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PROCESSING, PRODUCTS, AND FOOD SAFETY |
* Veterinary Medicine Teaching Hospital and Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan, 402; and The Branch Institute of Animal Drug Inspection, Animal Health Research Institute, Council of Agriculture, Executive Yuan, Taiwan, 350
1 Corresponding author: ccchou{at}nchu.edu.tw
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: pigeon microdialysis • amoxicillin • enzyme-linked immunosorbent assay
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-amino-p-hydroxybenzyl penicillin, AMX) is a medium-spectrum β-lactam antibiotic used to treat bacterial infections caused by gram-positive bacteria, including Haemophilus influenzae, Neisseria gonorrhoea, Escherichia coli, pneumococci, streptococci, and certain strains of staphylococci, and a limited range of gram-negative organisms (Handsfield et al., 1973; Neal, 2002). Amoxicillin acts by inhibiting the cross-linkage between the linear peptidoglycan polymer chains that make up the bacterial cell walls (Handsfield et al., 1973). It is usually the drug of choice within the class because it is better absorbed than other β-lactam antibiotics following oral administration. In the pigeon, AMX is effective against Streptococcus bovis (Soenens et al., 1998), E. coli, and some Salmonella (Dorrestein et al., 1987) and Haemophilus species (Carter et al., 1991), but has little effect on Mycoplasma and Chlamydia (Talaber, 2004). The suggested antimicrobial dosage for the pigeon is 150 to 200 mg/L of drinking water (Harrison et al., 1994) or 15 to 20 mg/kg of BW and could be given in breeding and racing seasons (Talaber, 2004). Amoxicillin is absorbed quickly from the gut after oral administration; its bioavailability ranges from 50 to 80% (Dorrestein et al., 1987; Escudero et al., 1998; Soenens et al., 1998). Various methods have been used for the detection of AMX or antibiotics with the β-lactam ring. Many of the methods have been updated recently, including microbiological assays (Popelka et al., 2005; Jiang et al., 2006), spectrophotometry assays (Duan et al., 2005; Li and Lu, 2006), and chromatographic analyses such as capillary electrophoresis (Perez et al., 2007) and HPLC with different detector modes (Cha et al., 2006; Li et al., 2006; Samanidou et al., 2006). However, very few ELISA have been designed specifically for AMX (Hill et al., 1992; Mayorga et al., 2002); most of the antibodies raised for detection of AMX were against various β-lactams and were developed decade(s) ago (Lapresle and Lafaye, 1985; Hill et al., 1992; Klein et al., 1993; Zhang et al., 1996; Benito-Pena et al., 2005). Although each detection method has its own strengths and limitations, the ELISA has the advantage of being simple, sensitive, inexpensive, and capable of detecting metabolites. When a drug is constantly overused, drug resistance may develop, and new drugs similar in structure are synthesized to replace the original drug. Developing a new ELISA is always desirable because each antibody produced against the immunogen may exhibit characteristic spectra of cross-reactivity that could specifically identify certain analogs with improved selectivity and sensitivity.
Microdialysis is an in vivo sampling method that was introduced for continuous monitoring of neurochemical events in the brains of small laboratory animal species (Bito et al., 1966). In view of its capacity to provide a direct measure of the free (unbound) concentration of drugs in extracellular fluid (de la Peña et al., 2000), microdialysis has been used to study the distribution and metabolism of drugs in vivo (Hansen et al., 1999; Chou et al., 2001) and to measure drug binding to serum proteins (Le-Quellec et al., 1994). In theory, dialysate samples may be collected from any tissue on a continual basis from a single animal, thereby reducing both data variability and the required number of experimental subjects. In addition, in a multiple-probe setting, microdialysis allows simultaneous determination and comparison of central and peripheral (tissue) pharmacokinetics (de la Peña et al., 2000). Recently, microdialysis has been increasingly used to investigate the distribution of antibiotics, including β-lactam compounds such as ampicillin, amoxicillin, cefaclor (de la Peña et al., 2001), ceftriaxone (Kovar et al., 1997), cefpodoxime (Liu et al., 2002), cefpirome (Joukhadar et al., 2002), and piperacillin (Tomasselli et al., 2003), in the muscle interstitial fluid of laboratory animals and in human subjects. However, to our knowledge, a proper microdialysis model has not been designed specifically for pharmacokinetic studies in pigeons. The reason microdialysis is increasingly popular in combination with antibiotic studies is that discrepancies usually arise between therapeutic levels of drugs in blood vs. in tissues, where the drugs are most needed to treat the infections. In many cases, the clinical outcome of therapy needs to be determined by the drug concentration in the tissue compartment in which the pharmacological effect occurs, rather than in the plasma. Inadequate tissue penetration of antibiotics can lead to therapeutic failure and bacterial resistance (Brunner et al., 2005). Pharmacokinetic evaluation of antibiotics should therefore include affected tissues rather than only in the serum. Owing to selective access to the target site for most antiinfective drugs, microdialysis provides pivotal information regarding the pharmacokinetic distribution of drugs and might become a reference technique to access the therapeutic effectiveness of an antibiotic drug.
