Aflatoxin (AF)-contaminated and fumonisin B1 (FB1)-contaminated (culture material from Fusarium moniliforme) diets were fed singly and in combination to growing cross-bred barrows. Six barrows (3 replicates of 2 each; mean body weight, 17.5 kg) per group were fed: 0 mg of AF and 0 mg of FB1/kg of feed (control); 2.5 mg of AF/kg of feed; 100 mg of FB1/kg of feed; or 2.5 mg of AF plus 100 mg of FB1/kg of feed for 35 days. The effects on production performance, serum biochemical, hematologic, immunologic, and pathologic measurements were evaluated. Body weight, gain, and feed consumption were significantly (P < 0.05) decreased by AF and AF plus FB1 diets. The FB1 diet decreased feed consumption, and although body weight was numerically decreased, it was not statistically significant. Aflatoxin increased serum gamma-glutamyltransferase (GGT) activity and total iron concentration and decreased urea nitrogen concentration and unsaturated iron-binding capacity. The FB1-alone diet increased serum GGT activity, whereas the AF plus FB1 diet increased serum aspartate transaminase, cholinesterase, alkaline phosphatase, and GGT activities, increased RBC count, triglycerides, and total iron concentrations, and decreased unsaturated iron-binding capacity and urea nitrogen concentration. For the most part, the effects of the AF plus FB1 diet on body weight and hematologic measurements could be considered additive. However, the effect of the AF plus FB1 diet on cholinesterase and alkaline phosphatase activities was greater than additive and was a synergistic response. One pig in the FB1-diet group and 2 pigs in the combination-diet group died. Postmortem lesions in pigs of the FB1-diet group consisted of ascites and increased liver weight. Observations at necropsy for pigs of the AF plus FB1-diet group consisted of hydrothorax, ascites, pulmonary edema, gastric erosions and ulceration, and increased liver and spleen weights. The AF diet increased relative liver weight and resulted in liver that was pale, rubbery, and resistant to cutting. Histologic lesions consisted of hepatic necrosis or degeneration, or both, with variable degrees of bile duct proliferation in barrows of the AF-diet groups. Renal tubular nephrosis was observed in barrows of the FB1 diet group, but this was not consistent in the AF plus FB1-diet group. Cell-mediated immunity, as measured by mitogen-induced lymphoblastogenic stimulation index, was decreased in barrows of the AF and FB1-diet groups, and values in barrows given the combination diet were significantly decreased from those in barrows given the single toxin diets. It was concluded that AF and FB1 (from culture material), singly or in combination, can adversely affect clinical performance, serum biochemical, hematologic, and immunologic values and induce lesions in growing barrows. For most of the variables we evaluated under our study conditions and dosages of toxins, measurements were affected more by the combination diet than by either single toxin diet, and the toxic responses could be described as additive or more than additive, particularly for induction of liver disease.

