Gitoxin

A Case Report of Accidental Intoxication following Ingestion of Foxglove Confused with Borage: High Digoxinemia without Major Complications.[Pubmed:31871798]

Case Rep Cardiol. 2019 Nov 29;2019:9707428.

Foxglove (Digitalis purpurea L.) leaves are frequently confused with borage (Borago officinalis L.), which is traditionally used as a food ingredient. Due to the presence of the cardiac glycosides, mostly diGitoxin, foxglove leaves are poisonous to human and may be fatal if ingested. A 55-year-old Caucasian woman complaining weakness, fatigue, nausea, and vomiting was admitted to the Emergency Department. Her symptoms started following consumption of a home-made savory pie with 5 leaves from a plant bought in a garden nursery as borage. Digoxinemia was high (10.4 mug/L). The patient was admitted to the cardiac intensive care unit for electrocardiographic monitoring. Two days after admission, a single episode of advanced atrioventricular (AV) block was recorded by telemetry, followed by a second-degree AV block episode. Plasma samples at day 11 were analysed by LC-MS spectrometry, and Gitoxin was identified suggesting that this compound may be responsible for the clinical toxicity rather than digoxin. In the case of Digitalis spp. poisoning, laboratory data should be interpreted according to the clinical picture and method of analysis used since a variety of glycosides, which are chemically similar to the cardioactive glycosides but without or with fewer cardiac effects, may be incorrectly recognized as digoxin by the test, giving misleading results.

Hellebrin and its aglycone form hellebrigenin display similar in vitro growth inhibitory effects in cancer cells and binding profiles to the alpha subunits of the Na+/K+-ATPase.[Pubmed:23621895]

Mol Cancer. 2013 Apr 26;12:33.

BACKGROUND: Surface-expressed Na+/K+-ATPase (NaK) has been suggested to function as a non-canonical cardiotonic steroid-binding receptor that activates multiple signaling cascades, especially in cancer cells. By contrast, the current study establishes a clear correlation between the IC50in vitro growth inhibitory concentration in human cancer cells and the Ki for the inhibition of activity of purified human alpha1beta1 NaK. METHODS: The in vitro growth inhibitory effects of seven cardiac glycosides including five cardenolides (ouabain, digoxin, diGitoxin, Gitoxin, uzarigenin-rhamnoside, and their respective aglycone forms) and two bufadienolides (gamabufotalin-rhamnoside and hellebrin, and their respective aglycone forms) were determined by means of the MTT colorimetric assay and hellebrigenin-induced cytotoxic effects were visualized by means of quantitative videomicroscopy. The binding affinity of ten of the 14 compounds under study was determined with respect to human alpha1beta1, alpha2beta1 and alpha3beta1 NaK complexes. Lactate releases and oxygen consumption rates were also determined in cancer cells treated with these various cardiac glycosides. RESULTS: Although cardiotonic steroid aglycones usually display weaker binding affinity and in vitro anticancer activity than the corresponding glycoside, the current study demonstrates that the hellebrin / hellebrigenin pair is at odds with respect to this rule. In addition, while some cardiac steroid glycosides (e.g., digoxin), but not the aglycones, display a higher binding affinity for the alpha2beta1 and alpha3beta1 than for the alpha1beta1 complex, both hellebrin and its aglycone hellebrigenin display ~2-fold higher binding affinity for alpha1beta1 than for the alpha2beta1 and alpha3beta1 complexes. Finally, the current study highlights a common feature for all cardiotonic steroids analyzed here, namely a dramatic reduction in the oxygen consumption rate in cardenolide- and bufadienolide-treated cells, reflecting a direct impact on mitochondrial oxidative phosphorylation. CONCLUSIONS: Altogether, these data show that the binding affinity of the bufadienolides and cardenolides under study is usually higher for the alpha2beta1 and alpha3beta1 than for the alpha1beta1 NaK complex, excepted for hellebrin and its aglycone form, hellebrigenin, with hellebrigenin being as potent as hellebrin in inhibiting in vitro cancer cell growth.

A structural view on the functional importance of the sugar moiety and steroid hydroxyls of cardiotonic steroids in binding to Na,K-ATPase.[Pubmed:23341448]

J Biol Chem. 2013 Mar 1;288(9):6602-16.

