then every hour until 8 h. Urine samples were quickly frozen and stored at –80°C until analysis.
Organ Perfusion Experiments. Livers from fed male Sprague-Dawley rats kept on standard rat chow (200–230 g) were perfused (Brunengraber et al., 1975) with recirculating Krebs-Ringer-bicarbonate buffer containing 4% bovine serum albumin (fraction V, fatty acid poor; Intergen, Purchase, NY) and 10 mM glucose. The bile duct was cannulated with PE 10 tubing (BD Biosciences, San Jose, CA) for bile collection. Throughout the 2-h experiment, sodium taurocholate (38 µmol/h) was infused into the perfusion reservoir to stimulate bile flow (Robins and Brunengraber, 1982). After 30 min of equilibration, a calculated amount of either PB, R,S-PHB, or PKB was added to the perfusate to set an initial concentration of 5 mM. The perfusion continued until 120 min. The pH of the perfusate was monitored and kept at 7.3 to 7.4 by adding 0.3 M NaOH. Samples of bile and perfusate were collected at regular intervals. For the assay of PKB, perfusate samples (2 ml) were treated immediately with 0.2 ml of 0.1 M NaB2H4 in 0.1 mM NaOH to convert unstable PKB to stable monodeuterated R,S-PHB. Bile samples were collected every 30 min. At the end of the experiment, the livers were quick-frozen with aluminum tongs precooled in liquid nitrogen.
Rat in Vivo Experiments. We tested the bioavailability of two PB esters as a means to deliver large amounts of PB without the corresponding sodium load. Overnight-fasted rats (330–400 g) were divided into six groups (5–7 rats per group) for the testing of three different PB preparations: Na-PB, glycerolmono-PB, glycerol-tri-PB, ribose-tetra-PB, glucose-penta-PB, and sorbitolhexa-PB. Each rat received one stomach gavage of the sodium salt or ester in an amount that delivered 2.15 mmol PB/kg. The weighed dose for each rat was mixed with 3 ml of Tween and administered to the rats through a stomach gavage needle.
Whole blood samples (100–200 µl) were taken at –5, 15, 30, 60, 150, 240, 330, 420, and 480 min from a small incision in a tail vein. Blood was collected in heparinized microcapillary tubes and centrifuged. The plasma (50–100 µl) was transferred to an Eppendorf tube and quick frozen.
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To further confirm the identity of the urinary metabolite detected by NMR, we synthesized unlabeled and R,S[2H5]PHB. The di-TMS derivative of synthetic R,S-PHB was analyzed by GC-MS in parallel with an extract of human urine (after PB ingestion) that had been reacted with TMS. In the sample derived from urine, we found a peak at the same retention time and with the same mass spectra (electron ionization and NH3-positive chemical ionization) as the standard of R,S-PHB. In addition, the NMR spectrum of synthetic PHB had the same chemical shifts as the material identified in human urine. This confirmed the identity of PHB in human urine but did not yield information about its chirality. The chirality of excreted PHB yields information on the mechanism of its formation (see below).
For the chromatographic separation of PHB enantiomers from the synthetic racemate, we tried various chiral hydroxyl derivatization reagents before selecting the combination of 1) methylation of the carboxyl group, and 2) reaction of the hydroxyl group with S-2-phenylbutyryl chloride (Powers et al., 1994). The expected derivatives of R- and S-PHB were well separated, and their order of elution was confirmed using a sample of R-PHB that we had synthesized. R-PHB elutes ahead of the S-derivative (Fig. 2).
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Chiral GC-MS analysis of the human urine samples (after PB ingestion) revealed that PHB is present as an enantiomeric mixture with 10% R-PHB and 90% S-PHB (Fig. 2). Figure 3 shows the time profile of (R+S)-PHB excretion in urine after an oral bolus of Na-PB. The excretion of (R+S)-PHB peaked at 120 to 240 min. Eight hours after ingestion of PB, the cumulative excretion of (R+S)-PHB (1.35 ± 0.13 mmol) amounted to 4.4 ± 0.56% of the PB dose.
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Small amounts of phenylacetone and 1-phenyl-2-propanol were identified in the urine samples (Table 1). Treatment of urine samples with ß-glucuronidase increased their PB + PA content. The total amount of PB + PA released by ß-glucuronidase amounted to 2.4 ± 0.3% of the PB dose.
