INTRODUCTION

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RACEHORSES UNDERGO TRAINING regimens of various intensities to improve fitness. The effects of training on the cardiovascular, respiratory, and musculoskeletal system in horses have been documented, but the effect of training on liver function in horses has not been studied. In other species, exercise training leads to variable effects on liver function (6, 7, 10, 16). Although biochemical tests such as serum enzyme (sorbitol dehydrogenase and alkaline phosphatase) activities may be sensitive indicators of liver disease, they cannot be used to evaluate liver function (2). Because liver function involves metabolism and clearance of toxins and exogenous substances, including most drugs, the hepatic clearance of certain drugs can be used to evaluate liver function. Such tests of hepatic clearance can reflect a single physiological determinant of clearance (19), such as the intrinsic drug-metabolizing capacity of the liver for poorly extracted drugs such as antipyrine.

Antipyrine has been effectively used in other animal species as a model substrate to measure changes in hepatic drug metabolism, to evaluate induction or inhibition of cytochrome P-450 microsomal enzyme systems, and to measure total body water (18), and antipyrine may be a suitable model drug to assess training-induced effects on hepatic function.

Antipyrine pharmacokinetics have been studied in horses (4, 8, 9). Antipyrine is predominantly cleared by hepatic metabolism, albeit poorly, and has a volume of distribution similar to total body water (8). Plasma clearance (Cl_p) of antipyrine is ~6.2 ml · min¹ · kg¹ and steady-state volume of distribution (Vd_ss) is ~860 ml/kg body weight (8). The dependence of the Cl_p of antipyrine on hepatic clearance of antipyrine suggests that altering the capacity of cytochrome P-450 enzymes to metabolize antipyrine may be reflected in changes in the Cl_p of antipyrine. Because training has been shown to increase cytochrome P-450 enzyme capacity in rats by increased mRNA synthesis of enzyme (6), we elected to study whether training-induced changes would be demonstrable in horses in vivo, because species-dependent differences in cytochrome P-450 systems have been documented (20). There is considerable evidence that antipyrine is metabolized by multiple cytochrome P-450 isoforms and that subtle training-induced changes in individual cytochrome P-450 systems may not be detected by examination of the Cl_p of antipyrine in isolation. Therefore, we also measured changes in the total urinary excretion of major antipyrine metabolites to detect potential changes in individual cytochrome P-450 systems.

To investigate the effects of training on hepatic oxidative metabolism in horses, we studied the pharmacokinetics of antipyrine in horses undergoing training (individualized for each horse) that was expected to be sufficient to significantly change each horse's maximal oxygen consumption (O_{2 max}) over a 5-wk period. In addition, we included a control (untrained) group of horses to examine possible changes in antipyrine pharmacokinetics as a function of time or repeated antipyrine administrations. If training has an effect on the hepatic clearance of drugs used in horses, veterinarians may need to alter dosages to accommodate for training effects. In addition, training may alter the urinary excretion of drugs and the subsequent detection of drugs in urine on race day.

	MATERIALS AND METHODS

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Study design. Antipyrine was injected intravenously in each of two groups of horses before and after a 5-wk period. During that 5-wk period, one group of horses underwent treadmill training while the other group was housed in a stall barn. All animal studies were conducted during the spring season. Plasma and urine samples were analyzed by HPLC for determination of concentrations of antipyrine and metabolites. Antipyrine pharmacokinetic parameter estimates were compared within and between each group to study the possible effects of exercise on antipyrine pharmacokinetics. The experiment was approved by The Ohio State University Institutional Laboratory Animal Care and Use Committee.

Animal studies. Thirteen healthy Standardbred mares, ranging from 6 to 9 yr of age and from 411 to 497 kg in weight, were used in these studies. They had spent the previous 3 mo in small paddocks (where they were observed to spend most daylight hours standing, walking, and occasionally cantering) or in box stalls (intermittently for no longer than 2 wk). All horses had been accustomed to treadmill exercise within 6-12 mo of this study. Horses were kept in box stalls for at least 14 days before antipyrine administration and were supplied with mixed grass hay ad libitum, crushed corn, trace mineral salt, and water. All horses were judged to be healthy based on a physical examination, clinical chemistry profile, and hemogram.

Measurement of O_{2 max} and the oxygen consumption ( O₂ )-speed relationship. The O_{2 max} and the relationship between O₂ and speed were determined for each horse during an incremental exercise test within 7 days of the experiment. The incremental exercise test consisted of each horse running on a treadmill (Sato, BIAB Industrial, Uppsala, Sweden) inclined at 4° for 90 s at 4 m/s, after which the treadmill speed was increased by 1 m/s every 90 s until the horse was unable to maintain its position on the treadmill. O₂ was measured every 10 s during the exercise test with the use of an open-circuit calorimeter (Oxymax-L; Columbus Instruments, Columbus, OH) as previously described (13). O_{2 max} was defined as the value at which O₂ reached a plateau despite further increases in speed. A plateau was defined as a change in O₂ of <4 ml · min¹ · kg¹ with an increase in speed.

