Pharmacokinetics and tissue disposition of meloxicam in beef calves after repeated oral administration.

The objective of this study was to investigate the pharmacokinetics and tissue disposition of meloxicam after repeated oral administration in calves. Thirteen male British × Continental beef calves aged 4 to 6 months and weighing 297-392 kg received 0.5 mg/kg meloxicam per os once daily for 4 days. Plasma meloxicam concentrations were determined in 8 calves over 6 days after first treatment. Calves were randomly assigned to be euthanized at 5, 10, 15 (n = 3/timepoint), and 19 days (n = 4) after final administration. Meloxicam concentrations were determined in plasma (LOQ= 0.025 µg/mL) and muscle, liver, kidney, and fat samples (LOQ = 2 ng/g) after extraction using validated LC-MS-MS methods. The mean (± SD) Cmax , Cmin , and Caverage plasma meloxicam concentrations were 4.52 ± 0.87 µg/mL, 2.95 ± 0.77 µg/mL, and 3.84 ± 0.81 µg/mL, respectively. Mean (± SD) tissue meloxicam concentrations were highest in liver (226.67 ± 118.16 ng/g) and kidney samples (52.73 ± 39.01 ng/g) at 5 days after final treatment. Meloxicam concentrations were below the LOQ in all tissues at 15 days after treatment. These findings suggest that tissue from meloxicam-treated calves will have low residue concentrations by 21 days after repeated oral administration.

Impact of oral meloxicam administered alone or in combination with gabapentin on experimentally induced lameness in beef calves.

This study examined the pharmacokinetics and analgesic effect of oral meloxicam (MEL) administered alone or in combination with gabapentin (GABA) in an experimental bovine lameness model. Eighteen male British × Continental beef calves aged 4 to 6 mo and weighing 297 to 392 kg were randomly assigned to receive either 1) 0.5 mg/kg lactose monohydrate placebo (PLBO; n = 6), 2) 0.5 mg/kg MEL (n = 6), or 3) 0.5 mg/kg MEL combined with 15 mg/kg GABA (MEL-GABA; n = 6) once daily for 4 d. The first treatment was administered 4 h after a chemical synovitis/arthritis was induced with injection of 15 mg amphotericin B into the left hind lateral distal interphalangeal joint. Changes in activity were evaluated continuously with pedometers. Contact force, contact area, contact pressure, impulse, and stride length were recorded once daily with a pressure mat and visual lameness scores were determined by a masked observer using a 5-point scale. Cortisol and drug concentrations were determined daily by immunoassay and HPLC-mass spectrometry, respectively. Outcomes were compared statistically using a random effects mixed model and analysis of covariance. There was a positive association between lameness scores and serum cortisol concentrations (P = 0.02) and a negative association between lameness score and step count (P < 0.0001), total force (P = 0.001), force applied to the lateral claw (P = 0.02), contact pressure (P = 0.005), and impulse of the lateral claw (P = 0.01). Step count was greater in MEL calves compared with PLBO (P = 0.008) and MEL-GABA (P = 0.04) calves. Impulse was greater in the MEL-GABA calves compared with the PLBO calves (P = 0.03). There was an inverse relationship between plasma MEL concentrations and lameness score (P = 0.02) and a positive association between MEL concentrations and force applied to the lateral claw (P = 0.03), total contact pressure (P = 0.03), and impulse on the lateral claw (P = 0.02). There was a tendency towards a positive association between GABA concentrations, total impulse, and impulse on the lateral claw (P = 0.08) and a negative associate between GABA concentrations and step count (P = 0.08). The results of this study suggest that MEL administered alone or in combination with GABA reduced the severity of lameness in calves following induction of lameness with amphotericin B. These findings have implications for developing analgesic protocols in lame calves that address both production and welfare concerns.

Comparative effects of castration and dehorning in series or concurrent castration and dehorning procedures on stress responses and production in Holstein calves.

