Detection of 11 carbamate pesticide residues in raw and pasteurized camel milk samples using liquid chromatography tandem mass spectrometry: Method development, method validation, and health risk assessment.

This study aimed to use ultra-high-performance liquid chromatography coupled to a triple-quadrupole mass spectrometer to detect 11 carbamate pesticide residues in raw and pasteurized camel milk samples collected from the United Arab Emirates. A method was developed and validated by evaluating limits of detection, limits of quantitation, linearity, extraction recovery, repeatability, intermediate precision, and matrix effect. Due to the high protein and fat content in camel milk, a sample preparation step was necessary to avoid potential interference during analysis. For this purpose, 5 different liquid-liquid extraction techniques were evaluated to determine their efficiency in extracting carbamate pesticides from camel milk. The established method demonstrated high accuracy and precision. The matrix effect for all carbamate pesticides was observed to fall within the soft range, indicating its negligible effect. Remarkably, detection limits for all carbamates were as low as 0.01 µg/kg. Additionally, the coefficients of determination were >0.998, demonstrating excellent linearity. A total of 17 camel milk samples were analyzed, and only one sample was found to be free from any carbamate residues. The remaining 16 samples contained at least one carbamate residue, yet all detected concentrations were below the recommended maximum residue limits set by Codex Alimentarius and the European Union pesticide databases. Nonetheless, it is worth noting that the detected levels of ethiofencarb in 3 samples were close to the borderline of the maximum residue limit. To assess the health risk for consumers of camel milk, the hazard index values of carbofuran, carbaryl, and propoxur were calculated. The hazard index values for these 3 carbamate pesticides were all below 1, indicating that camel milk consumers are not at risk from these residues.

Surveillance assessment for veterinary biocidal products in Korea: A laboratory investigation.

Veterinary biocides used in animal husbandry have the potential to cause human health concerns. Biocidal products for veterinary use, which contain pesticides approved in Korea, comprise 49 active ingredients within 234 products. Within 17 of these products there are 3 ingredients which are highly hazardous pesticides: coumaphos, dichlorvos and methomyl. In this study, the content of the active ingredients of 160 products sold domestically was investigated. Samples were collected for 119 biocidal products for veterinary use. These were analysed by high-performance liquid chromatography (HPLC) and gas chromatography (GC). Seventeen products were noncompliant (insufficient or excess quantity of active ingredients). The ingredients that were below the stated concentrations were amitraz, chlorpyrifos-methyl, cypermethrin, cyromazine, dichlorvos, fipronil, muscamone and trichlorfon. The ingredients that exceeded the stated concentrations were abamectin, fluvalinate and pyriproxyfen. The noncompliance rate in biocidal products for veterinary use was 9.19%. The results of this study show that three highly hazardous pesticides (coumaphos, dichlorvos and methomyl) and 10 active ingredients (abamectin, amitraz, chlorpyrifos-methyl, cypermethrin, cyromazine, fipronil, fluvalinate, muscamone, pyriproxyfen and trichlorfon) deviated from the stated concentrations. Thus, management plans should be established to ensure compliant veterinary drugs by post-distribution quality control, such as planning for regular inspection.

Biomarkers of oxidative stress as an assessment of the redox status of the liver in dogs.

