Mixture Of Deoxycholate Research Articles

Protein Nitrogen Determination by Kjeldahl Digestion and Ion Chromatography

We report development and validation of a simple, rapid, and accurate method for the quantitation of protein nitrogen, which combines Kjeldahl digestion and ion chromatography with suppressed conductivity detection and requires nanomolar amount of nitrogen in samples (≥10 μg protein). The mechanism of suppressed conductivity detection does not permit analysis of samples containing copper (present in Kjeldahl digestion solution) and aluminum (present in many vaccines as adjuvants) due to precipitation of their hydroxides within the suppressor. We overcame this problem by including 10 μM oxalic acid in Kjeldahl digests and in the eluent (30 mM methanesulfonic acid). The chromatography is performed using an IonPac CS-16 cation exchange column by isocratic elution. The method reduces the digestion time to less than 1 h and eliminates the distillation and titration steps of the Kjeldahl method, thereby reducing the analysis time significantly and improving precision and accuracy. To determine protein nitrogen in samples containing non-protein nitrogen, proteins are precipitated by a mixture of deoxycholate and trichloroacetic acid and the precipitates are analyzed after dissolving in KOH. The method is particularly useful for biological samples that are limited and can also be applied to food, environmental, and other materials.

Woman with hemorrhagic spots on her abdomen

Lipodissolve is a product that is composed of phosphatidylcholine and deoxycholate mixture, and other adjuvant. Lipodissolve injection seems to be performed in many countries for local fat reduction without any legal and scientific evidences of its safety and efficacy despite the US FDA warning.1 Herein, we report a case with agitation and metabolic acidosis following lipodissolve injection.

Wettability of psoralen powders: influence of bile salts on their contact angles and surface free energy components

The purpose of this work was to assess the surface free energy (SFE) of bare and bile-salt-precontacted pellet surfaces of psoralens (lipophilic molecules). This study was carried out according to the approach of van Oss et al. to the solid-liquid interfacial free energy using the Lifshitz-van der Waals component, electron acceptor and electron donor parameters of the SFE. With the help of a hydraulic press, powders of 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP) and 4,5',8-trimethylpsoralen (TMP) were compacted sufficiently to obtain smooth and glossy disc surfaces. One set of pressed discs was used directly after preparation and another set was treated with a bile salt (sodium cholate/sodium deoxycholate mixture) aqueous solution of 0.1 mol l −1 for 2 h at 37°C before droplet deposition. The surface free energies of bare and bile-salt-treated discs were determined by advancing contact angles using polar (water and formamide) and apolar (diiodomethane) standard liquids. The measurements were carried out at 21.5 ± 1.5°C using a surgical microscope fitted with a slanted mirror and equipped with a video camera linked to a computer. In addition, the surface and interfacial tensions of a biphasic system of octanol and water in which psoralens were solubilized were measured using the De Noüy ring method. The results were correlated with the lipophilicity and solubility of these drugs. The data of contact angle values of psoralen bare surfaces indicated the hydrophobicity order 8-MOP < 5-MOP < TMP. The percentages of Lifshitz-van der Waals ( γ s LW) components corroborated this. 8-MOP showed the same acidic and basic values ( γ s AB = 6.5 mJm −2, while γ s AB was equal to 1.8 and 0.4 mJm −2 for 5-MOP and TMP respectively. However, when the psoralens were treated with sodium cholate sodium deoxycholate ( NaC NaDC ) mixture, the presence of micelles on the surface modified the previous data by decreasing the contact angles with polar solvents and γ s LW. In contrast, the total surface free energy of all the psoralens increased, especially the electron onor component ( γ −) and γ s AB. When comparing the three psoralens, 5-MOP behaved peculiarly as it had the lowest γ s − (29.5 mJm −2) and γ s AB (21.4 mJm −2). Theoretical explanations concerning the reactivity of the psoralens vis à vis standard liquids were assumed. The results of this work enabled us to understand qualitatively and quantitatively the types of interaction between the molecules of each studied psoralen and to predict their behaviour when they permeate through a given biological membrane (e.g. human skin). From this work the adsorption of bile salts on to the hydrophobic surface was confirmed for the psoralen case. This result could constitute the beginning of a physicochemical explanation of the digestion step for orally taken psoralens.