The study objective was to compare serum cortisol as an acute stress measure, chute exit velocity as a behavioral measure, and ADG as an indicator of performance and well-being after castration, dehorning, or concurrent castration/dehorning of calves when performed in parallel and in series. Intact male Holstein calves, 3 to 4 mo, underwent sham handling before 2 procedures performed in series separated by 2 to 3 wk. In Period 1, calves were either dehorned by amputation, surgically castrated, concurrently castrated/dehorned, or served as nonsurgical controls (n = 10/treatment). In Period 2, calves that had been dehorned, castrated, or castrated/dehorned were then castrated, dehorned, or served as nonsurgical controls, respectively. Indicators of distress were measured after all procedures; ADG was assessed for 7 d after each procedure and over the 2 to 3 wk interim. Period 1 cortisol concentrations in dehorned calves were less than in castrated and castrated/dehorned calves at 120 min and from 50 to 240 min, respectively (P < 0.02). There was marginal evidence that cortisol concentrations were greater in castrated/dehorned than castrated calves at 60 min (P = 0.06). Period 2 cortisol concentrations were less in dehorned than castrated calves at 120 min (P = 0.005) but were greater from 360 to 480 min (P < 0.002). The Period 2 cortisol profile of control calves did not differ from the baseline obtained during sham handling, despite the intervening castration/dehorning in Period 1, suggesting that memory did not affect cortisol. The cortisol profile of castrated calves did not differ between periods except at 720 min, when Period 1 concentrations were greater than Period 2 (P = 0.02). Cortisol concentrations of calves dehorned in Period 2 were greater than those dehorned in Period 1 at 20 and 240 to 480 min (P < 0.05). In both periods, castrated calves exited the chute slower than dehorned calves (P < 0.05). The ADG did not differ between surgically treated calves in Period 1; in the interim, the ADG of castrated calves was greater than that of castrated/dehorned calves (difference ± SED, 1.4 ± 0.6 kg/d; P = 0.03), and in Period 2, the ADG of dehorned calves was less than castrated calves (1.8 ± 0.6 kg/d; P = 0.005). Our study supports both the common practice of concurrent castration/dehorning and the sequence of dehorning and castration. Delayed dehorning (vs. delayed castration) appeared to be more acutely stressful and more detrimental to ADG.

Effect of oral meloxicam on health and performance of beef steers relative to bulls castrated on arrival at the feedlot.

Castration in weaned calves is stressful and affects profitability by reducing ADG and increasing susceptibility to disease. This study evaluated the effect of meloxicam, a nonsteroidal anti-inflammatory drug (NSAID), on performance and health of calves received as steers compared with bull calves surgically castrated on arrival at the feedlot. British × Continental bulls (n = 145) and steers (n = 113; BW = 193 to 285 kg) were transported for 12 h in 3 truckloads (d 0), weighed, and randomly assigned to receive either lactose placebo (CONT; 1 mg/kg) or meloxicam (MEL; 1 mg/kg) suspended in water and administered per os, 24 h before castration. On d 1, bulls were surgically castrated (CAST) and steers were processed without castration (STR). Combinations of CONT/MEL and CAST/STR were allocated to 24 pens (6 pens per treatment) of 8 to 14 calves each. Pen was the experimental unit. Plasma meloxicam concentrations at the time of castration (d 1) were determined by HPLC-mass spectroscopy. Pen-level ADG, DMI, and G:F were estimated using BW obtained on d 0, 14, and 28 and weigh-back of feed. Individual animals were classified as sick based on a depression score of ≥2 on a 5-point scale and a rectal temperature of ≥39.8°C. On d 0, 1, and 14, calf chute temperament was evaluated using a 4-point scale. Data were analyzed using generalized linear mixed models and survival curve analyses. Castration reduced pen ADG (P < 0.001) and G:F (P < 0.001) from d 1 to 14, yet no effects (P > 0.45) were apparent by d 28. For all treatment groups, DMI increased with days on feed (P < 0.0001) but was less in CAST compared with STR calves (P < 0.016) throughout the study. From d 15 to 28, ADG increased (P = 0.0011) in CAST but not STR calves, and G:F decreased (P = 0.0004) in STR but not CAST calves. In CAST calves only, MEL treatment reduced the pen-level first pull rate (P = 0.04) and reduced bovine respiratory disease morbidity rate (P = 0.03). The frequency of chute escape behavior was greater on arrival and at castration in CAST vs. STR calves (P < 0.01) but not significantly different at d 14 (P = 0.22). Mean MEL concentrations at castration were no different between treated STR and CAST calves (P = 0.70). Meloxicam administration before castration in postweaning calves reduced the incidence of respiratory disease at the feedlot. These findings have implications for developing NSAID protocols for use in calves at castration with respect to addressing animal health and welfare concerns.