BACKGROUND: Oxidative stress is associated with a diverse group of liver disorders across species. OBJECTIVES: Determine whether glutathione (GSH) concentration in plasma and red blood cells correlates with liver GSH concentration in dogs and evaluate whether other markers of systemic oxidative stress, plasma vitamin E and urine 8-isoprostanes/creatinine (F2 -IsoPs/Cr) concentrations, correlate with liver GSH. ANIMALS: Thirty-four client-owned dogs undergoing clinically indicated liver biopsy and 15 healthy control dogs. METHODS: Prospective, observational cross-sectional study. Urine and blood were collected before liver biopsy. Plasma, erythrocyte, and liver GSH were measured using high performance liquid chromatography (HPLC); vitamin E was measured by HPLC, and F2 -IsoPs/Cr was measured by gas chromatography/mass spectrometry. RESULTS: All dogs were treated at the discretion of the attending clinician (24/34 received antioxidants; 4/34 fed therapeutic liver diet), which included dogs with primary or secondary liver disease (inflammatory (n = 21), metabolic (n = 9), vascular (n = 2), and neoplastic (n = 2)). Median GSH concentrations in plasma, erythrocyte, and liver were 0.18 mg/dL (range 0.14 to 0.56 mg/dL), 56.7 mg/dL (18.3 to 79.2 mg/dL), and 181 mg/dL (39.9 to 527 mg/dL), respectively. No significant correlations were found between liver GSH and erythrocyte GSH, plasma GSH, vitamin E, or F2 -IsoPs/Cr. Dogs undergoing clinically indicated liver biopsy had significantly higher urine F2 -IsoPs/Cr than did healthy controls (5.89 vs 2.98 ng/mg; P < .0001). CONCLUSIONS AND CLINICAL IMPORTANCE: Erythrocyte and plasma GSH are not indicative of liver GSH concentration in dogs. In addition, dogs undergoing clinically indicated liver biopsy have evidence of increased systemic oxidative stress compared to healthy controls.

Assessment of the long-acting ivermectin formulation in sheep: Further insight into potential pharmacokinetic interactions.

The aim of the current study was to evaluate the in vivo pharmacokinetic of ivermectin (IVM) after the administration of a long-acting (LA) formulation to sheep and its impact on potential drug-drug interactions. The work included the evaluation of the comparative plasma profiles of IVM administered at a single therapeutic dose (200 µg/kg) and as LA formulation at 630 µg/kg. Additionally, IVM was measured in different gastrointestinal tissues at 15 days posttreatment with both IVM formulations. The impact of the long-lasting and enhanced IVM exposure on the disposition kinetics of abamectin (ABM) was also assessed. Plasma (IVM and ABM) and gastrointestinal (IVM) concentrations were analyzed by HPLC with fluorescent detection. In plasma, the calculated Cmax and AUC0-t values of the IVM-LA formulation were 1.47- and 3.35-fold higher compared with IVM 1% formulation, respectively. The T1/2ab and Tmax collected after administration of the LA formulation were 2- and 3.5-fold longer than those observed after administration of IVM 1% formulation, respectively. Significantly higher IVM concentrations were measured in the intestine mucosal tissues and luminal contents with the LA formulation, and in the liver, the increase was 7-fold higher than conventional formulation. There was no drug interaction between IVM and ABM after the single administration of ABM at 15 days post-administration of the IVM LA formulation. The characterization of the kinetic behavior of the LA formulation to sheep and its potential influence on drug-drug interactions is a further contribution to the field.

Pharmacokinetics of levofloxacin after single intravenous, oral and subcutaneous administration to dogs.

The pharmacokinetic properties of the fluoroquinolone levofloxacin (LFX) were investigated in six dogs after single intravenous, oral and subcutaneous administration at a dose of 2.5, 5 and 5 mg/kg, respectively. After intravenous administration, distribution was rapid (T½dist 0.127 ± 0.055 hr) and wide as reflected by the volume of distribution of 1.20 ± 0.13 L/kg. Drug elimination was relatively slow with a total body clearance of 0.11 ± 0.03 L kg-1  hr-1 and a T½ for this process of 7.85 ± 2.30 hr. After oral and subcutaneous administration, absorption half-life and Tmax were 0.35 and 0.80 hr and 1.82 and 2.82 hr, respectively. The bioavailability was significantly higher (p Ë‚ 0.05) after subcutaneous than oral administration (79.90 vs. 60.94%). No statistically significant differences were observed between other pharmacokinetic parameters. Considering the AUC24 hr /MIC and Cmax /MIC ratios obtained, it can be concluded that LFX administered intravenously (2.5 mg/kg), subcutaneously (5 mg/kg) or orally (5 mg/kg) is efficacious against Gram-negative bacteria with MIC values of 0.1 µg/ml. For Gram-positive bacteria with MIC values of 0.5 µg/kg, only SC and PO administration at a dosage of 5 mg/kg showed to be efficacious. MIC-based PK/PD analysis by Monte Carlo simulation indicates that the proposed dose regimens of LFX, 5 and 7.5 mg/kg/24 hr by SC route and 10 mg/kg/24 hr by oral route, in dogs may be adequate to recommend as an empirical therapy against S. aureus strains with MIC ≤ 0.5 µg/ml and E. coli strains with MIC values ≤0.125 µg/ml.

