Cardiac arrhythmias, electrolyte abnormalities and serum cardiac glycoside concentrations in yellow oleander (Cascabela thevetia) poisoning - a prospective study.

BACKGROUND: Consumption of yellow oleander (Cascabela thevetia) is a popular method of intentional self-harm in South India. OBJECTIVES: The objectives of this study were to identify the cardiac arrhythmias and electrolyte abnormalities in yellow oleander poisoning and to identify the association between electrolyte abnormalities, cardiac glycoside concentrations at admission and the severity of cardiotoxicity. This study was also designed to identify clinical and biochemical parameters at presentation which predict serious arrhythmias and determinants of mortality. MATERIALS AND METHODS: This was a prospective study among 192 patients who attended our Emergency department after consuming yellow oleander seeds. Patients were monitored with serial ECGs. Serious cardiac arrhythmias included sinus bradycardia <40/min, sinus arrest/exit block, second or third degree AV block, atrial tachyarrhythmias and ventricular tachyarrhythmias. Serum sodium, potassium, magnesium, total calcium and cardiac glycoside concentrations were measured at presentation for all 192 patients. Serial estimation of cardiac glycoside concentration was done in 43 patients who presented within 24 hours of consuming at least five seeds. RESULTS: At presentation, 46 patients had serious arrhythmias and on follow-up, 11 developed new-onset serious arrhythmia. Sinus bradycardia (27%) was the most common arrhythmia followed by second-degree AV block (17%); multiple arrhythmias were observed in 18%. Digoxin effect in ECG correlated significantly with hyperkalemia. Mortality rate was 5%. Serum sodium, total calcium and magnesium levels did not correlate with cardiotoxicity. Cardiac glycoside concentration was of relatively modest clinical utility to discriminate patients with serious dysrhythmias (AUC: 0.719, 95% CI: 0.63-0.81). Prolonged PR interval and digoxin effect in ECG were significantly associated with an increased likelihood of serious dysrhythmias. Increase in 0.4 number of seed intake increased the odds of mortality by 1.5 times when all other independent variables were kept constant. CONCLUSION: Cardiac glycoside concentration at the time of presentation predicted the development of new-onset serious arrhythmias. Although serum potassium correlated significantly with cardiac glycoside concentration at admission and overall serious dysrhythmias, it did not predict the development of new-onset serious arrhythmia. On the whole, serious dysrhythmias were significantly associated with higher number of seeds ingested, hypotension at admission, PR interval prolongation, presence of digoxin effect in ECG, hyperkalemia and higher cardiac glycoside concentration. The independent determinants of mortality were larger number of seeds ingested and hypotension at admission. Cardiac glycoside concentration and hyperkalemia failed to be independent markers of serious dysrhythmias as well as mortality.

ASSOSIATION OF ENDOGENOUS CARDIOTONIC STEROIDS WITH SALT-SENSITIVITY OF BLOOD PRESSURE IN GEORGIAN POPULATION.

This investigation differentiates types of essential hypertension in a Georgian population as well as describes endogenous cardiotonic steroids in salt-sensitive and salt-resistant subjects. This case control study included 185 subjects: 94 cases with stage 1 essential hypertension (JNC7) naïve to antihypertensive treatment, and 91 controls. A salt-sensitivity test was used to dichotomize case and control groups into salt-sensitive and salt-resistant subgroups. Blood and urine samples were obtained to categorize participants as consuming high and low salt diets. Endogenous cardiotonic steroids, sodium and plasma-renin activity (PRA) were measured in both samples at the different sodium conditions. Determinants of circulating levels of endogenous sodium pump inhibitors were carried out using the ELISA and RIA methods; PRA was assessed by radioimmunoassay. Descriptive statistics were used to analyze the data. Differences in variables between sodium conditions were assessed using paired t-tests. Salt-sensitivity was found in 60.5% of the total population investigated, with a higher proportion in females. A statistically significant positive correlation was found between salt-sensitivity and age in females (r=0.262, p<0.01), and with 24-hour urine sodium concentration changes (r=0.334, p<0.01). A significant negative correlation was found between salt-sensitivity and PRA. At the high sodium condition, endogenous MBG and OU were high in salt-sensitive subjects compared to those who were salt-resistant. These compounds decreased with a low-salt diet in both salt-sensitive cases and controls but remained the same in salt-resistant individuals. The MBG and OU levels positively correlated with systolic blood pressure in salt-sensitive individuals but no variability was evident among salt-resistant subjects. Our results show that MBG and OU levels start to increase at the normotensive stage and sustained high concentrations can lead to elevated systolic blood pressure, a risk factor for arterial hypertension in salt-sensitive subjects.

