Lipid vesicle fusion induced by phospholipase C activity in model bile.

Using a system of phosphatidylcholine-cholesterol vesicles to model the vesicle phase of mammalian bile (1:1 molar ratio) we evaluated whether very small amounts of C. perfringens phospholipase C activity (0.5-6.5 nmol/min per ml) could lead to vesicle fusion, a precursor step for cholesterol precipitation in gallbladder bile. Quasielastic light scattering spectroscopy (QLS) was used to monitor vesicle growth and aggregation in model bile (0.89 mM total lipid) in the presence of phospholipase C. Vesicle growth over 2 h could be detected with phospholipase activity as little as 0.5 nmol/min per ml. Vesicle growth was sustainable over days in the absence of Ca2+ once as little as 3-7 mol% diacylglycerol had been generated as a result of the initial phospholipase C treatment. The presence of fusion intermediates was confirmed using transmission electron microscopy. In addition, kinetically slow vesicle fusion with intravesicle content mixing and minimal leakage was also confirmed by fluorescence spectroscopy using two populations of vesicles containing 5 mM TbCl3 or 50 mM dipicolinic acid. Efficient fusion (40% maximum fluorescence) was obtained at 30 min at 25 degrees C with phospholipase C activity. This level of enzyme activity approximates that found in human gallbladder bile (1.2 nmol/min per ml). We conclude that the hydrolysis products of phospholipase C activity can, in very small amounts (3-7 mol% diacylglycerol), lead to destabilization and fusion of cholesterol-saturated biliary vesicles. A reappraisal of the importance of phospholipase C hydrolysis products in the pathogenesis of cholesterol gallstones is warranted based on these observations.

Interconversions of lipid aggregates in rat and model bile.

The dynamic interchange of cholesterol and the phase transition between nonmicellar and micellar aggregates in rat and model bile were characterized with gel-permeation chromatography, quasi-elastic light scattering, turbidity measurements, and by radiolabeling lipid aggregates in bile. Cholesterol partitioned into either the micellar or nonmicellar phases independent of the lipid aggregate structure. In model bile, increasing bile salt concentrations led to a decrease in the relative proportion of nonmicellar aggregates beginning at 5 mM taurocholate (TC), while the relative cholesterol content of the nonmicellar fraction increased from 1.0 to 2.7 +/- 2.0 (means +/- SD). In rats, creation of a biliary fistula resulted in a decrease of bile salts from 41 to 4 mM. Mixed micelles increased from 25 to 120 A in radius, while nonmicellar aggregates increased from 180 to 800 A in radius. Addition of TC to model bile (cholesterol:lecithin = 1:1) vesicles with total lipid concentrations less than 7 mM yielded a progressive shift of vesicles (450 A) to mixed micelles (30 A). For mixtures with higher total lipid concentrations, addition of TC promoted substantial vesicle aggregation and resulted in formation of a third phase containing lipid aggregates larger in size than the initial vesicles. These results suggest that rapid exchange of cholesterol occurs in bile and that significant remodeling of vesicles can occur. These alterations in vesicles include both enrichment in cholesterol content and formation of larger aggregates during increases in bile salt concentration.

Baclofen overdose with cardiac conduction abnormalities: case report and review of the literature.

Cardiac conduction abnormalities and hypertension developed in a patient who ingested approximately 500 mg of baclofen (Lioresal). The patient also exhibited the more common features of baclofen overdose including coma, respiratory depression, hypotonia, and hyporeflexia. A review of the literature and a discussion of the interesting manifestations and treatment of baclofen overdose are included.

Urinary thromboxane B2 and prostaglandin E2 in the hepatorenal syndrome: evidence for increased vasoconstrictor and decreased vasodilator factors.

Vasodilatory prostaglandins function to maintain renal perfusion in patients with cirrhosis and ascites. To evaluate the potential contribution of the vasodilator prostaglandin E2 and the vasoconstrictor metabolite thromboxane B2 to the development of the hepatorenal syndrome, we measured urinary excretion of these products in 14 patients with hepatorenal syndrome and in control populations with acute or chronic liver or kidney failure. Radioimmunoassay measurements were confirmed by bioassay and by mass spectrometry. Prostaglandin E2 was decreased compared with healthy controls (2.2 +/- 0.3 vs. 6.3 +/- 0.8 ng/h, p less than 0.01) and compared with acute renal failure (9.6 +/- 2.1 ng/h) and with alcoholic hepatitis (9.2 +/- 3.3 ng/h). Thromboxane B2 concentration was normal in patients with alcoholic hepatitis (0.12 +/- 0.02 vs. 0.15 +/- 0.03 ng/ml) and minimally increased in acute renal failure (0.18 +/- 0.15 ng/ml), but markedly elevated in hepatorenal syndrome (0.69 +/- 0.15 ng/ml, p less than 0.001). Urinary thromboxane B2 concentration fell with improved renal function in 3 patients who survived. These data suggest an imbalance of vasodilator and vasoconstrictor metabolites of arachidonic acid in patients with the hepatorenal syndrome.

Purification and chemical characterization of human acidic alpha-D-mannosidase.

The acidic alpha-D-mannosidase (EC 3.2.1.24) has been purified from human placentae. Milligram quantities of the enzyme were obtained from several placentae, using a step-wise purification procedure which includes Con A-Sepharose treatment, acetone precipitation, heat treatment, DEAE-cellulose column chromatography and preparative disc electrophoresis. A high degree of purity of the purified enzyme was demonstrated by acrylamide gel electrophoresis, isoelectric focusing, and sedimentation equilibrium centrifugation. Immunological homogeneity of the preparation was demonstrated by a single precipitin line between the antiserum and purified, or partially purified enzyme preparation. The amino acid and carbohydrate composition of the enzyme was determined. The enzyme was found to be a glycoprotein containing 13.5% carbohydrate. The molecular weight of the enzyme was estimated at 205,000 +/- 18,400.

Dual effects of antidiuretic hormone on urinary prostaglandin E2 excretion in man.

To evaluate the relationship of renal prostaglandin synthesis to urine-concentrating mechanisms, 14 healthy subjects received antidiuretic hormone (ADH) and the nonpressor ADH analog desamino-d-arginine vasopressin (dD'AVP). Endogenous ADH was increased by water deprivation. Urinary prostaglandin E2 (PGE2) was measured by RIA and by bioassay. ADH, dD'AVP, and dehydration each reduced urinary volume by a similar amount (from 582 +/- 66 to an average of 141 +/- 13 ml/4 h) and similarly increased osmolality (from 231 +/- 13 to 721 +/- 31 mosmol/kg). Dehydration and dD'AVP reduced PGE2 from 44 +/- 4 to 25 +/- 5 and 30 +/- 5 ng/4 h, respectively (P less than 0.01), suggesting an inverse correlation of PGE2 with urine osmolality (r = -0.48; P less than 0.005). In contrast, ADH increased urinary PGE2 to 102 +/- 23 ng/4 h (P less than 0.02). Infusions of another vasoconstrictor peptide, angiotensin II, to six of the subjects doubled urine osmolality and also increased urinary PGE2 excretion. These data do not support the theory that the antidiuretic effect of ADH enhances PG synthesis; instead, the data indicate that ADH has two effects on PGE2 excretion: 1) stimulation, presumably by pharmacological pressor activity, and 2) inhibition by antidiuresis.