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Research ArticleArticle

Effects of Fibrates on the Glycine Conjugation of Benzoic Acid in Rats

Zoltán Gregus, Tibor Fekete, Éva Halászi, Ágnes Gyurasics and Curtis D. Klaassen
Drug Metabolism and Disposition November 1998, 26 (11) 1082-1088;

Abstract

In the course of glycine conjugation, benzoic acid is successively converted into benzoyl-CoA and benzoylglycine by mitochondrial enzymes (i.e. benzoyl-CoA synthetase and benzoyl-CoA/glycine N-acyltransferase, respectively), utilizing ATP, CoA, and glycine. Large doses of benzoate deplete CoA from the liver, suggesting that the supply of CoA may limit the capacity for glycine conjugation. Because fibrates are known to increase hepatic CoA synthesis, we examined whether treatment with fenofibrate or bezafibrate enhanced the capacity of rats to conjugate benzoic acid with glycine. Dietary administration of fenofibrate or bezafibrate (2.5 mmol/kg of feed, for 10 days) increased hepatic CoA levels 8–10-fold, while not affecting hepatic ATP levels; only fenofibrate elevated, albeit moderately, the concentration of glycine in liver. Hepatic mitochondria isolated from fibrate-fed rats, compared with those from controls, exhibited unchanged benzoyl-CoA synthetase activity but higher benzoyl-CoA hydrolase and lower benzoyl-CoA/glycineN-acyltransferase activities. Feeding with either fibrate increased liver mass by 50–60%. Control and fibrate-fed rats were administered benzoate at different doses, one to produce a large demand for CoA (i.e. 2 mmol/kg, iv) and two others to produce smaller demands for CoA (i.e. 1 mmol/kg or 2 mmol/kg plus glycine, iv). Fenofibrate-fed rats, and to a lesser extent bezafibrate-fed animals, exhibited increased glycine conjugation capacity, as indicated by faster disappearance of benzoate from the blood and appearance of benzoylglycine in the blood and urine, compared with controls; however, fibrates were not more effective in rats receiving the benzoate dose that produced the greatest demand for CoA. In contrast, benzoylglycine formation from benzoate (0.1–1 mM) was not enhanced in liver slices from fibrate-fed rats; moreover, it was lower than control levels in slices from bezafibrate-fed animals. Bezafibrate, but not fenofibrate, given to rats in a single dose (0.5 mmol/kg, ip) decreased the elimination and glycine conjugation of benzoate, indicating that bezafibrate is a direct inhibitor of glycine conjugation. In summary, fibrates influence glycine conjugation in a complex manner. Some fibrate-induced alterations (i.e.increased benzoyl-CoA hydrolase and decreased glycine transferase activities and direct inhibition by bezafibrate) can potentially hinder conjugation of benzoate with glycine, thus precluding conclusions regarding whether increased CoA availability enhances glycine conjugation. Fibrate-induced hepatomegaly appears to significantly contribute to the increased glycine conjugation capacity of rats treated with fenofibrate or bezafibrate.

A number of xenobiotic and endogenous carboxylic acids, such as benzoic, salicylic, and isovaleric acids, are eliminated and detoxified by conjugation with glycine (Tanaka and Isselbacher, 1967; Hutt and Caldwell, 1990; Tremblay and Qureshi, 1993). This conjugation involves sequential utilization of three cosubstrates and two enzymes. With benzoic acid as substrate, the process proceeds as follows: Benzoic acid+ATP+CoA →benzoyl­CoA+AMP+PP Equation 1Benzoyl­CoA+glycine →benzoylglycine+CoA Equation 2where PP is pyrophosphate. These reactions take place in the mitochondrial matrix and are catalyzed by benzoyl-CoA synthetase and benzoyl-CoA/glycine N-acyltransferase, respectively (Tremblay and Qureshi, 1993; Gatley and Sherratt, 1977). These enzymes exhibit high activity in both liver and kidney; however, its large mass makes the liver the predominant site of glycine conjugation.

Although conjugation with glycine plays a major role in the disposition of aspirin (Levy, 1965), which is probably the most widely used drug, control of glycine conjugation is still incompletely understood. The observation that large doses of benzoate deplete hepatic glycine and CoA in rats (Gregus et al., 1992) suggests that glycine conjugation capacity in vivo may be controlled by the hepatic availability of these cosubstrates. Administration of carboxylic acids that readily form CoA esters, such as valproic and lipoic acids, markedly lowers hepatic CoA levels and diminishes production of benzoylglycine from benzoate (Gregus et al., 1993a, 1996), supporting the hypothesis that a restricted supply of CoA may limit glycine conjugation. The role of cosubstrate availability in conjugations may also be assessed by examining how an increased supply of cosubstrates affects formation of the conjugate. Increasing the hepatic levels of glycine, either by experimental inhibition of the glycine cleavage system or by loading of rats with exogenous glycine, enhanced glycine conjugation of benzoic acid (Gregus et al., 1993b), confirming the hypothesis that benzoylglycine formation is controlled by the availability of glycine. It is unknown, however, whether increased availability of CoA would also promote glycine conjugation.

