Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane.

The zwitterionic detergent CHAPS, a derivative of the bile salts, is widely used in membrane protein solubilization. It is a "facial" detergent, having a hydrophilic side and a hydrophobic back. The objective of this work is to characterize the interaction of CHAPS with a cell membrane. To this aim, erythrocytes were incubated with a wide range of detergent concentrations in order to determine CHAPS partition behavior, and its effects on membrane lipid order, hemolytic effects, and the solubilization of membrane phospholipids and cholesterol. The results were compared with those obtained with the nonionic detergent Triton X-100. It was found that CHAPS has a low affinity for the erythrocyte membrane (partition coefficient K=0.06mM(-1)), and at sub-hemolytic concentrations it causes little effect on membrane lipid order. CHAPS hemolysis and phospholipid solubilization are closely correlated. On the other side, binding of Triton X-100 disorders the membrane at all levels, and has independent mechanisms for hemolysis and solubilization. Differential behavior was observed in the solubilization of phospholipids and cholesterol. Thus, the detergent resistant membranes (DRM) obtained with the two detergents will have different composition. The behaviors of the two detergents are related to the differences in their molecular structures, suggesting that CHAPS does not penetrate the lipid bilayer but binds in a flat position on the erythrocyte surface, both in intact and cholesterol depleted erythrocytes. A relevant result for Triton X-100 is that hemolysis is not directly correlated with the solubilization of membrane lipids, as it is usually assumed.

Isolation of acetylated bile acids from the sponge Siphonochalina fortis and DNA damage evaluation by the comet assay.

From the organic extracts of the sponge Siphonochalina fortis, collected at Bahía Bustamante, Chubut, Argentina, three major compounds were isolated and identified as deoxycholic acid 3, 12-diacetate (1), cholic acid 3, 7, 12-triacetate (2) and cholic acid, 3, 7, 12-triacetate. (3). This is the first report of acetylated bile acids in sponges and the first isolation of compound 3 as a natural product. The potential induction of DNA lesions by the isolated compounds was investigated using the comet assay in lymphocytes of human peripheral blood as in vitro model. The results showed that the administration of the bile acid derivatives would not induce DNA damages, indicating that acetylated bile acids are nontoxic metabolites at the tested concentrations. Since the free bile acids were not detected, it is unlikely that the acetylated compounds may be part of the sponge cells detoxification mechanisms. These results may suggest a possible role of acetylated bile acids as a chemical defense mechanism, product of a symbiotic relationship with microorganisms, which would explain their seasonal and geographical variation, and their influence on the previously observed genotoxicity of the organic extract of S. fortis.

The use of ASB-14 in combination with CHAPS is the best for solubilization of human brain proteins for two-dimensional gel electrophoresis.

Protein extraction is the most important step to reveal a proteome by Two-Dimensional Gel Electrophoresis. Usually, the urea/thiourea based standard protein extraction buffer (SB) is combined with detergents with the aim of achieving better resolution and solubilization of different classes of proteins. In order to produce better gels and achieve the greatest spot resolution of Human Brain Proteins, comparisons using 2-DE of extracted proteins from Human Brain Frontal Cortex with SB constituents (7M Urea, 2M Thiourea and 100mM DTT) were made, using different detergent compositions in the buffer. SB preparations in combination with CHAPS and ASB-14 as well as with ASB-16 (reported for the first time in 2-DE experiments) have been tested. Our results confirm that the most efficient solubilizing solution for 2-DE analysis of cytosolic and membrane Human Brain Proteins is SB combined with 4% CHAPS and 2% ASB-14.

Effect of surfactants and natural detergents on phosphatidylcholine synthesis in photoreceptor membranes.

The synthesis of phosphatidylcholine (PC) in rod outer segments (ROS) catalysed by lysophosphatidylcholine acyltransferase and phosphatidylethanolamine N-methyltransferase (PE N-MTase) was studied and the effects of natural (FA and lysophospholipids) and synthetic (Triton X-100, deoxycholate and CHAPS) surfactants was evaluated. In all experimental conditions used, incorporation of labelled oleate into lysophosphatidylcholine (lysoPC) was at least 40 times greater than oleate incorporation into any other lysophospholipid. Acylation of lysoPC was slightly affected by Triton X-100 and was totally inhibited in the presence of 10 mM sodium deoxycholate (NaDOC) or CHAPS. Below their critical micelle concentration (cmc) Triton X-100 and NaDOC stimulated acylation of all ROS lysophospholipids analysed. The activity of PE N-MTase was stimulated at detergent concentrations below the cmc and inhibited at concentrations above the cmc for all three detergents tested. The effect of FA with differing degree of unsaturation on PC synthesis was evaluated. Oleic acid (10 microM) inhibited methyl group incorporation into total PC, whereas from 100 microM onward, the methylating activity increased with preferential synthesis of PC. Docosahexaenoic acid, in turn, inhibited PE N-MTase activity at every concentration tested. These results suggest that PC synthesis in ROS membranes is modified by bioregulators and surfactants altering the physico-chemical state of the membrane.