Screening and Assessment of Low-Molecular-Weight Biomarkers of Milk from Cow and Water Buffalo: An Alternative Approach for the Rapid Identification of Adulterated Water Buffalo Mozzarellas.

Adulteration of Mozzarella di Bufala Campana with cow milk is a common fraud because of the high price and limited seasonal availability of water buffalo milk. To identify such adulteration, this work proposes a novel approach based on the use of species-specific, low-molecular-weight biomarkers (LMWBs). Liquid chromatography-tandem mass spectrometry screening analyses identified ß-carotene, lutein, and ß-cryptoxanthin as LMWBs of cow milk, while ergocalciferol was found only in water buffalo milk. Adulterated mozzarellas were prepared in the laboratory and analyzed for the four biomarkers. Combined quantification of ß-carotene and ergocalciferol enabled the detection of cow milk with a sensitivity threshold of 5% (w/w). The method was further tested by analyzing a certificated water buffalo mozzarella and several commercial products. This approach is alternative to conventional proteomic and genomic methods and is advantageous for routine operations as a result of its simplicity, speed, and low cost.

Pharmacokinetic assessment of the monepantel plus oxfendazole combined administration in dairy cows.

Monepantel (MNP) is a novel anthelmintic compound launched into the veterinary pharmaceutical market. MNP is not licenced for use in dairy animals due to the prolonged elimination of its metabolite monepantel sulphone (MNPSO2 ) into milk. The goal of this study was to evaluate the presence of potential in vivo drug-drug interactions affecting the pattern of milk excretion after the coadministration of the anthelmintics MNP and oxfendazole (OFZ) to lactating dairy cows. The concentrations of both parent drugs and their metabolites were measured in plasma and milk samples by HPLC. MNPSO2 was the main metabolite recovered from plasma and milk after oral administration of MNP. A high distribution of MNPSO2 into milk was observed. The milk-to-plasma ratio (M/P ratio) for this metabolite was equal to 6.75. Conversely, the M/P ratio of OFZ was 1.26. Plasma concentration profiles of MNP and MNPSO2 were not modified in the presence of OFZ. The pattern of MNPSO2 excretion into milk was also unchanged in animals receiving MNP plus OFZ. The percentage of the total administered dose recovered from milk was 0.09 ± 0.04% (MNP) and 2.79 ± 1.54% (MNPSO2 ) after the administration of MNP alone and 0.06 ± 0.04% (MNP) and 2.34 ± 1.38% (MNPSO2 ) after the combined treatment. The presence of MNP did not alter the plasma and milk disposition kinetics of OFZ. The concentrations of the metabolite fenbendazole sulphone tended to be slightly higher in the coadministered group. Although from a pharmacodynamic point of view the coadministration of MNP and OFZ may be a useful tool, the presence of OFZ did not modify the in vivo pharmacokinetic behaviour of MNP and therefore did not result in reduced milk concentrations of MNPSO2 .

Pharmacokinetics, milk penetration and PK/PD analysis by Monte Carlo simulation of marbofloxacin, after intravenous and intramuscular administration to lactating goats.

