Calcium Oxalate Formation Research Articles

Effect of dietary moisture and sodium content on urine composition and calcium oxalate relative supersaturation in healthy miniature schnauzers and labrador retrievers

The aim of this series of studies was to evaluate two possible feeding strategies as methods for reducing the risk of calcium oxalate (CaOx) formation in two breeds of healthy dog. The studies compared the effect of dietary moisture (Study 1) and dietary sodium (Na), (Study 2) on urine composition of labrador retrievers (LR) and miniature schnauzers (MS). A nutritionally complete dry dog food was fed to 16 dogs (eight LR, eight MS; Study 1) and 15 dogs (seven LR, eight MS; Study 2) for 24 days (Study 1), or 36 days (Study 2). The dogs were fed the diet alone (7% moisture, 0.06 g Na/100 kcal), or supplemented with deionised water to 73% moisture (Study 1), or dietary Na, to deliver 0.20 or 0.30 g Na per 100 kcal (Study 2). Urine pH, volume, specific gravity, and concentrations of 12 analytes were measured for each dog. Urinary relative supersaturations (RSS) with CaOx were calculated from these values. The effects of supplemental Na or water were established using t tests (Study 1) or analysis of variance, and multiple range tests (least significant difference) (Study 2); P<0.05 was considered significant. Increasing dietary moisture significantly increased total moisture intake ( P=0.001), and reduced urine specific gravity ( P=0.003), urinary oxalate concentration ( P=0.04), and CaOx relative supersaturation ( P=0.04) in the MS. Urinary parameters remained unchanged in the LR, indicating that feeding a high moisture diet may reduce the risk of CaOx formation in high-risk breeds. Increasing dietary Na led to production of urine with a significantly lower CaOx RSS in both breeds, indicating that sodium supplementation to dry diet formats may reduce the risk of CaOx formation. These feeding strategies should be considered when evaluating methods for preventing CaOx formation within high-risk groups.

Influence of urinary stones on the composition of a 24-hour urine sample.

It can be assumed that stones in the urinary tract continuously increase in size by incorporating material from urine. Consequently, urine will exhibit depleted concentrations of lithogenic constituents when urinary stones are present in the patient's urinary tract. To calculate the influence of the depletion effect, we considered two different models of stone growth. In the first model, the increase in stone size depends only on the urinary concentration of a lithogenic substance; the second model also considers the surface area of the growing stone. The case of only one kidney being affected by stone formation is considered separately. We discuss example calculations involving the formation of calcium oxalate. The calculated depletion effects are of a nonnegligible order of magnitude. Assuming both a measured oxalate concentration of, e.g., 0.37 mmol/L and a reasonable in vivo stone growing rate of 10 mm(3)/day, a relative underestimation of the real "in situ" oxalate concentration between approximately 21% (model 1) and approximately 42% (model 2) occurs. The depletion effect increases markedly with increasing stone growth rate. Metabolic status can be evaluated correctly only in patients who have been declared "stone-free", e.g., after stone removal. Because the expected stone-related depletion effect in most cases is of high clinical relevance, we recommend estimating the effect of the order of magnitude of the depletion on actual urinary composition.

Presence of lipids in urine, crystals and stones: Implications for the formation of kidney stones

Cell membranes and their lipids play critical roles in calcification. Specific membrane phospholipids promote the formation of calcium phosphate and become a part of the organic matrix of growing calcification. We propose that membrane lipids also promote the formation of calcium oxalate (CaOx) and calcium phosphate (CaP) containing kidney stones, and become a part of their stone matrix. Human urine, crystals of CaOx and CaP produced in the urine of healthy individuals, and urinary stones containing struvite, uric acid, CaOx and CaP crystals for the presence of membrane lipids were analyzed. Crystallization of CaOx monohydrate at Langmuir monolayers of dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylglycerol (DOPG), palmitoyloleoylphosphatidylglycerol (POPG) and dimyristoylphosphatidylglycerol (DMPG) was investigated to directly demonstrate that phospholipid assemblies can catalyze CaOx nucleation. Urine as well as CaOx and CaP crystals made in the urine and various types of urinary stones investigated contained some lipids. Urine of both CaOx and uric acid stone formers contained significantly more cholesterol, cholesterol ester and triglycerides than urine of healthy subjects. However, urine of CaOx stone formers contained more acidic phospholipids. The organic matrix of calcific stones contained significantly more acidic and complexed phospholipids than uric acid and struvite stones. For each Langmuir monolayer precipitation was heterogeneous and selective with respect to the orientation and morphology of the CaOx crystals. Crystals were predominantly monohydrate, and most often grew singly with the calcium rich (10-1) face toward the monolayer. The number of crystals/mm2 decreased in the order DPPG> DPPC and was inversely proportional to surface pressure and mean molecular area/molecule. Stone forming conditions in the kidneys greatly impact their epithelial cells producing significant differences in the urinary lipids between healthy and stone forming individuals. Altered membrane lipids promote face selective nucleation and retention of calcium oxalate crystals, and in the process become a part of the growing crystals and stones.