The Na,K-ATPase is specifically inhibited by cardiotonic steroids (CTSs) like digoxin and is of significant therapeutic value in the treatment of congestive heart failure and arrhythmia. Recently, new interest has arisen in developing Na,K-ATPase inhibitors as anticancer agents. In the present study, we compare the potency and rate of inhibition as well as the reactivation of enzyme activity following inhibition by various cardiac glycosides and their aglycones at different pH values using shark Na,K-ATPase stabilized in the E2MgPi or in the E2BeFx conformations. The effects of the number and nature of various sugar residues as well as changes in the positions of hydroxyl groups on the beta-side of the steroid core of cardiotonic steroids were investigated by comparing various cardiac glycoside compounds like ouabain, digoxin, diGitoxin, and Gitoxin with their aglycones. The results confirm our previous hypothesis that CTS binds primarily to the E2-P ground state through an extracellular access channel and that binding of extracellular Na(+) ions to K(+) binding sites relieved the CTS inhibition. This reactivation depended on the presence or absence of the sugar moiety on the CTS, and a single sugar is enough to impede reactivation. Finally, increasing the number of hydroxyl groups of the steroid was sterically unfavorable and was found to decrease the inhibitory potency and to confer high pH sensitivity, depending on their position on the steroid beta-face. The results are discussed with reference to the recent crystal structures of Na,K-ATPase in the unbound and ouabain-bound states.

HPLC method validation for Digitalis and its analogue by pulsed amperometric detection.[Pubmed:20800985]

J Pharm Biomed Anal. 2011 Jan 5;54(1):217-21.

We developed a highly sensitive and selective reversed-phase HPLC-pulsed amperometric detection (RP-HPLC-PAD) method for cardiac glycoside detection. Eight cardiac glycosides were completely separated within 45 min on a reversed-phase column using a water-acetonitrile gradient, and were detected using a PAD under NaOH alkaline conditions. The detection (S/N=3) and quantification (S/N=10) limits for the cardiac glycosides were 0.1-0.3 and 0.3-0.8 ng, respectively. The linear regression coefficient was 0.9962-0.9998 for concentrations of 1-25 mug/mL. Cardiac glycosides in the Digitalis purpurea leaf displayed intra- and inter-day precisions (RSDs) of <9.30% and average recoveries of 98.63-99.94%. The contents of Gitoxin, digitonin, and diGitoxin in the D. purpurea were 0.197, 0.11, and 0.379 mg/g for leaf dried at 60 degrees C, 0.058, 0.11, and 0.090 mg/g for leaf dried at ambient temperature, and N.D. (not detected), and 18.379 mg/g, N.D. for seed, respectively. We conclude that our method shows good precision and accuracy.

Simple coacervates of zein to encapsulate Gitoxin.[Pubmed:16814999]

Colloids Surf B Biointerfaces. 2006 Aug 1;51(1):39-43.

This work reports the use of simple coacervates of the hydrophobic protein zein to encapsulate Gitoxin, a cardiotonic glycoside. The microspheres obtained using ethanol, methanol, iso-propyl alcohol were characterized using viscosity index, scanning electron microscopy (SEM) and laser light scattering particle analyzer. Scanning electron micrographs indicated that the zein film was made of microspheres with diameter in the 1-1.5 microm range, which could be controlled. Sizes of Gitoxin-loaded zein microspheres changed little before and after release of the drug because of conglutination among zein microspheres. Release of Gitoxin from zein microspheres, were performed in vitro to investigate the mechanism of model drug release. The results show that the zein microspheres obtained using ethanol are best suited for use as a sustained-release form of Gitoxin. The microspheres may also be useful in drug targeting system since the diameter of the microspheres is appropriate for phagocytosis by macrophages. Both zein film and Gitoxin-loaded zein microsphere film were effective in suppressing platelet adhesion.

Effects of digoxin and gitoxin on the enzymatic activity and kinetic parameters of Na+/K+-ATPase.[Pubmed:15648655]

J Enzyme Inhib Med Chem. 2004 Oct;19(5):409-15.

Inhibition of Na+/K+-ATPase activity from human erythrocyte membranes and commercial porcine cerebral cortex by in vitro single and simultaneous exposure to digoxin and Gitoxin was investigated to elucidate the difference in the mechanism of the enzyme inhibition by structurally different cardiac glycosides. The drugs exerted a biphasic dose-dependent inhibitory effect on the enzyme activity in both tissues, supporting the existence of two sensitive Na+/K+-ATPase isoforms. The IC50 values for the low and high affinity isoforms were calculated from the inhibition curves using mathematical analysis. The Hill coefficient (n) fulfilled the relationship 1 < n < 3, suggesting cooperative binding of inhibitors to the enzyme. Kinetic analysis showed that digoxin and Gitoxin inhibited Na+/K+-ATPase by reducing the maximum enzymatic velocity (Vmax) and Km, implying an uncompetitive mode of interaction. Both the isoforms were always more sensitive to Gitoxin. The erythrocyte enzyme was more sensitive to the inhibitors in the range of low concentrations but the commercial cerebral cortex enzyme exerted a higher sensitivity in high inhibitors affinity concentration range. By simultaneous exposure of the enzyme to digoxin and Gitoxin in combinations a synergistic effect was achieved by low inhibitor concentrations. An antagonistic effect was obtained with erythrocyte membrane enzyme at high inhibitors concentration.