It has long been known that feeding rats clofibrate, a hypolipidemic drug, induces marked elevations in the hepatic content of total CoA, including nonesterified CoA (Miyazawa et al., 1975;Savolainen et al., 1977). This effect has been attributed to a clofibrate-induced increase in the hepatic activity of pantothenate kinase, the rate-limiting enzyme of CoA biosynthesis (Skrede and Halvorsen, 1979; Voltti et al., 1979). Some other fibric acid derivatives, including the more potent lipid-lowering drugs bezafibrate and fenofibrate, have been shown to be even more effective than clofibrate in increasing total CoA content and pantothenate kinase activity in rat liver (Halvorsen, 1983). Therefore, these fibrates were deemed to be useful as experimental tools for elucidating whether the hepatic availability of CoA, in addition to that of glycine, may control the glycine conjugation capacity of rats.

Specifically, this study was designed to determine whether prolonged feeding of rats with bezafibrate or fenofibrate (fig.1) facilitates conjugation of benzoic acid with glycine and, if it does, whether the enhanced conjugation results from increased availability of CoA in the liver. For this purpose, control and fibrate-treated rats were injected with sodium benzoate, and glycine conjugation of benzoic acid was monitored by quantifying (a) the disappearance of benzoic acid from blood, (b) the appearance of benzoylglycine in blood, and (c) the excretion of benzoylglycine in urine. Based on our previous study (Gregus et al., 1992), the load of the substrate (benzoate) was selected so that it would present a smaller demand for CoA (e.g. 1 mmol/kg benzoate or 2 mmol/kg benzoate plus glycine) or a larger demand for CoA (e.g. 2 mmol/kg benzoate). Furthermore, to establish whether the possible fibrate-induced changes in benzoylglycine formation are solely the result of greater availability of hepatic CoA or are ascribable to other factors, additional experiments were performed. These included (a) quantification of the hepatic concentrations of all cosubstrates (i.e. ATP, CoA, and glycine) and the activities of all enzymes (i.e. benzoyl-CoA synthetase, benzoyl-CoA hydrolase, and benzoyl-CoA/glycineN-acyltransferase) that are involved in glycine conjugation, (b) examination of the acute effects of the fibrates on glycine conjugation of benzoic acid in rats, and (c) quantification of glycine conjugation in liver slices taken from control and fibrate-fed rats.

Figure 1
Figure 1

Chemical structures of bezafibrate and fenofibrate.

Materials and Methods

Chemicals.

Sources of most chemicals used in the animal experiments and the analyses have been reported (Gregus et al., 1992,1993a). Bezafibrate and fenofibrate were gifts from Boehringer Mannheim (Vienna, Austria) and Richter Gedeon (Budapest, Hungary), respectively.

Animals and Treatments.

Male Wistar rats (LATI, Gödöllő, Hungary) weighing 270–330 g were used. The animals were housed at 23–26°C, with 55–65% relative humidity, on a 12-hr light/dark cycle. Tap water and laboratory chow (LATI) were provided ad libitum. For studies of the effects of prolonged treatment with fibrates, rats were fed for 10 days with ground laboratory chow or ground chow mixed with bezafibrate or fenofibrate, at a concentration of 2.5 mmol/kg of feed. For investigation of the acute effects of fibrates, rats were injected ip with bezafibrate or fenofibrate, dissolved in corn oil at a dose of 1 mmol/kg, 2 hr before benzoate administration. Control rats received corn oil (5 ml/kg, ip).

Glycine Conjugation in Rats.

Approximately 30 min before benzoate administration, control and fibrate-treated rats were hydrated by administration (by gavage) of 30 ml/kg saline solution containing 10 mM KCl, anesthetized with urethane (1.2 g/kg, ip), and maintained at 37°C by means of a heating lamp. The right carotid artery was cannulated with polyethylene-50 tubing for blood sampling. Through an incision in the lower abdominal wall, the urinary bladder was exposed and emptied by gentle manual compression. Sodium benzoate was administered in 10 ml/kg saline solution containing 7.5% mannitol, by injection into the right saphenous vein, at the doses indicated in figs.2-4. For groups of rats specified in figs. 2 and 3, as well as rats acutely pretreated with fibrates, glycine (5 mmol/kg in 5 ml/kg saline solution) was injected into the left saphenous vein 15 min before benzoate administration. After benzoate injection, blood (120–150 μl) was sampled from the carotid artery at 5, 10, 20, 40, 60, 90, and 120 min, for analysis of benzoate and benzoylglycine levels. To determine the rate of urinary excretion of benzoylglycine, urine was collected in 15-min periods for 2 hr by manually emptying the urinary bladder through the urethra. To prevent volume depletion and maintain urine flow, 2 ml/kg portions of 10% mannitol in saline solution were injected into the rats, via the indwelling carotid cannula, every 15 min. Under these conditions, the urine flow of rats was steady (120–150 μl/kg·min), yielding 0.6–0.8 ml of urine in each period, which was sufficient for accurate collection. Urine volumes were measured gravimetrically, taking 1.0 g/ml as the specific gravity. The urinary excretion rate of benzoylglycine was calculated as the product of urine flow and urinary concentration.