Reflections on current issues concerning the stability of parenteral nutrition mixtures

Reflections on current issues concerning the stability of parenteral nutrition mixtures

Determination of urinary calcium-oxalate formation risk with BONN-Risk-Index and EQUIL applied to a family.

Apart from environmental and acquired risk factors, a person's genetic predisposition may have a distinct influence on the probability of the onset of urolithiasis. To investigate the family related development of calcium oxalate, CaOx, crystallization risk, we studied urines from three generations of the same family. The paternal line has been suffering from CaOx-urolithiasis for at least two generations; no case of urolithiasis has been reported from the maternal line and the youngest generation.We applied the BONN-Risk-Index and the computer program EQUIL to determine the crystallization risk of each family member (n = 7). We clearly verified by probability calculations of the existence of the two risk groups within the family and showed that one of the siblings of the youngest generation may have inherited the stone-formation risk from its paternal relatives as this person clearly reflects a high risk pattern.

Effects of Tamm-Horsfall Protein and Albumin on the Inhibition of Free Radicals

Introduction: Oxalate in urine can cause tubular cellular damage by the production of free radicals. Then, cell death and cellular debris may promote the retention of calcium oxalate crystals and finally the formation of stones. The two most abundant urinary proteins, Tamm-Horsfall protein (THP) and albumin, were tested for the effects of antioxidants. Materials and Methods: By using xanthine-xanthine oxidase reaction, purified THP and albumin were tested for the inhibitory effect. OD₂₉₅ was used as a spectrophotometric method to measure the production of uric acid during the reaction. Results and Conclusions: Both proteins can inhibit the reaction of xanthine oxidase on xanthine, although the effect was decreased after enzymatic deglycosylation of sialic acid. Albumin has an IC₅₀ of 10.7 nM in native condition and 11.9 nM after deglycosylation, whereas THP has 69.6 nM in native condition and 102.0 nM in deglycosylated condition. The data indicates that THP and albumin have an antioxidant effect. Sialic acid in THP has partly an inhibitory effect and is associated with calcium oxalate formation. Studies have indicated that further investigation of the role of free radicals in the formation of urolithiasis and of sialic acid in protein function is needed.

Calcium oxalate crystals in tomato and tobacco plants: morphology and in vitro interactions of crystal-associated macromolecules.

Plants form calcium oxalate crystals with unique morphologies under well-controlled conditions. We studied the morphology of single calcium oxalate monohydrate (whewellite) crystals extracted from tomato and tobacco leaves. These crystals have a pseudotetrahedral shape. We identified the (101), (101) or (102), (110), and (hk0) faces as stable faces. The morphology is chiral with unique handedness. We also show that calcium oxalate monohydrate crystals isolated from tomato, tobacco, and bougainvillea leaves contain macromolecules rich in Gly, Glx, and Ser. Crystal-associated macromolecules extracted from tomato and tobacco influence the morphology of calcium oxalate monohydrate crystals grown in vitro, promoting preferential development of the [120] faces. Furthermore, crystal-associated macromolecules from tobacco promote nucleation of calcium oxalate monohydrate crystals, whereas model polypeptides do not have any significant effect on nucleation. These results imply an active role of the crystal-associated macromolecules in the formation of pseudotetrahedral shapes in vitro, and these properties may in part be responsible for the unique chiral morphology of the natural pyramidal-shaped crystals.

The relation of urinary Tamm-Horsfall-Protein on CaOx-crystallization under the scope of the Bonn-Risk-Index

In previous papers we introduced the Bonn-Risk Index (BRI) as a new method of evaluating an individual's actual risk of forming calcium oxalate (CaOx). A comparison of our results with the calculated urinary relative supersaturations (RS) with respect to CaOx, showed that samples with similar values of RS can have different values of BRI. We suggested that this may reflect the individual influence of the urinary macromolecular constituents which are not taken into account at the calculation of RS. To estimate the role of macromolecules on the value of BRI, we examined 45 unprepared 24-h urine samples from 24 persons (16 healthy subjects, eight CaOx stone-formers) with respect to BRI, RS, and the concentration of urinary Tamm-Horsfall-protein ([THP]). The crystallization experiments were carried out by the use of a laser-probe. Based on the data of BRI and RS, the effect of THP may shift from a minor promoter in stone-formers and persons "at risk", to an inert urinary constituent in healthy subjects and persons "without risk". However, the observed effects are small and at least in the latter group close to zero.

