Academic literature on the topic 'Endogenous digitalis-like factor (EDLF)'

Abstract We measured the concentration of endogenous digitalis-like factors (EDLFs) in milk or colostrum of women during nursing on different days after delivery. EDLF concentrations were assayed by a solid-phase RIA involving antidigoxin antibodies and by a radioreceptor assay (RRA) involving human placenta Na+/K(+)-ATPase. The mean (SD) EDLF concentrations as measured by RIA were 35.6 (19.4) ng of digoxin equivalents per liter in milk samples (n = 37) and 61.3 (12.5) ng/L in colostrum samples (n = 5); the mean EDLF concentration as measured by RRA in milk samples (n = 11) was 573 (717) ng/L (range 0-2098). EDLF concentration in milk is greater than circulating concentrations in healthy adults but is comparable with serum concentration in the third trimester of pregnancy. In milk and serum samples (n = 8) collected at the same time, heating and (or) extracting with Sep-Pak C18 cartridges before the RIA produced significantly different EDLF values from those in untreated serum (P less than 0.001) and milk (P = 0.035). EDLF in milk appeared to be not bound or weakly bound to milk protein, as indicated by the fact that boiling did not increase the digoxin-like immunoreactivity.

Abstract We describe the optimization and validation of a clinically feasible radioreceptor assay to detect endogenous digitalis-like factor(s) (EDLF) in human plasma and urine. The assay is based on the competitive replacement of 125I-labeled digoxin on human placenta membranes by ligands present in sample extracts. Digoxin and ouabain were used as calibrators. We also describe simple and effective methods for extraction and enrichment of EDLF from human plasma and urine. Assay sensitivity and precision were enhanced by using a sequential saturation technique with appropriate concentrations of tracer and receptors. Filtration was used to separate bound from free ligand. A two-step solid-state extraction with acetonitrile allowed the separation of two EDLFs with different polarity (EDLF-1 and EDLF-2) from the same plasma sample. A one-step solid-state extraction with methanol was suitable for urine. EDLF-1 and EDLF-2 in healthy adults were respectively 204 +/- 155 and 207 +/- 423 pmol/L ouabain equivalents, or 312 +/- 241 and 302 +/- 581 pmol/L digoxin equivalents. Plasma concentrations of EDLFs in newborns and pregnant women were higher than in healthy adults, and the concentrations in urine were higher than in plasma. Several cross-reactivity experiments showed that physiological concentrations of endogenous steroids and lipids did not inhibit binding, and supported the hypothesis that EDLFs are endogenous compounds other than the steroids and lipids also investigated.

ABSTRACT Plasma levels of an endogenous digitalis-like factor (EDLF) and atrial peptides were followed in pigs confined to metabolic cages during the development of deoxycorticosterone acetate (DOCA)-induced hypertension. During the first 2 days of DOCA treatment, urinary sodium excretion decreased and the plasma levels of renin and atrial peptide fell significantly. During this period, plasma levels of EDLF increased > 30-fold from a baseline of <0·25 to 9·72 nmol ouabain equivalents/l. Between days 2 and 5 of DOCA treatment, urinary sodium returned to preDOCA levels ('mineralocorticoid escape') and during this period significant increases of atrial peptide and mean arterial pressure (MAP) and a decrease in EDLF were found. Following mineralocorticoid escape there was a secondary rise in levels of EDLF and atrial peptide and both phenomena correlated with MAP (EDLF, r = 0·87, P< 0·05; atrial peptide, r = 0·9, P< 0·05) and with each other (r = 0·96, P < 0·05) over a 20-day period. Acute expansion of extracellular fluid volume before DOCA elicited significant increments in plasma EDLF and atrial peptide. Volume loading during chronic DOCA treatment increased plasma EDLF significantly whereas no response of atrial peptide was detected. These results suggest that DOCA affects the reactivity of mechanisms involved in the perception of and/or response to acute changes in volume status. However, neither EDLF nor atrial peptide appear to be viable candidates as direct mediators of mineralocorticoid escape. Finally, the nature of the changes found in EDLF and atrial peptide levels during DOCA treatment suggest that these factors are involved in the long-term control of blood pressure in this model of low renin hypertension. Journal of Endocrinology (1989) 122, 409–420