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Perfused Rat Liver Study. The metabolism of PB was studied in perfused rat livers by the addition of 5 mM PB to the recirculating perfusate. The time profile of the PB concentration was curvilinear (Fig. 4A). Plotting of the data under semilogarithmic coordinates yielded a linear relationship compatible with a first order kinetic process for PB uptake. The total uptake of PB amounted to 310 ± 16 µmol · (90 min–1) · liver–1.
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Figure 4B shows the time accumulation of metabolites derived from PB and released into the perfusate. Phenylacetylglycine is a known metabolite of PA in rats and dogs (Knoop, 1904; Ambrose and Sherwin, 1933; James et al., 1972). About 16% of the uptake of PB was accounted for by the production of R- and S-PHB, of which about 90% is the S-enantiomer. We also identified PtC, PKB, phenylacetone, and 1-phenyl-2-propanol, which, to our best review of the literature, have not been previously described as metabolites of PB. The identity of these metabolites was confirmed by GC-MS using standards we synthesized. Incubation of liver perfusate samples with ß-glucuronidase revealed the formation of PB and PA glucuronides, which accounted for 22 and 5% of the PB uptake, respectively (Fig. 4B; Table 1).
Figure 5 shows the time profile of biliary excretion of PB and its metabolites. At the end of the experiments, the concentrations of free PB, PA, and PHB in bile (2.14, 0.08, and 0.18 mM, respectively, Fig. 5) were 50 to 70% of the corresponding concentrations in the perfusate (3.11, 0.16, and 0.28 mM, respectively, Fig. 4). Alkaline treatment of the bile significantly increased the amounts of PB, PA, and PHB, presumably via hydrolysis of glucuronides. The total amount of free + alkali-released PB, PA, and PHB (Fig. 5, dark bars) amounted to 3.0 ± 0.6% of the amount of PB taken up by the liver from the perfusate.
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To compare the metabolism of the PHB enantiomers, one rat liver was perfused with 5 mM R,S-PHB added at 30 min. Figure 6 shows the GC-MS chromatograms of the assays of R- and S-PHB in the perfusate at 32 and 120 min. At 32 min, the perfusate contained equal concentrations of R- and S-PHB, which was expected since a racemic mixture of PHB had been used (Fig. 6A). However, at 120 min, the concentration of S-PHB had decreased much more than that of R-PHB (Fig. 6B). Using the internal standards of R,S-[2H5]PHB, we found that the uptake of R-PHB was immeasurable. In contrast, the uptake of S-PHB was 134 µmol. A very small amount of PKB (3 µmol) was found, derived presumably from the oxidation of R-PHB by R-ß-hydroxybutyrate dehydrogenase. At 120 min, the perfusate contained 55 µmol of PA resulting from the ß-oxidation of S-PHB.
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To test for the interconversion of R-PHB and PKB, another rat liver was perfused with 5 mM PKB added at 30 min. At 120 min, 358 µmol of PKB had disappeared from the perfusate, and 90% of this had appeared as phenylacetone, the product of PKB decarboxylation. Only traces of 1-phenyl-2-propanol were detected. Figure 7 shows the GC-MS chromatogram of the assays of R- and S-PHB in the perfusate at 120 min. Small amounts of R-PHB (2.3 µmol) and S-PHB (about 0.2 µmol) were detected. Table 1 presents a balance of the conversion of PB to metabolites in humans (urinary metabolites) and in perfused rat livers (metabolites released in the perfusate and bile).
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Bioavailability of PB Esters. Rats were gavaged with equivalent doses of Na-PB, glycerol-mono-PB, or glycerol-tri-PB suspended in Tween. The plasma concentrations of PB and PA were then followed for 8 h (Fig. 8). Table 2 shows the comparison of the areas under the curves (AUC; determined using the linear trapezoidal rule) for each of the compounds tested. Based on the AUCs for PB, the bioavailability of the glycerol monoester or triester are about 63 and 42%, respectively, of that of the sodium salt. However, the bioavailability of the glycerol-mono-PB ester is not significantly different from that of the sodium salt. The bioavailability of the glycerol-tri-PB ester is significantly lower than that of the sodium salt. In all cases, the concentrations of PB and PA had decreased to very low levels after 8 h of gavage. In fact, Na-PB and glycerol-mono-PB esters have the same Tmax at 30 min, with a similar Cmax for PB concentration in plasma, namely, 0.32 mM and 0.22 mM. However, for the glycerol-tri-PB, the Cmax is much lower, reaching a concentration of only 0.16 mM with its Tmax at 15 min. For all three preparations, the Tmax values for the PA metabolite are identical at 150 min, whereas their respective Cmax values are different, namely, 0.25 mM, 0.23 mM, and 0.14 mM for Na-PB, glycerol-mono-PB, and glycerol-tri-PB, respectively. Other rats were gavaged with equivalent doses of ribose tetra-PB, glucose penta-PB, or sorbitol-hexa-PB. Over the next 6 h, no PB or PA was detected in their plasma (not shown), suggesting that these compounds were not bioavailable.