Testing the predictability of the relative urinary supersaturation from the Bonn-Risk-Index for calcium oxalate stone formation.

When introducing a new parameter, it is necessary to compare the power of the new measure with already established ones. For a new method it is quite difficult to compete with established methods which have already ascertained sets of data over many years. A formal comparison of the new parameter with the actual "gold-standard" method can be a useful approach to reduce that problem. It cannot be expected that a new measure would reflect the "gold-standard" method in a simple proportionality. Therefore, it is important to find out the accuracy of the prediction of one parameter from the other, based on simple, e.g. linear, functions. A number of methods exist to determine the crystallization risk of calcium oxalate salts from urine. The most established method is the calculation of the relative urinary supersaturations with respect to these salts using the EQUIL-program, a program computing the equilibrium concentrations of complexes of primary cations and anions commonly found in urine. The Bonn-Risk-Index (BRI) is a new strategy for the evaluation of the risk of calcium oxalate formation, by performing crystallization experiments on native unprepared urine samples. Although the analytical and computational efforts of both approaches are quite different (relative supersaturation = high, BRI = low), the measurements revealed a considerable and significant linear relationship between the relative urinary calcium oxalate supersaturation, and BRI. We were, therefore, interested in predicting the relative supersaturation from the BRI and in the accuracy of this prediction.

Isolation of Medicago truncatula mutants defective in calcium oxalate crystal formation.

Plants accumulate crystals of calcium oxalate in a variety of shapes, sizes, amounts, and spatial locations. How and why many plants form crystals of calcium oxalate remain largely unknown. To gain insight into the regulatory mechanisms of crystal formation and function, we have initiated a mutant screen to identify the genetic determinants. Leaves from a chemically mutagenized Medicago truncatula population were visually screened for alterations in calcium oxalate crystal formation. Seven different classes of calcium oxalate defective mutants were identified that exhibited alterations in crystal nucleation, morphology, distribution and/or amount. Genetic analysis suggested that crystal formation is a complex process involving more than seven loci. Phenotypic analysis of a mutant that lacks crystals, cod 5, did not reveal any difference in plant growth and development compared with controls. This finding brings into question the hypothesized roles of calcium oxalate formation in supporting tissue structure and in regulating excess tissue calcium.

Assessing the relationship between the dry rot fungus Serpula lacrymans and selected forms of masonry

During this investigation into the relationship between the domestic dry rot fungus, Serpula lacrymans, and some non-woody building materials, it was found that S. lacrymans removed calcium, silicon and iron from sandstone, and calcium, sulphur and iron from traditional plaster. The sequestered elements were located on its hyphae, especially in the form of calcium oxalate. Degradation of the sandstone was implicit, but not obvious microscopically. In addition, S. lacrymans selectively transported iron from these building materials through its mycelial system. These findings lend further support to the theory that this wood-decaying fungus has an special dependence upon masonry in buildings.

Micro-archaeology of Engraved and Painted Rock Surface Crusts at Yiwarlarlay (the Lightning Brothers site), Northern Territory, Australia

The formation of calcium oxalate (whewellite) on the encrusted surfaces at Yiwarlarlay, both over engravings and off-art, was dated using laser and permanganate oxidation techniques and14C accelerator mass spectrometry. An age estimate of about 3160bp was obtained for the start of crust formation over a painted engraving. Microscopic evidence of previous episodes of painting, in the form of bright red and yellow iron-oxide layers, is also observed in the crusts suggesting that rock painting traditions extend back at least 3000 years. Therefore, for defining rock art chronologies we recommend the use of micro-archaeological investigations combined with the dating of carbon-bearing components in layered rock surface crusts. This study has shown that the encrusted surface deposits on stable rock ledges can provide information about past human painting activities, weathering processes and climatic events. Direct correlations can also be made between micro-archaeological data extracted from laminations in painted crust over engravings and archaeological and ethnographic information.

