Lytic effects of mixed micelles of fatty acids and bile acids J. A. LAPRE,
D. S. M. L. TERMONT,
A. K. GROEN,
AND R. VAN DER MEER
Department of Nutrition, Netherlands Institute for Dairy Research, 6710 BA Ede; and Department of Gastroenterology and Hepatology, Academic Medical Center, 1105 AZ Amsterdam, The Netherlands Lap&, J. A., D. S. M. L. Termont, A. K. Groen, and R. Van der Meer. Lytic effects of mixed micelles of fatty acids and bile acids. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G333-G337, 1992.-It has been hypothesized that bile acids and fatty acids promote colon cancer. A proposed mechanism is a lytic effect of these surfactants on colonic epithelium, resulting in a compensatory proliferation of colonic cells. To investigate the first step of this hypothesis, we studied the lytic activity of fatty acids and physiological mixtures of fatty acids and bile acids. Experiments were performed in both erythrocytes and cultured Caco-2 cells, a model system for intestinal epithelium. Fatty acids with a chain length of 10 C atoms or more were lytic, and the hemolytic activity increased in the order C10:0 < C18:0 < C16:0 < CZZO < C14Z0 < C18:1 = C18:2 but was not dependent on their critical micellar concentration. Addition of a sublytic, submicellar concentration of cholate resulted in the formation of highly lytic mixed micelles. Lytic activity of these mixed micelles was closely associated with their micellar aggregation as determined in parallel incubations using a fluorescent micellar probe. With use of identical concentrations of fatty acids and mixed micelles, lysis of erythrocytes was highly correlated (r > 0.95) with lysis of Caco-2 cells measured by either release of the apical membrane-marker alkaline phosphatase or the cytosolic marker lactate dehydrogenase. This indicates that the cytolytic activity of these surfactants is not cell-type dependent. Addition of bile acids in concentrations corresponding with the total bile acid concentration in human fecal water resulted in an increased lytic activity of fatty acids. The increase in lytic activity was lowest for the primary bile acid cholate and highest for the more hydrophobic, colonic bile acids deoxycholate and lithocholate. These results may contribute to a better knowledge of the relevant determinants of lytic activity of human fecal water. lactate dehydrogenase; alkaline phosphatase; Caco-2 cells; lytic activity; hemolysis; colon cancer DATA show a correlation between a high-fat intake and the incidence of colon cancer (7,34). It has been suggested that this effect is caused by a diet-dependent increase of bile acids and free fatty acids in colon (21, 33). Bile acids instilled intrarectally strongly promote colon cancer in tumor-induction models (24). Proliferation of colonic epithelium is drastically stimulated by bile acids and fatty acids in rodents (5,13, 31, 32), probably because of their intrinsic lytic properties (23, 3 1, 32). Also in humans dietary fat stimulates colonic proliferation (27). This hyperproliferation of coionic epithelium is generally accepted as an important biomarker of the risk for colon cancer (18). Recently, we have shown that lytic activity of bile acids (measured as lysis of erythrocytes) is strongly dependent on their structure and increases with increasing hydrophobicity (29). Lytic activity increases from the primary conjugated (duodenal) to the secondary unconjugated (colonic) bile acids and is associated with their micellar aggregation (29). The physiological relevance of EPIDEMIOLOGIC
0193~1857/92
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these studies has been ascertained in experiments with rats. A diet-induced increase of the bile acid concentration in fecal water results in a higher intestinal lytic activity (X5,16) and in a higher colonic cell proliferation (15). In these animal studies the concentration of bile acids in fecal water was higher than that of fatty acids (15, 16). However, in human fecal water the concentration of fatty acids is in excess of that of bile acids (1, 22, 28). Consequently, the fatty acid concentration in human fecal water might be a relevant determinant of luminal lytic activity in humans. For that reason, we studied the lytic properties of fatty acids in the absence and presence of bile acids using human erythrocytes as a model system for lytic activity. By measuring hemolysis as an endpoint of lytic activity, several studies (9,10,29) have shown that this model system can adequately be used for determining membrane damage by surfactants. To validate the physiological relevance of lysis of erythrocytes by surfactants, we studied the lytic effects of different kinds of surfactants on Caco-2 cells, which are derived from a human colon adenocarcinoma cell line. By culturing in a polarized monolayer, these cells provide an excellent model system to study lytic effects on the apical membrane of intestinal epithelial cells (30). To quantify lysis of these cells, we used both an apical membrane marker (alkaline phosphatase; 14), as well as a cytosolic marker (lactate dehydrogenase). MATERIALS
AND METHODS
Muterials. Human blood was obtained from healthy donors by venipuncture. Human erythrocytes were thoroughly washed and isolated exactly as described by Van der Meer et al. (29). Bile acids, fatty acids, and fat-free bovine serum albumin were acquired from Sigma Chemical (St. Louis, MO) and Fluka (Buchs, Switzerland) and were of the highest purity commercially available. The purity of bile acids and fatty acids used has been verified by gas chromatographic analysis. N-PhenylI-naphthylamine (NPN) was obtained from Eastman Kodak (Rochester, NY), and N-2-hydroxyethylpiperazine-N’2-ethanesulfonic acid (HEPES) from Sigma. Other chemicals used were of analytic grade. Methods. Fatty acids were dissolved in double-distilled water and saponified with potassium hydroxide. After saponification, the pH of the stock solutions was brought to 7.5-8.0. Concentrations of fatty acids were measured with a commercially available enzymatic kit (NEFA-C, Wako Chemicals, FRG). Sodium salts of bile acids were dissolved in double-distilled water. Lithocholate (LC) was solubilized by mixing it with deoxycholate (DC) at a molar ratio of I:3 (LC/DC). Concentrations of bile acids were measured using an enzymatic fluorescence method (Sterognost 3~ Flu, Nycomed, Oslo, Norway; Ref. 2). Lytic properties of fatty acids and mixtures of fatty acids and bile acids were studied using hemolysis of erythrocytes at a final hematocrit of 5%. To avoid peroxidation of the unsaturated fatty acids, the experiments with oleate and linoleate were performed under nitrogen. However, control experiments showed
