Investigation of the potential beneficial effects of supplemental polyunsaturated fatty acids and glycosaminoglycans on the risk factors for calcium oxalate kidney stone formation using theoretical, experimental and human models
Doctoral Thesis
2014
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University of Cape Town
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Introduction: Two hypotheses with regard to calcium oxalate (CaOx) renal stone formation were tested in this thesis. The first hypothesis is that fatty acid (FA) supplementation (n-6 and n-3) and chondroitin sulphate (CS) supplementation may reduce the plasma (FA) and urinary (FA and CS) risk factors for CaOx renal stone formation, and ultimately serve as therapeutic agents in the management of this disease. The notion that FAs may reduce plasma risk factors is based on previous studies which have shown that n-6 and n-3 FA supplementation reduces the concentrations of arachidonic acid, while the notion of an effect on urinary risk factors is based on reports of these supplements decreasing urinary calcium and/or oxalate excretion in animal and human studies. The notion of CS playing a role in reducing CaOx stone risk is based on its chemical structure which presents potential binding sites for calcium and magnesium. The second hypothesis is that black and white healthy South African subjects may respond differently to these dietary supplements and that these differences may provide insights which could account for the lower stone incidence in the former group compared to the latter. This hypothesis is based on the observation in many previous studies of different renal responses in the two race groups, to different dietary and supplemental challenges. FA’s and CS have not been previously investigated in this regard. Methods: These hypotheses were tested simultaneously by administering n-6 and n-3 FA supplements individually and in combination, and supplemental CS, to different groups of black and white healthy male subjects. For the FA studies, blood samples were analyzed for serum biomarkers (25-hydroxyvitamin 03 and triglycerides) for CaOx stone formation and FA profiles in plasma total phospholipids since arachidonic acid regulates calcium excretion. Urine samples were analyzed for individual CaOx stone risk factors, risk indices (Tiselius risk index and supersaturation (SS) of calcium oxalate, brushite and uric acid); crystallization experiments (metastable limit and crystal growth kinetics) were also conducted. For the CS studies, thermodynamic binding constants for calcium-CS and magnesium-CS complexes were determined by isothermal titration calorimetry. These constants were then used to model speciation in different urines using the computer program Joint Expert Speciation System (JESS), and to calculate supersaturation values under different urinary conditions. This was followed by in vitro crystallization experiments in which the effects of exogeneous CS on the CaOx metastable limit and CaOx crystallization kinetics were investigated in artificial and real urine samples. Finally, human studies were performed in which CS supplements were administered to subjects to test their efficacy on reducing the urinary risk factors for CaOx stone formation. Urines were analyzed and crystallization experiments were performed as described for the FA supplementation studies. Results: In the FA studies, favourable changes in the plasma CaOx stone risk factors were achieved by the supplementation of n-3 FA alone. Post-supplementation, the concentration of arachidonic acid in plasma total phospholipids was significantly reduced in both groups, thereby implying a reduction in urinary calcium excretion. However, FA supplementation had no positive effect on the urinary risk factors or on CaOx metastable limits and CaOx crystallization kinetics. In the CS studies, theoretical modelling showed that the reduction of ionized calcium concentrations can only be attainable at 100 times physiological concentrations of urinary CS and that the formation of the calcium-CS complex does not influence the urinary supersaturation of CaOx. The formation of the magnesium-CS complexes was unfavourable because it resulted in an increase in the concentrations of ionized oxalate, a risk factor for CaOx stone formation. The in vitro crystallization experiments showed that exogeneous CS at physiological and above physiological concentrations had no favourable effect on the metastable limit and crystal growth kinetics in all the tested urine samples. Finally, the human study showed that CS supplementation had no effect on urine chemistry and crystallization kinetics. Speciation calculations also showed that the SS values of CaOx and the concentrations of ionized calcium were not significantly changed by supplementation. Within groups, the effects of FA supplementation on the urinary risk factors were different. n-6 FA supplementation significantly increased magnesium and significantly decreased urate in the black group whereas in whites citrate, oxalate and potassium were significantly increased while ionized calcium was significantly reduced. In the n-3 FA study, magnesium was significantly increased and SS value of brushite was significantly decreased in whites. In the black group, there was no significant difference in the urinary risk factors after supplementation compared to baseline values. With regards to n-6 & n-3 FA supplementation, citrate was significantly increased while oxalate and SS CaOx were significantly decreased in the black group whereas in the white group, magnesium was significant increased. With regards to the CS study, SS values for brushite, tribasic calcium phosphate, hydroxylapatite and octacalcium phosphate decreased significantly in black subjects after CS supplementation, whereas the SS value for hydroxylapatite increased significantly in the white group. These effects could not be attributed to complexation of CS with calcium or magnesium. However, they are noteworthy because they are different in the two groups. Discussion: The results of these studies do not support the hypothesis that supplemental fatty acids or chondroitin sulfate have significant beneficial effects for reducing blood and urinary risk factors for calcium oxalate stone formation. Although the response to FA and CS supplementation was different between the two race groups, these findings did not provide new information to explain the difference in the incidence of calcium oxalate stone disease in the two population groups. The work described in this thesis provides a foundation for future studies in which CaOx stone patients, rather than healthy individuals are investigated.
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Gogwana, P. 2014. Investigation of the potential beneficial effects of supplemental polyunsaturated fatty acids and glycosaminoglycans on the risk factors for calcium oxalate kidney stone formation using theoretical, experimental and human models. University of Cape Town.