Milk antimicrobial residues are a serious concern for the dairy industry. Residues of the tetracycline family of antimicrobials have been reported in market milk by investigators, using radioimmunoassay and microbial receptor technology (hereafter referred to as the Charm II test). In response to these reports, an investigation was conducted to determine the potential of 3 extra-label routes of oxytetracycline (OTC) administration to cause milk residues above the Food and Drug Administration safe value of 30 parts per billion (ppb). Lactating Holstein cows were administered OTC once by use of 1 of 3 routes: IV at 16.5 mg/kg of body weight (n = 6); IM at 11 mg/kg (n = 6); and intrauterine (IU) at 2 g in 500 ml of saline solution/cow (n = 6). Duplicate milk samples were collected at the milking prior to drug administration and for the next 13 milkings at 12-hour intervals. Concentrations of OTC in milk samples were analyzed by use of the Charm II test for tetracyclines (limit of OTC detection, approx 5 ppb) and were compared with concentrations determined by use of a high-performance liquid chromatography (HPLC) method (lower limit of OTC quantitation, approx 2 ppb). The potential for milk OTC residues above the Food and Drug Administration safe value of 30 ppb after treatment was considerably greater for the IV and IM routes, compared with the IU route. Mean peak OTC concentrations in milk at the first milking after treatment for the HPLC and Charm II tests were approximately 3,700 to 4,200 ppb for the IV route, 2,200 to 2,600 ppb for the IM route, and 186 to 192 ppb for the IU route, respectively. Pharmacokinetic analysis, based on milk OTC concentrations, indicated that the area under the curve (AUC) and milk maximal concentration (Cmax) differed significantly (P < 0.001) among routes of administration. The AUC was similar for IV and IM administrations; values for both were greater than the AUC for IU administration. The Cmax was greatest for IV, intermediate for IM, and least for IU administration. There were significant (P less than or equal to 0.01) differences in AUC between assay methods (Charm II vs HPLC) for the IV route. Concentrations of OTC in milk determined by the Charm II test were often greater than those determined by HPLC. Administration of OTC to lactating cows via these routes is extra-label drug use. Failure to withhold the product from early milkings of cows administered OTC by the IV or IM route should be considered a potential cause of OTC residues in market milk. Milk from nearly all cows contained OTC (< 30 ppb), the Food and Drug Administration safe level, by 120 hours after OTC administration. Use of appropriate withholding times and antibiotic residue testing is indicated to avoid OTC residues.

A high-performance liquid chromatography method was developed for determination of flunixin in bovine plasma. The extraction procedure was easily performed and made it possible to detect low concentrations of flunixin with high accuracy. The limit of quantitation was 7 ng/ml (relative standard deviation = 18%, n = 10). The analytic method permits processing of 60 samples/d. Flunixin, as well as the internal standard (diclofenac sodium), belong to the group of nonsteroidal anti-inflammatory drugs, which are known to have a high degree of binding to plasma proteins. Therefore, an evaluation of several buffer systems was undertaken to optimize analytic conditions. Cattle were given 2.2 mg of flunixin meglumine/kg of body weight. In experiment 1, single injections were administered IV to q cow and IM to 1 heifer (7 days apart), and pharmacokinetic variables were calculated. The IV data were best described by a two-compartment model. The half-life after single IV or IM administration was around 4.0 hours. In experiment 2, the decreasing flunixin concentration was determined after the last of either 4 IM injections daily (n = 3 cows) or 2 IM injections daily (n = 3 cows) administered during a 14-day postpartum period. The half-life, determined between 48 and 96 hours after the last dose, was approximately 26 hours in both groups, and flunixin could be detected in plasma up to 8 days, on average. The protein binding of flunixin was studied, using the method of equilibrium dialysis. Flunixin was found to have a high degree of protein binding (ie, 99.4 +/- 0.2%) at a flunixin concentration in plasma of 3 to micrograms/ml. Differences in protein binding between cattle were not found.

