Digoxin-like Immunoreactivity Research Articles

Digifab and immunoneutralization of cardiotonic steroids in preeclampsia

Background. Elevated levels of Na + /K + -ATPase(NKA)-inhibitory cardiotonic steroids (CTS) including marinobufagenin (MBG) contribute to pathogenesis of preeclampsia (PE) via induction of vasoconstriction and of vascular stiffness. In PE, Digibind (affi nity-purifi ed digoxin antibody) exhibits benefi cial effects primarily due to immunoneutralization of CTS including MBG. Because Digibind is not available and the only other «clinically-usable» digoxin antibody is DigiFab (BTG International Ltd), we compared DigiFab and Digibind with respect to their ability to interact with CTS in PE plasma and to ex-vivo reverse PE-induced NKA inhibition. Objective. We compared effects of Digibind and Digifab on erythrocyte NKA from patients with PE and normotensive pregnant subjects. Using competitive fl uoroimmunoassays based on Eu-labeled DigiFab and Digibind we compared profi le of elution of digoxin-immunoreactive material following fractionation of PE plasma on reverse-phase HPLC column. Design and methods. 7 patients with PE (28 ± 2 years; gestational age, 39,0 ± 0,5 weeks, urinary protein excretion, 2,12 ± 0,46 g/24 hours, blood pressure 157 ± 5/94 ± 2 mm Hg) and 6 normotensive pregnant subjects (26 ± 1 years; gestational age, 37 ± 1 weeks, blood pressure 112 ± 2/75 ± 3 mm Hg) were studied. Results. PE was associated with substantial inhibition of erythrocyte NKA (1,47 ± 0,17 vs. 2,65 ± 0,16 umol Pi/mL/hr in control group, p < 0,001). At concentration 10 ug/mL (which mimics dose of Digibind administered in PE) both Digibind and DigiFab partially, but signifi cantly restored NKA activity (2,1 ± 0,2 and 2,05 ± 0,3 umol Pi/mL/hr, respectively). In competitive immunoassay Digibind and Digifab exhibited comparable cross-reactivity with MBG and endogenous ouabain. Following HPLC fractionation of plasma, both Digibind and Digifab detected PE-associated increase in CTS material in fractions 16–18; 176 vs. 75 pmoles for Digibind, control and preeclampsia, respectively; 221 vs. 70 pmoles for Digifab, control and PE, respectively. 4G4 anti-MBG monoclonal antibody detected preeclampsia-induced CTS increase (1056 vs. 421 pmoles) mainly in fraction 16. Conclusions. We conclude that: (i) Digifab and Digibind exhibit comparable cross-immunoreactivity with CTS; (ii) in patients with PE plasma levels of digoxin-like immunoreactivity are elevated proportionally when measured by both Digifab and Digibind and both antibodies reverse preeclampsia-induced NKA inhibition, and (iii) following HPLC fractionation of PE plasma, Digifab and Digibind interact with endogenous bufadienolides including MBG, rather than endogenous ouabain.

The New Enzyme-Linked Immunosorbent Digoxin Assay on the ADVIA Integrated Modular System® is Virtually Free From Oleander Interference

Despite known toxicity of oleander, this product is used in herbal preparations. Oleander interferes with various digoxin immunoassays. It is possible that a person taking digoxin also may take oleander-containing herbal products, and digoxin immunoassays interfering with oleander cannot be used for therapeutic monitoring of digoxin. Recently, Bayer Diagnostics introduced a new enzyme-linked chemiluminescent immunosorbent digoxin assay for application on the ADVIA IMS System (ECLIA-digoxin). We studied potential interference of oleander with this new digoxin assay and found that this assay is virtually free from oleander interference. When aliquots of drug-free serum pools were supplemented with ethyl alcohol extract of oleander leaf or pure oleandrin standard, we observed significant apparent digoxin concentration when measured by the fluorescence polarization immunoassay (FPIA) but minimal digoxin-like immunoreactivity using the ECLIA digoxin assay. Because cross-reactivity should be studied in the presence of primary analyte, we prepared 2 serum pools using sera from patients receiving digoxin. Then aliquots of first digoxin pool were supplemented with oleandrin standard and aliquots of second digoxin pool with oleander extract. We observed significant increases in apparent digoxin concentration in the presence of both oleandrin and oleander extract using the FPIA. However, we observed no statistically significant change in digoxin concentration when ECLIA digoxin assay was used, indicating that this assay is virtually free from oleander interference.

