Chemolysis of gallbladder debris left over after contact litholysis with methyl tert-butyl ether.

Rapid and safe gallbladder clearance from residual, post-MTBE stone debris is believed to be absolutely necessary to reduce stone recurrence after contact litholysis. Because the clearing effect of prokinetic agents is considered an uncertain postdissolution trial, we investigated by in vitro experiments whether and to what extent debris from various cholesterol and "mixed stones" could be removed by direct (topical) chemolysis. Debris from radiolucent cholesterol stones could be dissolved very easily, using the aqueous solvent S-01, composed of EDTA-2Na (1-2%), lauryl sulfobetaine-12 (0.1 M), and 0.1 M sodium carbonate/boric acid buffer, pH 9,5. Its dissolution capacity (DC) was 8.06 +/- 2.3 mg debris/ml and its dissolution efficacy (DE) was 16.2 +/- 4.6 mg debris/ml/hr. Debris from mixed, slightly to moderately calcified stones needed another treatment with S-05, composed of sodium citrate (0.25 M), lauryl sulfobetaine-12 (0.01 M), and citric acid. The initial pH was 5.2. The DC of S-05 ranged from 1.61 +/- 1.1 (debris enriched with Ca-phosphate) to 3.94 +/- 1.3 mg/ml (debris enriched with Ca-carbonate). Stones which did not respond immediately to MTBE because of a thin rim of inorganic or/and organic Ca salts could be made ready for MTBE litholysis by pretreatment with S-01 or S-05 or with a combination of both solvents. Debris containing large portions of biliary mucus could be dissolved most effectively by successive application of S-01 and S-06 (2 M urea).

Potential contribution of optional urease-positive bacteria to idiopathic urinary calcium stone formation. I. Expression of urease activity in bacteria from the urinary tract that are commonly classified as urease-negative.

The aetiology of calcium oxalate stones, which are commonly believed to be sterile, has not yet been fully elucidated. Recent bacteriological studies and investigations using the scanning electron microscope have also shown microorganisms to be present in this type of stone. These microorganisms were assumed not to be able to split urea. To list the most common urease-negative bacteria established in the human urinary system, we isolated apparently urease-negative microorganisms from a consecutive series of 58 urinary stone-forming patients by using standard selecting agars. Pure strains were incubated in an inductive medium lacking all sources of nitrogen except urea. Induction of urease activity was monitored by a test based on the reaction of phenol/hypochlorite with ammonium ions. This test revealed whether the urease negativity of a strain indicated by the selective agar was optional or absolute. All strains we investigated by this method and which were classified by standard methods as urease-negative we found produced urease activity which was clearly measurable, though it was often comparatively small. In the light of these results, the matrix theory of calcium oxalate stone development will need some modifications.

Potential contribution of optional urease-positive bacteria to idiopathic urinary calcium stone formation. II. Microlith formation kinetics in a fermenter model of the urinary tract infected by optional urease-positive microorganisms.

We investigated the effects of weak to moderate urease hydrolysis by optional urease-positive microorganisms in an artificial urine model enriched with calcium phosphate and calcium oxalate in respect of calcium stone formation. The incubation experiments were performed using a discontinuously running fermenter device to simulate the urinary system. The kinetics of cell division rates, pH and ammonium ion production were measured and correlated to crystallite appearance in the incubation medium. Qualitative analyses of the sediments revealed apatite. Investigations using light microscopy and scanning electron microscopy (SEM) confirmed the matrix effect of bacterial glycoproteins. It was shown that initiation of calcium oxalate stone formation is in all probability equally determined by matrix effects and by heteronuclear crystallization if the urinary tract is infected by optional urease-positive bacteria. When urinary inorganic phosphate is present, calcium phosphate nidi are always initially formed, and may subsequently be coated by calcium oxalate.

Mucin-like high molecular mass protein fractions from total pig gallbladder bile mucus, pig gallbladder wall mucus, and total human gallbladder bile mucus.

Native mucin-like complexes were obtained from both pig gallbladder bile and pig gallbladder wall mucus by precipitation, centrifugation, and gel permeation chromatography. Crude preparations by either dialysis (native mucus from bile, and native gallbladder wall mucus) or by precipitation (crude total bile mucus, and lipomucoid) were purified by gel permeation chromatography on Sephacryl S-300HR and Sephacryl S-500HR (Pharmacia). The elution profiles obtained with a reversibly denaturing and detergent-containing eluent showed the same pattern for all samples, although the amounts of the four main fractions differed somewhat. The excluded fraction with the highest carbohydrate portion had an apparent M(r) > 10(7). This fraction and the following included lipomucoid (in physiological solution tightly bound to fraction I), and an eluent-insoluble mucus portion from all samples were characterized by determination of the protein concentration, carbohydrates, sialic acids, and lipids, using standard methods. Sugar analysis was performed by gas-liquid chromatography. Human gallbladder bile was subjected to the same procedures of mucus precipitation and separation. Human gallbladder bile mucus showed identical behaviour to that of pig gallbladder bile mucus, and showed a very similar elution pattern in gel chromatography.

Experimental dissolution of pigment gallstone material using alkaline EDTA and adjuvant bile salts/non-bile salt detergents, thiols and urea, with respect to local chemolitholysis.

