Effects of inflammation-associated acute-phase response on hepatic and renal indices in the horse.

OBJECTIVES: To determine the effect of an acute soft tissue inflammatory response on biochemical and haematological indices of hepatic and renal function in the Thoroughbred horse. PROCEDURE: Soft tissue inflammation was induced in four Thoroughbred horses by intramuscular injections of Freund's complete adjuvant. The horses were clinically examined and blood and urine samples were collected before and after the adjuvant injections. Biochemical and haematological indices were measured in samples collected and used to determine the onset of the acute-phase response and to assess hepatic and renal function at this time. RESULTS: After adjuvant injection, significant increases (P < 0.01) in total white (13.1 +/- 1.4 x 10(9)/L) and neutrophil (10.2 +/- 1.2 x 109/L) cell counts, rectal temperature (39.7 +/- 0.5 degrees C) and various plasma protein concentrations, including fibrinogen (6.6 +/- 1.2 g/L), haptoglobin (1.3 +/- 0.1 g/L) and total protein (88.1 +/- 2.7 g/L), indicated the induction of an acute-phase response. This corresponded with significant reductions (P < 0.01) in the plasma elimination half-lives (t1/2 beta) sodium bromosulphthalein (3.13 +/- 0.05 to 2.82 +/- 0.07 min) and sodium sulphanilate (38.29 +/- 4.04 to 19.60 +/- 5.68 min) and reductions in the plasma activities of aspartate aminotransferase, glutamate dehydrogenase, creatine kinase, alkaline phosphatase, gamma glutamyl transferase; the urinary creatinine clearance ratios of sodium, chloride and potassium; and the urinary gamma glutamyl transferase-to-creatinine clearance ratios. (All values mean +/- SD.) CONCLUSIONS: The effects of the acute-phase response on indices of hepatic and renal function in the horse suggest that the disposition of pharmacological agents administered at this time may be altered and that indices of acute inflammation should be interpreted cautiously.

Stress response to chronic inflammation in the horse.

Five clinically healthy Thoroughbred geldings were injected with Freund's adjuvant 3 times to induce a chronic inflammatory response. Blood was collected at various times before and after adjuvant administration. Clinical responses (rectal temperature and general demeanor) were also monitored. Adjuvant injection induced increases in rectal temperature and plasma fibrinogen concentration (maximum levels measured were mean +/- s.d. 39.7 +/- 0.5 degrees C and 8.2 +/- 0.3 g/l, respectively), indicative of an inflammatory response. A mild clinical depression was also observed in the horses for 24 h after the first injection of adjuvant only. Plasma cortisol levels decreased significantly from control levels of mean +/- s.d. 187.7 +/- 24.3 nmol/l to a minimum of 80.2 +/- 22.1 nmol/l (P < 0.01) 9 days after the first injection of adjuvant. Conversely, plasma insulin levels increased after the first injection of adjuvant to a maximum (96.7 +/- 15.2 iu/ml; P < 0.01) 12 days later, while plasma glucose concentrations tended to decline. A control group of horses to rule out contemporary environmental influences on the physiological and biochemical indices measured was not included in this study. The results show that chronic inflammation in the horse depressed resting plasma cortisol concentrations.

The effect of the acute-phase response on in vitro drug metabolism and plasma protein binding in the horse.

The effect of the acute-phase response (APR) on the activity of the hepatic drug-metabolizing system (DMS) and on the binding of phenylbutazone to plasma proteins was investigated in the horse. An APR was induced by intramuscular injections of Freund's complete adjuvant in five horses and, five days later, these horses together with five clinically normal horses were shot and the right ventral lobe of each liver removed. The hepatic microsomal fractions from the liver samples were isolated and significantly lower (p < 0.01) concentrations of cytochromes P450 and b5 and activities of aniline-p-hydroxylase and aminopyrine N-demethylase (43%, 55%, 45% and 30%, respectively) were measured in the livers from the adjuvant-inflamed horses, compared to the controls. Phenylbutazone (PBZ) was administered intravenously (4.4 mg/kg) to a further four horses and plasma protein binding was measured by ultracentrifugation. Five weeks later, these horses were injected with Freund's complete adjuvant and the intravenous administration of PBZ (4.4 mg/kg) was repeated. Inflammation induced a significant increase (p < 0.01) in the unbound fraction of PBZ (5.2 +/- 0.5 as against 1.4 +/- 0.6%). These results suggest that the APR depresses the hepatic DMS and reduces the binding of PBZ to plasma proteins.