Figure 2
Figure 2

Effects of pretreatment with bezafibrate or fenofibrate on the disappearance of benzoate from blood.

Rats fed ground chow (control) or ground chow containing bezafibrate or fenofibrate (2.5 mmol/kg of feed) for 10 days were anesthetized with urethane and injected with sodium benzoate (1 or 2 mmol/kg, iv) at time 0. A group of rats was also given glycine (5 mmol/kg, iv) 15 min before benzoate administration. The urine flow was maintained during the experiment by periodic administration of mannitol, as described inMaterials and Methods. Values represent the mean ± SE for 6–10 rats. *, Significantly different (p < 0.05) from control.

Figure 3
Figure 3

Effects of pretreatment with bezafibrate or fenofibrate on the appearance in blood and excretion into urine of benzoylglycine.

Experimental conditions were as described for fig. 2. Values represent the mean ± SE for 6–10 rats. *, Significantly different (p < 0.05) from control.

Glycine Conjugation in Rat Liver Slices.

Slices from the livers of control and fibrate-fed rats were prepared basically as described by Barr et al. (1991), using equipment from Alabama Research and Development Corp. (Munford, AL) and Vitron, Inc. (Tucson, AZ). Briefly, cores (diameter, 8 mm) were prepared from the livers of exsanguinated rats with a tissue-coring tool attached to a tissue-coring press. The cores were sliced with a Krumdieck tissue slicer under ice-cold Krebs-Henseleit buffer (pH 7.4) that had been previously bubbled with 95% O2/5% CO2 gas at room temperature. Slices (18–22 mg of wet weight) were placed in pairs into roller inserts (type A), which were then inserted into 20-ml glass scintillation vials containing 1.7 ml of Krebs-Henseleit buffer (pH 7.4, 37°C). The vials were briefly gassed with 95% O2/5% CO2through holes in their caps and were preincubated at 37°C in a roller culture incubator in a 95% O2/5% CO2 atmosphere for 90 min. The inserts with the slices were then transferred to new vials containing 0–1 mM sodium benzoate, without or with glycine (5 mM), in 1.7 ml of Krebs-Henseleit buffer (pH 7.4, 37°C) and were incubated at 37°C in the roller culture incubator in a 95% O2/5% CO2 atmosphere for an additional 120 min. At the end of the incubation period, the slices were removed from the vials, blotted, and weighed. The slices and the incubation mixture were stored at −20°C until analysis for benzoic acid and benzoylglycine, as well as quantification of slice potassium contents (an indication of slice viability). Formation of benzoylglycine in the slices was calculated based on the amount of benzoylglycine in the incubation mixture, because pilot experiments indicated that the slices contained <5% of the total amount of glycine conjugate formed.

Enzymatic Assays.

To assay benzoyl-CoA synthetase, benzoyl-CoA hydrolase, and benzoyl-CoA/glycine N-acyltransferase activities, mitochondria were isolated from the livers of control and fibrate-fed rats as described in detail (Gregus et al., 1993a). Pilot studies testing the purity of mitochondrial fractions by electron microscopic analysis indicated that these fractions, prepared from control or fibrate-fed rats, were virtually free from contamination with peroxisomes. The resultant mitochondrial suspensions prepared for enzymatic analysis were stored in small aliquots at −20°C. During storage, the activities of the mitochondrial enzymes assayed were stable for at least 1 week. Before assays of the mitochondrial enzymes, a mitochondrial suspension containing 4 mg of protein/ml was prepared, and the mitochondria were solubilized by addition of Triton X-100 at a final concentration of 0.5% (v/v).

To assay benzoyl-CoA synthetase, 0.2 mg of solubilized mitochondrial protein was incubated at 37°C for 10 min in 500 μl of solution containing 100 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.5 mM MgCl2, 2 mM sodium benzoate, 5 mM ATP, and 0.5 mM CoA. The reaction was stopped with 50 μl of 4 M perchloric acid. After centrifugation, the supernatant was neutralized with 5 M potassium carbonate, degassed under reduced pressure, and used for HPLC analysis of benzoyl-CoA.

