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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Commercial preparations of clindamycin (Upjohn, Crawley, UK), gentamicin (SP Labo, Heist, Belgium) and ceftriaxone (HoffmannLa 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 MannWhitney U-test. P < 0.01 was considered to be significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Langer, K., Seidler, C. & Partsch, H. (1996). Ultrastructural study of the dermal microvasculature in patients undergoing retrograde intravenous pressure infusions. Dermatology 192, 1039.[ISI][Medline]
3 . Robibaro, B., Vorbach, H., Weigel, G., Weihs, A., Hlousek, M., Presterl, E. et al. (1998). Endothelial cell compatibility of glycopeptide antibiotics for intravenous use. Journal of Antimicrobial Chemotherapy 41, 297300.
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, 2358.[ISI][Medline]
5 . Griesmacher, A., Weigel, G., David, M., Schimke, I. & Mueller M. M. (1992). Influence of oxygen radical generating agents on eicosanoid metabolism of human endothelial cells. Thrombosis Research 65, 72131.[ISI][Medline]
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, 28192.[Medline]
7 . Halliwell, B. & Gutteridge, J. M. (1984). Oxygen toxicity, oxygen radicals, transition metals and disease. Biochemistry Journal 219, 114.[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, 274556.[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 , 275764.[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, 23127.
Received 11 October 1998; returned 11 January 1999; revised 16 February 1999; accepted 12 April 1999