Anti-tumour activity of Digitalis purpurea L. subsp. heywoodii.[Pubmed:14531018]

Planta Med. 2003 Aug;69(8):701-4.

Recent research has shown the anticancer effects of digitalis compounds suggesting their possible use in medical oncology. Four extracts obtained from the leaves of Digitalis purpurea subsp. heywoodii have been assessed for cytotoxic activity against three human cancer cell lines, using the SRB assay. All of them showed high cytotoxicity, producing IC50 values in the 0.78 - 15 microg/mL range with the methanolic extract being the most active, in non toxic concentrations. Steroid glycosides (gitoxigenin derivatives) were detected in this methanolic extract. Gitoxigenin and Gitoxin were evaluated in the SRB assay using the three human cancer cell lines, showing IC50 values in the 0.13 - 2.8 microM range, with the renal adenocarcinoma cancer cell line (TK-10) being the most sensitive one. Morphological apoptosis evaluation of the methanolic extract and both compounds on the TK-10 cell line showed that their cytotoxicity was mediated by an apoptotic effect. Finally, possible mechanisms involved in apoptosis induction by digitalis compounds are discussed.

Modifying specificity of antidigoxin antibodies using insertional mutagenesis.[Pubmed:12441388]

Protein Sci. 2002 Dec;11(12):2899-908.

Certain antibodies (Abs) elicited using the cardiac glycoside digoxin (digoxigenin tridigitoxoside) bind preferentially to analogs that differ from digoxin by substitutions on the cardenolide rings, the lactone, or by the presence or absence of attached sugars. Antibody 26-10 binds equally well to digoxin and diGitoxin, which differ only by the presence in the former and the absence in the latter of an hydroxyl group at C12. Other antidigoxin Abs, however, can distinguish between these ligands by three orders of magnitude in binding. Inspection of the structure of Fab 26-10 complexed with digoxin shows a gap in complementarity in the region between the digoxin O12 and LCDR3. We proposed that insertions in LCDR3 might result in Abs that bind diGitoxin preferentially. We produced libraries of mutants displayed on bacteriophage which were randomized at LCDR3 and contained LCDR3 insertions. Mutants were selected by panning against digoxin and analogs. The mutants bound diGitoxin preferentially up to 47-fold greater than digoxin. The mutants that bound well to diGitoxin demonstrated a consensus sequence including the substitution of Trp at position L:94. Using site-directed mutagenesis, the binding to diGitoxin was shown to be maximized by the combination of an insertion and L:Trp94 mutation, moving the L 94 side chain closer to digoxin. We also selected mutants that bound preferentially to Gitoxin, which, like diGitoxin, lacks the 12-hydroxyl, increasing relative binding to Gitoxin up to 600-fold compared to the unmutated Ab 26-10.

Complementary combining site contact residue mutations of the anti-digoxin Fab 26-10 permit high affinity wild-type binding.[Pubmed:11854275]

J Biol Chem. 2002 May 10;277(19):16365-70.

Antibody 26-10, obtained in a secondary immune response, binds digoxin with high affinity (K(a) = 1.3 x 10(10) M(-1)) because of extensive shape complementarity. We demonstrated previously that mutations of the hapten contact residue HTrp-100 to Arg (where H refers to the heavy chain) resulted in increased specificity for digoxin analogs substituted at the cardenolide 16 position. However, mutagenesis of H:CDR1 did not result in such a specificity change despite the proximity of the H:CDR1 hapten contact residue Asn-35 to the cardenolide 16 position. Here we constructed a bacteriophage-displayed library containing randomized mutations at H chain residues 30-35 in a 26-10 mutant containing Arg-100 (26-10-RRALD). Phage were selected by panning against digoxin, Gitoxin (16-OH), and 16-acetylGitoxin coupled to bovine serum albumin. Clones that retained wild-type Asn at position 35 showed preferred binding to Gitoxin, like the 26-10-RRALD parent. In contrast, clones containing Val-35 selected mainly on digoxin-bovine serum albumin demonstrated a shift back to wild-type specificity. Several clones containing Val-35 bound digoxin with increased affinity, approaching that of the wild type in a few instances, in contrast to the mutation Val-35 in the wild-type 26-10 background, which reduces affinity for digoxin 90-fold. It has therefore proven possible to reorder the 26-10 binding site by mutations including two major contact residues on opposite sides of the site and yet to retain high affinity for binding for digoxin. Thus, even among antibodies that have undergone affinity maturation in vivo, different structural solutions to high affinity binding may be revealed.

