THE MECHANISM OF BILE SALT-INDUCED HEMOLYSIS

CELLULAR & MOLECULAR BIOLOGY LETTERS

Volume 6, (2001) pp 881 Ó 895 Received 11 July 2001 Accepted 25 September 2001

THE MECHANISM OF BILE SALT-INDUCED HEMOLYSIS LUCYNA MRO WCZYN SKA and JO ZEF BIELAWSKI Department of Cytology and Histology, A. Mickiewicz University, Fredry 10, 61-701 Poznan, Poland

Abstract: The hemolytic activities of sodium deoxycholate (DChol) and its tauro-conjugate (TDChol) and glyco-conjugate (GDChol) were analysed. 50 % hemolysis occurred in 30 min at pH 7.3, at the concentrations of these detergents equal to 0.044, 0.042 and 0.040 % respectively. These values are below their critical micellar concentrations. Based on its kinetics, this hemolysis is classified as being of permeability type. The detergents increase the permeability of erythrocyte membranes to KCl, and colloid osmotic hemolysis occurs. The minimum of hemolytic activity of the three cholates is at about pH 7.5. A very high increase in hemolytic activity occurs at pHs below 6.8, 6.5 and 6.2 for DChol, TDChol, and GDChol, respectively. These values are close to the pKa for DChol (6.2), but much higher than the pKa for TDChol (1.9) and GDChol (4.8). It is therefore suggested that the increase in hemolytic activity is not a result of the protonation of the anionic groups of the cholates. At acidification below pH 6, the kinetics of DChol induced hemolysis change to the damage type characterised by nonselective membrane permeability. Such a transition is not observed in TDChol and GDChol induced hemolysis. It is therefore suggested that the change in the type of hemolysis depends on protonation of the anionic group of cholates. Key Words: Bile Salt, Detergent, Membrane, Erythrocyte, Hemolysis. INTRODUCTION Cholates are natural detergents, which facilitate the digestion and resorption of food in the alimentary tract [1, 2]. At high concentrations, detergents, show destructive activity on cell membranes, activity that results in cell injury [3-7].

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The damaging effects of bile salts 1 on cellular membranes are a well-established phenomenon. It has been implied that they play an important role in colon tumorigenesis [8-14] and in the pathogenesis of cholestatic liver injury [15-17]. The lytic activity of bile salts is strongly dependent on their chemical structure and increases with their increasing hydrophobicity [18]. The investigation of the mechanism of cholate interaction with cell membranes is therefore of utmost importance. The erythrocyte membrane is a convenient model for these investigations [4,6, 9,20]. It is possible and useful to compare and relate the hemolytic activity of bile salts to their widely comprehended cytotoxicity. The kinetics of hemolysis may differ depending on the kind of detergent and incubation conditions. However, two main types of hemolysis have been distinguished [21,22]. The permeability type is the effect of increased selective permeability of erythrocyte membrane to small molecules. As a result colloid osmotic hemolysis occurs. The damage type of hemolysis may be explained by the formation of large membrane perforations permeable to small molecules and macromolecules including hemoglobin [22]. In this paper, the kinetics of hemolysis induced by sodium deoxycholate and its glyco- and tauro-conjugates is described. MATERIALS AND METHODS Citrate-stabilised blood was obtained from a blood bank. Erythrocytes were washed three times in 160 mM NaCl, pH 7.3 at 4ęC (centrifugation for 20 min at 1000g) as described previously [22]. The hematocrit of the erythrocyte suspension was 50 %. The incubation medium was 160 mM KCl. 10 ml of the incubation media was preincubated with bile salt for 30 min in tubes of 14 mm inner diameter. The required pH of the solution was obtained by adding 160 mM KOH or HCl only. No buffer was added to avoid its potential effect on the colloid osmotic hemolysis. The incubation was started with the addition of erythrocytes at the final hematocrit of 0.15 %. The pH was controlled at the end of incubation. In some preliminary experiments, the cholate was added after the erythrocytes had been preincubated for 30 min. Under these conditions, the addition of cholate and the hemolysis did not change the pH of the suspension by more than 0.1. The light absorbance of the cell suspension was measured at λ = 590 nm wavelength at appropriate time intervals [22]. At this wavelength, the absorbance of lysed erythrocytes is very low and independent of pH. In an erythrocyte suspension, light absorption is a small fraction of absorbance. Most of the absorbance is due to light scattering, which is linearly related to the 1

Abbreviations: Sodium deoxycholate, DChol; Sodium glycodeoxycholate, GDChol; Sodium taurodeoxycholate, TDChol.