Effectiveness of activated charcoal and equilibrium dialysis in removing Asian, American, Siberian and Indian ginseng from human serum

Background Ginsengs are used by the general population worldwide and toxicity of various ginsengs has been reported. We studied the effectiveness of activated charcoal and in vitro equilibrium dialysis for removal of Asian, American, Siberian and Indian ginseng from human serum by measuring digoxin-like immunoreactivity using the fluorescence polarization immunoassay. Methods 1× PBS (phosphate buffered saline) or drug free serum pool was supplemented with Asian, American, Siberian or Indian ginseng extract in amount expected in overdose. The aliquots of supplemented buffer or serum pool were treated with activated charcoal (15 or 50 mg of activated charcoal/ml of buffer/serum) for 5, 10, 20 and 30 min and digoxin-like immunoreactivities were compared with the original specimens. Other drug free serum pools were also supplemented with various ginsengs and then passed through a small column packed with activated charcoal or subjected to in vitro equilibrium dialysis against phosphate buffer at pH 7.4. Results Complete removal of digoxin-like immunoreactivity from buffer solution or serum pool supplemented with various ginsengs can be achieved by treatment with activated charcoal. Moreover, when serum pools supplemented with various ginsengs were passed through columns packed with activated charcoal, we observed complete removal of digoxin-like immunoreactivity. In addition, significant removal of digoxin-like immunoreactivity was observed when serum pools supplemented with ginsengs were subjected to equilibrium dialysis for 24 h. Conclusions Removal of digoxin-like immunoreactivity from buffer solution or serum due to the presence of ginsengs can be achieved by treatment with activated charcoal in vitro but complete removal of digoxin-like activity from serum is not possible even after 24 h of equilibrium dialysis.

Effect of Brazilian, Indian, Siberian, Asian, and North American Ginseng on Serum Digoxin Measurement by Immunoassays and Binding of Digoxin-like Immunoreactive Components of Ginseng With Fab Fragment of Antidigoxin Antibody (Digibind)

We compared Brazilian, Indian, Siberian, Asian, and North American ginseng for potential interference with 3 digoxin immunoassays: fluorescence polarization (FPIA), microparticle enzyme (MEIA), and Tina-quant (Roche Diagnostics, Indianapolis, IN). We supplemented aliquots of a drug-free serum pool with ginseng extracts representing expected in vivo concentrations and overdose. We observed apparent digoxin-like immunoreactivity with FPIA, modest immunoreactivity with MEIA, and no apparent digoxin immunoreactivity with the Tina-quant with all ginsengs except Brazilian, which showed no immunoreactivity with any assay. When aliquots of serum pools prepared from patients receiving digoxin were supplemented with ginsengs, we observed falsely elevated digoxin values with FPIA, falsely lower digoxin values (negative interference) with MEIA, and no interference with the Tina-quant. Digoxin-like immunoreactive components of various ginsengs have moderate protein binding; monitoring free digoxin concentrations does not eliminate such interference. We also observed that Digibind (Burroughs Wellcome, Research Triangle Park, NC) can bind free digoxin-like immunoreactive components of ginsengs; such effects can be monitored by measuring apparent free digoxin concentrations. Indian, Asian, and North American ginsengs interfere with serum digoxin measurement by FPIA and MEIA; the Tina-quant is free of such interference. Digibind can bind free digoxin-like immunoreactive components of ginseng.