Drug administration. Antipyrine (Aldrich Chemical, Milwaukee, WI) was dissolved in 0.9% sodium chloride (Baxter Healthcare, Deerfield, IL) at a concentration of ~360 mg/ml and filtered through a 0.22-µm filter (Millex-GV; Millipore Products Division, Bedford, MA). A dose of 20 mg/kg was injected into the left jugular vein over 30-45 s.

Blood sampling. Before drugs were administered, an intravenous catheter (Angiocath; Becton Dickinson Vascular Access, Sandy, UT) was inserted into the right jugular vein. Blood samples were taken before administration of the drug and at 5, 10, 15, 20, 30, and 45 min and 1, 2, 3, 4, 5, 6, 7, and 8 h after antipyrine was administered.

Urine sampling. A Foley catheter was inserted into the urinary bladder of each mare. A urine sample was collected for 10-30 min before the drug was administered. After antipyrine was administered, urine samples were collected for 8 h. Hourly volumes were recorded, and 5- to 50-ml samples from each hourly collection were placed in acetate buffer (0.5 M sodium acetate; Jenneille Chemical, Cincinnati, OH) with 80 g/l sodium metabisulfite (Jenneille Chemical) in a 1:1 (vol/vol) ratio and kept at 70°C until analysis.

Training protocol. Horses were randomly allocated to two groups: group 1 underwent a training protocol; group 2 was untrained (control group) and was maintained in box stalls throughout the experiment. Group 1 horses underwent a training protocol consisting of treadmill exercise on 6 days/wk for 5 wk. Group 2 horses were untrained (control group) and were maintained in box stalls throughout the experiment. Exercise consisted of walking for 1 min at 2 m/s and then at 4 m/s for 1 min on a treadmill inclined at a 4^o slope. Treadmill speed was rapidly increased and maintained at a speed calculated to require 60% O_{2 max} for 10 min, at which point treadmill speed was increased to a speed calculated to require 90% O_{2 max} for 3 min. The horses then ran at 60% O_{2 max} for 2 min before walking at 2 m/s for 1 min. Total exercise time was 18 min, 13 min of which were at relatively high intensity. At all other times, the horses were in box stalls.

Determination of antipyrine concentrations in plasma. The normal-phase HPLC method previously described (8) was used to determine the concentration of antipyrine in methylene chloride extracts of alkalinized plasma samples, with the use of 4-aminoantipyrine as the internal standard.

Determination of concentrations of antipyrine, norantipyrine, and 4-hydroxyantipyrine in urine. The concentrations of antipyrine, norantipyrine, and 4-hydroxyantipyrine in urine samples were determined by a previously described reverse-phase HPLC analysis of acetonitrile extracts of acidified urine samples, with the use of phenacetin as the internal standard (8).

Pharmacokinetic analysis. Methods of analysis of plasma antipyrine concentration vs. time data and analysis of urinary excretion of antipyrine and metabolites data were identical to those previously reported (8).

Statistical analysis. Body weights, O_{2 max}, and blood lactate concentrations were compared within and between groups by using two-way ANOVA and post hoc Student t-tests for paired and independent data, as appropriate. Antipyrine Cl_p, Vd_ss, and renal excretion of drug and/or metabolite within and between groups were compared by using Wilcoxon signed-rank tests and Mann-Whitney rank-sum tests, respectively. A P value of <0.05 was considered significant.

	RESULTS

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Although 16 horses began the study, the results of two horses were not included because the horses became lame during training and were removed from the trial. Another horse (150) was initially included in group 2 and then was moved to group 1 for training. We were unable to perform O₂ tests on horse 46, as it became agitated when the face mask was placed on the face. Therefore, O_{2 max}- and O₂-speed regressions were not completed for this horse, and the speeds estimated to require 60 and 90% O_{2 max} for this horse were extrapolated from values calculated for other horses. Because blood lactate data were similar to those of the other horses, deviations in the initial protocol are not expected to have influenced the results. There were no significant differences between the trained and untrained group at the start of the study with respect to body weight (456 vs. 453 kg, respectively) or O_{2 max} (129 ± 9.5 vs. 121 ± 12.2 ml · min¹ · kg¹, respectively). In addition, there were no differences in Cl_p (6.0 vs. 5.0 ml · min¹ · kg¹, respectively) or Vd_ss (877 vs. 846 ml/kg, respectively) of antipyrine between groups at the start of the study.