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volume of intact erythrocytes and decreases abruptly on cell lysis. The method is very sensitive and enables measurements at very low hematocrit (low erythrocyte to detergent ratio). Bile salts were acquired from Sigma (St Louis, MO) and Fluka (Buchs, Switzerland). The other chemicals were of analytic grade as well. RESULTS The effect of DChol on the dependence of absorbance of erythrocyte suspension on incubation time, in isotonic KCl at pH 7.3, is presented in Fig. 1. Upon the addition of erythrocytes, there is an initial small rise in the absorbance of the suspension to a certain level. It is the result of the formation of a new steady state of permeable substances on both sides of the membrane [23]. The small shrinkage is independent of the presence of the detergent and is completed within about 3 min of incubation. In the absence of detergent, after the initial shrinkage, the absorbance remains constant for several hours. In the presence of DChol, a drop of absorbance follows the initial shrinkage. The rate of this drop is small at first and can be interpreted as a swelling of the erythrocytes. On exceeding the critical volume,cell lysis occurs and the rate of the drop in absorbance increases. Once all the erythrocytes are hemolysed, the absorbance reaches a constant level at its

Fig. 1. The influence of DChol concentration on the dependence of absorbance (A) of the erythrocyte suspension on the time of incubation (t) at pH 7.3 and 37ęC. The DChol concentrations in percentages × 10-3 are given in the figure.

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lowest level and the drop in absorbance is maximal. The rate of swelling and hemolysis increases with the increase in DChol concentration. However, all the erythrocytes are also hemolysed at low concentrations of DChol over an appropriately long period of time. The typical form of the curves, presented above, may be modified by incubation nditions. Between the initial shrinkage and the swelling of the erythrocytes a delay period may occur. The transition from swelling to hemolysis may be more or less evident. This form of the curves is indicative of the permeability type of hemolysis [22]. DChol increases the permeability of the erythrocyte membrane to KCl, and colloid osmotic uptake of KCl causes cell swelling and lysis. Therefore, the rate of hemolysis depends on the selective permeability to KCl induced by DChol. A convenient measure of the rate of hemolysis is the reciprocal of the time at which 50 % of the erythrocytes are hemolysed, or more conveniently expressed, the reciprocal of the time at which the absorbance of the erythrocyte suspension decreases to 50 % of the maximal decrease.

Fig. 2. The dependence of the rate of hemolysis (V) on DChol concentration at pH 7.3 and 37ęC.

The rate of hemolysis obtained in this way is a non-linear function of DChol concentration. However, the dependence of the logarithm of the rate of hemolysis on DChol concentration is linear (Fig. 2). The resistance of erythrocytes to DChol can be calculated from this relationship. It is defined as the concentration of DChol that hemolyses 50 % of erythrocytes in 30 min (the detergent concentration that induces hemolysis at the rate of 1/30 min). This


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value may be calculated by interpolation of the curve presented in Fig. 2. The results are presented in Table 1. The replacement of DChol with GDChol or TDChol shows a similar dependence of the absorbance of the erythrocyte suspension on the time of incubation to that in Fig. 1. In addition, the resistance of erythrocytes to each of these detergents is similar (Table 1). Tab. 1. The resistance of erythrocytes to cholates.

%

mM

n

DChol

0.0443 ± 0.00676

1.069 ± 0.163

10

GDChol

0.0398 ± 0.00433

0.845 ± 0.092

5

TDChol

0.0420 ± 0.01054

0.778 ± 0.195

6

The values are means from n measurements ± SD. 37ęC, pH 7.3.