The effect of inflammation on the disposition of phenylbutazone in thoroughbred horses.

The effect of inflammation on the disposition of phenylbutazone (PBZ) was investigated in Thoroughbred horses. An initial study (n = 5) in which PBZ (8.8 mg/kg) was injected intravenously twice, 5 weeks apart, suggested that the administration of PBZ would not affect the plasma kinetics of a subsequent dose. Two other groups of horses were given PBZ at either 8.8 mg/kg (n = 5) or 4.4 mg/kg (n = 4). Soft tissue inflammation was then induced by the injection of Freud's adjuvant and the administration of PBZ was repeated at a dose level equivalent to, but five weeks later than, the initial dose. Inflammation did not appear to affect the plasma kinetics or the urinary excretion of PBZ and its metabolites, oxyphenbutazone (OPBZ) or hydroxyphenylbutazone (OHPBZ) when PBZ was administered at 8.8 mg/kg. However, small but significant increases (P < 0.05) in total body clearance (CLB; 29.2 +/- 3.9 vs. 43.8 +/- 8.1 mL/ h.kg) and the volume of distribution, calculated by area (Vd(area); 0.18+/- 0.05 vs. 0.25 +/- 0.03 L/kg) or at steady-state (Vd(SS); 0.17 +/- 0.04 vs. 0.25 +/- 0.03 L/ kg), were obtained in horses after adjuvant injection, compared to controls, when PBZ was administered at 4.4 mg/kg which corresponded to relatively higher tissues concentrations and lower plasma concentrations (calculated) at the time of maximum peripheral PBZ concentration. Soft tissue inflammation also induced a significantly (P < 0.05) higher amount of OPBZ in the urine 18 h after PBZ administration but the total urinary excretion of analytes over 48 h was unchanged. These results have possible implications regarding the administration of PBZ to the horse close to race-day.

Disposition and urinary excretion of phenylbutazone in normal and febrile greyhounds.

Five days after the induction of acute systemic inflammation in greyhounds by intramuscular and subcutaneous injections of Freund's adjuvant, the hepatic concentrations of cytochromes P-450 and b5, the activities of the hepatic microsomal enzymes aniline p-hydroxylase and aminopyrine n-demethylase and the disposition and urinary excretion of phenylbutazone were determined. The mean plasma concentrations of phenylbutazone after intravenous administration were described by the bi-exponential equations: Cp = 144.2e-34.6t + 171.5e-0.104t for five normal greyhounds and Cp = 113.6e-16.13t + 163.1e-0.108t for five febrile greyhounds. The elimination half-lives, total body clearances and apparent volumes of distribution were 6.7 hours, 18.4 ml kg-1 hour-1 and 0.18 litre kg-1, for the normal greyhounds, and 6.4 hours, 19.5 ml kg-1 hour-1 and 0.18 litre kg-1, for the febrile greyhounds. There were no significant differences between the pharmacokinetic parameters describing the distribution and elimination of phenylbutazone, or between the quantities of phenylbutazone, oxyphenbutazone and hydroxyphenylbutazone excreted in the urine. In the febrile greyhounds, there were significant decreases in the hepatic microsomal concentrations of cytochromes P-450 and b5 and in the activities of aniline p-hydroxylase and aminopyrine n-demethylase.

Phenylbutazone in racing greyhounds: plasma and urinary residues 24 and 48 hours after a single intravenous administration.

The concentrations of phenylbutazone (PBZ), oxyphenbutazone (OPBZ) and gammahydroxyphenylbutazone (OHPBZ) in plasma and urine from 50 Greyhounds 24 and 48 h after the intravenous administration of a single dose of PBZ (30 mg/kg) were measured. The 24 h plasma concentrations of OPBZ and OHPBZ, the 48 h plasma concentration of OHPBZ and the 24 h urinary concentration of PBZ were normally distributed, while log transformations were required before the 24 h plasma concentration of PBZ and the 24 and 48 h urinary concentrations of OPBZ and OHPBZ became normally distributed. The 95%, 99%, 99.9% and 99.99% upper predicted confidence intervals for both 24 h and 48 h plasma and urinary concentrations demonstrated wide potential variation in the concentration of the analytes should PBZ be administered to Greyhounds. The 24 h plasma and urinary concentrations of PBZ were weakly correlated, but no similar relationship existed for OPBZ or OHPBZ. The urinary concentrations of each analyte were not affected by the trainer or sex of the Greyhound or the urinary pH. We conclude that it would be impossible to predict the timing of the PBZ administration or the plasma concentration of PBZ from the measurement of the concentration of PBZ in a single sample of urine.

