a Department of Internal Medicine II, Pulmonary Centre, Vienna; b Department of Pulmonary Medicine, University Hospital of Vienna; c Ludwig Boltzmann Institute for Cardiothoracic Surgery Research at the Institute of Laboratory Diagnostics, Kaiser-Franz-Josef-Spital, Vienna, Austria
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Abstract |
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Introduction |
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Materials and methods |
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Endothelial cells were prepared using human umbilical veins from umbilical cords recovered with verbal informed consent. Cells were isolated and cultured according to a modified standard procedure.4 Briefly, fresh human umbilical veins were filled with 0.1% collagenase solution and incubated at 37°C. The veins were then perfused with medium 199 (Sigma, St Louis, MO, USA) containing 20% bovine calf serum (HyClone, Road Logan, UT, USA). Cells were collected from the perfusate by centrifugation (300g, 4°C) and seeded into culture T-75 flasks precoated with human fibronectin (Upstate Biotechnology Inc., Lake Placid, NY, USA). Cells were cultured in medium 199 containing 20% bovine calf serum, 50 000 U/L penicillin streptomycin (Gibco, Paisley, UK), 50 mg low molecular weight heparin (Sigma) and 15 mg/L H-Neurext (endothelial cell growth supplement; Upstate Biotechnology Inc.). The confluent primary monolayers (c. 8 000 000 cells/flask) were washed and trypsinized. The cell suspensions were transferred into each well of a six well culture plate and cultivated for 4 days. Only cells from these first subcultures were used for the experiments. We used uninfected HUVECs because the causative microorganisms do not usually play a significant role at the site of antibiotic infusions.
Antibiotics
The commercially available preparations of erythromycin (Abbott, Chicago, IL, USA) and azithromycin (Pfizer Inc., New York, NY, USA) were dissolved in 10 mL water for injection and diluted further with 0.9% NaCl.
Incubation with azithromycin and erythromycin
For the experiments the culture medium was removed and the cell layers were washed gently with Dulbecco':s phosphate-buffered saline (Gibco). Thereafter, azithromycin and erythromycin solutions at a concentration of 2, 1 and 0.5 mg/mL were added to the endothelial cells and incubated for 20 or 60 min. Control experiments were performed using 0.9% NaCl. All incubations were carried out in a humidified incubator at 37°C and 5% CO2.
Determination of high-energy phosphates
The energy-rich phosphates were measured by means of HPLC.5 ATP, ADP and GTP were separated by injecting 100 µL of the neutralized supernatant on to a CNU-010 column (Chemcon, Vienna, Austria) using a KH2PO4 gradient. Buffer A consisted of 0.015 mol/L KH2PO4 (pH 3.45), and buffer B of 0.5 mol/L KH2PO4 (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 made at a wavelength of 254 nm.
The amount of formed ATP, ADP and GTP was determined by the ratio of its peak area in relation to the corresponding standards measured under the same conditions. The linear range for all three nucleotides was between 0.75 and 30 µmol/L. The results are given as nmol/million cells.
Statistical analysis
Data from eight different experiments are expressed as mean ± s.d. The statistical significance was determined by means of MannWhitney U-test. P < 0.001 (*) was considered to be significant.
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Results |
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A 60 min incubation of cells with 2 mg/mL azithromycin or erythromycin resulted in a rapid decrease of intracellular ATP to 4.1 ± 0.3 and 2.6 ± 0.4 nmol/million cells, respectively (Table). In addition, ADP was significantly decreased indicating a depletion of the ATP/creatine phosphate (CP) regeneration system.
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Effects of 1 mg/mL azithromycin and erythromycin on HUVEC
After incubation with 1 mg/mL azithromycin or erythromycin, the decrease of ATP/ADP and GTP levels after 60 min was less pronounced (Table). Intracellular high-energy triphosphates and the corresponding diphosphates did not decline after 20 min.
Effects of 0.5 mg/mL azithromycin and erythromycin on HUVEC
Purine nucleotide profiles were not affected during exposure to 0.5 mg/mL azithromycin or erythromycin. Even after 60 min of exposure ATP, ADP and GTP levels showed no significant difference compared with controls, indicating that no cellular damage occurred.
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Discussion |
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To investigate cellular impairment in response to azithromycin and erythromycin, intracellular purine content was measured after incubation with different concentrations of these macrolides. As shown in the Table, 1 mg/mL azithromycin and erythromycin led to an ATP decline compared with untreated HUVEC. This decrease, when combined with the data given in the Table
, can be considered as reversible since ATP levels can be restored by the action of creatine kinase using ADP as substrate and CP as phosphate donor. However, severe ATP depletion is irreversible and leads to endothelial cell death.9 Azithromycin and erythromycin at a dose of 2 mg/mL result in an ATP depletion that does not seem to be reversible via the CP/ADP regeneration system because of the strong decrease of intracellular ADP. The depletion of intracellular GTP might support this consideration and reflect a functional and structural alteration. A dilution to 0.5 mg/mL rendered the solutions more compatible to HUVEC and showed no significant difference in the purine nucleotide profiles compared with controls.
These data are in line with clinical observations that the occurrence of phlebitis could be reduced by diluting the commercially available preparations of erythromycin to a final concentration of 1 mg/mL.2,10 The importance of this in vitro study should be underlined, since no data exist on the influence of azithromycin on human endothelial cells.
In conclusion, based on our data we would like to draw clinicians' attention to the possibility that solutions of these macrolides may not be well tolerated, even if diluted according to the manufacturer's recommendation.
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Acknowledgements |
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Notes |
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References |
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Received 27 July 2001; returned 5 October 2001; revised 24 October 2001; accepted 26 October 2001