Quantitative determination of periplocymarin in rat plasma and tissue by LC-MS/MS: application to pharmacokinetic and tissue distribution study.

A simple, rapid and sensitive liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the determination of periplocymarin in biological samples was developed and successfully applied to the pharmacokinetic and tissue distribution study of periplocymarin after oral administration of periplocin. Biological samples were processed with ethyl acetate by liquid-liquid extraction, and diazepam was used as the internal standard. Periplocymarin was analyzed on a C18 column with isocratic eluted mobile phase composed of methanol and water (containing 0.1% formic acid) at a flow rate of 0.2 mL/min (73:27, v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer using positive-ion mode electrospray ionization in the selected reaction monitoring mode. The MS/MS ion transitions monitored were m/z 535.3→355.1 and 285.1→193.0 for periplocymarin and diazepam, respectively. Good linearity was observed over the concentration ranges. The lower limit of quantification was 0.5 ng/mL in plasma and tested tissues. The intra-and inter-day precisions (relative standard deviation) were <10.2 and 10.5%, respectively, and accuracies (relative error) were between -6.8 and 8.9%. Recoveries in plasma and tissue were >90%. The validated method was successfully applied to the pharmacokinetic and tissue distribution studies of periplocymarin in rats. Copyright © 2016 John Wiley & Sons, Ltd.

The iDigoxin Assay is More Sensitive than LOCI Digoxin Assay for Rapid Detection of Convallatoxin, the Active Cardiac Glycoside of Lily of The Valley.

OBJECTIVE: Lily of the valley is a poisonous plant due to the presence of the cardiac glycoside convallatoxin. We compared two immunoassays (LOCI digoxin assay and iDigoxin assay) for rapid detection of convallatoxin if present in human serum. MATERIALS AND METHODS: Aliquots of a drug free serum pool and a digoxin serum pool were supplemented with microliter amounts of lily of the valley extract or nanogram to microgram quantities of convallatoxin, followed by measurement of apparent digoxin concentrations using the LOCI and iDigxoin assays. RESULTS: Apparent digoxin concentrations were observed when aliquots of a drug free serum pool were supplemented with convallatoxin or lily of the valley extract using both assays but apparent digoxin concentrations were significantly higher using the iDigoxin assay. In addition, the interference of convallatoxin in serum digoxin measurement was also significantly higher using iDigxoin assay compared to the LOCI digoxin assay. CONCLUSIONS: The iDigxoin assay is more sensitive in detecting convallatoxin in human serum.

A validated LC-MS/MS assay for the simultaneous determination of periplocin and its two metabolites, periplocymarin and periplogenin in rat plasma: Application to a pharmacokinetic study.

A sensitive and reliable LC-MS/MS method was developed and validated for the simultaneous determination of periplocin and its two metabolites (periplocymarin and periplogenin) in rat plasma using psoralen as the internal standard (IS). After liquid-liquid extraction with ethyl acetate, chromatographic separation was performed on a C18 column with a 13 min gradient elution using 0.1% formic acid and acetonitrile as mobile phase at a flow rate of 0.3 mL/min. The detection was accomplished on a tandem mass spectrometer via an electrospray ionization (ESI) source by multiple reaction monitoring (MRM) in the positive ionization mode. The lower limits of quantitation (LLOQs) for periplocin, periplocymarin and periplogenin were 0.5, 1 and 0.1 ng/mL, respectively. The mean recoveries of the analytes and IS were higher than 67.7%. The proposed method was successfully applied to evaluating the pharmacokinetic studies of periplocin and its metabolites (periplocymarin and periplogenin) in rats after a single oral administration of periplocin at 50 mg/kg.

