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Cardiac glycosides inhibit the sodium pump. However, some studies suggest that nanomolar ouabain concentrations can stimulate the activity of the sodium pump. In this study, using the Na(+)/K(+)-ATPase of human erythrocytes, we compared the effect of digoxin, ouabain and an ouabain like-factor (OLF), on (86)Rb uptake. Ouabain concentrations below 10(-9) M significantly stimulate Rb(+) uptake, and the maximal increase above base-line values is 18 +/- 5% at 10(-10) M ouabain. No stimulation is observed in the same conditions by digoxin. OLF behaved like ouabain, producing an activation of Rb(+) flux at concentrations lower than 10(-9) M ouabain equivalents (14 +/- 3% at 10(-10) M). Western blot analysis revealed the presence of both alpha(1) and alpha(3) pump isoforms in human erythrocytes. Our data confirm the analogies between OLF and ouabain and suggest that Na(+)/K(+)-ATPase activation may be related to the alpha(3) isoform. In addition, we investigated whether ouabain at different concentrations was effective in altering the intracellular calcium concentration of erythrocytes. We found that ouabain at concentration lower than 10(-9) M did not affect this homeostasis., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Background: Endogenous ouabain (EO), which is structurally identical to the cardiac glycoside ouabain, has been isolated in human plasma. The substance EO has been implicated in states of volume expansion and in some types of arterial hypertension, especially as a factor of salt sensitivity of blood pressure. On the other hand, salt sensitivity has been described in low-renin hypertension. The aim of this study was to determine the response of plasma EO to acute sodium expansion in low-renin hypertension., Methods: We performed an acute intravenous sodium load (2 L of saline in 4 h) in 13 hypertensive subjects with low renin values (<0.65 ng/mL/h). We measured EO in plasma extracts by a radioimmunoassay and compared it with other endocrine parameters including atrial natriuretic peptide (ANP), aldosterone (ALDO), cortisol (CORT), adrenocorticotropic hormone (ACTH), and plasma renin activity (PRA)., Results: We found that EO showed only a mild nonsignificant decrease after saline infusion (from 938.8+/-218.8 pmol/L to 781.4+/-181.4 pmol/L ouabain equivalents), whereas ALDO and PRA significantly decreased (P<.0001; P<.05 respectively). The ANP significantly increased, as a marker of effective volume expansion (P<.01). At the end of the infusion, we found that EO was positively related to ACTH levels and to ALDO changes from baseline., Conclusions: Our results do not support the hypothesis that EO is stimulated in low-renin hypertension by acute salt-volume expansion. ACTH could be a factor modulating EO secretion in this condition.

Endogenous ouabain-like factor (OLF) has been detected in mammalian plasma, adrenal gland, and hypothalamus. We investigate whether cardiac tissue may also produce OLF. HPLC chromatographic separation of cardiac extracts showed that RIA-determined OLF activity coincided with the elution profile of exogenous ouabain and with the ability to inhibit 86Rb uptake in human erythrocytes. OLF activity was remarkably higher in excised hearts (3.94 +/- 0.84 pmol/g wet weight by RIA) than in rat blood (0.05 +/- 0.02 pmol/ml). Similar values were obtained in perfused working hearts, without significant changes over time from 5 to 30 minutes of aerobic perfusion. Significant OLF release in the perfusion buffer was also observed (0.54 +/- 0.05 pmoles over 30 minutes). In hearts subjected to 15 minutes of aerobic perfusion followed by 15 minutes of global myocardial ischemia OLF concentration was remarkably increased (8.59 +/- 1.13 versus 4.58 +/- 0.57 pmol/g wet weight by RIA, P < 0.01; an increase after ischemia was confirmed by the assay of 86Rb uptake). Our findings suggest that the rat heart is able to produce OLF, and that its concentration increases during ischemia. Myocardial OLF might modulate the Na/K-ATPase, producing relevant effects on ionic homeostasis and/or gene transcription.

