Introduction

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DCA³ has been shown to have numerous biological effects, ranging from desirable therapeutic effects in humans (lowering blood levels of lactic acid and glucose) (Stacpoole, 1989) to undesirable toxic effects in rats (e.g. testicular abnormalities, birth defects, and liver cancer) (Smith et al., 1992; Toth et al., 1992; DeAngelo et al., 1991; Stacpoole et al., 1998). DCA is being used in clinical trials to lower blood lactic acid levels in children with congenital lactic acidosis (Stacpoole et al., 1997) or with lactic acidosis resulting from severe malaria infection (Krishna et al., 1995). It has been used experimentally in humans to lower lactic acid levels during liver transplantation (Shangraw et al., 1994). The effective human therapeutic dose range is 12.5-37.5 mg/kg, administered twice-daily (Stacpoole et al., 1997). DCA is also an environmental contaminant, formed by chlorination of surface water and oxidation of chlorinated solvents. It has been found in chlorinated municipal drinking water at concentrations up to 0.160 mg/liter (Uden and Miller, 1983) and is a minor metabolite of the industrial solvents trichloroethylene and perchloroethylene (Jolley, 1985; Abbas and Fisher, 1997). It has been suggested that environmental exposure of humans to DCA may be hazardous, because DCA is a mouse carcinogen when administered in the drinking water for 60-104 weeks, at concentrations of 500-5000 mg/liter (Herren-Freund et al., 1987; Daniel et al., 1992). At present, however, the relevance of the rodent toxicological characteristics of DCA to its human toxicological profile is unknown.

Although the biological effects of DCA have been widely studied, there have been few studies of the human or rodent pharmacokinetics and metabolism of DCA. Studies of DCA pharmacokinetics in humans showed that DCA was rapidly and completely absorbed after oral administration, that the elimination half-life of DCA from blood increased after the first dose was given, and that this effect persisted for several weeks (Curry et al., 1985; Henderson et al., 1997). In Fischer 344 rats (mean body weight, 344 g) given single oral doses of 5, 20, or 100 mg of [1,2-¹⁴C]DCA/kg, 23-29% of the dose was recovered as CO₂ and 19-24% was excreted in urine (Larson and Bull, 1992). The urine contained 1-2% of the dose as DCA, and the remaining urinary radioactivity was reported as nonchlorinated organic acids. In another study, there was evidence for dose-dependent effects on the fate of DCA. Fischer 344 rats (180-240 g) given single oral doses of 28.2 or 282 mg of [¹⁴C]DCA/kg excreted 25-35% of the dose as CO₂ and 12-35% in urine; 20-36% of the dose was recovered in rat tissues (Lin et al., 1993). The rats that received 282 mg/kg excreted less of the ¹⁴C as CO₂ and more in urine, and the percentage of unmetabolized DCA in urine ranged from 0.6% of the dose for the 28.2 mg/kg group to 20% of the dose for the 282 mg/kg group (Lin et al., 1993). Other urinary metabolites were reported to be glyoxylate, glycolate, and oxalate, although these metabolites were not unequivocally identified (Lin et al., 1993). These previous studies suggested that dose and possibly size influence the fate of DCA in rats. The present study further investigated the disposition, metabolism, and elimination of DCA in rats. The objectives were to determine whether repeat doses of DCA altered the fate of DCA in rats, as in humans, to identify the urinary metabolites, and to examine the influence of body size and overnight fasting on the pharmacokinetics and metabolism of DCA.

