Intraoperator, intraobserver and interoperator variability of echocardiographic measurements in healthy foals.

REASON FOR PERFORMING STUDY: The repeatability of various echocardiographic measurements is unknown. OBJECTIVES: To determine the intraoperator, intraobserver and interoperator variability of echocardiographic measures in healthy foals. METHODS: Echocardiographic examinations were carried out on 6 healthy foals by 3 experienced echocardiographers. Intraoperator variability was determined by having a single echocardiographer obtain and measure images from 6 foals scanned on 3 consecutive days. Interoperator and intraobserver variability were determined by having 3 echocardiographers each obtain images from an additional 6 sedated foals. Within-day interoperator variability was determined by having each echocardiographer measure their own images. Intraobserver variability was determined by having a single echocardiographer measure images obtained by all 3 echocardiographers. The coefficient of variation (CV) and standard error were calculated for each measure. RESULTS: The variability for most measurements was either very low (CV < 5%) or low (CV = 5-15%). Measurements of right ventricular internal diameter (RVID) in systole and E-point to septal separation (EPSS) showed moderate (CV 15-25%) to high variability (CV > 25%) in all 3 categories. Measurements of the left ventricular ejection time (LVET) and velocity time integral from the right parasternal long axis view of right outflow tract in the fourth intercostal space showed moderate intraoperator variability. Measurements of the LVET, RVID in diastole and left atrial appendage (LAA) showed moderate interoperator variability and measurements of the RVID in diastole and acceleration time from the short axis view of the right outflow tract in the right third intercostal space showed moderate interobserver variability. CONCLUSION: The intraoperator, intraobserver and interoperatorvariabilities for most echocardiographic measurements in foals are low. POTENTIAL RELEVANCE: Most standard transthoracic echocardiographic measurements in foals have a low enough variability to warrant their use in serial clinical evaluations or experimental studies. Repeated measurements of RVID, EPSS, LVET and LAA should be interpreted with caution.

Cardiac troponin I concentrations in ponies challenged with equine influenza virus.

BACKGROUND: Myocarditis is thought to occur secondary to equine influenza virus (EIV) infections in horses, but there is a lack of published evidence. HYPOTHESIS/OBJECTIVES: We proposed that EIV challenge infection in ponies would cause myocardial damage, detectable by increases in plasma cardiac troponin I (cTnI) concentrations. ANIMALS: Twenty-nine influenza-naïve yearling ponies: 23 were part of an influenza vaccine study (11 unvaccinated and 12 vaccinated), and were challenged with 108 EID50 EIV A/eq/Kentucky/91 6 months after vaccination. Six age-matched healthy and unvaccinated ponies concurrently housed in a separate facility not exposed to influenza served as controls. METHODS: Heparinized blood was collected before and over 28 days after infection and cTnI determined. Repeated measures analysis of variance, chi-square, or clustered regression analyses were used to identify relationships between each group and cTnI. RESULTS: All EIV-infected ponies developed clinical signs and viral shedding, with the unvaccinated group displaying severe signs. One vaccinated pony and 2 unvaccinated ponies had cTnI greater than the reference range at 1 time point. At all other times, cTnI was < 0.05 ng/mL. All control ponies had normal cTnI. There were no significant associations between cTnI and either clinical signs or experimental groups. When separated into abnormal versus normal cTnI, there were no significant differences among groups. CONCLUSIONS AND CLINICAL IMPORTANCE: This study demonstrated no evidence of severe myocardial necrosis secondary to EIV challenge with 108 EID50 EIV A/eq/Kentucky/91 in these sedentary ponies, but transient increases in cTnI suggest that mild myocardial damage may occur.

Growth factor and receptor mRNA expression in the intestine of horses with large colon volvulus: a pilot study.