DigiFab interacts with endogenous cardiotonic steroids and reverses preeclampsia-induced Na/K-ATPase inhibition.

Elevated levels of endogenous Na/K-ATPase (NKA) inhibitors, cardiotonic steroids (CTSs) including marinobufagenin (MBG), contribute to pathogenesis of preeclampsia (PE) and represent a target for immunoneutralization by Digibind (Ovine Digoxin Immune Antibody, Glaxo-Smith Kline). Because Digibind is no longer commercially available, we studied whether DigiFab (BTG International Ltd, UK) can substitute Digibind for immunoneutralization of CTS in patients with PE. We compared DigiFab, Digibind, and anti-MBG monoclonal antibody (mAb) with respect to their ability to interact with CTS in PE plasma and to restore NKA activity in erythrocytes from patients with PE. Using immunoassays based on DigiFab, Digibind, and anti-MBG mAb, we studied the elution profile of CTS following high-performance liquid chromatography (HPLC) fractionation of PE plasma. Totally, 7 patients with mild PE (28 ± 2 years; gestational age, 39 ± 0.5 weeks; blood pressure 156 ± 5/94 ± 2 mm Hg) and 6 normotensive pregnant participants (28 ± 1 years; gestational age, 39 ± 0.4 weeks; blood pressure 111 ± 2/73 ± 2 mm Hg) were enrolled. Preeclampsia was associated with a substantial inhibition of erythrocyte NKA (1.47 ± 0.17 vs 2.65 ± 0.16 µmol Pi/mL per h in control group, P < .001). Ex vivo, at 10 µg/mL concentration, which is consistent with the clinical dosing of Digibind administered previously in PE, DigiFab and Digibind as well as anti-MBG mAb (0.5 µg/mL) restored erythrocyte NKA activity. Following HPLC fractionation of pooled PE and control plasma, PE-associated increase in CTS material was detected by Digibind (176 vs 75 pmoles), DigiFab (221 vs 70 pmoles), and anti-MBG mAb (1056 vs 421 pmoles). Therefore, because DigiFab interacts with CTS from PE plasma and reverses PE-induced NKA inhibition, it can substitute Digibind for immunoneutralization of CTS in patients with PE.

Pennogenin tetraglycoside stimulates secretion-dependent activation of rat platelets: evidence for critical roles of adenosine diphosphate receptor signal pathways.

BACKGROUND: Total steroidal saponins extracted from the rhizome of Paris polyphylla Sm. var. yunnanensis (TSSPs) have been demonstrated to promote hemostasis in vivo and induce platelet aggregation in vitro. Pennogenin tetraglycoside (Tg) has been identified as one of the active ingredients in TSSPs and can induce rat platelet aggregation. OBJECTIVE: To investigate the functional role of Tg in platelets and the signaling pathway mechanisms which mediate Tg-induced platelet aggregation. METHODS AND RESULTS: Using scanning electron microscopy, the turbidimetric method and flow cytometry, we demonstrated that Tg induces shape change and concentration-dependently induces aggregation, dense granule secretion and a-granule secretion in rat platelets. The activation characteristics were comprehensively confirmed using transmission electron microscopy. Apyrase and antagonists of the platelet adenosine diphosphate (ADP) receptors, P2Y1 and P2Y12, completely inhibited Tg-induced platelet aggregation, which was not sensitive to indomethacin or SQ29548 inhibition. Furthermore, ADP receptor antagonists inhibited Tg-induced a-granule secretion, and blockade of the P2Y1 receptor prevented Tg-induced platelet shape changes. Tg-induced dense granule secretion was not affected by ADP receptor antagonists or various various pharmacological inhibitors of the intracellular effectors involved in dense granule secretion signaling pathways. CONCLUSION: We identified that Tg directly induces platelet activation and demonstrated that Tg-induced platelet activation depends on dense granule secretion of ADP, which in turn activates the P2Y1 and P2Y12 receptor signaling pathways.

Method validation of a survey of thevetia cardiac glycosides in serum samples.

