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

Abstract

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...

Binding of digitoxin and some related cardenolides to human plasma proteins

“…Published data derived from nine patients maintained on 0.1 mg of digitoxin daily (9) and from eight normal subjects maintained on a daily dose of 0.5 mg of digoxin-81H (50) are presented in Table III. Because of its greater avidity for albumin, plasma concentrations of digitoxin were larger than those of digoxin and exhibited less fluctuation during the course of the day.…”

“…In some experiments, digitoxin in ethyl acetate extracts of bag contents and dialysate was also measured by the m-dinitrobenzene color reaction (9). No significant or systematic discrepancies between the isotopic measurements and the colorimetric data were encountered, and the calculated values for fraction of digitoxin bound agreed within 1%.…”

“…The methods employed in preparing and purifying the compound and establishing its chemical purityand specific activity have been presented previously (9).…”

“…No significant or systematic discrepancies between the isotopic measurements and the colorimetric data were encountered, and the calculated values for fraction of digitoxin bound agreed within 1%. To check on intactness of the digitoxin, randomly selected extracts were dried and chromatographed on paper in a solvent system containing benzene, methanol, and water (4:2: 1; v/v) for 6 hr or cyclohexane, dioxane, methanol, and water (4: 4: 2: 1) for 24 hr (9). Radioscans of the chromatograms showed single peaks corresponding to digitoxin.…”

“…Additional information on the association of digitoxin with the plasma proteins is needed since, as in the case of other compounds (6)(7)(8), such an association influences the distribution, metabolism, and excretion of the glycoside in man (9). The availability of pure crystalline human serum albumin and of digitoxin-'H with a high specific activity (9) facilitated reexamination of the problem.…”