Digoxin, a steroidal glycoside that inhibits Na⁺/K⁺-ATPase, is the most commonly prescribed cardiac medication in North America. Blood levels of this drug are routinely measured to reduce the risks of toxicity. Reports questioning the specificity of antisera used in radioimmunoassays for serum digoxin measurements began to appear after 1975¹ when plasma from patients with renal failure, not on glycoside therapy, showed false-positive digoxin levels. Since then, digoxin-like immunoreactive substances (DLIS) have been found in sera from patients with hepatic failure, hypertension, pre-eclampsia, in amniotic fluid and cord blood. Some of the highest values for DLIS have been detected in premature infants, where levels have often exceeded the therapeutic range (0.2-2.0 µg/L) for digoxin. Cord blood has been identified as a rich source of DLIS. Dahl et al² were the first to suggest that a circulating saluretic substance "endoxin", may cause hypertension in salt sensitive rats. Gruber et al³ reported on the existence of digoxin-like factor(s) in the plasma of volume-expanded dogs. Plasma from these dogs inhibited Na⁺/K⁺ATPase activity. A number of other studies have supported the concept that such digoxin-like factors may be of etiological significance in hypertension⁴. In view of these observations, a study was undertaken to isolate and fractionate DLIS from mixed cord blood and determine whether or not any of this digoxin-like material possessed Na⁺/K⁺-ATPase inhibitory properties. Cord blood collected in the Grace Hospital Maternity Unit (Vancouver, BC), was pooled and DLIS extracted using C₁₈,R-Sep Paks. Extracts were resolved by high performance liquid chromatography (HPLC) into several fractions containing digoxin equivalent immunoactivity as measured by radioimmunoassay (RIA). A number of steroids and bile acids (dehydroepi-androsterone-sulfate, cortisone, Cortisol, deoxycortisone, ∆⁴androstene-dione, progesterone and glycochenodeoxycholic acid) cross-reacted with digoxin antisera and had HPLC retention times similar to DLIS-containing fractions. The ability of HPLC generated DLIS positive cord blood fractions to inhibit Na⁺/K⁺-ATPase activity was determined in three different assay systems; red cell ⁸⁶Rb uptake canine kidney-Na⁺/K⁺-ATPase and red cell membrane-Na⁺/K⁺-ATPase. At least six fractions contained DLIS and inhibited Na⁺/K⁺-ATPase activity. Inhibition varied with the assay system used but none of the fractions inhibited ⁸⁶Rb uptake by erythocytes. One fraction (which eluted at 29 minutes) contained progesterone; 72% of the inhibitory activity present in this fraction was attributable to this steroid. Another inhibitory fraction co-eluted with dehydroepiandrosterone-sulfate (DHEAS-S). The only fractions found to inhibit both the red cell membrane and canine kidney Na⁺ /K⁺-ATPase enzymes eluted at 7 and 29 minutes. In summary, a number of digoxin-like immunoreactive substances were isolated from cord blood by HPLC fractionation and found to inhibit Na⁺/K⁺-ATPase activity. Inhibition varied with the assay system used. There was no apparent correlation between inhibition and digoxin immunoreactivity. Very large quantities (500 mL) of cord blood were extracted to demonstrate these properties. It remains to be determined whether or not DLIS isolated during the perinatal period is of physiological significance. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Wong, Shun-yun. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1989. / Bibliography: leaves 76-79.

