Application of microdialysis to cancellous bone tissue for measurement of gentamicin levels

Lars B. Stolle1,*, Magnus Arpi2, Peter Holmberg-Jørgensen3, Per Riegels-Nielsen4 and Johnny Keller3

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives: Knowledge concerning the distribution of antibiotics in bone tissue is valuable for pharmacokinetic and clinical use. Unfortunately, appropriate techniques are difficult to apply. We introduced microdialysis catheters to cancellous bone tissue for the investigation of gentamicin levels and compared the pharmacokinetics measured with values obtained from bone samples.

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Most antibiotics exert their effect inside the interstitial space, which is the site of surgical infections. It is therefore of great clinical importance to obtain the pharmacokinetic profile at this site, since effective treatment to inhibit bacterial growth demands concentrations that exceed the minimum inhibitory concentration.1

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Animals and anaesthetic

Eight pigs [females, Danish Landrace Breed, 46.4 kg (range 42–48)] 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 RR–1.

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).



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Figure 1. The standard curve for the investigation of gentamicin in cancellous bone. Data are presented as means±S.E.M. of four parallel determinations (r=0.98, P < 0.001).

 
Analysis

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Serum kinetics

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).



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Figure 2. Concentrations of gentamicin in cancellous bone. Filled squares, bone sample gentamicin; filled triangles, microdialysate gentamicin from the medial catheter; filled circles, microdialysate gentamicin from the lateral catheter. Points represent means±S.E.M. of eight animals.

 
Calibrations of the microdialysis

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Our main objective was to apply microdialysis to cancellous bone for measurements of gentamicin levels. Valid and reproducible methods for pharmacokinetic measurements in bone tissue are important. Bone samples are static, traumatic to harvest and represent a combination of blood, bone- and fibrous-connective tissue. As a result of ethical considerations, only limited amounts can be harvested from one patient. Finally, tissue homogenization is difficult to carry out and may be misleading.10 We used microdialysis to measure the free concentration of gentamicin in the interstitial fluid. For control of the microdialysis we chose bone samples and analysed them by an agar diffusion assay. The bioassay was preferred because it simplified the handling of bone samples and tissue homogenization was avoided.8,10

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Schering Plough A/S for their supply of gentamicin. We thank the Institute of Experimental Clinical Research, Aarhus University Hospitals, section Skejby Hospital for their financial support of the study.


    Footnotes
 
* Corresponding author. Tel: +45-89495511; Fax: +45-89496011; Email: stolle{at}iekf.au.dk


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Joukhadar, C., Klein, N., Frossard, M. et al. (2001). Angioplasty increases target site concentrations of ciprofloxacin in patients with peripheral arterial occlusive disease. Clinical Pharmacology and Therapeutics 70, 532–9.[ISI][Medline]

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, 69–72.

3 . Wahlig, H. & Dingeldein, E. (1980). Antibiotics and bone cements. Experimental and clinical long-term observations. Acta Orthopaedica Scandinavica 51, 49–56.

4 . Müller, M. (2000). Microdialysis in clinical drug delivery studies. Advanced Drug Delivery Reviews 45, 255–69.[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, 611–6.[CrossRef]

6 . Müller, M., Schmid, R., Georgopoulos, A. et al. (1995). Application of microdialysis to clinical pharmacokinetics in humans. Clinical Pharmacology and Therapeutics 57, 371–80.[ISI][Medline]

7 . Lorentzen, H., Kallehave, F., Kolmos, H. J. et al. (1996). Gentamicin concentrations in human subcutaneous tissue. Antimicrobial Agents and Chemotherapy 40, 1785–9.[Abstract]

8 . Dornbusch, K. (1978). Antibiotics in bone tissues. Methodological and practical aspects. Scandinavian Journal of Infectious Diseases Supplementum 14, 177–85.[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, 387–93.[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, 649–54.[ISI][Medline]





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