Neutralization of Free Digoxin-like Immunoreactive Components of Oriental Medicines Dan Shen and Lu-Shen-Wan by the Fab Fragment of Antidigoxin Antibody (Digibind)

Dan Shen and Lu-Shen-Wan, traditional Chinese medicines used as remedies for heart diseases, demonstrate digoxin-like immunoreactivity. The digoxin-like immunoreactive components of Lu-Shen-Wan show approximately 55% protein binding, while Dan Shen demonstrates concentration-dependent protein binding (68% bound at lower concentrations but only 25% bound at higher concentrations). Because Dan Shen and Lu-Shen-Wan can cause substantial toxic effects in patients, we studied the potential use of Digibind (Fab fragment of polyclonal antidigoxin antibody; Burroughs Wellcome, Research Triangle Park, NC) for neutralizing the pharmacologically active free fractions of Dan Shen and Lu-Shen-Wan. Drug-free serum pools were supplemented with Dan Shen or Lu-Shen-Wan to achieve apparent digoxin concentrations expected in severe overdoses. Aliquots of supplemented serum pools were supplemented further with aqueous Digibind solution to achieve final Digibind concentrations between 5 and 20 microg/mL (expected in vivo range in patients overdosed with digoxin and being treated with Digibind). We observed complete removal of the free apparent digoxin in the presence of Digibind for Dan Shen and Lu-Shen-Wan. Digibind binds free digoxin-like immunoreactive components of Dan Shen and Lu-Shen-Wan in vitro.

Neutralization of Free Digoxin-like Immunoreactive Components of Oriental Medicines Dan Shen and Lu-Shen-Wan by the Fab Fragment of Antidigoxin Antibody (Digibind)

Dan Shen and Lu-Shen-Wan, traditional Chinese medicines used as remedies for heart diseases, demonstrate digoxin-like immunoreactivity. The digoxin-like immunoreactive components of Lu-Shen-Wan show approximately 55% protein binding, while Dan Shen demonstrates concentration-dependent protein binding (68% bound at lower concentrations but only 25% bound at higher concentrations). Because Dan Shen and Lu-Shen-Wan can cause substantial toxic effects in patients, we studied the potential use of Digibind (Fab fragment of polyclonal antidigoxin antibody; Burroughs Wellcome, Research Triangle Park, NC) for neutralizing the pharmacologically active free fractions of Dan Shen and Lu-Shen-Wan. Drug-free serum pools were supplemented with Dan Shen or Lu-Shen-Wan to achieve apparent digoxin concentrations expected in severe overdoses. Aliquots of supplemented serum pools were supplemented further with aqueous Digibind solution to achieve final Digibind concentrations between 5 and 20 microg/mL (expected in vivo range in patients overdosed with digoxin and being treated with Digibind). We observed complete removal of the free apparent digoxin in the presence of Digibind for Dan Shen and Lu-Shen-Wan. Digibind binds free digoxin-like immunoreactive components of Dan Shen and Lu-Shen-Wan in vitro.

Activated charcoal is effective but equilibrium dialysis is ineffective in removing oleander leaf extract and oleandrin from human serum: monitoring the effect by measuring apparent digoxin concentration.

Accidental poisoning from oleander leaf or oleander tea can be life threatening. The authors studied the effectiveness of activated charcoal and equilibrium dialysis in removing oleander leaf extract and commercially available oleandrin as well as oleandrigenin, the active components of oleander plant, from human serum. Oleander leaf extract was prepared in distilled water and drug-free serum was supplemented with the extract. Then serum was treated with activated charcoal at room temperature and an aliquot was removed at 0 minutes, 10 minutes, 20 minutes, and finally 30 minutes to study the presence of oleander extract by measuring the apparent digoxin concentration using the FPIA for digoxin. The authors observed effective removal of oleander extract by activated charcoal. When the authors supplemented other drug-free serum pools with pure oleandrin or oleandrigenin and then subsequently treated them with activated charcoal, the authors observed complete removal of digoxin-like immunoreactivity at the end of 30 minutes' treatment. When drug-free serum pool supplemented with either oleander leaf extract, oleandrin, or oleandrigenin was passed through a small column packed with activated charcoal, the authors observed almost no apparent digoxin concentration following the passage through the column indicating that activated charcoal is very effective in removing oleander from human serum in vitro. In contrast, when serum pools containing either oleander leaf extract or oleandrin were subjected to equilibrium dialysis against phosphate buffer at pH 7.4, the authors observed no significant reduction in apparent digoxin concentration even after 24 hours. The authors conclude that activated charcoal is effective but equilibrium dialysis is ineffective in removing oleander leaf extract from human serum.