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-phenyl fatty acids used originally by Knoop as model compounds to elucidate the mechanism of fatty acid ß-oxidation (Knoop, 1904). He showed that after oral administration of PB to dogs, phenylacetylglycine (phenaceturic acid) is excreted in urine. This suggested that PB underwent one cycle of chain shortening, forming PA, which was then coupled with glycine. The metabolism of PB is different in humans, in whom PA conjugates with glutamine to form PAGN, rather than phenylacetylglycine (Ambrose and Sherwin, 1933). The mechanism of this conjugation was studied by Webster et al. (1976), who showed that it proceeds via phenylacetyl-CoA and is catalyzed by an acyl-CoA: L-glutamine N-acyltransferase.
In the 1980s, Brusilow and collaborators started using PA for the treatment of patients with inborn errors of the urea cycle (Batshaw et al., 1981). Later, PB was used as a precursor of PA because it did not have the foul smell of PA, and thus was more acceptable to patients (Brusilow, 1991; Batshaw et al., 2001). Most recently, PB has been used in various clinical trials for the treatment of malignancies, cystic fibrosis, sickle cell anemia, and thalassemia (Dover et al., 1994; Hoppe et al., 1999; Rubenstein and Zeitlin, 2000; Gilbert et al., 2001).
The use of large doses of Na-PB for therapeutic purposes has raised several issues. First, the recovery of known urinary metabolites of PB (PAGN + traces of PB and PA) was very variable, ranging from 50 to 90% of the ingested dose (Piscitelli et al., 1995, Comte et al., 2002). Since PB is known to have toxic effects at high doses, some of the unknown metabolites might contribute to its toxicity. It is also possible that part of the ingested PB was not absorbed in the gut and was excreted as such (or as secondary metabolites formed in the colon) in feces. Second, in cancer patients (and presumably other patients as well), the high sodium load associated with the treatment (0.4–0.6 g/kg) results in an expansion of the extracellular fluid, which could be dangerous in patients with cardiac or renal dysfunction.
When we learned of the under-recovery of the PB dose, we hypothesized that part of PB could be excreted in the form of PBGN, an analog of PAGN. Indeed, we previously identified PBGN in the urine of normal adult humans who had ingested small doses of PB (5 g) (Comte et al., 2002). Still, the total recovery of the PB dose as urinary PBGN + PAGN + PB + PA accounted for only about half of the dose. We thus looked for other metabolites of PB in the urine of the original subjects, concentrating on 1) compounds related to intermediates of fatty acid ß-oxidation, and 2) glucuronide conjugates. The compounds identified are shown in the scheme of PB metabolism presented in Fig. 9.
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Logical intermediates in the ß-oxidation of PB are 4-phenyl-trans-crotonyl-CoA, S-3-hydroxy-4-phenylbutyryl-CoA, 4-phenyl-3-ketobutyryl-CoA, and phenylacetyl-CoA. The latter is known to undergo partial hydrolysis to free PA, which accumulates transiently in body fluids after the ingestion of PB. We hypothesized that the other CoA intermediates could also undergo partial hydrolysis to free acids, which would then be excreted in urine. Although we identified S-3-hydroxy-4-phenylbutyrate (S-PHB), we were unable to find PKB or 4-phenyl-trans-crotonate in the urine of the human subjects who had ingested a small dose of PB (5 g). It is possible that these compounds are excreted after the ingestion of therapeutic doses of PB (0.4–0.6 g/kg).