A sensitive and specific liquid chromatography tandem mass spectrometry (HPLC-ESI(+)-MS/MS) procedure was developed and validated for the identification and quantification of thevetin B and further cardiac glycosides in human serum. The seeds of Yellow Oleander (Thevetia peruviana) contain cardiac glycosides that can cause serious intoxication. A mixture of six thevetia glycosides was extracted from these seeds and characterized. Thevetin B, isolated and efficiently purified from that mixture, is the main component and can be used as evidence. Solid phase extraction (SPE) proved to be an effective sample preparation method. Digoxin-d3 was used as the internal standard. Although ion suppression occurs, the limit of detection (LOD) is 0.27 ng/ml serum for thevetin B. Recovery is higher than 94%, and accuracy and precision were proficient. Method refinement was carried out with regard to developing a general screening method for cardiac glycosides. The assay is linear over the range of 0.5-8 ng/ml serum. Finally, the method was applied to a case of thevetia seed ingestion.

Simultaneous analysis of cardiac glycosides in blood and urine by thermoresponsive LC-MS-MS.

A new thermoresponsive polymer separation column was applied to simultaneous analysis of four cardiac glycosides (CGs) being widely used for the treatment of arrhythmias and heart failure in human blood and urine. This column is composed of an N-isopropylacrylamide polymer, the surface of which undergoes a reversible alteration from hydrophilic to hydrophobic by changing temperature. The chromatographic separation and retention times can be easily be controlled by adjusting the column temperature. As the column temperature was changed from 50 to 10 °C over 8 min, five CGs, including deslanoside, digoxin, methyldigoxin, digitoxin, and digitoxigenin (internal standard) were better resolved. Using these LC conditions, we analyzed four CGs in human whole blood and urine simultaneously by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Validation data as functions of recovery rates, linearity, accuracy, and precision were carefully tested; all were generally satisfactory. The detection limits for the four CGs in both matrices were 0.2-0.3 ng/mL. The method was applied to analysis of methyldigoxin and its main metabolite digoxin in whole blood and urine samples obtained from a deceased person in actual autopsy case. To our knowledge, this is the first report describing an LC-MS-MS method using a thermoresponsive column for analysis of drug(s). The inclusion of the thermoresponsive column in an LC-MS-MS technique seems to extend the possibility for simultaneous analysis of compounds of different properties, such as hydrophobic precursors and their hydrophilic metabolites in biological samples within limited analysis times.

[Mechanisms for linking high salt intake to vascular tone: role of Na(+) pump and Na(+)/Ca²(+) exchanger coupling].

Excessive salt intake is a major risk factor for hypertension. However, the underlying molecular relationship between salt and hypertension is not fully understood. Recently discovered cardiotonic steroids, such as endogenous ouabain and other steroids, have been proposed as candidate intermediaries. Plasma cardiotonic steroids are significantly elevated in patients with essential hypertension and in salt-dependent hypertensive animals. Generally, it is believed that cardiotonic steroids inhibit Na(+) pump activity and lead to an increase in the cytosolic Na(+) concentration. Cellular Na(+) accumulation raises the cytosolic Ca²(+) concentration through the involvement of Na(+)/Ca²(+) exchanger type 1 (NCX1). In isolated arteries from α2 Na(+) pump knockout mice (α2(+/-)), myogenic tone is increased, and NCX inhibitor normalizes the elevated myogenic tone in α2(+/-) arteries. The NCX inhibitor lowers arterial blood pressure in salt-dependent hypertensive rats but not in other types of hypertensive rats or in normotensive rats. Furthermore, smooth muscle-specific NCX1 transgenic mice are hypersensitive to salt, whereas mice with smooth muscle-specific knockout of NCX1 (NCX1(SM-/-)) have low salt sensitivity. These results suggest that functional coupling between the vascular α2 Na(+) pump and NCX1 is a critical molecular mechanism for salt-induced blood pressure elevation.

Hypertension, Na+/Ca2+ exchanger, and Na+, K+-ATPase.