Study on mechanism of action of Chinese medicine Chan Su: dose-dependent biphasic production of nitric oxide in trophoblastic BeWo cells

Background: Chan Su, a traditional Chinese medicine, is used for treating the heart diseases and other systemic illnesses. Our studies with animal model have revealed its role in increasing intracellular calcium concentration in cardiomyocytes. Nitric oxide (NO), a second messenger molecule, and its metabolites have been demonstrated to maintain and modulate multiple physiologic functions including the cardiovascular and reproductive systems. In order to explore the mechanism of action of Chan Su, we studied the ability of Chan Su to stimulate NO production in cultured trophoblastic BeWo cells. Materials and methods: BeWo cell is a cloned established cell line purified from human choriocarcinoma. These cells have some similarities in biological behavior with endothelial cells. Therefore, BeWo cell line may act as a model system for production of nitric oxide by Chan Su both in placenta and in cardiovascular tissue, and the results can easily be extrapolated to cardiomyocytes. Very small amount of ethanol extract of Chan Su was added to the cultured cells in KBM buffer and a chemiluminescence system was used for the measurement of nitric oxide. The amounts of Chan Su extract added to cultured cells were comparable to expected level of Chan Su in human serum after ingestion. We also repeated these experiments with bufalin, the active component of Chan Su. Results: The ethanol extract of Chan Su (5 and 10 μg/ml) significantly increased NO production up to 110% of basal control value, but higher concentrations (40 and 80 μg/ml) of Chan Su (as expected in an overdose) resulted in decreased NO production below the control level. This biphasic effect on nitric oxide production was also observed with bufalin, the active component of Chan Su responsible for its digoxin-like immunoreactivity. The presence of bufalin in Chan Su preparation was confirmed by thin layer chromatography (TLC) analysis. Conclusions: Chan Su as well as bufalin is able to modulate the production of NO in BeWo cell line.

Effect of the traditional Chinese medicines Chan Su, Lu-Shen-Wan, Dan Shen, and Asian ginseng on serum digoxin measurement by Tina-quant (Roche) and Synchron LX system (Beckman) digoxin immunoassays.

Chan Su, Lu-Shen-Wan, Dan Shen, and Asian ginseng are traditionally used to treat a number of conditions, including cardiovascular disease. All of these traditional Chinese medicines exhibit cardioactive properties. Digoxin is a cardioactive drug with a narrow therapeutic range (0.8-1.9 ng/mL). A patient taking digoxin may also take these Chinese medicines for their cardiotonic effects. Moreover, the active components of these medicines that are responsible for cardiotonic effects bear structural similarities to digoxin. Therefore, we studied the potential interference of these Chinese medicines with two digoxin immunoassays--the Tina-quant (Roche Diagnostics) and the Beckman (Synchron LX system)--and compared the values with the fluorescence polarization immunoassay (FPIA; Abbott Laboratories). When very small amounts (2-5 microL) of aqueous extract of Chan Su or Lu-Shen-Wan were added to drug-free serum, we observed high digoxin-like immunoreactivity with the FPIA. In contrast, when ethyl acetate extract of Dan Shen or microliter amounts of ginseng extract were added to drug-free serum, we observed modest digoxin-like immunoreactivity with the FPIA, but no apparent digoxin activity with the Roche and Beckman digoxin immunoassays. When aliquots of a digoxin pool prepared from patients receiving digoxin were supplemented with these Chinese medicines, we observed the most significant interference with the FPIA. The presence of endogenous digoxin-like immunoreactive substances can have additive effects with these Chinese medicines and falsely increase apparent digoxin levels by the FPIA. On the other hand, the Roche and Beckman assays were free from interference from DLIS but showed significant interference from Chan Su and Lu-Shen-Wan. We conclude that the FPIA showed the most significant interference from all four of the Chinese medicines we studied. However, the Roche and Beckman assays showed no interference from two (Dan Shen and Asian ginseng) of the four Chinese medicines we studied.