	Materials and Methods

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Chemicals. The two sources of [1-¹⁴C]DCA used involved the custom synthesis of NaDCA by Sigma Radiochemicals (St. Louis, MO) and the purchase of free acid from American Radiolabeled Chemicals (St. Louis, MO). The Sigma product had a specific activity of 5.5 mCi/mmol, a reported radiochemical purity of 99%, and a measured radiochemical purity of 97%. The American Radiolabeled Chemicals product had a specific activity of 55.5 mCi/mmol, and the reported and measured radiochemical purities were >99.9%. The free DCA was converted to the sodium salt by equimolar addition of NaOH. Na[1,2-¹³C-]DCA was custom-synthesized by Cambridge Isotope Laboratories (Cambridge, MA) as 100% isotope-enriched and was shown by GC/MS to be >99.5% chemically and isotopically pure. Unlabeled NaDCA (>99.8% chemically pure) purchased from TCI America (Portland, OR) was used to dilute the [¹⁴C]DCA to conserve radiolabeled compound. Carbosorb and Permafluor cocktails for use with the tissue oxidizer and Floscint II cocktail for use with the radiochemical detector were purchased from Packard Instruments (Chicago, IL). Ecolume scintillation cocktail was purchased from ICN (Costa Mesa, CA). DCA-glycine was synthesized by stirring 0.75 g of dichloroacetyl chloride with 1 g of glycine-t-butyl ester and 2 g of sodium carbonate, in 25 ml of acetonitrile, at 60°C for 2 hr. After cooling, the mixture was filtered and the filtrate was evaporated to dryness. The precipitate was suspended in ether and filtered, and the filtrate was allowed to evaporate in a hood. The product, DCA-glycine-t-butyl ester, was hydrolyzed in trifluoroacetic acid/water (9:1) to yield DCA-glycine. The structure of the DCA-glycine was confirmed by ¹H NMR (Jayanti, 1995). Phenylacetylglycine was prepared as described previously (James et al., 1972). All other chemicals used were of the purest grade available from Millipore (Woburn, MA), Fisher Scientific (Orlando, FL), Sigma Chemical Co. (St. Louis, MO), or Aldrich Chemical Co. (St. Louis, MO).

Animals. Male Sprague-Dawley rats were used in all studies. Most of the rats weighed 180-265 g and were 3-4 months of age, but one group of four larger rats (body weight, 580-690 g; age, 16 months) was also used. Rats were normally fed Purina laboratory chow ad libitum and maintained on a 12-hr light/dark cycle. In some studies of distribution, metabolism, and excretion, the chow was removed at 4 p.m. the day before dosing (fasted rats). All rats were allowed free access to water. DCA was administered by oral gavage of a solution of NaDCA in water. Doses were given between 10 a.m. and noon.

Preparation of Subcellular Fractions. Samples of liver (5 g) and kidney (1.5 g) were rinsed three times in 4 volumes of ice-cold 0.15 M KCl/0.05 M potassium phosphate, pH 7.4, and were then homogenized in 4 volumes of the ice-cold buffer. Subcellular fractions were prepared by differential centrifugation, as described previously (James and Little, 1983).

Analytical Methods. HPLC Analysis. Urine samples for each time point were filtered through a 0.45-µm nylon filter (Centrisart; Rainin Instruments) and analyzed by ion-pair reverse-phase HPLC. The following conditions were used: C₁₈ column (4.6 × 250 mm) with C₁₈ guard column (4.6 × 50 mm); 0.05-ml sample injection loop; isocratic mobile phase, 70% 0.005 M tetrabutylammonium sulfate (PICA, low UV; Waters)/30% methanol; flow rate, 1 ml/min; on-line detection, UV absorbance at 220 nm (Dynamax; Rainin Instruments) and radiochemical detection (Flo-one Beta; Packard Instruments). It was necessary to equilibrate the reverse-phase column with the ion-pair-containing mobile phase for at least 40 min before injection of samples, for reproducible analysis of DCA and metabolites. Standard solutions of DCA, as well as known and suspected DCA metabolites, were prepared in the mobile phase at concentrations of 0.2-2.5 mg/ml, and retention times were monitored by UV detection at 220 nm. The retention times of the standard compounds were as follows: glycine, 3.0 min; glyoxylate, 3.2 min; glycolate, 3.2 min; acetylglycine, 3.4 min; oxalate, 4.0 min; dichloroacetylglycine, 6.5 min; DCA, 7.1 min; hippuric acid, 10.2 min; phenylacetylglycine, 13.8 min; methyl-DCA, 15.8 min.