In order to enhance the dissolution capacity and the kinetics of topical solvents used in local pigment chemolitholysis, a series of dissolution experiments was performed with intact brown and black pigment stones and with standardized solutes such as pigment stone powder and compressed powder (static disc method). The basic dissolution medium was a 0.1 M boric acid/sodium carbonate buffer (pH 9.5), and the basic lytic agent was EDTA-4Na, working satisfactorily at 1-3 g/dl. It could be demonstrated that the dissolution efficiency of this basic solvent was enhanced significantly in the presence of a detergent (surfacant) and of urea. Among the detergents the zwitterionic (e.g., Sulfobetain-12) and the nonionic types (e.g., a polyoxyethylene ether like Lubrol PX) proved to be most effective. The adjuvant effect of the investigated thiols was disappointing. Only dithioerythritol/dithiothreitol and N-acetylcysteine showed any moderate, if consistent, lytic activity. The highest dissolution rates in dissolving compressed powder standards (disc method) were achieved with the ternary solvent (1% w/v EDTA/80 mM Lubrol PX/1 M urea, pH 9.5). Intact black pigment stones, well known as problematic candidates for chemolitholysis, could be largely dissolved up to approximately 70% of their initial weight. This was not merely a physical disintegration, but a chemical process.

The sugar spectrum of human cholesterol gallstones, mixed and pigment gallstones: combined quantitative analysis of neutral sugars, N-acetylhexosamines, hexuronic and N-acetylneuraminic acids by capillary gas-liquid chromatography.

Glycoproteins were investigated in different types of biliary calculi by a methanolysis procedure of the carbohydrate moiety, the formation of 2,6-dinitro-4-trifluoromethylbenzenesulphonate derivatives of N-acetylhexosamines and N-acetylneuraminic acid, followed by separation, identification and quantification of the liberated monosaccharides using capillary gas-liquid chromatography. This indirect method avoids several analytical problems caused by the presence of bile pigment derivatives; unless these are carefully removed they interfere strongly in the measurement of protein. The carbohydrate contents were much higher in pigment stones than in cholesterol and mixed stones. Rhamnose, a non-mammalian monosaccharide, was identified in most samples, but was significant increased in pigment stones, indicating a bacterial history of this stone type.

Colloid chemical aspects of pigment lithiasis in man: solubilization of calcium bilirubinate by bile salts (a preliminary report).

Calcium bilirubinate, an important constituent of pigment gallstones, can be solubilized in aqueous systems (including bile) to a considerable extent, depending on colloid mechanisms, strongly influenced by the actual ionic strength. Simple bile salt micelles protect colloid calcium bilirubinate against coagulation more effectively than do mixed bile salt-lecithin micelles, in particular when the latter are cholesterol saturated. The dissolved pigment does not enter the bile salt micelle. Pigment stone formation is believed to be due not merely to calcium bilirubinate overproduction, but also to pathological conditions leading to a coagulation of the colloid pigment in bile.

Investigations on common bile duct stones.

Common bile duct stones from 59 consecutive patients who underwent cholecystectomy more than 1 year before radiological detection of choledocholithiasis (group I) and from 31 consecutive patients with choledocholithiasis and gallbladder in situ (group II) were subjected to chemical analysis and compared with the composition of gallbladder stones from the same population (two consecutive biopsy and autopsy series, n = 343). In contrast to cholecystolithiasis, inorganic calcium salts were infrequent in choledocholithiasis while calcium bilirubinate and fatty acid calcium salts played an important role in the composition of common bile duct stones. This is of particular importance in terms of litholysis. With respect to stone pathogenesis, clinical and radiological data were analyzed, as was hepatic bile lipid composition. There was no significant difference in the lithogenic index (percent cholesterol saturation) in cholecystectomized and in non-cholecystectomized patients with choledocholithiasis. The most important cause of stone recurrence in the common duct after surgery was incrustation of unabsorbed suture material, which was the case in 30.5% of group-I patients.

Dynamics of Galactolipids and Plastids in Nonphotosynthetic Cells of Glycine max Suspension Cultures : A Morphological and Biochemical Study.

The age-dependent interrelationship of galactolipids and plastids in heterotrophic cell suspension cultures of Glycine max (soybean) was studied with regard to aging of nonphotosynthetic cells. Cells were propagated in the dark and under illumination with white light, and were harvested at days 7 (end of logarithmic phase), 14, and 21 (extended stationary phase). Electron microscopy revealed in dark-grown cells a proliferating decay of the amyloplast-type plastids, which could be correlated to a decrease of galactolipids. This trend was dramatically reversed in irradiated cultures, where the plastids of day 21 cells appeared rejuvenated. A concomitant increase of galactolipid content in the cells was observed, yet chlorophyll synthesis and photosynthetic activity were not induced. The dynamics of galactolipid contents did not correlate with total lipid contents in dark-grown as well as in irradiated cultures. [(3)H]Galactose served as a radioactive probe for the subcellular localization of galactolipids by electron microscopic autoradiography. Apart from plastids, galactolipids may also be constituents of the plasma membrane. The results render the heterotrophic cell suspension culture a suitable model to study the impact of senescence on plastids of nonphotosynthetic cells.