Litiasis renal oxalocálcica. Avances en la comprensión de su etiología y mejoras en la metodología analítica para su estudio

by Muñoz Martínez, José Antonio

Abstract (Summary)
Urolithiasis is a common disease, which will affect 12% of the European Union population along their life, characterized by high prevalence and incidence, high morbidity and high rates of recurrence. Nowadays, multifactorial origin is accepted for the lithogenic process: the main phenomena is the supersaturation of several compounds in urine that might crystallise forming solid concretions, influenced by the scarcity of crystallization inhibitors, the presence of crystallization promoters and morphoanatomic factors of the urinary tract. The recurrence rate during five years lies between 44-75%, independently that the lithiasic episode is being solved spontaneously or by urologic intervention. This fact is due to the persistence of the urinary alteration which causes the renal stone formation. Such alterations are susceptible to be diagnosed by calculi and urine characterization and treated by dietetic modifications, pharmacological treatment or both, decreasing the aggressiveness of the disease and, consequently, the number of recurrences. At the moment, main efforts in the urolithiasis field should be addressed to reduce incidence and recurrence rates, especially if we consider the lower costs associated to prophylactic treatments and prevention of this disease against to those of calculi extraction. In this sense, better understanding of lithiasic etiology and better efficacy in the diagnosis of lithogenic alterations are required to increase efficacy in the prevention of urolithiasis. Under this context, the main objectives of the present thesis include the development of efficient analytical methodologies for urolithiasis markers, the characterization of diary and stational fluctuations in urinary composition regarding lithogenic risk and the effect of various chemical substances (and its mixtures) on calcium oxalate crystallization. Development of simple analytical methodologies for urolithiasis markers Accurate evaluation of urinary abnormalities in lithiasic patients is known to have vital importance in the reliable diagnosis and treatment of urolithiasis. Thus, the development of more efficient analytical methodologies for urolithiasis markers is crucial for the accurate characterization of urolithiasis, especially if we consider the well-known drawbacks of some existing analytical methods. Moreover, some urinary parameters (i.e. phytic acid and pyrophosphate) are not routinely analysed probably because of the scarcity of efficient methodologies for their determination, despite its knowledge is most valuable for diagnostic and treatment purposes. In this sense, a need for efficient analytical methodologies offering relevant information for urolithiasis management was identified. Research in this field has been focused on 6 urolithiasis markers: oxalate, citrate, uric acid, creatinine, phytic acid and pyrophosphate. Main drawbacks associated to existing methodologies for these analytes include individual determination, large and tedious sample treatment (often requiring dilution, filtration, centrifugation, pH adjusting, incubation, purification, hydrolysis, derivatization) with consequent large times of analysis, lack of specificity or the need of especial component (enzymes) which could be rather expensive in some cases (oxalate). The development of more efficient, multicomponent and, thus, faster analytical methodologies involved a method for analysis of urinary metabolites (oxalate, citrate, creatinine and uric acid) by capillary zone electrophoresis and a method for the determination of phytic acid and pyrophosphate in urine by inductively coupled plasma mass spectrometry. Validation parameters were evaluated in both cases, indicating appropriate lineal ranges and limits of detection, lack of interferences and good accuracy and precision, thus, demonstrating their applicability. Main advantages of these methods (ease of sample treatment, low requirements of reagents and sample amounts, multicomponent analysis capabilities, short times of analysis and high sample throughput, adequate accuracy and precision) make feasible its implementation in clinical laboratories for a better diagnosis of urolithiasis illness, being the main associated drawback the limited (but increasing) availability of the required instrumentation. Characterization of diary and stational fluctuations in lithogenic risk In the second part of this thesis, the study of the diary and stational fluctuations of urinary composition revealed important temporal variations producing relevant peaks of increased risk for the pathologic crystallization of calcium oxalate. In this sense, the identification of periods with increased risk of lithogenesis is of interest in the therapeutic treatment design, by trying to reduce the supersaturation of urine or to increase the inhibitory capacity of given urine by dietary changes of pharmacological treatment at a stage when they could be more effective. With respect to circadian course, relevant fluctuations in the supersaturation / promotion / inhibition system for crystallization in urine, related with some of the urolithiasis-related measured urinary parameters (volume/h, calcium, oxalate, magnesium, phytic acid) were found, leading to a variation on the lithogenic risk during 24 hours. An increased risk period for calcium oxalate crystallization was identified at the nocturnal rest period, thus indicating that 24-hour urines do not reflect properly the crystallization properties of urine. Moreover, application of multivariate statistical analysis (PCA and HCA) to fractionated 24-h urines revealed that nocturnal urines can be differentiated from the diurnal ones by their lithogenecity, also indicating some homogeneity in urinary composition of such samples. Both facts, lithogenecity naturally increased at night and homogeneity in urinary composition, suggested that analysis of nocturnal urines can represent an objective for more appropriate evaluation of calcium oxalate lithogenic risk. With respect to stational fluctuation in urinary composition, remarkable variations were found indicating an increased risk of calcium oxalate crystallization in the summer period, provoked by both a decrease in diuresis with an associated increased supersaturation and a non-increased inhibitory