An in-depth look into a well-known herbal drug: Fingerprinting, isolation, identification, and content estimation of saponins in different Strophanthus seeds.

Seeds of Strophanthus species are known as a source of rapid-acting cardenolides. These water-soluble glycosides are listed as the sole critical constituents of this raw herbal drug. A non-standard cardioprotective medication with ouabain-containing oral remedies has become popular in Europe as a result of the withdrawal of corresponding registered drugs from the market. However, the bioequivalence of pure ouabain solutions, tinctures, and home-made extracts from Strophanthus seeds is unknown. Thus, this study aimed to update the information on the composition of Strophanthus seeds used for this purpose. The distribution of two main saponins and about 90 previously unreported compounds, tentatively identified as saponins in eleven Strophanthus species, was systematically evaluated by ultra-high-performance liquid chromatography mass spectrometry (UHPLC-MS) and -MS/MS. Seeds of S. gratus were selected to isolate the dominant unreported triterpenoids, bidesmosides of echinocystic and oleanolic acid. Their structures were established by HRMS, MS/MS, as well as by NMR techniques. The total saponin content, estimated by UHPLC-MS, was up to 1%. The detected saponins could influence the peroral bioavailability of hardly absorbable Strophanthus cardenolides and exhibit their own activity. This finding may be relevant when Strophanthus preparations (containing both saponins and cardiac glycosides) are used, particularly when homemade preparations are administered.

Endogenous ouabain in cardiovascular function and disease.

An endogenous ouabain has been isolated and conclusively identified from several mammalian tissues, including human plasma, by a number of independent laboratories. Substantial evidence from independent laboratories in several continents is consistent with an adrenal source for most if not all of the circulating endogenous ouabain. Accumulating evidence suggests that circulating levels of endogenous ouabain in humans are modulated by dietary salt and chronic volume status. Endogenous ouabain is linked significantly with vascular function in hypertension and likely impacts the pathogenesis of heart and renal failure. Moreover, the molecular mechanism of endogenous ouabain-linked hypertension involves the sodium pump/sodium-calcium exchange duet. The outstanding analytical issues include the elucidation of distal events in the biosynthetic pathway for endogenous ouabain and identification of molecular mechanisms that regulate its secretion and clearance.

Quantification of the Na+/K(+)-pump in solubilized tissue by the ouabain binding method coupled with high-performance gel chromatography.

Membrane-bound Na+/K(+)-ATPase purified from dog kidney outer medulla was solubilized with octaethylene glycol n-dodecyl ether (C12E8) and incubated with [3H]ouabain in the presence of NaCl. ATP and MgCl2 for 10 min at 0 degrees C. The resulting enzyme was separated, by high-performance gel chromatography executed at 0.2 degrees C. Mainly into its (alpha beta)2-diprotomer and alpha beta-protomer, which both bound stoichiometrically to [3H]ouabain. The amounts of ouabain that bound to the tissue itself and its microsomes could be estimated in the same way, as [3H]ouabain was found to bind only to the diprotomer and protomer they possessed. The amounts of ouabain that bound to them in the solubilized state were at least 5-times higher than those that did so when they were non-solubilized, suggesting that the surfactant rendered the enzyme accessible to ouabain. When the solubilized tissue (138 mg ml-1 wet tissue) was reacted with ouabain in the presence of 0.1 M NaCl and 4.8 mM MgCl2 for 10 min at 0 degrees C, maximal ouabain binding was attained in the presence of 18.3 microM [3H]ouabain, 1.2 mM ATP and 3 to 5 mg ml-1 C12E8, which was common to the outer medulla and human colon cancer cells. The present method enabled the pump number in protein and tissue samples in the range 7.2 x 10(-9) (purified pump) to 1.5 x 10(-12) (cancer tissue) mol/mg protein to be estimated within 2 h.

Ouabain is secreted by bovine adrenocortical cells.

