Endothelial cell compatibility of clindamycin, gentamicin, ceftriaxone and teicoplanin in Bier's arterial arrest

H. Vorbacha,*, B. Robibarob, C. Armbrustera, M. Attenederd, M. Reiterc, M. Hlousekc, R. Schaumannc and A. Georgopoulosc

a Department of Internal Medicine II, Pulmonary Centre, University Hospital of Vienna, Vienna, Austria b Department of Pulmonary Medicine, University Hospital of Vienna, Vienna, Austria c Department of Infectious Diseases, University Hospital of Vienna, Vienna, Austria d Department of Medical Angiology, University Hospital of Vienna, Vienna, Austria


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In patients with infected diabetic foot lesions, and gangrenous, peripheral, occlusive arterial disease, it is important to achieve high concentrations of antibiotics in the tissues, as the extent of amputation is often influenced by the presence of infection. Local transvenous pressure injection of antibiotics, in Bier's arterial arrest, allows high local tissue concentrations to be attained in the extremities. Information on the endothelial compatibility of antibiotics in high concentrations combined with the effect of reperfusion injury following tissue hypoxia is lacking. To evaluate the effect of clindamycin, gentamicin, ceftriaxone and teicoplanin injected in Bier's arterial arrest, on endothelial cells, an in-vitro model using human umbilical venous endothelial cells (HUVEC) has been devised. The intracellular levels of purine nucleotides, reflecting DNA/RNA synthesis, energy production and signal transduction of these cells were measured by means of high-performance liquid chromatography. Incubation of cells with 10 mg/mL clindamycin, gentamicin, ceftriaxone and teicoplanin for 20 min resulted in no significant decline of intracellular purines. Levels of purines obtained after exposure of the cells to 0.1 mmol/L hydrogen peroxide (H2O2), to simulate reperfusion injury, were not significantly different from those obtained from cells allowed to recover after antibiotic exposure. These findings indicate that the infusion of high doses of antibiotics, during Bier's arterial arrest, is compatible with maintenance of endothelial cell function, even in the presence of increased free radical activity, provided the exposure is limited to 20 min.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the treatment of patients with infected diabetic gangrene and neuropathic foot ulcers, it is important to achieve high tissue concentrations of antibiotics, the extent of amputation often being influenced by the presence of infection. 1,2 The technique of local transvenous pressure injection of antibiotics (Bier's arterial arrest (TVA-Bier)), allows higher local tissue concentrations to be attained than those reached after intravenous or intra-arterial infusions. 1 Burgmann et al. described the procedure in the following manner. After venous puncture at the dorsum pedis, veins are emptied by raising the leg. A tourniquet placed round the thigh, above the knee, is inflated to 300 mm Hg to achieve arterial arrest. A second tourniquet placed above the ankle is inflated to 200 mm Hg to reduce the primary distribution of the injected volume. After lowering the leg to the horizontal, the antibiotic is injected into the vein and arterial arrest maintained for 20 min. 1 In clinical situations in which antibiotics are unable to reach infected diabetic foot ulcers, TVA-Bier has proved an efficient technique. 1,2 There are no data on the endothelial compatibility of antibiotics administered under such conditions. As previously shown for fluoroquinolones and glycopeptide antibiotics, exposure to high concentrations may lead to endothelial cell damage, the extent of which is dependent on dose and time. 3,4 Furthermore, free oxygen radicals, generated during reperfusion after oxygen deficiency, have been reported to have a damaging effect on vascular endothelium. 5,6 In this study, the possible damage to endothelial cells was investigated using H2O2 to model reperfusion injury. Halliwell & Gutteridge 7 described the formation of free oxygen radicals (hydroxyl radical, superoxide radical) by H2O2, via iron-catalysed reactions. To test the tolerance of antibiotics and reactive oxygen species an in-vitro system was devised using human umbilical venous endothelial cells (HUVEC). The effects of clindamycin, gentamicin, ceftriaxone and teicoplanin on tissue cultures was investigated, and reperfusion injury was simulated by subsequent incubation with 0.1 mM H2O2.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Endothelial cells were prepared for tissue culture, using human umbilical veins, by a modified standard procedure. 8,9

Commercial preparations of clindamycin (Upjohn, Crawley, UK), gentamicin (SP Labo, Heist, Belgium) and ceftriaxone (Hoffmann–La Roche, Basel, Switzerland) were diluted with 0.9% NaCl. Teicoplanin (Merrell Dow Pharma GmbH, Rüsselsheim, Germany) was dissolved in water for injection and diluted further with 0.9% NaCl, to yield final concentrations of 10 mg/mL.

