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

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 (12-20). 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 (22-27). 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 (28-41) or the administration of radioisotopically labeled compounds (42-53). Bioassay with the chick or duck * embryo heart is sensitive to 0.005 to 2 pug of car-denolide or glycoside, depending on the source of the biologic extract and the degree of purification (28-32, 41), and even smaller quantities of glyco-side 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 bio-logic extracts …