Digitoxigenin Monodigitoxoside Research Articles

Cytotoxic and cytostatic effects of digitoxigenin monodigitoxoside (DGX) in human lung cancer cells and its link to Na,K-ATPase

Cardiac glycosides (CGs) are natural compounds widely used to treat several cardiac conditions and more recently have been recognized as potential antitumor agents. They are known as Na,K-ATPases ligands, which is a promising drug target in cancer. In this study, the short and long-lasting cytotoxic effects of the natural cardenolide digitoxigenin monodigitoxoside (DGX) were evaluated against two non-small cell lung cancer lines (A549 and H460 cells). It was found that DGX induced cytotoxic effects in both cells and the apoptotic effects were more pronounced on H460 cells. In long-term analysis, using the clonogenic and the cumulative population doubling (CPD) assays, DGX showed a reduction of cell survival, after 15days without re-treatment. To better understand DGX effects in A549 cells, several assays were conducted. In cell cycle analysis, DGX caused an arrest in S and G2/M phases. This compound also increased the number of cells in subG1 phase in a concentration- and time-dependent manner. The presence of β-galactosidase positive cells, large nucleus and flattened cells indicated senescence. Additionally, DGX inhibited Na,K-ATPase activity in A549 cells, as well as in purified pig kidney and in human red blood cell membrane preparations, at nanomolar range. Moreover, results of molecular docking showed that DGX binds with high efficiency (−11.4Kcal/mol) to the Na,K-ATPase (PDB:4HYT). Taken together, our results highlight the potent effects of DGX both in A549 and H460 cells, and disclose its link with Na,K-ATPase inhibition.

Inhibition of cell proliferation, invasion and migration by the cardenolides digitoxigenin monodigitoxoside and convallatoxin in human lung cancer cell line

Cardiac glycosides consist of a large family of naturally derived compounds that are clinically used to treat congestive heart failure, and also present anticancer properties. In this study, the cytotoxic effects of two cardenolides, digitoxigenin monodigitoxoside (DGX) and convallatoxin (CON) were screened in four human tumour cell lines. Both compounds showed anti-proliferative effects in all tumour cells, at nanomolar concentrations. Since the human lung cancer cell line A549 was the most sensitive, we investigated the anti-proliferative, anti-migratory and anti-invasive effects of these cardenolides. DGX and CON reduced A549 cell migration, being able to reduce more than 90% of cell invasion. Their effects on the expression of key regulators of metastatic mechanism showed decreased levels of MMP-2, MMP-9 and p-FAK. Both compounds also presented low toxicity for healthy cells. Finally, this work provides the first insights into the effects of these cardenolides on key steps of lung cancer metastasis.

Effects of Digitoxigenin, Digoxigenin, and Various Cardiac Glycosides on Cardenolide Accumulation in Shoot Cultures ofDigitalis lanata

Various cardenolide genins and cardenolide glycosides were administered to light-grown and dark-grown Digitalis lanata shoot cultures to investigate conversion reactions related to the formation and rearrangement of the sugar side chain of Digitalis glycosides. Digitoxigenin was converted to digitoxigen-3-one, 3-epidigitoxigenin, and digoxigenin. In addition, various cardiac glycosides were formed, including mono-glycosides with glucose, glucomethylose, fucose, and digitalose, as well as the corresponding diglycosides, all containing a terminal glucose. Digitoxosylated cardenolides were not formed, although the light-grown shoot cultures were capable of producing these compounds. Exogenous cardenolide fucosides were not converted into cardenolide digitoxosides. Administration of evatromonoside (digitoxigenin monodigitoxoside) did not force the formation of cardenolide di- or tridigitoxosides. Our results support the hypothesis that cardenolide fucosides and digitoxosides are formed via different biosynthetic routes and that cardenolide genins can be fucosylated but not digitoxosylated, indicating that digitoxosylation may only occur at an earlier stage in the cardenolide pathway.