Here we describe a sensitive AMX ELISA that differed in specificity from previously reported immunoassays. In addition, the usefulness of this assay was demonstrated by monitoring immunoreactive AMX levels in the blood and muscles of pigeons with samples obtained from a microdialysis model designed for fowl species.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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AMX ELISA
To establish the assay, AMX-ovalbumin (OVA) conjugate was first prepared as described above for AMX-BSA. Optimal antibody and conjugate concentrations were determined by the checkerboard method (Chard, 1987). In brief, AMX-OVA was diluted (1:500-, 1:1,000-, 1:2,000-, and 1:4,000-fold) with coating buffer (0.5 M carbonate-bicarbonate buffer, pH 9.6). A 96-well polystyrene microtiter plate (Nalge Nunc, Naperville, IL) was coated with 100 µL/well of the diluted AMX-OVA conjugate across rows and kept at 4°C overnight or left to react for 2 h at 37°C. Plates were then rinsed 3 times with 400 µL of PBST (PBS with 0.05% Tween 20). Amoxicillin antibodies to be assayed were diluted (1:200-, 1:400-, 1:800-, 1:1,600-, 1:3,200-, and 1:6,400-fold) with PBS and 100 µL/well of diluted AMX antibody was added to the plate across columns and incubated for 1 h at 37°C. After washing 3 times with PBST, peroxidase conjugated goat-antirabbit IgG antibody (1:2,000 dilution) was added at 100 µL/well and allowed to incubate at 37°C for an additional hour. The plate was again washed 3 times with 400 µL/well of PBST, and 100 µL/well of 3,3',5,5'-tetramethybenzidine (TMB) was added to each well to initiate a color change. The plate was read at 30 min after color initiation. It was determined that this was the optimal length of time to allow the color formation reaction to reach equilibrium, eliminating the need for an acid solution to stop the process. A Dynex MRX microplate reader (Dynex Technologies, Billingshurst, West Sussex, UK) was used to measure optical density (OD) at 650 nm. The antibody and AMX-OVA combination that gave rise to an OD of 1.5 was chosen as the optimal total binding. Chemicals in this section, including OVA, peroxidase conjugated goat-antirabbit IgG, and TMB, were purchased from Sigma Ltd.; Tween 20 was purchased from Merck Co. (Darmstadt, Germany).