Ouabain is a specific inhibitor of the sodium pump. This steroid has been found in the mammalian circulation in significant amounts and may be of adrenal origin. Secretion of ouabain from adrenal cells has been little studied and the purpose of the present work was to determine the adrenal distribution of ouabain, aldosterone and cortisol, and to characterize the effects of ACTH and angiotensin II on the secretion of these steroids in primary cultures of bovine adrenocortical cells. In fresh bovine adrenals, the cortical to medullary ratios for aldosterone, cortisol and ouabain were 14, 4.25 and 2.5, respectively. All three steroids were detected in elevated amounts in the conditioned medium of primary cultures of adrenocortical cells. Reverse phase HPLC of the secreted ouabain immunoreactivity showed it was isopolar with commercial ouabain. In the presence of 10 nM ACTH or angiotensin II, the secretion of all three steroids increased significantly with similar time courses. The stimulated secretion of ouabain exceeded the intracellular content of this steroid in either control or activated cells by 3-5 fold. The amount of angiotensin II stimulated ouabain secretion was greater from cells incubated in larger volumes. These results show that ouabain is enriched in the bovine adrenal cortex, and is secreted by primary cultures of these cells. The secretion of ouabain is increased by ACTH and angiotensin II, is due to either de novo synthesis or transformation of an intracellular precursor that is not overtly immunoreactive, and is feedback regulated by either ouabain itself or a cosecreted factor. These cells may be useful to study stimulus-secretion coupling and the biosynthetic pathway of ouabain.

Is ouabain an authentic endogenous mammalian substance derived from the adrenal?

Ouabain has recently been reported to be an endogenous mammalian substance released by the adrenal cortex and present in normal plasma. We have attempted to confirm and extend this observation. Using a ouabain radioimmunoassay developed in this laboratory, we fractionated by high-performance liquid chromatography (HPLC) normal human plasma from healthy volunteers to determine the presence of ouabain immunoreactivity and compare this immunoreactivity with authentic ouabain. In most subjects no ouabain immunoreactivity that coeluted with authentic ouabain was observed. Some subjects had ouabain-immunoreactive material present at low levels, but it was largely attributable to cross-reactivity with diverse substances found not to be ouabain. Similar results were obtained after analysis of plasma collected from 10 patients entering a medical intensive care unit. Studies of serum-free medium conditioned by bovine adrenocortical cells showed some ouabain immunoreactivity. To determine whether this material might be a steroid product of cholesterol side-chain cleavage, we performed chemical blockade of steroidogenesis, which effectively suppressed progesterone production by these cells but had no consistent effect on ouabain immunoreactivity in this medium. Stimulation of steroidogenesis with 22-R-OH-cholesterol in bovine adrenocortical cells did not produce any increase in the ouabain immunoreactivity present in conditioned medium. Subsequent HPLC studies of ouabain immunoreactivity in bovine adrenocortical cell-conditioned medium indicated that authentic ouabain did not account for most of the ouabain immunoreactivity in serum-free medium. Studies with bovine adrenocortical cells incubated in a minimal salt and glucose medium indicated a small peak of immunoreactivity that may correspond to authentic ouabain.(ABSTRACT TRUNCATED AT 250 WORDS)

An endogenous ouabain-like factor associated with hypertensive pregnant women.

Levels of a ouabain-like factor (OLF) were measured in amniotic fluid from 49 undigitalized third trimester pregnant women by means of its cross-reactivity in a digoxin RIA and its inhibition of ouabain-sensitive [Na,K]ATPase. The results from these 2 assays were significantly correlated (P less than 0.05). Of the women included in this study, 25 had blood pressures considered normal for their gestational age, while 24 had developed during their current pregnancy blood pressures judged to be elevated. When levels of OLF in the amniotic fluids from the normotensive and hypertensive pregnant women were compared, significantly higher levels were present in the hypertensive group for both assays (P less than 0.002). Further, there was a significant correlation between the diastolic blood pressures of these women at the time of amniocentesis and the amniotic fluid OLF levels determined by either assay (P less than 0.002). These results are consistent with OLF having a role in hypertensive complications of pregnancy.

Stress-induced elevation of ouabainlike compound in rat plasma and adrenal.