Preparation and antigenic properties of digitoxin-bovine serum albumin conjugates linked at the digitoxose C-3' and C-3" positions.

In order to obtain specific antisera to digitoxin, four new types of hapten-bovine serum albumin (BSA) conjugates were synthesized from digitoxin. The haptens were linked to the carrier protein through hemisuccinate and hemisuccinylglycine bridges at the C-3' and C-3" positions in the digitoxose chain. The antisera were prepared by immunizing rabbits with each digitoxin-BSA conjugate and the properties of the antisera were investigated by RIA with 3H-labeled digitoxin. Among these antisera, the antiserum raised against digitoxin 3'-hemisuccinate-BSA conjugate possessed high specificity for digitoxin, exhibiting only minor cross-reactions with digitoxigenin bisdigitoxoside (4.0%), dihydrodigitoxin (2.8%), digoxin (2.4%), digitoxigenin monodigitoxoside (0.23%) and digitoxigenin (< 0.05%).

Differential stability of drug-metabolizing enzyme activities in primary rat hepatocytes, cultured in the absence or presence of dexamethasone

The effects of primary hepatocyte culture on the rat cytochrome P450-dependent monooxygenase system and several conjugating enzyme activities were examined using a culture system similar to those used for evaluation of chemicals as potential genotoxins. Cytochrome P450 and cytochrome b 5 contents progressively decreased throughout the 72-h culture period to < 25% of initial values, whereas cytochrome P450 reductase rapidly decreased by 50% during attachment, but then remained stable. Cytochrome P450-dependent testosterone hydroxylase activities decreased more rapidly in culture than did cytochrome P450 content reaching < 50% of attachment levels by 24 h. Cytochrome P450IIIA immunoreactive protein decreased at a similar rate to testosterone-6β-hydroxylase. Activated UDP-glucuronyltransferase activities towards 1-naphthol and testosterone declined more slowly over the 72 h than cytochrome P450 and remained at 50–60% of initial values at 72 h. UDP-glucuronyltransferase activity towards digitoxigenin monodigitoxoside (DIG) did not decrease during culture. Glutathione- S-ransferase and sulfotransferase activities also declined during the 72 h at rates which appeared to be isozyme-dependent. Addition of 1 μM dexamethasone (DEX) to the culture medium increased UDP-glucuronyltransferase activity towards DIG, cytochrome P450 reductase and testosterone-6β-hydroxylase activities up to 2.5-, 2.0- and 7-fold, respectively and induced cytochrome P450IIIA immunoreactive protein(s) in the hepatocytes after 24 and 48 h of culture; DEX was less effective at the 72 h time-point. DEX treatment also significantly accelerated the decreases in glutathione- S-transferase activities and in sulfotransferase activities towards 1-naphthol and estrone. Thus, it appears that primary rat hepatocytes cultured under standard conditions, not only rapidly lose their monooxygenase capabilities, but also some of their capacity for conjugation.

Characterization of paracetamol UDP-glucuronosyltransferase activity in human liver microsomes

A specific high performance liquid Chromatographie assay has been developed for the measurement of paracetamol glucuronide formation by the microsomal fraction of human liver. The procedure has been used to characterize paracetamol glucuronidation kinetics in human liver microsomes and to assess the substrate specificity of the paracetamol UDP-glucuronosyltransferase (UDPGT) activity. Paracetamol glucuronidation followed Michaelis-Menten kinetics, suggesting the involvement of a single form of UDPGT, or possibly two or more forms of UDPGT with similar affinities for paracetamol, in this reaction. Mean apparent K m and V max values were 7.37 ± 0.99 mM and 4.76 ±1.35 nmol/min/mg, respectively. Addition of the non-ionic detergent Brij 58 to microsomal incubations resulted in approximately 50% activation of microsomal paracetamol UDPGT-activity. This contrasts to the approximately three-fold activation of 4-methylumbelliferone, morphine and 4-nitrophenol glucuronidation observed following Brij 58 treatment of human liver microsomes. The glucuronidated xenobiotics chloramphenicol, digitoxigenin monodigitoxoside, 4-hydroxybiphenyl, 4-methylumbelliferone, morphine, 1-naphthol and 4-nitrophenol were screened for inhibitory effects on paracetamol glucuronidation. Of these compounds, only digitoxigenin monodigitoxoside and 1-naphthol were found to cause significant inhibition of paracetamol UDPGT activity. Along with the results of previous studies of the kinetics and inhibitor profile of human liver glucuronidation reactions (Miners et al., Biochem Pharmacol 37: 665–671, 1988 and 37: 2839–2845, 1988), these data indicate that the model glucuronidated substrates paracetamol, morphine and 4-methyllumbelliferone may be used to differentiate at least four human liver UDPGT isozyme activities.