It has been reported that canrenone, which is used in hypertensive therapy as an antialdosteronic drug, may also act as a blocker of ouabain effects. Several studies suggest that human plasma contains an endogenous ouabain-like factor similar to ouabain, which may be increased in hypertension, in pregnancy, and in the neonatal state. This study evaluated (1) the effect of canrenone on Na+/K(+)-ATPase in relation to ouabain in human placental membranes and erythrocytes by 3H-ouabain binding assay; (2) the capacity of canrenone (10 microM) to reverse the inhibition of Na+/K(+)-ATPase by ouabain and by ouabain-like factor (from umbilical cord plasma) in human erythrocytes employing a 86Rb uptake assay. Increasing concentrations of canrenone (0-350 microM) partially competed with 3H-ouabain binding in placental membrane (40%) and erythrocytes (60%). Scatchard plot from radioreceptor assay in placental membrane showed that ouabain and canrenone compete for the same binding site. In erythrocytes, canrenone completely reversed the inhibition caused by ouabain (5 x 10(-9) M) and ouabain-like factor (2 x 10(-9) M ouabain equivalents). A reduction of inhibition of about 50% was observed with ouabain and ouabain-like factor respectively at a concentration of 5 x 10(-8) M and 2 x 10(-8) M (ouabain equivalents). Our results thus provide evidence that canrenone, at therapeutical concentrations, is a partial competitive agonist of ouabain and of ouabain-like factor in human placental membranes and erythrocytes.

Background: Much evidence has been accumulated that human plasma contains digitalis-like factor(s) with Na/K ATPase inhibitor properties. Increased concentrations of ouabain-like factor (OLF) have been reported in patients with moderate to severe hypertension and in patients with overt congestive heart failure due to dilated cardiomyopathy., Aim: The presence of circulating OLF has not been investigated in borderline to mild hypertension or in the early stage of dilated cardiomyopathy., Methods and Results: The study population consisted of 18 normal volunteers, 24 patients with borderline to mild hypertension, 47 patients with asymptomatic left ventricular dysfunction (ALVD) due to dilated cardiomyopathy and 26 patients with cardiac arrhythmias but normal left ventricular function. OLF values (pM ouabain equivalent) were assayed in extracted plasma, using a radioimmunoassay for ouabain. OLF was, respectively, 29.4+/-20.6 pM in normal controls, 39.1+/-23.8 pM in hypertensives, 35+/-18 pM in patients with cardiac arrhythmias, 52.3+/-25.8 pM in ALVD patients not treated with digoxin and 64.6+/-29.6 pM in ALVD patients treated with digoxin. Patients with ALVD, both treated and not treated with digoxin, had OLF significantly higher (P<0.05) than all the other groups. In patients with ALVD no correlation between OLF and left ventricular ejection fraction was observed. In the hypertensive group no correlation between OLF and both diastolic and systolic pressure was found., Conclusion: Increased concentrations of OLF were observed in patients with left ventricular dysfunction due to dilated cardiomyopathy, before the occurrence of overt heart failure, suggesting that OLF may be an early marker of the disease.

Na+/K+ATPase is a transport membrane protein which contains the functional receptor for digitalis compounds. In this work we compare the inhibition curves of Na+/K+ATPase measured by the inhibition of 86Rb uptake in human red blood cells by cardiac glycosides and by an endogenous digitalis like factor (EDLF) extracted from human newborn cord blood. The curves of Na+/K+TPase inhibition show a monophasic shape for ouabain, strophantidin, digitoxin, proscillaridin and EDLF whereas a biphasic shape for ouabagenin, digoxin, digoxigenin and digitoxigenin. All the drugs are potent inhibitors of erythrocyte Na+/K+ATPase with an IC50 ranging from 1.8 x 10(-9) M to 1.4 x 10(-11) M for the higher affinity binding site and from 1.8 x 10(-6) M to 5.5 x 10(-9) M for the lower affinity site. Digitoxigenin is the most active showing the higher active site at 1.4 x 10(-11) M. Ouabain and digoxin have higher affinity compared with their corresponding genins, while digitoxigenin shows a binding site with higher affinity than the respective cardiac glycosides. The increased affinity of the drugs to Na+/K+ATPase may be related to a lipophilic region in correspondence of the carbons 10, 9, 11, 12, 13 of the steroid nucleus, situated in the opposite side with respect of the C-OH-14. The comparison of the inhibition curves and the HPLC profile of newborn EDLF and of the investigated cardenolides suggest that EDLF may be a compound identical or very similar to ouabain.

