Enzymatic Basis for Epimerization of Cardiotonic Steroids at Carbon 3 in Rat Liver<sup>*</sup>

Repke, K.; Samuels, Leo T.

doi:10.1021/bi00893a016

Cited by 31 publications

(10 citation statements)

References 38 publications

(41 reference statements)

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“…In tests of acute cardiotoxicity, however, digitoxin is 2.6-2.8 times more potent on a molar basis than the genin (45,46). The mechanism whereby the digitoxose residues potentiate the activity of the genin is unknown, although it has been attributed to increased water solubility and cell penetrability (47) and to protection of the genin from metabolic attack and inactivation at the 3/3-hydroxyl position (48). The increase in potency conferred on the genin by the sugars does not appear to vary with the number of digitoxose residues in the saccharide side chain; the monodigitoxoside is as potent as the tridigitoxoside, digitoxin (45,46).…”

Section: Resultsmentioning

confidence: 99%

Binding of digitoxin and some related cardenolides to human plasma proteins

Lukas¹,

Martino²

1969

J. Clin. Invest.

136

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A B S T R A C T Tritium-labeled digitoxin, digitoxigenin, digoxin, and digoxigenin of established purity and chemcal authenticity were used to study the binding of these compounds to human plasma proteins. 97% of digitoxin in plasma was nondialyzable. Continuous flow paper electrophoresis of plasma containing digitoxin and dialysis experiments in which human serum albumin competed for the glycoside with plasma or plasma protein fractions demonstrated that digitoxin was almost exclusively bound by albumin. Equilibrium dialyses revealed that the interaction was characterized by a single binding site on the albumin molecule and an association constant of 9.62 X 10' liter/mole at 370C. At 1VC the association constant was 4.64 X 10' liter/mole. The interaction therefore was endothermic; the gain in enthalpy of 3.5 kcal/mole and the free energy change of -7.06 kcal/ mole was derived from a large change in entropy of 33.8 cal/mole per 'K. The direction of these thermodynamic changes suggested the formation of a hydrophobic bond between digitoxin and albumin. Quenching of the fluorescence of albumin by digitoxin indicated that the conformation of albumin was altered by the binding process.Digitoxigenin, its mono-and didigitoxosides, digoxin, and digoxigenin competed with digitoxn for its binding site on albumin. The affinity of the mono-and didigitoxosides for the site was equal to that of digitoxin, but that of digitoxigenin was only one-third as great. The ability of the digitoxose residues of the glycosides to enhance binding to albumin was also observed with digoxin, Presented in part at the 28th Scientific Sessions of the American Heart Association, Miami Beach, Fla., 16 October 1965.Dr. De Martino was formerly a Postdoctoral Research Fellow of the National Heart Institute.Received for publication 15 November 1968 and in revised form 6 February 1969. which was more extensively bound by the protein than digoxigenin.At concentrations of 2 jg/ml or less in plasma, only 23% of digoxin was bound. Albumin, which interacted with digoxin with an apparent association constant of 9 X 102 liter/mole at 370C, was entirely responsible for the binding. Lowering the temperature from 370 to 1VC decreased the fraction of digoxin bound to albumin by two-thirds.The marked difference in avidity of digitoxin and digoxin for serum albumin is reflected by the higher plasma concentrations, lower rate of urinary excretion, and longer half-time of digitoxin as compared to those of digoxin when these compounds are administered to man.

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Section: Resultsmentioning

confidence: 99%

Binding of digitoxin and some related cardenolides to human plasma proteins

Lukas¹,

Martino²

1969

J. Clin. Invest.

136

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“…Stepwise hydrolysis of the trisaccharide moiety of either glycoside to the didigitoxoside (60,64), monodigitoxoside (60), and genin (60,61) has been demonstrated. In the liver, either of the genins may undergo epimerization to the physiologically inactive 3a-hydroxy form, which is then conjugated with glucuronic or sulfuric acid (61,63). The quantitative significance of these reactions has not been defined.…”

Section: Resultsmentioning

confidence: 99%

“…Separation of digitoxin from digoxin, digoxigenin didigitoxoside, digoxigenin monodigitoxoside, digoxigenin, digitoxigenin di-digitoxoside, digitoxigenin monodigitoxoside, and digitoxigenin, compounds that have been proposed to result from the metabolic degradation of digitoxin in man (41,42,(60)(61)(62)(63)(64), was accomplished by preacetylation chromatography. Thus, in the cyclohexane, dioxane, methanol, water (4: 4: 2: 1) system, digitoxin migrated 16 cm from the origin in 24 hours, whereas digoxin, digoxigenin, and compounds of intermediate polarity, the di-and monodigitoxosides of digoxigenin, were arrayed between 2 and 6 cm.…”

Section: Methodsmentioning

confidence: 99%

Double isotope dilution derivative assay of digitoxin in plasma, urine, and stool of patients maintained on the drug.

