Investigations of trace and toxic elements of kidney stones from two different Sudanese areas by µ-PIXE using Nuclear Microprobe (NMP).

In this study 14 kidney stones (oxalate and phosphate stones) collected from two Sudanese hospitals Particle Induced X ray Emission (µ-PIXE). This technique was used to investigate the matrix as well as the trace element compositions of kidney stones on a microscopic scale using 1.5MeV proton energy. Significant differences in elements content across the groups were found by applying statistical methods. Trace elements such as P,Ca, Fe, Ni, Cu, Zn, Se, Sr, Br, and Pb showed a possible linear relationship of the mean profile for trace elements for each group of stones. This study shows that micro analysis of urinary stones can provide complementary information on patients' exposure to epidemiological risk factors such as geography, diet and drinking water.

Experimental determination of multiple thermodynamic and kinetic risk factors for nephrolithiasis in the urine of healthy controls and calcium oxalate stone formers: does a universal discriminator exist?

Nephrolithiasis is thought to be governed by urinary thermodynamic and kinetic risk factors. However, identification of one or more of these factors which consistently and unambiguously differentiates between healthy subjects (N) and calcium oxalate (CaOx) renal stone patients (SF) remains elusive. The present study addresses this challenge. 24 h urines were collected from 15 N and 10 SF. Urine compositions were used to compute thermodynamic risk indices including urinary ratios, quotients and supersaturation (SS) values, while CaOx metastable limits (MSL) were determined experimentally. Crystallisation kinetics was determined by measuring rates of particle formation (number, volume, size) using a Coulter counter multisizer (CC) and a Coulter flow cytometer (FC). Particle shapes were qualitatively differentiated by FC and were viewed directly by scanning electron microscopy. Several urinary composition ratios and risk quotients were significantly different between the groups. However, there were no significant differences between CaOx MSL or SS values. Using transformed FC data, the rate of CaOx crystallisation in SF was significantly greater than in N. This was not supported by CC measurements. There were no significant differences between the groups with respect to particle size or CaOx crystal growth rates. Single and aggregated CaOx dihydrate crystals were observed in both groups with equal frequency and there were no differences in the kinetic properties of these deposits. A few CaOx monohydrate crystals were observed in SF. Although several risk factors were found to be significantly different between the groups, none of them were consistently robust when compared to other cognate factors. Arguments were readily invoked which demonstrated inter-factor inconsistencies and conflicts. We suspect that a unique discriminatory factor, such as any of those which we investigated in the present study, may not exist.

Characterization of human kidney stones using micro-PIXE and RBS: a comparative study between two different populations.

The micro-PIXE and RBS techniques are used to investigate the matrix as well as the trace elemental composition of calcium-rich human tissues on a microscopic scale. This paper deals with the spatial distribution of trace metals in hard human tissues such as kidney stone concretions, undertaken at the nuclear microprobe (NMP) facility. Relevant information about ion beam techniques used for material characterization will be discussed. Mapping correlation between different trace metals to extract information related to micro-regions composition will be illustrated with an application using proton energies of 1.5 and 3.0 MeV and applied to a comparative study for human kidney stone concretions nucleation region analysis from two different population groups (Sudan and South Africa).

Methods for measuring crystallization in urolithiasis research: why, how and when?

Whereas crystalluria does not distinguish between kidney stone formers and healthy people and thus can be considered a physiologic event, kidney stone formation is a pathologic incident and reflects a specific form of biomineralization. Both single urinary crystals as well as whole kidney stones form under exquisite control of organic macromolecules. Simple crystal formation in the urinary tract is distinguished from stone formation in the kidney by the process of particle retention. The latter occurs either because nucleated crystals strongly aggregate to particles too large to pass freely through the tubules ('free particle' theory), or because crystals become abnormally adherent to tubular cell surfaces ('fixed particle' theory). Since it is impossible to mimic all the processes involved in stone formation in vitro, it is highly important to carefully chose a specific crystallization process for in vitro studies, and to select the most appropriate experimental conditions for measuring the chosen process as reliably as possible. This overview aims at critically reviewing the principles of currently available assay systems for studying crystallization processes involved in stone formation. Consensus is reached by the experts that no in vitro system really mimics what happens in renal stone formation, but that carefully designed in vitro studies will always play an important part in urolithiasis research. For such studies, it is highly important to exactly control the appropriate experimental conditions that are relevant to a specific crystallization process under investigation. Practical guidelines for researchers working with crystallization systems are provided, and it is concluded that international efforts should be made to standardize the terminology, to agree on a set of basic experimental parameters (temperature, pH, artificial urine composition), and to adopt simple tests or conditions are reference points for quality and comparative control.