An automated surface plasmon resonance-based biosensor system has been used to detect endogenous and exogenous digitalis-like factors (EDLF) in the pmolar range in real time. EDLF was purified from umbilical cord blood. EDLF has been suggested to play a role in hypertension and in perinatal adaptation. Highly specific polyclonal anti-ouabain antibodies showed a high affinity binding capacity for ouabain, ouabagenin and strophantidin with an IC50 value of 5 x 10(-10) M, 7.0 x 10(-10) M and 2 x 10(-8) M, respectively. EDLF cross-reacted with antibodies and its concentration in plasma at IC50 was around 50 pmol ouabain equivalent. This study shows the potential usefulness of the biosensor technology for biomolecular interaction analysis. The features of this technology (fully automated, measured in real time, sharpened response) offer several advantages compared with a traditional immunoassay like radioimmunoassay (RIA) in the detection of digitalis compounds in human fluids.

An unique endogenous digitalis-like factor (EDLF) has been previously purified from human newborn cord plasma and its differential effects tested on the three well defined functional isoforms (alpha1, alpha2 and alpha3) of the alpha subunits of Na+/K+-ATPase in rat. EDLF specifically inhibits the enzymatic activity. It differs from ouabain by three criteria: a preincubation with the membranes is required for full activity, no effect on the rat cerebral alpha3 isoform and a steep dose-response curve with the same apparent potency for rat alpha2 and alpha1 isoforms of high (10(-7) M) and low affinity (3 x 10(-5) M) for ouabain. These results indicate that the Na+/K+-ATPase inhibitor involved in the regulation of sodium and body fluid volume and present in neonate and adult human plasmas is distinct from ouabain.

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.

It is widely but not unanimously accepted that one or more endogenous digitalis-like factors (EDLF) circulate in human plasma. In this paper we provide confirmatory evidence that newborn plasma contains a factor with immunological and biological properties similar to ouabain and demonstrate that this factor may be present also in the adult. In fact, we obtained in newborn and adult plasma extracts, identical HPLC elution profiles of ouabain-like immunoreactivity and 86Rb erythrocyte uptake inhibitory activity with a major peak corresponding to the retention time of ouabain. The fact that immunoreactivity and biological digitalis-like activity in the peak are due to an identical substance is strongly supported by the correlation between RIA and 86Rb uptake inhibitory values observed in the purified fractions. Finally, the strong correlation between immunoreactivity observed in plasma samples after simple SepPak C18 extraction and after additional HPLC suggests that less purified samples may be assayed for screening purposes. However, for a more quantitative assessment, this simple extraction method needs a subsequent HPLC purification for eliminating an overestimation of values due to cross-reacting impurities.