Kinetics, dose response, tachyphylaxis and cross-tachyphylaxis of vascular leakage induced by endotoxin, zymosan-activated plasma and platelet-activating factor in the horse.

Vascular leakage induced by intradermal injection of endotoxin, zymosan-activated plasma (ZAP) and platelet-activating factor (PAF) was measured in nine Thoroughbreds using 125-iodine human serum albumin (125I-HSA) as a marker in the blood. ZAP and PAF produced dose-dependent increases in vascular permeability with the maximum occurring within the first 15 min after injection. The vascular leakage induced by endotoxin was also dose-dependent, but the maximum occurred 2 h after intradermal injection. Intradermal sites previously injected with endotoxin were refractory to a second injection of endotoxin for up to 5 days. However, sites injected with endotoxin and re-injected with either ZAP or PAF remained responsive with increased vascular leakage compared to saline injected control sites re-injected with either ZAP or PAF. Diminished response to endotoxin challenge may contribute to the poor prognosis of endotoxaemia in the horse.

Exercise-induced connective tissue turnover and lipid peroxidation in horses.

Four unfit thoroughbred horses were exercised on a treadmill twice, 5 weeks apart. Exercise consisted of stepwise increments in treadmill speed up to a maximum of 12 m s-1 and then maintained at this speed until the horses were fatigued. Two of the horses were administered phenylbutazone (4.4 mg kg-1) intravenously immediately before the first exercise period and the other two horses immediately before the second exercise period. Clinical observation revealed stiffness of gait and palpable soreness over the lumbar-sacral region in the horses 24 h after the exercise concluded. Mean plasma aspartate aminotransferase and creatine kinase activities and urinary creatinine clearance did not change as a result of exercise. The mean urinary excretion and clearance of hydroxyproline significantly increased in the 24 h following exercise (P < 0.001). A concurrent increase in the urinary excretion of malondialdehyde also occurred (P < 0.001). Prior administration of phenylbutazone did not affect hydroxyproline or malondialdehyde excretion or clearance, nor did it appear to reduce the severity of soreness after exercise. The results indicate that lipid peroxidation and the excretion and clearance of hydroxyproline increase when unfit thoroughbreds are strenuously exercised on a treadmill.

Detection of bicarbonate administration (milkshake) in standardbred horses.

Total plasma carbon dioxide (TCO2) concentrations were measured in Standardbred horses to determine criteria to discriminate between normal horses and horses with excessive TCO2 concentrations on raceday. TCO2 concentrations from stabled horses were distributed normally with a mean of 30.2 mmol/L and a standard deviation of 1.2 (n = 192) while pre-race TCO2 concentrations were not normally distributed. The results indicate that about 50 horses per million are likely to have TCO2 concentrations greater than or equal to 35 mmol/L and that it is extremely unlikely that a normal horse would have a resting TCO2 concentration above 36 mmol/L. These values were associated with sensitivities of 67% and 59%, respectively, and with a specificity of 100%. TCO2 concentrations were relatively stable in blood samples stored at 4 degrees C for 4 days, whereas the TCO2 in specimens stored at room temperature (25 degrees C) and at ambient temperature (16-28 degrees C) declined progressively over 5 days. The accuracy and precision of the Beckman EL-ISE Auto Analyser were acceptable and within the manufacturers specified range. Paired specimens analysed using a Beckman EL-ISE Auto Analyser and a Kodak Dry Chemistry Analyser were not significantly different. However, the measurements made using the Kodak Dry Chemistry Analyser averaged 0.5 mmol/L higher than those analysed on the Beckman EL-ISE. The significance of these sources of variation in TCO2 concentration in relation to the testing of horses for 'milkshake' administration are discussed.

Free radical oxidation products in plasma and synovial fluid of horses with synovial inflammation.

Free radical oxidation products, namely conjugated dienes, ultraviolet fluorescence (excitation 325 nm, emission 395 nm) and visible fluorescence (excitation 360 nm, emission 460 nm) were measured in equine synovial fluid exposed to free radicals in vitro and in the plasma and synovial fluids of horses with synovial effusions. The synovial effusions were induced by intra-articularly administered carrageenin (0.3 ml, 1%), which rarely resulted in clinical lameness. The free radicals were generated in vitro by mixtures of iron and ethylene diamine tetra acetate (Fe/EDTA) or mixtures of hypoxanthine and xanthine oxidase (HX/XO). The conjugated diene concentrations and intensity of ultraviolet fluorescence were negligible in plasma and synovial fluid specimens. No increase resulted from incubation of synovial fluids with either a free radical generating system or as a result of the induced inflammation. The intensity of visible fluorescence did not increase in specimens incubated with Fe/EDTA. However, the intensity of visible fluorescence increased in specimens incubated with HX/XO, in synovial effusions induced by carrageenin, in plasma and in synovial fluids aspirated from saline injected controls. The results indicate that the intensity of visible fluorescence of equine synovial fluid increases after exposure to free radicals and during synovitis in the horse, suggesting a possible role for free radicals in the pathogenesis of equine inflammatory joint disease.