The main objectives of this study were (i) to evaluate the serum pharmacokinetic behaviour and milk penetration of marbofloxacin (MFX; 5 mg/kg), after intravenous (IV) and intramuscular (IM) administration in lactating goats and simulate a multidose regimen on steady-state conditions, (ii) to determine the minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) of coagulase negative staphylococci (CNS) isolated from caprine mastitis in Córdoba, Argentina and (iii) to make a PK/PD analysis by Monte Carlo simulation from steady-state pharmacokinetic parameters of MFX by IV and IM routes to evaluate the efficacy and risk of the emergence of resistance. The study was carried out with six healthy, female, adult Anglo Nubian lactating goats. Marbofloxacin was administered at 5 mg/kg bw by IV and IM route. Serum and milk concentrations of MFX were determined with HPLC/uv. From 106 regional strains of CNS isolated from caprine mastitis in herds from Córdoba, Argentina, MICs and MPCs were determined. MIC90 and MPC90 were 0.4 and 6.4 µg/ml, respectively. MIC and MPC-based PK/PD analysis by Monte Carlo simulation indicates that IV and IM administration of MFX in lactating goats may not be adequate to recommend it as an empirical therapy against CNS, because the most exigent endpoints were not reached. Moreover, this dose regimen could increase the probability of selecting mutants and resulting in emergence of resistance. Based on the results of Monte Carlo simulation, the optimal dose of MFX to achieve an adequate antimicrobial efficacy should be 10 mg/kg, but it is important take into account that fluoroquinolones are substrates of efflux pumps, and this fact may determine that assumption of linear pharmacokinetics at high doses of MFX may be incorrect.

Pharmacokinetic and pharmacodynamic evaluations of a 10 mg/kg enrofloxacin intramuscular administration in bearded dragons (Pogona vitticeps): a preliminary assessment.

Enrofloxacin (E) is commonly used in veterinary medicine. It is necessary to perform pharmacokinetic/dynamic studies to minimize the selection of resistant mutants of bacteria and extend the efficacy of antimicrobial agents. Eight healthy adult Pogona vitticeps were assigned into two groups of equal size and treated with a single intramuscular injection of E at 10 mg/kg. Blood samples were withdrawn at different scheduled times for each group, and rectal swabs were collected. E and ciprofloxacin (active metabolite) blood concentrations were quantified by an HPLC validated method, while the in vitro antimicrobial susceptibility was evaluated by the Kirby-Bauer disc diffusion susceptibility test. The pharmacokinetic profiles of E gave similar pharmacokinetic parameters irrespective of the collection time schedule. Bacteria isolation showed the presence of both E. coli, Salmonella enterica subspecies enterica and subspecies 3a, Proteus spp., and Pseudomonas spp. The majority of isolated colonies were sensitive to E, but the treatment did not reduce the number of bacteria in faeces. Results suggest that E is able to reach blood concentrations high enough to kill susceptible bacteria (MIC < 0.9 µg/mL), but at the same time does not significantly affect intestinal bacteria.

Quantification of malachite green in fish feed utilising liquid chromatography-tandem mass spectrometry with a monolithic column.

The purpose of this study was to develop a rapid and sensitive method for the quantification of malachite green (MG) in fish feed using LC-ESI-MS/MS with a monolithic column as stationary phase. Fish feed was cleaned using ultrasonic assisted liquid-liquid extraction. The separation was achieved on a Chromolith® Performance RP-18e column (100 × 4.6 mm) using gradient mobile phase composition of methanol and 0.1% formic acid at the flow rate of 1.0 ml min⁻¹. The analyte was ionised using electrospray ionisation in positive mode. Mass spectral transitions were recorded in selected reaction monitoring (SRM) mode at m/z 329.78 → m/z 314.75 with a collision energy (CE) of 52% for MG. The system suitability responses were calculated for reproducibility tests of the retention time, number of theoretical plates and capacity factor. System validation was evaluated for precision, specificity and linearity of MG. The linearity and calibration graph was plotted in the range of 15.0-250 ng ml⁻¹ with the regression coefficient of >0.997. The lower limits of detection and quantification for MG were 0.55 and 1.44 ng ml⁻¹, respectively, allowing easy determination in fish feed with accuracy evaluated as a percentage recovery of 92.1% and precision determined as % CV of < 5. The method was also extended to the determination of MG in an actual fish feed. The sensitivity and selectivity of LC-ESI-MS/MS using monolithic column offers a valuable alternative to the methodologies currently employed for the quantification of MG in fish feeds.

Tetracycline residues in porcine stomach after administration via drinking water on a swine farm.