Recent observations demonstrate the presence of neurosteroids and their rapid increase in response to acute stress. In view of a steroidal nature of ouabainlike compound, we tested the hypothesis that ouabainlike compound may participate in a homeostatic response to acute stress. Male Wistar rats were subjected to acute stress by swimming in water (22 degrees C) for 10 minutes. The levels of ouabainlike compound in plasma, hypothalamus, pituitary, and adrenal at 10, 40, and 70 minutes (n = 8 for each) after the end of swim stress were compared with nonstressed control levels (n = 10). Ouabainlike compound was measured by a radioimmunoassay for ouabain. Plasma levels of corticosterone and catecholamines were also measured. Plasma corticosterone concentrations increased rapidly at 10 minutes (P < .01) and then declined. A trend for a rise in plasma catecholamines was found at 10 minutes. Adrenal levels of ouabainlike compound concomitantly increased at 10 minutes (P < .01, control: 58.9 +/- 5.9 pmol ouabain equivalents per gram; 10 minutes: 92.5 +/- 4.8; 40 minutes: 47.3 +/- 9.6; 70 minutes: 45.1 +/- 6.3). In contrast, the response of plasma ouabainlike compound was slow and doubled at 40 minutes (P < .01, control: 115 +/- 12 pmol ouabain equivalents per liter; 10 minutes: 132 +/- 23; 40 minutes: 226 +/- 53; 70 minutes: 117 +/- 16). Ouabainlike compound levels in hypothalamus and pituitary remained unaltered. These findings suggest that ouabainlike compound may function as a stress hormone.

Ouabain-like factor quantification in mammalian tissues and plasma: comparison of two independent assays.

The resolution of controversies that concern the detectability of an endogenous ouabain-like factor (OLF) in mammalian tissues and plasma was approached by the application of a standardized method for its extraction and quantification. Two independent assays were used to quantify the OLF: (1) a radioimmunoassay, which used a polyclonal anti-ouabain antiserum, and (2) a radioenzymatic assay based on the inhibition of dog kidney Na+,K+-ATPase. Plasma and tissues were obtained from the Milan hypertensive strain (MHS) and the Milan normotensive strain (MNS) of rats and from healthy human volunteers. Results indicate that (1) a single high-performance liquid chromatography (HPLC) fraction identical to that of ouabain was identified by both assay methods in the rat hypothalamus and hypophysis and in both rat and human plasma; (2) dilution curves of OLF and standard ouabain were parallel and with a similar Kd, both in radioimmunoassay (3 nmol/L) and ATPase assay (14 nmol/L); (3) after HPLC, OLF was similarly quantified by the two methods in the hypothalamus, hypophysis, adrenals, and plasma of rats and in human plasma; (4) OLF was present in larger amounts in the hypothalamus, hypophysis, and plasma of MHS rats than that of MNS rats; (5) the HPLC fraction of human plasma was quantified similarly by both assays (range, 60 to 150 pmol/L); (6) recovery of standard ouabain in pre-HPLC plasma extracts was approximately 90%; and (7) pre-HPLC OLF concentrations in human plasma ranged between 0.05 and 0.75 nmol/L. Rat cerebral tissues and both rat and human plasma contained measurable amounts of OLF, which were quantified similarly by radioimmunoassay and ATPase assay, both before and after HPLC fractionation. The increased MHS tissue and plasma levels of OLF are in keeping with the pathogenetic role of this factor in MHS hypertension.

Elevation of ouabainlike compound levels with hypertonic sodium chloride load in rat plasma and tissues.

A major biologically active endogenous digitalis-like factor in the mammalian body may be an isomer of ouabain (ouabainlike compound, OLC). However, the exact role of OLC in sodium homeostasis is still unclear, and acute isotonic volume expansion does not enhance the secretion of OLC. We tested the hypothesis that OLC may be more important in the response to acute hypertonic NaCl load rather than isotonic volume expansion. We injected intraperitoneally 2 mL of 20% NaCl solution into male Wistar rats (n=34) and measured OLC levels in plasma, hypothalamus, pituitary, and adrenal at baseline (n=10) and 1, 2, and 4 hours (n=8 for each). In response to hypertonic NaCl loading, plasma Na-K ratio was elevated at 2 and 4 hours (P<.01). OLC levels in pituitary increased (P<.01) at 1 hour. Thereafter, plasma OLC levels increased at 2 and 4 hours (P<.05; basal, 75+/-11 pmol/L [+/-SEM]; 1 hour, 55+/-11; 2 hours, 130+/-24; 4 hours, 156+/-20). Concomitantly, OLC levels in adrenal increased at 2 and 4 hours (P<.01; basal, 1.7+/-0.2 pmol/g; 1 hour, 4.5+/-0.9; 2 hours, 5.0+/-0.7; 4 hours, 6.8+/-2.2). A significant correlation was observed between OLC levels in plasma and adrenal (P<.05). Plasma Na-K ratio positively correlated with OLC levels in plasma (r=.51, P<.01) and adrenal (r=.48, P<.01). Similar injection of physiological saline solution or hypertonic sucrose solution in physiological saline did not increase OLC levels in plasma and tissues. These findings indicate the elevation of OLC levels in plasma, pituitary, and adrenal in response to acute hypertonic NaCl load in rats and suggest that OLC may be involved in the response to the hypernatremic state.