Kinetic and inhibitor studies of 4-methylumbelliferone and 1-naphthol glucuronidation in human liver microsomes

The glucuronidation kinetics of 4-methylumbelliferone (4MU) and 1-naphthol (1NP) have been investigated in human liver microsomes to determine the validity of using these compounds as probes for specific UDP-glucuronosyltransferase (GT) activities in human liver. 4MU glucuronidation followed Michaelis-Menten kinetics, whereas 1NP glucuronidation kinetics were biphasic. Cross inhibition studies were performed with 4MU and 1NP to determine the relationship between 4MU glucuronidation and the two phases of 1NP glucuronidation. 4MU glucuronidation was competitively inhibited by 1NP but 4MU inhibited only the high affinity component of 1NP glucuronidation. There was good agreement between the apparent K m values for 4MU and the high affinity component of 1NP glucuronidation and their respective apparent K i values determined in the cross inhibition studies. These data suggest that the same form(s) of human liver GT is involved in 4MU glucuronidation and the high affinity component of 1NP glucuronidation. A number of compounds known to be specific substrates for purified rat liver GTs were screened for inhibitory effects on 4MU glucuronidation in human liver microsomes. 4-Nitrophenol, 2-aminophenol and androsterone inhibited 4MU glucuronidation whereas bilirubin, chloramphenicol, digitoxigenin monodigitoxoside, morphine, oestrone and testosterone had no effect. 4-Nitrophenol and 2-aminophenol were competitive inhibitors of 4MU glucuronidation but the inhibition of 4MU glucuronidation by androsterone followed atypical kinetics. Overall, the substrate specificity of the human liver 4MU/high affinity 1NP-GT activity appears to be broadly similar to that of the 3-methylcholanthrene inducible rat hepatic microsomal GT.

Studies on the pregnenolone-16α-carbonitrile-inducible form of rat liver microsomal cytochrome P-450 and UDP-glucuronosyltransferase

Treatment of rats with pregnenolone-16α-carbonitrile (PCN) markedly induces rat liver microsomal cytochrome P-450p and UDP-GT-dt 1, a glucuronosyltransferase active towards the digitoxin metabolite, digitoxigenin monodigitoxoside. The present study characterizes the regulation of these two enzymes in rats treated with different xenobiotics. Like PCN, treatment of rats with dexamethasone, spironolactone, troleandomycin or erythromycin estolate markedly induced both UDP-GT-dt 1 and cytochrome P-450p (measured as erythromycin demethylase and testosterone 2β-, 6β-, 15β-, and 18-hydroxylase activities). However, compared to PCN and dexamethasone, both troleandomycin and erythromycin estolate preferentially induced cytochrome P-450p, whereas spironolactone preferentially induced UDP-GT-dt 1. Treatment of rats with the polychlorinated biphenyl mixture, Aroclor 1254, increased both cytochrome P-450p and UDP-GT-dt 1 activity to about 40% of that in liver microsomes from rats induced with PCN or dexamethasone. Treatment of rats with phenobarbital or chlordane caused a relatively small increase in cytochrome P-450p and UDP-GT-dt 1 activity. Neither enzyme was induced by treatment of rats with 3-methylcholanthrene, rifampin or digitoxin. The induction of cytochrome P-450p and UDP-GT-dt 1 by PCN followed similar dose-response curves. Although cytochrome P-450p and UDP-GT-dt 1 are differentially affected by the age and the sex of rats, the enzymes responded similarly, but not identically, to xenobiotic treatment. This suggests that cytochrome P-450p and UDP-GT-dt 1 are co-inducible but not coordinately regulated.