Hypertension is the most prevalent risk factor for stroke, myocardial infarction, or end-stage renal failure. The critical importance of excess salt intake in the pathogenesis of hypertension is widely recognized, but the mechanisms whereby salt intake elevates blood pressure have puzzled researchers. Recent studies using Na+/Ca2+ exchange inhibitors and genetically engineered mice provide evidence that vascular Na+/Ca2+ exchanger type 1 (NCX1) is involved in the development of salt-dependent hypertension. Endogenous cardiac glycosides, which may contribute to salt-dependent hypertension, seem to be necessary for NCX1-mediated hypertension. Intriguingly, studies using knock-in mice with modified cardiac glycoside binding affinity of Na+,K+-ATPases provide a clear demonstration that this cardiac glycoside-binding site plays an important role in blood pressure regulation. Taken all together: (1) endogenous cardiac glycosides are secreted after high salt intake; (2) these cardiac glycosides inhibit Na+,K+-ATPase in vascular smooth muscle cells; (3) this inhibition results in the elevation of local Na+ on the submembrane area; and (4) this elevation of local Na+ facilitates Ca2+ entry through NCX1, resulting in vasoconstriction. This proposed pathway may have enabled us to explain how to link dietary salt to hypertension.

Molecular insights into uremic cardiomyopathy: cardiotonic steroids and Na/K ATPase signaling.

Patients with chronic renal failure develop a "uremic" cardiomyopathy characterized by diastolic dysfunction, left ventricular hypertrophy, fibrosis, and systemic oxidant stress. Patients with chronic renal failure also are known to have increases in the circulating concentrations of endogenous cardiotonic steroids (also referred to as endogenous digitalis-like substances.) Endogenous cardiotonic steroids produce reactive oxygen species as part of the signal cascade induced by binding to the plasmalemmal Na/K-ATPase in patients, and this signal cascade appears capable of inducing several key pathophysiologic features of uremic cardiomyopathy. In addition, these patients develop both fibrosis and oxidant stress without a known mechanism. In this review we highlight data supporting the hypothesis that endogenous cardiotonic steroids are a key molecular component involved in the diastolic dysfunction, left ventricular hypertrophy, fibrosis, and systemic oxidant stress associated with chronic kidney disease.

Diverse effects of stress and additional adrenocorticotropic hormone on digitalis-like compounds in normal and nude mice.

Digitalis-like compounds (DLC) are steroidal hormones that are synthesized in, and released from, the adrenal gland, whose regulation may be directed by the hypothalamic-pituitary-adrenal (HPA) axis. Increasing evidence points to antitumour properties of these compounds and we hypothesized that the establishment of tumours in athymic nude mice may be facilitated by an abnormal synthesis or secretion of DLC. To explore this hypothesis, DLC concentrations were determined in the plasma, and in adrenal and hypothalamic tissues of nude compared to normal mice under basal conditions, and 30 min after a stress stimulus (i.p. injection of 100 micro l saline) with or without additional adrenocorticotropic hormone (ACTH) 1 micro g/per animal. Simultaneously, plasma corticosterone and serum adrenocorticotropic hormone (ACTH) concentrations were analysed. The basal DLC concentrations were similar in the plasma and the hypothalamus of both strains, whereas the basal adrenal DLC concentration was significantly lower in the nude mice compared to normal mice. The stress stimulus induced in normal mice a significant increase in DLC concentrations in the adrenal gland, the plasma and the hypothalamus. However, in nude mice, it caused an increase only in the adrenal gland and the hypothalamus, whereas the plasma DLC concentration was not affected. In both strains, the administration of ACTH in addition to injection stress did not provoke a further increase in DLC concentrations while inducing a significant increase in plasma corticosterone concentration. Regardless of the applied stimulus, the nude mice expressed significant lower DLC concentrations in the adrenal gland and the plasma compared to normal mice. The low basal adrenal DLC concentration in nude mice and their impaired DLC response towards stress- and ACTH stimulation both support an involvement of DLC in tumorigenesis.

Toxicokinetics of cotyledoside following intravenous administration to sheep.

