Identification and preliminary characterization of two human digitalis-like substances that are structurally related to digoxin and ouabain.

In order to characterize the structure of endogenous digitalis-like immunoreactive factor (DLIF), we utilized peritoneal dialysis fluid from patients with chronic renal failure as a source of endogenous digitalis-like immunoreactive factor (DLIF), and subjected it to one-step ion exchange chromatography, followed by one step reverse HPLC. Crude dialysis fluid contained 0.09 ng/ml of DLIF, and using Amberlite XAD-2 chromatography we extracted 110 ng of DLIF from 800 ml of dialysis fluid. By applying this partially purified DLIF to our HPLC system, we discerned three peaks of DLIF activity, with retention times of 34, 58 and 63 minutes. The first peak overlapped the elution profile of ouabain, and the third peak co-eluted precisely with digoxin. The second DLIF peak was not in proximity to any of the digitalis-like markers employed. Thus, our results indicate that DLIF isolated from peritoneal dialysis fluid exists in three distinct forms, one of which resembles ouabain, and one which is identical to digoxin.     

Digitalis-like activity in human plasma. Purification, affinity, and mechanism

A factor having digitalis-like characteristics has been isolated from human plasma and its mechanism of action compared with the commonly used cardenolide, ouabain. The purification scheme involved dialysis of human plasma, lyophilization of dialysate, extraction of methanol-soluble components, and flash evaporation, followed by preparative, semipreparative, and analytical scale reverse-phase chromatography. One peak of biologically active material was obtained and shown to possess digitalis-like activity in assays of sodium pump activity, receptor binding, and Na,K-ATPase activity. Results from (i) the determination of the ligand conditions supporting binding, (ii) kinetics of association and dissociation from the Na,K-ATPase, (iii) affinity titration, (iv) selectivity, and (v) competition studies, when taken together, show that the endogenous digitalis-like factor is a specific inhibitor of the sodium pump that stabilizes the E2P form of the enzyme in a manner analogous to ouabain. The endogenous digitalis-like factor binds competitively in or near the receptor site for cardiac glycosides with an apparent affinity 8-20-fold greater than any known cardioactive steroid. The presence of digitalis-like activity in the circulation of individuals with no known intake of these compounds suggests that the material characterized here is an endogenous counterpart to the cardenolides. This factor may regulate sodium pump activity and provide a rationale for the existence of gene and tissue-specific forms of the Na,K-ATPase having distinct sensitivity to the cardenolides.     

Evidence for the existence of the same endogenous digitalis-like factor in several mammalian species

1. Endogenous digitalis-like activity was studied comparatively in four mammalian species: guinea pig, dog, cow and rat. 2. Water extracts were prepared from guinea pig, dog, cow and rat hearts and assayed by ouabain radioreceptorassay, digoxin radioimmunoassay and digitoxin radioimmunoassay. Extracts were further analysed by fractionation by gel permeation chromatography with Sephadex G-25. 3. A similar behaviour was observed with the four species in the three assays. Extracts displaced tritiated ouabain binding to its receptor and labeled digoxin analogue binding to antidigoxin antibodies in a competitive manner. Displacement of labeled digitoxin analogue to antidigitoxin antibodies did not follow Michaelis-Menten kinetics. IC50 ratios between assays were similar for the four species studied. 4. Extracts from the four species exhibited a similar pattern when fractionated with Sephadex G-25. Endogenous digoxin-like immunoreactivity eluted after the salts, suggesting that the active material is of a molecular weight of less than 1000. 5. Results suggest that a similar endogenous factor endowed with digitalis-like characteristics is present in all mammalian species.     