Simultaneous analysis of digitoxin and its clinically relevant metabolites using high-performance liquid chromatography and radioimmunoassay

A specific assay for determining the urinary excretion of unchanged digitoxin and its metabolites is described. The procedure includes solvent extraction of uring at pH 8.5, reversed-phase high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA) of equivalent fractions. Confidence limits showed good linearity and precision of recovery and high sensitivity, accuracy and specificity. Cross-reactivities were high for digitoxigenin (DGTN) and digitoxigenin bisdigitoxoside (Bis-DGTN), they were low for digitoxigenin monodigitoxoside (Mono-DGTN) or digoxin when [ 125I] digitoxin RIA was used. The interference of endogenous compounds in urine in the RIA was overcome by using HPLC. Compared with results reported in the literature, the urinary recovery of unchanged digitoxin was lower, being only 8.11 ± 1.51% of the dose administered. Amounts of 6.52 ± 1.31% were excreted hydrolysed as Bis-DGTN, Mono-DGTN, DGTN or C 12-hydroxylated as digoxin.

Digitoxin metabolism by liver microsomal cytochrome P-450 and UDP-glucuronosyltransferase and its role in the protection of rats from digitoxin toxicity by pregnenolone-16α-carbonitrile

The aim of the present study was to investigate whether the mechanism by which pregnenolone-16α-carbonitrile (PCN) protects rats from digitoxin toxicity was dependent on the induction of liver microsomal cytochrome P-450 p and/or the UDP-glucuronosyltransferase active toward digitoxigenin monodigitoxoside (UDP-GT-dt 1). Evidence is presented that suggests troleandomycin is a selective inhibitor of cytochrome P-450 p in vivo, based on the pattern of inhibition observed when zoxazolamine paralysis time and hexobarbital sleeping time were measured in rats treated with different cytochrome P-450 inducers. A single dose of troleandomycin completely reversed the ability of PCN to protect rats from digitoxin toxicity, establishing the importance of cytochrome P-450 p induction in the protective effect of PCN. The postpubertal decline in constitutive cytochrome P-450 p levels in female but not male rats was paralleled by a female-specific, age-dependent decline in the rate of digitoxin sugar cleavage (i.e., digitoxosyl oxidation of digitoxin to 15′-dehydrodigitoxin and digitoxosyl cleavage to digitoxigenin bisdigitoxoside). This resulted in a marked sex difference in the rate of digitoxin sugar cleavage catalyzed by liver microsomes from mature rats (male/female ~ 6). However, no sex difference in digitoxin toxicity was observed in either immature or mature rats. In contrast to cytochrome P-450 p, liver microsomal UDP-GT-dt 1 activity increased dramatically with age in both male and female rats (mature/immature ~ 10). However, no age differences in digitoxin toxicity were observed in rats of either sex. The results indicate that cytochrome P-450 p and UDP-GT-dt 1 can be independently regulated in rat liver and that large changes in the constitutive levels of these microsomal enzymes have no effect on digitoxin toxicity. This suggests that the induction of cytochrome P-450 p and UDP-GT-dt 1 does not fully account for the mechanism by which PCN protects rats from digitoxin toxicity.

Induction of digitoxigenin monodigitoxoside UDP-glucuronosyltransferase activity by glucocorticoids and other inducers of cytochrome P-450p in primary monolayer cultures of adult rat hepatocytes and in human liver.