REASONS FOR PERFORMING STUDY: Growth factors (GF) are important for maintenance and repair of intestinal mucosal structure and function, but there have been no studies investigating growth factor (GF) or growth factor receptor (GF-R) mRNA expression in the intestine of horses with large colon volvulus (LCV). OBJECTIVES: (1) To determine mRNA expression for epidermal growth factor (EGF), EGF receptor (EGF-R), insulin-like growth factor-I (IGF), IGF receptor (IGF-R), vascular endothelial growth factor (VEGF) and VEGF receptor (VEGF-R) in the intestine of horses with an LCV compared to normal intestine. (2) To measure the correlation between histological intestinal injury and mRNA expression. METHODS: In 5 horses, samples were collected from the mid-jejunum (small intestine, SI), pelvic flexure (PF) and right dorsal colon (RDC) prior to creation of the LCV (NORM), 1 h following creation of the LCV (ISCH) and 1 h following correction of the LCV (REPER). In 2 clinical cases of LCV, samples were collected from the PF and RDC. Samples were assessed histologically for the amount of intestinal injury. The mRNA expressions of growth factors and receptors were determined using qRT-PCR. RESULTS: VEGF and VEGF-R mRNA expression was greater in horses with an LCV compared to NORM. Expression of IGF-R mRNA increased in the SI during ISCH and REPER. CONCLUSION AND POTENTIAL RELEVANCE: The increase compared to NORM in VEGF and VEGF-R mRNA expression in horses with LCV may be important in early intestinal healing and may also explain, in part, the increase in vascular permeability in horses with a LCV. Expression of IGF and IGF-R in the SI warrants further investigation and may be important for understanding post operative complications in horses with SI lesions.

Endurance exercise is associated with increased plasma cardiac troponin I in horses.

REASON FOR PERFORMING STUDY: Information is lacking regarding the influence of long distance exercise on the systemic concentration of cardiac troponin I (cTnI) in horses. OBJECTIVES: To determine if the concentration of cTnI in horses competing in 80 and 160 km endurance races increases with exercise duration and if cTnI concentrations can be correlated with performance data. METHODS: Blood samples for the measurement of cTnI and 3 min electrocardiogram recordings were obtained from horses prior to, during and after completion of 80 and 160 km endurance races at 3 ride sites during the 2004 and 2005 American Endurance Ride Conference competition seasons. RESULTS: Full data sets were obtained from 100 of the 118 horses. Endurance exercise was associated with a significant increase in cTnI over baseline in both distance groups. Failure to finish competition (poor performance) was also associated with an increased cTnI concentration over baseline at the time of elimination when data from both distances were combined. Other than one horse that developed paroxysmal atrial fibrillation, no arrhythmias were noted on the 3 minute ECG recordings that were obtained after endurance exercise in either distance group. CONCLUSIONS: Systemic concentrations of cTnI increase in endurance horses competing in both 80 and 160 km distances. Although final cTnI concentrations were significantly increased over their baseline values in horses that failed to finish competition, the degree of increase was not greater than the increase over baseline seen in the horses that successfully completed competition. The clinical significance of increased cTnI in exercising horses could not be ascertained from the results of this study. POTENTIAL RELEVANCE: These data indicate that cardiac stress may occur in horses associated with endurance exercise. Future studies utilising echocardiograpy to assess cardiac function in horses with increased cTnI are warranted.

Right ventricular pressure dynamics and stress echocardiography in pharmacological and exercise stress testing.

REASONS FOR PERFORMING STUDY: There is interest in using pharmacological stress testing (PST) as a substitute for exercise stress testing (EST) to evaluate cardiac function in horses. OBJECTIVES: To compare the effect of PST and EST on right ventricular pressure dynamics and stress echocardiography. METHODS: Five horses completed a PST and EST in a randomised crossover design. High fidelity pressure transducers were placed in the right ventricle. Continuous pressure signals were digitally collected and stored, and dP/dtmax, dP/dtmin and tau calculated from these measurements. ECGs were recorded continuously for 20 h. Echocardiography was performed prior to EST and PST, during and after PST, and immediately post EST. Plasma cardiac troponin I concentrations were measured pre- and 3-4 h post stress testing. For PST, 5 microg/kg bwt glycopyrrolate i.v. followed after 10 min by 5 microg/kg bwt/min dobutamine infusion over 10 min was given. EST consisted of a 2 min gallop at 110% speed required to elicit VO2max. RESULTS: Both EST and PST resulted in a significant increase in right-ventricular dP/dtmax and dP/dtmin over baseline (P<0.05) and a significant decrease in tau compared with baseline (P<0.05). EST dP/dtmax and dP/dtmin were significantly greater than PST dP/dtmax and dP/dtmin (P<0.05) and EST tau was significantly less than PST tau (P<0.05). Two minutes post EST and 5 min post PST dP/dtmax were not significantly different, but were significantly less than end-EST and during PST. Tau was also not significantly different between post EST and post PST, but was significantly decreased end-EST compared with during PST. FS were not significantly different between PST and post EST, but during PST and post EST all FS were significantly higher than baseline. Cardiac troponin I concentrations were significantly elevated post PST and were greater than post EST. The clinical relevance of this is unknown. CONCLUSIONS: PST had a similar, although less marked effect on the cardiac parameters related to right-ventricular pressure dynamics and a similar effect on echocardiography as exercise stress testing. POTENTIAL RELEVANCE: PST deserves further evaluation in normal horses and those with cardiac disease, and may be complementary to EST to better identify exercise-induced cardiac dysfunction.

