A critical appraisal of the hypothesis that urine is a saturated equilibrium with respect to stone-forming calcium salts.
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Calcium Oxalate↗
Biomedical subjects
J P Kavanagh
Publications and source records attributed to J P Kavanagh.
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.
Calcium Oxalate↗
Calcium oxalate crystallization kinetics studied by oxalate-induced turbidity in fresh human urine and artificial urine.
We have studied the kinetics of oxalate-induced turbidity in fresh human urine and artificial urine. Assays are performed in 96-well plates, which allows many oxalate concentrations to be studied, repeatedly, in a short time. The metastable limit is defined in terms of the lowest oxalate concentration that gives a rate of change of attenuance significantly greater than the control. Interpretation of rates above this limit is based on ln/ln plots of initial rates against added oxalate concentration. This approach has a good theoretical basis, is well supported by our results and gives a turbidity rate index that is related to the product of the growth rate constant and a factor relating to the number and characteristics of the heteronuclei responsible for initiation of crystallization. This interpretation is posited upon the assumptions that second-order crystallization kinetics occur in unseeded urine when supersaturation exceeds the metastable limit and that aggregation during the initial phase of crystallization does not significantly contribute to changes in turbidity. Metastable limits of urine from healthy volunteers corresponded to a calcium oxalate supersaturation ratio of approx. 10. The turbidity rate index was higher in human urine than in artificial urine. The metastable limit, based on either oxalate concentration or supersaturation, for induction of calcium oxalate crystallization in normal human urine is higher than is likely to be found in normal subjects in vivo. The shape of the relationship between the metastable limit (based on oxalate concentration) and calcium concentration emphasizes the benefit of achieving a low urine calcium concentration. Comparison of the turbidity rate indices for human and artificial urine suggests that the role of nucleation promoters is more dominant than that of growth inhibitors.
Calcium↗
Calcium oxalate crystallization kinetics at different concentrations of human and artificial urine, with a constant calcium to oxalate ratio.
The effect of in vitro dilution of artificial urine or human urine on the crystallization of calcium oxalate was examined in a mixed suspension, mixed product removal crystallization system. Direct growth inhibition by components of artificial urine was not significant and supersaturation was the dominant factor in determining crystal nucleation and growth rates. Dilution of human urine caused a decrease in crystal growth rate that was independent of the input calcium and oxalate concentrations, suggesting that dilution of growth inhibitors could be physiologically more important than any reduction in supersaturation. This loss of growth inhibition was counteracted by a reduction in nucleation promotion, with the net effect that the mass of crystals declined. Correlation of crystallization measurements with urinary concentration (osmotic pressure) confirmed these observations, with a negative relationship for growth rate and a positive relationship for nucleation rate and suspension density. Increasing the concentration of urine shifts the crystallization balance from low nucleation/high growth to high nucleation/low growth. Calcium oxalate crystalluria in healthy urine is therefore less likely at early stages of urine development in the nephron and the likelihood can be further reduced by increased fluid output. Our results suggest that lowering the heterogeneous nucleation activity by dilution is more than sufficient to override the loss of growth inhibition.
Calcium↗
Enlargement of a lower pole calcium oxalate stone: a theoretical examination of the role of crystal nucleation, growth, and aggregation.
BACKGROUND AND PURPOSE: Nucleation, growth, and aggregation are considered to be the principal crystallization mechanisms in stone development. It is important to understand their relative significance so that appropriate experimental models can be used, and to identify the best therapeutic targets. The aim of this study was to explore a simplistic model of precipitation and aggregation and to determine the impact of these processes on crystal size density (CSD) and stone growth. METHODS: The computer model represents a lower pole calcium oxalate stone and takes into account the limitations imposed by the amount of available oxalate. RESULTS: Stone enlargement is a result of direct precipitation onto the stone or aggregation of suspended crystals. About one third of the available oxalate needs to be precipitated to form a clinically significant stone within a realistic time frame. If all crystallization is directly at the stone surface, then crystal growth rates are limiting for stones less than about 1 mm, and aggregation of suspended particles would be required for significant enlargement. For crystals in suspension, the CSD is taken to result from two mixed-suspension, mixed-product removal crystallizers operating in series. Using realistic values for nucleation and growth rate, the CSD can be consistent with precipitation of a significant proportion of the available oxalate. This CSD is modified by aggregation, which is assumed to be proportional to the number and volume of crystals in each size range, and further modified by considering the aggregation to be shared by stone enlargement and polymerization in suspension. CONCLUSION: The variables in the model are the probability of aggregation, its rate, and the distribution between aggregation in suspension and onto the stone. Under some circumstances, the bimodal CSD of crystalluria seen in stone forming patients can be reproduced.