ELISA Standard Curve and Cross-Reactivity
To establish the standard curves, microtiter plates were coated with diluted AMX-OVA (1:1,000) and washed as described above. Amoxicillin stock standards (10 concentrations at 500, 100, 20, and 4 µg/mL; 800, 160, 32, 6.4, and 1.28 ng/mL; and 256 pg/mL, diluted in PBS, milk, normal bovine serum, and normal bovine urine) were added at 50 µL to each well containing 50 µL of diluted AMX antibody (1:1,600). Plates were then incubated at 37°C for 1 h and washed 3 times with 400 µof PBST, followed by addition of peroxidase conjugated goat-antirabbit IgG antibody (1:2,000 dilution) as described previously for the checkerboard method and allowed to incubate at 37°C for 1 h. The plate was then washed again with 400 µof PBST, and 100 µ/well of TMB was added to each well to initiate a color change, which was read at 30 min by an ELISA reader measuring OD at 650 nm. Immunologic cross-reactivity between AMX and 9 pharmacologically or structurally related analogs [ampicillin, 6-aminopenillinic acid (6-APA), carbenicillin, cloxacillin, dicloxacillin, oxacillin, penicillin G, cefadroxil, and cefazolin] was assessed by the ability of the test compounds to inhibit the reaction between the AMX-OVA and anti-AMX antibody. This was performed by substituting various concentrations of the test analogs (2 mg/mL; 200, 20 and 2 µg/mL; 200, 20 and 2 ng/mL; and 200 pg/mL diluted in PBS) for the AMX standards in the above ELISA procedure. Standard curves for AMX and the various analogs were generated and the cross-reactivity was determined by averaging the calculated IC25, IC50, and IC75 (concentration at which 25, 50, 75% of AMX-OVA binding was inhibited) over the AMX concentration required to achieve an equivalent inhibition (IC25/50/75 drug/IC25/50/75 AMX).
The sensitivity of the assay was determined according to Hayashi et al. (2004). The lowest concentration in the projected standard curve that showed less than 30% variation was determined as the limit of detection (sensitivity) of this assay. To assess the accuracy of the assay, the established ELISA was used to determine AMX at 2 nominal concentrations (10 µg/mL and 800 ng/mL) in PBS, bovine serum, urine, and milk. The accuracy was represented by the recovery calculated by the equation: Recovery (%) = (estimated concentration/nominal concentration) x 100%. Intra- and interday variations were assessed at 5 concentrations (10, 1, 0.1, 0.01, and 0.001 µg/mL). All experiments were performed in triplicate for 5 plates.
Microdialysis Apparatus and Sample Collection
The microdialysis system was assembled entirely from commercially available equipment and supplies (Figure 1
). Linear-type microdialysis probes with a 10-mm membrane length (LM-10, molecular weight cut-off = 6 kDa, Bioanalytical System Inc., Lafayette, IN) were used in all subjects. Following implantation of the probes (see below) in the left pectoral and femoral muscles, each probe was connected to a gas-tight microliter syringe (1 mL) with small-bore polyethylene tubing (0.12 mm i.d., CMA Microdialysis Inc., Stockholm, Sweden). Probes were perfused independently with sterile 0.9% (wt/vol) sodium chloride solution (normal saline, Tai-Yu Chemical and Pharmaceutical Co. Ltd., Tao-Yan, Taiwan) by battery-powered syringe pumps (801, Univentor Ltd., Zejtun, Malta). Following the initial wash-out period of 30 min at 5 µL/min, probes were perfused at a flow rate of 1 µ/min throughout the experiment and presterilized polyethylene tubing (1 m) was used to connect the lines of each probe to a collection vial. A quantity of 25 mg/kg of AMX was administered into the right femoral muscles. Dialysate fractions were collected at preset intervals (10, 20, 30, and 60 min) for 12 h in glass microvials (300-µL capacity) after AMX administration. The delay time associated with dead volume of the tubing was determined and accounted for in all data calculations. Blood samples (50 µL) were collected in parallel to the collection of dialysate via intermittent venipuncture at 10, 20, 30, 40, and 50 min, and at 1, 2, 3, 4, 5, 6, 7, and 8 h post-AMX injection. Blood samples were diluted 10-fold to obtain enough volume for triplicate wells in the assay and for accurate conversion of the drug concentration using the "middle" linear range of the standard curve. Microvials containing dialysate samples were covered tightly and stored at 4°C before analysis on the following day.
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). Inlet and outlet lines were connected, and the probe was flushed as described above. Following implantation, the anesthetization was terminated and microdialysis probes were perfused with sterile saline for a minimum of 1 h to ensure equilibration before AMX administration.