A study of crystal matrix extract and urinary prothrombin fragment 1 from a stone-prone and stone-free population.

South African blacks are immune to urinary calculi whereas whites have an incidence rate similar to that reported in Western societies. Urinary prothrombin fragment 1 (UPTF1) and the crystal matrix extract (CME) from which it is derived have been shown to be potent inhibitors of crystal growth and aggregation in undiluted human urine. The objective of the present study was to isolate CME and UPTF1 from the urines of black and white subjects in order to assess whether either might contribute to the black population's relative stone immunity. CME was isolated from freshly precipitated calcium oxalate (CaOx) crystals and a crystallization study was conducted in synthetic urine. Coulter Counter, 14C-oxalate deposition, and scanning electron microscopy data demonstrated that the extracts from both race groups strongly inhibited CaOx nucleation. The extract derived from the black subjects inhibited nucleation to a greater extent than that from the whites. A phase conversion from COM to COD in the presence of the extracts, in support of the inhibitory effect of CME, was also observed. Purified UPTF1 isolated from both groups' CME was subjected to rigorous biochemical characterization involving matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, protein sequencing by Edman degradation, and amino acid analyses. No differences in molecular weight or amino acid sequence and composition were identified. It is suggested that the more potent inhibitory activity of the extract derived from the black subjects might be related to this group's relative stone immunity.

Trace metal excretion in patients with homozygous hypercholesterolaemia.

In patients with familial hypercholesterolaemia regular therapeutic apheresis is acknowledged to have long-term benefit. A previously unrecognised complication of such intervention is the development of anaemia that reflects a sub-optimal dietary iron intake coupled with accelerated loss of this trace metal in the fluid discarded after each procedure. Additional contributions result from enhanced urinary excretion as a result of chelation to citrate used as an anticoagulant and frequent blood sampling. The underlying pathophysiologic process appears to be reduced deformability. We now document similar and significant losses of zinc, copper and chromium in these circumstances. In the case of the latter three elements, no associated clinical syndromes have thus far been identified, probably because deficiency states are less well-recognised than that due to iron loss and, additionally, because critical reductions are avoided by their replenishment during a normal food intake. These studies are, nevertheless, relevant since they are the basis for recommending prophylactic supplementation during this form of management.

Relative hyperoxaluria, crystalluria and haematuria after megadose ingestion of vitamin C.

BACKGROUND: Long-term or high-dosage consumption of vitamin C may play a role in calcium oxalate kidney stone formation. The present study was undertaken to determine the biochemical and physicochemical risk factors in a male subject who developed haematuria and calcium oxalate crystalluria after ingestion of large doses of ascorbic acid for 8 consecutive days. METHODS: Twenty-four-hour urine samples were collected before and during the ascorbic acid ingestion period as well as after the detection of haematuria. A special procedure was implemented for urine collections to allow for oxalate, ascorbate and other urinalysis. Oxalate was determined in the presence of EDTA to prevent in vitro conversion to ascorbic acid, whereas ascorbate itself was determined by manual titration in a redox method using the dye dichlorophenolindophenol. Urinalysis data were used to compute calcium oxalate relative supersaturations and Tiselius risk indices, whereas scanning electron microscopy was used to examine urinary deposits. RESULTS: Oxalate excretion increased by about 350% during ascorbate ingestion before haematuria. Ascorbate concentrations also increased dramatically but appeared to reach a plateau maximum. Increasing calcium excretion was accompanied by decreasing potassium and phosphate values. The calcium oxalate relative supersaturation and Tiselius risk index increased during vitamin C ingestion and large aggregates of calcium oxalate dihydrate crystals were observed by scanning electron microscopy immediately after the detection of haematuria. CONCLUSION: High percentage metabolic conversion of ascorbate to oxalate in this subject caused relative hyperoxaluria and crystalluria, the latter manifesting itself as haematuria. Clinicians need to be alerted to the potential dangers of large dose ingestion of vitamin C in some individuals.

The effect of ascorbic acid ingestion on the biochemical and physicochemical risk factors associated with calcium oxalate kidney stone formation.