Benzoyl-CoA/glycine N-acyltransferase activity was assayed according to the method of Webster (1981), by using solubilized mitochondria containing 0.2 mg of protein. This assay is based on release of CoA in the presence of glycine and quantification of the released CoA in the incubation mixture by reaction with 5,5′-dithiobis(2-nitrobenzoic acid), using continuous detection of absorbance at 412 nm. Benzoyl-CoA hydrolase activity of solubilized mitochondria was assayed under similar conditions, except that glycine was omitted from the incubation. For all enzymatic assays, product formation was linear with respect to time and mitochondrial protein concentration in the incubation medium.

Chemical Analyses.

Benzoic acid and benzoylglycine levels in blood and urine, as well as in the incubation mixtures from liver slices, were quantified by the HPLC procedure of Bachmann et al. (1986), as specified earlier (Gregus et al., 1993a). For quantification of adenine nucleotides, CoA, glycine, and carnitine in the livers of control and fibrate-fed rats, the rats were anesthetized with urethane (1.5 g/kg, ip). The livers of urethane-anesthetized rats were exposed, and the left lobe was freeze-clamped with aluminum blocks precooled in liquid nitrogen. The liver samples were stored in liquid nitrogen until analysis. Adenine nucleotides, CoA, glycine, and carnitine were extracted with perchloric acid from freeze-clamped liver samples, as described (Gregus et al., 1993a). An aliquot of the freshly prepared extract was used immediately for analysis of adenine nucleotides and CoA, whereas other aliquots were stored at −20°C for analysis of glycine and carnitine. Adenine nucleotides and glycine, the latter after dansylation by the procedure of Saller and Czupryna (1989), were quantified by HPLC analysis based on the methods of Dills and Klaassen (1985) and Fürst et al. (1990), respectively. CoA and carnitine were assayed enzymatically according to the methods of Michal and Bergmeyer (1974) and Pearson et al. (1974), respectively. Protein concentrations of the mitochondrial suspensions were determined by the biuret method Gornallet al. (1949).

For quantification of benzoyl-CoA produced in the benzoyl-CoA synthetase assay, 20-μl samples of the deproteinized, neutralized, and degassed incubation mixtures were subjected to HPLC analysis. The HPLC procedure used a Chromsil-C18 analytical column (250 × 4.6 mm, 6 μm) preceded by a guard column (30 × 4.6 mm) packed with Chromsil-C18 (10 μm), eluent consisting of 0.2 M KH2PO4 (pH 6.0)/methanol (50:50) delivered at a rate of 1.0 ml/min, and a variable-wavelength detector set at 261 nm.

For quantification of the potassium contents of liver slices, the slices were individually sonicated in 1 ml of water, and the resultant homogenates were deproteinized with perchloric acid and centrifuged. Potassium in the supernatants were measured with a flame photometer, after appropriate dilution with water.

Pharmacokinetic Analysis.

Although it is premature to interpret the pharmacokinetic behavior of benzoic acid, the apparent t1/2,Vd ,1and CLb were calculated at each dose, assuming linear kinetics. The Kel and the blood concentration extrapolated to time 0 (C0) for benzoic acid were calculated as the y intercept and slope, respectively, of the least-squares regression line of the natural logarithm of the blood concentration vs. time plot. The apparentVd , eliminationt1/2, and CLb were calculated using the equations Vd = dose/C0, t1/2 = 0.693/Kel, andCLb =Kel·Vd .

Statistics.

Data were analyzed by one-way analysis of variance. Duncan’s new multiple-range test was used to compare the means. A value ofp < 0.05 was considered significant.

Results

Glycine Conjugation in Rats Fed Fibrates.

To study glycine conjugation of benzoate under conditions of quantitatively different demands for CoA, rats were injected with 1 mmol/kg benzoate, 2 mmol/kg benzoate, or glycine plus 2 mmol/kg benzoate. Fig. 2 demonstrates the disappearance of injected benzoate from blood, whereas fig. 3 shows the appearance of the formed benzoylglycine in blood and its excretion in urine in control rats and in rats fed bezafibrate or fenofibrate for 10 days. Pharmacokinetic parameters of benzoate elimination in these animals are presented in table 1.

View this table:
Table 1

Effects of pretreatment with bezafibrate or fenofibrate on pharmacokinetic parameters of benzoate elimination in rats

Treatment with either fibrate promoted the disappearance of benzoic acid from blood (fig. 2), increased its Keland CLb , and shortened itst1/2 (table 1). Fenofibrate was consistently more effective than bezafibrate in increasing benzoate elimination. Relatively little difference appeared in the effects of fibrates with respect to the dosing conditions of benzoate. However, fenofibrate treatment tended to facilitate the elimination of benzoic acid less in rats receiving 2 mmol/kg benzoate than in the other two groups of animals. For example, fenofibrate increasedKel and CLb by only 45 and 50%, respectively, in the rats given 2 mmol/kg benzoate, whereas it increased these parameters by 95 and 68% in the rats injected with 1 mmol/kg benzoate and by 150 and 71% in the animals receiving 2 mmol/kg benzoate with glycine (table 1).