Pharmacokinetics of oral meloxicam in ruminant and preruminant calves.

The pharmacokinetics of oral meloxicam has been studied in ruminant, but not preruminant calves. Oral meloxicam was administered at 0.5 mg/kg to six ruminant calves via gavage (RG); to six preruminant calves via gavage (PRG); and to six preruminant calves via suckling in milk replacer (PRF). Plasma drug concentrations, determined over 120-h postadministration, were analyzed by compartmental and noncompartmental methods. The rate of drug absorption was faster (P<0.01) in PRF (0.237±0.0478/h) than RG calves (0.0815±0.0188/h), while absorption in PRG calves (0.153±0.128/h) was not different from other groups. C(max) was lower (P=0.03) in PRF (1.27±0.430 µg/mL) than in PRG calves (2.20±0.467 µg/mL), while C(max) of RG calves (1.95±0.955 µg/mL) was not different from other groups. V/F was higher in PRF calves (365±57 mL/kg) than either PRG (177±63 mL/kg, P<0.01) or RG (232±83 mL/kg, P=0.01) calves. These observations were likely due to differences in bioavailability, physiological maturity, and timing of the drug delivery into different compartments of the ruminant gastrointestinal tract. Results suggest that an adjustment in meloxicam dose may be necessary when administered with milk replacer.

Effect of intravenous sodium salicylate administration prior to castration on plasma cortisol and electroencephalography parameters in calves.

Nociception is an unavoidable consequence of many routine management procedures such as castration in cattle. This study investigated electroencephalography (EEG) parameters and cortisol levels in calves receiving intravenous sodium salicylate in response to a castration model. Twelve Holstein calves were randomly assigned to the following groups: (i) castrated, untreated controls, (ii) 50 mg/kg sodium salicylate IV precastration, were blood sampled at 0, 5, 10, 20, 30, 45, 60, 90, 120, 150, 180, 240, 360, and 480 min postcastration. The EEG recording included baseline, castration, immediate recovery (0-5 min after castration), middle recovery (5-10 min after castration), and late recovery (10-20 min after castration). Samples were analyzed by competitive chemiluminescent immunoassay and fluorescence polarization immunoassay for cortisol and salicylate, respectively. EEG visual inspection and spectral analysis were performed. Statistical analyses included anova repeated measures and correlations between response variable. No treatment effect was noted between the two groups for cortisol and EEG measurements, namely an attenuation of acute cortisol response and EEG desynchronization in sodium salicylate group. Time effects were noted for EEG measurements, cortisol and salicylates levels. Significant correlations between cortisol and EEG parameters were noted. These findings have implications for designing effective analgesic regimens, and they suggest that EEG can be useful to monitor pain attributable to castration.

Modeling of electroosmotic and electrophoretic mobilization in capillary and microchip isoelectric focusing.