Lukas¹,

Peterson²

1966

J. Clin. Invest.

103

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Although the cardiac glycosides have been used extensively since Withering published his observations on the effects of foxglove nearly two hundred years ago (1), their distribution and metabolism and the cellular basis of their action in man are unknown. The lack of sufficiently sensitive, chemically specific methods of assay has hampered studies of the biologic behavior of these compounds.The sensitivity of available spectrophotometric and fluorometric methods is limited to 2 ,ug or more, and none of them are molecularly specific, since they involve structural components common to all cardenolides and many glycosides. Thus, production of color depends on the action of various reagents on either the lactone ring (2-11) or the 2-deoxy sugar residues (12)(13)(14)(15)(16)(17)(18)(19)(20). The a,,8-unsaturated lactone ring is also responsible for absorption of ultraviolet light at a wavelength of 217 mp (21). Fluorescence results from the action of strong acids on the steroid segment of the molecule and is attributable to dehydration and oxidation products and the formation of accessory ring structures (22)(23)(24)(25)(26)(27). All these reactions are subject to interference by numerous substances.Most current information regarding the metabolism of the cardiac glycosides is derived from studies employing bioassay methods (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41) embryo heart is sensitive to 0.005 to 2 pug of cardenolide or glycoside, depending on the source of the biologic extract and the degree of purification (28)(29)(30)(31)(32)41), and even smaller quantities of glycoside labeled with 14carbon (48-50) or tritium (51-53) can be detected. Neither method, however, is intrinsically specific.Bioassay measures not only the parent compound but also its active metabolites, as well as other cardioactive substances in the sample. Similarly, radioactivity in samples from a subject given a radioisotopically labeled glycoside may represent the original compound, metabolites, or degradation products commonly formed during extraction of submicrogram quantities of steroids. Isolation of the glycoside from its metabolic products by partition with solvents (43) and by paper (39) or column (45) chromatography before bioassay or radioassay has improved specificity, but precision is impaired unless means are incorporated to correct for losses during these procedures. An additional disadvantage of the direct administration of radioisotopic compounds to human subjects is that it can be used only for acute studies.The double isotope dilution derivative method is one of the most sensitive, precise, and chemically specific methods for measuring steroids in biologic extracts (54-57). As little as 0.2 m~ug of aldosterone in peripheral plasma can be determined (56). The method has proved to be applicable to digitoxin. This paper describes the techniques employed and presents data on digitoxin in the plasma, urine, and stool of patients treated chronically with this glycoside. MethodsPrinciple of assay. A quantity of tritiu...

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“…The 3 position of these sterols has proven to be an important site of enzymatic transformation (2), long thought to be under control of an unknown enzyme (3). After oxidation of the Sf3-OH group of the genins, cardiac activity is decreased by more than 90%.…”

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confidence: 99%

Digitalis metabolism and human liver alcohol dehydrogenase.

Frey¹,

Vallée²

1980

Proc. Natl. Acad. Sci. U.S.A.

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Human liver alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.1) catalyzes the oxidation of the 3d-OH group of digitoxigenin, digoxigenin, and gitoxigenin to their 3-keto derivatives, which have been characterized by high-performance liquid chromatography and mass spectrometry. These Our earliest kinetic studies of partially purified human liver alcohol dehydrogenase (LADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) showed its broad substrate specificity (1), encompassing, inter alia, aldehydes, primary and secondary aliphatic and aromatic alcohols, polyenoates, and sterols.We have found recently that highly purified human LADH inactivates digoxin, digitoxin, and gitoxin, glycosides employed widely in the treatment of cardiac failure. Specifically, human LADH catalyzes the oxidation of the 33-OH group of digoxigenin, digitoxigenin, and gitoxigenin, the genins of digoxin, digitoxin, and gitoxin, to their 3-keto derivatives.The 3 position of these sterols has proven to be an important site of enzymatic transformation (2), long thought to be under control of an unknown enzyme (3). After oxidation of the Sf3-OH group of the genins, cardiac activity is decreased by more than 90%. Moreover, LADH is the only cytosolic enzyme in human liver, the known site of genin metabolism, to catalyze this oxidation, which is the first major step in the abolition of the pharmacological action of these cardiac glycosides. Subsequent enzymatic conversion of the 3-keto to the 3a-OH group abolishes activity. The participation of human LADH in the oxidation of ethanol and of the genins, the active principles of digitalis, has important biochemical, therapeutic, and toxicological implications because it establishes a direct link between the metabolisms of these important drugs. The finding is a result of our efforts to delineate the substrate specificities, to identify and characterize the human isoenzymes, and to further explore the biochemical basis for the metabolism and pathology of ethanol. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. METHODS

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Enzymatic Basis for Epimerization of Cardiotonic Steroids at Carbon 3 in Rat Liver^*

Cited by 31 publications

References 38 publications

Binding of digitoxin and some related cardenolides to human plasma proteins

Binding of digitoxin and some related cardenolides to human plasma proteins

Double isotope dilution derivative assay of digitoxin in plasma, urine, and stool of patients maintained on the drug.

Digitalis metabolism and human liver alcohol dehydrogenase.

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Enzymatic Basis for Epimerization of Cardiotonic Steroids at Carbon 3 in Rat Liver*

Cited by 31 publications

References 38 publications

Binding of digitoxin and some related cardenolides to human plasma proteins

Binding of digitoxin and some related cardenolides to human plasma proteins

Double isotope dilution derivative assay of digitoxin in plasma, urine, and stool of patients maintained on the drug.

Digitalis metabolism and human liver alcohol dehydrogenase.

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Enzymatic Basis for Epimerization of Cardiotonic Steroids at Carbon 3 in Rat Liver^*