Calcium Oxalate↗
Growth inhibition of prostate cell lines in vitro by phyto-oestrogens.
OBJECTIVE: To assess a range of phyto-oestrogens as moderators of growth and metabolism in several prostate cell lines. MATERIALS AND METHODS: Four prostate cell lines (PNT-1/A, PNT-2, PC-3 and DU145) were challenged with different doses of five phyto-oestrogens (biochanin A, daidzein, genistein, genistin and nordihydroguaiaretic acid) over 3 days in culture. Cell proliferation was assessed by incorporation of 5-bromo-2'-deoxyuridine (BrdU) and metabolic activity by cleavage of a tetrazolium salt (XTT). RESULTS: Growth and metabolism were inhibited with all compounds and cell lines (e.g. the dose for 50% inhibition of proliferation of PC-3 cells by genistein was 38 micromol/L); differences in the patterns of results suggested that different mechanisms operated, but there was no evidence for any synergistic activity on the inhibition of cell proliferation. CONCLUSION: These results offer further support for the hypothesized role of phyto-oestrogens as dietary protectors against prostatic cancer.
Cell Division↗
Kinetics of circulating TNF-alpha and TNF soluble receptors following surgery in a clinical model of sepsis.
The interrelationship between cytokines and their natural antagonists in patients with systemic sepsis are incompletely understood. We have followed the changes in serum levels of TNF-alpha and the two soluble receptors (TNF-sr) in a clinical model of post-operative sepsis. Serial blood samples were taken in patients undergoing percutaneous nephrolithotomy (PCNL) starting pre-operatively and continuing for 24 h thereafter. The levels of TNF-alpha and TNF-sr were raised in patients who became clinically septic and correlated well with the severity of sepsis (using the APACHE III score). In septic patients there was no difference in the pattern of changes in the two types of receptor (TNF-sr55 and TNF-sr75). However, in non-septic patients TNF-sr75 was higher in those with endotoxaemia than those without. This difference was not observed with TNF-sr55 which suggests a different mechanism of release or degree of sensitivity for the two soluble receptors. Regardless of severity of illness, the levels of all three molecules (TNF-alpha and the two receptors) appeared to start rising at about the same time point. The peak TNF-alpha level was reached earlier (2-4 h) than that of the two TNF-sr (4-8 h). The relative rise in TNF-alpha was greater than that of the soluble receptors and this difference was even more marked in those with more severe sepsis. The relationship between peak TNF-alpha and peak TNF-sr was non-linear and the concentration of each TNF-sr appeared to plateau at the higher levels of TNF-alpha. This suggests the exhaustion of a limited pool or saturation of the rate of release. Taken together, these results suggest sepsis develops because of delayed and insufficient secretion of TNF-sr compared with TNF-alpha.
Antigens, CD↗
Do "Venflon" sideport valves leak?
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Humans↗
Isocitric and citric acid in human prostatic and seminal fluid: implications for prostatic metabolism and secretion.
Human prostatic secretion is remarkably rich in citric acid but the mechanisms to account for this accumulation are not well understood. One factor may be the extent of citrate oxidation to isocitrate, catalyzed by aconitase. The citrate-to-isocitrate ratio will help characterize the relative significance of this reaction in prostatic production and secretion of citrate. Isocitric acid and citric acid were measured in samples of seminal fluid and expressed prostatic secretion (EPS). A constant ratio between citrate and isocitrate of about 33:1 was found (r = 0.93, P < 0.0001) despite the wide variation in concentrations. Citrate ranged from 1 to 180 mM in EPS and from 13 to 50 mM in seminal fluid while isocitrate varied between 0 to 4.8 mM in EPS and from 0.4 to 1.5 mM in seminal fluid. Isocitrate is present in EPS and semen at much higher levels than found in most other animal or plant tissues or fluids and may be actively secreted by the same mechanism as citrate. The high citrate to isocitrate ratio of about 33:1, compared to the expected value of about 10:1, supports suggestions that citrate to isocitrate oxidation by aconitase is a rate limiting step in prostatic citrate metabolism. A low aconitase activity will therefore play a significant role in enabling accumulation of high citrate levels in prostatic epithelia and acini.