Correlations between exercising arterial blood gas values, tracheal wash findings and upper respiratory tract abnormalities in horses presented for poor performance.

REASONS FOR PERFORMING STUDY: There are limited data on the correlations between arterial blood gas (ABG) values, tracheal wash (TW) cytology and upper respiratory tract (URT) abnormalities. OBJECTIVES: To identify horses with abnormal exercising ABG, and compare the proportions of horses with abnormal ABG and TW cytology, mucus or URT dysfunction with those with normal ABG results and abnormal TW cytology, mucus or URT dysfunction. METHODS: Medical records of 813 horses presenting to the treadmill facility that had a complete treadmill examination, including ABG analysis, TW and URT endoscopy were selected. Diagnoses, ABG results, TW cytology and URT endoscopy were compared. RESULTS: Two hundred and eleven horses met the study criteria of a complete treadmill examination and could have ABG evaluated. There were no significant differences in the age distribution of horses having normal and abnormal ABG or upper respiratory tract (URT) examinations. There was a significantly higher percentage of geldings with abnormal ABG analysis. In the horses with abnormal URT examinations, there were no differences in the proportion of horses having mucus vs. no mucus. However, in the horses with normal URT, there were a higher percentage of horses with visible mucus in the group with abnormal ABG analysis. The majority of horses had abnormal TW cytology and evidence of prior EIPH, with no differences in proportions between the groups. CONCLUSIONS: Because such a large percentage of horses had evidence of inflammation and/or evidence of prior EIPH on TW cytology, it was not possible to determine the effect of these findings on gas exchange. Mucus was present in a larger percentage of cases with abnormal ABG analysis and normal URT examinations, suggesting that the presence of mucus may affect gas exchange. Standardbreds may be more likely to have abnormal gas exchange than Thoroughbreds. A larger number of horses is needed to determine the significance of these findings. POTENTIAL RELEVANCE: Abnormal TW cytology and endoscopic visualised mucus may contribute to impairment of gas exchange, but they do not specifically predict abnormal ABG analysis.

Dynamic pharyngeal collapse in racehorses.

REASON FOR PERFORMING STUDY: Dynamic pharyngeal collapse (PC) is a condition seen in racehorses that can be career-ending. OBJECTIVES: To characterise and grade PC and describe the effects of PC on athletic performance. METHODS: Medical records were reviewed for 828 horses, of which 49 (6%) records were identified as horses with a primary diagnosis of PC. Tapes of video-endoscopy of the pharynx during exercise were reviewed. Each video recording was assigned a grade (0-4) reflecting the degree of PC and a classification for severity of upper airway obstruction. Earnings per race prior to diagnosis of PC were compared to earnings per race after diagnosis of PC for all horses, as well as performance index (PI). Available exercising arterial blood gases were reviewed for horses with PC. RESULTS: There were 35 (80%) Thoroughbreds (TB), and 9 (20%) Standardbreds (STD). 32 (73%) had a history of making an upper respiratory noise. 4 (9%) grade 1 PC, 8 (18%) grade 2 PC, 26 (59%) grade 3 PC, and 6 (14%) grade 4 PC. Seven (16%) horses were classified as mild PC, 18 (41%) as low-moderate PC, 14 (32%) as high-moderate PC, and 5 (11%) as severe PC. Of 30 horses 11 had abnormally decreased PaO2 and 8 horses had abnormally elevated PaCO2. A significant decrease was found in earnings per race prediagnosis when compared to post diagnosis earnings per race in horses > or =4 years of age (P = 0.003). A significant decrease was also observed for earnings per race prediagnosis when compared to post diagnosis earnings per race in horses with grade 3 PC (P = 0.03) No significant differences were observed in PI before or after diagnosis of PC. CONCLUSIONS: There was a trend for PC to be observed in more TB than STD, and more males than females compared to the general hospital population. Horses with PC significant had decreases in arterial oxygenation. Racing records after a diagnosis of PC in all horses > or = 4 years of age suggesting that older horses have a guarded prognosis for continued success. POTENTIAL RELEVANCE: This study provides a classification system for dynamic pharyngeal collapse and suggests that older racehorses (> or = 4 years of age) diagnosed with PC and all horses with grade 3 PC have a poor prognosis for return to previous level of performance.