0 1992 the American
Physiological
Society
G333
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G334
LYTIC
EFFECTS
OF MIXED
that no difference in lytic activity occurred during our shortterm incubations when the experiments were carried out aerobically. After a preincubation of 10 min, erythrocytes were added to tubes containing increasing concentrations of fatty acids and/or bile acids, HEPES (final concentration 50 mM and pH 7.4), and NaCl (to maintain a constant ionic strength of 150 mM). KC1 was used to correct for the amount of potassium in the fatty acid stock solutions, thus maintaining a constant potassium concentration in the incubation of 60 mM. Control experiments showed that this potassium concentration did not affect hemolysis (data not shown). After 15 min of incubation at 37”C, the tubes were centrifuged for 1 min at 10,000 g (Eppendorf 5415). After dilution of the supernatants with double-distilled water, hemolysis was determined by measuring the absorbance at 540 nm. In control experiments it appeared that neither bile acids nor fatty acids affected the absorbance of hemoglobin at this wavelength. Percent hemolysis was calculated by comparing the absorbance of the samples with that of an identical amount of erythrocytes lysed in double-distilled water, which represents 100% hemolysis. Monolayers of Caco-2 cells were grown in microwell plates as described by Velardi et al. (30). After postconfluent cultures were washed with phosphate-buffered saline (pH 7.4), cells were incubated for 45 min at 37°C with solutions containing surfactants as described above. At the end of the incubation time, the medium was collected and the cells were treated with 0.1% Triton X-100. Lactate dehydrogenase (LDH) activity was measured according to Mitchell et al. (ZO), and alkaline phosphatase (ALP) activity according to Bessey et al. (3) in both medium as well as in Triton X-loo-treated cells. Fat-free bovine serum albumin (final concentration 0.6%) was added to prevent interference of surfactants with the spectrophotometric assay of LDH activity. %Lysis was calculated as enzyme activity in medium divided by the total enzyme activity (medium + Triton X-100).
The apparent termined under by using 2 PM scribed by Brito of at least three
critical micellar concentration (CMC) was dethe same conditions, but in the absence of cells, of the fluorescent micellar probe NPN, as deand Vaz (4). Results are given as means t SD experiments.
RESULTS
Figure 1 shows the concentration dependence of the hemolytic effect of laurate (C&J. At low concentrations laurate was not lytic, but above a certain threshold con-
MICELLES
centration significant lysis was observed. The concentration at which 2% lysis occurred is defined as the critical lytic concentration (CLC). In addition, we used the lytic concentration required for 50% lysis (LC,,) as a second parameter to define the lytic curve. To investigate whether addition of bile acids enhances the lytic effect of fatty acids, we used 5 mM of the primary bile acid cholate. Addition of this submicellar concentration of cholate (29) to increasing concentrations of laurate caused a shift of both CLC and LCSO to lower values, which indicates an increased lytic activity (Fig. 1). In the absence of laurate, no lytic activity was observed, which indicates that 5 mM cholate alone did not induce lysis. Figure 2 shows the lytic potential, which is the reciprocal of L&, for a range of fatty acids. No lytic effects were observed with &, and the lytic potential increased from C&o to C1A.0with increasing chain length. The longchain saturated fatty acids (C16.0 and Cl& were less lytic than the medium-chain fatty acids. Oleate and linoleate showed a higher lytic potential than stearate. Figure 2 also shows that the lytic potential of all fatty acids >C10.0 was drastically increased by the addition of a sub&c concentration (5 mM) of cholate. To determine whether this increase in lytic potential is due to the formation of mixed micelles, we determined the CMC of these mixtures using a fluorescent probe. In contrast to bile acids (29), the CLC of fatty acids was not associated with their micellar aggregation (Fig. 3). Addition of a submicellar concentration of cholate induced the formation of mixed micelles. Thus, at concentrations below their individual CMCs, the combination of bile acids and fatty acids induced the formation of mixed micelles. The CLC of these mixed micelles corresponded with their CMC (Fig. 3), which is analogous to the relationship observed for bile acids (29). This indicates that the lytic activity of the bile acid-fatty acid mixtures is caused by mixed micelles. Table I gives the results of the comparison between the effects of surfactants on lysis of Caco-2 cells and on lysis of erythrocytes. It is clearly shown that the different surfactants (bile acids alone, fatty acids alone, and combinations of fatty acids and bile acids) had comparable lytic effects on erythrocytes and Caco-2 cells. Moreover, the
- cholate + 5 mM cholate
0.5
’ 1.0 t 1 CLC LCg
1.5
T
n m
2.0 C12:0 OnM)
in absence Fig. 1. Concentration dependence of lytic effect of laurate CLC, critical and presence of sublytic concentration of cholate (5 mM). lytic-concentration required for 2% lysis; LC&, concentration required for 50% lysis.