Inhibitory effect of combinations of digoxin and endogenous cardiotonic steroids on Na+/K+-ATPase activity in human kidney membrane preparation

AimsCardiac glycosides have been extensively used in the treatment of congestive heart failure for more than 200 years. Recently, cardenolides and bufadienolides were isolated from mammalian tissue and are considered as a new class of steroidal hormones. The aim of the present work was to characterize the interaction between the most clinical used cardiac glycoside digoxin and the cardiac glycosides known to exist endogenously, i.e., ouabain, marinobufagin and telocinobufagin, on human kidney Na⁺/K⁺-ATPase.Main methodsInhibition of Na⁺/K⁺-ATPase activity from crude membrane preparations of human kidney was performed using increasing concentrations of the drugs alone or mixtures of ouabain:digoxin, telocinobufagin:digoxin and marinobufagin:digoxin in a fixed ratio 1:4, 2:3 and 3:2, respectively. The colorimetric method of Fiske and Subbarow was used to measure the inorganic phosphate released.Key findingsAnalyses of inhibition curves showed that the experimental curves for all combinations were superimposed on the theoretical additive curves indicating that an additive effect occurs among distinct cardenolides and bufadienolides combinations on the human α1β1 Na⁺/K⁺-ATPase protomer.SignificanceConsidering the extensive use of digoxin in the treatment of heart failure and the recent findings that endogenous cardiac glycosides may have altered levels in many diseases, including heart failure, the demonstration of additive effect between cardiac glycosides can help in the understanding of recent clinical observations, including that lower than usual doses of cardiac glycosides are necessary for decreasing mortality in these patients.     

Doxorubicin cardiotoxicity: possible role of digoxin in its prevention.

Twenty-nine patients with gynaecological cancers who received over 400 mg of doxorubicin were monitored electrocardiographically to determine whether cardiac glycosides countered the adverse effects of high total doses of doxorubicin. Minor electrocardiographical changes were noted in five out of six patients who were not receiving a cardiac glycoside and four out of six who were receiving ouabain, and none of the 16 who were receiving digoxin. One other patient on digoxin stopped taking it and developed cardiomyopathy. One patient on ouabain also developed cardiomyopathy. So far nine patients on digoxin have received between 550 and 1000 mg/m2 of doxorubicin without ill effect. Cardiac glycosides are thought to prevent doxorubicin cardiomyopathy by competitively inhibiting doxorubicin at its receptor sites, but ouabain has a much shorter half life than doxorubicin and its metabolites and so is less effective than digoxin.     

Interaction of cardiodigin, endogenous inhibitor of Na+,K+-ATPase, with antidigoxin and antidigitoxin antibodies

The present study was undertaken to further characterize the immunoreactivity of cardiodigin, digitalis-like factor present in mammalian tissues. Guinea-pig heart extracts purified by reverse-phase low pressure and high pressure liquid chromatographies were analysed for their cross-reactivity with antidigitoxin and antidigoxin antibodies. The putative digitalis-like factor showed an affinity about 10 times higher for antidigoxin antibodies than for antidigitoxin antibodies. EC50 ratios (digoxin/digitoxin assay) determined at two purification steps were different from those of digoxin and digitoxin. These results show that cardiodigin presents molecular determinants recognizable by antidigoxin and antidigitoxin antibodies but that it is a chemical entity different from these well known cardioactive steroids.     

Studies on cardioactive steroids. III. Characterization of different cardiac glycosides by their effects on contractility and rhythmicity at different extracellular potassium concentrations.