Pharmacokinetics and disposition of clenbuterol in the horse.

The pharmacokinetics of clenbuterol (CLB) following a single intravenous (i.v.) and oral (p.o.) administration twice daily for 7 days were investigated in thoroughbred horses. The plasma concentrations of CLB following i.v. administration declined mono-exponentially with a median elimination half-life (t(1/2k)) of 9.2 h, area under the time-concentration curve (AUC) of 12.4 ng.h/mL, and a zero-time concentration of 1.04 ng/mL. Volume of distribution (V(d)) was 1616.0 mL/kg and plasma clearance (Cl) was 120.0 mL/h/kg. The terminal portion of the plasma curve following multiple p.o. administrations also declined mono-exponentially with a median elimination half-life (t(1/2k)) of 12.9 h, a Cl of 94.0 mL/h/kg and V(d) of 1574.7 mL/kg. Following the last p.o. administration the baseline plasma concentration was 537.5 +/- 268.4 and increased to 1302.6 +/- 925.0 pg/mL at 0.25 h, and declined to 18.9 +/- 7.4 pg/mL at 96 h. CLB was still quantifiable in urine at 288 h following the last administration (210.0 +/- 110 pg/mL). The difference between plasma and urinary concentrations of CLB was 100-fold irrespective of the route of administration. This 100-fold urine/plasma difference should be considered when the presence of CLB in urine is reported by equine forensic laboratories.

Tissue distribution of clenbuterol in the horse.

Plasma and tissue concentrations of clenbuterol (CLB) were determined following oral (p.o.) administration of 1.6 microg/kg twice daily (b.i.d.) for 2 weeks. Horses were administered the last dose on morning of day 15, killed at 0.25, 24, 48, and 72 h post-administration. At 0.25 h, the highest tissue concentrations of CLB were found in the liver (16.21 ng/g), lung (6.48 ng/g), left ventricle (4.99 ng/g), kidney (3.35 ng/g), bronchi (2.56 ng/g), right ventricle (2.08 ng/g), and eye fluids (1.09 ng/g) all of which were higher than that of plasma (1.10 ng/mL). The elimination half-lives (t(1/2k)) for CLB in tissues ranged from 21.2 to 56.3 h, the longest were in the eye fluids (56.9 h), spleen (21.2 h), cerebrum (27.1 h), cerebellum (21.5) and cecum (23.7 h). The t(1/2k) for plasma was 10.9 h. Tissue/plasma ratios of liver (14.7), lung (5.9), left ventricle (4.6), kidney (3.1), bronchi, (2.3) and right ventricle (1.9) were high at 0.25 h and remained elevated up to 72 h. Accumulation and sustained high concentration of CLB relative to plasma in these tissues contributed to the prolonged elimination and the ability to quantify CLB in plasma and urine for a prolonged period.

Effects of mild forelimb lameness on exercise performance.