The effects of bile salt hydrophobicity on model bile vesicle morphology

The addition of bile salts to vesicles supersaturated with cholesterol induces cholesterol precipitation, an important step in the formation of cholesterol gallstones. To investigate the effects of bile salt hydrophobicity on vesicle morphology, vesicles obtained from supersaturated model bile by density gradient ultracentrifugation, were incubated with mixtures of deoxycholate (DC) and ursodeoxycholate (UDC) with a constant total bile salt concentration of 30 mM but with a varying hydrophobicity index ranging from −0.31 (UDC alone) up to + 0.72 (DC alone) depending on the composition of the mixture. Five days after addition of bile salts to vesicles, cholesterol precipitation was determined microscopically and incubation samples were again subjected to ultracentrifugation to assess the lipid distribution among residual vesicles, mixed micelles, and cholesterol crystals. Structure and size of the isolated residual vesicles were studied by freeze fracture electron microscopy. The control, and samples exposed to hydrophilic bile salt mixtures, consisted of unilamellar vesicles of which more than 75% had a diameter of 50–80 nm. After addition of increasingly hydrophobic bile salt mixtures, multilamellar vesicles with progressively greater diameters (up to 1300 nm) were found, suggesting that vesicle fusion and aggregation took place and might hence be important in the cholesterol precipitation process. Accordingly, crystallization was positively correlated with bile salt hydrophobicity. We conclude that cholesterol crystallization from vesicles depends on the hydrophobicity of the bile salts added, and apparently occurs from fused or aggregated vesicles of extended magnitude and with a multilamellar constitution.

A Novel Cytosolic Component, p40 phox, of Respiratory Burst Oxidase Associates with p67 phox and Is Absent in Patients with Chronic Granulomatous Disease Who Lack p67 phox

Cytosolic components of human neutrophils, p47 phox and p67 phox , deficiencies of which lead to chronic granulomatous disease (CGD), potentiate respiratory burst oxidase translocating from cytosol to membrane upon cell stimulation. In this report we describe a novel cytosolic component, p40 phox , which consistently behaves with p67 phox through immunoprecipitation and column works, and is missing in patients with CGD who lack p67 phox . Although actin has been reported to be involved in O 2 − generation, the p40 phox profile did not correspond to that of actin. The tight association between p40 phox and p67 phox was not affected by treatment with a mixture of deoxycholate and Nonidet P-40, until subjected to SDS-PAGE. Addition of recombinant p67 phox to cytosol did not produce any additional p40 phox in the immunoprecipitate, unlike the additive increment in the band of p67 phox . These results suggest that p40 phox forms a complex with p67 phox in a molar ratio of 1:1, without any free p40 phox in the cytosol.

NADPH-dependent reduction of ubiquinone-1 associated with the superoxide-forming oxidase of pig polymorphonuclear leucocytes

NADPH-dependent ubiquinone-1 reductase activity was present in the phagocytic vesicles of pig polymorphonuclear leucocytes. The apparent K m-value of the reductase for NADPH was 29 μM which is similar to that of the NADPH-dependent superoxide formation. Increase of the quinone-reductase activity by increasing the concentrations of ubiquinone-1 was associated with the decrease of the superoxide forming activity, the rate of the NADPH oxidation being constant independent of the quinone concentration. p-Chloromercuribenzoate inhibited both superoxide formation and reduction of the quinone, whereas low concentrations of cetyltrimethylammonium bromide which inhibit the superoxide formation did not inhibit the reduction of the quinone. The reduction of 2,6-dichlorophenolindophenol which has been shown not to be inhibited by both inhibitors. The quinone-reductase activity could be extracted with a mixture of deoxycholate and Tween 20 which extracts the superoxide forming activity. The observations indicate that a region of the superoxide-forming NADPH oxidase between a mercurial-sensitive site and a site sensitive to the cationic detergent is responsible for the reduction of ubiquinone.

NADPH-dependent reduction of 2,6-dichlorophenol-indophenol by the phagocytic vesicles of pig polymorphonuclear leucocytes.

NADPH-dependent 2,6-dichlorophenol-indophenol (DCIP) reductase activity in the homogenate of phagocytosing pig polymorphonuclear leucocytes was twice that of the resting cells and the activity in the phagocytic vesicles corresponded to the activity increment due to phagocytosis. The apparent Km value of the reductase activity in the vesicles for NADPH was 30 microM, which is similar to that of the NADPH-dependent superoxide (O2-) formation. Increasing the DCIP reductase activity by increasing the DCIP concentration caused a decrease in the O2- -forming activity, the NADPH oxidation rate being constant and independent of the dye concentration. p-Chloromercuribenzoate and cetyltrimethylammonium bromide at low concentrations inhibited the O2- -forming activity of the vesicles without inhibiting the DCIP reductase. Quinacrine inhibited both O2- formation and DCIP reduction. The DCIP reductase activity could be extracted with a mixture of deoxycholate and Tween-20, which extracts the O2- -forming activity. The reductase activity in the extract was enhanced 2-fold by the addition of FAD, and its apparent Km was 0.085 microM. These results indicate that the NADPH-dependent DCIP reductase activity of the phagocytic vesicles is catalysed by a flavin-containing component of the O2- -forming system.