Endogenous digitalis-like factor(s), originally proposed as a vasoconstrictor natriuretic hormone, was discovered in fetal and neonatal blood accidentally because it cross-reacts with antidigoxin antibodies (ADAs). Early studies using immunoassays with ADA identified the digoxin-like immuno-reactive factor(s) (EDLF) in maternal blood as well, and suggested it originated in the feto–placental unit. Mammalian digoxin-like factors have recently been identified as at least two classes of steroid compounds, plant derived ouabain (O), and several toad derived bufodienolides, most prominent being marinobufagenin (MBG). A synthetic pathway for MBG has been identified in mammalian placental tissue. Elevated maternal and fetal EDLF, O and MBG have been demonstrated in preeclampsia (PE), and inhibition of red cell membrane sodium, potassium ATPase (Na, K ATPase (NKA)) by EDLF is reversed by ADA fragments (ADA-FAB). Accordingly, maternal administration of a commercial ADA-antibody fragment (FAB) was tested in several anecdotal cases of PE, and two, small randomized, prospective, double-blind clinical trials. In the first randomized trial, ADA-FAB was administered post-partum, in the second antepartum. In the post-partum trial, ADA-FAB reduced use of antihypertensive drugs. In the second trial, there was no effect of ADA-FAB on blood pressure, but the fall in maternal creatinine clearance (CrCl) was prevented. In a secondary analysis using the pre-treatment maternal level of circulating Na, K ATPase (NKA) inhibitory activity (NKAI), ADA-FAB reduced the incidence of pulmonary edema and, unexpectedly, that of severe neonatal intraventricular hemorrhage (IVH). The fall in CrCl in patients given placebo was proportional to the circulating level of NKAI. The implications of these findings on the pathophysiology of the clinical manifestations PE are discussed, and a new model of the respective roles of placenta derived anti-angiogenic (AAG) factors (AAGFs) and EDLF is proposed.

SummaryThe existence of endogenously produced, digoxin-like factor(s) is clear. The implications of die presence of this circulating substance are substantial for the practice of emergency medical care. Clearly, EDLF plays an important role in the generation of dysrhythmias associated with an AMI. Treatment with AA could become routine early in the course of management of some patients with AMI and in die treatment of some forms of hypertension

Ouabainlike compound (OLC) has recently been identified as a likely mammalian endogenous digitalis-like factor (EDLF) from human plasma. In this study, plasma levels of OLC were determined to assess the role of OLC in a model known as volume-expanded, reduced renal mass (RRM)-saline (S) hypertension in rats with use of a newly developed radioimmunoassay for ouabain. In the first experiment, at 3 wk after subtotal nephrectomy and drinking 1% saline solution, sysolic blood pressure (SBP) of 18 rats with reduced renal mass (RRM-S rats) was significantly higher than in 17 sham-operated saline-drinking control (C-S) rats [154 +/- 4 (SE) vs. 132 +/- 2 mmHg; P < 0.01]. Plasma OLC levels were 355 +/- 68 pmol/l in RRM-S rats, sevenfold higher than in C-S rats (54 +/- 4 pmol/l; P < 0.01). In the second experiment, we measured plasma OLC levels of 10 RRM-S, 12 sham-operated control (C), and 10 subtotally nephrectomized rats drinking distilled water (RRM rats). Concomitant with a marked increase in blood pressure (203 +/- 5 mmHg), RRM-S rats showed significantly higher plasma OLC levels compared with C and RRM rats (RRM-S 114 +/- 24, C 47 +/- 11, and RRM 52 +/- 9 pmol/l; P < 0.05). In both experiments, plasma OLC levels correlated significantly with SBP (P < 0.05). These findings suggest that plasma OLC shows a similar behavior to that of EDLFs or Na(+)-K(+)-adenosinetriphosphatase inhibitors reported in previous publications and may play a role in hypertensive mechanisms in rats with RRM and excess Na intake.