Fenofibrate treatment resulted in increases in blood concentrations of benzoylglycine during the first 1 hr after injection of benzoate (fig.3, top), as well as in the urinary excretion rates of the glycine conjugate (fig. 3, bottom). Such effects of bezafibrate appeared to be more moderate (fig. 3) or were not observed (fig. 3, bottom left). The dose of benzoate had little influence on the effect of fibrates. For example, fenofibrate increased the urinary excretion rate of benzoylglycine at 45–60 min after administration of benzoate by 50, 37, and 62% in rats injected with 1 mmol/kg benzoate, 2 mmol/kg benzoate, and 2 mmol/kg benzoate with glycine, respectively (fig. 3, bottom).

Hepatic Cosubstrates and Enzymes Involved in Glycine Conjugation in Rats Fed Fibrates.

Table 2 presents the liver weights and the hepatic concentrations of cosubstrates required for glycine conjugation (i.e. ATP, CoA, and glycine) for control and fibrate-fed rats. Additional data obtained in the analyses of cosubstrates (i.e. concentrations of ADP, AMP, and the sum of acetyl-CoA and oxidized CoA, as well as the hepatic level of carnitine, which influences the availability of CoA) are also presented.

View this table:
Table 2

Effects of treatment with bezafibrate or fenofibrate on liver weight and hepatic concentrations of adenine nucleotides, CoA, glycine, and carnitine

Of the cosubstrates required for glycine conjugation, it was only CoA for which the hepatic level was elevated dramatically, i.e.8.1-fold by bezafibrate and 9.9-fold by fenofibrate (table 2). The two fibrates increased the concentration of carnitine in the liver to very similar extents (7.2-fold). Fenofibrate, but not bezafibrate, induced a slight increase in the concentration of glycine (+23%), whereas the hepatic level of ATP remained unchanged in rats treated with either fibrate. The specific liver weight was increased significantly by both bezafibrate (+49%) and fenofibrate (+60%).

The activities of the enzymes involved in glycine conjugation in solubilized mitochondria isolated from the livers of control and fibrate-fed rats are presented in table3. Treatment with either fibrate did not significantly influence the activity of benzoyl-CoA synthetase, the enzyme catalyzing the first step in benzoylglycine formation. However, both fibrates lowered (−20%) the activity of benzoyl-CoA/glycineN-acyltransferase, which catalyzes the second step of the conjugation. In addition, both bezafibrate and fenofibrate induced increases in the activity of benzoyl-CoA hydrolase (+65% and 76%, respectively), which can counteract the formation of benzoylglycine.

View this table:
Table 3

Effects of treatment with bezafibrate or fenofibrate on the activities of enzymes involved in benzoylglycine formation in solubilized mitochondria isolated from rat liver

Glycine Conjugation in Rats Acutely Injected with Fibrates.

To determine whether alterations in benzoylglycine formation induced by fibrate feeding may also result from direct effects of the fibrates on glycine conjugation, the acute effects of ip injected fenofibrate and bezafibrate (0.5 mmol/kg) were also examined. Fig. 4 indicates that administration of a single dose of fenofibrate 2 hr before benzoate did not significantly influence the elimination of benzoate and the formation of benzoylglycine. In contrast, in rats injected with bezafibrate, compared with vehicle-treated controls, disappearance of benzoic acid from blood was delayed (fig. 4, left), as were the initial increases in the blood levels (fig. 4, top right) and the urinary excretion rate of benzoylglycine (fig. 4,bottom right). Acute administration of bezafibrate diminished the Kel of benzoic acid from 2.75 to 1.64 hr−1 (p < 0.05) and decreased the maximal urinary excretion rate of benzoylglycine by 46%.

Figure 4
Figure 4

Acute effects of bezafibrate and fenofibrate on the disappearance of benzoate from blood and the appearance in blood and excretion into urine of benzoylglycine.

Corn oil (5 ml/kg, control), bezafibrate (1 mmol/kg), or fenofibrate (1 mmol/kg) was injected ip into rats 2 hr before the administration of benzoate. After urethane anesthesia, the rats were injected with glycine (5 mmol/kg, iv) and, 15 min later (time 0), with sodium benzoate (1 mmol/kg, iv). Urine flow was maintained by periodic administration of mannitol, as described in Materials and Methods. Values represent the mean ± SE of 6–9 rats. *, Significantly different (p < 0.05) from control.

Glycine Conjugation in Liver Slices from Rats Fed Fibrates.