The chiral GC-MS assay we used to identify S-PHB (Powers et al., 1994) revealed that the R-enantiomer accounted for only about 10% of the total PHB excreted by humans. The probable origin of this compound would involve hydrolysis of PKB-CoA to PKB, which would then be reduced to R-PHB by R-ß-hydroxybutyrate dehydrogenase (Fig. 9). We did not identify PKB in the human urine, possibly because it undergoes spontaneous decarboxylation similar to acetoacetate (Pedersen, 1929). Indeed, in the subjects' urine, we found phenylacetone, which derives from the presumably spontaneous decarboxylation of PKB. In addition, we found measurable amounts of 1-phenyl-2-propanol, which is also formed from the reduction of phenylacetone in microsomes (Coutts et al., 1981). The absence of 1-phenyl-2-propanol in the urine before PB ingestion confirms its origin from PB. Note that phenylacetone and 1-phenyl-2-propanol are also products of amphetamine catabolism (Shiiyama et al., 1997; Lee et al., 1999). It is not known whether phenylacetone and 1-phenyl-2-propanol contribute to the toxic side effects of amphetamine.
In searching for metabolites of PB, we looked for glucuronides since it was known that benzoate, which is also used for the treatment of urea cycle disorders, is eliminated in part as a glucuronide (Riddick, 1998). Small amounts of PB and PA glucuronides were indirectly identified in human urine by the increases in PB and PA concentrations induced by incubating the urine with ß-glucuronidase.
The total recovery of PB and its metabolites in human urine accounted for only 62% of the ingested dose. Part of the missing fraction could still be disposed of as unknown urinary metabolites. Also, one cannot exclude the possibility that some PB and/or PB metabolites are excreted in feces. We do not know whether PB glucuronide excreted in bile is entirely hydrolyzed in the gut with complete reabsorption of the PB moiety of the glucuronide.
To gain more insight into the metabolism of PB that does not involve glutamine conjugation (which is specific to humans and primates), we perfused rat livers with recirculating perfusate containing an initial PB concentration of 5 mM. To ensure that PKB (which could possibly be formed in these experiments) would not undergo spontaneous decarboxylation, we treated some of the samples with NaB2H4. This converts PKB to R,S-[2H]PHB, which can then be differentiated from the original PHB by GC-MS. This technique has been used previously to protect acetoacetate and ß-ketopentanoate prior to analysis (Des Rosiers et al., 1988; Leclerc et al., 1995).
The uptake of PB by rat liver [310 ± 16 µmol · (90 min–1) · liver–1 or roughly 0.34 µmol · min–1 · g–1] is of the same magnitude as the uptake of long-chain fatty acids such as oleate (Brunengraber et al., 1978). The apparent first order character of PB uptake shows that it was not saturated at 5 mM substrate (Fig. 4A). Perfusion of rat livers with PB resulted in the production of PKB and PtC. Although these compounds have not been found in human urine after ingestion of a small dose of PB, the presence of phenylacetone and R-PHB is indirect evidence for the formation of PKB (Fig. 9). It is quite possible that, after ingestion of large therapeutic doses of PB, humans would excrete measurable amounts of PKB and PtC.
Sixteen percent of the uptake of PB by the perfused rat liver was released in the form of PHB, 90% of which was the S-enantiomer, similar to what we found in humans. To probe the origin of the R-PHB enantiomer, one liver was perfused with 5 mM PKB, and the perfusate was analyzed for the presence of PHB enantiomers. As expected, most of the PHB generated from PKB was in the R form (Fig. 7), which most likely derived from the action of R-ß-hydroxybutyrate dehydrogenase on PKB (Fig. 9). Presumably, the production of small amounts of R-PHB in humans who ingested PB can also be attributed to the reduction of PKB. Note that the bacterial R-ß-hydroxybutyrate dehydrogenase used in the enzymatic assay of acetoacetate and R-ß-hydroxybutyrate does not use PKB or R,S-PHB as a substrate.
Rat livers perfused with 5 mM PB produced large amounts of PB and PA glucuronides, corresponding to 22% and 5% of the PB uptake, respectively. These glucuronides were formed presumably from free PB and PA, since glucuronidation of carboxylates does not require activation to a CoA ester. The difference in the fractions of the PB uptake recovered as PB versus PA glucuronide results probably from the low concentration of PA derived from PB (Fig. 4).
