IMAGING SERVICES NORTH Boston Spa, Wetherby West Yorkshire, LS23 7BQ www.bl.uk This PDF was created from the British Library’s microfilm copy of the original thesis. As such the images are greyscale and no colour was captured. Due to the scanning process, an area greater than the page area is recorded and extraneous details can be captured. This is the best available copy > .nt ^ *r • itìceiltion isr-^ra: copyiight ot tills thesis rests witJi its author. " • • ^ ^ _ This copy of the thesis has been supplied on condition that anyone who consults it is • K upderstood to recognise that its copyright rests ^th its autlwr and that no quotation from toe thesis and no information derived from it ^ y be published X without the author’s prior [tten consent. - ■ Jli^ P ;?,934/?7 LtVACK V^- CíÁa^ f^oJ^ \)c~ Pr^ Z ( o S Of Loffíy»^ ' ( C N f \ ^ ' CONTENTS PAGE N O . 1. Abstract....................................................... 1 2. Acknowledgements............................................. (xiv) 3. Chapter 1 LITERATURE REVIEW ......................................... 2 1:1 The Significance of Plant-induced Calcinosis.......... 5 1:2 Endocrine Control of Calcium Metabolism............. 15 1:3 The Relationships Between the Vitamin D Endocrine 3 System and the Known Calcinogenic Plants •••••« ....... AO S u m n a r y ................................................ 55 A, Chapter 2 GENERAL METHODS ............................................. 57 2:1 Preparation of Extracts of Trisetum flavescens Using Aqueous and Organic Solvents .................. 58 2:2 Techniques for the Analysis of Plasoia Minerals and Alkaline Phosphatase............................. 55 2:3 Liquid Scintillation Counting ........................ 71 2:A Bone Culture Technique .......... ..................... yy 5. Chapter 3 The Biological Activity of Organic Soluble Extracts of Trisetum flavescens........... ................. 8A Experiment 1 Demonstration of the Ability of Organic Solvent Soluble Extracts of Trisetum flavescens to Promote Radiophosphate Uptake from the Ileum .................... 86 ^eriment 2 An Investigation into the Effects of a Purified Organic Extract of Trisetum flavescens on Radiocalcium Transport Across the Duodenum ............ 98 PAGE N O . 5. Chapter 3 Contd/... Experiment 3 The Effects of a Purified, Organic Soluble, Extract of Trisetum flavesoena on Bone Cultured ^ vitro ......................................... i q 4 6. Chapter A The Biological Activity of Aqueous Soluble Extracts of Trisetum flavescens ...................... 113 Experiment 1 A Comparison of the Effects of a Crude Aqueous Extract and a Partially Purified Aqueous Extract of Trisetum flavescens on Vitamin D^-Deficient Chicks...... 115 Experiment 2 Evidence for the Potentiation of Biologically Active Substances by the G u t ............... 120 Experiment 3 Ultra-violet Spectroscopy of Aqueous Extracts of Trisetum flavescens Following Sephadex Chromatography................................... j25 Experiment A(i) The "Strontium Chick” Model ............. 129 Experiment A(ii) Experiment to Determine the Effects of a Crude Aqueous Extract of Trisetum flavescens in the "Strontium C h i c k " ...................... 133 périment 5 A Comparison of the Effects of "Peak 3" and "Peak A" on Radio-calcium Uptake from the Gut, in the "Strontium Chick" .................................. 137 Experiment 6 (i) The Effects of "Peak A" on Plasma Calcium and Phosphate in Vitamin D^-Deficient Chicks.... lAA ^eperiment 6(ii) Examination of the Effect of A" on Plasma Calciiim and Phosphate in Vitamin IKreplete Chicks .................................. 151 (ii) PAGE NO. 6. Chapter 4 Contd/... Experiment 7 An Investigation into the Relative Effects of "Peak 4" and Vitamin on Ileal Radio-Phosphate Uptake ......................... 157 Experiment 8 The Effect of Partially Purified Aqueous Extracts of Trisetum flavescens on Bone Cultured ^ vitro .................................... 7. Chapter 5 Partial Purification and Characterisation of Experiment 1 Evidence that the Biologically Active Eluate from the Liquid Chromatography 1:1 Examination of the Ultra-*violet 1:4 Polarimetry ............................... Experiment 2 Evidence that the Phosphataemic Fraction Contains no Substances Structurally Similar to Vitamin ......................... 2:3 Comparison Between Vitamin D and the 3 Phosphataemic Fraction Using X-ray (iii) PAGE NO. 198 TJST OF TABLES PAGE NO. Table 1 The Distribution of Pastoral Calcinosis ................ 13 Table 2 Composition of Standard Calcium Solutions .............. 66 Table 3 Relationship Between Level of Formic Acid and Quench ... 73 Table A Table Showing Optimum Level of Solubilised Tissue for C o u n t i n g .............................................. 