Vesicular Phase Research Articles

Effect of phospholipase C on cholesterol solubilization in model bile: A concanavalin A-binding nucleation-promoting factor from human gallbladder bile

Human bile contains a phospholipase C activity. To examine its pathophysiological importance, the effect of phospholipase C on the dynamics of lipid solubilization and nucleation (cholesterol crystal formation) were investigated in model bile. Phospholipase C from gallbladder bile from patients with gallstones was partially purified by competitively eluting from a concanavalin A (con A)-Sepharose (Sigma, St. Louis, MO) column and incubating with Pronase (Calbiochem, Behring Diagnostics, La Jolla, CA). Phospholipase C activity was resistant to Pronase digestion. When this fraction (concentrated to half the original volume) was mixed with model bile (1:1, vol/vol), a transfer of cholesterol and phospholipid from the micellar to the vesicular phase and an accelerate nucleation time were found concomitant with phospholipid hydrolysis. These effects were prevented by inhibiting the phospholipase C activity with ethylenediaminetetraacetic acid. To confirm that the results were caused by phospholipase C activity and not some other nucleation-promoting factor within the biliary con A preparation, model bile was incubated with bacterial phospholipase C. An identical cascade of events to that found with the partially purified biliary enzyme was observed. Further purification of the con A-bound proteins on DEAE-Sephadex (Pharmacia, Uppsala, Sweden) did not resolve any separate nucleation-promoting activity to that associated with phospholipase C activity. In conclusion, this study has identified phospholipase C as a/the con A nucleation-promoting activity in human gallbladder bile and has characterized a possible molecular mechanism by which cholesterol nucleation is stimulated by this fraction.

High Resolution 1H Nuclear Magnetic Resonance Spectroscopy (NMRS) to Determine the Distribution of Cholesterol (CHOL) and Phospholipid (PL) between the Micellar and Vesicular Phases of Human Bile in Vitro: Pilot Studies

High Resolution 1H Nuclear Magnetic Resonance Spectroscopy (NMRS) to Determine the Distribution of Cholesterol (CHOL) and Phospholipid (PL) between the Micellar and Vesicular Phases of Human Bile in Vitro: Pilot Studies

Distribution of cholesterol between vesicles and micelles in human gallbladder bile: Influence of treatment with chenodeoxycholic acid and ursodeoxycholic acid

The present study aimed at determining the relative distribution of cholesterol between the vesicular and micellar phases in gallbladder bile of gallstone patients (n = 23) and gallstone-free subjects (n = 7). Nine of the gallstone patients were treated with chenodeoxycholic acid and seven were treated with ursodeoxycholic acid, 15 mg/kg/day, for 3 wk before cholecystectomy. The vesicular and micellar fractions in bile were separated by sucrose density gradient ultracentrifugation, and a clear separation between the two phases was obtained. The vesicles were further identified by quasielastic light scattering spectroscopy and appeared to be of a uniform size with a mean hydrodynamic radius of 760 Å. The proportion of cholesterol in the vesicular fraction was significantly higher in the untreated gallstone group (40% ± 4%) compared with the gallstone-free (28% ± 3%), ursodeoxycholic acid (28% ± 3%) and chenodeoxycholic acid (18%± 4%) groups. Despite a low cholesterol saturation of bile in the latter three groups (88% ± 12%, 51% ± 9% and 65% ± 5%, respectively), a considerable part of the biliary cholesterol was carried in the vesicular fraction. The cholesterol/phospholipid ratio in the vesicular fraction averaged between 0.49 and 0.58 in the gallstone, gallstone-free and chenode-oxycholic acid groups, whereas the ursodeoxycholic acid group had a significantly lower ratio of 0.24. The cholesterol/phospholipid ratio was about 0.15 in all micellar fractions. The nucleation time of bile from the gallstone group was short (2 ± 1 days) compared with the gallstone-free, chenodeoxycholic acid and ursodeoxycholic acid groups (23 ± 3, 24 ± 6 and 14 ± 3 days, respectively). These observations give further support to the concept that the mechanism for gallstone dissolution is complex and different for chenodeoxycholic acid and ursodeoxycholic acid. (HEPATOLOGY 1991;13:104–110).