Our dynamic capillary electrophoresis model which uses material specific input data for estimation of electroosmosis was applied to investigate fundamental aspects of isoelectric focusing (IEF) in capillaries or microchannels made from bare fused-silica (FS), FS coated with a sulfonated polymer, polymethylmethacrylate (PMMA) and poly(dimethylsiloxane) (PDMS). Input data were generated via determination of the electroosmotic flow (EOF) using buffers with varying pH and ionic strength. Two models are distinguished, one that neglects changes of ionic strength and one that includes the dependence between electroosmotic mobility and ionic strength. For each configuration, the models provide insight into the magnitude and dynamics of electroosmosis. The contribution of each electrophoretic zone to the net EOF is thereby visualized and the amount of EOF required for the detection of the zone structures at a particular location along the capillary, including at its end for MS detection, is predicted. For bare FS, PDMS and PMMA, simulations reveal that EOF is decreasing with time and that the entire IEF process is characterized by the asymptotic formation of a stationary steady-state zone configuration in which electrophoretic transport and electroosmotic zone displacement are opposite and of equal magnitude. The location of immobilization of the boundary between anolyte and most acidic carrier ampholyte is dependent on EOF, i.e. capillary material and anolyte. Overall time intervals for reaching this state in microchannels produced by PDMS and PMMA are predicted to be similar and about twice as long compared to uncoated FS. Additional mobilization for the detection of the entire pH gradient at the capillary end is required. Using concomitant electrophoretic mobilization with an acid as coanion in the catholyte is shown to provide sufficient additional cathodic transport for that purpose. FS capillaries dynamically double coated with polybrene and poly(vinylsulfonate) are predicted to provide sufficient electroosmotic pumping for detection of the entire IEF gradient at the cathodic column end.

Modeling of the impact of ionic strength on the electroosmotic flow in capillary electrophoresis with uniform and discontinuous buffer systems.

A new dynamic computer model permitting the combined simulation of the temporal behavior of electroosmosis and electrophoresis under constant voltage or current conditions and in a capillary which exhibits a pH-dependent surface charge has been constructed and applied to the description of capillary zone electrophoresis, isotachophoresis, and isoelectric focusing with electroosmotic zone displacement. Electroosmosis is calculated via use of a normalized wall titration curve (mobility vs pH). Two approaches employed for normalization of the experimentally determined wall titration data are discussed, one that considers the electroosmotic mobility to be inversely proportional to the square root of the ionic strength (method based on the Gouy-Chapman theory with the counterion layer thickness being equal to the Debye-Hückel length) and one that assumes the double-layer thickness to be the sum of a compact layer of fixed charges and the Debye-Hückel thickness and the existence of a wall adsorption equilibrium of the buffer cation other than the proton (method described by Salomon, K.; et al. J. Chromatogr. 1991, 559, 69). The first approach is shown to overestimate the magnitude of electroosmosis, whereas, with the more complex dependence between the electroosmotic mobility and ionic strength, qualitative agreement between experimental and simulation data is obtained. Using one set of electroosmosis input data, the new model is shown to provide detailed insight into the dynamics of electroosmosis in typical discontinuous buffer systems employed in capillary zone electrophoresis (in which the sample matrix provides the discontinuity), in capillary isotachophoresis, and in capillary isoelectric focusing.

Impact of electroosmosis on isotachophoresis in open-tubular fused-silica capillaries: analysis of the evolution of a stationary steady-state zone structure by computer simulation and experimental validation.

A dynamic computer model for simulation of open-tubular capillary electrophoresis that includes in situ calculation of electroosmosis along the fused-silica capillary column has been applied to the characterization of an anionic isotachophoretic system in presence of a cathodic electroosmotic flow. For each column segment, electroosmosis is calculated with the use of a wall mobility, the voltage gradient and the degree of dissociation of the silanol surface groups of the capillary wall. Then, the bulk capillary flow is taken to be the average of all of the segment flows and considered to represent a plug flow. This simple approach enables the combined simulation of the temporal behavior of an isotachophoretic zone structure in presence of electroosmosis. For a model anionic isotachophoretic configuration at pH 6, simulation data reveal the asymptotic formation of a stationary steady-state zone configuration in which electrophoretic and electroosmotic zone displacements are opposite and of equal magnitude. The position of the stationary boundaries are predicted to be dependent on the selected wall pK and mobility values. For two different instruments, qualitative agreement between experimental data and simulation results obtained with a wall pK between 5 and 6 is demonstrated. However, for the two experimental setups, significant differences in electroosmotic pumping (i.e. wall mobility values) are noted.