In vivo digoxin-like immunoreactivity in mice and interference of Chinese medicine Danshen in serum digoxin measurement: elimination of interference by using a chemiluminescent assay

Background: Danshen, a traditional Chinese medicine used in the management of cardiovascular diseases, is available without prescription in the US. Because Danshen is used to treat cardiovascular diseases, we studied the potential interference of Danshen with serum digoxin measurement using various immunoassays. Methods: Blood was collected 1 day before and then 1 and 2 h after feeding mice with Danshen. The apparent digitoxin activities were measured by the fluorescence polarization immunoassay (FPIA). We also added microliter amounts of Danshen extract to digoxin pools prepared from patients receiving digoxin. The digoxin concentrations were measured using the fluorescence polarization immunoassay (FPIA), microparticle enzyme immunoassay (MEIA) and chemiluminescent assay (CLIA). The observed values were compared with original values. We also fed mice with Danshen. Results: We observed measurable digoxin-like immunoreactivity in sera of mice after feeding with Danshen. We also observed falsely lower digoxin concentrations (negative interference) when MEIA was used for digoxin measurement. However, serum digoxin concentrations were falsely elevated with FPIA. We observed no interference of Danshen in serum digoxin measurement using the CLIA. Conclusions: Danshen appears to contain digoxin-like immunoreactivity but does not interfere with serum digoxin measurement when CLIA was used.

Positive and Negative In Vitro Interference of Chinese Medicine Dan Shen in Serum Digoxin Measurement

Dan Shen, a traditional Chinese medicine used in the management of cardiovascular diseases, is now available without prescription in the United States from Chinese herbal stores. We demonstrated digoxin-like immunoreactivity of Dan Shen in vitro. Because Dan Shen is used to treat cardiovascular disease, we studied potential interference of Dan Shen with serum digoxin measurement. Addition of microliter quantities of Dan Shen extract to digoxin pools prepared from patients receiving digoxin resulted in falsely elevated serum digoxin concentrations (positive interference) as measured by the fluorescence polarization immunoassay for digoxin (Abbott Laboratories, Abbott Park, IL). More interestingly, serum digoxin concentrations were falsely lowered (negative interference) when measured by the microparticle enzyme immunoassay, also marketed by Abbott Laboratories. Taking advantage of poor protein binding of digoxin (25%) and high protein binding of digoxin-like immunoreactive components of Dan Shen, we further demonstrated that the positive and negative interference of Dan Shen in serum digoxin measurement can be eliminated by monitoring the free digoxin concentration.

Falsely elevated serum digitoxin concentrations due to cross-reactivity of water-extractable digitoxin-like immunoreactivity of Chinese medicine Chan SU: elimination of interference by use of a chemiluminescent assay

Chinese medicines are available without prescription in health food stores. One such Chinese preparation, Chan SU, is used as a cardiotonic agent. Digoxin-like immunoreactivity of Chan SU has been reported in the past. In this report we demonstrated significant digitoxin-like immunoreactivity of Chan SU. For example, when a 20-μl aliquot of an aqueous extract of Chan SU (2 mg/ml) was added to drug-free serum, the observed digitoxin-like immunoreactivity was 51.40 ng/ml by the fluorescence polarization assay. In contrast, a new chemiluminescent assay for digitoxin did not show any immunoreactivity. When very small amount of aqueous extract of Chan SU was added into serum containing digitoxin, the observed digitoxin concentrations were falsely elevated when measured by the fluorescence polarization immunoassay (FPIA), but did not change significantly when measured by the chemiluminescent immunoassay (CLIA). Significant digitoxin-like immunoreactivity was also observed (FPIA) in mice after feeding with Chan SU. Because bufalin, cinobufotalin and cinobufagin are major components of Chan SU, digitoxin-like immunoreactivity of these purified compounds was also studied. Bufalin was identified as the major digitoxin-like immunoreactive compound responsible for most of the interference in serum digitoxin measurement using the FPIA.

Digoxin-like immunoreactivity in early infantile death.

