R. Azoury

Colocalization of cholesterol and hydroxyapatite in human atherosclerotic lesions

SummaryCholesterol and calcium phosphate, the latter in the form of hydroxyapatite, accumulate in atherosclerotic lesions. In this report, we demonstrate that these organic and inorganic constitutents of lesions can accumulate together, closely associated in crystal agglomerates. Using the fluorescent cholesterol probe, filipin, we identified unesterified cholesterol that was associated with calcium granules in tissue sections of lesions. We also have shown that small crystallites of cholesterol can associate with preformed hydroxyapatite crystals in vitro. Scanning electron microscopy couple with energy-dispersive X-ray analysis demonstrated the physical association of many small crystallites of cholesterol with larger crystals of hydroxyapatite. These small crystallites of cholesterol associated with hydroxyapatite stained with filipin. This contrasted with the lack of filipin staining of unassociated larger cholesterol crystals or hydroxyapatite alone. How cholesterol and calcium come to be closely associated in crystal agglomerates within atherosclerotic lesions remains to be determined.

Rapid precipitation of apatite from ethanol-water solution

Abstract Calcium phosphate was precipitated by simultaneous mixing of 9mM–18mM calcium chloride and 5.4mM–10.8mM sodium dihydrogen phosphate at pH = 7.4. After periods of several minutes an amorphous phase was present, as expected. Ethanol added to the initial solutions, at 0.245 mol fraction, effected the crystallization on hydroxyapatite crystals. In the presence of fluoride, a well characterized apatite phase was obtained after one minute.

Inhibition of calcium oxalate crystallization by glutamic acid: Different effects at low and high concentrations

Abstract The effects of L-aspartic acid, alanine and L-glutamic acid on the crystallization of calcium oxalate were followed by two methods: measurements of the changes in calcium ion concentrations and determination of crystal size distribution in the calcium oxalate precipitate. The changes in calcium ion concentration were detected by a specific calcium electrode and are expressed as Discriminating Index (DI) values. Median size and number of crystals per milliliter were determined by Particle Data Counter. Glutamic acid in the range of 0–200 ppm was added to solutions of CaCl 2 which were subsequently mixed with solutions of Na 2 C 2 O 4 to yield CaC 2 O 4 precipitation. At relatively high concentrations of glutamic acid, namely 20–200 ppm, the precipitation of calcium oxalate is retarded. However, at low concentrations, 2.5–20 ppm oxalate precipitation is enhanced. The two independent methods yield well-correlated results. Both L-aspartic acid and alanine did not affect the precipitation of calcium oxalate at similar experimental conditions.

A method for discrimination between calcium oxalate kidney stone formers and normals.

Calcium ion concentration versus time was measured in solutions containing admixture of 10 per cent tested urine of normals and stone-formers, during induced calcium oxalate precipitation. A Discriminating Index was formulated by statistical analysis of the data. It was found that stone-formers and normals differ significantly with respect to the measurements and the Discriminating Indices. An equation to evaluate the odds of stone-forming based on results of an individual test has been derived. The Discrimination Index may be recommended as a diagnostic tool.

Crystallization processes using reverse osmosis

Abstract Supersaturation is one of the main factors controlling the growth of crystals in solution. Narrow crystal size distribution is desirable but often is difficult to achieve. This is mainly because of difficulties which occur in the generation and maintenance of the required levels of supersaturation. In reverse osmosis processes, water is removed selectively from salt solution. While the water recovery proceeds, the residual solution can become supersaturated with respect to dissolved salts. In this study reverse osmosis hollow-fibre membranes have been employed as a means of generating supersaturation in a plug flow system and precipitating calcium oxalate. Significant levels of supersaturation were generated in the reject flow, the level of supersaturation being a function of the residence time of the solution within the hollow fibres. Crystallization and precipitation processes subsequently occurred. The crystals which had been formed in the concentrated flow were characterized by employing size distribution measurement, X-ray diffraction and scanning electron microscopy (SEM). The narrow size distribution and the similarity observed in the SEM micrographs (taken during different experiments) appear to indicate reproducible and constant conditions prevailing in the system; there was a constant generation and maintenance of the supersaturation driving force necessary for salt precipitation.

Reversal of trends in impurity effects on crystallization parameters

Abstract The effects of impurities at varying concentrations on crystallization parameters were studied in two continuous systems. Lead ions affected production rate, median size, growth rates and nuclei density of potassium chloride crystals differently at low and high concentrations of Pb 2+ . The same pattern was evident in calcium oxalate/glutamic acid system. The supersaturation levels in both systems showed also reversal of trends as a function of steady increase of impurities concentration. Literature survey includes papers in which fluctuations of crystallization parameters with increasing impurity concentrations were reported but not commented upon and reports in which various explanations were suggested. Reversal of trends of supersaturation values with increase of impurity concentration was recorded. The presented phenomena propound the existence of at least two mechanisms of impurities effects.

