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Abstract

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.

Item Type:	Thesis - PhD
Authors/Creators:	Williams, Gethin
Keywords:	Phosphocitrate, Calcification, Phosphates, Fentanyl
Copyright Holders:	The Author
Copyright Information:	Copyright 1981 the author - The University is continuing to endeavour to trace the copyright owner(s) and in the meantime this item has been reproduced here in good faith. We would be pleased to hear from the copyright owner(s).
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