Williams_whole_thesis_ex_pub_mat.pdf (10.86 MB)
Phosphocitrate : its chemical synthesis, characterization, natural occurrence and role in calcifying systems
thesisposted on 2023-05-27, 15:23 authored by Williams, Gethin
A chemical synthesis of phosphocitrate (PC) was devised to facilitate investigation into its possible natural occurrence. Concomitantly, detection systems for characterising PC were developed utilizing thin layer chromatography, electrophoresis, isotachophoresis, and enzymatic and chemical PC assays. Synthesis of PC was effected by condensing 2-cyanoethyl phosphate (CEP) and triethyl citrate, followed by alkaline hydrolysis. CEP was considered to be the reagent-of-choice because of the ease of preparing [ 32P]-CEP from [32P]- inorganic phosphate enabling the production of [32P]-PC. An ion-exchange chromatographic system was developed to provide ultimate purification of PC, and it was characterised by 1H, 13C and 31P NMR spectroscopy. PC was demonstrated to inhibit potently hydroxyapatite (HAP) formation being more powerful than ATP and pyrophosphate. PC was also found to inhibit strongly calcium oxalate formation, its presence modifying crystal morphology. The unique structure of PC led to the proposal of a structure-activity relationship for inhibitors. Secondary factors influencing inhibitor power were elucidated and two groups of inhibitors were recognised. Type I inhibitors, which included PC, appeared to be crystal growth inhibitors, whereas Type II inhibitors were proposed to act by a range of mechanisms. The demonstration of a synergistic interaction between Type , I and Type II inhibitors was used to support the claim that in physiological fluids, PC could act as a potentiating agent. Comparison of a purified fraction from tissue samples with authenticated PC showed that PC occurs in kidney and liver of the rat and rabbit whilst other studies indicated that small amounts of PC were present in rabbit bone and human urine. More detailed studies indicated that PC was present, at a level of ca. 1 nmol/mg mitochondrial protein, in mitochondria, a subcellular organelle known to concentrate calcium. Administration of [ 32 P]-PC demonstrated that PC Was stable in blood but rapidly hydrolysed in kidney. Further, PC was rapidly taken up by bone, consistent with its physicochemical properties. These results suggested that although PC itself might be useful in treating some disorders of calcium metabolism, for other disorders, such as urolithiasis, a more stable analogue may be desirable. The nature of such analogues was discussed. Further experimentation indicated PC was compartmentalised within mitochondria and preliminary, work demonstrated that it could be biosynthesized there under the impetus of calcium loading. Data supported the proposal that compartmentalisation protected PC from the extramitochondrial lytic activity. The role of PC as a mitochondrial agent stabilising calcium phosphate deposits as amorphous spherules rather than HAP needles was described, and the effect of this stabilisation on membrane integrity, enzyme activity and calcium mobilisation was also detailed along with specific roles for PC in particular tissues, 'especially bone. Avenues for future research were presented' and possible roles of PC in the aetiology and therapy of some calcium metabolism disorders discussed.
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