The aim of this study was to determine if the level of digoxin-like immunoreactivity in post-mortem sera obtained from infants differs according to the cause of death and if the level is related to age, post-mortem interval, cardiac pathology or adrenal weight. Twelve infants whose deaths were attributed to sudden infant death syndrome (SIDS), and 11 infants who died from other causes, had blood sampled between 3 to 53 hours post-mortem from their right atrial cavity. Digoxin-like immunoreactivity was measured, using a specific and sensitive digoxin radioimmunoassay, and was detected in 7 of the infants who died of SIDS and 7 of those who died from other causes. The highest levels were seen in two patients who died from meningococcal sepsis and haemorrhage, hyperpyrexia and encephalopathy syndrome, respectively. No correlation was detected between the digoxin-like immunoreactivity level, gender, age at death, post-mortem interval or cardiac pathology. Digoxin-like immunoreactivity levels correlated with adrenal weight. It is concluded that digoxin-like immunoreactivity is frequently found in infant sera, but levels are not specific to and are no higher in SIDS infants than infants dying of other conditions.

Digitoxinlike and digoxinlike immunoreactivitis in sera of patients with uremia and liver disease as measured by fluorescence polarization immunoassays: poor correlation between digitoxinlike and digoxinlike immunoreactivities.

Digoxinlike immunoreactive substances (DLIS) cross-react with antidigoxin antibodies and falsely elevante total digoxin levels. Cross-reactivity of DLIS with various immunoassays for digoxin has been extensively studied in the past. The digitoxin molecule differs from digoxin by having one extra hydroxyl group in the aglycone ring. Therefore, DLIS may also falsely elevate total digitoxin concentrations. However, in the past, limited studies have shown the presence of digitoxinlike immunoreactive substances (DTLIS) in cord blood and sera of neonates. We compared DLIS and DTLIS concentrations in sera of patients with uremia, liver disease, and hypoalbuminemia. We found measurable DLIS concentrations in 11 of 45 patients by the fluorescence polarization immunoassay (FPIA) for digoxin (range, 0.20-0.89 ng/ml digoxin equivalent). These patients did not receive any digoxin or digitoxin. Using the FPIA, we also found elevated DTLIS concentrations in 10 of these 45 patients (range, 2.88-21.24 ng/ml digitoxin equivalent). Given the narrow therapeutic range of digitoxin (15-30 ng/ml), this cross-reactivity is significant. Elevated concentrations of DTLIS in sera falsely elevated the measured concentrations of digitoxin (positive interference) when known amounts of digitoxin were added to such sera. Interestingly, we found a poor correlation between DLIS and DTLIS concentrations in sera of patients with liver disease and uremia (r = 0.58), with some patients having no measurable DLIS activity but measurable DTLIS activity and vice versa. Digoxin showed only 4-8% cross-reactivity against digitoxin antibody with a wide range of concentration. Some proposed DLIS compounds (nonesterified fatty acids, cholic acid, lysophospholipid, and DHEA-sulfate) did not show any cross-reactivity with the digitoxin assay at a concentrations much higher than the physiologic range. We conclude that DTLIS activity is present in patients with liver disease and uremia, and the correlation between DLIS activity and DTLIS activity is poor. Moreover, some proposed DLIS do not explain the DTLIS activity detected in serum.

Evidence for presence of a reduced form of digoxin-like immunoreactive factor (dihydro-DLIF) in mammalian tissues

Digoxin-like immunoreactive factor (DLIF) from adrenal glands is an endogenous ligand structurally related to the plant-derived cardiac glycoside digoxin. Cardiac glycosides regulate the activity of the sodium pump and thus play key roles in disease processes involving regulation of ion transport. We now report the discovery of an endogenous dihydro-DLIF analogous to dihydrodigoxin. We used HPLC, ultraviolet spectrophotometry, and cross-reactivity with two antibodies, one specific for digoxin and one for dihydrodigoxin, to support the hypothesis that dihydro-DLIF contains a chemically reduced lactone ring. The spectral absorbance maximum for dihydro-DLIF is at 196 nm, identical to dihydrodigoxin. DLIF and dihydro-DLIF are 975- and 2588-fold less immunoreactive than digoxin and dihydrodigoxin for their respective antibodies. The molar ratio of dihydro-DLIF to DLIF is approximately 5.3 in bovine adrenocortical tissue and approximately 0.38 in human serum. Dihydrodigoxin (reduced lactone ring) added to microsomes isolated from bovine adrenal cortex produced a 4.5-fold increase in digoxin-like immunoreactivity (oxidized lactone ring) after 3 h of incubation. The biotransformation is likely mediated by a cytochrome P-450 NADPH-dependent process. Our findings demonstrate the presence of a dihydro-DLIF in mammals and suggest a metabolic route for synthesis of endogenous DLIF in mammalian tissue.