8:0
16:0
18:0
18:2
Fig. 2. Lytic effects of fatty acids measured as hemolysis and expressed as lytic potential (l/LC& in absence and presence of sublytic concentration of cholate (5 mM). Bars represent SD (n = 3).
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LYTIC fatty
q
acids
mixed
/ y =x
micelles
/
w
10
OF MIXED
acids
o bile l
EFFECTS
c + c12:o
/
l
c / O/
/
/
oclo:o
/
gV 0 d GDC + c12:o
1
9
,.
DC + C12.0 .
o c12:o
/ /
7
c12:o
+ c o c14:o
/ ,/cl4:0 0.1
16:0
+ c
I 1
+ ’
I 10
1 100 CMC (mM>
Fig. 3. Correlation between critical lytic concentration (CLC) and critical micellar concentration (CMC) for bile acids, fatty acids, and mixtures of bile acids and fatty acids. Note that scales are logarithmic. C, cholate; GDC, glycodeoxycholate; DC, deoxycholate. Mixed micelles consist of different fatty acids combined with 5 mM cholate (CMC and CLC expressed as millimolar fatty acid). For combinations C + C120, GDC + C12.0, and DC + C12.0 different bile acids were combined with 300 PM la&ate (CMC and CLC expressed as millimolar bile acid).
Table 1. Lytic effects of bile acids, fatty acids, and mixtures of bile acids and fatty acids on Caco-2 cells and on erythrocytes Lysis,
%
Surfactant
Caco-2
cells
Erythrocytes LDH
No surfactants C holate 16.5 mM Go., lb.0 mM 20.0 mM C 12:o 0.6 mM 1.4 mM C 1o:o + 5 mM 5.0 mM 10.0 mM C lcj:o + 5 mM 0.3 mM 0.6 mM
oto 99k5 l&O 102tll l&O 79A19
release
ALP
release
6tl
It1
96&l
96tl
6~~1 89k3
2t2 98t3
6tl 5921
3t2 5024
14t2 103t3
lOt5 106t2
G335
MICELLES
time (15 min for erythrocytes vs. 45 min for Caco-2 cells) indicates that erythrocytes are more sensitive to surfactant-induced lysis compared with Caco-2 cells. Finally, to examine the effect of bile acid structure on the lytic activity of mixed micelles, we used an increasing concentration of oleate combined with 250 PM cholate, deoxycholate, or a mixture of deoxycholate and lithocholate in a molar ratio of 3:l. Control experiments showed that 250 PM of these bile acids in the absence of oleate is not lytic. This bile acid concentration corresponds with the total bile acid concentration in human fecal water (1, 22, 28), and oleate is one of the most predominant longchain fatty acids in human fecal water (unpublished data). As shown in Fig. 4, a change in bile acid structure caused a significant change in lytic potential. Addition of the primary bile acid cholate had little or no effect on oleate-induced lytic potential, whereas the secondary bile acid deoxycholate increased its lytic potential. The addition of the mixture of deoxycholate and lithocholate (predominantly present in the colon) induced the highest lytic potential. Comparable results were obtained using laurate as a model for a medium-chain fatty acid and palmitate as long-chain saturated fatty acid. For instance, addition of 250 PM cholate has little or no effect on the lytic potential of laurate or palmitate, whereas 250 PM of deoxycholate plus lithocholate stimulated lytic potential of laurate from 0.83 to 3.0 mM-l and of palmitate from 0.53 to 1.1 mM-l. DISCUSSION
The results of the present study show that the lytic potential of fatty acids is at least of the same magnitude as that of bile acids (9,10,29). Buset et al. (6) have shown that palmitate, oleate, and linoleate decrease the viability of isolated colonocytes as quantified by [3H] thymidine incorporation. This is probably due to the lytic effects of these fatty acids as observed in our present study. In our experiments using an incubation temperature of 3’7”C, lytic activity of the saturated fatty acids increased with
DC/LC
cholate 3021 94t2
DC
cholate 54tlO 8Ok5
57t6 80*13
65t16 93t5
Values are means t SD; n = 3 incubations. Lysis of Caco-2 cells was measured by lactate dehydrogenase (LDH) release and by alkaline phosphatase (ALP) release, and hemolysis by release of hemoglobin. Correlation coefficient between hemolysis and LDH release was 0.97 (hemolysis = 1.03 LDH + 0.9), between hemolysis and ALP release 0.96 LDH release and ALP (hemolysis = 0.91 ALP - 7.2), and between = 0.89 ALP - 5.0). release 0.99 (LDH
J
control
high correlation coefficients of hemolysis compared with Caco-2 lysis (r > 0.95) indicated that the lytic effects are similar in both cell types under these conditions. However, it should be noted that the difference in incubation
b
ncreasing
hydrophobicity
Fig. 4. Effect of 250 PM cholate (C), deoxycholate (DC), and deoxycholate-lithocholate (DC/LC) ( mo 1ar ratio 3:l) on lytic potential of oleate. Bars represent SD (n = 4).