In the trained group, body weights decreased an average of 5% [from 456 ± 25.8 (SD) to 432 ± 24.2 kg]. In the untrained group, body weights decreased an average of 2%, from 453 ± 30.6 to 445 ± 30.7 kg. Body weight decreases in the trained group were significantly different from those in the untrained group (P < 0.005). O_{2 max} increased after training by 15%, which was significantly different from an increase of 2% in the untrained group (P = 0.004; Fig. 1). Changes in the treadmill velocity needed to produce a V_La4 in the trained group were significantly greater after 5 wk than in the untrained group (9 vs. 4%, respectively; P = 0.035; Fig. 1). Initially V_La4 was 5.6 ± 0.76 and 4.8 ± 0.52 m/s in the trained and untrained groups, respectively. Changes in blood [La]_max were not significantly different between groups (P = 0.836).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Maximal oxygen consumption (O2 max) and velocity to obtain a blood lactate concentration of 4 mmol/l (VLa4) for horses before and after a 5-wk period of either training (n = 7 mares) or box stall rest (n = 6 mares).

Mean plasma concentrations of antipyrine vs. time in both groups (Fig. 2) were representative of data from each horse. Cl_p of antipyrine significantly increased (from 5.5 to 6.4 ml · min¹ · kg¹, before and after training, respectively; P = 0.031). In the untrained group, Cl_p was significantly less after 5 wk of box rest (5.0 vs. 4.2 ml · min¹ · kg¹; P = 0.031; Table 1 and Fig. 3). Percent differences in Cl_p before and after the training period were significantly different between the trained group (median, +16%) and the untrained group (median, 12%; P = 0.001). When Cl_p was not normalized for body weight (that is, expressed as liters per minute), similar differences in the two groups were still observed. There was no apparent relationship between the relative change in O_{2 max} during training or rest and the corresponding relative change in Cl_p of antipyrine (r < 0.4).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Mean plasma concentration of antipyrine vs. time data for horses after administration of antipyrine (20 mg/kg iv) before and after a 5-wk period of either training (Trained, n = 7 mares) or box stall rest (Untrained, n = 6 mares).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Plasma clearance (Clp), steady-state volume of distribution (Vdss), and elimination half-life of antipyrine for horses before and after a 5-wk period of either training (n = 7 mares) or box stall rest (n = 6 mares). * P < 0.05 compared with initial values.

Median Vd_ss of antipyrine increased as a result of training (from 813 to 881 ml/kg; P = 0.047) and decreased significantly in the untrained group (from 846 to 745 ml/kg; P = 0.031; Table 1 and Fig. 3). Percent differences in Vd_ss after the 5-wk period were significantly different between the trained group (+8%) and the untrained group (12%; P = 0.008). Similar results were seen when Vd_ss was not normalized for body weight. Elimination half-lives were not significantly different from each other, either within a group or between groups (P > 0.7; Table 1 and Fig. 3).

There was no effect of training or rest on the percent difference in the excretion of norantipyrine, antipyrine, 4-hydroxyantiyprine, or total excretion of metabolites (Table 2).

	DISCUSSION

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The purposes of our study were to determine the effects of an intense 5-wk exercise training period on hepatic function by assessing changes in the hepatic clearance and oxidative metabolism of antipyrine. We demonstrated that 5 wk of training at submaximal intensities led to a 15% increase in O_{2 max} and a 9% increase in V_La4. This was associated with a 16% increase in Cl_p of antipyrine and an 8% increase in Vd_ss of antipyrine; this suggests that both hepatic function and total body water are elevated by training. These changes were significantly greater than those seen in the untrained group. Other studies have demonstrated increases in O_{2 max} during training (12, 14), but an untrained control group was not included in those studies.

Cl_p of antipyrine increased in the trained group by ~7% and decreased in the untrained group by ~12%. We suggest that training led to an increase in the oxidative metabolic capacity of the liver to metabolize antipyrine and that a decrease in this capacity occurred in the other group of horses as a result of box rest. The endogenous biochemical stimuli that induce hepatic oxidative capacity during training have not been identified. Expression of cytochrome P-450 proteins can be induced by various factors, including age, gender, disease, and exposure to chemicals, that lead to increased numbers of enzyme molecules and capacity for metabolism by that isozyme. An inducing agent may produce a specific pattern of isozyme synthesis rates, resulting in different isozyme expression and individual changes in rates of metabolism by different isozymes (17). There is evidence that hepatic mRNA activity for cytochrome P-450 isoform enzymes changes during training in rats (6). This would result in changes in the maximal capacity of the cytochrome P-450 enzyme pathway, resulting in altered ability of the liver to metabolize certain drugs. In addition, it has been shown that the activity of oxidative enzymes in skeletal muscle (such as citric synthase) increases with training of horses (1). The training-induced increase in the Cl_p of antipyrine observed in our study demonstrates that training can alter hepatic enzyme function in horses and that the effects of training are not limited to the cardiorespiratory or musculoskeletal systems.