Naproxen (+ 6-methoxy-[alpha - methyl]- 2-naphthalene acetic acid) is a nonsteroidal anti-inflammatory drug that is used for the treatment of inflammatory conditions in horses. We developed a model that describes the drug's disposition and renal excretion, including synovial fluid disposition and elimination after IV administration in horses. The plasma disposition, after IV administration of 5 mg/kg of body weight, was described by a two-compartment model; mean +/- SD distribution and elimination half-lives were 1.42 +/- 0.42 and 8.26 +/- 2.56 hours, respectively. Plasma concentration of naproxen after IV administration of 5 mg/kg was 55.3 +/- 13.5 and 0.61 +/- 0.42 mg/L at 5 minutes and 48 hours after its administration, respectively. Steady-state volume of distribution was 0.163 +/- 0.053 L/kg, and area under the plasma concentration time-curve was 372.1 +/- 128.2 mg/h/L The peak synovial fluid concentration of 12.68 +/- 12.39 mg/L was measured at 6 hours, and decreased to 0.71 +/- 0.38 mg/L at 36 hours after naproxen administration. The decrease of naproxen concentration in synovial fluid paralleled that in plasma. The appearance half-life of naproxen in synovial fluid was 4.64 hours, and the elimination half-life was 6.73 hours. Total body clearance was 0.015 +/- 0.006 L/h/ kg. The percentage of plasma protein binding was 97.0 +/- 2.9% at plasma concentrations between 5 and 100 mg/L. This was significantly (P < 0.05) higher than the percentage of binding at plasma concentrations of 0.5, 1, and 500 mg/L, which was 75.2 +/- 11.8%. Most of the drug was excreted as glucuronidated naproxen and unconjugated desmethylnaproxen. The recovery of naproxen and all metabolites in urine at 36 hours was 64.6 +/- 7.2% of the total dose. Of this total, 39.6 +/- 10.3% and 8.5 +/- 7.9% were glucuronidated naporoxen and desmethylnaproxen, respectively; 0.3 +/- 0.1% and 16.6 +/- 7.9% were free naproxen and desmethylnaproxen, respectively.

Using 7 penicillins (amoxicillin, ampicillin, methicillin, penicillin G, oxacillin, cloxacillin, and dicloxacillin), simultaneous and direct determination of residual penicillins in biological samples was carried out by use of bioassay and high-performance liquid chromatography with spectrophotometric or fluorometric detectors. By use of assay medium seeded with penicillin-sensitive Micrococcus luteus (ATCC No. 9341) as a test organism, we were able to detect penicillins even at low concentrations. All penicillins treated with 10 U of penicillinase/ml did not produce inhibition zones by disk testing, even at a concentration of 100 micrograms of penicillin/ml/assay plate. Using a mobile phase of acetonitrile:methanol:0.01M KH2PO4 (19:11:70, v/v/v; pH, 7.1), standard solutions of the penicillins were separated from each other by use of high-performance liquid chromatography analysis, producing symmetric peaks without tailing, each of which had a characteristic retention time. Simultaneous detection of residual penicillins in bovine serum, kidneys, and liver, for the 5 penicillins for which analysis was possible by use of the UV method, yielded recovery rates from 71.4 to 102.3%; for the 2 amino-penicillins, amoxicillin and ampicillin, which could only be detected by use of the fluorometric method, recovery rate ranged from 72.9 to 103%.

A radioimmunoassay test for tetracyclines (Charm II) was compared with high-pressure liquid chromatography (HPLC) for detection of oxytetracycline (OTC) residues in milk samples from individual lactating cows. Oxytetracycline was administered by 1 of 3 routes (IV, IM, or intrauterine) to 21 lactating dairy cows. A total of 292 duplicate milk samples were collected from milkings before and through 156 hours after OTC administration. Concentration of OTC in these samples was determined by use of the Charm II test and an HPLC method with a lower limit of quantitation, approximately 2 ng of OTC/ml. Samples were also classified with respect to presence of OTC residues relative to the FDA safe concentration (less than or equal to 30 ng/ml), using the Charm II (by control point determination) and HPLC methods. There was a significant (P less than or equal to 0.05) difference between test methods in classification of milk samples with respect to presence or absence of OTC at the FDA safe concentration. A total of 48 of the 292 test results (16.4%) did not agree. Using the HPLC test results as the standard with which Charm II test results were compared, 47 false presumptive-violative test results and 1 false presumptive-nonviolative Charm II test result (a sample containing 31 ng of OTC/ml, as evaluated by HPLC) were obtained. The samples with false presumptive-violative Charm II results contained (less than or equal to 30 ng of OTC/ml, as evaluated by HPLC. In some respects, the Charm II test performed appropriately as a screening test to detect OTC residues in milk samples from individual cows. However, the tendency for the test to yield presumptive-violative test results at OTC concentrations lower than the FDA safe concentration (as evaluated by HPLC), suggests that caution should be exercised in using the test as the sole basis on which a decision is made to reject milk. As indicated by the manufacturer, presumptive-violative Charm II test results should be confirmed by additional testing Although not specifically evaluated, the tendency for misclassification of milk samples as presumptive-violative by the Charm II test may or may not occur in commingled milk, compared with milk samples from individual cows.