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G336
LYTIC
EFFECTS
OF MIXED
the increase in the number of carbon atoms, which reflects an increasing hydrophobicity. A similar relationship between bile acid hydrophobicity and lytic activity has been reported previously (29). However, palmitic and stearic acids were less lytic than would have been expected from their chain length. This anomalous behavior is probably due to the fact that our incubation temperature of 37°C is below the critical temperature of these long-chain fatty acids (17), so that their lytic activity is decreased because of gel formation (8, 17). As shown by Csordas and Rybczynska (12), this may drastically decrease the fatty acid-induced lysis of erythrocytes. The observed increase in lytic activity with unsaturation of fatty acids (Fig. 2) is in accordance with this explanation, because unsaturation of fatty acids decreases their critical temperature ( 17). The similarity of CMC and CLC values for bile acids and mixed micelles (Fig. 3) shows that hydrophobicity and micellar aggregation are important determinants of their cytolytic effect. This indicates that lysis is due to disruption of the cellular membrane integrity by comicellization of membrane lipids (9, 29). Whether the same mechanism holds for the lytic effects of fatty acids per se is at present not clear. For instance, comparison of our CMC value of 7.1 mM for laurate, which is similar to the value of 7.5 mM published earlier (1 I), with the CLC value of 0.73 mM indicates that there is no association between micellar aggregation of fatty acids and fatty acidinduced lysis of erythrocytes. It should be noted that this lack of association cannot be taken as evidence that lysis is mediated by fatty acid monomers. For instance, the sigmoidal concentration dependence of fatty acid-induced lysis (Fig. 1) may reflect that aggregation of fatty acids is required for its lytic effects. This may indicate that this lytic activity is due to dimers or oligomers with a low aggregation number. Apparently our fluorescent probe does not intercalate with these small structures, but this requires further investigation. The present study showed that mixed micelles are formed at physiological concentrations below their individual CMC. We are not aware of other studies that show associations between submicellar concentrations of bile acids and fatty acids, but similar associations between submicellar concentrations of lysophospholipids and bile salts have recently been reported (25). At present no information on the structure of these aggregates is available. Our results indicate that the formation of mixed micelles of fatty acids and bile acids drastically stimulated lytic activity. A bile acid concentration of 250 PM of secondary bile acids such as deoxycholate or a mixture of deoxycholate and lithocholate increased the lytic activity of fatty acids (laurate, palmitate, and oleate). However, addition of 250 VM of a primary bile acid such as cholate had almost no effect on lytic activity. These results indicate that changes in the hydrophobic-hydrophilic balance of bile acids in fecal water may have a drastic effect on intestinal lytic activity. The significant effects of hydrophobicity of fatty acids and bile acids on micellar aggregation and cytotoxicity may be relevant for the integrity of the intestinal mucosa. The present results show that the lytic effects of these
MICELLES
surfactants on Caco-2 cells and erythrocytes are highly correlated (Table 1) and thus support and extend our recent findings (30). Our resu Its indicate that Caco-2 cells, compared with erythrocytes, are slightly more resistant to surfactant- 1nduced lysis. This can be explained by the high content of glycolipids in the apical membrane of enterocytes (26), because it has been shown that differences in membrane glycolipids could affect the resistance to bile-acid-induced lysis (10). Our results indicate that lysis of enterocytes can be determined with the same accuracy by measuring the release of either an apical (ALP) or a cytosolic marker (LDH). Finally, we have recently demonstrated that a diet-induced increase in lytic activity of rat fecal water correlated highly with in vivo colonic proliferation (15). This also indicates that the in vitro experiments reported in this paper are of relevance to a proper understanding of the molecular determinants of luminal lytic activity. Lipkin and Newmark (19) found that dietary calcium supplementation reduces the proliferation in colonic biopsies of humans at high risk of colon cancer. Using the same study design, Van der Meer et al. (28) found that luminal lytic activity is decreased by oral calcium. In that study, the total bile acid concentration in fecal water (-250 PM) is not changed by talc ium supplementation, but the fatty acid concentration in fecal water is decreased (28). The predominant bile acid in fecal water is deoxycholate (-125 PM; Ref. l), and the predominant long-chain fatty acids are C16-0 (150 ,uM), C18.0 . (150 PM), data). As shown in Fig. and cm1 (190 PM) (unpublished 4, the physiologically relevant combination of oleate with low concentrations of the colonic bile acids deoxycholate and lithocholate drastically increased lytic activity. Therefore, these results may offer a molecular explanation for the calcium-dependent inhibition of lytic activity of fecal water because of the concomitant decrease in fatty acid concentration. An additional effect of calcium supplementation on the hydrophobic-hydrophilic balance of bile acids in fecal water is now under investigation. In conclusion, fatty acids, bile acids, and mixtures of fatty acids and bile acids have similar lytic effects on erythrocytes and Caco-2 cells. Mixtures of fatty acids and bile acids synergistically increase lytic activity because of mixed micelle formation. The lytic activity of these mixed micelles is drastically increased by the amount of fatty acids as well as by increasing hydrophobicity of bile acids. These mixed micelles may be important determinants of lytic activity of human fecal water. Therefore, the results of the present study may contribute to a proper understanding of the mechanism of dietary effects on luminal lytic activity. The authors thank H. Van den Berg and E. Strootman for technical assistance. This study was supported by European Community Grant 663/W1.7 and by Dutch Research Organization Grant 900-562-078. Address for reprint requests: J. A. Lapre, Netherlands Institute for Dairy Research (NIZO), PO Box 20, 6710 BA Ede, The Netherlands. Received
19 September
1991; accepted
in final
form
28 April
1992.