It is well recognized that one of the difficulties in the search for endogenous ligand(s) with digitalis-like properties (endogenous digitalis-like factor(s), EDLF) in mammals has been the lack of a unique, specific method for the accurate measurement of EDLF. Using C18 solid-phase extracts of plasma from normal adults and various patient groups, and purified extracts from umbilical cord plasma by affinity resin chromatography and HPLC, different methods to measure EDLF were evaluated. These were: (a) a human placenta radioreceptor assay (RRA) developed on the premise that competition for cardiac glycoside receptors was an absolute requirement for EDLF; (b) the inhibition of 86Rb uptake in human erythrocytes to estimate the potassium transport by the sodium pump; (c) an enzyme immunoassay specific for ouabain recently introduced in the market (DuPont Ouabain EIA Reagent Pack). The human placenta RRA was found to have the same ease of application as immunoassay, but could have major advantages in detecting active molecules, being "biologically more meaningful". Ouabain immunoreactivity correlated with EDLF values obtained by RRA, but in some instances the two assays were completely unrelated. Moreover, the high specificity of the DuPont antibody for ouabain (< 3% cross reactivity with digoxin) could be disadvantageous to detect EDLF not strictly resembling ouabain. The 86Rb uptake inhibition method correlated with RRA for EDLF purified by HPLC. It tested the complete enzymatic cycle and could therefore better reflect the in-vivo inhibitory activity of EDLF. However, it appeared not suitable for the routine EDLF evaluation in clinical studies since it was susceptible to sample osmolarity and required daily isolation of human erythrocytes possibly from the same donor. Results of the present study demonstrate that every assay has its limitations, and would suggest the use of multiple assays for EDLF detection.

Several lines of evidence suggest the existence, in mammalian body fluids and tissue extracts, of an as yet unidentified endogenous digitalis-like factor with potential cross-immunoreactivity with digitalis. Using anti-digoxin antibodies, we found preliminary immunohistochemical evidence of digoxin-like immunoreactivity in several human tissues. Strong reactivity was found in the adrenal cortex, which may thus represent a site of production of this endogenous factor.

Elevated levels of endogenous digoxin-like immunoreactivity have been reported in the body fluids of premature and full-term infants as well as in term pregnancy, in the amniotic fluid, and in human milk. Several lines of evidence suggest that these factors could also have biological properties in common with digitalis: i.e., they could represent truly endogenous digitalis-like factor(s). In recent years we succeeded in partially purifying this factor from umbilical cord blood, which represents an easily available source of this factor. The inhibitory activity of this factor on 86Rb uptake could be neutralized by antidigoxin antibodies (Fab fragments) and provided, for the first time, direct evidence of an association between digoxin-like immunoreactivity and biological digitalis-like activity. In addition, these antibodies could be used for immunoaffine chromatography as a purification step before separation by high-performance liquid chromatography. Preliminary experiments suggest that this endogenous compound has both a tissue and an isoenzyme selectivity and is not a well-known steroid (testosterone, progesterone, 17-OH progesterone, cortisol, dehydroepiandrosterone sulfate, and estradiol).

We have previously shown that antidigoxin antibodies may neutralize partially purified endogenous digitalis like factor(s) present in newborn (umbilical cord) plasma. We here report on the preparation of an immunoaffinity chromatographic system (high affinity digoxin-binding antibodies (Fab fragments) bound covalently to Sepharose) for the purification of endogenous digitalis like factor(s). Neonate plasma extract loses all its biological digitalis-like activity (erythrocyte 86Rb uptake inhibition) after absorption on Sepharose coupled to Fab fragments but not after absorption on uncoupled Sepharose. Endogenous digitalis like factor(s) absorbed to Sepharose coupled to Fab fragments can be eluted by methanol. Subsequent HPLC separation indicate that at least two molecular species with digitalis-like properties are retained by antibodies bound to Sepharose and can be recovered with methanol.