Tetracycline is a broad-spectrum antibiotic used to treat infections in swine. The maximum residue levels of tetracycline in pork stomach tissue in Russia, Europe, and the United States are 10, 200, and 2,000 ppb, respectively. This difference in accepted safety levels may be the reason why stomach tissues that the United States exports continue to be residue violators in overseas markets. In this study, 30 pigs at two different stages of production (weanling and finisher) were treated with tetracycline at 22 mg/kg of body weight per day for a total of 5 days via a water medicator. Blood samples were collected at 0, 72, 78, 96, and 102 h after the start of medication. The medication was stopped at 120 h, and blood samples were again collected at 126, 144, 168, 192, and 216 h after exposure. Five animals were slaughtered for stomach tissue 0, 24, 48, 96, and 192 h after the drug was flushed from the water line. All blood and tissue samples were analyzed by high-performance liquid chromatography-UV methods. The tetracycline levels in plasma were below the level of detection after the U.S.-labeled withdrawal time of 4 days. The stomach tissue residues averaged 671.72, 330.31, 297.77, 136.36, and 268.08 ppb on withdrawal days 0, 1, 2, 4, and 8, respectively. Using the U.S. Food and Drug Administration tolerance limit method and a population-based pharmacokinetic model with Monte Carlo simulation, a withdrawal interval was estimated. This study demonstrated that tetracycline residues are still detectable in the stomach tissues after the established United States withdrawal time of 4 days. These residue levels may explain why stomach tissues tested in Russia and Europe show positive residues for tetracycline, even though the meat may pass inspection here in the United States prior to export.

Assessment of adrenocortical activity by non-invasive measurement of faecal cortisol metabolites in dromedary camels (Camelus dromedarius).

The aim of this study was to determine whether glucocorticoid production could be monitored non-invasively in dromedary camels by measuring faecal cortisol metabolites (FCMs). Five Sudanese dromedaries, two males and three females, were injected with a synthetic adrenocorticotropic hormone (ACTH) analogue. Blood samples were collected pre- and post-ACTH injection. Faeces were sampled after spontaneous defecation for five consecutive days (2 days before and 3 days after ACTH injection). Baseline plasma cortisol values ranged from 0.6 to 10.8 ng/ml in males and from 1.1 to 16.6 ng/ml in females, while peak values after ACTH injection were 10.9-41.9 in males and 10-42.2 ng/ml in females. Peak blood cortisol values were reached between 1.5 and 2.0 h after ACTH injection. The concentration of FCMs increased after ACTH injection in the faeces of both sexes, although steroid levels peaked earlier in males [24 h; (286.7-2,559.7 ng/g faeces)] than in females [36-48 h; (1,182.6-5,169.1 ng/g faeces)], reflecting increases of 3.1-8.3- and 4.3-8-fold above baseline levels. To detect chromatographic patterns of immunoreactive FCMs, faecal samples with high FCM concentrations from both sexes were pooled and subjected to reverse phase high performance liquid chromatography (RP-HPLC). RP-HPLC analysis revealed sex differences in the polarity of FCMs, with females showing more polar FCMs than males. We concluded that stimulation of adrenocortical activity by ACTH injection resulted in a measurable increase in blood cortisol that was reliably paralleled by increases in FCM levels. Thus, measurement of FCMs is a powerful tool for monitoring the adrenocortical responses of dromedaries to stressors in field conditions.

Voluntary ingestion of antiparasitic drugs emulsified in honey represents an alternative to gavage in mice.

The oral route is the most frequently used method of drug intake in humans. Oral administration of drugs to laboratory animals such as mice typically is achieved through gavage, in which a feeding needle is introduced into the esophagus and the drug is delivered directly into the stomach. This method requires technical skill, is stressful for animals, and introduces risk of injury, pain and morbidity. Here we investigated another method of drug administration. The benzimidazole derivative albendazole was emulsified in commercially available honey and administered to mice by voluntary feeding or gavage. Mice that received albendazole by either gavage or honey ingestion had virtually identical levels of serum albendazole sulfoxide, indicating that uptake and metabolism of albendazole was similar for both administration techniques. In addition, dosing mice with the albendazole-honey mixture for 8 wk had antiparasitic activity comparable to earlier studies using gavage for drug administration. Compared with gavage, voluntary ingestion of a drug in honey is more rapid, less stressful to the animal, and less technically demanding for the administrator. Because of its low cost and ready availability, honey presents a viable vehicle for drug delivery.