In the present paper, the naturally occurring glycosides digitoxin, gitoxin, 16-acetyl-gitoxin, digoxin, cymarol, ouabain, and proscillaridin, and the semi-synthetic 16-epi-gitoxin and 16-acetyl-16-epi-gitoxin are investigated as to their inotropic action and their effects on rhythmicity at isolated spontaneously beating atria of the guinea-pig heart in dependence on the variation of the potassium concentration of the nutritive fluid ([K+]0: 1.34, 2.68, and 5.36 mM resp.). The major results are as follows. 1. Effects of raising [K+]0 from 1.34 to 2.68 mM: The range of the inotropically effective concentrations as well as the size of the maximum inotropic action are more or less strongly improved with all glycosides. The glycoside concentrations required to get inotropic maximum had to be increased to a high degree with proscillaridin and digoxin. The mean arrhythmia percentage occurring at the inotropic maximum is either decreased (gitoxin, 16-epi-gitoxin, digoxin, proscillaridin), unchanged (digitoxin, 16-acetyl-16-epi-gitoxin) or even increased (16-acetyl-gitoxin, cymarol, ouabain). The inotropic value is improved to a high extent with gitoxin only. 2. Effect of raising [K+]0 from 2.68 to 5.36 mM: The range of the inotropically effective concentrations is extended (digitoxin and cymarol) or diminished (proscillaridin), but remains essentially unchanged with most glycosides. The size of the maximum inotropic effect is increased with digoxin, ouabain and 16-epi-gitoxin, but decreased significantly with digitoxin and proscillaridin. The glycoside concentrations required to produce the inotropic maximum are essentially unchanged with the exception of 16-epi-gitoxin, 16-acetyl-gitoxin and ouabain. The mean arrhythmia percentage at the maximum inotropic effect is dramatically reduced with digoxin, cymarol and proscillaridin. The inotropic value is improved with all glycosides except digitoxin. 3. Evaluation of the various glycosides: When judged on the basis of the range of inotropically effective concentrations, the maximum inotropic effect, the mean arrhythmia percentage at the inotropic maximum and the inotropic value, the best first three glycosides include 16-epi-gitoxin and digoxin. 16-Epi-gitoxin and its 16-acetate show that most favourable relationship between the effect on contractility and rhythmicity. The cause of the differential actions of the structurally-different glycosides on contractility and rhythmicity is hypothesized to be due to divergences in structure and/or conformation of the receptor areas of (Na+ + K+)-ATPase of contractile and excitable cells.     

Preparation of Biologically Transformed Raw Material of Woolly Foxglove (Digitalis lanata Ehrh.) and Isolation of Digoxin Therefrom

A biotechnological approach is proposed for anaerobic conservation of aerial parts of woolly foxglove, followed by air–sun drying of the biologically transformed raw material. During the conservation, primary glycosides of foxglove undergo complete conversion into secondary glycosides, with no further transformations. A simple method is described for preparing an enriched glycoside fraction from the transformed raw material (yield, 3.6%) and for isolating highly purified digoxin from this fraction (yield, 0.06% of the starting raw material); other secondary glycosides can also be isolated.     

Preparation of biologically transformed raw material from woolly foxglove Digitalis lanata Ehrh and isolation of digoxin from it

A biotechnological approach is proposed for conservation of a terraneous part of woolly foxglove under anaerobic conditions with a subsequent air-sun drying of the biologically transformed raw material. During the conservation primary foxglove glycosides completely convert to secondary ones which do not transform further. A simple method is described for preparation from the transformed raw material of an enriched glycoside fraction with the yield of 3.6% and for isolation from this fraction of highly purified digoxin with the yield of 0.06% of the starting raw material, and the other secondary glycosides can be also isolated.     

Is the endogenous digitalis-like factor the link between hypertension and metabolic disorders as diabetes mellitus, obesity and acromegaly?

Endogenous factors cross-reacting with antidigoxin antibodies have been found in several tissues and body fluids of animals and humans, using commercially available digoxin radioimmunoassay or enzyme immunoassay methods. The chemical characteristics of these endogenous factors are, at present, unknown, although it has been suggested that they could be substances with low molecular weight. Experimental studies and theoretical considerations indicate that endogenous digitalis-like factors (DDLFs), in addition to the ability to react with antibodies, might also bind to the specific cellular receptor of the cardiac glycosides and thus inhibit the membrane Na+/K(+)-ATPase (sodium pump). Therefore, EDLF can be an endogenous modulator of the membrane sodium-potassium pump and several authors have suggested that EDLF could play a role in the regulation of fluids and electrolytes, muscular tone of myocardial and also in the pathogenesis of arterial hypertension. In this review, the authors discuss the hypothesis that, in metabolic diseases such as diabetes mellitus, obesity and acromegaly, the sodium retention and volume expansion, possibly due to exaggerated sodium intake, and/or exogenously induced peripheral hyperinsulinemia and high levels of growth hormone, could trigger a sustained release of EDLF, which in turn increases the blood pressure.     

Effect of Na(+), K(+)-pump inhibitors on the sensory ganglia neurite growth

Inhibitors of Na(+), K(+)-pump belonging to the class of cardiac glycosides were investigated in organotypic tissue culture of dorsal root ganglia cells of 10-12 days old chicken embryos. The data obtained show that the application of cardiac glycosides (strophantin K and digoxin) in a wide range of concentrations controls the neurite growth in sensory neurons in the dose-dependent manner. It was shown, that at the concentrations of cardiac glycoside exceeding 1 x 10(-6) M the growth of neurites was totally inhibited. Our data indicate that cardiac glycoside have the down-regulation effect on the neurite growth. The data obtained indicate that the Na(+), K(+)-ATPase is involved in the control of the process of neurite growth as a signal transducer.     