We have recently proposed that glucocorticoids induce cytochrome P-450p, a liver microsomal hemoprotein originally isolated from rats treated with the antiglucocorticoid pregnenolone 16 alpha-carbonitrile (PCN), through a mechanism that involves a stereospecific recognition system clearly distinguishable from the classic glucocorticoid receptor (Schuetz, E. G., Wrighton, S. A., Barwick, J. L., and Guzelian, P. S. (1984) J. Biol. Chem. 259, 1999-2012). We now report that digitoxigenin monodigitoxoside UDP-glucuronosyltransferase (DIG UDP-glucuronosyltransferase), a liver microsomal enzyme activity induced by PCN in rats, is also inducible, as is P-450p, in primary monolayer cultures of adult rat hepatocytes. DIG UDP-glucuronosyltransferase activity closely resembled reported characteristics of induction of P-450p in its time course of induction, concentration-response relationships, exclusivity of induction by steroids with glucocorticoid properties, unusual rank order of potency of glucocorticoid agonists, unusually high ED50 for induction by glucocorticoids, enhanced induction rather than inhibition by anti-glucocorticoids in the presence of glucocorticoids, and finally, induction by nonsteroidal inducers of P-450p. DIG UDP-glucuronosyltransferase activity was also readily detected in human liver microsomes and was elevated in two patients who had received inducers of P-450p. We conclude that the liver enzymes controlled by the postulated PCN recognition system include not only P-450p but also one or more UDP-glucuronosyltransferases.

Effects of butylated hydroxyanisole on hepatic glucuronidation capacity in mice

The present study has examined biochemical mechanisms by which butylated hydroxyanisole (BHA) increases the glucuronidation of xenobiotics. Male and female Swiss Webster mice received BHA in the diet (1% w/w) for 10 days (600 to 800 mg/kg/day). Hepatic UDP-glucuronosyltransferase activities were increased toward specific substrates in native and detergent-activated microsomes. In general, BHA increased glucuronidation toward group 1 substrates (1-naphthol and 4-nitrophenol) 36 to 141% whereas no changes were found with a group 2 (chloramphenicol) or a group 3 substrate (digitoxigenin monodigitoxoside). Also, activities toward unclassified substrates (estrone, acetaminophen, and diethylstilbestrol) were increased (29 to 139%) by BHA. BHA treatment also increased hepatic UDP-glucuronic acid content (two- to threefold) by increasing UDP-glucose concentration (30 to 50%) and enhancing UDP-glucose dehydrogenase activity (300 to 400%). BHA had a more pronounced effect in female than male mice when data were expressed as activity or amount per liver because BHA treatment selectively increased the liver weight in female mice. In conclusion, BHA increases the capacity for glucuronidation in mice by elevating both UDP-glucuronosyltransferase activities and UDP-glucuronic acid concentration in liver.

On the substrate specificity of the digitoxigenin monodigitoxoside conjugating UDP-glucuronyltransferase in rat liver

The aim of the present study was to investigate the specificity of the UDP-glucuronosyltransferase (EC 2.4.1.17) involved in the conjugation of digitoxigenin monodigitoxoside. By in vitro assays with detergent activated liver musomes it was found that (1) digitoxigenin monodigitoxoside is by far the best substrate of all cardenolides and cardenolide digitoxosides tested. (2) In the presence of saturating UDP-glucuronate concentrations an apparent K m of 5.8 μM was obtained from linear Lineweaver-Burk plots together with a V max of about 150 pmoles/mg musomal protein/min. (3) Neither phenobarbital nor polycyclic aromatic hydrocarbons caused a considerable induction of the enzyme without change of the apparent K m , but spironolactone did. (4) The conjugation of the substrate (4 μM) could only be inhibited by the 3'-epi-digitoxoside of digitoxigenin. (5) 25–50 μM substrate inhibited only the conjugation of the 3'-epimer and that of digoxigenin monodigitoxoside. It is suggested that there is a form of glucuronyltransferase which specifically conjugates digitoxigenin monodigitoxoside.