Identification of endogenous ouabain in culture supernatant of PC12 cells.

OBJECTIVE: Ouabain-like factor (OLF), assayed as ouabain-like immunoreactivity (OLI), is thought to represent an endogenous digitalis-like factor. We found increased plasma OLI during the surgical removal of a pheochromocytoma. The elution volume of the OLI extracted from plasma and the pheochromocytoma tissue was the same as that for authentic ouabain, using reverse phase high-performance liquid chromatography. The present study was performed to characterize OLF from the culture supernatant of a rat pheochromocytoma cell line, PC12 cells. DESIGN: OLI from culture supernatant and chromatographic fractions were assayed by a sensitive enzyme-linked immunosorbent assay for ouabain. PC12 cells, subcultured in RPMI 1640 with 10% horse serum and 5% fetal bovine serum, were washed, and then cultured in Iscove's modified Dulbecco's medium (Life Technologies, Rockville, Maryland, USA) with 0.4% bovine serum albumin (without serum). Progesterone was added to augment the production or secretion of OLI. The conditioned medium was acidified to dissociate the binding protein, and OLI was purified by five steps of octadecylsilane (ODS) column chromatography. The structural identity of this OLI was determined by liquid chromatography and mass spectrometry (LC/MS). RESULTS: OLI in the culture medium increased after addition of progesterone in a dose-dependent manner. The concentration in the culture medium was approximately double of that in homogenized PC12 cells. After five rounds of ODS column chromatography, approximately 100 ng of OLI was purified from 21 of culture supernatant, without fetal calf serum, in the presence of progesterone. The molecular size of purified OLI was found to be identical to authentic ouabain, based on analysis by LC/ MS. CONCLUSION: Mammalian cells originating from a rat pheochromocytoma cell line were found to produce and/or secrete OLF by the addition of progesterone.

Effects of intracerebroventricular administration of 6-hydroxydopamine on ouabain-like immunoreactivity in plasma and the hypothalamo-pituitary axis in rats.

OBJECTIVE: To examine the role of central mechanisms on the production and release of an ouabain-like factor, the effects of intracerebroventricular injections of 6-hydroxydopamine on the tissue content and on the plasma level of the ouabain-like factor were determined in rats. METHODS: The vehicle (0.1% ascorbic acid in 0.9% saline) and 6- hydroxydopamine (250 micrograms/rat) were injected into the left lateral ventricle in ether-anaesthetized Wistar rats. Hypothalamus, pituitary, adrenal and venous blood was sampled 24h and 7 days later. The procedure was repeated using another rat group 7 days later. Characteristics of immunoreactive ouabain-like factor were determined by a combination of high-performance liquid chromatography and a highly sensitive enzyme-linked immunosorbent assay for ouabain. The level of the ouabain-like factor in these tissues and in plasma extracts measured by the enzyme-linked immunosorbent assay was compared between the two groups receiving 6-hydroxydopamine and the vehicle. RESULTS: Twenty-four hours after the intracerebroventricular injections of 6-hydroxydopamine, the ouabain-like factor level in the pituitary, hypothalamus and plasma had decreased significantly, whereas the ouabain-like factor level in the adrenal had not changed. The content of noradrenaline in the hypothalamus was also decreased markedly 7 days later and the content of ouabain-like factor in the pituitary remained low. On liquid chromatography the elution pattern of the ouabain-like factor in plasma and in tissue extracts coincided with that of authentic ouabain. CONCLUSIONS: Intracerebroventricular treatments with 6-hydroxydopamine elicited decreases in ouabain-like factor contents in the pituitary, the hypothalamus and the plasma. These results suggest that the production and release of ouabain-like factor are closely associated with the brain, particularly the hypothalamus-pituitary axis, and that noradrenergic or dopaminergic neurons, or both, play a key role in this mechanism.