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LYTIC
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EFFECTS
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D. S. M. L. TERMONT,
A. K. GROEN,
AND R. VAN DER MEER
Department of Nutrition, Netherlands Institute for Dairy Research, 6710 BA Ede; and Department of Gastroenterology and Hepatology, Academic Medical Center, 1105 AZ Amsterdam, The Netherlands Lap&, J. A., D. S. M. L. Termont, A. K. Groen, and R. Van der Meer. Lytic effects of mixed micelles of fatty acids and bile acids. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G333-G337, 1992.-It has been hypothesized that bile acids and fatty acids promote colon cancer. A proposed mechanism is a lytic effect of these surfactants on colonic epithelium, resulting in a compensatory proliferation of colonic cells. To investigate the first step of this hypothesis, we studied the lytic activity of fatty acids and physiological mixtures of fatty acids and bile acids. Experiments were performed in both erythrocytes and cultured Caco-2 cells, a model system for intestinal epithelium. Fatty acids with a chain length of 10 C atoms or more were lytic, and the hemolytic activity increased in the order C10:0 < C18:0 < C16:0 < CZZO < C14Z0 < C18:1 = C18:2 but was not dependent on their critical micellar concentration. Addition of a sublytic, submicellar concentration of cholate resulted in the formation of highly lytic mixed micelles. Lytic activity of these mixed micelles was closely associated with their micellar aggregation as determined in parallel incubations using a fluorescent micellar probe. With use of identical concentrations of fatty acids and mixed micelles, lysis of erythrocytes was highly correlated (r > 0.95) with lysis of Caco-2 cells measured by either release of the apical membrane-marker alkaline phosphatase or the cytosolic marker lactate dehydrogenase. This indicates that the cytolytic activity of these surfactants is not cell-type dependent. Addition of bile acids in concentrations corresponding with the total bile acid concentration in human fecal water resulted in an increased lytic activity of fatty acids. The increase in lytic activity was lowest for the primary bile acid cholate and highest for the more hydrophobic, colonic bile acids deoxycholate and lithocholate. These results may contribute to a better knowledge of the relevant determinants of lytic activity of human fecal water. lactate dehydrogenase; alkaline phosphatase; Caco-2 cells; lytic activity; hemolysis; colon cancer DATA show a correlation between a high-fat intake and the incidence of colon cancer (7,34). It has been suggested that this effect is caused by a diet-dependent increase of bile acids and free fatty acids in colon (21, 33). Bile acids instilled intrarectally strongly promote colon cancer in tumor-induction models (24). Proliferation of colonic epithelium is drastically stimulated by bile acids and fatty acids in rodents (5,13, 31, 32), probably because of their intrinsic lytic properties (23, 3 1, 32). Also in humans dietary fat stimulates colonic proliferation (27). This hyperproliferation of coionic epithelium is generally accepted as an important biomarker of the risk for colon cancer (18). Recently, we have shown that lytic activity of bile acids (measured as lysis of erythrocytes) is strongly dependent on their structure and increases with increasing hydrophobicity (29). Lytic activity increases from the primary conjugated (duodenal) to the secondary unconjugated (colonic) bile acids and is associated with their micellar aggregation (29). The physiological relevance of EPIDEMIOLOGIC
0193~1857/92
$2.00
Copyright
these studies has been ascertained in experiments with rats. A diet-induced increase of the bile acid concentration in fecal water results in a higher intestinal lytic activity (X5,16) and in a higher colonic cell proliferation (15). In these animal studies the concentration of bile acids in fecal water was higher than that of fatty acids (15, 16). However, in human fecal water the concentration of fatty acids is in excess of that of bile acids (1, 22, 28). Consequently, the fatty acid concentration in human fecal water might be a relevant determinant of luminal lytic activity in humans. For that reason, we studied the lytic properties of fatty acids in the absence and presence of bile acids using human erythrocytes as a model system for lytic activity. By measuring hemolysis as an endpoint of lytic activity, several studies (9,10,29) have shown that this model system can adequately be used for determining membrane damage by surfactants. To validate the physiological relevance of lysis of erythrocytes by surfactants, we studied the lytic effects of different kinds of surfactants on Caco-2 cells, which are derived from a human colon adenocarcinoma cell line. By culturing in a polarized monolayer, these cells provide an excellent model system to study lytic effects on the apical membrane of intestinal epithelial cells (30). To quantify lysis of these cells, we used both an apical membrane marker (alkaline phosphatase; 14), as well as a cytosolic marker (lactate dehydrogenase). MATERIALS
AND METHODS
Muterials. Human blood was obtained from healthy donors by venipuncture. Human erythrocytes were thoroughly washed and isolated exactly as described by Van der Meer et al. (29). Bile acids, fatty acids, and fat-free bovine serum albumin were acquired from Sigma Chemical (St. Louis, MO) and Fluka (Buchs, Switzerland) and were of the highest purity commercially available. The purity of bile acids and fatty acids used has been verified by gas chromatographic analysis. N-PhenylI-naphthylamine (NPN) was obtained from Eastman Kodak (Rochester, NY), and N-2-hydroxyethylpiperazine-N’2-ethanesulfonic acid (HEPES) from Sigma. Other chemicals used were of analytic grade. Methods. Fatty acids were dissolved in double-distilled water and saponified with potassium hydroxide. After saponification, the pH of the stock solutions was brought to 7.5-8.0. Concentrations of fatty acids were measured with a commercially available enzymatic kit (NEFA-C, Wako Chemicals, FRG). Sodium salts of bile acids were dissolved in double-distilled water. Lithocholate (LC) was solubilized by mixing it with deoxycholate (DC) at a molar ratio of I:3 (LC/DC). Concentrations of bile acids were measured using an enzymatic fluorescence method (Sterognost 3~ Flu, Nycomed, Oslo, Norway; Ref. 2). Lytic properties of fatty acids and mixtures of fatty acids and bile acids were studied using hemolysis of erythrocytes at a final hematocrit of 5%. To avoid peroxidation of the unsaturated fatty acids, the experiments with oleate and linoleate were performed under nitrogen. However, control experiments showed