Experimental and theoretical dynamics of isoelectric focusing: IV. Cathodic, anodic and symmetrical drifts of the pH gradient.

The production of anodic, cathodic and symmetrical drifts of a pH 3.5-10 gradient formed by isoelectric focusing in polyacrylamide gels is demonstrated experimentally by manipulation of the electrolyte concentrations. Experimental behavior is reproduced by computer simulation of a model mixture of 15 hypothetical carrier ampholytes whose pIs span the pH range 3-10. The mechanism which produces the drifts is elucidated and approaches to minimize such drifts are discussed. The data suggest why most experimentally observed drifts are cathodic.

The use of metal ion-supplemented buffers to enhance the resolution of peptides in capillary zone electrophoresis.

The potential of metal ion-containing buffers to enhance the resolution of peptides in capillary zone electrophoresis was evaluated. The impact of adding Cu(II) and Zn(II) salts to electrophoresis buffers is shown to affect the migrational behavior of several dipeptides containing histidine. Interaction with a metal ion differentially decreases the electrophoretic mobilities of peptides which comigrate in the absence of metal ions, thus causing their separation. This effect is obtained at low pH where the large net charge on the samples yields short analysis times. The dependence of the resolution on Zn(II) concentration is presented for two different samples. The influence of the background buffer is discussed.

Experimental and theoretical description of the isotachophoretic behavior of serum albumin.

The isotachophoretic behavior of serum albumin is examined for three anionic and one cationic electrolyte systems by (i) computer simulation, (ii) capillary isotachophoresis (ITP) and (iii) continuous flow ITP. The theoretical relationship between pH of the leading electrolyte and the steady state protein plateau concentration is presented for one of the anionic systems. With leading ion concentrations of the order of 10 mM, experimental protein plateau concentrations of 1.3-2.3% w/v are obtained. The computer predictions are approximately half these values.

Isotachophoretic zone formation of serum albumin in different free fluid electrophoresis instruments.

The isotachophoretic behavior of a model protein, serum albumin, was examined (i) by computer simulation, (ii) by capillary isotachophoresis in HPE 100 and Tachophor 2127, (iii) by continuous flow isotachophoresis in Elphor VaP 22 and the BIO-STREAM Separator and (iv) by recycling isotachophoresis in an apparatus of our own design. Variations in monitored zone shapes can be explained by differences in engineering aspects and fluid stabilization principles of the instruments.

The impact of boundary conditions in free flow field step electrophoresis.

The impact of the type of membrane used to separate the electrolyte compartments from the separation cell for continuous flow field step electrophoresis is examined using both experimental and computer simulation data. It is shown that the distinct transport characteristics of ion exchange membranes and dialysis membranes may have an effect on the stability of the pH and conductivity gradients created by the buffer system. These effects are interpreted using computer simulation data. The limitations of the model used to portray the transport characteristics of ion exchange membranes are discussed as are the experimental conditions which provide the greatest stability for field step electrophoresis.

Computer simulation and experimental validation of the electrophoretic behavior of proteins.

A mathematical model of the electrophoretic behavior of proteins is presented. The Debye-Hückel-Henry theory is used for the description of protein mobility, which has the important result of making net mobility a function of ionic strength. A net charge vs pH relationship and a diffusion coefficient are required to describe a specific protein. The model is employed for the computer simulation of three distinct electrophoretic modes: isoelectric focusing, isotachophoresis, and zone electrophoresis. The validity of the model is tested by comparing simulation with experimental data. Excellent qualitative agreement was found.

Characterization of synthetic carrier ampholytes by repetitive scanning of the electric field during focusing.

This paper reports the utilization of a potential gradient array detector for monitoring the dynamics of the electric field during isoelectric focusing. Transient and steady state electric field profiles are presented for synthetic carrier ampholyte mixtures with a wide (approximately 3-10) pH range. Two available commercial products (Ampholine and Pharmalyte) and a laboratory synthesized mixture (PEHA ampholytes) are compared. The formation of conductivity gaps and their migration toward the cathode in extended experiments (cathodic drift) can be visualized with this system.