Mild lameness is considered a performance-limiting problem that may escape detection until it worsens, and is considered the primary reason for reduced racing performance. The kinematics changes associated with a lame horse at the trot have been demonstrated previously, but the metabolic cost of these alterations in their gait have not been demonstrated. Six fit Thoroughbred horses with an established VO2max participated in 4 trials using a randomised cross-over design study, separated by 10-14 days. The horses were tested with one of 4 trial conditions: lead forelimb lameness (LL); off-lead forelimb lameness (OL); bilateral forelimb lameness (BL) or no lameness (NL). Lameness was induced by sole pressure from a modified shoe that resulted in a consistent slight head nod at a trot in a straight line while jogging in hand. Lameness was adjusted to provide a lameness that would be quantified as a 1-2/5 on the grading system recommended by the AAEP. Each trial consisted of 4 different levels of exercise intensity at speeds equivalent to 30, 60, 80 and 110% of an individual's speed required to elicit VO2max. Stride parameters, oxygen consumption (VO2), carbon dioxide production (VCO2), electrolytes, plasma lactate, glucose and PCV/TP were measured prior to exercise, at each exercise level and after exercise. A multiway ANOVA with repeated measures was utilised to examine possible effects of individual horse, lameness, and exercise intensity on measured parameters. Significance was set at alpha = 0.05. For horses exercising at the maximum intensity, VO2 was significantly lower for both of the single-leg lamenesses (LL or OL) when compared to NL or BL (mean +/- s.e. 165.6 +/- 2.5, 164.7 +/- 3.0, 175.8 +/- 2.4 and 170.9 +/- 2.1 ml O2/min/kg bwt, respectively). Blood lactate concentrations were not significantly different among the treatment groups. However, lactate accumulation rates computed as the change with time in lactate concentration at the highest exercise intensity were significantly higher for LL and OL than for NL and BL (7.8 +/- 03, 83 +/- 0.2, 4.1 +/- 0.2 and 4.7 +/- 0.3 mmol/min, respectively). Exercise intensity had significant effects on all of the measured parameters, but there were no other significant differences due to treatment. These results suggest that metabolic energy transduction is affected by even mild unilateral forelimb lamenesses.

EIPH: postrace endoscopic evaluation of Standardbreds and Thoroughbreds.

The incidence and severity of exercise-induced pulmonary haemorrhage (EIPH) in the 2 most commonly raced horse breeds, Thoroughbreds (TB) and Standardbreds (STD), were studied, with particular interest in the possible influence of frusemide (F) and/or the breed (or running gait) on EIPH. The appearance of blood within the trachea was semi-quantified using a published 5-point system, with zero assigned when no blood was observed, and numbers 1-4 assigned with increasing amounts of blood. Considering each endoscopic examination as a separate event, approximately 75% of the postrace endoscopic examinations had blood-scores of 1, 2, 3, or 4, regardless of breed or F administration. For horses examined twice, the chances of finding blood-scores of 1 or greater in either of the examinations increased to approximately 95%. All horses examined 3 or more times had endoscopic blood-scores of 1 or greater following one or more races, again, irrespective of the breed or F administration. Mean +/- s.e. 'blood scores' were 1.5 +/- 0.1 and 1.8 +/- 0.2 for TB, and 1.4 +/- 0.2 and 1.2 +/- 0.1 for STD racing with and without prerace F, respectively. Therefore, there was no apparent effect of breed (or possibly racing gait) on EIPH, and no differences in the incidence or severity of EIPH were observed between horses with or without prerace frusemide administration.

Dynamic upper airway changes and arterial blood gas parameters during treadmill exercise.

The primary goal was to investigate the relationship between dynamic upper airway abnormalities and arterial blood gas tensions during exercise. Horses that completed a high-speed treadmill examination consisting of upper-airway videoendoscopy, blood gas evaluation and electrocardiogams and, postexercise, echocardiograms and tracheal washes, were included. An age-matched group of fit, healthy Thoroughbreds, trained to run on a high-speed treadmill, served as controls for blood gas values at specific exercise speeds. One hundred and nineteen horses completed the treadmill examination. Sixty (50%) were Thoroughbreds (TB), 51 (43%) Standardbreds (STD) and 8 (7%) other breeds. Mean +/- s.d. age TB 3.8 +/- 2.2 years and STD 4.0 +/- 1.7 years, with no gender predilection. Fifty-four horses (45%) had abnormal upper respiratory tract (URT) abnormalities alone or in combination with abnormalities in another body system. Thirty-eight (70%) were TB, 14 (26%) were STD and 2 (4%) were other breeds. Of these, 24 (45%) had exercising PaO2 values significantly lower than those observed in healthy TB. Nineteen (35%) horses also had significantly elevated exercising PaCO2. Only 14 (12%) horses had abnormal clinical findings in the URT alone, and of these, only 3 (21%) had an abnormally low PaO2 and/or elevated PaCO2. Multiple URT abnormalities were more commonly associated with abnormal exercising blood gases than were single disorders, but pharyngeal collapse (PC) was much more commonly associated with abnormal values if only one disorder was detected. Fifty-five percent (n = 65) of all cases admitted had no evidence of URT disease. Twenty-two (35%) were TB and 37 (57%) were STD. Twenty (31%) of these had abnormally low PaO2 and 14 (22%) had elevated PaCO2 values. Seventy percent (14) of the horses with abnormal PaO2 were STD, while almost 80% (11) of the horses with elevated PaCO2 were STD. These data suggest that dynamic URT dysfunction can adversely affect gas exchange during exercise. While multiple abnormalities were more commonly associated with gas exchange problems than were single disorders, pharyngeal collapse, either alone or in combination with other URT problems, was the disorder most frequently associated with blood gas abnormalities. Additionally, URT disease was more commonly seen in TB, and the proportion of URT diagnoses in horses with abnormal blood gases reflected this percentage, while STD without URT disease had a much higher incidence of abnormal blood gases than did TB without URT abnormalities.