Fig. 3. The influence of pH on the dependence of the absorbance (A) of erythrocyte suspension on the time of incubation (t) in the presence of 0.03 % DChol at 37ęC. The values of pH are given in the figure.

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pH is an important factor that modifies the hemolytic activity of DChol. To study the effect of pH on hemolysis, erythrocytes were exposed to media of different pH in the presence and absence of DChol. The suspensions of various pH were obtained by the addition of an appropriate volume of 160 mM HCl or KOH to the incubation medium before erythrocytes were added. The final pH was measured at the end of the measurements of absorbance. The dependence of absorbance on incubation time measured at various pH and constant DChol concentration is presented in Fig. 3. The low DChol concentration (0.03 %) facilitated the measurements of rapid hemolysis at low pH. The influence of pH on the rate of hemolysis in the presence of DChol is presented in Fig. 4. The minimal rate of hemolysis occurs at pH 7.7. Higher alkalisation shows a small increase in the rate of hemolysis. On the other hand, the acidification of the suspension below 7.0 greatly increases the hemolytic activity of DChol. The absorbance-time curve is changed on transition from pH 6.8 to 6.6 (Fig. 3). At pH 6.8 and higher, all the curves are similar to those presented in Fig. 1. At pH 6.6 and below, the initial shrinkage of erythrocytes is absent and the absorbance decreases immediately. There are two reasons for it. At acidic pH, the approach of the steady state of permeable ions causes a small initial swelling instead of initial shrinkage, both in the absence and presence of detergents [23]. The other reason is the very rapid hemolysis due to the high increase in the hemolytic activity of DChol at acidic pH. Therefore the following measurements at low pH were done at much lower DChol concentration.

Fig. 4. The influence of pH on the rate of hemolysis (V) in the presence of 0.03 % DChol


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A large decrease in pH causes spontaneous hemolysis without the addition of detergent and makes the interpretation of the results difficult. However, at pH 5.7 the rate of Dchol induced hemolysis is very high and spontaneous hemolysis without the addition of detergent is absent. Apparently at acidic pH, the absorbance-time curves are changed (Fig. 5). These modifications are best evident at lower temperature. Therefore, the measurements presented in Fig. 5 were taken at 15ęC. In these conditions, the contact of DChol with the erythrocytes causes a rapid drop in absorbance. The rate of this drop diminishes gradually to zero within about 15 min, and afterwards the absorbance remains constant for several hours. It means that some of the erythrocytes are hemolysed within 15 min, while the other cells remain intact. At DChol concentration higher than 0.001 % all the erythrocytes are hemolysed.

Fig. 5. The influence of DChol concentration on the dependence of absorbance (A) of the erythrocyte suspension on the incubation time (t) at pH 5.7 and 15ęC. The DChol concentrations in percentages × 10-4 are given in the figure.

The dependence of the final drop in absorbance on DChol concentration presented in Fig. 6 shows a sigmoidal pattern. The kinetics of hemolysis presented in Figs 5 and 6 are indicative of the damage type of hemolysis [22]. At 37ęC and pH 5.3 the rapid drop in absorbance may be followed by a slower decrease to the maximal value (Fig. 7). This later decrease might be interpreted as a slower permeability type of hemolysis of the cells remaining after the damage type of hemolysis.

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Fig. 6. The influence of DChol concentration on the decrease in absorbance (dA) of the erythrocyte suspension during 20 min incubation at pH 5.7 and 15ęC.

Fig. 7. The influence of DChol concentration on the dependence of absorbance (A) of the erythrocyte suspension on the incubation time (t) at pH 5.3 and 37ęC. DChol concentrations in percentage × 10-5 are given in the figure.


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Fig. 8. The influence of TDChol concentration on the dependence of absorbance (A) of the erythrocyte suspension on the time of incubation (t) at pH 7.3 and 37ęC. The TDChol concentrations in percentage × 10-3 are given in the figure.