Calcium decreases urinary oxalate

The effects of calcium supplementation on urinary oxalate excretion was tested in 9 normal subjects, 4 males and 5 females between 23 and 49 years of age. In a crossover study 800 mg calcium was orally administered as active absorbable algal calcium (AAACa) (A) and calcium carbonate (B), and compared with non-calcium containing placebo (C). Calcium, oxalate, osmolality, creatinine and pH were measured in the first three morning urine samples and Ca/osmolality, Ca/osmolality/body weight, Ca/creatinine and oxalate/osmolality were calculated to correct for urine dilution. Ca x oxalate product was also calculated and Ca oxalate crystal in the sediment was microscopically examined and semiquantitatively estimated as -, +, ++, and +++ expressed as 0, 1, 2 and 3 respectively. Urinary Ca excretion was similar in A and B, but significantly larger than C, regardless of the method of correction for dilution. Urinary oxalate excretion tended to be lower in A than in B and C. Urine pH was similar among all three groups. Ca x oxalate product was higher in C than in A and B. AAACa, unlike calcium carbonate, appeared to decrease urinary oxalate excretion and Ca x oxalate product more efficiently than Ca carbonate, suggesting a possibility of inhibiting the formation of Ca x oxalate kidney stones. Formation of calcium oxalate was also tested in vitro by adding oxalate to urine samples and aqueous calcium solution.

Calcium oxalate crystalluria in a goat

Recognition of the diverse shapes of various urine crystals is necessary for their accurate identification. Calcium oxalate dihydrate crystals most commonly have an octahedral or envelope shape. Calcium ions and oxalic acid may form calcium oxalate crystals in urine. Calcium oxalate dihydrate crystalluria is a risk factor for urolith formation.

Influence of nitrogen source on the solubilization of natural gypsum (CaSO 4. 2H 2O) and the formation of calcium oxalate by different oxalic and citric acid-producing fungi

The ability of six fungi to solubilize natural-occurring gypsum was tested in vitro. The solubilization process was monitored by the production of a clear zone (halo) around or underneath the growing colony on Czapek-Dox agar containing 0.5% (w/v) gypsum (CaSO4. 2H2O). Aspergillus niger, Penicillium bilaii, P. simplicissimum and Paxillus involutus displayed differential solubilization activities depending on the supplemented nitrogen source. Colonies grown on nitrate-containing medium showed the ability to solubilize gypsum, but when ammonium (at equivalent nitrogen) was used there was a significant reduction in solubilization. It was found that liquid cultures of nitrate-grown fungi produced substantial amounts of oxalic acid, whereas in ammonium-containing medium oxalic acid was only detected in small amounts. The production of citric and gluconic acid under these experimental conditions was low in both media, although the involvement of citric acid in gypsum solubilization is possible. Coriolus versicolor and Phanaerochaete chrysosporium did not exhibit any solubilization activity in nitrate- or ammonium-containing medium. Additionally these two fungi excreted small quantities of oxalic acid in both media with no citric acid being produced in liquid medium. Concomitant with the solubilization process and inside the clear solubilized zone, A. niger, Pax. involutus and P. bilaii produced crystals of differing shapes and abundance depending on the fungal strain. No crystals were produced by P. simplicissimum. These crystals were identified as calcium oxalate based on HPLC analysis and energy-dispersive X-ray microanalysis. Abundance of these crystals was found to be correlated with both oxalic acid production and the acidity of the medium. It is concluded that gypsum solubilization was predominantly achieved by both oxalic acid, which was accompanied by formation of calcium oxalate crystals, and citric acid production rather than the acidity of the medium. The results are discussed in relation to the significance of such an activity in agricultural applications, e.g. land reclamation, as well as the possible roles played by these fungi in mineral cycling.

Beverages, diet, and prevention of kidney stones.

Beverages, diet, and prevention of kidney stones.

Fungal Production of Citric and Oxalic Acid: Importance in Metal Speciation, Physiology and Biogeochemical Processes