Evaluation of the In Vitro Recovery and AMX Protein Binding
The in vitro recovery of AMX was determined at 3 concentrations (1 µg/mL, 500 ng/mL, and 100 ng/mL in normal saline) and 3 flow rates (0.5, 1, and 2 µL/min) for the LM-10 probe. The probe was immersed in AMX solution in a beaker and perfused with normal saline. The recovery was calculated by dividing the AMX concentration in the perfusate, determined by the developed ELISA, over the designated AMX concentration in the beaker. A brief study was carried out to delineate the extent of AMX binding to pigeon serum proteins. Amoxicillin was spiked in the fresh pigeon serum at a final concentration of 1 µg/mL and incubated (12 h at 4°C) before the estimation of free drug concentrations (CF) by microdialysis at a flow rate of 1µL/min. The percentage of drug bound to protein (B) was calculated from the relationship:
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where CT is the total concentration of drug in serum (1 µg/mL) and CF is the drug concentration in dialysate divided by fractional drug recovery.
Pharmacokinetic Analyses and Statistics
Pharmacokinetic parameters were determined for AMX in the blood and the pectoral and femoral muscles of the pigeon by using commercialized pharmacokinetic-pharmacodynamic modeling software (WinNonLin version 1.1, Pharsight Corp., Mountain View, CA). Compartmental analysis, which assumes the existence of first-order kinetics, was used to fit data for drug concentrations in blood and muscles vs. time. Data were evaluated for statistical differences between the 2 sampling methods and between pectoral and femoral muscles by using Student’s t-test. A P-value of <0.05 was accepted as a statistically significant difference.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and the working ranges of the assay spanned 4 orders of magnitude (Table 1 and Figure 2). Addition of normal bovine serum, urine, and milk caused no interference; however, addition of normal bovine serum and milk caused a slight left shift in the standard curve (Figure 2). Because the slope of the standard curve was not significantly affected by drug-free urine, serum, or milk, it was concluded that the assay was suitable for detection of AMX immunoreactivity in urine, serum, and milk samples.
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). The accuracy of the assay was expressed by recovery (%). The mean recoveries of AMX in different matrices ranged from 92.7 ±5.6% in serum to 109.8 ±6.4% in milk (Table 3).
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. Using this ELISA, we determined that the anti-AMX antibody cross-reacted highly with penicillin G (77.39%), cross-reacted moderately with ampicillin, oxacillin, and cloxacillin (56.87, 51.41, and 48.79%, respectively), and cross-reacted minimally with dicloxacillin (7.4%). Cefadroxil and cefazolin had insignificant (<1%) cross-reactivity with the developed AMX antibody. The chemical structures of AMX and its analogs and their cross-reactivities are summarized in Table 4 and Figure 4.
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. The recoveries were found to be inversely related to the flow rate but remained relatively constant over 3 different concentrations. The optimal flow rate of 1 µL/min was chosen thereafter in view of its acceptable combination of recovery and sample volume. In addition, a preliminary study (data not shown) confirmed that drug passage through the dialysis membrane exhibited bidirectional symmetry, such that the fraction of drug recovered from an isotonic medium (recovery mode) was equivalent to the fraction of drug that diffused from the perfusion fluid into the external drug-free medium (delivery mode). This result provided a rational basis for using the delivery method as a way to measure AMX recovery in vivo for microdialysis probes placed within the pectoral and femoral muscles of pigeons. The brief study that was carried out to delineate the extent of AMX binding to pigeon serum protein indicated that the binding was not high, at 27.9 ±5.7%.
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. The levels of AMX in intermittent venipuncture exhibited a quick rise to a peak concentration of 4.74 µg/mL approximately 30 min after injection. The peak concentrations of AMX in pectoral (0.97 µ/mL) and femoral (1.6 µg/mL) muscles occurred later, at approximately 60 min. Concentrations of AMX in both the pectoral and femoral muscles appeared to decrease gradually to a concentration of 0.3 to 0.5 µ/mL during the subsequent 8-h monitoring period. Amoxicillin achieved peak concentrations in muscles that were significantly lower than the peak AMX concentration in venous blood. Pharmacokinetic analysis of AMX concentrations in the muscles revealed statistically significant differences in the apparent half-life (t
), peak time (Tmax), and peak concentration (Cmax) between the 2 muscle locations and from those obtained in blood (Table 6). The lowest blood concentration of AMX (at 8 h) remained above 0.6 µg/mL, indicating that a 10-fold dilution was appropriate to maintain an accurate concentration conversion because the diluted, lowest concentration (60 ng/mL) was well within the linear working range of the standard curve (Figure 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and AMX at low concentrations could be successfully detected in urine, serum, and milk samples. The broad spectrum of drugs detected and the high sensitivity made this method particularly useful as a screening technique for AMX in biological fluids.