Purification of fibroblast growth factor-2 from human placenta using tri(n-butyl)phosphate and sodium cholate.

Tri(n-butyl)phosphate (TNBP) and sodium cholate (SC) mixtures have been used to inactivate lipid-enveloped viruses like HIV and hepatitis B. We exploited the use of this combination to purify fibroblast growth factor-2 (FGF-2) from human placenta. Human placentas were extracted in the presence of 0.3% TNBP/0.2% SC and the clarified homogenate was adsorbed to S-Sepharose. The active fractions were further loaded onto a heparin-Sepharose column and purified FGF-2 was eluted with 2.0 M NaCl. FGF-2 purified this way was indistinguishable from FGF-2 purified without TNBP/SC in the extraction step in terms of yield, specific activity and biological response. The lipid-enveloped vaccinia virus was used in a parallel experiment to evaluate the inactivation capacity of our protocol. Under the conditions described here, the combined use of TNBP/SC did not eliminate but reduced significantly the number of vaccinia virus PFUs by log 2-3.

Phospholipid solubilization during detergent extraction of rhodopsin from photoreceptor disk membranes.

The solubilization of rhodopsin and phospholipids from disks prepared from bovine retinal rods was studied using five different detergents. The relative amounts of rhodopsin and lipid extracted during membrane solubilization differed dramatically with the nature of the surfactant; the two nonpolar detergents, Emulphogene (polyoxyethylene-10 tridecylether) and octylglucoside, removed more protein than lipid; two bile salt-related detergents, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (Chaps) and taurocholate, released relatively more lipid than protein; and digitonin, which shares characteristics with both groups of detergents, extracted more lipid per mole of rhodopsin than the former two but less than the latter two. Solubilization was temperature-dependent with all five detergents, though particularly so with octylglucoside: concentrations adequate for the total micellation of disks at 23 degrees C were ineffectual at 4 degrees C. In total solubilizates of disks, the amount of lipid recovered in rhodopsin-lipid-detergent micelles showed a closer correlation with the critical micellar concentration (CMC) than with the chemical nature of the detergent (octylglucoside > taurocholate > Chaps > digitonin > Emulphogene). The higher the CMC, the larger the amount of lipid associated to the solubilized rhodopsin and the larger the amount of lipid reassociated to rhodopsin upon surfactant dilution. For all five detergents, the lipid progressively extracted from disks during solubilization was relatively richer in phosphatidylcholine (PC) than the lipid in the original membranes. The lipid which tended to be associated with rhodopsin in protein-lipid-detergent mixed micelles was also consistently richer in PC than that present in lipid-detergent micelles. Bleaching of solubilized rhodopsin decreased the amount of lipid in protein-lipid-detergent micelles. Rhodopsin photolytic transitions were faster in nonionic than in bile salt-related detergents.

Endothelium inactivation in in vitro perfused vascular beds. Comparison of methods.

In order to choose the best procedure to inactive the endothelium from vascular beds perfused in vitro, we compared four methods: perfusion with sodium deoxycholate 0.3% for 30 sec; 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate 0.3% (CHAPS) for 2.5 min; collagenase 0.2% for 15 min, and distilled water for 10 min, using the mesenteric arterial bed (MAB) of the rat. The effectiveness of the treatments used to inactivate the endothelium was assessed functionally by using acetylcholine and sodium nitroprusside and histologically using light microscopy. Phenylephrine was used to test the contractile properties of the preparations after each treatment. After collagenase, distilled water, and CHAPS treatment, a potentiated response to phenylephrine was observed, whereas sodium deoxycholate treatment did not modify phenylephrine-induced responses. Acetylcholine-induced responses were reduced by collagenase (60% reduction), CHAPS (30% reduction), and distilled water (52% reduction) treatment, and sodium deoxycholate completely abolished acetylcholine-induced responses. Except after collagenase treatment, smooth muscle relaxant responses were not altered. Medial smooth muscle cells displayed an unchanged morphology, appearing similar to those in control mesenteric arterial beds, except for collagenase and distilled water. Despite the fact that sodium deoxycholate treatment completely abolished acetylcholine-induced response, endothelial cells were still found. No treatment totally removed endothelial cells. In conclusion, we suggest that sodium deoxycholate treatment is the best procedure to inactivate endothelial cells from vascular beds perfused in vitro since it completely abolished endothelium-dependent relaxation and did not interfere with smooth muscle vasodilating and contracting properties.