Following tissue culture preparation, the culture medium was removed and the cell layers were washed with Dulbecco's phosphate-buffered saline (DPBS) (Gibco, Paisley, UK). Antibiotic solutions (10 mg/mL) were added to the endothelial cells and incubated for 20 min. After removing the antibiotic solutions, the cell layers were washed and incubated with DPBS containing 0.1 mM H2O2 for 60 min. Control cells were pretreated with 0.9% NaCl and subsequently incubated with either 0.1 mM H2O2 or DPBS alone. All incubations were carried out in a humidified incubator at 37°C in 5% CO2.

Energy-rich phosphates were measured by high-performance liquid chromatography (HPLC). 10 Adenosine 5' triphosphate (ATP), adenosine 5' diphosphate (ADP), guanosine 5' triphosphate (GTP) and guanosine 9 diphosphate (GDP) were separated by injecting 100 µL of the neutralized supernatant onto a CNU-010 column (Chemcon, Vienna, Austria) using a KH2PO4 gradient. Buffer A consisted of KH2PO4 0.015 mol/L (pH 3.45), and buffer B of KH2PO4 0.5 mol/L (pH 3.45). A linear gradient rising from 0% B to 100% B in 40 min was used, with a total running time of 60 min and an equilibrium delay of 8 min. The flow rate was 1.2 mL/min and the detection was performed at a wavelength of 254 nm.

Amounts of ATP, ADP, GTP and GDP generated were quantified by determining the ratio of peak areas in relation to corresponding standards. The linear range for all four nucleotides was between 0.75 and 30 µmol/L. Results are expressed as nmol/10 6 cells.

Data are expressed as mean ± S.D. The statistical significance was determined by means of the Mann–Whitney U-test. P < 0.01 was considered to be significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The Table shows the effects of clindamycin, gentamicin, ceftriaxone and teicoplanin solutions at concentrations of 10 mg/mL, after exposure time of 20 min, on intracellular ATP and ADP content. After antibiotic exposure the cells were incubated with PBS or 0.1 mM H2O2 for 60 min. Control cells were treated with 0.9% NaCl, followed by PBS or 0.1 mM H2O2 for 60 min. Levels of purine production indicated that the antibiotic solutions tested were well tolerated in the concentration used, for a treatment period of 20 min. The subsequent addition of 0.1 mM H2O2 did not significantly affect the intracellular purine nucleotide levels (Table). Our results demonstrate that both the total ATP and GTP content remained unchanged, and that the ATP/ADP and GTP/GDP ratio stayed constant, indicating that degradation had not taken place (Table). There was no significant difference between HUVEC preparations allowed to recover for 1 h after the high dose antibiotic treatment and those submitted to subsequent simulation of reperfusion.


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Table. Intracellular purine levels of human umbilical venous endothelial cells, post exposure to antibiotics ± H2O2
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
The use of TVA-Bier has been shown to permit high local antibiotic tissue concentrations to be attained in patients with peripheral occlusive arterial disease or diabetic foot lesions. Burgmann et al. found median local tissue concentration enhancing factors of 18.3 for clindamycin and 9.4 for gentamicin when TVA-Bier was used. (The tissue concentration enhancing factor equals the ratio of tissue concentration of an antibiotic achieved by TVA-Bier to that achieved by iv injection. 1) As aminoglycosides are bactericidal antibiotics, higher concentrations result in faster elimination of the bacterial inoculum, and suppression of local infection.