Induction studies on the functional heterogeneity of rat liver UDP-glucuronosyltransferases

Differential induction with phenobarbital (PB) and 3-methylcholanthrene (3-MC) suggests at least two functionally distinct UDP glucuronosyltransferases (UDP-GT) which have different acceptor selectivities. One form is induced by 3-MC and preferentially conjugates group 1 acceptors, such as p-nitrophenol and 1-naphthol. Another UDP-GT is induced by PB and glucuronidates group 2 aglycones, morphine and chloramphenicol. To further study this functional heterogeneity, male Sprague-Dawley rats were pretreated with the following microsomal enzyme inducers: 7,8-benzoflavone (BF); benzo( a)pyrene (BP); 3-MC; 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD); butylated hydroxyanisole (BHA); isosafrole; PB; pregnenolone-16α-carbonitrile (PCN); trans-stilbene oxide (TSO). The effect of induction on UDP-GT activity was determined with nine acceptors. Conjugation of group 1 aglycones, naphthol and p-nitrophenol, was increased by 3-MC (185 and 80%, respectively) whereas PB was ineffective. Conjugation of group 2 acceptors, morphine and chloramphenicol, was stimulated by PB (120 and 250%, respectively) while 3-MC had little effect. BP and TCDD enhanced glucuronidation of group 1 aglycones. ISF and TSO induced conjugation of both acceptor groups but were more effective for group 2. BF and BHA had negligible effects on UDP-GT activity. Since glucuronidation of valproic acid was increased only by PB and TSO treatment, this aglycone is probably a group 2 acceptor. Conjugation of digitoxigenin-monodigitoxoside (DIG) was stimulated by PB (200%) and PCN (1200%). PCN did not induce glucuronidation of group 1 acceptors but did have a slight effect on group 2 aglycones (130 and 40% for chloramphenicol and morphine, respectively). The 12-fold increase in DIG conjugation by PCN pretreated rats suggests that PCN may induce another group (form) of UDP-GT which preferentially glucuronidates DIG. Differential induction of UDP-GT activities within each group of acceptors indicates possible additional heterogeneity of the transferase.

Glucuronidation of digitalis glycosides by rat liver microsomes: Stimulation by spironolactone and pregnenolone-16α-carbonitrile

In the present studies, glucuronidation of [ 3H]digoxigenin monodigitoxoside and [ 3H]digitoxigenin monodigitoxoside has been investigated in vitro using rat liver microsomes. During a 40-min incubation period, the conjugation of digitoxigenin monodigitoxoside was five times greater than of digoxigenin monodigitoxoside at a substrate concentration of 5 μm and three times greater at a 20 μM substrate concentration. No difference in conjugation rates was observed between male and female rats, with either substrate. Pretreatment with spironolactone (100 mg/kg/day for 3 days) stimulated the conjugation of both substrates in females, but no significant changes were observed in males with either substrate. Pretreatment (75 mg/kg/day) with pregnenolone-16α-carbonitrile (PCN) caused enhanced glucuronidation of both substrates in both sexes. The conjugation rates in the PCN-pretreated rats were approximately twice those of the spironolactone-pretreated rats. The possible role of this increased conjugation in the protective effect of spironolactone and PCN against digitalis toxicity is discussed.

Impaired biliary excretion of digitoxin and its metabolites after treatment with polychlorinated biphenyls