Cotyledoside, a bufadienolide cardiac glycoside, was administered intravenously to sheep in 2 studies. In experiment 1, sheep (n = 4) received 0.0135 mg/kg daily on 5 consecutive days and in the 2nd experiment, sheep (n = 4) received 0.027 mg/kg as a single dose. Jugular blood was collected at different time intervals and kinetic parameters were determined. The data fitted a 1-compartmental model. In both experiments a short half-life (t1/2) and mean residence time (MRT), a relative small volume of distribution (Vd(ss)) and rapid clearance were calculated. In the 1st experiment, t1/2 and MRT increased significantly (P < 0.007) from Day (D) 0 to D4. It is suggested that the rapid decline in plasma cotyledoside concentrations in sheep denotes rapid distribution of cotyledoside to the tissues or extracellular spaces and possible accumulation at the biophase.

Acute yellow oleander (Thevetia peruviana) poisoning: cardiac arrhythmias, electrolyte disturbances, and serum cardiac glycoside concentrations on presentation to hospital.

OBJECTIVE: To describe the cardiac arrhythmias, electrolyte disturbances, and serum cardiac glycoside levels seen in patients presenting to hospital with acute yellow oleander (Thevetia peruviana) poisoning and to compare these with published reports of digitalis poisoning. DESIGN: Case series. SETTING: Medical wards of Anuradhapura District General Hospital, Sri Lanka, and coronary care unit of the Institute of Cardiology, National Hospital of Sri Lanka, Colombo, the national tertiary referral centre for cardiology. PATIENTS: 351 patients with a history of oleander ingestion. MEASUREMENTS: ECG and blood sample analysis on admission. RESULTS: Most symptomatic patients had conduction defects affecting the sinus node, the atrioventricular (AV) node, or both. Patients showing cardiac arrhythmias that required transfer for specialised management had significantly higher mean serum cardiac glycoside and potassium but not magnesium concentrations. Although there was considerable overlap between groups, those with conduction defects affecting both sinus and AV nodes had significantly higher mean serum cardiac glycoside levels. CONCLUSIONS: Most of these young previously healthy patients had conduction defects affecting the sinus or AV nodes. Relatively few had the atrial or ventricular tachyarrhythmias or ventricular ectopic beats that are typical of digoxin poisoning. Serious yellow oleander induced arrhythmias were associated with higher serum cardiac glycoside concentrations and hyperkalaemia but not with disturbances of magnesium.

Identification and quantification of cardiac glycosides in blood and urine samples by HPLC/MS/MS.

Cardiac glycosides (CG) are of forensic importance because of their toxicity and the fact that very limited methods are available for identification of CG in biological samples. In this study, we have developed an identification and quantification method for digoxin, digitoxin, deslanoside, digoxigenin, and digitoxigenin by high-performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS). CG formed abundant [M + NH4]+ ions and much less abundant [M + H]+ ions as observed with electrospray ionization (ESI) source and ammonium formate buffer. Under mild conditions for collision-induced dissociation (CID), each [M + NH4]+ ion fragmented to produce a dominant daughter ion, which was essential to the sensitive method of selected reaction monitoring (SRM) quantification of CG achieved in this study. SRM was compared with selected ion monitoring (SIM) regarding the effects of sample matrixes on the methodology. SRM produced lower detection limits with biological samples than SIM, while both methods produced equal detection limits with CG standards. On the basis of the HPLC/MS/MS results for CG, we have proposed some generalized points for conducting sensitive SRM measurements, in view of the property of analytes as well as instrumental conditions such as the type of HPLC/MS interface and CID parameters. Analytes of which the molecular ion can produce one abundant daughter ion with high yield under CID conditions may be sensitively measured by SRM. ESI is the most soft ionization source developed so far and can afford formation of the fragile molecular ions that are necessary for sensitive SRM detection. Mild CID conditions such as low collision energy and low pressure of collision gas favor production of an abundant daughter ion that is essential to sensitive SRM detection. This knowledge may provide some guidelines for conducting sensitive SRM measurements of very low concentrations of drugs or toxicants in biological samples.

Circulating bufodienolide and cardenolide sodium pump inhibitors in preeclampsia.