A single H:CDR3 residue in the anti-digoxin antibody 26-10 modulates specificity for C16-substituted digoxin analogs.[Pubmed:11391021]

Protein Eng. 2001 Apr;14(4):287-96.

We constructed Fab libraries of bacteriophage-displayed H:CDR3 mutants in the high-affinity anti-digoxin antibody 26-10 to determine structural constraints on affinity and specificity for digoxin. Libraries of mutant Fabs randomized at five or 10 contiguous positions were panned against digoxin and three C16-substituted analogs, Gitoxin (16-OH), 16-formylGitoxin and 16-acetylGitoxin. The sequence data from 83 different mutant Fabs showed highly restricted consensus patterns at positions H:100, 100a and 100b for binding to digoxin; these residues contact digoxin in the 26-10:digoxin co-crystal structure. Several mutant Fabs obtained following panning on digoxin-BSA showed increased affinity for digoxin compared with 26-10 and retained the wild-type (wt) Trp at position 100. Those Fabs selected following panning on C16-substituted analogs showed enhanced binding to the analogs. Replacement of H:Trp100 by Arg resulted in mutants that bound better to the analogs than to digoxin. This specificity change was unexpected, as C16 lies on the opposite side of digoxin from H:CDR3. Substitution of wt Trp by Arg appears to alter specificity by allowing the hapten to shift toward H:CDR3, thereby providing room for C16 substituents in the region of H:CDR1.

Phage display-selected sequences of the heavy-chain CDR3 loop of the anti-digoxin antibody 26-10 define a high affinity binding site for position 16-substituted analogs of digoxin.[Pubmed:11060305]

J Biol Chem. 2001 Mar 16;276(11):8149-58.

The heavy-chain CDR3 region of the high affinity (K(a) = 1.3 x 10(10) M(-)1) anti-digoxin monoclonal antibody 26-10 was modified previously to shift its specificity, by substitution of tryptophan 100 by arginine, toward binding analogs of digoxin containing substitutions at position 16. To further change specificity, two 5-mer libraries of the randomly mutagenized phage-displayed 26-10 HCDR3 region (positions 94-98) were panned against digoxin-bovine serum albumin (BSA) as well as against 16-acetylGitoxin-BSA. When a mutant Fab that binds 16-substituted analogs preferentially was used as a parent sequence, clones were obtained with affinities for digoxin increased 2-4-fold, by panning on digoxin-BSA yet retaining the specificity shift. Selection on 16-acetylGitoxin-BSA, however, resulted in nine clones that bound Gitoxin (16-OH) up to 150-fold higher than the wild-type 26-10, due to a consensus mutation of Ser(H95) to Gly(H95). The residues at both position H95 (serine) and position H100 (tryptophan) contact hapten in the crystal structure of the Fab 26-10-digoxin complex. Thus, by mutating hapten contact residues, it is possible to reorder the combining site of a high affinity antibody, resulting in altered specificity, yet retain or substantially increase the relative affinity for the cross-reactive ligand.

A non-fatal case of intoxication with foxglove, documented by means of liquid chromatography-electrospray-mass spectrometry.[Pubmed:11005196]

J Forensic Sci. 2000 Sep;45(5):1154-8.

The non-fatal self-poisoning of a 36-year-old female patient, who ingested a concoction of foxglove (Digitalis Purpurea), is presented. On the admission, initial symptoms were nausea and vomiting, abdominal pain, and cardiovascular shock with sinus bradycardia. Blood and urine were assayed for 17 cardiotonic hetorosides, using a highly specific LC-MS procedure. Serum and urine specimens were collected over five days and analyzed by liquid chromatography-electrospray-mass spectrometry (LC-ES-MS). This accurate procedure allowed the determination of the digitalis glycosides and their metabolites in serum and urine. The serum concentrations of digitalis glycosides were maximum on the first day (Gitoxin 13.1 ng/mL, diGitoxin 112.6 ng/mL, digitoxigenin 3.3 ng/mL, and digitoxigenin mono-digitoxoside 8.9 ng/mL) and decreased over five days. We observed a peak gitaloxin level (112.6 ng/mL) on the fifth day only. After administration of atropine as well as dimeticone, alginic acid, and metoclopramide, health status improved. The peak urine concentrations were reached at hour 30 and were respectively 91.3 and 69.9 ng/mL for gitaloxin and diGitoxin, while those of digitoxigenin, digitoxigenin mono-digoxoside and Gitoxin were lower (respectively 0.7, 1, and 5.6 ng/mL). The patient was discharged on the fifth day when there were no residual symptoms.