GC and GC/MS Analysis. The DCA concentrations in plasma from rats that had received unlabeled DCA were measured by the GC method of Chu et al. (1992). Plasma samples from rats that had received [1,2-¹³C]DCA were analyzed for DCA by GC/MS, using a Hewlett-Packard 5890 Series II Plus GC system, a 5972A mass-selective detector, and a Vectra multimedia VL2 4/66 computer with ChemStation software (Yan et al., 1997). GC/MS was also used to verify the identity of metabolites present in urine. Urine or plasma samples were spiked with 4-chlorobutyric acid as an internal standard and were derivatized by heating with an equal volume of a 14% solution of boron trifluoride in methanol at 115°C for 15 min. The methylated derivatives were extracted into methylene chloride. A portion of the methylene chloride extract was injected onto a Carbowax column (HP-Wax, 30 m × 0.25 mm with 0.15-µm film thickness; phase ratio, 420), with helium carrier gas (flow rate, 1.21 ml/min) and an inlet pressure of 9 psi. The GC system temperature was maintained at 35°C for 4 min, followed by a linear gradient to 100°C at 3°C/min and then to 240°C at 50°C/min. The temperature was maintained at 240°C for 8 min. Under these conditions, the retention times of the methyl esters of DCA and metabolites were as follows: DCA, 10.8 min; glyoxylate, 11.2 min; oxalate, 11.7 min; acetylglycine, 17.2 min; hippuric acid, 20.2 min; phenylacetylglycine, 21.2 min. For quantitation of DCA in plasma, single ions of molecular mass 59 and 60, corresponding to the -¹²COOCH₃ and -¹³COOCH₃ fragments, respectively, were monitored for the peak with a retention time matching that of authentic methylated DCA. Standard curves were developed with methylated [¹³C]DCA standard under identical conditions and were used to quantitate the plasma peaks, as described previously for human studies (Yan et al., 1997). Each of the urinary metabolites was identified by determining the GC retention time and by matching the complete mass spectrum of the ¹³C-labeled methylated metabolite to the mass spectrum of the methylated authentic ¹²C standard. The presence of ¹³C in each metabolite peak was confirmed by comparing the ¹³C/¹²C ratios in the molecular ion (for methylated glycine conjugates of benzoic, acetic, and phenylacetic acids) or the -COOCH₃ fragment (for oxalate and glyoxylate) with the natural abundance of ¹³C in unlabeled metabolite standards. If the ratio was higher than expected for the natural abundance of ¹³C, it was assumed that the metabolite arose from [¹³C]DCA.

NMR Analysis. One sample of urine from a rat that had received a DCA dose containing a mixture of 99% [1,2-¹³C]DCA and 1% [1-¹⁴C]DCA (final specific radioactivity, 0.555 mCi/mmol) was analyzed by ¹H and ¹³C NMR spectroscopy. HPLC analysis, with radiochemical detection, of this urine sample showed that 70.5% of the urinary DCA-derived ¹⁴C was in the form of an unidentified metabolite with a retention time of 10.5 min. This urine sample also contained 0.5% parent DCA, 18.6% oxalate, and 9.6% other polar metabolites and had 300 µg of DCA-molar equivalents/0.6 ml of urine. To adjust magnetic field homogeneity and to maintain a stable field-frequency lock, the urine sample (0.6 ml) was mixed with D₂O (0.15 ml), and this solution was examined in a 5-mm tube. The D₂O contained a trace of 3-(trimethylsilyl)propionate-2,2,3,3-d₄ sodium salt, serving as a chemical shift reference. For both ¹H and ¹³C studies, the sample was analyzed (spinning at 20 Hz) with a Varian Unity-300 spectrometer (magnetic field, 7 T; ¹H frequency, 300 MHz), using a 5-mm Nalorac Z-Spec broad-band probe regulated at 25°C (Center for Structural Biology, University of Florida). The ¹H and ¹³C spectra were each referenced to the 3-(trimethylsilyl)propionate-2,2,3,3-d₄ sodium salt resonance at 0.0 ppm.

Pharmacokinetic Modeling. Pharmacokinetic modeling was conducted with the PCNONLIN program (Statistical Consultants, Lexington, KY). The initial data analysis was performed using the RSTRIP program (MicroMath, Salt Lake City, UT). After the initial analysis with the RSTRIP program, DCA plasma levels were fitted to one-, two-, and three-compartment models, using the nonlinear regression program PCNONLIN (version 3.0). AIC, where AIC = N·ln(SSE) + 2p (where N is the number of observations, p is the number of model parameters, and SSE is the residual sum of squares), was used to compare the three models (Akaike, 1978; Akcay and Rose, 1980; Torres-Molina et al., 1992). The model with the lowest AIC would be the most efficient. It was found that some of the data best fit a one-compartment pharmacokinetic model and other sets best fit a two-compartment model. The pharmacokinetic parameters were obtained using the appropriate model for each rat.