OBJECTIVE: To determine plasma levels of the endogenous bufodienolide Na+/K+ ATPase inhibitor, marinobufagenin-like factor (MBG), in normotensive pregnancy and in preeclampsia, to compare changes of MBG with that of ouabain-like compound (OLC), and to characterize the purified MBG immunoreactive factor from preeclamptic plasma. DESIGN AND METHODS: Consecutive sample study. The levels of MBG and OLC compounds were measured in extracted plasma by solid phase fluoroimmunoassays. MBG and ouabain immunoreactive materials were partially purified from preeclamptic plasma via reverse-phase high-performance liquid chromatography (HPLC) and studied for their ability to cross react with MBG and ouabain antibodies, and to inhibit the Na+/K+ ATPase from human mesenteric arteries. Vasoconstrictor effect of authentic MBG was studied in isolated rings of human umbilical arteries. RESULTS: In 11 nonpregnant control individuals, plasma concentrations of MBG and OLC were 0.190+/-0.04 nmol/l and 0.297+/-0.037 nmol/l, respectively. In the third trimester of noncomplicated pregnancy (n = 6), plasma MBG increased (0.625+/-0.067 nmol/l, P<0.05), and OLC did not (0.32+/-0.07 nmol/l). In 15 patients with preeclampsia, plasma levels of both MBG and OLC increased dramatically (2.63+/-0.10 nmol/l and 0.697+/-0.16 nmol/l, respectively, P<0.01 versus both control groups). When fractionated by reverse phase HPLC, OLC was eluted by 18% acetonitrile, and MBG by 48% acetonitrile. Serially diluted samples of MBG and OLC immunoreactive materials from HPLC fractions reacted with MBG and ouabain antibody in solid phase immunoassay in a concentration dependent fashion. Authentic MBG caused contractile responses of isolated rings of human mesenteric arteries in a concentration-dependent manner. Similarly to the authentic MBG, HPLC purified MBG immunoreactive material from preeclamptic plasma inhibited Na+/K+ ATPase purified from human mesenteric artery. CONCLUSIONS: Our observations demonstrate the coexistence of two endogenous cardiotonic steroids in preeclamptic plasma, a more polar OLC and a less polar MBG-like compound. Substantial increases in plasma OLC and MBG immunoreactivity in preeclampsia, along with the vasoconstrictor properties of authentic MBG and Na+,K+ ATPase inhibitory activity of human MBG immunoreactive factor, suggest, that in preeclampsia, plasma concentrations of MBG are enough to substantially inhibit the sodium pump in cardiovascular tissues, and are in accordance with the views attributing endogenous digitalis-like factors a pathogenic role in the preeclamptic hypertension.

A specific binding protein for cardiac glycosides exists in bovine serum.

Searching for a binding protein in blood, which may be involved in the specific transport of cardiac glycosides to their receptor sites on the sodium pump, we isolated a cardiac glycoside-binding protein (CGBG) of 26 kDa from the globulin fraction of bovine serum by affinity chromatography and on a ouabain-Sepharose 4B column by a purification factor of 5000. The cardiac glycoside-binding globulin was labeled specifically and covalently by the protein-reactive digoxigenin derivative HDMA (N-hydroxysuccimidyldigoxigenin-3-O-methylcarbonyl-epsilon-+ ++aminocapro ate). Even very high concentrations of other steroids, such as estrogen, testosterone, progesterone, and cortisone, did not prevent HDMA-labeling (at 5 and 100 nM) of CGBG, but the cardenolides ouabain and digoxin or the bufadienolide proscillaridin A did so. CGBG is a homodimer of two 26-kDa subunits forming disulfide bonds, since HDMA labeling of a protein of 53 kDa was observed in SDS-polyacrylamide gel electrophoresis when beta-mercaptoethanol was absent during SDS denaturation. The N-terminal amino acid sequence K-D-V-Y-R-A-P-D-G-T-Q-S-A showed no sequence similarity with proteins recorded in gene and protein sequence data banks. A 90-kDa cytosolic CGBG exists in bovine kidneys and reacts with antibodies against CGBG. Binding of ouabain to the cardiac glycoside-binding globulin was monitored by quenching of intrinsic tryptophan fluorescence. Such studies reveal two negatively cooperative ouabain binding sites with Kd' of 1.52 nM and Kd' = 75 nM and with an interaction factor of 50 using a Koshland-Némethy-Filmer model. The demonstration of a cardiac glycoside-binding globulin in plasma is consistent with the recent finding of endogenous cardiac glycosides in mammals.