The present study was undertaken to determine the effect of ingestion of large doses of vitamin C on urinary oxalate excretion and on a number of other biochemical and physicochemical risk factors associated with calcium oxalate urolithiasis. A further objective was to determine urinary ascorbate excretion and to relate it qualitatively to ingested levels of the vitamin and oxalate excretion. Ten healthy males participated in a protocol in which 4 g ascorbic acid was ingested for 5 days. Urines (24 h) were collected prior to, during and after the protocol. The urine collection procedure was designed to allow for the analysis of oxalate in the presence and absence of an EDTA preservative and for the analysis of ascorbic acid by manual titration using 2,6 dichlorophenolindophenol. Physicochemical risk factors such as the calcium oxalate relative supersaturation and Tiselius risk index were calculated from urine composition. The results showed that erroneously high analytical oxalate levels occur in the asence of preservative. In the preserved samples there was no significant increase in oxalate excretion at any stage of the protocol. Ascorbate excretion increased when vitamin C ingestion commenced but levelled out after 24 hours suggesting that saturation of the metabolic pool is reached within 24 hours after which ingested ascorbic acid is excreted unmetabolized in the urine. While transient statistically significant changes occurred in some of the biochemical risk factors, they were not regarded as being clinically significant. There were no changes in either the calcium oxalate relative supersaturation or Tiselius risk index. It is concluded that ingestion of large doses of ascorbic acid does not affect the principal risk factors associated with calcium oxalate kidney stone formation.

The influence of South African mineral water on reduction of risk of calcium oxalate kidney stone formation.

OBJECTIVES: This study was undertaken to identify a South African mineral water containing relatively high concentrations of calcium and magnesium and to investigate its effect on urinary biochemical and physicochemical risk factors associated with calcium oxalate kidney stone formation. DESIGN: The study followed a change-over design in which each subject followed a randomised sequence of three water-drinking protocols involving their normal diet, a calcium and magnesium-rich mineral water and a mineral water deficient in these elements. SETTING: University of Cape Town. SUBJECTS: 54 volunteers without any previous history of stone disease (27 men, 27 women) in the age group 21-35 years and 31 with a history of calcium oxalate kidney stones (24 men, 7 women) in the age group 25-45 years participated in the study. OUTCOME MEASURES: Both mineral waters favourably altered several risk factors. However, the effect of the calcium- and magnesium-rich water was shown to be significantly greater as it altered a larger number of these factors and induced several unique changes that were not achieved by the other water. CONCLUSIONS: The risk of calcium oxalate stone formation can be significantly reduced by consumption of mineral water which is rich in calcium and magnesium.

Effect of mineral water containing calcium and magnesium on calcium oxalate urolithiasis risk factors.

Calcium oxalate kidney stone formers are invariably advised to increase their fluid intake. In addition, magnesium therapy is often administered. Recently, a prospective study showed that a high dietary intake of calcium reduces the risk of symptomatic kidney stones. The present study was performed to test whether simultaneous delivery of these factors--high fluid intake, magnesium ingestion and increased dietary calcium--could reduce the risk of calcium oxalate kidney stone formation. A French mineral water, containing calcium and magnesium (202 and 36 ppm, respectively) was selected as the dietary vehicle. Twenty calcium oxalate stone-forming patients of each sex as well as 20 healthy volunteers of each sex participated in the study. Each subject provided a 24-hour urine collection after ingestion of mineral water over a period of 3 days; after a suitable rest period the protocol was repeated using local tap water (Ca: 13 ppm, Mg: 1 ppm). In addition, 24-hour urines were collected by each subject on their free diets. The entire cycle was repeated at least twice by each subject. Several risk factors (excretion of oxalate; relative supersaturations of calcium oxalate, brushite and uric acid; calcium oxalate metastable limit; oxalate:magnesium ratio and oxalate:metastable limit ratio) were favourably altered by the mineral water and tap water regimens but the former was more effective. In addition, the mineral water protocol produced favourable but unique changes in the excretion of citrate and magnesium as well as in the relative supersaturation of brushite which were not achieved by the tap water regimen. To the contrary, tap water produced an unfavourable change in the magnesium excretion. The group which benefitted most were male stone formers in whom 9 risk factors were favourably altered by the mineral water protocol. It is concluded that mineral water containing calcium and magnesium, such as that used in this study, deserves to be considered as a possible therapeutic or prophylactic agent in calcium oxalate kidney stone disease.