capacity. These results indicate the need for both maintaining adequate hidratation levels during the summer and increasing the inhibitory activity of urine by dietary changes or pharmacological treatment in those patients with higher risks of recurrence. Effect of various substances (and its mixtures) on calcium oxalate crystallization In the third part of this thesis, the effect of several substances (and its mixtures) on calcium oxalate crystallization were studied. A test of urinary lithogenic risk was used to follow the crystallization of calcium oxalate from artificial urine in presence of several substances (phytate, pyrophosphate, citrate, chondroitin sulphate and some trace metals including Zn2+, Fe3+, Cu2+, Sn2+, Pb2+, Al3+, Ni2+, Cd2+ y Mn2+), which were assayed in physiological concentrations. Several substances such as phytate, pyrophosphate, citrate and, chondroitin sulphate were proved to significantly decrease the calcium oxalate crystallization, both in artificial and natural lithogenic urines. However, the existence of relevant interactions between these substances and their effect on modulating the inhibition capacity of calcium oxalate crystallization were also examined, indicating interactions between different urinary inhibitors of calcium oxalate crystallization and incipient crystals in urine can result either in synergic, additive or negative effects on the overall inhibition of insoluble urinary salts. Synergistic effects occur when substances with high affinity for calcium (i.e. phytate + pyrophosphate mixtures) are selectively adsorbed on actives sites of calcium oxalate crystallization, disturbing the crystal surface more effectively than the mere additive effect of the individual inhibitors. Negative effects take place when a substance with high affinity for calcium (i.e. phytate) is in presence of high amounts of another substance with lower affinity (i.e. citrate), resulting in a effective displacement of the former from actives sites of calcium oxalate crystallization, thus decreasing the overall inhibition below the expected additive effect. Finally, merely additive effects seem to occur when the substances involved in the inhibition have similar affinities for actives sites of calcium oxalate crystallization and/or they are present in urine in concentrations that do not exceed considerably from each other. As a consequence of the related effects, it can be concluded that the crystallization inhibitory capacity of urine could not be assigned to the inhibitory capacity of each individual substance but also to the possible interactions between these substances in the complex media of urine containing incipient crystals. In this sense, the abundant likely interactions of this compound with other components of urine (complex formation, precipitation, chemisorption on crystal surface, displacement from crystal surface) can determine the inability of a given substance to disturb the development of a specific insoluble salt. For example, the interactions of physiological concentrations of citrate with urinary phytate, can limit the high inhibitory potential of phytate, with the consequent disadvantage in prevention of calcium oxalate crystallization. On the other hand, physiological concentrations of trace metals in urine do not have a significant influence on calcium oxalate crystallization. In this sense, Fe3+, which is the unique trace metal that exhibit an intrinsic high inhibitory capacity of calcium oxalate crystallization at physiological concentrations, even increased by concomitant presence of phytate and pyrophosphate, is probably unable to act as a powerful inhibitor in presence of physiological concentrations of citrate, as a consequence of the negative effect produced for citrate and Fe3+ mixtures, due to the formation of highly stable complexes in solution without inhibitory activity. Because of the scarce knowledge of such interactions, further investigation in this concern will contribute to best assessment on designing prophylactic treatments with inhibitors. Evaluation of nocturnal urine as most appropriate urinary specimen for study of urolithiasis Finally, efficacy in the diagnosis of lithogenic alterations by the use of nocturnal urines as reference urinary specimen for study of urolithiasis was preliminary evaluated. In this sense, the main objective was to obtain a simplified scheme for a first simple and accurate diagnostic of urinary lithogenic alterations, also overcoming the known limitations of current schemes based on 24-hours urines. Nocturnal urines were able to effectively discriminate the main lithogenic factors of oxalocalcic lithiasis, mainly related to deficient inhibitory capacity (phytic acid, pyrophosphate and citrate concentrations) for calcium oxalate monohydrate and high urinary pH and high calciuria for calcium oxalate dihydrate. Thus, nocturnal urines can represent an objective for more appropriate evaluation of calcium oxalate lithogenic risk, that can be useful and suitable in diagnosing and monitoring of preventive therapy. This analysis, against the 24-hours urinalysis, can also simplify the necessary metabolic investigations in patients, being easy its implementation in clinical practice, with relevant advantages related to greater representativity with respect to lithogenic risk and easy of sample collection by the patient. In this sense, further study (now under development) is required before implementation of nocturnal urines for urolithiasis evaluation in order to establish new reference values for lithogenic risk assessment based on nocturnal urines, to extend the study to the other main kinds of lithiasis (phosphate and uric acid) and to compare the diagnostic power of nocturnal urines with respect to 24-hours specimens. The different contributions of this thesis could deserve for a better understanding of lithiasic etiology and better efficacy in the diagnosis of lithogenic alterations, both required to increase efficacy in the evaluation and monitorization of urolithiasis and to decrease incidence and recurrence rates by adoption of preventive measures.
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Bibliographical Information:

Advisor:Valiente Malmagro, Manuel

School:Universitat Autónoma de Barcelona

School Location:Spain

Source Type:Master's Thesis

Keywords:403 departament de quimica


Date of Publication:11/22/2004

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