Vascular leakage induced by histamine, bradykinin, serotonin and prostaglandin E2 in greyhounds.

Vascular leakage induced by intradermal injection of histamine, bradykinin and serotonin alone and co-injected with prostaglandin E2 was measured in Greyhounds using 125iodine-labelled human serum albumin (125I-HSA) as a marker in the blood. Histamine and bradykinin produced dose-dependent vascular leakage. At equimolar concentrations, histamine was more than twice as potent as bradykinin. Serotonin did not induce vascular leakage and was irritant. Prostaglandin E2 did not induce significant vascular leakage (maximum 5 microL) when injected alone, but when co-injected with histamine and bradykinin, the vascular leakage of both histamine and bradykinin was increased. This effect was more pronounced if lower concentrations of histamine and bradykinin were injected. The induced vascular leakage was greatest during the first five minutes of lesion development for histamine, during the second five minutes of lesion development for bradykinin, and the synergistic effect of prostaglandin E2 was maximal during the third five minute period of lesion development.

Assessment of histamine, bradykinin, prostaglandins E1 and E2 and carrageenin as vascular permeability agents in the horse.

The vascular leakage induced by histamine, bradykinin, serotonin and prostaglandin E1 and E2 was assessed. The test agents were injected intradermally into the shaved thoracic skin of horses and the vascular leakage estimated either semi-quantitatively by recording the diameter of the lesions or by measuring the actual volume of extravasated plasma in microliters using iodine-125-labelled human serum albumin (125I-HSA) as a marker in the blood plasma. Using the latter method, the vascular leakage induced by carrageenin and the effect of coadministered prostaglandins E1 and E2 upon the vascular leakage of both histamine and bradykinin were also investigated. No obvious lesions resulted when serotonin (10(-2) mol/l) was injected but histamine and bradykinin produced circular lesions which increased in diameter for approximately 30 min. The size of the lesions and volume of extravasated plasma was dose dependent. On a molar basis, bradykinin (10(-6) mol/l, 10(-5) mol/l) was more potent than histamine but they were equipotent at 10(-4) mol/l. The size of the lesions induced by carrageenin were independent of their anatomical location on the thorax. Except for the second hour, the hourly volume of vascular leakage increased until the fifth hour when the experiment was concluded. The maximum vascular leakage resulting from the injection of prostaglandin E1 or E2 (1, 10, 100 or 1000 ng) was 7 microliters but when co-administered with bradykinin (10(-6) mol/l), the volume of leaked plasma increased from 29 to 78 microliters. No synergy was observed when either prostaglandin was co-administered with histamine (10(-5) mol/l).

Copper salicylate and copper phenylbutazone as topically applied anti-inflammatory agents in the rat and horse.

Topically applied copper phenylbutazone, phenylbutazone, copper salicylate, salicylate and dimethylsulfoxide glycerol (80:20) were investigated as anti-inflammatory agents in rats and horses. Dimethylsulfoxide and glycerol (80:20) or dimethylsulfoxide, ethanol and glycerol (60:20:20) were used as the drug solvents. Subcutaneously administered carrageenin was used to induce inflammatory oedema, either in the paws of rats or the alar fold of the horse. The severity of the oedema and the anti-inflammatory effect of the drugs were assessed by measuring changes in the paw or alar-fold diameters. Copper salicylate and copper phenylbutazone were effective inhibitors of the inflammatory oedema in both species, but dimethylsulfoxide:glycerol (80:20) was not. In the rat, copper salicylate and copper phenylbutazone were superior anti-inflammatory agents compared to either salicylate or phenylbutazone, respectively. Following the topical application of four times the recommended daily dose of copper phenylbutazone to the horse for 5 days, minor skin irritation occurred and trace concentrations of phenylbutazone (maximum 0.6 microgram/ml) and negligible concentrations of oxyphenbutazone and gamma-hydroxyphenylbutazone were detected in the plasma.