Statistical Analysis. Statistical analyses of the experimental data were performed with the general linear models procedure of the Statistical Analysis System (SAS Institute, 1989). Feeding status and number of doses were modeled as separate main effects. A cross-products term was included to assess potential interactions. In no instance did the interaction term have a significant impact on the comparison of main effects; therefore, the reported analyses are of the main effects only. The outcome variables were the percentage of the dose excreted as ¹⁴CO₂, the total percentage of the dose excreted in urine, and the percentage of the dose excreted as each of the separated metabolites or metabolite groups.

	Results

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Excretion in Expired Air. The extent of metabolism of DCA and the routes of excretion of DCA metabolites varied with rat size, preexposure to DCA, and feeding status. A major route of elimination of radioactivity from [¹⁴C]DCA was in expired air as ¹⁴CO₂, as shown in fig. 1 and table 1. The percentage of the radioactive dose excreted as CO₂ was quantitated in the young adult rats and varied with access to food and number of DCA doses. It was not possible to quantitate ¹⁴CO₂ excretion by the large fed rats given two doses of DCA, because they were too large for the metabolism cages. For rats that were allowed free access to food, 17-26% of the radiolabeled dose was excreted as CO₂ in 24 hr. Less ¹⁴CO₂ was expired in 24 hr by fed, repeatedly dosed rats, compared with fed, singly dosed rats, and the initial rate of CO₂ excretion was lower (fig. 1). In the first 6 hr after receiving a single dose of [¹⁴C]DCA, fed rats excreted 2.93 ± 0.43% of the dose/hr as CO₂, whereas repeatedly dosed rats excreted 1.67 ± 0.32%/hr (significantly different, p < 0.05).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Cumulative excretion of 14CO2 in the expired air of young adult rats given one (upper) or two (lower) doses of [14C]DCA (50 mg/kg) by oral gavage. Points, mean ± SD (N = 3 or 4/treatment group). The fasted animals excreted more 14CO2 in 24 hr than did fed animals in both groups (p < 0.001). The initial rate of excretion of 14CO2 was faster in the fed, repeatedly dosed rats (2.93% of the dose/hr in the first 6 hr) than in the fed, singly dosed rats (1.67%/hr in the first 6 hr) (p < 0.05).

Urinary Excretion and Identification of Urinary Metabolites. There were major differences between the young adult rats and the large rats with respect to the percentage of the dose excreted in urine over 24 hr and the percentage of the dose excreted as parent DCA (fig. 2, table 2). Access to food did not affect the percentage of the dose excreted in urine for the young adult rats; therefore, the data for total urinary excretion of ¹⁴C were pooled for singly dosed rats and for repeatedly dosed rats (fig. 2). All rats excreted approximately 7% of the dose in urine in the first 6 hr. The rate of urinary excretion decreased after 6 hr for the young adult rats, whereas the large rats continued to excrete DCA and metabolites in urine, in an almost linear manner, up to 24 hr (fig. 2).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Cumulative excretion of 14C in urine of rats given one or two 50 mg/kg doses of [14C]DCA. There were no differences between fed and fasted rats in the rate or extent of urinary excretion; therefore, the results are shown based on the number of doses received. Points, mean ± SE. Between 6 and 24 hr, there were significant differences in the rate and extent of urinary excretion in the large rats, compared with the young adult rats (p < 0.05).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   HPLC chromatograms for representative urine samples from rats given [14C]DCA (50 mg/kg) under different conditions of access to food and prior administration of DCA. Reverse-phase, ion-pair HPLC was performed as described in Materials and Methods. The traces shown are from the radiochemical detector, set to detect 14C. i-iii, traces from young adult rats (body weight, 180-265 g); iv, trace from a large rat (body weight, 690 g). i, Singly dosed rat fasted overnight, 0-3-hr urine sample; ii, repeatedly dosed rat fasted overnight, 3-6-hr urine sample; iii, repeatedly dosed rat with free access to food, 0-3-hr urine sample; iv, repeatedly dosed large rat with free access to food, 0-6-hr urine sample. The positions of DCA and some metabolites are indicated. D, DCA peak; U1, unknown metabolite 1; U2, hippuric acid; U3, phenylacetylglycine. The peaks eluting before 6 min include oxalate, glyoxylate, glycine, and acetylglycine. See Materials and Methods for a complete list of retention times.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Proton-decoupled NMR spectrum of urine from a rat dosed with a 99:1 mixture of [13C]DCA and [14C]DCA (50 mg/kg). The urine sample used was collected 3-6 hr after the dose. HPLC analysis showed that 70% of the radioactivity in the urine sample comigrated with hippurate and 18.6% comigrated with oxalate. Peaks corresponding to the 13C-labeled (*) and unlabeled (natural abundance) carbons in hippuric acid and oxalic acid are numbered as shown in the structures.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   GC/MS analysis of urine from a rat given one 50 mg/kg dose of a 99:1 mixture of [13C]DCA and [14C]DCA. The sample was collected 3-6 hr after the dose and methylated as described in Materials and Methods. Upper, GC trace for the methylated urine sample, obtained under conditions described in Materials and Methods. Middle, mass spectrum of the peak at 20.8 min (hippurate). Lower, mass spectrum of the peak at 21.2 min (phenylacetylglycine). The peak at 17.3 min represents acetylglycine (mass spectrum not shown). Insets, expansions of the molecular ion peaks, showing the M+2 ions that arise from the 13C enrichment of the glycine.