0 1992 the American
Physiological
Society
G333
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G334
LYTIC
EFFECTS
OF MIXED
that no difference in lytic activity occurred during our shortterm incubations when the experiments were carried out aerobically. After a preincubation of 10 min, erythrocytes were added to tubes containing increasing concentrations of fatty acids and/or bile acids, HEPES (final concentration 50 mM and pH 7.4), and NaCl (to maintain a constant ionic strength of 150 mM). KC1 was used to correct for the amount of potassium in the fatty acid stock solutions, thus maintaining a constant potassium concentration in the incubation of 60 mM. Control experiments showed that this potassium concentration did not affect hemolysis (data not shown). After 15 min of incubation at 37”C, the tubes were centrifuged for 1 min at 10,000 g (Eppendorf 5415). After dilution of the supernatants with double-distilled water, hemolysis was determined by measuring the absorbance at 540 nm. In control experiments it appeared that neither bile acids nor fatty acids affected the absorbance of hemoglobin at this wavelength. Percent hemolysis was calculated by comparing the absorbance of the samples with that of an identical amount of erythrocytes lysed in double-distilled water, which represents 100% hemolysis. Monolayers of Caco-2 cells were grown in microwell plates as described by Velardi et al. (30). After postconfluent cultures were washed with phosphate-buffered saline (pH 7.4), cells were incubated for 45 min at 37°C with solutions containing surfactants as described above. At the end of the incubation time, the medium was collected and the cells were treated with 0.1% Triton X-100. Lactate dehydrogenase (LDH) activity was measured according to Mitchell et al. (ZO), and alkaline phosphatase (ALP) activity according to Bessey et al. (3) in both medium as well as in Triton X-loo-treated cells. Fat-free bovine serum albumin (final concentration 0.6%) was added to prevent interference of surfactants with the spectrophotometric assay of LDH activity. %Lysis was calculated as enzyme activity in medium divided by the total enzyme activity (medium + Triton X-100).
The apparent termined under by using 2 PM scribed by Brito of at least three
critical micellar concentration (CMC) was dethe same conditions, but in the absence of cells, of the fluorescent micellar probe NPN, as deand Vaz (4). Results are given as means t SD experiments.
RESULTS
Figure 1 shows the concentration dependence of the hemolytic effect of laurate (C&J. At low concentrations laurate was not lytic, but above a certain threshold con-
MICELLES
centration significant lysis was observed. The concentration at which 2% lysis occurred is defined as the critical lytic concentration (CLC). In addition, we used the lytic concentration required for 50% lysis (LC,,) as a second parameter to define the lytic curve. To investigate whether addition of bile acids enhances the lytic effect of fatty acids, we used 5 mM of the primary bile acid cholate. Addition of this submicellar concentration of cholate (29) to increasing concentrations of laurate caused a shift of both CLC and LCSO to lower values, which indicates an increased lytic activity (Fig. 1). In the absence of laurate, no lytic activity was observed, which indicates that 5 mM cholate alone did not induce lysis. Figure 2 shows the lytic potential, which is the reciprocal of L&, for a range of fatty acids. No lytic effects were observed with &, and the lytic potential increased from C&o to C1A.0with increasing chain length. The longchain saturated fatty acids (C16.0 and Cl& were less lytic than the medium-chain fatty acids. Oleate and linoleate showed a higher lytic potential than stearate. Figure 2 also shows that the lytic potential of all fatty acids >C10.0 was drastically increased by the addition of a sub&c concentration (5 mM) of cholate. To determine whether this increase in lytic potential is due to the formation of mixed micelles, we determined the CMC of these mixtures using a fluorescent probe. In contrast to bile acids (29), the CLC of fatty acids was not associated with their micellar aggregation (Fig. 3). Addition of a submicellar concentration of cholate induced the formation of mixed micelles. Thus, at concentrations below their individual CMCs, the combination of bile acids and fatty acids induced the formation of mixed micelles. The CLC of these mixed micelles corresponded with their CMC (Fig. 3), which is analogous to the relationship observed for bile acids (29). This indicates that the lytic activity of the bile acid-fatty acid mixtures is caused by mixed micelles. Table I gives the results of the comparison between the effects of surfactants on lysis of Caco-2 cells and on lysis of erythrocytes. It is clearly shown that the different surfactants (bile acids alone, fatty acids alone, and combinations of fatty acids and bile acids) had comparable lytic effects on erythrocytes and Caco-2 cells. Moreover, the
- cholate + 5 mM cholate
0.5
’ 1.0 t 1 CLC LCg
1.5
T
n m
2.0 C12:0 OnM)
in absence Fig. 1. Concentration dependence of lytic effect of laurate CLC, critical and presence of sublytic concentration of cholate (5 mM). lytic-concentration required for 2% lysis; LC&, concentration required for 50% lysis.