Distribution of cholesterol between vesicles and micelles in human gallbladder bile: Influence of treatment with chenodeoxycholic acid and ursodeoxycholic acid

The present study aimed at determining the relative distribution of cholesterol between the vesicular and micellar phases in gallbladder bile of gallstone patients (n = 23) and gallstone-free subjects (n = 7). Nine of the gallstone patients were treated with chenodeoxycholic acid and seven were treated with ursodeoxycholic acid, 15 mg/kg/day, for 3 wk before cholecystectomy. The vesicular and micellar fractions in bile were separated by sucrose density gradient ultracentrifugation, and a clear separation between the two phases was obtained. The vesicles were further identified by quasielastic light scattering spectroscopy and appeared to be of a uniform size with a mean hydrodynamic radius of 760 A. The proportion of cholesterol in the vesicular fraction was significantly higher in the untreated gallstone group (40% +/- 4%) compared with the gallstone-free (28% +/- 3%), ursodeoxycholic acid (28% +/- 3%) and chenodeoxycholic acid (18% +/- 4%) groups. Despite a low cholesterol saturation of bile in the latter three groups (88% +/- 12%, 51% +/- 9% and 65% +/- 5%, respectively), a considerable part of the biliary cholesterol was carried in the vesicular fraction. The cholesterol/phospholipid ratio in the vesicular fraction averaged between 0.49 and 0.58 in the gallstone, gallstone-free and chenodeoxycholic acid groups, whereas the ursodeoxycholic acid group had a significantly lower ratio of 0.24. The nucleation time of bile from the gallstone group was short (2 +/- 1 days) compared with the gallstone-free, chenodeoxycholic acid and ursodeoxycholic acid groups (23 +/- 3, 24 +/- 6 and 14 +/- 3 days, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of 1H-NMR to determine the distribution of lecithin between the micellar and vesicular phases in model bile.

Biliary cholesterol/phospholipid vesicles play an important role in the pathogenesis of gallstone disease. A prerequisite for the study of the lipid composition and stability of these vesicles is a reliable method to quantify the amount of vesicular lipid. In the present report we show that NMR can be used to determine the distribution of biliary lecithin between the micellar and vesicular phases. The relatively large size of the vesicles leads to such a broadening of the lipid resonances that they are no longer visible in high resolution 1H-NMR spectra. Since micelles are much smaller, lipid present in the micellar phase does give rise to sharp peaks in 1H-NMR spectra. Micellar lecithin can easily be quantified in these spectra. The resonances of cholesterol are masked by the closely related bile acid that is present in a much higher concentration. By determining the difference between chemically and NMR estimated lecithin, the distribution of this phospholipid between the micellar phase and vesicular phase can be assessed. We have compared the results of NMR with gel permeation and density gradient ultracentrifugation. Using standard fractionation conditions, both gel permeation and density gradient ultracentrifugation lead to an underestimation of vesicular lecithin, the difference being minor at relatively high total lipid concentrations (10 g/dl) but large in diluted model bile. We conclude that 1H-NMR can be used to determine the distribution of lecithin in model bile.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of lipid in the electron transfer between NADPH-cytochrome P-450 reductase and cytochrome P-450 from mammalian liver cells