Right ventricular pressure dynamics during exercise: relationship to stress echocardiography.

Thorough evaluation of myocardial function remains difficult to evaluate under exercising conditions. This study described right ventricular (RV) pressure dynamics during and immediately following exercise. Nine Thoroughbreds without evidence of cardiac disease completed treadmill exercise at 110% of the speed necessary to elicit VO2max while RV pressures were recorded. RV pressure dynamics were calculated at rest, maximal speed and at 10 s intervals for 2 min after exercise. Stress echocardiography was performed at rest and within 120 s after exercise. Mean dP/dtmax and dP/dtmin values were significantly greater at maximal speed and up to 30 s immediately postexercise than at rest and all time points from 60 to 120 s postexercise. Mean dP/dtmax and dP/dtmin were not significantly different from resting values after 60 s postexercise. Tau (the time constant for ventricular relaxation) decreased significantly with exercise, but was not significantly different from rest at time points from 60 to 120 s following exercise. Mean % fractional shortening (FS) increased postexercise; however, the coefficient of variability was large. Wall motion indices also showed large variability postexercise. These temporal changes in normal horses suggest that exercising RV pressure dynamics may provide a better estimation of cardiac function during exercise than postexercise stress echocardiography.

Evaluation of the i-STAT hand-held chemical analyser during treadmill and endurance exercise.

This study examined the accuracy and precision of a hand-held, chemical analyser, i-STAT, in measuring selected blood constituents which may be of use in the diagnosis and management of metabolic disorders found in exercising horses. Venous blood samples were taken from 3 Thoroughbred geldings, fit and trained to exercise on a treadmill, both before and after exercise at a speed sufficient to elicit VO2max. The samples were analysed both with the i-STAT and with in-house analysers to compare the values of pH, partial pressure of oxygen (PO2), partial pressure of carbon dioxide (PCO2), bicarbonate (HCO3), lactate and base excess (BE). The i-STAT demonstrated acceptable accuracy and precision for determination of pH, PO2 and PCO2, and lactate. We concluded that these parameters could be reliably evaluated by the i-STAT. The i-STAT was further evaluated at veterinary checkpoints during a 60 and 100 mile endurance ride. Because a built-in thermostat prevents function when the temperature of the analyser is outside the optimum range (16-30 degrees C), it was necessary to insulate the i-STAT from extreme ambient temperatures. As this portion of the study was conducted in warm temperatures, the appropriate insulation was to maintain the i-STAT in an ice-cooled container except during actual blood analyses. Further investigation into the feasibility of using hand-held chemical analysers at the veterinary checkpoints during endurance rides is recommended.

Disposition, elimination, and bioavailability of phenytoin and its major metabolite in horses.