In order to study the effects of polychlorinated biphenyls (PCB), potent inducers of microsomal drug-metabolizing enzymes, on the elimination rate of digitoxin (DT-3), the bile of rats were collected for 4 hr after a single dose of tritiated digitoxin ([ 3H]DT-3). In comparison to normal rats PCB caused a decrease of elimination by 50% whereas phenobarbital (PB) increased the rate by 36%. After extracting the bile with CHCl 3, measurements of radioactivity revealed that PCB-treated rats excreted only 3.2% of the dose as CHCl 3-soluble compounds. The corresponding fraction of both normal and PB-pretreated rats contained about 10% of the dose. The increased DT-3 elimination produced by PB was due to the large amount of water-soluble metabolites (nearly 24% of the dose). Normal and PCB-pretreated rats excreted about 10% water-soluble metabolites. Further analysis of metabolites showed: (1) In addition to the known metabolites (digoxin, bis-, and monodigitoxosides of both digitoxigenin and digoxigenin) the CHCl 3-soluble fraction contained a more lipophilic fraction. It consisted mainly of the dehydro-bis-digitoxoside of digitoxigenin. (2) Free genins could not be found. (3) Digitoxigenin monodigitoxoside was the main substrate for conjugating enzymes. (4) Nearly 30% of water-soluble metabolites were cardenolide acids presumably formed by hydrolysis of the butenolide. The results suggest that after PCB treatment the decreased DT-3 elimination rate is caused at least in part by an impaired digitoxoside cleavage.

Stimulation of the glucuronidation of digitoxigenin-monodigitoxoside by liver homogenates from spironolactone-pretreated rats

Pretreatment of rats with spironolactone (SPL) produces an increased formation of the glucuronide conjugates of digitoxigenin-mono-digitoxoside (DMD) present in the bile after administration of digitoxin. For the digitoxin series of cardiac glycosides, DMD is the preferred substrate for in vitro conjugation with glucuronic acid by rat liver homogenates. The present experiments were performed to determine whether SPL pretreatment could produce an inceased glucuronidation of DMD by rat liver homogenates. Three different substrate concentrations (3, 30, and 100 nmol DMD/g of liver homogenate) were incubated under identical conditions with homogenates from control and SPL-pretreated rats. At 3.0-nmol. treatment with SPL produced a slight, but significant, increase in the production of aqueous alcohol-soluble metabolites of DMD by the experimental homogenates. With 30 nmol, treatment with SPL produced a 1.57-fold increase in the formation of aqueous alcohol-soluble metabolites of DMD. At 100 nmol, treatment with SPL produced a 3.44-fold increase in the formation of the aqueous alcohol-soluble metabolites of DMD. Treatment of these aqueous alcohol-soluble fractions (30-nmol substrate incubations only) with β-glucuronidase converted nearly 99% of the activity to a nonconjugate fraction. For both the control and the SPL-treated groups, greater than 92% of this nonconjugate fraction was determined by high-pressure liquid chromatography (HPLC) to be identical to the parent substrate (i.e., DMD). Thus, by selective enzyme treatment of the apparent conjugate and subsequent HPLC identification of the released products, the aqueous alcohol-soluble metabolites were identified as glucuronide conjugates of DMD. Pretreatment with spironolactone increased the rate of glucuronidation of DMD by rat liver homogenates.

Mass spectrometry of the trimethylsilyl derivatives of some cardiac aglycones and monoglycosides

AbstractMass spectra of the trimethylsilyl (TMS) ethers of digitoxigenin, digoxigenin, digitoxigenin monodigitoxoside, digoxigenin monodigitoxoside and peruvoside were obtained. Ions appearing at m/e 170 and m/e 183 are proposed to be indicative of the enol TMS ether moiety as confirmed by appropriate shifts in the mass spectrum of digitoxigenin derivatized with perdeuterotrimethylsilylating reagent. The TMS ether derivatives of the monoglycosides produced abundant molecular ions and the base peaks in the spectra represent fragments of the sugar portion of the molecule.

強心配糖体の代謝と分布に関する研究 (第3報)

The extent of binding of digitoxin and its metabolites to bovine albumin or rat serum was determined by the method of equilibrium dialysis or ultrafiltration. The results were as follows:1) It was found that the binding effect of bovine albumin or rat serum was most marked with digitoxigenin monodigitoxoside, less with digitoxin and digitoxigenin didigitoxoside, and least with digoxin.2) With increasing concentration of digitoxin, digitoxigenin didigitoxoside or didigitoxigenin monodigitoxoside the binding proportion decreased.3) In a phosphate buffer of pH 6.4-9.2, there was no obvious effect of pH on the drug-protein binding.