1. The anaerobic NADPH-reduction of the isozymes cytochrome P-450 LM2 and LM4 was used as a functional tool to study the component interaction in reconstituted monooxygenase systems in dependence on different phospholipids. 2. The isozymes were shown to exhibit similar lipid interaction. The lipids generally favour a catalytically active 1:1 complex formation between reductase and cytochrome P-450 as the rate-determining unit in electron transfer. 3. The cytochrome P-450 reduction proceeds in a biphasic reaction. In dilauroyl phosphatidylcholine (DLPC)-reconstituted systems the amount of the fast reduction psi 1 is stoichiometrically limited by the reductase in deficit: psi 1 corresponds to the 1:1 complex formation capability of the reductase. 4. In vesicle-reconstituted systems an 'overstoichiometric' reductase cycling is observed which gives rise to a significantly increased amount of fast reduction psi 1. Reductase cycling is proposed to occur in protein clusters of cytochrome P-450 and reductase in deficit. 5. The dissociation constant KRP of the functionally active reductase-cytochrome P-450 complex has been determined by means of the amount of psi 1 (DPLC) and the rate constant kapp 1 (vesicles) of the fast reduction as a measure of the complex formation in dependence on the protein molar ratio. Taking into account the actual protein concentration in the vesicular lipid phase, KRP in vesicles has been calculated to be about 3 orders of magnitude increased in comparison to DLPC-reconstituted systems. 6. Vmax data reveal almost the same catalytic activity of both reconstitution modes, which justifies DLPC-reconstitution in model investigations. The vesicle-specific increased accumulation of reduced cytochrome P-450 in the steady state as originated by reductase cycling may offer the physiological advantage of an increased capacity of cytochrome P-450 for synergistic substrate conversion via cytochrome b5.

Effect of cholesterol nucleation-promoting activity on cholesterol solubilization in model bile.

Human bile contains a factor with cholesterol nucleation-promoting activity that binds to concanavalin A-Sepharose. In this study we have investigated the effect of this activity on the dynamics of lipid solubilization in supersaturated model bile. A concanavalin A binding protein fraction of human bile was mixed with model bile and the effect on the distribution of cholesterol and phospholipid between mixed micelles and phospholipid/cholesterol vesicles was studied by means of density gradient ultracentrifugation. The nucleation-promoting activity containing fraction induced a transfer of cholesterol and phospholipid from the micellar to the vesicular phase. This led to a decrease in the density of the vesicular fraction. We have also studied the effect of promoting activity on the nucleation time of an isolated vesicle fraction. A decrease of the nucleation time of 10.7 +/- 1.3 to 2.3 +/- 0.3 days was observed. In conclusion, a concanavalin A binding protein fraction from human bile stimulated cholesterol nucleation via a double effect; it increased the amount of vesicular cholesterol and phospholipid, and it also directly induced nucleation of cholesterol from the vesicles.

Visualization by freeze fracture, in vitro and in vivo, of the products of fat digestion.

The technique of freeze fracture was used to visualize triglyceride (TG) hydrolysis and the production of lipolytic products (LPs) in vitro and in vivo in the presence of bile salts (BS). Three systems were investigated: pure lipolytic products (oleic acid and monoolein) in the presence of a pure bile salt (taurodeoxycholate (TDC)), lipolytic products produced from TG by pancreatic lipase in the presence of a variety of bile salts, and lipolytic products produced in the intestine of the killifish, Fundulus heteroclitus, after fat feeding. In vitro, lamellae (4-5 nm thick with 0-8-nm water spacings) appeared on the surface of TG droplets in all preparations with LP/BS molar ratios of 1.5 or greater and spherical vesicles (diameter range, 20-130 nm) were produced from these lamellae. With model killifish bile (taurocholate-cholate 1:1) at LP/BS ratios between 1.5 and 4, homogeneous vesicles or particles (mean diameter, 23.8 nm) were produced by lipase at pH 6.9. In vivo, lamellar product phases also occurred after fat feeding. The smallest visible LP/BS structures by freeze fracture electron microscopy were approximately 20 nm globular particles. Large disc-shaped micelles either were not present or were below the resolution limit of the replica (approximately 10 nm). The dominant aggregated lipolytic product phase was composed of multiple layers of rough-textured lamellae. No evidence of cubic structure was seen. These results show that lamellar and vesicular lipolytic product phases can be intermediates in intestinal fat digestion. However, no evidence for the direct endocytotic absorption of these product phases by the intestinal microvillus membrane was found.