The production of organic acids by fungi has profound implications for metal speciation, physiology and biogeochemical cycles. Biosynthesis of oxalic acid from glucose occurs by hydrolysis of oxaloacetate to oxalate and acetate catalysed by cytosolic oxaloacetase, whereas on citric acid, oxalate production occurs by means of glyoxylate oxidation. Citric acid is an intermediate in the tricarboxylic acid cycle, with metals greatly influencing biosynthesis: growth limiting concentrations of Mn, Fe and Zn are important for high yields. The metal-complexing properties of these organic acids assist both essential metal and anionic (e.g. phosphate) nutrition of fungi, other microbes and plants, and determine metal speciation and mobility in the environment, including transfer between terrestrial and aquatic habitats, biocorrosion and weathering. Metal solubilization processes are also of potential for metal recovery and reclamation from contaminated solid wastes, soils and low-grade ores. Such 'heterotrophic leaching' can occur by several mechanisms but organic acids occupy a central position in the overall process, supplying both protons and a metal-complexing organic acid anion. Most simple metal oxalates [except those of alkali metals, Fe(III) and Al] are sparingly soluble and precipitate as crystalline or amorphous solids. Calcium oxalate is the most important manifestation of this in the environment and, in a variety of crystalline structures, is ubiquitously associated with free-living, plant symbiotic and pathogenic fungi. The main forms are the monohydrate (whewellite) and the dihydrate (weddelite) and their formation is of significance in biomineralization, since they affect nutritional heterogeneity in soil, especially Ca, P, K and Al cycling. The formation of insoluble toxic metal oxalates, e.g. of Cu, may confer tolerance and ensure survival in contaminated environments. In semi-arid environments, calcium oxalate formation is important in the formation and alteration of terrestrial subsurface limestones. Oxalate also plays an important role in lignocellulose degradation and plant pathogenesis, affecting activities of key enzymes and metal oxido-reduction reactions, therefore underpinning one of the most fundamental roles of fungi in carbon cycling in the natural environment. This review discusses the physiology and chemistry of citric and oxalic acid production in fungi, the intimate association of these acids and processes with metal speciation, physiology and mobility, and their importance and involvement in key fungal-mediated processes, including lignocellulose degradation, plant pathogenesis and metal biogeochemistry.

Solubilization of natural gypsum (CaSO 4.2H 2O) and the formation of calcium oxalate by Aspergillus niger and Serpula himantioides

The natural dihydrate form of calcium sulphate, CaSO4.2H2O (gypsum), particularly found in gypsiferous soils and in certain building constructions, was found to be effectively solubilized by both Aspergillus niger and Serpula himantioides. When the fungi were grown on malt extract agar (MEA) medium amended with up to 1% (w/v) of gypsum, solubilization activity was evident by the appearance of a clear zone (haol) around and/or under the growing colonies. The haloes were found subsequently to enclose concentric rings of newly formed crystalline precipitate. The pH profile of the medium underneath the growing mycelium of A. niger was not affected by the presence of gypsum, while the S. himantioides pH profile displayed a slight reduction in pH values when grown on gypsum-containing media compared with the control. Electron microscopy and X-ray microanalysis of the crystals revealed the formation of calcium-containing crystals with the characteristic morphology typical of different forms of calcium oxalate. The complexing activity of fungal-produced organic acids with calcium was suggested to be the prevalent mechanism of solubilization with the production of oxalic acid resulting in precipitation of oxalate. Such activity resulted in the release of available sulphate which increased over the incubation period. The concentrations of sulphate released from 0.5% (w/v) gypsum after growth of A. niger and S. himantioides for 8 d were 14·7±0·7 and 7·4±0·9 μmol cm-3 respectively. These results are discussed in the light of their relevance to land reclamation and plant nutrition, as well as the weathering of building materials containing gypsum.

Microscopical studies of the tissue structure of konjac tubers

The tissue structures of 2-year-old konjac tubers were investigated using a variable pressure scanning electron microscope equipped with an energy-dispersive X-ray spectrometer and an optical microscope. Konjac mannan (KM) cells are distributed all over the tuber and the spaces between the KM cells are filled with parenchyma. The KM cells are covered with scale-like cell walls and the size of the cells varies from 160 to 650 μm. Other kinds of particles with different sizes are observed in the honeycombed cells around the KM cells. The small grains relate to protein granules and the spherical particles (~12 μm in diameter), which are composed of small granules (1–3 μm in diameter), are starch. The tuber also contains potassium and calcium in the parenchyma. Most calcium is found in the needle-like crystals in the form of calcium oxalate. Such crystals are also found in 1 year and seed tubers.

Intratubular crystallization events.

Can urolithiasis start as an intratubular event? Under severe hyperoxaluric conditions in animal models at least crystal formation can. Recently models have been presented that assess the chances of crystal formation under more normal conditions. These models describe changes in fluid composition as this passes through the nephron, these conditions being simulated in in vitro experiments. It appears that under naturally occurring intratubular conditions calcium-salt crystallization takes place within the time tubular fluid normally spends in the nephron. Precipitation starts with a calcium-phosphate phase under conditions found in the thin lambs. This crystalline phase then (partly) dissolves when collecting duct conditions are used, thereby inducing formation of calcium oxalates. Under these conditions the latter increase in size by way of crystal growth and agglomeration. Large particle formation and cell adhesion can eventually result in particle retention and subsequent stone formation. Viewing urolithiasis as originally an intratubular event has consequences for in vitro experiments and treatments, which are discussed in this paper.