Effects of two endogenous Na +,K +-ATPase inhibitors, marinobufagenin and ouabain, on isolated rat aorta

Previously, we reported that the venom of Bufo marinus toad contains a Na +,K +-ATPase inhibitor with potent vasoconstrictor activity. In the present study, using thin-layer chromatography in Silicagel 60 F 254 + 366, we separated a vasoactive substance from a mixture of steroids from Bufo marinus venom. Based on chromatographic mobility of this substance and typical color reaction after its vizualization with SbCl 3, we identified it as a previously described steroid, marinobufagenin. Vasoconstrictor and Na +,K + pump inhibitory properties of marinobufagenin were studied in isolated rat aortic rings and compared with those of ouabain. Ouabain (10–100 μmol·l −1) produced weak vasoconstriction, which was blocked by 2 μmol·l −1 phentolamine. 10 μmol·l −1 ouabain stimulated, and at higher concentrations inhibited, the Na +,K + pump. 2 μmol·l −1 phentolamine abolished the activating effect of 10 μmol·l −1 ouabain on the Na +,K + pump, but did not alter the inhibitory action of higher concentrations of ouabain. By contrast, marunibufagenin elicited rapid and strong vasoconstriction and inhibited ouabain-sensitive 86Rb uptake. Antidigoxin antibody antagonized the vasoconstrictor responses to marinobufagenin, but not to ouabain. 2 μmol·l −1 phentolamine did not alter the constrictor effect of marinobufagenin. In solid-phase digoxin immunoassay, marinobufagenin demonstrated higher digoxin-like immunoreactivity than ouabain.

Alterations of membrane properties in erythrocytes of salt hypertensive sabra rats

This study was designed to investigate the effects of a hypertensive stimulus, high salt intake, in hypertension-prone (SBH) and -resistant (SBN) Sabra rats on erythrocyte Na + content (Na i +), Ca 2+ influx and cytosolic Ca 2+ concentration ([Ca 2+] i). The relationships of these parameters to plasma lipids, circulating digoxin-like immunoreactivity and membrane microviscosity, determined by the fluorescence anisotropy of trimethylamino-diphenylhexatriene (TMA-DPH) and diphenylhexatriene (DPH), were also evaluated. Erythrocytes of SBH rats were characterized by increased [Ca 2+] i, unchanged Ca 2+ influx and reduced Na + i. There were no significant differences in the plasma digoxin-like immunoreactivity between the two strains. High-salt intake decreased membrane microviscosity (DPH anisotropy) in SBH rats but did not alter the above parameters. Erythrocyte [Ca 2+] i correlated positively with diastolic blood pressure and negatively with erythrocyte Na + i. Membrane dynamics evaluated by the two fluorescent probes did not correlate with [Ca 2+] i, Ca 2+ influx or Na + i whereas DPH anisotropy was inversely related to blood pressure. These relationships were independent of plasma cholesterol or triglycerides. It can be concluded that 1) similarly to earlier observations in essential hypertension and spontaneously hypertensive rats, erythrocyte [Ca 2+] i correlates positively with blood pressure in salt-dependent hypertension, and 2) increased erythrocyte Na + content need not be a hallmark of hypertension.

Effect of endogenous digoxin-like factor and digoxin antibody on myocardial Na+, K+-pump activity and ventricular arrhythmias in acute myocardial ischaemia in rats

