1 Institute of Experimental Clinical Research and 2 Department of Clinical Microbiology, Aarhus University Hospital, Skejby Hospital, Brendstrupgaardvej, 8200 Aarhus; 3 Department of Orthopaedic Surgery, Aarhus University Hospital, Aarhus Hospitals, Nørrebrogade 44, 8000 Aarhus C; 4 Department of Orthopaedic Surgery, Esbjerg/Varde Hospital, Østergade 8, 6700 Esbjerg, Denmark
Received 2 January 2004; returned 8 March 2004; revised 26 March 2004; accepted 14 April 2004
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: After two microdialysis catheters had been inserted into cancellous bone, eight pigs received an intravenous bolus of 240 mg of gentamicin. Microdialysates and bone samples were obtained over a period of 6 h and drug concentrations were measured.
Results: The area under the curves of the two microdialysates and bone samples were 1569, 1721 and 1533 mg·min/L (ANOVA, P=0.81). Reproducibility of the measurements from the microdialysates was defined as the mean ratio of AUC6/catheter no. 1/AUC6/catheter no. 2. This ratio was 1.02.
Conclusions: Microdialysis is a suitable, relatively non-invasive and reproducible technique for dynamic and quantitative measurement of gentamicin levels in experimental research.
Keywords: experimental research , bioassays , bone samples , pharmacokinetics
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For decades, pharmacokinetics in bone tissue was investigated through analysis of bone samples or exudates.2,3 Microdialysis, which is minimally invasive, is a technique that allows dynamic and continuous in vivo sampling. The principle is to introduce a semi-permeable membrane into the tissue of choice. The membrane is perfused with a liquid that equilibrates with the fluid outside the membrane by diffusion in both directions, thus enabling dynamic measurements to be made.4 Recently, the distribution of gentamicin in cortical bone was investigated and no differences were found between values obtained from bone samples and microdialysates.5
The aim of this study was to introduce microdialysis to cancellous bone tissue for the measurement of gentamicin and compare the results to values obtained from bone samples.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Eight pigs [females, Danish Landrace Breed, 46.4 kg (range 4248)] were included in the study. All animals underwent surgery under general anaesthesia.5
Operative technique
Two holes with a diameter of 1.1 mm and depth of 15 mm were drilled at an angle of 90° into cancellous bone in the right tibia, and the microdialysis catheters were inserted into the channels. No post-operative exudation was observed after 1 h. In order to let the tissue recover from insertion trauma, a period of 1 h was allowed before starting the experiment. The positions of the catheters were controlled by autopsy.
Drugs
All animals received a bolus of 240 mg gentamicin (Garamycin; Schering-Plough A/S) intravenously. Gentamicin was chosen because of its low protein binding, and it has recently been investigated by microdialysis.57
Microdialysis equipment
CMA 70 Microdialysis Brain Catheters (CMA/Microdialysis, Solna, Sweden) and CMA 107 Microdialysis Infusion Pumps (CMA/Microdialysis) were used in this study. Flow rates of the pumps were 1 µL/min and isotonic saline with 1% albumin was chosen as perfusate.
Calibration of the probe
In vivo relative recovery of gentamicin was attained according to retrodialysis.5 For this, two premanufactured concentrations of gentamicin (4.4 and 8.7 mg/L) plus 1% albumin were added to the perfusate. The in vivo relative recovery was calculated as follows: RR = 1 (Cout/Cin) where Cout is the outlet concentration (mg/L) and Cin is the inlet concentration (mg/L) of gentamicin. The tissue concentration was defined as: Ctissue=100 x Cout x RR1.
Dialysates were collected at 30 min and every hour until 6.5 h. Since dialysates are time averaged over the collection interval, these values were translated into concentrations at a single point by assuming that the concentration obtained is the actual concentration at the mid-point of the time interval.
Tissue and serum samples
Bone samples were taken from the left tibia with a Coombs Bone Drill. The drill was placed perpendicular to the bone and standardized samples of 4 mm diameter and 5 mm height were obtained, weighing 54.1 mg (3.1). Periosteum and cortical bone were removed from all samples. Blood was collected from a sheath placed in the external jugular vein. All samples were collected at 15 min and every hour until 6 h. Samples were immediately frozen to 80°C.
Assays of bone specimens
Concentrations of gentamicin in bone biopsies were measured by a direct agar diffusion technique using Staphylococcus epidermidis ATCC 12228.8,9 Standard curves were constructed by adding known amounts of gentamicin to freeze-dried bone biopsies, dry weight 33.2 mg (1.9) free of antibiotics. One standard curve was constructed from means ±S.E.M. of four parallel determinations (r=0.98, P<0.001) (Figure 1).
|
All microdialysates and serum samples were analysed on a commercial Abbott Drug Analyser.
Pharmacokinetic data and statistics
Data presented are means (S.E.M.). Paired t-test was carried out for comparison of values obtained from the two microdialysis catheters. One-way analysis of variance (ANOVA) was carried out for comparison of microdialysates, bone samples and serum values of gentamicin. A P value below 0.05 was considered significant. The pharmacokinetic measure used was the area under the curve from 0 to 6 h (AUC6) and was calculated by the trapezoid method. The tissue penetration was defined as AUCtissue/AUCserum.