8:0
16:0
18:0
18:2
Fig. 2. Lytic effects of fatty acids measured as hemolysis and expressed as lytic potential (l/LC& in absence and presence of sublytic concentration of cholate (5 mM). Bars represent SD (n = 3).
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LYTIC fatty
q
acids
mixed
/ y =x
micelles
/
w
10
OF MIXED
acids
o bile l
EFFECTS
c + c12:o
/
l
c / O/
/
/
oclo:o
/
gV 0 d GDC + c12:o
1
9
,.
DC + C12.0 .
o c12:o
/ /
7
c12:o
+ c o c14:o
/ ,/cl4:0 0.1
16:0
+ c
I 1
+ ’
I 10
1 100 CMC (mM>
Fig. 3. Correlation between critical lytic concentration (CLC) and critical micellar concentration (CMC) for bile acids, fatty acids, and mixtures of bile acids and fatty acids. Note that scales are logarithmic. C, cholate; GDC, glycodeoxycholate; DC, deoxycholate. Mixed micelles consist of different fatty acids combined with 5 mM cholate (CMC and CLC expressed as millimolar fatty acid). For combinations C + C120, GDC + C12.0, and DC + C12.0 different bile acids were combined with 300 PM la&ate (CMC and CLC expressed as millimolar bile acid).
Table 1. Lytic effects of bile acids, fatty acids, and mixtures of bile acids and fatty acids on Caco-2 cells and on erythrocytes Lysis,
%
Surfactant
Caco-2
cells
Erythrocytes LDH
No surfactants C holate 16.5 mM Go., lb.0 mM 20.0 mM C 12:o 0.6 mM 1.4 mM C 1o:o + 5 mM 5.0 mM 10.0 mM C lcj:o + 5 mM 0.3 mM 0.6 mM
oto 99k5 l&O 102tll l&O 79A19
release
ALP
release
6tl
It1
96&l
96tl
6~~1 89k3
2t2 98t3
6tl 5921
3t2 5024
14t2 103t3
lOt5 106t2
G335
MICELLES
time (15 min for erythrocytes vs. 45 min for Caco-2 cells) indicates that erythrocytes are more sensitive to surfactant-induced lysis compared with Caco-2 cells. Finally, to examine the effect of bile acid structure on the lytic activity of mixed micelles, we used an increasing concentration of oleate combined with 250 PM cholate, deoxycholate, or a mixture of deoxycholate and lithocholate in a molar ratio of 3:l. Control experiments showed that 250 PM of these bile acids in the absence of oleate is not lytic. This bile acid concentration corresponds with the total bile acid concentration in human fecal water (1, 22, 28), and oleate is one of the most predominant longchain fatty acids in human fecal water (unpublished data). As shown in Fig. 4, a change in bile acid structure caused a significant change in lytic potential. Addition of the primary bile acid cholate had little or no effect on oleate-induced lytic potential, whereas the secondary bile acid deoxycholate increased its lytic potential. The addition of the mixture of deoxycholate and lithocholate (predominantly present in the colon) induced the highest lytic potential. Comparable results were obtained using laurate as a model for a medium-chain fatty acid and palmitate as long-chain saturated fatty acid. For instance, addition of 250 PM cholate has little or no effect on the lytic potential of laurate or palmitate, whereas 250 PM of deoxycholate plus lithocholate stimulated lytic potential of laurate from 0.83 to 3.0 mM-l and of palmitate from 0.53 to 1.1 mM-l. DISCUSSION
The results of the present study show that the lytic potential of fatty acids is at least of the same magnitude as that of bile acids (9,10,29). Buset et al. (6) have shown that palmitate, oleate, and linoleate decrease the viability of isolated colonocytes as quantified by [3H] thymidine incorporation. This is probably due to the lytic effects of these fatty acids as observed in our present study. In our experiments using an incubation temperature of 3’7”C, lytic activity of the saturated fatty acids increased with
DC/LC
cholate 3021 94t2
DC
cholate 54tlO 8Ok5
57t6 80*13
65t16 93t5
Values are means t SD; n = 3 incubations. Lysis of Caco-2 cells was measured by lactate dehydrogenase (LDH) release and by alkaline phosphatase (ALP) release, and hemolysis by release of hemoglobin. Correlation coefficient between hemolysis and LDH release was 0.97 (hemolysis = 1.03 LDH + 0.9), between hemolysis and ALP release 0.96 LDH release and ALP (hemolysis = 0.91 ALP - 7.2), and between = 0.89 ALP - 5.0). release 0.99 (LDH
J
control
high correlation coefficients of hemolysis compared with Caco-2 lysis (r > 0.95) indicated that the lytic effects are similar in both cell types under these conditions. However, it should be noted that the difference in incubation
b
ncreasing
hydrophobicity
Fig. 4. Effect of 250 PM cholate (C), deoxycholate (DC), and deoxycholate-lithocholate (DC/LC) ( mo 1ar ratio 3:l) on lytic potential of oleate. Bars represent SD (n = 4).