Increasing evidence indicates the presence of endogenous digitalis-like compound(s) in human body fluids. In this preliminary report, we describe a study of the partial purification by HPLC of these compounds in the plasma of neonates (who have particularly high concentrations of this substance) and adults. Plasma samples from neonates (cord blood) and adults, lyophilized and extracted with methanol, were applied on a 300 x 3.9 mm C18 Nova Pak column and eluted with a mobile phase of acetonitrile/methanol/water (17/17/66 or 14/14/72 by vol) and, after 30 min, with 100% methanol. We assayed eluted fractions for inhibitory activity of 86Rb uptake and for digoxin-like immunoreactivity. The elution profile revealed a first peak of inhibitory activity of 86Rb uptake at the beginning of the chromatography; another peak was eluted with the 100% methanol. The two peaks also cross-reacted with antidigoxin antibodies. Because the second peak could possibly reflect the nonspecific interference of various lipophilic compounds, we focused our attention on the first peak. For these fractions dose-response curves for 86Rb uptake and for displacement of digoxin were parallel, respectively, to those of ouabain and digoxin, suggesting similarities of digoxin-like immunoreactive substance to cardiac glycosides. Similar chromatographic profiles were also obtained for plasma from adults, suggesting that the endogenous glycoside-like compound(s) in the neonate may be the same as those in the adult.

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

Endogenous factors with biological and immunological activity similar to cardiac glycoside drugs (endogenous digitalis-like factors; EDLF) have been found in several tissues and body fluids of animals and humans. Detectable EDLF concentrations were found in blood and urine extracts of adults (normal healthy controls, hypertensive patients and salt-loaded healthy subjects), while higher levels were generally observed in plasma samples of pregnant women, newborns, and patients with renal insufficiency. The chemical characteristics of this endogenous factor are, at present, unknown, although it has been suggested that EDLF could be a substance with low molecular weight. Experimental studies and theoretical considerations suggest that EDLF, in addition to the ability to react with antibodies, might also bind to the specific cellular receptor of the cardiac glycosides and thus inhibit the membrane Na+/K(+)-ATPase (sodium pump). Therefore, it has been suggested that EDLF is an endogenous modulator of the membrane sodium-potassium pump, and that it could play a role in the regulation of fluids and electrolytes, in the myocardial muscular tone and also in the pathogenesis of hypertension.

The existence of endogenous cardiac glycoside-like compounds and their property of being recognized by anti-digoxin antibodies is still a matter of controversy. In order to investigate this problem, endogenous digoxin-like immunoreactivity (measured by RIA) and digitalis-like radioreceptor activity (measured by displacement of 3H-ouabain from erythrocyte membranes) were assessed in plasma extracts of normal adults, pregnant women and newborns. These three groups were chosen because of their known widely different levels of digoxin-like immunoreactivity. Compared to adults, newborns and pregnant women had significantly higher levels not only of immunoreactivity but also of displacement of 3H-ouabain binding, the latter being due, according to Scatchard analysis, to a decrease of the affinity of ouabain to its cellular receptor rather than to its maximal binding capacity. Furthermore, immunoreactivity and binding displacement correlated significantly. Our data indicate that one (or more) compounds with cardiac glycoside-like properties (both immunological and at the receptor level) are present in the plasma of newborns and pregnant women, and confirm the idea that radioimmunological methods may be useful in studying endogenous inhibitors of the sodium pump.

1. To investigate endogenous cardiac glycoside-like compounds in plasma and their ability to inhibit the sodium pump, digoxin-like immunoreactivity [digoxin-like immunoreactive substance(s), DLIS] and 86Rb uptake by erythrocytes were measured in plasma extracts from normal adults, hypertensive adults and neonates. 2. DLIS levels in neonate plasma extracts were significantly higher than those found for normotensive or hypertensive adults. No difference was observed between normotensive and hypertensive subjects. DLIS was significantly increased when boiled plasma was extracted. 3. Extracts of boiled neonate and adult plasma inhibited 86Rb uptake. Instead, when boiling was omitted, no detectable inhibition was found in extracts of plasma from normotensive or hypertensive adult subjects. When present, the inhibition resulted from a depression of the ouabain-sensitive (sodium-pump-mediated) component, and, for the boiled neonate plasma only, also of the ouabain-resistant component. When the data from the different groups were pooled, a statistically significant inverse relationship between DLIS and erythrocyte 86Rb uptake was observed. Furthermore, in a subgroup of samples in which determinations were made before and after boiling in the same samples, an inverse correlation was found between changes in DLIS and changes in ouabain-sensitive (but not ouabain-resistant) 86Rb uptake. 4. Plasma extracts incubated with albumin at a physiological concentration significantly decreased (by approximately 20%) the inhibition of 86Rb uptake observed. 5. These findings support the existence of one or more endogenous compounds which both bind to antidigoxin antibodies and inhibit transmembrane cation transport. Part of this inhibition may, however, not involve the sodium pump. Furthermore, this chemically unidentified substance(s) may be bound to plasma proteins which partly reduce its action in vivo.