In order to study the influence of the hypothalamic-pituitary-adrenal axis on the levels of endogenous digitalis-like substances (EDLS) in plasma and urine, eight healthy subjects (25–40 years old) were given dexamethasone 1 mg orally and tetracosactide (an ACTH analog) 0.25 mg iv, on separate occasions. The circulating levels of EDLS, TSH, PRL and AVP following administration of either test drug, and under control conditions, were measured by a RIA for digoxin and specific RIAs for each hormone. Plasma cortisol was measured by liquid chromatography. The area under the curve (AUC) of hormone levels between 08.00 and 09.30 was used for data comparisons. Urine was collected before and after each test dose, and analysed for cortisol levels by gas chromatography/mass spectrometry, and for digitalis-like activity both by RIA and by a bioassay measuring 86Rb-uptake into red blood cells. Dexamethasone suppressed the AUC of plasma and urine levels of cortisol (p=0.0001 and p<0.01, respectively) and immunoreactive EDLS (p=0.0007 and p<0.01), as well as serum levels of TSH (p=0.0002) and PRL (p=0.001), but did not alter AVP levels. The biological digitalis-like activity in the urine measured by the 86Rb-uptake assay was decreased, but not to a statistically significant degree. ACTH increased the levels of cortisol in plasma (p=0.0001) and urine (p<0.01) and the immunoreactive EDLS in plasma (p = 0.03), but not in urine. There were no effects of ACTH on TSH, PRL or AVP. There are alternative explanations for the discrepancy between the effects on EDLS levels in plasma and urine: methodological difficulties in quantitating EDLS, the doses of dexamethasone and ACTH used for the adrenal function tests, and that other hypothalamic or pituitary factors than ACTH may contribute to a significant degree in the regulation of EDLS levels. Taken together, the results of the present study support the hypothesis that EDLS is of adrenal origin, rather than hypothalamic or pituitary.

The sodium pump (SP or Na+,K+-ATPase) is a membrane embedded protein complex that pumps 3 sodium ions out and 2 potassium ions into the cell per cycle and in so doing creates a cell membrane electrochemical potential. The membrane potential is critical for any functional cell. In the vasculature, reduction in the voltage potential causes vascular smooth muscle contraction and a narrowing of blood vessels (vasoconstriction) which can lead to increased blood pressure (hypertension). Substantial research over the past several decades has provided a vast amount of research on SP inhibitors, sometimes called endogenous digitalis-like factors (EDLF). Increased levels of these factors have been implicated in many hypertensive disorders including preeclampsia (PE), a life-threatening complication of pregnancy. It has been demonstrated that EDLF might be a causative factor in the pathophysiology of hypertension in PE. In order to elucidate EDLF production and regulation in PE, We developed a radioimmunoassay (RIA) measuring EDLF that could be applied to serum from pregnant women, placental homogenate and placental tissue culture. This assay employs Digibind, a commercially available Fab fragment derived from polyclonal antidigoxin antibodies that cross reacts with EDLF, as the primary antibody. Using Digibind RIA, we demonstrated that placenta is a source of EDLF production and regulation. Moreover, the identification of an inhibitor, ketoconazole and a substrate, 17-hydroxyprogesterone of the synthetic pathway of EDLF in placenta proved that this pathway shares steps with the steroid synthetic pathway. Some potential regulatory agents which have elevated levels in PE or be associated in PE and thus are thought to mediate PE, such as hydrogen peroxide, tumor necrosis factor-α (TNF-α) and hypoxia have also been demonstrated to be stimuli of EDLF production in placenta. These findings are helpful to the further study on EDLF synthesis and regulation in placenta. Once we elucidate the mechanisms, it could be easier to provide deeper insights into the pathogenesis of PE and subsequently develop earlier diagnosis and effective prevention of or therapeutic approaches to PE.

The mechanisms mediating the hypertension of preeclampsia (PE) are unclear. Endogenous digitalis-like factors (EDLFs) are specific sodium pump (SP) inhibitors implicated in essential and experimental hypertension, but they have not been fully explored in the setting of PE. This study uses a digoxin antibody Fab fragment to address the question of whether such factors are present and increased in PE, to investigate a possible treatment of PE, and to isolate and characterize all EDLFs present in PE. Sera and placenta from women with PE did show a significant increase in SP inhibition in comparison to women with normal pregnancy and Digibind® was found to bind EDLFs and essentially block or reverse SP inhibition. Sera were collected in a Phase II, double-blind, placebo controlled clinical study in which women with severe preeclampsia were dosed with Digibind®, as a therapeutic, and the SP activity measured. Sera and placenta from women with PE was also investigated for their inhibitory effects on the SP. Known candidates for EDLFs were investigated for their SP inhibitory effects, as well as how digitalis antibody immune Fab fragments, Digibind® and DigiFab™, bound them and affected the SP activity. Digibind® is also a sufficient affinity material used to isolate and purify PE EDLFs. Additionally, the placentas of preeclamptic women have high levels of similar EDLFs. These studies provide evidence for the existence of EDLFs that circulate in women with PE, and Digibind® is an effective and novel tool to bind, isolate and purify EDLFs in PE.