A cross-over study was performed in 6 healthy mixed-breed dogs and 4 healthy Beagles. Diazepam was administered per rectum to Beagles (0.5 mg/kg of body weight) and mixed-breed dogs (2 mg/kg), and IV (0.5 mg/kg) to both groups of dogs. Each dog received the drug by both routes, with a 1-week washout period between dosages. After diazepam administration, blood samples were collected to measure plasma concentration of diazepam and its active metabolites, desmethyldiazepam and oxazepam, by use of reverse-phase high-performance liquid chromatography (HPLC). Systemic availability was assessed by comparing the area under the curve for diazepam metabolites for each route of administration. Mean (+/- SD) diazepam concentrations in plasma after rectal administration were low in comparison with those obtained after IV administration, with systemic availability of only 7.4 (+/- 5.9) and 2.7 (+/- 3.2)% for the high and low dose, respectively. However, diazepam was converted to its metabolites within minutes after administration. Accounting for the total concentration of benzodiazepines (diazepam plus desmethyldiazepam and oxazepam) in plasma, systemic availability was 79.9 (+/- 20.7) and 66.0 (+/- 23.8)% for the high and low dosage, respectively. After IV administration, diazepam concentration decreased, with a half-life of only 14 to 16 minutes, but desmethyldiazepam and oxazepam concentrations decreased more slowly, with a half-life of 2.2 to 2.8 hours and 3.5 to 5.1 hours, respectively. Each of the metabolites is reported to have anticonvulsant activity. After rectal administration of the high dose, mean total benzodiazepine concentration was above 1.0 micrograms/ml within 10 minutes and was maintained above this concentration for at least 6 hours. We conclude that diazepam is absorbed after rectal administration in dogs, and that the pharmacologic effects are probably caused by the active metabolites, not the parent drug. Samples also were analyzed by use of a nonspecific commercial benzodiazepine fluorescence polarization immunoassay (FPIA). Correlation between the FPLA and HPLC assay was strongest for diazeparn (R2 = 0.84), weak for desmethyldiazepam (R2 = 0.09), and nonexistent for oxazepam. We conclude from a comparison of assays that HPLC is preferred over the FPLA method for measuring benzodiazepines in dogs.

Objective--To study whether end products of 2 pathways of anaerobic energy metabolism, lactate and purines, that accumulate in the blood after intense exercise indicate any relation to exercise performance. Design--Venous blood samples were taken within 1 and 15 minutes after a trotting race of 2,100 m. Animals--16 Clinically healthy Standardbred trotters. Procedure--Blood and plasma lactate concentrations were measured by enzymatic analyzer, and purines, uric acid and allantoin, were determined by high-performance liquid chromatography. The concentrations of metabolites were then correlated to racing time and individual performance indexes that are annually calculated from the percentage of winnings, placings, and starts rejected, average earnings per start, and the racing record. Results--Blood lactate concentration immediately and calculated cell lactate concentration immediately and 15 minutes after the race correlated positively (P < 0.05 to P < 0.01) with the individual performance indexes. Plasma lactate concentration was not correlated to the individual performance indexes. Uric acid concentration, immediately and 15 minutes after the race, was negatively correlated (P < 0.05) to the individual performance indexes, and a positive relation (P < 0.05) was found between the highest concentration of uric acid and the racing time. Concentration of allantoin immediately or 15 minutes after the race did not have any significant correlation to the individual performance indexes. Conclusions--Accumulation of lactate in the blood, which was greater in the superior performing horses, may prove to be an useful predictor of anaerobic capacity. The results also indicate that the loss of purine nucleotides was less in the superior performing horses, although further studies are needed to confirm this.