Superoxide production by stimulated equine polymorphonuclear leukocytes--inhibition by anti-inflammatory drugs.

Polymorphonuclear leukocytes (PMNLs) were isolated from an inflammatory exudate induced in the intercarpal joints of horses by an administration of carrageenin. Their superoxide production at rest and following stimulation with either serum-treated zymosan (STZ) or phorbol myristate acetate (PMA) was measured by cytochrome-c reduction. Stimulation of the cells increased the cytochrome-c reduction 10-15 times that of resting cells. The maxima were 20 nmol of reduced cytochrome-c per 10(6) cells per ml at 120 min (STZ) and 35 nmol of reduced cytochrome-c per 10(6) cells per ml at 60 min (PMA). The maximum inhibition of the cytochrome-c reduction by superoxide dismutase (Palosein) was 83.6% (STZ stimulation) and 72.1% (PMA stimulation). The non-steroidal anti-inflammatory drugs, phenylbutazone, salicylic acid, aspirin, sodium salicylate in addition to D-penicillamine and dimethylsulfoxide caused dose-dependent inhibition of the cytochrome-c reduction when the cells were stimulated by PMA. The maximum inhibitions were 64% and 36% for aspirin (10(-2) M), 32% and 17% for phenylbutazone (10(-3) M), 15% and 31% for dimethylsulfoxide (6.4 x 10(-1) M), 32% and 19% for salicylic acid (10(-2) M), 0% and 17% for sodium salicylate (10(-2) M) and 2.2% and 2.5% for D-penicillamine (10(-4) M) when the cells were stimulated by STZ and PMA, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of palosein (superoxide dismutase) and catalase upon oxygen derived free radical induced degradation of equine synovial fluid.

The effect of oxygen derived free radicals (ODFR) upon the specific viscosity of equine synovial fluid was studied. ODFR were generated either by a mixture of ferrous iron and EDTA (Fe/EDTA) or by a mixture of hypoxanthine and xanthine oxidase (HX/XO). Incubation of the synovial fluid with both free radical generating systems decreased its specific viscosity. When the synovial fluid was incubated with Fe/EDTA the specific viscosity of the synovial fluid was reduced rapidly. By 2 mins, it was 53 +/- 3 per cent of the original specific viscosity and by 30 mins it was reduced to 39 +/- 5 per cent. In the HX/XO system, the specific viscosity was 75 +/- 4 per cent of the original specific viscosity at 10 mins and by 50 mins it was reduced to 55 +/- 3 per cent. Palosein (superoxide dismutase) was an effective inhibitor of the free radical induced reduction of the viscosity of the synovial fluid when the free radicals were generated with HX/XO but not with Fe/EDTA. Catalase was moderately effective as an inhibitor of reduction in specific viscosity of the synovial fluid when the free radicals were generated by either system. Only minor synergy resulted when mixtures of Palosein and catalase were tested for inhibition of Fe/EDTA induced reduction in the specific viscosity of equine synovial fluid. The results indicate that Palosein may protect equine synovial fluid from the effects of the superoxide radical (O2-) but not from the hydroxyl radical (OH.).(ABSTRACT TRUNCATED AT 250 WORDS)

Acute phase response in horses: changes in plasma cation concentrations after localised tissue injury.

An acute phase reaction was elicited in four horses to which Freund's adjuvant was administered intramuscularly. The localised inflammation was accompanied by changes in the plasma concentrations of copper, iron and zinc. The plasma copper concentration, the plasma ceruloplasmin copper concentration and the ceruloplasmin oxidase activity in the plasma steadily increased to a maximum 24 days after the administration of the adjuvant. At this time, the plasma copper concentration was 2.2 micrograms/ml, a 90 per cent increase over the baseline concentration. The ratio of the concentration of plasma ceruloplasmin copper to plasma copper remained constant, indicating that the non-ceruloplasmin bound copper component of the plasma is also an acute phase reactant in the horse. The plasma zinc and iron concentrations decreased to 59 per cent and 30 per cent of their respective baseline concentrations and the severity of the inflammation appeared to influence the plasma concentrations of each metal. Weak correlations between the plasma fibrinogen concentration and the plasma copper and zinc concentrations of 25 horses with plasma fibrinogen concentrations of 5 g/litre or greater indicated that a single measurement of plasma copper concentration is not useful in the diagnosis of non-specific inflammatory disorders of the horse. However, the results suggest that the plasma copper concentrations in serial samples may be used to monitor the resolution of inflammatory disorders in the horse.