Aminoglycosides induce fragility of human red cell membrane: an in vitro study.

OBJECTIVES: It is well-known that aminoglycosides are ototoxic and nephrotoxic. Recent advances in pharmacology research suggest that the red cell used as a carrier of aminoglycosides. This study aimed to find the effect of aminoglycosides on the human red cell membrane using osmotic fragility test.
MATERIALS AND METHODS: This study was conducted in Rizgari Teaching Hospital in Erbil, Iraq. The effect of aminoglycosides, namely gentamicin, amikacin, and spectinomycin, on human red cells was investigated. The effects of aminoglycosides were evaluated by osmotic fragility test using fresh human blood in the presence of aminoglycosides in concentrations of 10-160 μg/mL.
RESULTS: The results showed that aminoglycosides drugs shifted the osmotic fragility curve to some extent, and this effect was well observed with spectinomycin. The hemolysis did not depend on the concentration of aminoglycosides. The concentration of sodium chloride to induced 50% hemolysis is higher in presence of gentamicin, amikacin and spectinomycin (at 160 μg/mL) than corresponding control and this account to an increment in hemolysis percents of 1.88, 1.5 and 1.06%, respectively.
CONCLUSION: Aminoglycosides induce human red cell membrane fragility in a concentration-independent manner.

Introduction

Several mechanisms are involved in drugs-induced immune hemolytic anemia.[1] One of these mechanisms is the formation of a trimolecular immune complex. This complex consisted of the drug, red cell membrane antigen, and an antibody (which directed against the antigen formed by a drug and red cell membrane).[1] Such mechanism is reported with beta-lactams including penicillines,[2] and cephalosporins[34] by which these drugs bind firmly with red cell membrane. Immune hemolytic anemia is also caused by other antimicrobials such as tetracyclines.[56] Aminoglycosides exhibit a great efficacy against aerobic gram-negative bacteria. The antibacterial effects of these agents are related to their binding to the 30S ribosomal subunit leading to disruption of protein synthesis. Their toxicity includes ototoxicity, nephrototoxicity and inhibition of the neuromuscular junction that may result in flaccid muscle paralysis. Several mechanisms are linked with these toxicities including: (1) Selective uptake of aminoglycosides by transporter system or by endocytosis,[7] (2) generation of reactive oxygen species,[8] (3) interference with potassium channels activity,[9] and (4) interaction with cations like magnesium and calcium.[10]

Assessment of cell membrane function can be achieved using osmotic fragility test that measures the degree of red cell hemolysis at different sodium concentrations. This test can be utilized to assess the free radical defense mechanism. The peroxidation process of unsaturated bonds of membrane lipids caused by peroxyl radical led to fragility and lysis of red cells.[11]

Cueff et al. demonstrated that the higher intra-red cell concentration of calcium results in a progressive increase in red cell fragility, which indicates that the calcium itself induced red cell membrane lysis.[12]

The aim of this study was to investigate the effect of aminoglycosides; gentamicin, amikacin and spectinomycin on the red cell membrane; to identify aminoglycosides induced red cell hemolysis.

Materials and Methods

The study was conducted in the laboratories of Rizgari Teaching Hospital in Erbil (Hawler), Iraq during 2012. It was approved by the local Scientific Committee of College of Pharmacy at Hawler Medical University. The venous blood samples were obtained from healthy male volunteers (a total number of 12 participants), and a consent was obtained from each individual enrolled in the study. None of them was a smoker or had a history of alcohol intake. The medical history of volunteers revealed no evidence of familial hereditary hemolytic anemia or previous history of acquired hemolytic anemia, hypertension, diabetes mellitus or renal failure. Venous blood samples were obtained and osmotic fragility test was done in the presence of the solvent or the pharmaceutical preparation of aminoglycosides. Three pharmaceutical preparations of each drug, commercially available in vials, namely gentamicin, amikacin and spectinomycin, purchased from the local sources were used in osmotic fragility tests, at final concentrations of 0, 20, 40, 80, 160 μg/mL. The final concentrations of sodium chloride (NaCl) used in osmotic fragility test were 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.75 and 0.85% (w/v) as percent weight to volume ratio. Osmotic fragility test was performed by the method described in laboratory tests (Tietz, 1995) by using the heparinized whole blood samples mixed with increasing concentration of buffered salt solution (NaCl) followed by incubation at room temperature for 30 min. After incubation with drugs or solvent (vehicle), solutions were centrifuged (1000 rpm for 10 min) and the hemoglobin released from the erythrocytes was measured at 540 nm (the constituents of buffered NaCl include; NaCl 90 g; Na2HPO4 13.65 g, NaH2PO4.2H2O 34 g dissolved in 1 L distilled water).