The presence of an endogenous digoxin-like immunoreactive substance(s) (DLIS) has been reported in the plasma and urine of experimental animals and humans. This substance might have a role in arterial hypertension. Although the chemical structure of DLIS is at present unknown, several studies indicate that DLIS has a low molecular weight and is reversibly bound in serum to carrier proteins. We have investigated the carrier protein of DLIS in chromatographic studies, using neonate plasma eluted on a Sephadex G200 column. The chromatographic profile of digoxin-like immunoreactivity showed a major peak with a molecular weight of approximately 60,000 daltons and a smaller peak eluted after the salt region. When the major peak was chromatographed on a Sephadex G100 column only one single peak was obtained, which closely coincided with the elution peak of albumin. Chromatographic separation of neonate plasma on a Sephadex G25 revealed a major post-salt immunoreactive peak. When these fractions were incubated with purified human albumin and separated again on the Sephadex G25, a large immunoreactive peak corresponding to albumin was again found while the immunoreactivity after salt completely disappeared. Our data therefore strongly suggest that the endogenous digitalis-like factor is carried in plasma by albumin.

Evidence has been provided on the increased presence, in essential hypertension, of endogenous digitalis-like factor(s) [DLIS, digoxin-like immunoreactive substance(s)] able to cross-react with antidigoxin antibodies and to inhibit the membrane-bound sodium-potassium pump. An inhibition of the sodium pump could lead, in smooth muscle cells, to an increase of intracellular calcium ions and to an increase of total peripheral resistances. In this study the relation between plasma levels of DLIS and the acute hypotensive effect of a calcium antagonist (nifedipine) has been evaluated in a group of borderline to severe hypertensive patients and in a control group of normotensive subjects. The results obtained confirm that the hypotensive effect of nifedipine is related to pretreatment blood pressure and show, only in hypertensive patients, a significant relation of DLIS with both pretreatment blood pressure and blood pressure decrement induced by nifedipine. These findings are compatible with a possible role of DLIS in modulating cellular calcium handling.

In an attempt to confirm the presence of endogenous substances with cardiac glycoside-like activity, the biological and immunological cardiac glycoside-like activity was measured by a sensitive solid-phase radioimmunological assay (RIA), two radioreceptor assays (RRA), and a 86Rb uptake method in normal subjects and in some pathophysiological conditions characterized by sodium retention and volume expansion. Significant concentrations of digoxin-like immunoreactive substances (DLIS) were measured in plasma (or serum) of normal subjects while significantly higher levels were found in pregnant women, newborns and in patients with renal impairment, and in some with essential hypertension. Concentrations in urine of normal adults or newborns were several times higher than in plasma. The results obtained by RIA correlated with those obtained by RRA and 86Rb uptake methods. In 88 normal subjects, DLIS excretion rates (overnight urine collection) in men were significantly higher than in women (68.6 +/- 23.6 pg/min vs 50.9 +/- 21.0 pg/min, p less than 0.01). The DLIS excretion rates correlated with creatinine, Na and K urinary excretion rates, and also with the subjects' body weight, height, body mass index, and systolic blood pressure. These findings confirm the presence of endogenous substances with immunological and biological activity similar to cardiac glycosides in human body fluids and also confirm the hypothesis that these endogenous factors may be involved in fluid and electrolyte regulation in man. In addition, the present data indicate that urinary excretion of DLIS is dependent on body mass and renal glomerular filtration.