Training also led to an increase of ~8% in the Vd_ss of antipyrine in our study, presumably because of an increase in the amount of total body water or an increase in total body water relative to body weight (liters/kg body weight). Conversely, box rest led to a 12% decrease in antipyrine Vd_ss. Training-induced changes in body mass, percent lean body mass, or body fluid compartments have not been studied in horses, except for plasma volume. Plasma volume increased 20-30% after training (14, 15), but our study is the first to document increases in total body water (the putative Vd_ss of antipyrine) as a result of training.

Elimination half-life is dependent on Vd_ss and Cl_p, and this relationship can be expressed as The combined increase in the Cl_p and Vd_ss of antipyrine had a minimal effect on the elimination half-life of antipyrine in our study; therefore, plasma concentrations of antipyrine were minimally affected by training. Nevertheless, the training-induced increases in Cl_p and Vd_ss may have practical consequences. Calculations of loading doses of drugs are dependent on Vd_ss, and calculations of maintenance doses are dependent on Cl_p. For therapeutic drugs with kinetic parameters similar to those for antipyrine, both the loading dose and maintenance dose may have to be increased in animals that are exercise trained.

Antipyrine was selected as the model drug for examination of hepatic oxidative metabolism because of its pharmacokinetic properties, including low hepatic extraction, metabolism by numerous cytochrome P-450 pathways, and rapid excretion of metabolites into urine. In the horse, antipyrine is metabolized to at least two metabolites, norantipyrine and 4-hydroxyantipyrine (8). The urinary excretion of both of these metabolites was studied, because production of metabolites was not sufficient to lead to measurable plasma concentrations of major antipyrine metabolites. Demethylation and hydroxylation are involved in the production of these metabolites; therefore, at least two different cytochrome P-450 pathways must be involved in the metabolism of antipyrine. There is evidence in other animal species that certain factors (including other drugs, diet, and environmental factors) can induce individual cytochrome P-450 pathways differently, but this differential induction may be subtle and may not be detected when only plasma concentrations of antipyrine are studied. Administration of 3-methylcholanthrene to rats increased the formation rate of 4-hydroxyantipyrine and norantipyrine but decreased the formation of 3-hydroxymethylantipyrine (11). However, administration of either phenobarbitone or antipyrine leads to increased formation rate of norantipyrine but not 4-hydroxyantipyrine or 3-hydroxymethylantipyrine (5). In our study, we demonstrated a training-induced increase in antipyrine Cl_p but did not demonstrate increased formation rates of 4-hydroxyantipyrine or norantipyrine. Therefore, although training increased antipyrine clearance, we did not identify an increase in any one pathway.

Unlike antipyrine recovery in other animal species, only a small percentage of the dose of antipyrine administered is recovered as antipyrine or metabolites in urine of horses (8). This species difference and the considerable interindividual variability in metabolite excretion observed in our studies may make it difficult to detect changes in the urinary excretion of antipyrine metabolites in horses. The biliary excretion of antipyrine and its metabolites in horses has not been investigated, but excretion is likely to be a major route of elimination of antipyrine. Training-induced increases in Cl_p of antipyrine may be reflected in changes in biliary excretion rather than in urinary excretion of antipyrine metabolites.

This model has some limitations. We used a relatively brief period (5 wk of training) and a constant relative amount of work/wk. However, this period and intensity of training were sufficient to cause training effects, as shown by increases in O_{2 max} and V_La4. In addition, the period of acclimation to box stalls was only 2 wk before antipyrine administration. Our horses had minimal paddock activity before this acclimation period, but there is evidence to suggest that horses detrain very slowly and may require many months of rest before they are considered unfit (3). We suggest that, to avoid potential variability caused by previous training, horses should be detrained for extended periods of time before drugs are administered in further studies. To account for these potential confounding variables, we included a control group of horses that were kept in box stalls for the 5-wk period. Over this period of rest, antipyrine Cl_p and Vd_ss of antipyrine decreased in these horses, thus demonstrating an effect of inactivity on hepatic function and total body water. Interestingly, significant changes in O_{2 max} and V_La4 were not seen during this 5-wk rest period.

Although training altered the hepatic clearance and Vd_ss of antipyrine, it would be unrealistic to predict the effects of training on other drugs that are eliminated by hepatic clearance. The hepatic clearance of other drugs that differ greatly from antipyrine with respect to distribution, metabolism, and excretion would not be expected to correlate closely with the hepatic clearance of antipyrine. The purpose of this study was to determine whether exercise training could influence the hepatic metabolism of a model substance, thereby demonstrating that training could affect hepatic function in horses. Caution must be used in extrapolation of these results to predict the training-induced effects on the hepatic clearance of other drugs in horses, and such extrapolation was not the purpose of this study.