Elimination of DCA from Plasma. There was no effect of feeding on the plasma uptake or elimination of DCA, so data for rats allowed free access to food were pooled with data for rats restricted from food overnight. GC and GC/MS methods gave similar results for DCA concentrations. DCA was very rapidly absorbed after oral administration (table 3, fig. 6). The peak DCA plasma concentrations varied among groups of rats and were considerably higher for the large, repeatedly dosed rats than for the young adult rats. The rate of elimination of DCA from plasma varied markedly between the rat groups receiving single and repeat doses and between young adult and large rats receiving repeat doses (fig. 6). The elimination half-life for singly dosed rats was 0.11 ± 0.05 hr, that for repeatedly dosed, young adult rats was 5.4 ± 1.8 hr, and that for repeatedly dosed, large rats was 9.7 ± 1.9 hr. Other pharmacokinetic parameters are shown in table 3.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Plasma elimination of DCA after one or two oral doses of 50 mg/kg. Upper, young adult rats given one dose; middle, young adult rats given two doses; lower, large rats given two doses. The plasma concentrations of DCA were measured by GC or GC/MS with single-ion monitoring (see Materials and Methods) and are shown as mean values from four or five rats. The pharmacokinetic parameters are summarized in table 3.

Tissue Distribution of Radioactivity. Between 25 and 42% of the administered [¹⁴C]DCA dose remained in the body after 24 hr (table 1). Liver, muscle, skin, and the gastrointestinal tract contained most of the remaining radioactivity. Although there was interindividual variability in the amounts left in tissues, there were no significant between-group differences in these values. The percentage of radioactivity in the livers of the repeatedly dosed, large rats was very variable, ranging from 6 to 15.6% of the dose.

	Discussion

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Excretion of DCA as CO₂. Previous studies with Fischer 344 rats showed that expired CO₂ was a major route of excretion of DCA (Larsen and Bull, 1992; Lin et al., 1993). Preliminary investigations in this laboratory with Sprague-Dawley rats also showed that a large percentage of the DCA dose was expired as CO₂ (Jayanti et al., 1994). In a recent report, Gonzalez-Leon et al. (1997) showed that, in Fischer 344 rats, the rate and extent of conversion of [¹⁴C]DCA to ¹⁴CO₂ were dependent on dose and pretreatment with DCA. Rats exposed to 2 g/liter DCA in the drinking water before being given a radiolabeled dose of 5, 20, or 100 mg/kg DCA excreted CO₂ more slowly than did rats that were not pretreated. Over a 24-hr period, less total DCA was excreted as CO₂ by pretreated rats given 20 or 100 mg/kg DCA, but for rats given the lowest dose (5 mg/kg DCA) the total amount of CO₂ excreted in 24 hr was similar to that for control rats. The access to food of the Fischer 344 rats was not stated.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Scheme showing the possible routes for formation of CO2 from 1-[14C]DCA. *, 14C position; THF, tetrahydrofolate; PDH, pyruvate dehydrogenase.