ChemInform Abstract: Kinetics and Mechanism of Transamination Reaction of L‐Phenylalanine with Hydrophobic Pyridoxal in Vesicular and Micellar Phases.

AbstractThe metal ion catalyzed transamination of the pyridoxal derivative (I) with Phe is more effective in vesicular (→ (V)) than in CTAB micelles due to differences in the microenvironmental properties.

Vesicle aggregation in model systems of supersaturated bile: relation to crystal nucleation and lipid composition of the vesicular phase.

The presence of small vesicles composed of phospholipid and cholesterol has recently been demonstrated in super-saturated model and in dilute native human biles by several groups using differing methods. Among compositional factors shown to favor spontaneous vesicle formation and prolong the cholesterol monohydrate nucleation time in model bile systems are dilution, a raised cholesterol saturation index (CSI), and a low bile salt/phospholipid ratio. Time-lapse video-enhanced microscopy of a series of model bile systems representing systematically designed variations in the above factors revealed strong evidence for an essential linkage between antecedent vesicle aggregation and subsequent crystal nucleation. Stability of vesicles was inversely related to their degree of cholesterol saturation, i.e., the greater the degree of vesicular cholesterol saturation, the less their stability (metastability). Instability of vesicles was reflected by their early aggregation followed by rapid cholesterol crystal nucleation. The lowest degree of vesicular cholesterol saturation was found in dilute systems which also exhibited the greatest metastability despite a high degree of cholesterol solubility (raised CSI). Conversely, the more concentrated and least metastable systems exhibited both rapid vesicle aggregation and rapid onset of crystal nucleation. These systems, while influenced by the other compositional factors, were found to have a high degree of vesicular cholesterol saturation, i.e., cholesterol/phospholipid molar ratio = 2.0. An additional finding was the extreme variability in the proportionate distribution of total solution cholesterol distributed to the vesicular phase, i.e., from zero to as high as 37%. Higher solute concentration, raised bile salt/lecithin ratio, and raised CSI were interactive and almost equally capable of increasing the proportionate amount of cholesterol in the vesicular phase. In conclusion, lipid compositional differences in model bile systems drastically affect the cholesterol saturation of spontaneously formed phospholipid-cholesterol vesicles. This effect, in turn, exerts a potent influence upon the metastability of vesicles, subsequently affecting the cholesterol crystal nucleation time.

Kinetics and mechanism of transamination reaction of L-phenylalanine with hydrophobic pyridoxal in vesicular and micellar phases

A hydrophobic pyridoxal derivative quaternized at the pyridyl nitrogen with a double-chain segment (PL +2C16) was embedded in the single-walled vesicle of NN-dihexadecyl-Nα-[6-(trimethyl-ammonio)hexanoyl]-L-alaninamide bromide (N+C5Ala2C16), and the pyridoxal moiety was fixed in the hydrogen-belt domain of the vesicle. While the transamination of L-phenylanine (L-Phe), a hydrophobic α-amino acid, with PL +2C16 in the vesicle and the hexadecyltrimethylammonium bromide (CTAB) micelle proceeded slowly to afford the pyridoxamine derivative (PM +2C16) and β-phenylpyruvic acid, addition of metal ions to the equilibrium mixture of the aldimine Schiff's base (ASB), PL +2C16, and L-Phe caused acceleration of the overall transamination rate. The transmination was most effectively catalysed by copper(II) ions in the N+C5Ala2C16 vesicle and the CTAB micelle. The catalytic activity of copper(II) ions was so enhanced as to allow significant accumulation of the carbanion chelate, derived from the ASB chelate by α-hydrogen removal, as an intermediate in the aldimine–ketimine isomerization. The reactivity of the overall copper(II)-catalysed transamination was greater in the vesicle than in the micelle and primarily controlled by the collapse ratio of the copper(II)–carbanion species as clarified by detailed kinetic analysis.