Reproducibility
Reproducibility was evaluated as intra-animal variability, which was expressed by the AUC6/medial probe/AUC6/lateral probe ratio.
Ethics
All surgical procedures were carried out under the approval and guidelines of the Danish Ministry of Justice, Animal Experimentation Inspectorate.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The peak concentration was 33 mg/L (2.6). The AUC6 was 3357 mg·min/L (212). Six hours after the bolus of gentamicin, the concentration (C6) was 3.3 mg/L (0.3).
Bone sample kinetics
Peak concentration was 6.9 mg/L (1.8) and C6 was 2.0 mg/L (1.4) (Figure 2). The AUC6 was 1533 mg·min/L (198). The tissue penetration was 0.47 (0.07).
|
In vivo relative recoveries of gentamicin were 39.6% (3.7) and 41.5% (3.1) for the medial and lateral catheters, respectively (P=0.78).
Microdialysate kinetics
Peak concentrations for the medial (mc) and lateral (lc) catheters were 6.7 mg/L (0.8) and 6.5 mg/L (1.1), respectively (P=0.66). C6,mc was 3.8 mg/L (0.6) and C6,lc was 3.5 mg/L (0.4) (P=0.52). The AUC6,mc was 1569 mg·min/L (198) and AUC6,lc was 1721 mg·min/L (248) (P=0.64). The tissue penetration of the medial and lateral catheter was 0.48 (0.07) and 0.54 (0.1), respectively (P=0.61). Reproducibility of the measurements was 1.02 (0.17).
The AUC6 obtained from the two microdialysates and bone samples did not differ (P=0.81). No differences were found in the tissue penetration obtained from microdialysates and bone samples (P=0.79). The AUC6 of serum was higher compared with microdialysates and bone samples (P=0.001).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
When we compared the area under the curve, values of gentamicin obtained from bone samples and microdialysates did not differ. Furthermore, the reproducibility of the microdialysates was high (AUC6/medial probe/AUC6/lateral probe ratio = 1.02). This indicated that microdialysates were representative of interstitial concentrations. Our results seemed more convincing compared with results obtained by application of microdialysis to cortical bone for measurements of gentamicin. The AUC6 values obtained from microdialysates and bone samples slightly differed (P=0.07) in cortical bone and the reproducibility was much lower (AUC6/medial probe/AUC6/lateral probe ratio = 1.12).5 The pharmacokinetic values obtained were comparable to measurements of gentamicin in subcutaneous tissue in humans.6,7 One explanation could be the high amount of bone marrow and blood inside cancellous bone tissue. Theoretically, we expected a higher flow around the membrane and this made the tissue more sensitive for dialysis. It might explain the high tissue penetration observed and the higher reproducibility found compared to cortical bone tissue.5
The investigation of antibiotics in bone tissue remains a difficult task. Microdialysis has the advantage of permitting continuous sampling on the same individual and allowing measurements of the free, unbound and active concentration of the antibiotic inside the target tissue. Only this free tissue concentration is responsible for effective treatment.4 Microdialysis seems ideal for dynamic measurements of gentamicin in cancellous bone tissue in experimental research.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Riegels-Nielsen, P., Espersen, P., Holmich, L. R. et al. (1995). Collagen with gentamicin for prophylaxis of postoperative infection. Staphylococcus aureus osteomyelitis studied in rabbits. Acta Orthopaedica Scandinavica 66, 6972.
3 . Wahlig, H. & Dingeldein, E. (1980). Antibiotics and bone cements. Experimental and clinical long-term observations. Acta Orthopaedica Scandinavica 51, 4956.
4 . Müller, M. (2000). Microdialysis in clinical drug delivery studies. Advanced Drug Delivery Reviews 45, 25569.[CrossRef][ISI][Medline]
5 . Stolle, L. B., Arpi, M., Holmberg-Jørgensen, P. et al. (2003). In situ gentamicin concentrations in cortical bone tissue: an experimental study using microdialysis in bone. Acta Orthopaedica Scandinavia 74, 6116.[CrossRef]
6 . Müller, M., Schmid, R., Georgopoulos, A. et al. (1995). Application of microdialysis to clinical pharmacokinetics in humans. Clinical Pharmacology and Therapeutics 57, 37180.[ISI][Medline]
7 . Lorentzen, H., Kallehave, F., Kolmos, H. J. et al. (1996). Gentamicin concentrations in human subcutaneous tissue. Antimicrobial Agents and Chemotherapy 40, 17859.[Abstract]
8 . Dornbusch, K. (1978). Antibiotics in bone tissues. Methodological and practical aspects. Scandinavian Journal of Infectious Diseases Supplementum 14, 17785.[Medline]
9 . Georgopoulos, A. (1978). A simple micro agar diffusion method for the determination of antibiotic concentrations in blood and other body fluids. Zentralblatt fur Bakteriologie 242, 38793.[ISI]
10 . Heimdahl, A., Cars, O., Hedberg, M. et al. (1988). A micromethod for determination of antimicrobial agents in bone. Drugs Under Experimental and Clinical Research 14, 64954.[ISI][Medline]
|