A brain Na+, K+-ATPase inhibitor (endobain E) enhances norepinephrine release in rat hypothalamus.

We have shown that synaptosomal membrane Na+, K+-ATPase activity is stimulated or inhibited by norepinephrine according to the presence or absence of a brain soluble fraction. Gel filtration of such soluble fraction has allowed the separation of two fractions, peaks I and II, able to stimulate and inhibit Na+, K+-ATPase activity, respectively. Peak II behaves much like ouabain, which has suggested the term endobain. From peak II, a subfraction termed II-E (endobain E), which highly inhibits Na+, K+-ATPase, has been separated by anionic exchange chromatography in a Synchropack AX-300 column. We determined the in vitro effect of endobain E obtained from rat cerebral cortex on neuronal norepinephrine release by incubating rat hypothalamic tissue in the presence of [3H]norepinephrine. Neuronal norepinephrine release was quantified as the factor above basal [3H]norepinephrine released to the medium at experimental and three post-experimental periods. Endobain E was found to increase norepinephrine release in a concentration-dependent fashion, reaching 200%, equivalent to the effect achieved with 400 microM ouabain. Ouabain effect persisted along three post-experimental periods whereas that of endobain E remained only during the first post-experimental period. These results led us to conclude that endobain increases norepinephrine release in hypothalamic neurons at the presynaptic nerve ending level, an effect resembling that of ouabain. It is postulated that endobain E may enhance catecholamine availability in the synaptic gap, leading to an increase in noradrenergic activity.

Relation of left ventricular function and Na,K-pump concentration in suspected idiopathic dilated cardiomyopathy.

The possible relation between Na-K-pump concentration and left ventricular (LV) function was studied in 24 patients with suspected idiopathic dilated cardiomyopathy. This was done by measurement of 3H-ouabain binding to biopsies obtained during left-sided heart catheterization. In all patients light microscopy of biopsies was compatilel with dilated cardiomyopathy. Nineteen patients had impaired LV function as defined by NYHA/WHO and a Na,K-pump concentration of 331 +/- 19 pmol/g wet weight, whereas 5 patients had normal LV function and a Na,K-pump concentration of 559 +/- 62 pmol/g wet weight (p less than 0.001). The correlation between Na,K-pump concentration and ejection fraction was highly significant n = 24, r = 0.81, p less than 0.001). There was no correlation between volume fraction of collagen tissue and Na,K-pump concentration in the biopsies (n = 24, r = -0.08, p less than 0.80), indicating that the decrease in Na,K-pump concentration with dilated cardiomyopathy is not the simple outcome of increased fibrosis in the myocardium. The results indicate that the decrease in Na,K-pump concentration may be of importance for myocardial dysfunction and suggest a simple biochemical assessment of dilated cardiomyopathy by measurement of 3H-ouabain binding.

The uptake of cardiac glycosides by intestinal smooth muscle of the guinea-pig in relation to digitalis receptors.

1. The accumulation and release of (3)H-digitoxin, (3)H-digoxin and (3)H-ouabain by isolated guinea-pig intestinal smooth muscle has been studied and compared with a pharmacological action due to inhibition of the sodium pump.2. The uptake of labelled cardiac glycosides can be described by means of an exponential function. The t of uptake was similar for the three compounds and did not depend on the concentration.3. Analysis of the curve relating the uptake of cardiac glycosides at equilibrium to the bath concentration enabled a non-saturable and a saturable binding site to be distinguished.4. In contrast to the uptake observations, the onset of the pharmacological effect was dependent on the concentration, and furthermore the t((1/2)) for this effect was shorter.5. The release of cardiac glycosides proceeded more slowly than the uptake.6. The uptake of a labelled glycoside was reduced in the presence of another glycoside. The amount of displaceable glycoside was nearly equivalent to the capacity of the saturable binding site.7. The significance of these results is discussed.

Cat assay for the emetic action of digitalis and related glycosides (digitoxin, digoxin, lanatoside C, ouabain and calactin).