Figure 9 summarizes the proposed metabolism of PB, including the new compounds identified in this report. The proportion of PB and PA glucuronides is much smaller in humans compared with perfused rat livers (Table 1). Note that the humans were overnight-fasted and, therefore, had presumably low portal vein glucose concentrations, and fairly low glycogen concentrations in the liver. In contrast, the livers from rats fed ad libitum were perfused with a high glucose concentration (10 mM), a concentration likely to occur in the portal vein during the absorption of digested food. Liver glucokinase has a high Km for glucose (10 mM); thus, the uptake of glucose is practically proportional to the portal glucose concentration in the physiological range. Since the production of glucuronides uses glucose 1-phosphate derived from glucose phosphorylation and from glycogenolysis, the rate of glucuronidation may be partially dependent on the glucose concentration in the portal vein. We could not find in the literature reports on studies on the influence of portal vein glucose concentration on rates of xenobiotic glucuronidation in intact livers. We found one report on the stimulation of acetaminophen glucuronidation in humans fed a high-carbohydrate diet compared with a low-carbohydrate diet (Pantuck et al., 1991). Although the stimulation of glucuronidation can result from a high glucokinase flux or a high rate of glycogen cycling, both mechanisms would operate after the ingestion of a high-carbohydrate diet.
Our data raise several clinically relevant questions. First, if patients with urea cycle defects ingest their PB dose with a high-carbohydrate meal, or if decompensated patients are infused with intravenous glucose and PA, would a large fraction of the PB/PA dose be excreted as glucuronides, i.e., compounds that do not carry nitrogen out of the organism? Second, if patients ingest their PB dose with a high-fat meal, would competition between PB and dietary long-chain fatty acids for ß-oxidation result in excretion of side products (PHB, PKB, PtC), which are also non-nitrogenous? Either of these scenarios would decrease the clinical effectiveness of PB therapy. Third, independent of the composition of the diet, could saturation of ß-oxidation by PB itself lead to the excretion of these same side products? Fourth, could the accumulation of ß-oxidation side products such as PtC explain episodes of toxicity encountered occasionally during treatment with PB (Hoppe et al., 1999; Carducci et al., 2001; Gilbert et al., 2001; Gore et al., 2002)? Although we were unable to find reports of phenylcrotonate toxicity, this compound is present in toxic pesticides (Dinocap). Other side products of PB metabolism, phenylacetone and 1-phenyl-2-propanol, are also formed during amphetamine intoxication (Shiiyama et al., 1997; Lee et al., 1999), but it is not clear whether they contribute to the toxicity of amphetamine or phenylbutyrate. The production of phenylacetone and 1-phenyl-2-propanol may be increased under conditions associated with stress and stimulation of lipolysis. It is well known that stimulation of lipolysis raises the concentration of plasma free fatty acids, thus increasing the competition for ß-oxidation.
In conclusion, by presenting our preliminary data, we hope to draw the attention of clinicians to the potential interference between the metabolism of PB and the metabolism of dietary carbohydrates and fatty acids. It might be advisable to administer PB in multiple doses between meals, rather than with meals. Also, the potential toxicity of side products of PB metabolism needs to be thoroughly investigated. From the metabolic balances of Table 1, it is clear that a substantial fraction of the metabolites of PB has not yet been identified (38% in humans and 26% in our perfused rat liver experiments). Carnitine derivatives of PB and PA, as well as compounds resulting from hydroxylation of the benzene ring of PB, might be formed, and we are presently searching for them.
Lastly, the sodium-free glycerol-mono-PB ester (Fig. 8; Table 2) could be considered for patients who might be harmed by a large sodium load and/or by the causticity of sodium PB. Since this ester is a tasteless solid compound, it could be incorporated in processed foods and ingested as snacks in between meals. The above questions need to be investigated in detailed clinical and animal studies.
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Footnotes |
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1 Abbreviations used are: PB, 4-phenylbutyrate; PAGN, phenylacetylglutamine; PA, phenylacetate; PBGN, 4-phenylbutyrylglutamine; PHB, 3-hydroxy-4-phenylbutyrate; PtC, 4-phenyl-trans-crotonate; PKB, 4-phenyl-3-ketobutyrate; TMS, trimethylsilyl; GC-MS, gas chromatography-mass spectrometry; SW, sweep width; TD, data points; COSY, correlation spectroscopy; HSQC, heteronuclear single-quantum coherence; AUC, area under the curve. 
Address correspondence to: Henri Brunengraber, Department of Nutrition, Room 280, Case Western Reserve University, 11000 Cedar Rd., Cleveland OH 44106-7139. E-mail: hxb8{at}cwru.edu
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