Microanalysis of calcium-rich human kidney stones at the NAC nuclear microprobe.

Previous studies on the role of trace elements (TE) in the development of calcium oxalate (CaOx) human kidney stones by nuclear microprobe (NMP) investigated the mechanisms and role of TE in the buildup of urinary CaOx concretions. In the present work, microanalysis of the previously reported series of recurrent human kidney stones was further expanded. Interest was focused on determining levels of directional variability in elemental concentrations of Ca and TE throughout selected micro-regions of single stones by Dynamic Analysis (DA).

Studies on the role of urinary macromolecules in urolithiasis: review of methodologies and a proposal for a standard reference crystallization system.

In this study, urine from a calcium oxalate kidney stone former was ultrafiltered (10 kD cut-off). Crystallization was induced in the ultrafiltrate and retentate fractions as well as in a sample of the whole urine. The progress of crystallization was monitored by Coulter Counter and flow cytometry techniques. (The latter has not been used in studies of the role of urinary macromolecules in urolithiasis). Deposited crystals were examined by scanning electron microscopy. Results indicated that urinary macromolecules in this subject are inhibitors of nucleation and aggregation. These results agree with the findings of some workers but disagree with those of others. Indeed, studies on the role played by urinary macromolecules in promoting or inhibiting urolithiasis have failed to produce consistent findings. Examination of the literature reveals that a wide variety of experimental techniques and crystallization systems have been used in these studies and that this might be the cause of the inconsistencies. Based on reported experiences and those of the present study, a standard reference crystallization system is proposed. The key elements of this system involve the use of real urine, ultrafiltration, continuous crystallizer equipment, Coulter Counter procedures and scanning electron microscopy.

Re-evaluation of the "week-end effect" data: possible role of urinary copper and phosphorus in the pathogenesis of renal calculi.

Early morning urinary concentrations of 10 elements which had demonstrated a "week-end effect" in a previous study, were subjected to a normalization procedure thereby allowing a re-assessment of their potential role in urolithiasis. After transformation of each concentration to a weighted proportion of the total concentration on each day, only Cu and P values were significantly different for kidney stone formers and healthy controls on all three days indicating that these elements may play a role in the pathogenesis of renal calculi. The results obtained in this study demonstrate that a more meaningful picture of the possible differences in the urinary concentrations of stone formers and normal controls might emerge if "proportional" rather than "raw" concentrations are compared.

Effect of urinary macromolecules and chondroitin sulphate on calcium oxalate crystallization in urine.

After filtration and ultrafiltration (10 kD) of 24 hour urine specimens from 12 healthy male subjects, calcium oxalate crystallization was induced in the filtered (FILTD) and ultrafiltered (UF) fractions by administration of a sodium oxalate load. In addition crystallization was also induced in UF fractions to which physiological quantities of chondroitin sulphate (CHON) had been added (UF+CHON). The rate of calcium oxalate crystallization was determined by measuring the rate at which turbidity increased. Crystal numbers and sizes were measured with a Malvern particle size analyzer and by scanning electron microscopy. Crystallization rates, crystal numbers and crystal sizes were generally lower in UF fractions than in FILTD fractions suggesting that urinary macromolecules are promoters of calcium oxalate crystallization. No increase in crystallization rate, crystal numbers or sizes occurred when chondroitin sulphate was added to UF fractions, indicating that the promoter activity of urinary macromolecules is not due to this particular glycosaminoglycan. On the contrary, crystallization rates were qualitatively lower in UF+CHON fractions than in UF fractions alone, suggesting a possible inhibitory role for chondroitin sulphate in real urine. Scanning electron microscopy revealed that while aggregates were present in UF and FILTD fractions, they were absent in UF+CHON fractions. This observation indicates that chondroitin sulphate might be an inhibitor of calcium oxalate crystal aggregation in real urine.

Urinary macromolecules are promoters of calcium oxalate nucleation in human urine: turbidimetric studies.