Fig. 9. The influence of pH on the rate of hemolysis (V) in the absence (squares) and in the presence (circles) of 0.04 % TDChol at 15ęC.

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The high increase in DChol activity at pH around 6.0 was previously observed by other authors [18, 24-26]. They show the similarity of this pH with the pKa of DChol. According to their interpretation, the protonation of the anionic group of DChol increases the solubility of this detergent in the membranes. In consequence, the hemolytic activity of the detergent is much higher. To verify this interpretation, the influence of pH on the hemolytic activity of glyco- and tauro-conjugated deoxycholates were examined. The pKa of these compounds, 4.8 and 1.9 respectively, are much lower than that of deoxycholate, which is equal to 6.2 [27]. At neutral pH the kinetics of hemolysis induced by TDChol, presented in Fig. 8, do not differ significantly from those obtained for DChol (Fig. 1). Similar results were obtained for GDChol (data not shown). The difference in the resistance of erythrocytes to the three detergents is also small (Table 1). The rate of hemolysis induced by TDChol increases with decreasing pH (Fig. 9). Similar results were obtained for GDChol. The high increase is at pH below 6.5 and 6.2 for TDChol and GDChol respectively. These values are not much different from those obtained for DChol shown in Fig. 4. However, they are much higher than the pKa of GDChol and TDChol. It is therefore evident that the high increase in the hemolytic activity of GDChol and TDChol on acidification is not simply a consequence of the protonation of the acidic groups of the two detergents. There is however, an essential difference in the response of erythrocytes to the two conjugated deoxycholates. Acidification does not change the kinetics of hemolysis from the permeability to the damage type. Negative results were obtained in several experiments performed in order to demonstrate the damage type of hemolysis induced by GDChol and TDChol on acidification featuring the incubation conditions shown in Figs 5 and 7. Therefore, it can be suggested that the transition from the formation of selective channels to large perforations is the result of the protonation of the acid group of DChol. DISCUSSION The effect of the interaction of a detergent with erythrocytes depends on the concentration of that detergent in the medium. At low concentrations, the absorbed detergent changes the lipid organisation within the membrane and the resistance to osmotic hemolysis increases [28-31]. At higher concentrations cell lysis occurs [3-6, 32-34]. It is known that some detergents form channels across the membrane selectively permeable for substances of low molecular weight [35-37]. Due to the colloid osmotic uptake of solutes, the erythrocytes swell. On exceeding the critical volume, hemolysis occurs [38, 39]. Some detergents, at low concentration, form large perforations of membrane permeable nonselectively for substances of low as well as high molecular weight including hemoglobin. In this case, swelling does not precede lysis [22]. The kinetics of

Fig. 9. The influence of pH on the rate of hemolysis


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hemolysis induced by DChol and its conjugates at neutral and alkaline pH indicate the permeability type of hemolysis. The resistance of human erythrocytes to DChol in slightly alkaline media, presented in this paper, is significantly lower than calculated from results obtained with the use of other methods [20, 40]. This difference might be explained by the much lower hematocrit of the erythrocyte suspension in our experiments and a higher detergent/erythrocyte ratio at any given detergent concentration. It has been demonstrated that the lytic activities of cholates occur at concentrations above CMC [4-6, 13, 14, 41]. Few investigations suggest the possibility of hemolysis induced by cholates at monomeric concentrations [42, 43]. Our results clearly support the latter opinion. The discrepancy can be again explained by the differing hematocrit values in the experiments of other authors. In the pioneering studies, Coleman et al. [44-46] showed a correlation between the chemical structure of bile salts and their cytotoxicity. It has been well demonstrated that the lytic activity of cholates is strongly dependent on their hydrophobicity [4-6, 13, 14, 24, 37, 41, 47]. The flip-flop of bile salt monomers and oligomers across membrane bilayers increases with their increasing hydrophobicity [26, 48]. The similar hemolytic activity of DChol and its glycoand tauro-conjugates concurs with these findings, as the hydrophobicity index for these three cholates is similar [18]. The lytic activity of cholates is largely modified by pH [24, 48-51]. Our results support this finding. Protonation of the anionic group of DChol increases its solubility in lipids and in consequence, the lytic activity of the detergent is higher [24-26, 52, 53]. Correlation between the pKa of DChol and the pH of a high increase in the hemolytic activity of this detergent concurs with this finding. However, the results obtained with TDChol and GDChol do not fit this hypothesis. The pH of high activation of hemolysis for both of these bile salts are similar to that for DChol. The pKa of the conjugates is much lower than that for DChol [27]. It means that not only protonation but also some other properties of the molecules of the cholates and the cell membrane components determine the change in hemolytic activity during acidification. However, the transition of kinetics from permeability to damage type on acidification to pH of about 6 probably depends on the protonation of the anionic group of DChol. In consequence it occurs in hemolysis induced by DChol (pKa = 6.2) and is absent in that induced by TDChol (pKa = 1.9) and GDChol (pKa = 4.8). The possibility of the existence of two separate mechanisms of membrane damage, one attributed to bile salt and one to bile acid was suggested earlier [51]. A decrease in temperature inhibits the permeability type of hemolysis [19, 22] and stimulates the damage type [22]. We speculate that changes in temperature can therefore alter the participation of both of the mechanisms of hemolysis induced by DChol. These features are arguments for the simultaneous presence