1. A titration assay with two end points is described for comparison of the emetic and lethal potencies of digitalis-like drugs.2. A drug was infused at constant rate to a conscious, unrestrained cat, through an indwelling venous cannula. At the moment of vomiting the cat was rapidly anaesthetized and infusion continued at the same rate until the moment of cardiac arrest.3. With very slow and very fast infusions, the emetic and lethal doses tended to rise. In the range between these extremes (which varied from drug to drug) they were independent of time.4. The observations could be accounted for by analogue computation, assuming that the drugs entered an initial pool and were distributed at finite rates to receptors in the CNS (vomiting centre) and heart.5. Half times of metabolic loss derived from this computation for digitoxin, digoxin and ouabain (17, 9.9 and 1.8 h, respectively) were in the same ratio as the threefold longer half times reported for these drugs in man.6. When measured with infusion rates in the time independent range, the ratio of lethal to emetic doses did not vary between the drugs studied. All caused vomiting at 40% of the lethal dose.7. From a review of the literature, the emetic and cardiotoxic actions of digitalis-like drugs appear inseparable and probably share a common biochemical mechanism.8. It is concluded that foreseeable improvements in digitalis-like drugs are small and would depend on the elimination of any local emetic effect on gut receptors which they may have.

Plasma concentration, uptake by liver, and biliary excretion of tritiated cardiac glycosides in the isolated perfused guinea-pig liver.

1. Investigations were carried out on isolated perfused guinea-pig livers. Different doses of tritiated ouabain, digoxin, and digitoxin were added to the perfusion medium and the subsequent plasma elimination, hepatic uptake, and biliary excretion quantitatively measured. After the perfusion, extracts of liver, bile and plasma were subjected to thin layer chromatography in order to detect the radioactively labelled glycosides and their metabolites.2. The ouabain concentration in the plasma approached the equilibrium stage within 45 minutes. At this time 40% of the administered dose had been taken up by the liver, and no further elimination occurred. The elimination curve for ouabain followed a simple exponential function. After 1 h the tissue medium (T/M) ratio was approximately 3. In bile hardly any radioactivity could be detected. Ouabain was therefore not excreted by the liver.3. Up to 80% of the digitoxin was eliminated from the plasma within 4 hours. The elimination of radioactive material for the dose range studied could be described by a hyperbolic function. The T/M ratio in the liver varied with time. At the beginning it was as high as 10 and after 4 h reduced to approximately 3. After 45-60 min the concentration of radioactive material in the bile was 500 times as high as that in the plasma. Almost 70% of the administered radioactivity was excreted with the bile within 4 hours. At the end of the perfusion almost all the identifiable substances in plasma and bile were polar metabolites, as shown by thin layer radiochromatography.4. Digoxin behaved similarly to digitoxin.5. The findings led to the following hypothesis: uptake of cardiac glycosides into the liver cells occurs by a passive diffusion process and is related to their lipid solubility. On the other hand excretion in the bile occurs in general if polar metabolites are formed in the liver cells.

Is the circulating ouabain-like compound ouabain?

The evidence is very strong for a circulating inhibitor of the sodium, potassium ATPase in volume-expanded hypertension. Recently, this inhibitor was isolated from human plasma and identified as ouabain. We are reporting our results using a very specific and sensitive immunoassay for ouabain with which we were unable to detect or able to detect only very low levels of circulating immunoreactive ouabain. Immunoassay of 5 mL of human and rat plasma, incubation fluid from bovine and human adrenal cell cultures extracted using a C-18 solid phase column, and HPLC separation did not detect a peak corresponding to ouabain. This procedure could easily detect authentic ouabain added to these extracts at a concentration slightly below that reported to be present by others. The extract from the adrenal cultures had clearly detectable sodium, potassium ATPase using an assay based on inhibition of tritiated ouabain binding to human red cells. Extraction of bovine adrenals detected a very small amount of immunoassayable ouabain which did not elute at a time corresponding to that of ouabain. This study indicates that the postulated sodium, potassium ATPase inhibitor that circulates in plasma is not ouabain, but it is likely to be structurally similar to ouabain, as it appears to cross-react with some antibodies against ouabain.

Endogenous ouabain secretion in man is not regulated by ACTH.