OBJECTIVE: To determine pharmacokinetics and excretion of phenytoin in horses. ANIMALS: 6 adult horses. PROCEDURE: Using a crossover design, phenytoin was administered (8.8 mg/kg of body weight, IV and PO) to 6 horses to determine bioavailability (F). Phenytoin also was administered orally twice daily for 5 days to those same 6 horses to determine steady-state concentrations and excretion patterns. Blood and urine samples were collected for analysis. RESULTS: Mean (+/- SD) elimination half-life following a single IV or PO administration was 12.6+/-2.8 and 13.9+/-6.3 hours, respectively, and was 11.2+/-4.0 hours following twice-daily administration for 5 days. Values for F ranged from 14.5 to 84.7%. Mean peak plasma concentration (Cmax) following single oral administration was 1.8+/-0.68 microg/ml. Steady-state plasma concentrations following twice-daily administration for 5 days was 4.0+/-1.8 microg/ml. Of the 12.0+/-5.4% of the drug excreted during the 36-hour collection period, 0.78+/-0.39% was the parent drug phenytoin, and 11.2+/-5.3% was 5-(phydroxyphenyl)-5-phenylhydantoin (p-HPPH). Following twice-daily administration for 5 days, phenytoin was quantified in plasma and urine for up to 72 and 96 hours, respectively, and p-HPPH was quantified in urine for up to 144 hours after administration. This excretion pattern was not consistent in all horses. CONCLUSIONS AND CLINICAL RELEVANCE: Variability in F, terminal elimination-phase half-life, and Cmax following single or multiple oral administration of phenytoin was considerable. This variability makes it difficult to predict plasma concentrations in horses after phenytoin administration.

Quantification of phenytoin and its metabolites in equine plasma and urine using high-performance liquid chromatography.

A reliable and sensitive method for the extraction and quantification of phenytoin (5,5'-diphenylhydantoin), its major metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) and minor metabolite, 5-(m-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) in horse urine and plasma is described. The method involves the use of solid-phase extraction (SPE), liquid-liquid extraction (LLE), enzyme hydrolysis (EH) and high-performance liquid chromatography (HPLC). The minor metabolite, 5-(m-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) was not present in a reliably quantifiable concentration in all samples. The new method described was successfully applied in the pharmacokinetic studies and elimination profile of phenytoin and p-HPPH following oral or intravenous administration in the horse.

Hypoxic pulmonary vasoconstriction is modified by P-450 metabolites.

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P-450 4A (CYP4A) metabolite of arachidonic acid (AA) in human and rabbit lung microsomes and is a dilator of isolated human pulmonary arteries (PA). However, little is known regarding the contribution of P-450 metabolites to pulmonary vascular tone. We examined 1) the effect of two mechanistically distinct omega- and omega1-hydroxylase inhibitors on perfusion pressures in isolated rabbit lungs ventilated with normoxic or hypoxic gases, 2) changes in rabbit PA ring tone elicited by 20-HETE or omega- and omega1-hydroxylase inhibitors, and 3) expression of CYP4A protein in lung tissue. A modest increase in perfusion pressure (55 +/- 11% above normoxic conditions) was observed in isolated perfused lungs during ventilation with hypoxic gas (FI(O(2)) = 0.05). Inhibitors of 20-HETE synthesis, 17-oxydecanoic acid (17-ODYA) or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), increased baseline perfusion pressure above that of vehicle and amplified hypoxia-induced increases in perfusion pressures by 92 +/- 11% and 105 +/- 11% over baseline pressures, respectively. 20-HETE relaxed phenylephrine (PE)-constricted PA rings. Treatment with 17-ODYA enhanced PE-induced contraction of PA rings, consistent with inhibition of a product that promotes arterial relaxation, whereas 6-(20-propargyloxyphenyl)hexanoic acid (PPOH), an epoxygenase inhibitor, blunted contraction to PE. Conversion of AA into 20-HETE was blocked by 17-ODYA, DDMS, and hypoxia. CYP4A immunospecific protein confirms expression of CYP4A in male rabbit lung tissue. Our data suggest that endogenously produced 20-HETE could modify rabbit pulmonary vascular tone, particularly under hypoxic conditions.

The effects of frusemide on racing times of Standardbred pacers.