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of two types of transport system induced by DChol. By changing the incubation conditions, the domination of one of them takes place. Acknowledgments: The authors gratefully thank Halina Ke sa MSc for her valuable technical assistance in conducting the experiments. REFERENCES 1. Hofmann, A. F. and Roda, A. Physicochemical properties of bile acids and their relationship to biological properties: an overview of the problem. J. Lipid Res. 25 (1984) 1477-1489. 2. Thompson, M. B. Bile acids in the assessment of hepatocellular function. Toxicol. Pathol. 24 (1996) 62-71. 3. Coleman, R., Lowe, P. J. and Billington, D. Membrane lipid composition and susceptibility to bile salt damage. Biochim. Biophys. Acta 599 (1980) 294-300. 4. Heuman, D. M., Pandak, W. M., Hylemon P.B. and Vlahcevic, Z. R. Conjugates of urodeoxycholate protect against cytotoxicity of more hydrophobic bile salts: in vitro studies in rat hepatocytes and human erythrocytes. Hepatology 14 (1991) 920-926. 5. Velardi, A. L. M., Groen, A. K., Oude Elefrink, R. P., Van der Meer, J. R., Palasciano, G. and Tytgat, G. N. J. Cell type-dependent effect of phospholipid and cholesterol on bile salt cytotoxicity. Gastroenterology 101 (1991) 457-464. 6. Sagawa, H., Tazuma, S. and Kajiyama, G. Protection against hydrophobic bile salt-induced cell membrane damage by liposomes and hydrophilic bile salts. Am. J. Physiol. 264 (1993) 835-839. 7. Shiao, Y. J., Chen, J. Ch., Wang, Ch. N. and Wang, Ch. T. The mode of action of primary bile salts on human platelets. Biochim. Biophys. Acta 1146 (1993) 282-293. 8. Rafter, J. J., Child, P., Anderson, A. M., Alder, R., Eng, V. and Bruce, W. R. Cellular toxicity of fecal water depends on diet. Am. J. Clin. Nutr. 45 (1987) 559-563. 9. Lipkin, M. Biomarkers of increased susceptibility to gastrointestinal cancer: new application to studies of cancer prevention in human subjects. Cancer Res. 48 (1988) 235-245. 10. Laprä , J. A., De Vries, H. T., Termont, D. S. M. L., Kleibeuker, J. H., De Vries, E. G. E. and Van der Meer, R. Mechanism of protective effect of supplemental dietary calcium on cytolytic activity of fecal water. Cancer Res. 53 (1993) 248-253. 11. Latta, R. K., Fiander, H., Ross, N. W., Simpson, C. and Schneider, H. Toxicity of bile acids to colon cancer cell lines. Cancer Lett. 70 (1993) 167-173.


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