Use of TVA-Bier could potentiate increased free radical activity. A period of hypoxia is followed by reperfusion, possibly leading to the formation of reactive oxygen species. 7 To study the possible impairment of endothelial cell function in vitro after antibiotic treatment, H2O2 was applied to the tissue culture preparation after removal of the antibiotic solutions. 7 Concentrations of H2O2 of up to 1 mmol/L have been reported, generated by stimulated neutrophils in the immediate vicinity of the endothelium. 5 The Table shows the effects of the antibiotics tested on intracellular ATP and ADP content. Exposure to a concentration of 0.1 mmol H2O2 did not significantly affect intracellular purine nucleotide levels. The detection of intracellular levels of adenine and guanine nucleotides similar to those found in control cells indicated that endothelial metabolism was intact. The importance of the stability of the ATP/ADP ratio should be emphasized. Decreasing ATP levels could be regenerated by the action of creatine kinase, using ADP as a substrate and phosphocreatine as a phosphate donor. As GTP plays an important role in DNA/RNA synthesis, G-protein coupled signal transduction and glycosylation of membrane proteins, both functional and structural alterations of the endothelium seem unlikely.

This study is the first to demonstrate that high doses of antibiotics are compatible with the maintenance of endothelial cell integrity, and that pretreated cells challenged with free oxygen radicals are resistant to oxidative damage. The use of clindamycin, gentamicin, ceftriaxone and teicoplanin, as agents for local transvenous pressure infusion, does not compromise endothelial cell metabolism given an exposure time of 20 min. Maintenance of the integrity of the endothelial cell layer is important, as the loss of the inner stratum of the blood vessel leads to blood coagulation, extravascular migration of leucocytes and uncontrolled proliferation of smooth muscle cells, further impairing the perfusion of the extremity. Our in-vitro data support the clinical usefulness of the TVA-Bier technique.


    Notes
 
* Correspondence address: Department of Internal Medecine II, Pulmonary Centre, Sanatoriumstrasse 2, 1140 Vienna, Austria. Tel: +43-1-91060-42008; Fax. +43-1-9112901; E-mail: vorbach{at}austriaone.at Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Burgmann, H., Georgopoulos, A., Graninger, W., Koppensteiner, R., Maca, T., Minar, E. et al. (1996). Tissue concentration of clindamycin and gentamycin near ischaemic ulcers with transvenous injection in Bier's arterial arrest. Lancet 348, 781–3.[ISI][Medline]

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4 . Vorbach, H., Weigel, G., Robibaro, B., Schaumann, R., Hlousek, M., Beil, B. J. et al. (1997). Endothelial cell compatibility of fluoroquinolone solutions for intravenous use. International Journal of Clinical Pharmacology and Therapeutics 35, 235–8.[ISI][Medline]

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6 . Schimke, I., Griesmacher, A., Weigel, G., Holzhutter, H. G. & Muller, M. M. (1992). Effects of reactive oxygen species on eicosanoid metabolism in human endothelial cells. Prostaglandins 43, 281–92.[Medline]

7 . Halliwell, B. & Gutteridge, J. M. (1984). Oxygen toxicity, oxygen radicals, transition metals and disease. Biochemistry Journal 219, 1–14.[ISI][Medline]

8 . Jaffe, E. A., Nachman, R. L., Becker, C. G. & Minck, C. R. (1973). Culture of human endothelial cells derived from umbilical vein: Identification by morphologic and immunologic criteria. Journal of Clinical Investigation 52, 2745–56.[ISI][Medline]

9 . Jaffe, E. A., Hoyer, L. W. & Nachman, R. L. (1973). Synthesis of antihaemophilic factor antigen by cultured human endothelial cells. Journal of Clinical Investigation52 , 2757–64.[ISI][Medline]

10 . Griesmacher, A., Weigel, G., Seebacher, G. & Muller, M. M. (1997). IMP-dehydrogenase inhibition in human lymphocytes and lymphoblasts by mycophenolic acid and mycophenolic glucuronide. Clinical Chemistry 43, 2312–7.[Abstract/Free Full Text]

Received 11 October 1998; returned 11 January 1999; revised 16 February 1999; accepted 12 April 1999