Preeclampsia (PE) is a multisystem disorder that contributes to maternal and fetal mortality and morbidity worldwide. It is characterized by de-novo hypertension and proteinuria or other maternal organ damage after 20 weeks of gestation. Evidence suggested that endogenous digitalis-like factor (EDLF) contributes to the pathogenesis of PE, and that the potential source of EDLF is the placenta. EDLF can inhibit the sodium pump (SP) specifically and may lead to hypertension, it has also been associated with hypoxia, oxidative stress and other abnormalitites present in PE.We studied whether normal human placenta responded to SP inhibition casued by EDLF with a change in abundance of lipids in the placental cytosol, and whether there was a characteristic set of lipid changes that could serve as a signature for EDLF exposure if there were such changes. Placenta tissues from 20 normal pregnancies were incubated for 48 hr in the presence and absence of ouabain, a widely studied EDLF, followed by tissue homogenization, lipid extraction, and the study of lipids using a mass spectrometery (MS) based lipidomics approach. 1207 lipidomic markers were surveyed by paired Student t-test, among which 26 markers had significantly different abundances between cases and control at the FDR=0.05 level. A set of 8 lipidomic markers were selected by a statistical model built with a sparse partial least squares discriminant analysis method (sPLS-DA) and a bootstrap procedure. All eight markers were then chemically characterized and partially identified using tandem MS. These markers might be used to identify placentas that have been previously exposed to EDLF in return.Endogenous peptides and small proteins might contribute to the pathophysiology of various diseases. Therefore, we investigated the potential peptidomic profile of placenta tissues in response to EDLF exposure as well. Placenta tissues from 20 normal pregnancies were incubated for 25 hr with and without the addition of ouabain, followed by homogenization, protein depletion, and the study of the peptides by a LC-MS based peptidomics approach. 275 peptidomic markers were evaluated by Student t-test. A set of 8 markers was chosen using a logistic regression model build with the Akaike information criterion (AIC). However, no peptidomics markers or set of markers showed specific, statististically significantly different changes in abundances between cases and controls after applying a false discovery rate (FDR) correction or using more conservative methods to overcome over-fitting. Using an optimal sPLS- DA, cross-validation studies and logistic regression models, we also found that the addition of any peptidomic marker to the previously selected lipidomic profile was unlikely to help identify placentas that had been exposed to EDLF.Alzheimer's disease (AD) is the most common form of dementia and the number of AD cases worldwide is currently estimated to be 36 million. The exact pathogenesis of AD remainsiielusive and available therapeutic strategies can only delay its progession temporarily. Several hypotheses have been proposed regarding the pathophysiology of AD and the beta-amyloid (Aβ) hypothesis is considered the core mechanism. However, the majority of studies concerning AD, or AD biomarkers specifically, have ignored a potentially important variable that is gender, despite reported gender differences in the risk of developing AD, the risk factors, clinical symptoms and CSF biomarkers of the disease, among many other aspects.We analyzed data obtained from a previous study of diagnostic serum lipid biomarkers for AD with the consideration of potential gender difference. Firstly, we studied the interaction between gender and disease stage using analysis of variance (ANOVA) and analysis of covariance (ANCOVA). Lipid markers that showed statistically significant interaction were selected after applying a FDR correction. Secondly, using a lasso logistic regression model with binary classification (control vs. all AD stages), we identified gender-specific markers and found different coefficient estimates for different genders as well. Lastly, we build a new ordinal model with the addition of a gender-specific marker using a Bayesian lasso probit ordinal regression model. The predictive performance of the new model was found to be statistically significantly better than the previous model which was built without the consideration of gender.In conclusion, we successfully discovered, chemically characterized lipidomic markers indicative of EDLF exposure in placenta and detected gender-specific lipid markers for AD.