Cardiovascular activity of a methanolic extract of Digitalis purpurea spp. heywoodii.[Pubmed:10940580]

J Ethnopharmacol. 2000 Aug;71(3):437-42.

The paper deals with the effects of a glycosidal extract of Digitalis heywoodii, ssp. of Digitalis purpurea L., (Schrophulariaceae) grown in Badajoz (Spain), on isolated cardiac auricle of rabbits, urinary excretion of rats, as well as its emetic effect in pigeons. These effects using vehicle (propylene glycol-ethanol-water, 40:10:50) and digoxin as standards are presented. The extract at concentrations of 20 and 40 microg/ml produced an increase in the contraction force of auricles in a dose-dependent way. At doses of 15 and 30 mg/kg a slight diuretic and natriuretic effect was observed. The active dose range for emesis was 0.5-4 mg/kg and a decrease of the emesis time within 10 min of injection in dose-dependent manner was obtained. The pharmacological activity of the extract is related to Gitoxin derivatives (digitalinum verum and strospeside), the most abundant compounds obtained from the leaves of Digitalis purpurea spp. heywoodii.

Multiresidue screen for cardiotoxins by two-dimensional thin-layer chromatography.[Pubmed:10637052]

J Agric Food Chem. 2000 Jan;48(1):60-4.

A two-dimensional thin-layer chromatographic method was developed for the qualitative determination of the cardiotoxins oleandrin, Gitoxin, diGitoxin, gitoxigenin, and grayanotoxins I, II, and III in gastrointestinal contents (stomach, rumen, colon, and cecum contents), feces, and plant material. The cardiotoxins were extracted with dichloromethane. The extract was cleaned up by charcoal and reverse phase solid-phase extraction columns. Analysis was performed by two-dimensional thin-layer chromatography on silica gel plates and visualized by aluminum chloride followed by chloramine T spray. The method detection limits were 0.05 microg/g for oleandrin, 0.1 microg/g for Gitoxin, and 0.2 microg/g for the other toxicants in gastrointestinal contents and feces and were 5 times higher in plant material. Four replicate fortifications of bovine rumen contents, bovine feces, and alfalfa at these levels were all well recovered. The diagnostic utility of the method was tested by analyzing samples submitted to the veterinary toxicology laboratory.

Monoclonal antibodies that distinguish between two related digitalis glycosides, ouabain and digoxin.[Pubmed:10510376]

J Immunol. 1999 Oct 15;163(8):4360-6.

The exogenous digitalis glycosides, ouabain and digoxin, have been widely used in humans to treat congestive heart failure and cardiac arrhythmias. Several reports have also pointed to the existence of endogenous ouabain- and digoxin-like compounds, but their precise roles in mammalian physiology and various disorders of the circulation are not clear. In an attempt to produce specific Abs for the purification and identification of endogenous ouabain-like compounds, somatic cell fusion was used to produce mAbs specific for ouabain. Our attempts to produce ouabain-specific mAbs were unsuccessful when ouabain was coupled to exogenous proteins such as bovine gamma-globulins, BSA, and human serum albumin. However, when ouabain was coupled to an Ab of A/J mice origin and the same strain of mouse was used for immunization with ouabain-Ab conjugate, three Abs (1-10, 5A12, and 7-1) specific for ouabain were obtained. In assays of fluorescence quenching and saturation equilibrium with tritiated ouabain, Ab 1-10 exhibited 200 nM affinity for ouabain. These three mAbs are distinguished from existing Abs to ouabain and digoxin by their specificity for ouabain and lack of cross-reactivity with digoxin. Specificity studies showed that the loss of cross-reactivity was correlated with the presence of a hydroxyl group at either position 12beta (digoxin) or 16beta (Gitoxin) of the steroid ring. These Abs can be used to develop assays for detection and characterization of ouabain-like molecules in vivo.