Sex, digitalis, and the sodium pump

Foxglove and its constituents therapeutic agent digitalis have been used for centuries for the treatment of heart failure. All digitalis-like cardiotonic steroids enhance heart contraction through a mechanism involving the inhibition of the Na(+),K(+)- ATPase. Recently, Rathore and colleagues reported that sex-based differences may exist in the efficacy of digoxin for the treatment of heart failure. The authors of the study found that female patients exhibited increased risk of death associated with digoxin therapy, whereas male patients appeared to have no increased risk of death related to digoxin therapy. Blaustein and colleagues delve into the report and discuss possible explanations for these findings, suggest alternative ones, and advocate for enrolling greater numbers of women in clinical studies.     

Antidigoxin antibodies neutralize the effect of newborn endogenous digitalis-like factor on erythrocyte 86Rb uptake.

Increasing evidence indicates the existence of endogenous digitalis like factor(s) (EDLF). We recently reported on the partial purification of an EDLF from newborn (cord) blood which possesses both digoxin-like immunoreactivity and the ability to inhibit the cell membrane sodium pump measured as the inhibitory activity on erythrocyte 86Rb uptake. We here report that high affinity digoxin-binding antibodies (Fab fragments; Digibind, Burroughs Wellcome Co.) are capable of neutralizing the inhibitory activity on erythrocyte Rb uptake not only of digoxin but also of ouabain and of partially purified newborn EDLF. These results provide, to our knowledge for the first time, direct evidence that antidigitalis antibodies may cross-react with one or more circulating substances which share antigenic determinants with digoxin and ouabain and possess endogenous digitalis-like properties, strongly suggesting that these antibodies may be useful tools both for the assay of EDLF and for the study of its biological effects.     

Selection of peptidic mimics of digoxin from phage-displayed peptide libraries by anti-digoxin antibodies

Since the initial report of the development of methodology to generate high-affinity digitalis-specific (digoxin) antibodies, these antibodies have proven extremely useful tools to monitor digoxin levels in digitalized patients and, as Fab fragments, to reverse toxic digoxin effects in life-threatening digoxin overdoses. These antibodies (both digoxin-specific and ouabain-specific) have been used extensively by investigators for the identification and characterization of putative endogenous digitalis-like factors. In this study, we used two well-characterized mouse anti-digoxin monoclonal antibodies (mAbs), designated 26-10 and 45-20, as binding templates with which to select short bacteriophage-displayed (pIII protein inserted) peptides that are capable of binding to these mAbs and mimicking the conformational structure of digoxin. Selective enrichment from two phage-displayed random peptide libraries enabled us to isolate and identify distinct 15 and 26 amino acid residue peptide inserts that bind with high avidity and idiotypic specificity to the selecting mAbs. Among these displayed inserts a subset was identified whose mAb binding is inhibited by digoxin and whose corresponding synthetic peptides inhibit phage binding. They, therefore, appear to bind at the mAbs digoxin-binding sites. These data provide the first clear evidence that short polypeptides can serve as surrogates for the low molecular mass hapten digoxin.     

Reduction of digoxin to 20R-dihydrodigoxin by cultures of Eubacterium lentum

The anaerobic bacterium Eubacterium lentum, a common constituent of the intestinal microflora, inactivates digoxin by reducing the unsaturated lactone ring. Reduction of the cardiac glycoside by growing cultures of E. lentum ATCC 25559 proceeded in a stereospecific manner, with the 20R-dihydrodigoxin constituting more than 99% of the product formed. This is in contrast to the 3:1 ratio of 20R and 20S epimers formed in the chemical catalytic hydrogenation. Formation of the reduced glycosides proceeded quantitatively when an overall concentration of 10 micrograms/ml was added to the cultures. E. lentum did not hydrolyze the digitoxose sugars from C-3 of the parent glycoside. However, the synthetically prepared sugar-hydrolyzed metabolites (digoxigenin, digoxigenin monodigitoxoside, and digoxigenin bisdigitoxoside) were reduced to the corresponding dihydro metabolites. Repetition of the experiments with a feces sample from a volunteer who was known to be a converter of digoxin to dihydrodigoxin gave results identical to those obtained with pure E. lentum cultures.     