Electrophoresis: mathematical modeling and computer simulation.

A mathematical model of electrophoretic separation processes has been developed and adapted for computer simulations. The model is used to predict the characteristic behavior of a variety of electrophoretic techniques from a knowledge of chemical equilibria and physical transport phenomena. The model provides a unifying basis for a rational classification of all electrophoretic processes.

Mathematical modeling and computer simulation of isoelectric focusing with electrochemically defined ampholytes.

A mathematical model of isoelectric focusing at the steady state has been developed for an M-component system of electrochemically defined ampholytes. The model is formulated from fundamental principles describing the components' chemical equilibria, mass transfer resulting from diffusion and electromigration, and electroneutrality. The model consists of ordinary differential equations coupled with a system of algebraic equations. The model is implemented on a digital computer using FORTRAN-based simulation software. Computer simulation data are presented for several two-component systems showing the effects of varying the isoelectric points and dissociation constants of the constituents.

Bacteriophage T4-coded dihydrofolate reductase: synthesis, turnover, and location of the virion protein.

Dihydrofolate reductase plays a dual role in bacteriophage T4, first, as an enzyme of thymidylate metabolism, and second, as a protein component of the tail baseplate. Antibody to the purified enzyme has been used to study its synthesis and intracellular turnover. The antibody specifically precipitates one protein from T4D-infected cell extracts. This has been identified as dihydrofolate reductase, although the polypeptide molecular weight (22,000) is lower than that earlier determined for this enzyme. The protein comigrates on gels with pY, a genetically undefined protein component of the baseplate. However, it is not pY, for pY is synthesized late in infection, whereas virtually no dihydrofolate reductase synthesis occurs later than 10 min after infection at 37 degrees C. Dihydrofolate reductase, once formed, is neither degraded nor converted to proteins of higher or lower molecular weight. Thus, it is probably incorporated into virions at the same molecular weight as that of the soluble enzyme. 125I-radiolabeled antibody binds to the wedge substructure of the baseplate, and this binding is blocked by preincubation with purified T4 dihydrofolate reductase. Thus, the enzyme protein seems to be a component of the wedge.

Bacteriophage T4 virion dihydrofolate reductase: approaches to quantitation and assessment of function.

This paper is concerned with the physiological role(s) of T4 phage-coded dihydrofolate reductase, which functions both in DNA precursor metabolism and as a virion protein. (i) We have detected enzyme activity in noninfectious particles produced under restrictive conditions by gene 11 mutants. This supports the conclusion of Kozloff et al. (J. Virol. 16:1401-1408, 1975) that the protein lies in the baseplate, covered by the gene 11 protein. (ii) We have obtained further evidence for virion dihydrofolate reductase as the target for neutralizing activity of T4 dihydrofolate reductase antiserum and as a determinant of the heat lability of the virion. This derives from our observation that the reductases specified by T4B and T4D differ in several properties. (iii) We have investigated several anomalous properties of T4 mutants bearing deletions that reportedly extend into or through the frd gene, which codes for dihydrofolate reductase. Evidence is presented that the deletions in fact do not extend through frd. These strains direct the synthesis of material that cross-reacts with antiserum to homogeneous dihydrofolate reductase. Moreover, they are all quite sensitive to the phage-neutralizing effects of this antiserum. In addition, they are restricted by several of the hospital strains, wild-type strains of Escherichia coli supplied by the California Institute of Technology group. (iv) We have attempted to detect dihydrofolate reductase among early-synthesized proteins present in T4 tails. Two such proteins are seen, one of which is evidently the gene 25 product and one that is a bacterial protein. Quantitation of our electrophoretic technique has allowed determination of the number of molecules of some T4 tail components present per virion. (v) Finally, we have compared the T4 dihydrofolate reductase with the corresponding enzyme specified by two plasmids conferring resistance to trimethoprim (Skold and Widh, J. Biol. Chem. 249:4324-4325, 1974). Although the enzymes are similar in some properties, they differ in several important respects, including immunological activity.