Tissue Distribution of DCA and Metabolites. Radioactivity from DCA was widely distributed in tissues of rats sacrificed as early as 1 hr after the dose (table 1). Liver and muscle were major initial distribution sites, and liver retained radioactivity from DCA for at least 24 hr after the dose. Kidneys also exhibited high concentrations of radioactivity, but because of their size the percentage of the dose was small. At the 1-hr time point, both liver and kidney cytosolic fractions had the highest concentrations of radioactivity, consistent with the localization of an enzyme for conversion of DCA to glyoxylate (Lipscomb et al., 1995; James et al., 1997). Liver microsomes and mitochondria had similar percentages of radioactivity, whereas in the kidney microsomes had very little radioactivity from DCA (table 4). To date no microsomal pathways of metabolism of DCA have been identified. It is likely that DCA metabolites formed in the cytosol, such as glycine, are incorporated into newly synthesized microsomal proteins. At this point, there is no evidence that the radioactivity in hepatic proteins is derived from adducts with intact DCA or chlorinated metabolites, rather than from incorporation of the DCA carbons into the carbon pool.

Identification of Glycine Conjugates as Urinary Metabolites of DCA. This study is the first to report that glycine conjugates are urinary metabolites of DCA. Based on the positive identification of hippurate (HPLC retention time, NMR spectrum of urine, and GC/MS results for methylated urine) and phenylacetylglycine (HPLC retention time and GC/MS results) as urinary metabolites of DCA, table 2 reports the percentage of the dose excreted in urine as hippuric acid and phenylacetylglycine, with the glycine coming from DCA. Unknown urinary metabolite U1 may also be a glycine conjugate, possibly p-hydroxyhippurate, but it was not present in large enough amounts for identification in the subset of rats given doubly labeled [¹³C]DCA and [¹⁴C]DCA. Unlike other xenobiotic carboxylic acids, which are often excreted as glycine or glucuronide conjugates, we found no evidence that DCA itself directly forms either glycine or glucuronide conjugates. Glycine arising from the metabolism of DCA to glyoxylate (fig. 8) appears to be available to glycine N-acyltransferase, which is located in the mitochondrial matrix (James and Bend, 1978; Kelley and Vessey, 1993). Some of the glyoxylate formed in the hepatic cytosol (James et al., 1997) may be transported to the mitochondria and transaminated in this organelle. Alternatively, glycine formed in another cellular compartment may be taken up by mitochondria, where some is decarboxylated and some is used in glycine conjugation. Studies with liver and kidney homogenates from mice have shown that glyoxylate, as a glycine precursor, is as effective as glycine in supporting hippurate synthesis from benzoic acid in vitro (Qureshi et al., 1989). Other studies have shown that the availability of glycine limits the formation of hippurate from benzoic acid (Beliveau and Brusilow, 1987), perhaps because the hepatic concentration of glycine is close to the K_M for glycine (3 mM) of benzoyl-CoA/glycine N-acyltransferase (Gregus et al., 1992; Nandi et al., 1979). The apparent K_M for glycine of rat renal phenylacetyl-CoA/glycine N-acyltransferase is 20 mM (James and Bend, 1978), suggesting that glycine availability is critical for formation of phenylacetylglycine from phenylacetic acid.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Pathways for DCA metabolism in rats that were verified in this study.

Urinary Excretion of DCA and Metabolites. The importance of urine as a route of elimination of DCA varied with rat size, feeding status, and prior exposure to DCA. With young adult rats given one dose of DCA, <1% of the dose was excreted unchanged in urine; with large rats given two doses of DCA, an average of 20% of the second DCA dose was excreted unchanged in urine (table 2). With young adult rats given two doses of DCA, the excretion of unchanged DCA in urine varied with feeding status but was <5% of the second dose. The finding of extensive urinary excretion of unchanged DCA, together with reduced plasma elimination of DCA, suggests that DCA metabolism is very slow in the large rats.

Pharmacokinetics of DCA in Rats. As has been found in humans (Stacpoole et al., 1998), the present study showed that a single dose of 50 mg/kg DCA dramatically impaired the elimination of a subsequent DCA dose in young adult male Sprague-Dawley rats. Although no DCA was detectable in singly dosed rats by 12 hr after the dose, rats given a second dose the next day had detectable levels of DCA in plasma up to 24 hr after the dose (fig. 6). Another striking finding from the present study was that the pharmacokinetics of DCA in large rats given two doses of DCA were very different from those in similarly treated young adult rats. The peak plasma concentrations were 5-fold higher in the large rats, and the elimination half-life was slowed from 5.4 to 9.7 hr. The urinary excretion of unchanged DCA was also different in the large rats (see above). It is not clear whether the observed differences in DCA metabolism in the two repeatedly dosed groups were the result of size or age, because the large rats used were both older and larger.