Reduction of digoxin to 20R-dihydrodigoxin by cultures of Eubacterium lentum.

The anaerobic bacterium Eubacterium lentum, a common constituent of the intestinal microflora, inactivates digoxin by reducing the unsaturated lactone ring. Reduction of the cardiac glycoside by growing cultures of E. lentum ATCC 25559 proceeded in a stereospecific manner, with the 20R-dihydrodigoxin constituting more than 99% of the product formed. This is in contrast to the 3:1 ratio of 20R and 20S epimers formed in the chemical catalytic hydrogenation. Formation of the reduced glycosides proceeded quantitatively when an overall concentration of 10 micrograms/ml was added to the cultures. E. lentum did not hydrolyze the digitoxose sugars from C-3 of the parent glycoside. However, the synthetically prepared sugar-hydrolyzed metabolites (digoxigenin, digoxigenin monodigitoxoside, and digoxigenin bisdigitoxoside) were reduced to the corresponding dihydro metabolites. Repetition of the experiments with a feces sample from a volunteer who was known to be a converter of digoxin to dihydrodigoxin gave results identical to those obtained with pure E. lentum cultures.     

Endogenous Digitalis-Like Na+, K+-ATPase Inhibitors,and Brain Function

Digitalis-like compounds are recently identified steroids synthesized by the adrenal gland, which resemble the structure of plant cardiac glycosides. These compounds, like the plant steroids, bind to and inhibit the activity of the Na⁺, K⁺-ATPase. The possible function of the endogenous digitalis-like compounds has to be evaluated in view of the presence of different isoforms of the Na⁺, K⁺-ATPase, which differ in their sensitivity to digitalis. This review focuses on recent published data on the Na⁺, K⁺-ATPase inhibitors, the digitalis-like compounds, regarding their structure, biosynthesis and secretion from the adrenal gland, physiological role and pathological implications in diseases such as hypertension and depression. Emphasis is given to studies describing the involvement of these compounds in brain function.     

Evidence that mammalian lignans show endogenous digitalis-like activities

Enterolactone, a lignan that has been identified in biological samples from man and several mammals, shares with ascorbic acid and cardiac glycosides a gamma-butyrolactone. It displaces 3H-ouabain from its binding sites on cardiac digitalis receptor and inhibits, dose dependently, the Na+, K+-ATPase activity of human and guinea-pig heart. The time dependence of this inhibition resembles that of dihydroouabain, a cardiac glycoside in which the lactone ring does not contain conjugated double bonds. The active concentrations of enterolactone as inhibitor of Na+,K+-ATPase are in the 10(-4) M range and, at those concentrations, the cross-reactivity with antidigoxin antibodies is low. Lignans may contribute to the putative digitalis-like activity found in tissues, blood and urine of several mammals including man.     

Studies on digoxin--14C-acetate incorporation in to digoxin and degenerative changes in the brain in rats administered digoxin

The human hypothalamus produces an endogenous membrane Na+-K+ ATPase inhibitor digoxin. Digoxin is a steroidal glycoside and could be synthesised by the isoprenoid pathway. The other metabolites of the isoprenoid pathway are cholesterol, dolichol and ubiquinone. We have tried to find out the extent of incorporation of 14C acetate into digoxin in rat brain. The effects of digoxin administration on the rat brain was also studied. The results show that the percentage incorporation of 14C acetate into digoxin is low but detectable. The maximum incorporation was observed for cholesterol, followed by dolichol and finally ubiquinone. The histopathological changes observed after digoxin administration were focal degeneration of the ganglion cells in the cerebrum and cerebellum. The carbohydrate components of the glycoproteins were reduced and the concentration of serotonin, dopamine, and epinephrine showed a significant increase. The role of digoxin in mediating neuronal cell death is discussed.