The selectivity of an ELISA is characteristic of the specific antibody raised against the target compounds and is therefore of great importance insofar as it reflects the nature of the substances that are able to be detected by ELISA tests. On the basis of our results, it appeared that the developed polyclonal AMX antibody recognized some key multiple epitopes on the penicillin molecules (Table 4 and Figure 4). For instance, ampicillin, which differed in only 1 oxygen molecule at the R1 substituent with AMX, shared only 56.87% cross-reactivity with AMX, suggesting that the R1 position was a critical determinant for antibody recognition. On the other hand, the R4 position showed significant but differential effects in cross-reactivity based on the substitution group. Penicillin G, oxacillin, cloxacillin, and carbenicillin, which also had 1 hydrogen substitute at the R1 position like ampicillin but differed in the R4 position, had variable cross-reactivities, ranging from 77 to 36%. The significant cross-reactivity drop of dicloxacillin from oxacillin and cloxacillin suggested that the R2 substituent with a chloride significantly reduced the antibody recognition (from 49% to 7%), whereas the R3 substituent of the same molecule (chloride) posed virtually no change to the cross-reactivity (Table 4 and Figure 4). These results pointed to a greater recognition of the R1 and R2 groups for this antibody in terms of cross-reactivity to AMX. Although cefadroxil, the first generation of cephalosporin, shared identical groups of R1, R2, R3, and R4 with AMX, the 6-membered dihydrothiazine ring (instead of the thiazolidine ring in penicillins) that diffused with the β-lactam ring resulted in a significantly reduced cross-reactivity (to only <1 %). A similar result was found with cefazolin, which could lead to the conclusion that first-generation cephalosporins should exhibit low cross-reactivity to this AMX antibody because of replacement of the thiazolidine ring. Although other newer generation cephalosporins were not tested in this study, on the basis of the current analysis of structure-reactivity, it seemed plausible that drugs such as ceftiofur, cephalothin, or cephaloridine, which all have a significant modification at the R1 position and replacement of the thiazolidine ring, should have low cross-reactivity to the developed AMX antibody, making this ELISA comparatively selective to the most often used β-lactams. Nevertheless, it should be noted that the overall cross-reactivity is the result of multiple antibody recognition at multiple sites with different cross-reactive significances. Therefore, it may not be possible to explain or predict all cross-reactivities by substitutions at 1 or 2 sites. Nevertheless, compared with the 2 previously developed antibodies against AMX, both studies failed to provide suitable characterization of their antibodies in terms of the cross-reactivity to structurally similar β-lactam analogs. For those antibodies produced against ampicillin, cloxacillin, and 6-APA that also cross-reacted to AMX, their cross-reactivity patterns were very different from ours. For example, the ampicillin-KLH antibody (Dietrich et al., 1998), although detecting AMX completely (108% at more than 100% cross-reactivity), detected penicillin G (30% cross-reactivity) and oxacillin (14% cross-reactivity) at much lower sensitivities; the cloxacillin-HSA antibody (Usleber et al., 1994) cross-reacted only to oxacillin and dicloxacillin, but not to other β-lactams; and the 6-APA-KLH antibody (Benito-Pena et al., 2005) detected AMX and oxacillin-like compounds at much lower sensitivities (2 to 12% cross-reactivity). These comparisons indicate that the antibody raised and the ELISA developed here exhibited great sensitivity and a characteristic spectrum of selectivity, and therefore represented a novel addition to the existing antibody-based assays for semiquantitative analysis of AMX and certain β-lactams.