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by Paul Li Wai Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Includes bibliographical references. / Acknowledgements --- p.i / Summary --- p.ii / Index to Figures --- p.V / Index to Tables --- p.vii / Abbreviations --- p.viii / CONTENTS / Chapter Chapter 1 --- INTRODUCTION --- p.1 / Chapter Chapter 2 --- LITERATURE REVIEW : SALT AND HYPERTENSION / Chapter 2.1. --- Summary of evidence linking salt and hypertension --- p.4 / Chapter 2.1.1. --- Epidemiological studies --- p.4 / Chapter 2.1.2. --- Dietary intervention studies --- p.7 / Chapter 2.1.3. --- Experimental studies --- p.9 / Chapter 2.2. --- Cellular sodium transport --- p.10 / Chapter 2.2.1. --- The Sodium Pump --- p.10 / Chapter 2.2.2. --- Defects in sodium transport in hypertension --- p.14 / Chapter 2.3. --- Hypothesis linking salt to the pathogenesis of hypertension --- p.15 / Chapter 2.4. --- Evidence for the presence of natriuretic Hormone --- p.18 / Chapter 2.4.1. --- Indirect evidence --- p.18 / Chapter 2.4.2. --- Direct evidence --- p.18 / Chapter 2.4.3. --- The source and properties of natriuretic hormone --- p.20 / Chapter 2.4.4. --- Other natriuretic factors --- p.21 / Chapter Chapter 3 --- REGULATION OF THE SODIUM PUMP / Chapter 3.1. --- General introduction --- p.24 / Chapter 3.2. --- Regulation of the sodium pump by intracellular sodium --- p.24 / Chapter 3.3. --- "Effects of ethanol on Na+,K+-ATPase activity" --- p.26 / Chapter 3.4. --- "Effects of potassium depletion on Na+,K+-ATPase activity" --- p.27 / Chapter 3.4.1. --- In vivo studies of sodium pump regulation by potassium --- p.27 / Chapter 3.4.2. --- In vitro studies of sodium pump regulation by potassium --- p.29 / Chapter 3.5. --- Effects of cardiac glycosides on the sodium pump --- p.30 / Chapter 3.5.1. --- In vivo studies of sodium pump regulation by cardiac glycosides --- p.31 / Chapter 3.5.2. --- In vitro studies of sodium pump regulation by cardiac glycosides --- p.33 / Chapter 3.6. --- Effects of dietary salt on the sodium pump --- p.35 / Chapter 3.6.1. --- Acute effects of salt-loading --- p.35 / Chapter 3.6.2. --- Chronic effects of salt-loading --- p.36 / Chapter Chapter 4 --- AIMS OF THE STUDY --- p.39 / Chapter Chapter 5 --- MEASUREMENT OF THE SODIUM PUMP ACTIVITY / Chapter 5.1. --- General introduction --- p.41 / Chapter 5.2. --- The measurement of sodium pump activity --- p.42 / Chapter 5.2.1. --- The sodium pump transport activity --- p.42 / Chapter 5.2.2. --- Quantitation of the number of sodium pump sites --- p.45 / Chapter 5.2.3. --- The measurement of enzyme activity --- p.47 / Chapter (a) --- Introduction --- p.47 / Chapter (b) --- Preparation of tissues and detergent treatment --- p.48 / Chapter (c) --- Measurement of ATPase activity by measuring the rate of release of inorganic phosphate --- p.49 / Chapter (d) --- The coupled-enzyme assay --- p.53 / Chapter (e) --- The K+-stimulated 3-0-MFPase assay --- p.54 / Chapter Chapter 6 --- METHODS - ESTABLISHMENT AND EVALUATION / Chapter 6.1. --- Chemicals --- p.57 / Chapter 6.2. --- "Measurement of Na+,K+-ATPase activity by the rate of release of inorganic phosphate" --- p.58 / Chapter 6.3. --- "Automated coupled-enzyme assay of Na+,K+-ATPase activity" --- p.62 / Chapter 6.4. --- "The measurement of Na+,K+-ATPase activity by the potassium-stimulated 3-0-MFPase assay" --- p.67 / Chapter 6.5. --- Determination of protein concentration --- p.70 / Chapter 6.6. --- Statistical analysis --- p.73 / Chapter 6.7. --- Results --- p.73 / Chapter 6.7.1. --- Evaluation of the inorganic phosphate release method --- p.73 / Chapter 6.7.2. --- Evaluation of the coupled-enzyme method --- p.78 / Chapter 6.7.3. --- Evaluation of the K+-stimulated 3-0-MFPase method --- p.89 / Chapter 6.7.4. --- Evaluation of the protein determination method --- p.94 / Chapter 6.8. --- Discussion --- p.96 / Chapter Chapter 7 --- "THE EFFECTS OF DIGOXIN TREATMENT ON Na+,K+-ATPase ACTIVITY OF DIFFERENT TISSUES" / Chapter 7.1. --- Introduction --- p.101 / Chapter 7.2. --- Materials and Methods --- p.103 / Chapter 7.2.1. --- Animals and diets --- p.103 / Chapter 7.2.2. --- Drugs --- p.103 / Chapter 7.2.3. --- Pharmacokinetics of digoxin --- p.103 / Chapter 7.2.4. --- The digoxin regimes --- p.104 / Chapter 7.2.5. --- Preparation and deoxycholate treatment of tissue homogenates --- p.105 / Chapter 7.2.6. --- "Measurement of Na+,K+-ATPase activity" --- p.107 / Chapter 7.2.7. --- Digoxin radioimmunoassay --- p.107 / Chapter 7.2.8. --- Measurement of plasma electrolytes --- p.109 / Chapter 7.3. --- Results --- p.110 / Chapter 7.3.1. --- The pharmacokinetics of digoxin in the rat --- p.110 / Chapter 7.3.2. --- Plasma digoxin levels --- p.110 / Chapter 7.3.3. --- Effects of digoxin treatment on body weight --- p.113 / Chapter 7.3.4. --- Effects of digoxin treatment on plasma electrolytes --- p.117 / Chapter 7.3.5. --- "Effects of digoxin treatment on tissue Na+,K+-ATPase activity" --- p.119 / Chapter 7.4. --- Discussion --- p.129 / Chapter Chapter 8 --- THE SALT-LOADING EXPERIMENT / Chapter 8.1. --- Introduction --- p.140 / Chapter 8.2. --- Materials and Methods --- p.142 / Chapter 8.2.1. --- Animals --- p.142 / Chapter 8.2.2. --- The salt-loading protocol --- p.142 / Chapter 8.2.3. --- Preparation of crude tissue homogenates --- p.143 / Chapter 8.2.4. --- "Measurement of Na+,K+-ATPase activity" --- p.144 / Chapter 8.2.5. --- Analysis of urinary electrolytes --- p.144 / Chapter 8.2.6. --- Measurements of body weight and wet weight of kidney --- p.145 / Chapter 8.3. --- Results --- p.145 / Chapter 8.3.1. --- Effects of salt loading on the body weight --- p.145 / Chapter 8.3.2. --- Effects of salt loading on 24-hour urinary sodium excretion --- p.148 / Chapter 8.3.3. --- Effects of salt loading on the wet weight of kidney --- p.152 / Chapter 8.3.4. --- "Effects of salt loading on tissue Na+,K+-ATPase activity" --- p.152 / Chapter 8.4. --- Discussion --- p.163 / Chapter Chapter 9 --- CONCLUSIONS AND FUTURE WORK --- p.175 / REFERENCES --- p.183

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No description available.

Thesis (Ph. D.)--Victoria University (Melbourne, Vic.), 2007. / Includes bibliographical references.

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 200-242). Also available on the World Wide Web.

Thesis (M. App. Sc.)--Victoria University (Melbourne, Vic.), 2006. / Includes bibliographical references.

Zugl.: Giessen, Univ., Diss., 2005.