74 Table 5 The Optimum Level of Lumen Contents per Vial ........... 75 Table 6 Dosing Regimen ............................................ 87 Table 7 Effects Upon Blood Parameters of Chicks Treated with Various Organic Extracts of Trisetum flavescens ........ 90 Table 8 To Show the Results for the Neasurenents of the Transport 32 of Phosphate Across the Gut Wall as the Result of Administration of Organic Solvent-Soluble Extracts of Trisetum flavescens ...................................... 93 Table 9 Dosing Regimen ........................................... 99 Table 10 Plasma Calcium and Phosphate levels in Vitamin D^-Deficient Chicks Treated with an Active, Organic Solvent Soluble Extract of Trisetum flavescens and untreated controls. ..100 PAGE N O . Table 11 45 Duodenal Calcium Transport in Vitamin D-deficient Chicks Treated with Either Vitamin or an Organic Soluble Extract of Trisetum flavescens ........................... jqj Table 12 Treatment Regimen for Cultured Bones ............. ....... 105 Table 13 45 Calcium Released from Pre-labelled Calvariae for each Treatment....................................... 107 Table 14 • 40 Medium Total Calcium, Inorganic Phosphate and Glucose Levels of Bones Cultured in vitro ........... 109 Table 15 Dosing Regimen ............................................. ^^5 Table 16 The Effects of Two Aqueous Extracts of Trisetum flavescens on Plasma Levels of Calcium, Inorganic Phosphate and Alkaline Phosphatase in Vitamin IHdeficient Chicks ............... 117 Table 17 Dosing Regimen ............................................ 121 Table 18 Effects of Dosing Site on Plasma Calcium and Phosphate Levels 122 Table 19 Effects of Vitamin D^ and Calcitriol on Several Plasma Constituents in Strontium Treated Chicks 131 (Vi) PAGE NO . Table 20 The Effect of a Crude Aqueous Extract on Plasma Calcium, Inorganic Phosphate and Radiocalcium Transport ............................................ ^^4 Table 21 Dosing Regimen ................................. Table 22 The Effects of "Peak 3" and "Peak A" on Plasma Calcium, Phosphate and Strontium Levels .................. 139 Table 23 Table to Show the Effects of "Peak 3" and "Peak A" on Duodenal Calcium Transport in the Chick ................ lAO Table 2A Treatment of Experimental Groups .......... 144 Table 25 Plasma Calcium and Phosphate Changes in Chicks Dosed with "Peak A" ............................. ^45 Table 26 Changes in Plasma Calcium and Phosphate in Response to Oral Administration of "Peak A" .............. 152 Table 27 Dosing Regimen............................................. X58 Table 28 The Effects of Vitamin D^ and "Peak A" upon Plasma Calcium, Plasma Phosphate and Radio-Phosphate Absorption . 159 (vii) PAGE NO. Table 29 Dosing Regimen ............................................ Table 30 Sampling Times of Tissue Culture Medium ................. 165 Table 31 Table to Show the Effects on Medium Calcium and Phosphate Levels of "Peak 3" and "Peak 4" and Glucose Utilisation.................. ........ ...... 165 Table 32 The Effects of "Peak 4" l^on Radiocalcium Release from Pre-labelled Bones ......................... 167 Table 33 Ratios of Absorption Maxima Intensity of Fractions Obtained from a Sephadex G15 Column and a Sançle of Partially Purified Trisetum flavescens Extract Containing the Phosphataemic Principle................... .............. 174 Table 34 Table to Show Relative Peak Heights of Fractions Obtained from a Preparative Sephadex G25 Column, in the Region of the Phosphataemic Principle .................................. 175 (viii) PAGE N O . Table 35 Choice of a Suitable Mobile and Stationary Phase ....... 180 Table 36 Reagents Used in the Characterisation of "Peak A" Fractions...................................... i82 Table 37 Sample Positions .......................................... 191 Table 38 Comparison of the Physiological Effects of the Three Calcinogenic Factors of Trisetum flavescens with Whole Lyophilised Plant L e a f ............................. 215 (ix) PAGE NO. LIST OF FIGURES Figure I The Pathway of Biosynthesis of Vitamin D Metabolites............ 16 Figure 2 The Calcium Homeostatic Cycle ............................ 20 Figure 3 The Elution Profile at 280nm of a Crude Aqueous Extract of Trisetum flavescens Following Sephadex Chromatography........................ 61 Figure 4 Steps in the Preparation of an Organic Soluble Extract .......................................... 63 Figure 5 p-Nitrophenol Standard Curve............................... 70 Figure 6 Histogram Showing Levels of Radiophosphate Absorbed by the Ileum of Vitamin D^deficient 3 Chicks Dosed with Organic Soluble Plant Extracts ......... 95 Figure 7 The Effects of Organic Extracts of Trisetum flavescens on ^^Calcium Release from Bone