Assessment of plasma uracil-to-dihydrouracil concentration ratio as an indicator of dihydropyrimidine dehydrogenase activity in clinically normal dogs and dogs with neoplasia or renal insufficiency.

OBJECTIVE: To determine and compare the ratio of uracil (U) to dihydrouracil (UH(2)) concentrations in plasma as an indicator of dihydropyrimidine dehydrogenase activity in clinically normal dogs and dogs with neoplasia or renal insufficiency. ANIMALS: 101 client- and shelter-owned dogs. PROCEDURES: Study dogs included 74 clinically normal dogs, 17 dogs with neoplasia, and 10 dogs with renal insufficiency. For each dog, a blood sample was collected into an EDTA-containing tube; plasma U and UH(2) concentrations were determined via UV high-performance liquid chromatography, and the U:UH(2) concentration ratio was calculated. Data were compared among dogs grouped on the basis of sex, clinical group assignment, reproductive status (sexually intact, spayed, or castrated), and age. RESULTS: Mean ± SEM U:UH(2) concentration ratio for all dogs was 1.55 ± 0.08 (median, 1.38; range, 0.4 to 7.14). In 14 (13.9%) dogs, the U:UH(2) concentration ratio was considered abnormal (ie, > 2). Overall, mean ratio for sexually intact dogs was significantly higher than that for neutered dogs; a similar difference was apparent among males but not females. Dogs with ratios > 2 and dogs with ratios ≤ 2 did not differ significantly with regard to sex, clinical group, reproductive status, or age. CONCLUSIONS AND CLINICAL RELEVANCE: Determination of the U:UH(2) concentration ratio was easy to perform. Ratios were variable among dogs, possibly suggesting differences in dihydropyrimidine dehydrogenase activity. However, studies correlating U:UH(2) concentration ratio and fluoropyrimidine antimetabolite drug tolerability are required to further evaluate the test's validity and its appropriate use in dogs.

Assessment of diurnal urinary cortisol excretion in Asian and African elephants using different endocrine methods.

Longitudinal urine samples were collected from Asian and African elephants to assess sample processing and immunoassay techniques for monitoring adrenal activity. Temporal profiles of urinary cortisol measured by RIA and EIA, with and without dichloromethane extraction, were similar; all correlation coefficients were >0.90. However, based on regression analyses, cortisol immunoactivity in extracted samples was only 72-81% of that of unextracted values. Within assay technique, RIA values were only 74-81% of EIA values. Collection of 24-hr urine samples demonstrated a clear diurnal pattern of glucocorticoid excretion, with the lowest concentrations observed just before midnight and peak concentrations occurring around 0600-0800 hr. These results indicate that elephants fit the pattern of a diurnal species, and that glucocorticoid production is affected by a sleep-wake cycle similar to that described for other terrestrial mammals. Cortisol can be measured in both extracted and unextracted urine using RIA and EIA methodologies. However, unexplained differences in quantitative results suggest there may be sample matrix effects and that data generated using different techniques may not be directly comparable or interchangeable.

Assessment of a von Frey device for evaluation of the antinociceptive effects of morphine and its application in pharmacodynamic modeling of morphine in dogs.

OBJECTIVE: To assess the use of a von Frey device as a mechanical nociceptive stimulus for evaluation of the antinociceptive effects of morphine in dogs and its potential application in the pharmacodynamic modeling of morphine in that species. ANIMALS: 6 healthy Beagles. PROCEDURE: von Frey thresholds were measured in all dogs before and at intervals after they received no treatment (control dogs) and i.v. administration of morphine sulfate (1 mg/kg; treated dogs) in a crossover study. The von Frey device consisted of a rigid tip (0.5 mm in diameter) and an electronic load cell; the operator was unaware of recorded measurements. RESULTS: Application of the von Frey device was simple and well tolerated by all dogs and caused no apparent tissue damage. No significant changes in thresholds were detected in the control dogs at 8 hourly measurements, indicating a lack of acquired tolerance, learned aversion, or local hyperalgesia. When assessed as a group, treated dogs had significantly high thresholds for 4 hours following morphine administration, compared with baseline values; individually, thresholds decreased to baseline values within (mean +/- SE) 2.8 +/- 0.6 hours. The maximal effect (change from baseline values) was 213 +/- 43%, and the plasma morphine concentration to achieve 50% maximal effect was 13.92 +/- 2.39 ng/mL. CONCLUSIONS AND CLINICAL RELEVANCE: Data suggest that, in dogs, evaluation of the antinociceptive effect and pharmacodynamic modeling of a dose of morphine sulfate (1 mg/kg, i.v.) can be successfully achieved by use of a von Frey device.