The formation of benzoylglycine by liver slices obtained from control and fibrate-fed rats and incubated in the presence of sodium benzoate (0.1–1 mM), with or without glycine, is depicted in fig.5. Compared with liver slices from control rats, the slices from fibrate-fed rats produced less benzoylglycine. However, decreases in glycine conjugation were significant only in the bezafibrate-treated rat liver slices incubated with benzoate in the absence of glycine (fig. 5, left).

Figure 5
Figure 5

Effects of pretreatment with bezafibrate or fenofibrate on benzoylglycine formation in rat liver slices incubated in the presence of benzoate, with or without glycine.

Liver slices were obtained from rats fed ground chow (control) or ground chow containing bezafibrate or fenofibrate (2.5 mmol/kg of feed) for 10 days. After a 90-min preincubation, the slices were transferred into medium containing benzoate at the given concentrations, without or with glycine (5 mM), and were incubated for an additional 120 min, to determine the amount of benzoylglycine appearing in the incubation mixture. Bars, mean + SE of eight separate incubations of liver slice pairs taken from four rats. *, Significantly different (p < 0.05) from control.

Discussion

CoA is a cosubstrate in the first reaction of glycine conjugation, which is thought, on the basis of in vitro studies, to control the rate of benzoylglycine formation from benzoate when glycine is present in excess (Gatley and Sherratt, 1977). Whether the supply of CoA limits glycine conjugation capacity in vivo is uncertain; however, some findings suggest that it might. For example, rats injected with benzoate at ≥0.5 mmol/kg exhibited significant decreases in hepatic CoA levels and reduction of the benzoylglycine formation capacity (Gregus et al., 1992). The concentration of CoA in the livers of such rats was lowered to and even well below the KM of CoA (i.e. 35 μM) for an acyl-CoA synthetase in rat liver mitochondria (Garland et al., 1970) that is functionally similar to benzoyl-CoA synthetase, for which the KM of CoA is unknown. Furthermore, administration of either valproic acid or lipoic acid decreased both hepatic CoA concentrations (<20 nmol/g) and the capacity of rats to form benzoylglycine (Gregus et al., 1993a, 1996). Nevertheless, because lipoic acid and a metabolite of valproic acid also inhibited one or both of the enzymes catalyzing glycine conjugation of benzoic acid, these findings could not be unequivocally interpreted in favor of the hypothesis that CoA supply limits glycine conjugation capacity in vivo. Therefore, a further attempt was made to test this hypothesis by assessing the effect of increased hepatic CoA levels on the capacity of rats to conjugate benzoic acid with glycine.

The present work was prompted by a report indicating that repeated administration of bezafibrate and fenofibrate markedly increased the synthesis of CoA and the level of total (i.e. esterified plus nonesterified) CoA in the livers of rats (Halvorsen, 1983). It was expected that these drugs would also increase the hepatic levels of nonesterified CoA, i.e. the cosubstrate in benzoyl-CoA formation, because such an effect had been shown for clofibrate, their less effective progenitor (Miyazawa et al., 1975; Savolainenet al., 1977). Indeed, the selected fibric acid derivatives, when fed to rats, elevated the concentrations of nonesterified CoA in the liver by as much as 8–10-fold (table 2). In addition, like clofibrate (Paul et al., 1986), both bezafibrate and fenofibrate markedly increased the hepatic concentrations of carnitine (table 2). Theoretically, this would facilitate transport of fatty acids into the mitochondria, where the fatty acids would utilize CoA for their β-oxidation, thereby diminishing intramitochondrial CoA available for other processes, such as glycine conjugation. Although we did not analyze the subcellular distribution of CoA in the liver, it has been reported that clofibrate markedly increases not only the tissue levels but also the mitochondrial contents of both total and nonesterified CoA in rat liver (Berge et al., 1983; Brass and Ruff, 1992). Therefore, it is most likely that the clofibrate congeners used in this study also induce accumulation of nonesterified CoA in hepatic mitochondria.

This study demonstrated that fenofibrate and (less effectively) bezafibrate, when administered in the diet for 10 days, did enhance the capacity of rats to conjugate benzoic acid with glycine. Glycine conjugation is the only significant mechanism for benzoate elimination in rats (Bridges et al., 1970; Gregus et al., 1992). Therefore, the more rapid disappearance of benzoic acid from blood (fig. 2) and the pharmacokinetic parameters indicating accelerated blood clearance of benzoate in fibrate-fed rats, compared with controls (table 1), represent circumstantial evidence for increased benzoylglycine formation in these animals. The higher blood levels and urinary excretion rates of benzoylglycine in rats receiving fibrate-containing diets, compared with control animals (fig. 3), corroborate these findings. However, some observations, such as the lack of increased urinary excretion of benzoylglycine in bezafibrate-fed rats injected with 1 mmol/kg benzoate (fig. 3,bottom left), indicate that bezafibrate probably also adversely affects the renal excretion of the glycine conjugate.