Contribution of vesicular and micellar carriers to cholesterol transport in human bile.

A nonmicellar, bile salt-independent mode of cholesterol transport in human bile involving phospholipid vesicles was recently reported by our group. In the present study, we have investigated the relative contribution of the phospholipid vesicles and mixed bile salt-phospholipid micelles to cholesterol transport in human hepatic and gallbladder biles. The vesicles (ca 800 A diameter) were demonstrated by quasi-elastic light scattering (QELS) in fresh bile and after chromatography. Gel filtration under conditions that preserved micellar integrity demonstrated that biliary cholesterol was associated with both vesicles and micelles. At low bile salt concentration, the vesicular phase was predominant and most of the cholesterol was transported by it. With increasing bile salt concentrations, a progressive solubilization of the vesicles occurred with a concomitant increase in the amount of cholesterol transported by micelles. The vesicular carrier may be of particular biological significance for cholesterol solubilization in supersaturated biles.

Vesicular and ulcerative infections of the mouth and oropharynx.

Many infectious diseases involve the mouth and oropharynx. Classification by age of patient, type of lesion, and local or systemic involvement provides the clinician with important diagnostic information. Differential diagnosis rests on adequate history and physical examination. Lesions with vesicular phases are usually viral; ulcerative lesions may be bacterial, viral, fungal, mycobacterial, or spirochetal. Easily performed staining techniques and cultures usually provide useful diagnostic information. Other studies, such as biopsy, serologic evaluation, and electron microscopy may occasionally be necessary.

Solitary Cysts of the Kidney

THE causative factors involved in the production of solitary cysts of the kidney have been subject to considerable discussion for a long period of time. Cysts of the kidney in general have long been recognized by anatomists. In fact, they were described by Fabry of Hilden who died in 1624, and have been studied by other distinguished anatomists, including Willis and Morgagni. However, the exact etiology has never been satisfactorily determined. Etiologic Theories.—One etiologic theory explains the development of the cyst on the basis of a mechanical blockage of the collecting tubules, with subsequent retrodilatation. Other authors contend that it is due to a retention incident to interstitial nephritis. A third theory states that the cyst may be caused by a persistence of the “anlagen or rudiments of uriniferous tubules in the vesicular phase of development and their later expansion.” Still another hypothesis is that failure of the S-shaped anlagen of uriniferous tubules to unite with the straight collecting ducts may be the causative factor. The failure of the uriniferous tubules to join the collecting ducts of higher orders (later generations) after having become detached from such ducts of a lower order is also considered as involved in the production of cysts. The belated appearance of disconnected cavities in a uriniferous tubule which had remained solid during its formation has also been presented as an instrumental factor. Finally, the secondary compression of collecting or secretory tubules caused by local inflammatory processes, as congenital syphilis, is considered by others to offer a satisfactory explanation for the production of cysts. Recently, Hepler (1) has experimentally produced such cysts in the kidney of a rabbit by fulgurating a papilla and ligating the artery which supplied the same pyramid. He thus produced an obstruction to urinary secretion and an anemia of the obstructed secretory tissue. Symptomatology.—In many cases, the cyst is relatively small and does not interfere with renal function, and hence produces no symptoms. In such instances, the finding of the cyst is coincidental at necropsy. When the cyst enlarges it may attain sufficient size to exert pressure on adjacent organs, or the presence of a mass may be noted by the patient. Occasionally a dull dragging or aching in the loin or hypochondrium may be experienced. As a general rule, urinary symptoms do not predominate, although in rare instances hematuria has been reported. Diagnosis.—As stated previously, the small cysts are asymptomatic; no symptoms referable to the urinary tract occur and their presence is not suspected. The larger cysts are usually confused with hydronephrosis, polycystic kidney, ovarian cysts, and occasionally renal neoplasm. The roentgenogram may show the outline of the cyst, especially if it arises from the lower pole of the kidney and has attained moderate size.