Hemolysis was expressed as a percentage, and 100% hemolysis was determined from the absorbance of the distilled water (0% NaCl). The NaCl concentrations that induce hemolysis in 20% (CH2O), 50% (CH5O) and 80% (CH8O) were calculated from the percentage of hemolysis in buffered salt solutions at various concentrations. The cut-off point that the NaCl concentration did not induce hemolysis (CHO) was also calculated. The osmotic fragility curve was constructed with Microsoft Excel, and the regression equation of the best line was calculated to determine CHO, CH2O, CH5O, and CH8O. The standard deviation for each data point was calculated by using the same program.

Results

Figure 1 shows that gentamicin at 160 μg/mL shifts the osmotic fragility test slightly to the left while at low concentration (at 20 μg/mL) shifts slightly the curve toward the right side. Amikacin shifts the curve of osmotic fragility toward the right side (i.e. hemolysis of red cells at high NaCl concentration compared with control) at 160 μg/mL [Figure 1]. The effect of spectinomycin on the human red cell fragility is more obvious than gentamycin and amikacin. It shifts the osmotic fragility curve toward the right side at higher tested concentrations [Figure 1].

Figure 1

Effects of gentamicin (a), amikacin (b) and spectinomycin (c) on the osmotic fragility of red cells and the cut-off point of sodium chloride that does not induce hemolysis (d)

Table 1 shows that the concentration of NaCl that induced hemolysis in 20, 50 and 80% in presence or absence of cephalosporins. These percents were chosen because they represent the straight line of sigmoid shape of fragility test. The significance of these values was to explore the effective concentration aminoglycosides to induce hemolysis in 50% of red cells and to show whether the hemolysis was concentration-dependent. The results clearly demonstrated that the hemolysis did not depend on the concentration of aminoglycosides. Table 1 shows that the concentration of NaCl to induced 50% hemolysis is higher in presence of gentamicin, amikacin and spectinomycin (at 160 μg/mL) than corresponding control and this account to an increment in hemolysis percents of 1.88, 1.5 and 1.06%, respectively. Figure 1 shows that the cut-off point of NaCl concentration that does not induce hemolysis (CHO) is higher with amikacin compared with gentamicin. Further, it is lower with low concentration of any aminoglycosides as compared with a high concentration.

Table 1

The concentration of NaCl (%) at which aminoglycosides induced corresponding percentage of hemolysis in human red cells

Discussion

The results of this study show that aminoglycosides in different loading concentrations produce slight effect on the red cell membrane. Such effect does not follow a concentration-dependent pattern, and it points to the direct effect of these compounds. Therefore, this effect is completely different from the ototoxic or nephrotoxic effects of aminoglycosides in which the aminoglycosides are up-taken by hair or renal tubule cell via specific mechanisms.[1314] It is well known that aminoglycosides interfere with calcium entry into the cells.[9] Such mechanism does not explain the effect of aminoglycosides on red cell membrane. Furthermore, there is evidence that these compounds are involved in the generation of reactive oxygen species[15] and again such mechanism does not explain the findings reported in this study as the experiments were carried in vitro in absence of other pro-oxidant substances. The fragile effect of aminoglycosides on the red cell membrane does not mean that these compounds can induce autoimmune hemolytic anemia[16] because this study was designed in vitro and there is no antibody directed to the antigen of aminoglycosides or of the red cells. The effect of spectinomycin is obvious compared with gentamicin or amikacin. This finding indicated that the damaging effect of aminoglycosides is related to the chemical structure of the drug.[17]

This finding is important because red cells are used as a carrier system for targeting the drugs.[18] Therefore, in such situation, spectinomycin should be avoided, whereas the gentamicin or amikacin are preferable. It concludes that the aminoglycosides induce direct red cell membrane fragility in a concentration-independent manner.