Steroid production in the ovary of the gray mullet (Mugil cephalus) during stages of egg ripening

Abstract Steroid production in the ovary of the mullet, Mugil cephalus , during several stages of gonadal development and following spawning, were examined. Pieces of ovarian tissue were incubated with labeled androstenedione and the steroid metabolites isolated by thin-layer and gas-liquid chromatographic methods and identified by derivative formation and recrystallization to constant specific activity. The results indicate that with increasing development of the ovary there occurs a reduction in the concentration of 5α-reduced steroids, 5α-androstane-3α, 17β-diol, and 3α-hydroxy-5α-androstan-17-one, and a concomitant increase in the δ-4, 3-oxosteroids; the ratio of 5α-reduced to 4-ene-3-oxosteroids declining to about one-seventh of the original value. This ratio is not changed following spawning. During all stages of gonadal development, an active 11β-hydroxylase is present in the ovary producing 11β-hydroxyandrost-4-ene-3, 17-dione, 11β-hydroxytestosterone, and 11-ketostestosterone. Only the concentration of 11-ketotestosterone increases with advancing gonadal development.

Co-crystallization of cholesterol and calcium phosphate as related to atherosclerosis

Abstract Calcification of atherosclerotic plaques occurs very frequently and aggravates the disease. In biological systems, epitaxial relationships between crystal structures may be important in nucleating the deposit of a solid phase. The biologically preferred calcium phosphate species, apatite, and cholesterol crystal have structurally compatible crystallographic faces which allow epitaxial growth of one crystal upon another. The present study describes a new approach to explore, in vitro, the crystallization processes of calcium phosphate (CaP) with cholesterol (CS) and cholestanol (CN) which are related to atherosclerosis. Aqueous solutions containing calcium and phosphate ions or CaP crystals as hydroxyapatite were added into saturated ethanolic solutions of CS or CS and 10% CN. After precipitation, crystals were collected and analyzed by nuclear magnetic resonance (NMR), infra-red (IR), X-ray, scanning electron microscope (SEM-LINK), differential scanning calorimeter (DSC) and atomic absorption. The principal result is the well-formed crystals precipitation when an aqueous solution and CaP seed crystals were added to saturated solutions of CS and 10% CN. Cholesterol-cholestanol dihydrate (CC2W) crystals precipitated in the presence of CaP seeds were compared to the CC2W crystals obtained without the mineral compound. The results of this comparison indicate a special link between crystals of CaP and CC2W, and support the epitaxial relationship between the two kinds of crystals. The potential of CC2W crystals to be precipitated by CaP seed crystals prove likewise the possible significant role of the cholestanol metabolite in the process of cholesterol crystallization and calcification in the arteries.

Evaluation of the relative inhibitory potential of fractionated urinary macromolecules

SummaryUltrafiltration membranes of 10,000 d, 1,000 d and 500 d were used to remove urinary macromolecules from the urine of normal subjects and from the urine of stone forming patients. The filtrated urines were examined for their residual inhibitory potential for calcium-oxalate precipitation, by the discrimination method of Sarig et al. (D.I. test). The results of testing the filtrate were complementary to the information gained by analyses of retentates obtained in successive ultrafiltration. The method has an inherent advantage because the manipulation of solids retained on membranes may inadvertantly modify their inhibitory potential. At least two distinct groups of inhibitors were found in 20 normal urines. The first group has MW above 10,000 d while the second group of inhibitors has MW in the range of 500–1,000 d. The mean of the D.I. values increased dramatically from the normal range (<0.6) to the stone former range (>1.1) (p<0.001) after the 500 d filtration. Some of the normal urines, even after the 500 d filtration, still had a degree of inhibitory potential. This inhibitory potential may be related to the inorganic compounds which were found in the urines. The inhibitory activity of macromolecules with MW above 10,000 d and below 500 δ was negligible in 7 stone formers (SF) urines. The relative contribution of 500–1,000 d macromolecules is the highest both in SF and normal urines. Conclusions: 1) inhibitors in human urine are of wide range in MW; 2) stone formers and normals differ in the level of inhibitor activity at all MW ranges; especially in above 10,000 d and below inhibitors.

May enzyme activity in urine play a role in kidney stone formation

SummaryIt has been found that calcium oxalate stone formers have low UGOT and UGPT activity compared to healthy individuals. The urine of 23 stone formers and 19 controls has been tested for combined UGOT and UGPT activity. The effect of L-aspartic acid, alanine and L-glutamic acid on calcium oxalate precipitation has been tested. Only L-glutamic acid exerted a significant retardation effect at physiological concentrations. As GPT and GOT convert alanine and aspartic acid respectively into glutamic acid, a possible mechanism of retardation of kidney stone formation involving enzyme steps via glutamic acid creation in situ is suggested.