Seven hundred and eighty-eight Standardbred pacers competing in 8378 races at one racetrack were analysed to determine the effects of the administration of prerace frusemide on racing times (RT). Frusemide was administered i.v. 4 h before the race to pacers diagnosed with exercise-induced pulmonary haemorrhage (EIPH). Of the pacers, starting in the 1997 racing season, 32.5% received prerace frusemide. This study demonstrated that administration of frusemide prior to racing significantly decreased RT. There was an overall significant decrease (P<0.00001) in RT of 0.67 s. The overall RT for horses, geldings, and females, were mean +/- s.e 117.91 +/- 0.06, 118.20 +/- 0.03 and 118.86 +/- 0.04, respectively. RT progressively decreased until age 6 and increased thereafter. Horses, geldings and females ran a mean of 0.46, 0.31 and 0.74 s faster, respectively, with prerace administration of frusemide. This decrease in RT following prerace administration was most pronounced in younger pacers. In this study, a greater percentage of older pacers received prerace frusemide; however, the effect of frusemide on RT was decreasing with age. Prerace venous acid-base screening was performed in 2729 of the pacers competing. Pennsylvania Harness Racing Commission Regulations disqualify Standardbreds from racing with a base excess of over 10 and 12 mmol/l for Standardbreds without and with prerace administration of frusemide. The prerace venous acid-base levels were not significantly related to RT and, for those Standardbreds also sampled following the race, there was no correlation between pre- and postrace acid-base status.

Production of 20-HETE and its role in autoregulation of cerebral blood flow.

In the brain, pressure-induced myogenic constriction of cerebral arteriolar muscle contributes to autoregulation of cerebral blood flow (CBF). This study examined the role of 20-HETE in autoregulation of CBF in anesthetized rats. The expression of P-450 4A protein and mRNA was localized in isolated cerebral arteriolar muscle of rat by immunocytochemistry and in situ hybridization. The results of reverse transcriptase-polymerase chain reaction studies revealed that rat cerebral microvessels express cytochrome P-450 4A1, 4A2, 4A3, and 4A8 isoforms, some of which catalyze the formation of 20-HETE from arachidonic acid. Cerebral arterial microsomes incubated with [(14)C]arachidonic acid produced 20-HETE. An elevation in transmural pressure from 20 to 140 mm Hg increased 20-HETE concentration by 6-fold in cerebral arteries as measured by gas chromatography/mass spectrometry. In vivo, inhibition of vascular 20-HETE formation with N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS), or its vasoconstrictor actions using 15-HETE or 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE), attenuated autoregulation of CBF to elevations of arterial pressure. In vitro application of DDMS, 15-HETE, or 20-HEDE eliminated pressure-induced constriction of rat middle cerebral arteries, and 20-HEDE and 15-HETE blocked the vasoconstriction action of 20-HETE. Taken together, these data suggest an important role for 20-HETE in the autoregulation of CBF.

Effects of chronic pulmonary overcirculation on pulmonary vasomotor tone.

BACKGROUND: A model of shunt-induced pulmonary hypertension was used to study the effects of pulmonary overcirculation on endothelial nitric oxide synthase (eNOS) and cytochrome P450-4A (cP450-4A) vasodilatory mechanisms and related hemodynamic responses. METHODS: An aortopulmonary shunt was constructed in 6-week-old piglets (n = 7, sham-operated controls n = 8). Hemodynamic measurements were made 4 weeks later under serial experimental conditions: baseline (fractional concentration of oxygen, 0.4); inhaled nitric oxide, 25 ppm (INO); hypoxia (fractional concentration of oxygen, 0.14); hypoxia + INO; N(omega)-nitro-L-arginine methylester (L-NAME 30 mg/kg intravenously, competitive NOS inhibitor); and L-NAME + INO. Lung protein levels of eNOS and cP450-4A and NOS activity were compared between groups. RESULTS: Shunted animals had a higher baseline pulmonary artery pressure (p < 0.05). L-NAME resulted in a greater increase in pulmonary vascular resistance in shunted animals (150% +/- 26% shunt versus 69% +/- 14% control; p = 0.01). The INO administered during baseline conditions decreased pulmonary vascular resistance only in control animals (p < 0.05). Protein levels of eNOS and NOS activity were similar in both groups; however, cP450-4A protein levels were decreased in the shunted group (p = 0.02). CONCLUSIONS: The NO production was preserved in shunted animals but they demonstrated greater vasodilatory dependence on NO, evidenced by an exaggerated increase in pulmonary vascular resistance after NOS inhibition. Loss of the cP450-4A vasodilatory system may be the driving force for NO dependency in the shunted pulmonary circulation.