Citrates↗
Does fish oil benefit stone formers?
The possibility that dietary fish oil supplementation may benefit patients with hypercalciuric urolithiasis by decreasing calcium excretion and enhancing protective mechanisms has been studied in rats and humans. In experiments on rats in metabolic cages, fish oil inhibited experimental nephrocalcinosis induced by intraperitoneal calcium gluconate. There were no significant changes in urinary biochemistry. In a clinical study on 18 hypercalciuric recurrent stone patients fish oil significantly decreased urinary calcium excretion. This effect was accompanied by decreases in the excretion of magnesium and citrate. Oxalate excretion and urinary fibrinolytic activity were unchanged. Overall, fish oil had a limited impact on the risk profile for recurrent urolithiasis.
Adult↗
Crystallization kinetics of calcium oxalate in fresh, minimally diluted urine: comparison of recurrent stone formers and healthy controls in a continuous mixed suspension mixed product removal crystallizer.
A reproducible method has been developed for studying calcium oxalate crystallization from fresh, minimally diluted (92%) urine with the mixed suspension mixed product removal continuous crystallization technique. All samples were adjusted to give the same starting calcium and oxalate concentrations. Twenty-one recurrent male stone formers were compared with twenty-two healthy controls. There was no difference in crystal growth rates but crystal nucleation rates were much higher in the control group (p = 0.003). Using growth rate and nucleation rate results, the amount of crystalline material in suspension was shown to be lower in the urine from stone formers, and therefore the equilibrium supersaturation in the crystallizer was lower in the control group (p = 0.001). We propose that the ability of a healthy person's urine to maintain a lower supersaturation is a crucial protective factor distinguishing non-stone formers from stone formers.
Calcium Oxalate↗
Pyrophosphate in synovial fluid and urine and its relationship to urinary risk factors for stone disease.
Inorganic pyrophosphate (PPi) measurement in urine and synovial fluid has been established using the PPi-dependent phosphorylation of fructose-6-phosphate and subsequent reduction of dihydroxyacetone phosphate by NADH. The assay is linear up to 200 mumol/L, easy to perform and gives results comparable to more complex methods. Daily urinary output of PPi was independently related to both age (P = 0.0014) and sex (P = 0.0002). Men had higher values than women and older individuals excreted greater amounts. Male stone formers, younger than 45 years, had lower values than age matched male controls (P = 0.012). Younger female stone formers also tended to have lower values. In stone formers' urine significant and independent correlations were found of PPi excretion with urine volume (P = 0.004) and with phosphate excretion (P = 0.008). Oxalate excretion and that of other urine constituents and the degree of supersaturation with common stone-forming salts were not correlated with PPi. PPi excretion was markedly elevated in the urine of two patients with hypophosphatasia. The PPi concentration in synovial fluid from painful, swollen knee joints was elevated, but unrelated to the presence or absence of PPi or urate crystals.
Adolescent↗
Methods for the study of calcium oxalate crystallisation and their application to urolithiasis research.
Many methods have been used to study calcium oxalate crystallisation. Most can be characterised by changes in supersaturation during the experiment, which may increase, remain constant or decay. Their ability to quantify various aspects of crystallisation often reflects the extent to which nucleation, growth and aggregation can be measured independently, when two or three of these processes may be occurring simultaneously. The mixed suspension, mixed product removal technique reaches a steady state supersaturation, is a good model for intrarenal crystallisation and allows both growth and nucleation rates to be measured. Using 92% urine and comparing control urines with samples from recurrent stone formers no difference in growth rates was found but the controls had higher nucleation rates (p = 0.003) and lower supersaturations (p = 0.001). In parallel crystallisers running simultaneously, heparin or hyaluronic acid addition to 92% urine was studied. Both macromolecules increased growth rates, decreased nucleation rates and increased supersaturation (p < 0.05). The steady state supersaturation achieved in this system may be an important determinant of stone forming potential. The ability to reach a lower urinary supersaturation by increased nucleation may be a crucial protective factor distinguishing non stone formers from stone formers.