It has been suggested that endogenous ouabain-like substance (OLS) is of adrenal origin and the secretion of OLS may be ACTH dependent. To determine if OLS is influenced by the pituitary-adrenal axis, we studied the effect of adrenal stimulation (0.25 mg Synacthen) and suppression (1 mg Dexamethasone) on two separate groups of nine subjects. Serum OLS was measured by a radioimmunoassay (RIA) developed in our lab, and cortisol and ACTH were measured by commercial assay kits. Dexamethasone significantly (P< 0.001) suppressed serum cortisol and ACTH concentrations, without effecting endogenous OLS concentration (0.64+/-0.17 vs 0.85+/-0.18nmol/l). Synacthen increased the concentration of cortisol in serum (p < 0.001) over the test period; OLS concentration, again, remained unchanged (0.45+/-0.04 vs 0.43+/-0.05 nmol/l). In further studies, serum concentrations of cortisol and OLS were compared between left (LAV) and right (RAV) adrenal veins with that from the inferior vena cava (IVC). Concentration of cortisol in the LAV and RAV was five-fold greater than that in IVC. However, there was no difference in OLS concentration at the corresponding sites. In addition, serum OLS concentrations in patients having undergone bilateral adrenalectomy or diagnosed with Addison's disease (0.62+/-0.19 nmol/l) were similar to concentrations in healthy subjects (0.67+/-0.21 nmol/l). Examination of bovine adrenal, liver, kidney, heart and human placenta demonstrated that OLS content of bovine adrenal was comparable with other tissues analysed. HPLC studies of human serum and bovine adrenal gland produced identical elution profiles, resolving a single peak which coincided with the retention time observed for standard ouabain. We conclude that the adrenal is unlikely to be the source of endogenous OLS, the secretion of which appears to be independent of ACTH.

Liquid chromatography mass spectrometric analysis of ouabainlike factor in biological fluid.

Ouabainlike factor (OLF), assayed as ouabainlike immunoreactivity (OLI), is a probable endogenous digitalislike factor (EDLF). Liquid chromatography/mass spectrometry (LC/MS) is one of the most highly sensitive tools for obtaining structural information regarding low-molecular weight materials in a target compound, and to measure the concentrations of these materials. We have previously reported that OLI can be isolated from the culture supernatant of the rat pheochromocytoma cell line, PC12, by several reverse-phase chromatography and LC/MS techniques. The present study was performed to characterize OLF from biological fluids such as plasma and culture supernatant of PC12 cells by LC/MS. The previous applications of LC/MS to OLI in plasma have been limited to structural identification at the final stages of isolation, in which the starting volume of plasma has been over 10 I. In the present study, we tried to minimize the volume of plasma, and to develop a new preclearing step to gain adequate LC/MS characterization using MS/MS analysis. The plasma was acidified, and OLI was purified by ODS column chromatography. OLI in chromatographic fractions from plasma was assayed by a sensitive enzyme-linked immunosorbent assay for ouabain. After Sep-Pak treatment and two rounds of ODS column chromatography, OLI was identified from 80 ml of plasma. The structure of the purified OLI was identical to authentic ouabain and digoxin, as assessed by LC/MS. In conclusion, we identified the chemically or structurally clarified ouabain and digoxin as the circulating form in plasma by LC/MS.

Centrally mediated enhancement of ouabain cardiotoxicity by angiotensin II in dogs.

The effect of angiotensin II (Ang II) infusion on the doses of ouabain required to induce ventricular arrhythmias and death was investigated in anesthetized dogs. Tritiated ouabain was infused alone i.v. (1 microgram/kg per min) to establish the control toxic doses and the level of ouabain in the heart at death. When Ang II and ouabain were co-infused i.v. the doses of ouabain needed to induce arrhythmias and death were significantly reduced. Spinal transections performed at C-1 prior to drug infusions prevented the effect of Ang II to enhance the toxicity of ouabain. Thus, the action of Ang II to augment ouabain toxicity appeared to be related to its effects on the sympathetic nervous system. To confirm that Ang II was acting within the brain rather than at peripheral sites of the sympathetic system, Ang II was infused into the vertebral artery of spinal cord intact dogs. The infusion rate of Ang II needed to produce a significant enhancement of ouabain toxicity was much lower when given into the vertebral artery than into the femoral vein. These data indicate that Ang II enhances the cardiotoxicity of ouabain via an action produced within the brain and mediated by the sympathetic nervous system.