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G336
LYTIC
EFFECTS
OF MIXED
the increase in the number of carbon atoms, which reflects an increasing hydrophobicity. A similar relationship between bile acid hydrophobicity and lytic activity has been reported previously (29). However, palmitic and stearic acids were less lytic than would have been expected from their chain length. This anomalous behavior is probably due to the fact that our incubation temperature of 37°C is below the critical temperature of these long-chain fatty acids (17), so that their lytic activity is decreased because of gel formation (8, 17). As shown by Csordas and Rybczynska (12), this may drastically decrease the fatty acid-induced lysis of erythrocytes. The observed increase in lytic activity with unsaturation of fatty acids (Fig. 2) is in accordance with this explanation, because unsaturation of fatty acids decreases their critical temperature ( 17). The similarity of CMC and CLC values for bile acids and mixed micelles (Fig. 3) shows that hydrophobicity and micellar aggregation are important determinants of their cytolytic effect. This indicates that lysis is due to disruption of the cellular membrane integrity by comicellization of membrane lipids (9, 29). Whether the same mechanism holds for the lytic effects of fatty acids per se is at present not clear. For instance, comparison of our CMC value of 7.1 mM for laurate, which is similar to the value of 7.5 mM published earlier (1 I), with the CLC value of 0.73 mM indicates that there is no association between micellar aggregation of fatty acids and fatty acidinduced lysis of erythrocytes. It should be noted that this lack of association cannot be taken as evidence that lysis is mediated by fatty acid monomers. For instance, the sigmoidal concentration dependence of fatty acid-induced lysis (Fig. 1) may reflect that aggregation of fatty acids is required for its lytic effects. This may indicate that this lytic activity is due to dimers or oligomers with a low aggregation number. Apparently our fluorescent probe does not intercalate with these small structures, but this requires further investigation. The present study showed that mixed micelles are formed at physiological concentrations below their individual CMC. We are not aware of other studies that show associations between submicellar concentrations of bile acids and fatty acids, but similar associations between submicellar concentrations of lysophospholipids and bile salts have recently been reported (25). At present no information on the structure of these aggregates is available. Our results indicate that the formation of mixed micelles of fatty acids and bile acids drastically stimulated lytic activity. A bile acid concentration of 250 PM of secondary bile acids such as deoxycholate or a mixture of deoxycholate and lithocholate increased the lytic activity of fatty acids (laurate, palmitate, and oleate). However, addition of 250 VM of a primary bile acid such as cholate had almost no effect on lytic activity. These results indicate that changes in the hydrophobic-hydrophilic balance of bile acids in fecal water may have a drastic effect on intestinal lytic activity. The significant effects of hydrophobicity of fatty acids and bile acids on micellar aggregation and cytotoxicity may be relevant for the integrity of the intestinal mucosa. The present results show that the lytic effects of these
MICELLES
surfactants on Caco-2 cells and erythrocytes are highly correlated (Table 1) and thus support and extend our recent findings (30). Our resu Its indicate that Caco-2 cells, compared with erythrocytes, are slightly more resistant to surfactant- 1nduced lysis. This can be explained by the high content of glycolipids in the apical membrane of enterocytes (26), because it has been shown that differences in membrane glycolipids could affect the resistance to bile-acid-induced lysis (10). Our results indicate that lysis of enterocytes can be determined with the same accuracy by measuring the release of either an apical (ALP) or a cytosolic marker (LDH). Finally, we have recently demonstrated that a diet-induced increase in lytic activity of rat fecal water correlated highly with in vivo colonic proliferation (15). This also indicates that the in vitro experiments reported in this paper are of relevance to a proper understanding of the molecular determinants of luminal lytic activity. Lipkin and Newmark (19) found that dietary calcium supplementation reduces the proliferation in colonic biopsies of humans at high risk of colon cancer. Using the same study design, Van der Meer et al. (28) found that luminal lytic activity is decreased by oral calcium. In that study, the total bile acid concentration in fecal water (-250 PM) is not changed by talc ium supplementation, but the fatty acid concentration in fecal water is decreased (28). The predominant bile acid in fecal water is deoxycholate (-125 PM; Ref. l), and the predominant long-chain fatty acids are C16-0 (150 ,uM), C18.0 . (150 PM), data). As shown in Fig. and cm1 (190 PM) (unpublished 4, the physiologically relevant combination of oleate with low concentrations of the colonic bile acids deoxycholate and lithocholate drastically increased lytic activity. Therefore, these results may offer a molecular explanation for the calcium-dependent inhibition of lytic activity of fecal water because of the concomitant decrease in fatty acid concentration. An additional effect of calcium supplementation on the hydrophobic-hydrophilic balance of bile acids in fecal water is now under investigation. In conclusion, fatty acids, bile acids, and mixtures of fatty acids and bile acids have similar lytic effects on erythrocytes and Caco-2 cells. Mixtures of fatty acids and bile acids synergistically increase lytic activity because of mixed micelle formation. The lytic activity of these mixed micelles is drastically increased by the amount of fatty acids as well as by increasing hydrophobicity of bile acids. These mixed micelles may be important determinants of lytic activity of human fecal water. Therefore, the results of the present study may contribute to a proper understanding of the mechanism of dietary effects on luminal lytic activity. The authors thank H. Van den Berg and E. Strootman for technical assistance. This study was supported by European Community Grant 663/W1.7 and by Dutch Research Organization Grant 900-562-078. Address for reprint requests: J. A. Lapre, Netherlands Institute for Dairy Research (NIZO), PO Box 20, 6710 BA Ede, The Netherlands. Received
19 September
1991; accepted
in final
form
28 April
1992.
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LYTIC
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EFFECTS
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