Calcium Oxalate↗
Do stone formers have lower urinary fibrinolytic activity than controls?
A comparison of urinary fibrinolytic activity between 31 stone formers and 50 controls failed to demonstrate any significant difference and so failed to confirm an earlier independent observation. This may be due to methodological and design differences between the 2 studies, but does suggest reservations about the significance of lowered urinary fibrinolytic activity as a risk factor for renal stone disease. Dietary information from those taking part in the study suggests that a reduction of meat intake by stone formers might be associated with increased urinary fibrinolytic activity. This might account for the discrepancy between the 2 studies, in which case this observation could be of significance.
Creatinine↗
Calcium oxalate crystallisation kinetics and the effects of calcium and gamma-carboxyglutamic acid.
gamma-carboxyglutamic acid (GLA) is an amino acid with a high affinity for calcium. It is found in urine both as the free amino acid and incorporated into proteins such as osteocalcin. Free and bound GLA have been reported to be found at higher concentrations in the urine of stone formers than controls. We have investigated the effect of GLA and calcium, at physiological levels, on the crystallisation of calcium oxalate using a mixed suspension mixed product removal continuous crystalliser. GLA caused very significant changes in the crystallisation kinetics, but the effect was dependent on the calcium concentration. At 4 mM calcium, GLA decreased the growth rate and increased the nucleation rate; at 12 mM the reverse occurred. At all concentrations of calcium tested, GLA caused a significantly increased crystal mass to be produced. Our evidence supports the hypothesis that GLA modifies calcium oxalate crystallisation and could be a promoter of stone formation in vivo, particularly at moderately elevated levels of calcium excretion.
1-Carboxyglutamic Acid↗
Ratio of post-prostatic massage urinary zinc concentration to initial urinary zinc concentration. An improved method of assessing prostatic function.
Various methods have been used clinically to assess prostatic function. We evaluated a number of ways of using zinc in post-prostatic massage urine (VB3) and found that the ratio of VB3 zinc concentration to initial urinary zinc concentration was a good indicator of prostatic secretory activity. This ratio can differentiate hormonally treated patients with carcinoma of the prostate from those untreated and from those with benign prostatic hypertrophy. The specificity is increased for individuals by repeat samples. Comparison of samples before and after resection showed no loss of function and no difference was found between groups treated with orchiectomy or LHRH agonist (Zoladex). The VB3/VB1 zinc concentration could be used in a prospective study of hormonal treatments directed against the prostate, where it could give objective evidence of a fall in secretory function of the gland.
Humans↗
Urogastrone-epidermal growth factor concentrations in prostatic fluid of normal individuals and patients with benign prostatic hypertrophy.
High concentrations (272 +/- 33 ng/ml) of urogastrone-epidermal growth factor were measured in prostatic fluid from normal males by a specific radioimmunoassay. Significantly lower concentrations (155 +/- 24 ng/ml) were observed in the prostatic fluid of patients with benign prostatic hypertrophy than in the age-matched normal controls (2P less than 0.01). The growth factor content of seminal fluid was accounted for by the contribution of prostatic fluid. Immunochemical studies failed to show evidence of synthesis within the gland nor could high affinity receptors for the protein be demonstrated in membrane preparations of the gland.
Chromatography, Gel↗
Does sucrose damage kidneys?
There is evidence to suggest that sucrose ingestion can cause renal parenchymal changes as well as increasing the urinary saturation index for calcium oxalate. Ten stone formers and 10 normal subjects received 250 gm of sucrose daily over a period of 7 days. Observations on the risk factors for calcium stone formation and urinary N-acetyl-B-glucosaminidase (NAG), a marker of renal tubular cell damage, were made. Oxalate excretion increased. Urinary calcium levels were unchanged but the pattern of response was different between the two groups, as with magnesium and phosphate. NAG was spontaneously higher in the patient group and increased significantly after sucrose ingestion in both groups.
Acetylglucosaminidase↗