Calcium oxalate crystallization was induced in the filtered, ultrafiltered (10 kDa) and retentate fractions of 24-h urine specimens obtained from 15 male controls and 10 male stone formers, by administration of an aqueous sodium oxalate challenge to each test solution. Crystallization rates were followed by monitoring of the increase in turbidity in these fractions as a function of time. A laboratory nephelometer, previously calibrated against a Coulter counter, was used for this purpose. In addition, to facilitate interpretation of turbidity data, a Malvern particle size analyzer was used to determine crystal sizes and numbers in control urines. Crystallization rates, crystal numbers and crystal sizes were generally lower in ultrafiltered fractions than in filtered or retentate fractions, indicating that urinary macromolecules are promoters of calcium oxalate nucleation. Data for stone formers suggest that the urinary macromolecules in this group may be more potent nucleation promoters than those in controls.

Crystalluria in marathon runners. IV. Black subjects.

Crystal sizes (scanning electron microscopy) and distributions (Coulter Counter) as well as 24-h urinary sodium (Na) and calcium (Ca) excretions (flame atomic absorption) were determined in a group of black South African runners immediately after a marathon and again 3 weeks later. White runners and black and white control subjects were included in the study. Particle volume-size histograms for black controls and black runners were identical. There was no significant difference in the Na excretion of all the groups. However, while urinary Ca excretion was significantly raised in white runners relative to white controls, Ca excretion in black runners was unchanged relative to their controls. It is postulated that the lower rates of urinary Ca excretion may result from lower rates of Ca resorption from bone in response to the cyclical loading of running in black marathon runners. The results of this study suggest that black marathon runners are not prone to the same increased risk of renal stone formation as are white runners.

Influence of ageing, pH and various additives on crystal formation in artificial urine.

In order to investigate the effect of various factors on urinary crystallization processes, a series of five experiments was carried out using an artificial urine (AU) in a rotary evaporator. The influence of ageing, pH and organic, inorganic and potential inhibitory additives formed the basis of the study. Precipitates were characterized by X-ray powder diffraction, scanning electron microscopy and energy dispersive X-ray analysis. In the ageing experiment, AU aliquots, adjusted to various pH values, were allowed to stand for several days and were not evaporated. Calcium oxalate monohydrate (COM) was formed at low pH, while whitlockite, apatite and struvite occurred at pH greater than 7. In the second experiment, AU aliquots at various pH values, were evaporated. Similar results to those of series 1 were recorded but, in addition, calcium oxalate trihydrate (COT) precipitated in the pH range 3 to 6.5 and brushite at pH greater than 5.5. In series 3, uric acid, creatinine and urea were included in AU aliquots (pH 5.5) which were subjected to evaporation. Uric acid promoted the formation of uric acid dihydrate; however, when present with creatinine, dihydrate formation was inhibited. Urea appeared to inhibit precipitation. In the fourth experiment, MgO, methylene blue and chondroitin sulphate A were independently included in the AU (pH 5.5). Precipitates of calcium oxalate mono-, di- and trihydrates were obtained. In the final experiment fluoride aliquots of variable concentrations were included in the AU (pH 5.5 and 6.5). COT crystals of superior quality to those observed in control solutions were obtained.

Crystalluria in marathon runners. III. Stone-forming subjects.

In order to investigate further the possible relationship between urinary stone formation and marathon running, the crystalluria in seven male, stone forming runners was characterized. Particle size distribution curves (Coulter counter) and crystal number, size and morphology (scanning electron microscopy) were measured. These studies suggest that urinary stone formation may be accelerated in those subjects with previous histories of renal stone formation but that the nature of the crystalluria is favourably affected by an increase in fluid intake. The presence of large quantities of mucoid material in the urine of "natural" stone formers and its absence in the urine of stone-forming runners is cited as evidence for the existence of different aetiological mechanisms in these groups. It is concluded that while natural stone formers may be at chronic risk of stone formation due to pathological factors, marathon runners may be at acute risk due to factors associated with long distance running itself.

A probable case of prehistoric kidney stone disease from the northern Cape Province, South Africa.

During the excavation of isolated graves along the north shore of the Orange River in the Cape Province of South Africa, a skeleton was uncovered that showed two large calcareous deposits near the lumbar region of the vertebral column. The individual was an adult female of about 55 years of age at the time of death. The calcified residue did not in any way resemble the external or internal anatomical form of a kidney, but X-ray powder diffraction analysis and scanning electron microscopy identified the material as apatite (Ca10(PO4)6 (OH)2), a common constituent of human urinary calculi. It is postulated that the bilateral calcification was the result of a chronic renal disorder. Although it is rarely possible to identify the cause of death from skeletal remains alone, the disorder as seen in this specimen would appear to have been very severe and may well have been the ultimate cause of death.