These findings raise the question of whether the fibrate-induced increases in hepatic CoA concentrations and benzoylglycine formation in rats are causally related. To provide a positive answer to this question, one should demonstrate that the fibrates (a) enhance glycine conjugation to a greater extent when the demand for CoA is especially large and/or when the supply of CoA is thought to predominantly determine the rate of conjugate formation, (b) do not induce other changes that might also enhance the capacity of rats to conjugate benzoic acid with glycine, and (c) do not induce changes that could adversely affect glycine conjugation and thus could at least partially confound the potential stimulatory effect of increased CoA availability on glycine conjugation.

The three dosing conditions for benzoate applied in this study should have provided quantitatively different demands for CoA in the livers of rats. We showed that hepatic CoA concentrations declined by 60 min to 40 and 14% of control values in rats injected iv with benzoate at doses of 1 and 2 mmol/kg, respectively (Gregus et al., 1992). As indicated by experiments with isolated hepatocytes (Cyret al., 1991), loading with glycine counteracts benzoate-induced CoA depletion, because glycine facilitates formation of benzoylglycine from benzoyl-CoA, with concomitant release of CoA. Therefore, it can be concluded that a greater demand for CoA was created in rats receiving 2 mmol/kg benzoate alone than in the other two groups of rats. Consequently, if CoA availability does limit glycine conjugation capacity, one would expect that fibrate-induced increases in hepatic CoA supply should promote benzoylglycine formation comparatively more in rats receiving 2 mmol/kg benzoate than in those receiving 1 mmol/kg benzoate or glycine plus 2 mmol/kg benzoate. This expectation was clearly not fulfilled. Moreover, both fibrates increased elimination of benzoate the least in rats treated with 2 mmol/kg benzoate without glycine (fig. 2 and table 1). However, the degree of CoA demand may not solely determine the response of benzoylglycine formation to increased CoA supply. It is likely, for example, that limited supply of both CoA and glycine limits glycine conjugation capacity at high benzoate doses (Gregus et al., 1992); therefore, the effect of increased CoA availability cannot be fully manifested in enhanced benzoylglycine formation until the limiting influence of restricted glycine supply is removed. This speculation appears to be supported by the observation that fenofibrate tended to produce larger increases in Keland CLb values for benzoate (table 1), as well as in blood concentrations and urinary excretion rates of benzoylglycine (fig. 3), in rats that had been loaded with glycine before receiving benzoate (2 mmol/kg) than in those that had not received glycine.

Fibrates are pleiotropic agents that activate a subtype of peroxisome proliferator-activated receptors and induce morphological alterations (e.g. hypertrophy and hyperplasia of hepatocytes, marked proliferation of peroxisomes, and a slight increase in the number but a decrease in the volume of mitochondria), overexpression of various enzymes (such as those involved in fatty acid oxidation in the peroxisomes and mitochondria), and underexpression of some other proteins in the livers of rodents (Lock et al., 1989; Meijeret al., 1991; Moody et al., 1992; Schoonjanset al., 1997; Motojima et al., 1997). Therefore, it was important to determine whether fibrates bring about alterations that may contribute to or confound the potential facilitating effect of fibrate-induced CoA accumulation in the liver on glycine conjugation in rats.

Among the fibrate-induced alterations, in addition to increased hepatic CoA supply, that could facilitate glycine conjugation is the slight but significant increase in hepatic glycine concentration found in fenofibrate-fed rats (table 2). In theory, this effect of fenofibrate might also explain, in part, the finding that fenofibrate enhanced benzoate elimination (fig. 2 and table 1) and benzoylglycine excretion (fig. 3) more than did bezafibrate, because the latter did not elevate hepatic glycine levels (table 2). Nevertheless, the fenofibrate-induced increase in hepatic glycine levels is unlikely to contribute significantly either to its effect to promote glycine conjugation in rats or to its superiority over bezafibrate in this respect. This conclusion is based on the observation that fenofibrate enhanced glycine conjugation of benzoate, and enhanced it more than did bezafibrate, not only in rats receiving no exogenous glycine but also in glycine-loaded rats (figs. 2 and 3 and table 1).

Because the liver is the most active organ in glycine conjugation, the 50–60% increase in its mass in fibrate-fed rats (table 2) could also contribute to the increased capacity of these animals to conjugate benzoate with glycine. To discount the effect of hepatomegaly, glycine conjugation of benzoic acid was also studied in liver slices prepared from untreated and fibrate-fed rats. The finding that slices from the latter animals failed to produce more benzoylglycine (moreover, slices from bezafibrate-treated rats formed significantly less conjugate in the absence of added glycine) than did liver slices from the control rats (fig. 5) suggests that fibrate-induced liver enlargement is responsible for the increased glycine conjugation capacity of rats maintained on fibrate-containing diets. In addition, this observation would also refute the hypothesis that increased CoA supply enhances glycine conjugation capacity, unless fibrates also induced changes that could counteract the potential stimulatory effect of greater CoA availability. We have obtained evidence for three changes of that sort.