Although microdialysis is a well-established technique, its application has been mostly focused on laboratory animals and mammals. Very few studies have used microdialysis to evaluate muscle or other tissue concentrations of AMX until recently (Marchand et al., 2005; Sawchuk et al., 2005). This study described and validated the first microdialysis model for continuous sampling of drugs in vivo from the soft tissue (skeletal muscles) of pigeons. The capacity to measure levels of drugs and drug metabolites simultaneously by in vivo microdialysis in multiple tissues afforded investigators with a powerful and less invasive approach for conducting pharmacokinetic studies in pigeons and possibly other poultry species. In the present study, the pigeon was injected with a dose (25 mg/kg, i.m.) of AMX, and drug concentrations in the blood were measured by the developed ELISA. In view of the semiquantitative nature of ELISA and the small number of animals used, pharmacokinetic analysis of the drug in the pigeon was not the main purpose of the study. Nevertheless, estimates of pharmacokinetic parameters and the peak blood concentration and time (5 µg/mL at 20 to 30 min) agreed closely with results (5.8 µg/mL at 24 min) published previously for pigeons after i.m. injection of AMX at the same 25 mg/kg dosage (Escudero et al., 1998). At a higher i.m. dosage (100 mg/kg), a higher peak concentration (28.8 µg/mL) at a similar peak time (30 min; Dorrestein et al., 1987) or a similar peak concentration (5.04 µ/mL) at a slightly delayed peak time (54 min) after an oily suspension (Dorrestein et al., 1986) were reported previously. Both of these agreed reasonably well with the current results because a higher peak concentration from a higher dosage and a reduced peak concentration or delayed peak time from an oily suspension were expected in those situations. On the other hand, the serum elimination half-life (t1/2) of 2.3 h in this study was longer than the 1.5 h in the study by Escudero et al. (1998) and the 0.55 h in other i.m. studies using a higher dosage (100 mg/kg). The difference in elimination half-life could be partially explained by the difference in dosages or dosage formations (Dorrestein et al., 1986) and the different models used for pharmacokinetic analysis. A terminal β t1/2 could be longer than if the elimination were to be presented as a single t1/2, which discounted the distribution phase ( t1/2). Although it has been suggested that the number of animals needed in preclinical pharmacokinetic studies can be substantially reduced by applying the microdialysis technique (Fettweis and Borlak, 1996; de la Peña et al., 2000), larger sample sizes are always desirable to further validate these similarities and differences.
To our knowledge, tissue pharmacokinetic data of AMX were not available for the pigeon and were also rare for avian species. Therefore, despite small sample sizes, the muscle kinetic data in the current study represented 1 of very few studies available regarding the disposition and elimination of AMX in pigeon tissue and could be a useful reference for other avian species. In view of the limited published information, tissue pharmacokinetic comparisons based on a similar dosage and administration route were difficult. After daily oral applications of AMX (20 mg/kg) to chickens for 6 d, Lashev and Semerdzhiev (1983) suggested that tissue levels of AMX did not surpass the serum levels and that the therapeutic levels of AMX were retained better in the liver, kidney, lungs, and muscles than in the spleen, heart, and brain. After 4 consecutive daily subcutaneous injections of AMX (20 mg/kg) to turkeys, AMX concentrations were greatest in muscle 1 d after treatment ceased, with a subsequent rapid decline. The drug was undetectable in the liver and kidney 10 d after the final dosing (Tomasi et al., 1996). The available results, although fragmented, suggested good tissue (muscle) penetration of AMX in avian species and shorter muscle residual time than in the liver and kidney. In the current study, AMX concentrations rose quickly in blood following i.m. administration of 25 mg/kg of AMX, whereas muscle concentrations of AMX exhibited significant delays (at 60 min) and lower peak concentrations (0.9 µg/mL in pectoral muscle and 1.6 µg/mL in femoral muscle; Figure 5). The tissue penetration and distribution of antibiotics is of great importance, because many infections occur in the tissue. Amoxicillin concentrations obtained from femoral muscle were significantly higher than concentrations in pectoral muscle. The apparent half-lives of AMX in pectoral and femoral muscles were increased by 26 and 71%, respectively. A significant pharmacokinetic difference was found between the 2 muscle sites (Table 6). A potential explanation for the plasma-to-tissue and femoral-to-pectoral gradient of AMX was that for certain analytes, unrestricted diffusion across capillaries cannot be taken for granted. Physiological