Effect of phospholipid methylation on calcium transport and (Ca 2+ + Mg 2+)-ATPase activity in kidney cortex basolateral membranes

Basolateral membranes isolated from hog kidney cortex, enriched 12- to 15-fold in (Na + + K +)-ATPase activity, were 80% oriented inside-out as determined by assay of oubain-sensitive (Na + + K +)-ATPase activity before and after opening of the membrane vesicle preparation with a mixture of deoxycholate and EDTA. In these membrane preparations 80% of total phosphatidylethanolamine was accessible to trinitrophenylation by trinitrobenzenesulfonic acid at 4°C, while at 37°C all of phosphatidylethanolamine fraction was chemically modified. Phospholipase C treatment resulted in hydrolysis of 80% phosphatidylethanolamine, 40% phosphatidylcholine and 35% of phosphatidylserine. Sphingomyelinase treatment resulted in 20% hydrolysis of sphingomyelin, presumably derived from right-side-out oriented vesicles. Results indicate that phosphatidylethanolamine is oriented exclusively on the outer leaflet of the lipid bilayer of inside-out oriented vesicles. Methylation of phospholipids in basolateral membranes with S- adenosyl[methyl- 3 H] methionine resulted in the three successive methylation of ethanolamine moiety of phosphatidylethanolamine to phosphatidylcholine. The K m for S- adenosylmethionine was 1·10 −4 M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg 2+ was without any effect between pH 5 and 10. Basolateral membranes incubated in the presence of methyl donor, S- adenosylmethionine , exhibited increased (12–15%) (Ca 2+ + Mg 2+)-ATPase activity and increased ATP-dependent uptake of calcium. ATP-dependent calcium uptake in these vesicles was insensitive to oligomycin and ouabain but was abolished completely by 50 μM vanadate. The increase in ATP-dependent calcium uptake was due to an increase in V max and not due to a change in K m for Ca 2+. Preincubation of membranes with S- adenosylhomocysteine , a methyltransferase inhibitor, abolished the stimulatory effect of phospholipid methylation on calcium uptake. Phospholipid methylation at both low and high pH did not result in a change in bulk membrane fluidity as determined by the fluorescence polarization of diphenylhexatriene. These results suggest that phospholipid methylation may regulate transepithelial calcium flux in vivo.

The simultaneous preparation of basolateral and brush-border membrane vesicles from guinea-pig intestinal epithelium, and the determination of the orientation of the basolateral vesicles

A rapid method is described for the simultaneous preparation of both membranes of guinea-pig enterocytes, using simple differential centrifugation techniques. Basolateral membranes were purified on a Percoll gradient and the final yield of (Na + + K +)-ATPase was 12.4% of the original activity with an enrichment factor of 12.6-fold. Purification of the brush-border fraction was achieved by a calcium-precipitation technique. The yield of alkaline phosphatase was 18.9% of the original activity with an enrichment of 17.5-fold. Both fractions could be obtained within 3 h of the original homogenization. The characteristics of the preparations were checked by negative-staining electron microscopy and by the determination of glucose uptake. The orientation of the basolateral vesicles was determined by measuring the Mg 2+-ATPase and (Na + + K +)-ATPase activities and the [ 3H]ouabain binding before and after treatment of the preparation with a mixture of deoxycholate and EDTA which transforms the vesicles into sheets. There was a 60% rise in (Na + + K +)-ATPase activity and ouabain binding, but no change in Mg 2+-ATPase activity. It was therefore concluded that 60% of the original preparation consisted of inside-out vesicles and 40% of membrane sheets.

Influence of Mixtures of Taurocholate, Fatty Acids, and Monolein on the Toxic Effects of Deoxycholate in Rat Jejunum in Vivo

The influence of mixtures of taurocholate (TC), oleic acid (OA), caprylic acid (CA), and monolein (MO) on the toxic effects of deoxycholate (DC) in rat jejunum have been investigated using both a closed loop and perfusion technique. DC induced net secretion of water and electrolytes, inhibited glucose transport and transmural potential difference (PD), and inactivated mucosal "total" and (Na+ -K+)-adenosine triphosphatase. Secretion was reversed to absorption when the instilled or perfused solutions were composed of mixtures of DC, TC and OA; substitution of MO or CA for OA produced a similar effect. DC-induced inhibition of PD, glucose absorption, and mucosal adenosine triphosphatase activity was abolished when DC was mixed with TC and OA. Oleic acid emulsions had no effect on secretion induced by DC. Absorption of DC was inhibited from mixed micellar solutions (TC, OA, DC) but not from pure micellar solutions (TC, DC). These results indicate that the presence of taurocholate and fatty acids or monolein within the intestinal lumen markedly modify a number of the toxic effects of DC on jejunal function. The clinical effects of DC on intestinal function in man may therefore depend on the relative concentrations of other bile salts and lipids within the intestinal lumen.