Feeding of fenofibrate or bezafibrate increased the activity of benzoyl-CoA hydrolase while decreasing the activity of benzoyl-CoA/glycine N-acyltransferase in rat liver mitochondria (table 3). Enhancement of other mitochondrial acyl-CoA hydrolases by prolonged treatment with clofibrate and its congeners has been reported (Berge et al., 1984; Urrea and Bronfman, 1996). Although induction of enzymes by peroxisome proliferators is common, repressed synthesis of certain proteins has also been reported (Motojima et al., 1977); therefore, diminished activity of glycine N-acyltransferase is not without example. These fibrate-induced alterations in enzymatic activities might negatively affect benzoylglycine formation, because they would enhance reversal of the first reaction by benzoyl-CoA hydrolase and diminish the rate of glycine conjugate formation by benzoyl-CoA glycine/N-acyltransferase in the second reaction of glycine conjugation.

Bezafibrate appears to have yet another negative effect; direct inhibition of benzoylglycine formation was observed in rats acutely injected with the drug (fig. 4). Because fenofibrate did not exhibit such an effect, this inhibitory action of bezafibrate might be responsible, at least in part, for the findings that bezafibrate feeding enhanced benzoate elimination and benzoylglycine formation less than did fenofibrate treatment (figs. 2 and 3 and table 1) and that liver slices from bezafibrate-fed rats, but not fenofibrate-fed animals, produced significantly less benzoylglycine than did slices from control rats (fig. 5). The mechanism by which bezafibrate inhibits glycine conjugation is not known. However, it is interesting to note a chemical difference between the two fibrates we used, namely that fenofibrate is a carboxylic acid ester, whereas bezafibrate is a free carboxylic acid (fig. 1). It is likely, therefore, that after bezafibrate administration the concentration of this acidic drug in the liver is higher than the concentration of fenofibric acid after fenofibrate administration, because formation of fenofibric acid requires enzymatic hydrolysis of the ester fenofibrate (Caldwell, 1989). Several carboxylic acids, especially lipophilic ones, are inhibitors of medium-chain acyl-CoA synthetase (Kasuya et al., 1996). In addition, fibric acids can form CoA esters and thus consume CoA (Lock et al., 1989). Further investigations are needed to determine whether these mechanisms are relevant in the direct inhibition of glycine conjugation by bezafibrate.

In summary, prolonged administration of fenofibrate and bezafibrate dramatically increased the hepatic content of CoA and also enhanced the conjugation of benzoic acid with glycine in rats, but not in rat liver slices. It appears certain that fibrate-induced hepatomegaly contributes to the enhancement of benzoylglycine formation in vivo. Fibrates also induced alterations (i.e. increased activity of benzoyl-CoA hydrolase, decreased activity of benzoyl-CoA/glycine transferase, and direct inhibitory effects of bezafibrate) that could negatively affect glycine conjugation of benzoic acid and thus preclude conclusions regarding whether increased CoA availability enhances benzoylglycine formation. Therefore, this study revealed that fibrates influence glycine conjugation in a complex manner, but it failed to provide conclusive evidence regarding the limiting role of CoA supply in glycine conjugation capacity.

Acknowledgments

Electron microscopic examination of mitochondrial fractions was carried out by Dr. László Komáromy (Department of Biology, University Medical School of Pécs), for which he is gratefully acknowledged. We thank Katalin Gyulai for preparation of the manuscript and Angéla Schön and Ágnes Somos for technical assistance.

Footnotes

  • Send reprint requests to: Zoltán Gregus, M.D., Ph.D., D.Sc., Department of Pharmacology, University Medical School of Pécs, Szigeti út 12, H-7643 Pécs, Hungary. E-mail:gregus{at}apecs.pote.hu

  • This report is based on work sponsored by the Hungarian-United States Science and Technology Joint Fund, in cooperation with the Ministry of Social Welfare in Hungary and the United States Department of Health and Human Services. Financial support was also received from the Hungarian National Scientific Research Foundation (OTKA) and the United States Public Health Service (Grant ES03192).

  • Abbreviations used are::
    Vd
    volume of distribution
    CLb
    blood clearance
    Kel
    elimination rate constant
    • Received March 23, 1998.
    • Accepted June 9, 1998.

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Effects of Fibrates on the Glycine Conjugation of Benzoic Acid in Rats

Zoltán Gregus, Tibor Fekete, Éva Halászi, Ágnes Gyurasics and Curtis D. Klaassen
Drug Metabolism and Disposition November 1, 1998, 26 (11) 1082-1088;
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