factors that determine blood-to-interstitium transfer, such as local blood flow, local capillary density, and capillary permeability, may all have influences. Site-related differences in probe recovery should always be considered (Chou et al., 2001). In addition, local changes secondary to implantation of the probe could promote unequally elevated drug concentrations in muscle extracellular fluids. Placement of the probe within the muscle could cause local irritation and inflammation that change capillary permeability or the muscle microenvironment, leading to altered drug delivery or retention. Although the sample size in the current study was rather small and the basis for the differences between the 2 muscle sites is unknown, our results underscored the importance of monitoring drug levels directly at the potential tissue sites of action. Measurement of AMX concentration in muscle tissue may be necessary to provide better insight into the overall therapeutic effectiveness of this drug and its residual period. In developing the in vivo microdialysis model, its suitability to carry out measurements of AMX in pigeon skeletal muscles was tested. The pectoral and femoral muscles were chosen in an attempt to simulate the study of the 2 most frequently edible tissues in avian species. Anatomical factors also supported selection of these muscles, including the parallel orientation of myofibrils as well as the superficial location of the muscle, which facilitates reliable straightforward insertion (suturing) of the probe. A previous study suggested that many technical problems (reduced dialysate flow, probe breaks, etc.) could be avoided if probes were inserted parallel rather than orthogonal to the orientation of the muscle myofibrils (Chou et al., 2001). Although the reason for this difference is not entirely clear, we assume that probes oriented parallel to the myofibril long axis experience less physical stress associated with changes in muscle length. On the basis of these findings, we concluded that the microdialysis procedure described here represents a suitable method for the performance of pharmacokinetic studies in poultry species. Furthermore, microdialysis-based measurements offer a distinct advantage over traditional venipuncture techniques insofar as interactions with the experimental subject are greatly reduced and multiple tissues may be sampled simultaneously. These advantages could be truly significant for studies designed to elucidate effects wherein interactions between the experimenter and experimental subjects must be minimized. In theory, dialysate samples may be collected from any tissue on a continual basis from a single animal, thereby reducing both data variability and the required number of experimental subjects while maintaining satisfactory statistical significance. This is especially desirable for studies in rare animal species. Because of the fragile nature and small diameter of pigeon blood vessels, microdialysis in the blood was not performed. Although the current design prevented us from direct assessment of free AMX in the blood, it should be noted that in theory, significant differences between direct venipuncture and blood microdialysis are likely with a drug that is highly bound to plasma proteins. In such cases, the free drug concentration in the microdialysate would be expected to differ substantially from the total drug concentration in venipuncture samples because of the inability of bound drug molecules to cross the dialysis membrane (Bailey, 1998; Chou et al., 2001). Our protein-binding study indicated that AMX is not highly bound to pigeon plasma proteins (28%); therefore, it is more likely that the AMX concentration-time curves by microdialysis would be similar to the current results from venipuncture.
In conclusion, an immunoassay was developed for the detection of AMX as well as other β-lactam analogs in serum, urine, and milk samples. The ELISA exhibited great sensitivity and a characteristic spectrum of selectivity. In addition to AMX detection, we developed and validated a reliable in vivo sampling technique for pigeons based on tissue microdialysis. Results from this investigation demonstrated the practicality of using the first in vivo microdialysis in pigeons and revealed significant time-dependent differences in the free concentrations and pharmacokinetics of AMX in skeletal muscles and in the blood. The procedure allowed for continuous measurement of free AMX levels in soft tissues and underscored the advantages of this method for evaluating drug kinetics in muscles. This approach should be readily adaptable for use in other fowl species and could improve our understanding of clinical pharmacokinetics for effective therapy as well as for the capacity to estimate drug residues in specific tissues of interest.
Received for publication April 23, 2007. Accepted for publication December 4, 2007.
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