Vasoconstrictive effect of bile acids on isolated human placental chorionic veins.

Intrahepatic cholestasis of pregnancy (ICP) is a rare complication of late pregnancy associated with a high rate of premature delivery, antepartum fetal death and fetal hypoxia. Since the principal biochemical feature of ICP is a marked elevation of maternal serum bile acid levels, a role of these substances in the pathophysiology of fetal complications has been suggested. In this study, the effect of bile acids on isolated human placental chorionic veins is described. High concentrations of bile acids, especially cholic acid, have a dose-dependent vasoconstrictive effect, which suggests that these substances could exert a detrimental effect on the fetus by increasing the resistance in chorionic veins through a vasospasm of the placental chorionic surface. An abrupt reduction of the oxygenated blood flow at the level of the placental chorionic plate may cause an acute impairment of the fetal perfusion and oxygenation, leading to fetal asphyxia. This is the first report that provides experimental evidence of the possible role of bile acids in those mechanisms that trigger fetal asphyxia in pregnancies complicated by ICP.

Effects of bile salts on energy metabolism and acid secretion by the isolated toad gastric mucosa.

The effects of bile salts on energy metabolism and acid secretion were investigated in the amphibian gastric mucosa in vitro. Serosal exposure to deoxycholate (0.2-2 mM), cholate (2-8 mM), or taurocholate (4-80 mM), at pH 7.4, decreased acid secretion depending on the concentration and time of exposure. Mucosal application of cholate and taurocholate at pH 5.6 caused a more pronounced reduction in the apparent rate of acid secretion. Oxygen uptake and substrate oxidations were significantly inhibited by bile salts in a dose-dependent manner. At pH 7.4, 1 mM deoxycholate inhibited the respiration by 49% and the rates of oxidation by 51%, 52%, 78%, 74%, and 54% of control values, for glucose, pyruvate, succinate, acetate, and butyrate, respectively. Cholate and taurocholate were found to be less potent than deoxycholate. Tissue adenosine triphosphate concentration was decreased by 13%, 57%, and 67% with 0.5, 1, and 2 mM deoxycholate, respectively. We believe the impairment of energy metabolism could be involved in the mechanism of bile salt injury to the gastric mucosa.

In vivo cholesterol removal from liver microsomes induces changes in fatty acid desaturase activities.

Rats fed a 1% cholesterol and 0.5% cholate diet for 21 days were transferred to a sterol-free diet after variable periods of time. The effect of cholesterol removal on liver microsomal composition and fatty acid desaturases was studied. Some changes were already observed after 1 day. However, after 21 days of a sterol-free diet, the cholesterol content of liver microsomes decreased as well as that of phosphatidylcholine. So did the cholesterol/phospholipid ratio. Phosphatidylinositol, phosphatidylserine and sphingomyelin slightly increased along with time. The total fatty acid composition was altered by a decrease in monounsaturated acids and an increase in the saturated acids, palmitic and stearic acids. The arachidonic acid content rose. A similar pattern of change was found in the fatty acid composition of the main phospholipids: phosphatidylcholine and phosphatidylethanolamine. delta 9-Desaturase activity steadily decreased along with cholesterol removal, whereas delta 5- and delta 6-desaturase activities were enhanced towards the end of the removal period. The microsomal membrane became more 'fluid', according to the decrease of fluorescence anisotropy of the 1,6-diphenyl-1,3,5-hexatriene incorporated into the membrane.

[Effects of choledochal perfusion with biliary acid solutions on activity of the sphincter of Oddi (author's transl)]. / Efeitos da perfusão do colédoco com soluções de acidos biliares sobre a atividade do esfincter de Oddi.

The changes of sphincter of Oddi's resistance, induced by choledochal perfusion of conjugated (taurocolic) and non-conjugated (colic) biliary acid solutions, in anesthetized dogs, were studied. The perfusions were made at a constant flow and intracholedochal pressures were registered. The mean number of contractions per minute, the mean maximal pressures and the mean minimal pressures in each study periods were analysed. The choledochal perfusion with the biliary acids solutions induced a slight but significative increase in sphincteric resistance. After 15 minutes, the perfusion with colic acid solution induced maximal pressures significantly more elevated than the ones observed with taurocolic acid solution. The non-conjugated solution induced a pressure tracing significantly distinct from the tracing observed with the conjugated acid solution. No changes in resistance were observed with a 2% NaCl solution. This implies that the observed changes in resistance were not related to osmotic stimulation of the sphincter of Oddi.