The aim was to study whether a circulating sodium pump inhibitor (endogenous digoxin-like factor) contributes to the genesis of early ventricular arrhythmias in acute myocardial ischaemia in rats. Effects of digoxin antibody (260 micrograms.kg-1) on the incidence of ventricular arrhythmias, plasma digoxin-like immunoreactivity (DELFIA immunoassay), Na+, K+, and Mg2+ ions, and activity of the ouabain sensitive Na+, K(+)-pump in different regions of myocardium have been studied in propranolol naive and propranolol pretreated rats exposed to acute coronary artery ligation. Adult male Wistar rats were divided into six experimental groups: (1) saline pretreated controls; (2) saline pretreated coronary artery ligated rats; (3) coronary artery ligated rats pretreated with 260 micrograms.kg-1 digoxin antibody; (4) propranolol pretreated controls; (5) propranolol pretreated rats with acute myocardial ischaemia; (6) rats with acute myocardial ischaemia pretreated with both propranolol and digoxin antibody. Acute myocardial ischaemia in saline pretreated rats was associated with a twofold increase of plasma digoxin-like immunoreactivity and ventricular arrhythmias, but did not lead to changes in myocardial Na+, K(+)-pump activity. Pretreatment of coronary artery ligated rats with digoxin antibody reduced the total duration of ventricular tachycardia and ventricular fibrillation during a 15 minute postligation period from 201 (SEM 34) to 46(18) seconds (p < 0.002) but did not alter activity of the myocardial Na+, K(+)-pump. In rats pretreated with propranolol, acute myocardial ischaemia was associated with a twofold inhibition of the Na+, K(+)-pump in left atrial and left ventricular myocardium, and with a 69% increase in plasma K+ concentration. Administration of digoxin antibody to propranolol pretreated coronary artery ligated rats in parallel with the antiarrhythmic effect prevented the increase in plasma K+ concentration and inhibition of Na+, K(+)-pump in the left atrial, but not the left ventricular myocardium. A circulating digoxin-like factor contributes to the pathogenesis of myocardial ischaemia induced ventricular arrhythmias. As propranolol pretreatment of coronary artery ligated rats inhibited the Na, K(+)-pump in myocardium, the inhibitory effect of endogenous digoxin-like factor on Na+, K(+)-ATPase was probably masked in propranolol naive animals by the stimulatory action of catecholamines on Na+, K(+)-ATPase described previously.

Digitalis-like and vasoconstrictor effects of endogenous digoxin-like factor(s) from the venom of Bufo marinus toad.

Digitalis glycoside-like properties of the Bufo marinus toad crude venom and one of its constituents, bufalin, were studied in various assay systems. In concentrations 0.3-30 micrograms/ml crude venom increased the contractility of isolated electrically driven rat atria, constricted rat aortic rings, inhibited ouabain-sensitive Na+,K(+)-ATPase in rat erythrocytes and the Na+,K(+)-pump in rat aorta, and cross-reacted with antidigoxin antibody from the dissociation enhanced lanthanide fluoroimmunoassay (DELFIA). These effects were unaffected by adrenoceptor blockers and the 5-HT antagonist, deseril, but were blocked by antidigoxin antibody. Bufalin (10-30 microM) increased myocardial contractility and inhibited Na+,K(+)-ATPase in rat erythrocytes similarly to crude Bufo marinus venom. In rat aorta bufalin showed weak and delayed vasoconstrictor activity which was antagonized by 2 microM phentolamine, and had a biphasic effect on the Na+,K(+)-pump; 0.5-1.0 microM bufalin stimulated the pump, while higher concentrations inhibited its activity. Although the effects of bufalin were blocked by antidigoxin antibody, bufalin showed very low digoxin-like immunoreactivity in the DELFIA. These observations suggest that, in addition to bufalin, Bufo marinus venom contains at least one more digitalis-like steroid with significant intrinsic vasoconstrictor activity which, unlike bufalin, constricts the blood vessels acting directly via inhibition of the sodium pump in the vascular smooth muscle membrane.

Digoxin-like immunoreactivity may contribute to hyperinsulinemia-associated hypertension in patients with glucose intolerance.

The role of endogenous digitalis-like factors in the pathogenesis of the hypertension associated with impaired glucose tolerance was investigated by measuring plasma digoxin-like immunoreactivity (DLI). Mean blood pressure correlated significantly with the obesity index, serum insulin-like immunoreactivity (IRI), and plasma DLI concentrations in subjects with impaired glucose tolerance (IGT). Plasma DLI concentrations also correlated significantly with the obesity index and serum IRI concentrations. Because increased insulin has been proposed to promote sodium reabsorption, sodium retention in turn has presumably caused an increase of natriuretic, digitalis-like factors reflected by the increased plasma DLI concentrations in patients with IGT. Consequently, increased DLI may contribute to the elevated arterial pressure in patients with hyperinsulinemia.