Renal evaluation in the healthy green iguana (Iguana iguana): assessment of plasma biochemistry, glomerular filtration rate, and endoscopic biopsy.

Plasma biochemistry, iohexol clearance, endoscopic renal evaluation, and biopsy were performed in 23 clinically healthy 2-yr-old green iguanas (Iguana iguana). Mean (+/- SD) values for packed cell volume (30 +/- 3%), total protein (62 +/- 7 g/L, 6.2 +/- 0.7 g/dl), albumin (25 +/- 2 g/L, 2.5 +/- 0.2 g/dl), globulin (37 +/- 6 g/L, 3.7 +/- 0.6 g/ dl), total calcium (3.0 +/- 0.2 mmol/L, 12.0 +/- 0.7 mg/dl), ionized calcium (1.38 +/- 0.1 mmol/L), phosphorus (1.32 +/- 0.28 mmol/L, 4.1 +/- 0.9 mg/dl), uric acid (222 +/- 100 micromol/L, 3.8 +/- 1.7 mg/dl), sodium (148 +/- 3 mmol/L or mEq/ L), and potassium (2.6 +/- 0.4 mmol/L or mEq/L) were considered within normal limits. Values for urea were low (< 1.4 mmol/L, < 4 mg/dl) with 70% of samples below the detectable analyzer range. After the i.v. injection of 75 mg/ kg iohexol into the caudal (ventral coccygeal or tail) vein, serial blood collections were performed over 32 hr. Iohexol assays by high-performance liquid chromatography produced plasma iohexol clearance graphs for each lizard. A three-compartment model was used to fit area under the curve values and to obtain the glomerular filtration rate (GFR) using regression analysis. The mean GFR (SD) was 16.56 +/- 3.90 ml/kg/hr, with a 95% confidence interval of 14.78-18.34 ml/kg/hr. Bilateral endoscopic renal evaluation and biopsy provided tissue samples of excellent diagnostic quality, which correlated with tissue harvested at necropsy and evaluated histologically. None of the 23 animals demonstrated any adverse effects of iohexol clearance or endoscopy. Recommended diagnostics for the evaluation of renal function and disease in the green iguana include plasma biochemical profiles, iohexol clearance, endoscopic examination, and renal biopsy.

Integrated pharmacological assessment of flubendazole potential for use in sheep: disposition kinetics, liver metabolism and parasite diffusion ability.

Flubendazole (FLBZ) is a broad spectrum benzimidazole methylcarbamate anthelmintic widely used in poultry and swine. However, there is no information available on the pharmacological behaviour of FLBZ in ruminants. The work reported here was addressed to evaluate the potential of FLBZ for use in sheep. The integrated assessment included evaluation of FLBZ and metabolites plasma disposition kinetics, liver metabolism and ex vivo ability to diffuse into the cestode parasite Moniezia benedeni. In a cross-over kinetic study, six healthy Corriedale sheep were treated with FLBZ by intravenous (i.v.) (4% solution) and intraruminal (i.r.) (4% suspension) administrations at the same dosage (5 mg/kg) with a 21-day washout period between treatments. Blood samples were collected between 0 and 72 h post-treatments. Sheep liver microsomes were incubated with 40 microm FLBZ and specimens of the cestode parasite M. benedeni, collected from untreated animals, were incubated (5-120 min) with FLBZ and its reduced (R-FLBZ) metabolite (5 microm). Samples of plasma, microsomal incubations and parasite material were prepared and analyzed by high-performance liquid chromatography to measure FLBZ and its metabolites. FLBZ parent drug showed a fast disposition being detected in the bloodstream up to 36 h after its i.v. administration. Both R-FLBZ and hydrolyzed FLBZ (H-FLBZ) metabolites were recovered in plasma as early as 5 min after the i.v. treatment in sheep. The plasma AUC ratios for R-FLBZ and FLBZ (AUC(R-FLBZ)/AUC(FLBZ)) were 4.07 i.v. and 5.55 i.r., respectively. R-FLBZ achieved a significantly higher (P < 0.01) C(max) value (0.14 microg/mL at 17.3 h post-treatment) than that observed for the parent drug FLBZ (0.04 microg/mL at 14.4 h post-treatment). Low plasma concentrations of FLBZ parent drug were measured between 6 and 48 h, and only trace concentrations of H-FLBZ were detected during a short period of time after the i.r. treatment. Consistently, sheep liver microsomes metabolized FLBZ into its reduced metabolite at a rate of 9.46 +/- 2.72 nmol/mg/h. Both FLBZ and R-FLBZ demonstrated a similar ability to quickly diffuse through the tegument of the cestode parasite. The data on FLBZ pharmacological behaviour presented here contribute to evaluate its potential to be developed as an anthelmintic for broad spectrum parasite control in ruminants.

Assessment of lipid peroxidation and serum vitamin E concentration in dogs with immune-mediated hemolytic anemia.

OBJECTIVE: To determine plasma malondialdehyde (MDA) and serum vitamin E concentrations in dogs with immune-mediated hemolytic anemia (IMHA) and healthy control dogs. SAMPLE POPULATION: Serum and plasma samples from 36 dogs with IMHA and 40 healthy control dogs. PROCEDURE: Blood samples were collected from all study dogs. Plasma MDA concentrations were measured by use of a commercial colorimetric assay, and serum vitamin E concentrations (alpha-, gamma, and delta-tocopherol concentrations) were measured via high-performance liquid chromatography. RESULTS: Plasma MDA concentrations were significantly higher in the dogs with IMHA than in the control dogs. Compared with control dogs, serum alpha-, gamma-, and &tocopherol concentrations were significantly lower in the IMHA-affected dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated a state of oxidative stress and reduced antioxidant reserve in dogs with IMHA; this finding provides support for further investigation of the potential benefits of antioxidant treatment in dogs with this disease.

Fecal corticosterone assessment in the epaulette shark, Hemiscyllium ocellatum.

The present study examined the feasibility of measuring the steroid hormone corticosterone in fecal extracts of epaulette sharks, Hemiscyllium ocellatum. Six immature, captive-raised epaulette sharks (four females and two males) were obtained from two different zoos and were maintained in a closed-system, 530-liter aquarium. After a one-month adaptation, fecal samples were collected daily from each animal for 33 days. Five-day sets of samples were pooled within animals to insure sufficient material for analysis. Fecal hormone extraction was achieved using repeated cycles of dichloromethane and aqueous washes. The levels of corticosterone were measured by reverse-phase high-performance liquid chromatography (HPLC). Corticosterone presence in HPLC eluent peaks from fecal extracts was determined by comparison of the elution pattern of corticosterone standard with the elution patterns of fecal extracts with and without the addition of tritiated corticosterone or exogenous, unlabeled corticosterone. Exclusive presence of corticosterone in HPLC eluent peaks presumed to be corticosterone was determined by nuclear magnetic resonance mass spectrometry. Corticosterone levels, calculated from a 10-point standard curve, ranged from 1.2 to 20.9 ng/g feces across all sharks, with 92.3% of values being < or =13.5 ng/g. Within individuals, the lowest average for corticosterone levels across 33 days was 2.6+/-0.4 ng/g feces, and the highest average was 8.4+/-2.2 ng/g feces. This study demonstrated that corticosterone was extractable from and reliably measurable in fecal extracts of epaulette sharks. This is the first evidence of this hormone in epaulette sharks and the first report of fecal corticosterone in elasmobranchs.