Digoxin concentrations were measured in serum samples from 102 patients with renal impairment who were receiving digoxin therapy. Many patients had values that differed widely on several c...

An Endogenous Digoxin-Like Substance in Patients with Renal Impairment

We report the presence of an immunoreactive digoxin-like substance in blood from third trimester pregnant women. The sera from 51 women in the third trimester of pregnancy were analyzed by 4 commercially available digoxin RIAs. None of these patients was receiving digoxin. Digoxinimmunoreactivity was detected in all patients by 3 of 4 assays. The measured values, in nanograms per ml digoxin equivalent, were (mean ± SD): method A, 0.27± 0.05; method B, 0.28 ± 0.07; method C, 0.01 ± 0.01; and method D, 0.15 ± 0.06. Method B measured values greater than 0.50 ng/ml in sera from 5 patients. Digoxin immunoactivity was not detectable 24 h postpartum, suggesting a half-life in serum of 6 h or less. Exogenous digoxin added to these serum samples resulted in quantitatively additive increments above the endogenous measured levels. Three of 4 digoxin RIAs did not distinguish between true digoxin and the endogenous substance present in the sera of thirdtrimester pregnant patients. Preliminary evidence suggests that th...

Endogenous digoxin-immunoreactive substance in human pregnancies.

We previously identified endogenous digoxinlike immunoactivity in serum from pregnant women, newborn infants, and patients in renal failure. This activity is due to an endogenous factor(s) that cross-reacts with antibodies raised against digoxin. Using serum from the above sources as well as serum and urine from normal individuals, we further characterized these immunoreactive factors. The factors are water soluble, heat stable, and neutral in molecular charge. That isolated from serum has an apparent mol wt of 200 daltons, as estimated by membrane partitioning. The factor from urine has twice this apparent mol wt, an apparent higher affinity for the digoxin antisera, and is less resistant to acid hydrolysis. It may represent a conjugated metabolite of the factor from serum. The immunoactive factor in serum is noncovalently bound to serum protein, and we describe methods for estimating total, weakly protein- bound (i.e. heat-dissociable), tightly protein-bound (i.e. not heat-dissociable), and unbound (fre...

Protein Binding of Endogenous Digoxin-Immunoactive Factors in Human Serum and Its Variation with Clinical Condition

Accumulating experimental evidence suggests that natriuresis in response to intravascular volume expansion is promoted by an endogenous regulator of Na+,K+-adenosine triphosphatase (ATPase). Efforts to purify this substance by a number of laboratories have as yet been unsuccessful. The properties of partially purified inhibitors from plasma, urine, and tissue often fail to possess the characteristics thought to be consistent with those of a physiological regulator. These include potency (Ki of approximately 1 nM), reversibility of inhibition, specificity for Na+,K+-ATPase, and responsiveness to relevant physiological stimuli. Two rather different candidate substances, extracted from urine and hypothalamus, have been purified to a high degree. Neither is a peptide, and both are of low molecular weight and resistant to acid hydrolysis. The substance from urine is rather nonpolar and interacts with digoxin-specific antibodies, while that from hypothalamus is polar and does not appear to share epitopes with the cardiac glycosides. On the serosal surface of the toad urinary bladder, the hypothalamic substance causes a reversible inhibition of Na+ transport, inhibits rubidium uptake in red blood cells by acting on the membrane's exterior surface, inhibits binding of ouabain to purified Na+,K+-ATPase, and reversibly inhibits hydrolysis of adenosine 5'-triphosphate by the enzyme with a Ki of 1.4 nM. The hypothalamic inhibitor may be differentiated from ouabain by their respective ionic requirements for optimal inhibition of enzymatic activity, and although both ouabain and the hypothalamic inhibitor fix Na+,K+-ATPase in its E2 conformation, the hypothalamic inhibitor does not promote phosphorylation of the enzyme by inorganic phosphate in the presence of Mg2+. Ionic requirements for inhibition also differentiate the hypothalamic inhibitor from vanadate ion, as does the inhibitor's activity in the presence of norepinephrine. Further enzymological and physiological studies will be facilitated by structural characterizations of the inhibitory substances and by the availability of a method to measure their concentrations in physiological fluids.

The search for a hypothalamic Na+,K+-ATPase inhibitor.

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.

An Endogenous Ouabain-Like Factor Associated with Hypertensive Pregnant Women*

In previous studies we have demonstrated a natriuretic factor of small molecular weight (<1,000 Daltons) in the serum and urine of salt loaded subjects. This factor isolated from salt loaded animals inhibits the Na-K-ATPase enzyme system. In addition, the natriuretic material isolated from plasma of salt-loaded dogs was shown to bind to specific digoxin antibodies. It was therefore suggested that a digitalis-like endogenous natriuretic factor (endoxin) is released in response to saline loading. In the present study we therefore investigated the presence of such an endogenous natriuretic digitalis-like activity in the urine of healthy volunteers during high salt intake. Using Sephadex G-25 for chromatographic separation of urine a material elutes as a single peak in the natriuretic post-salt fraction IV which is specifically bound to digoxin antiserum complex. Mean peak activity amounted to 1.55±0.48 ng/ml digoxin equivalents. We further purified the natriuretic material by immunoprecipitation with the digoxin antiserum complex. This purification procedure resulted in a more than 10-fold increase in specific natriuretic activity from 2.7±0.4 to 30.4±5.8 µEq Na+·min−1·mg−1. Thus the digitalis-like natriuretic activity previously observed in the plasma of saline loaded dogs is also present in the urine of healthy subjects during high dietary salt intake. Immunoprecipitation may offer a meaningfull tool for further isolation and identification of the natriuretic hormone(s).

E. Weiler

Papers

Digoxin-like natriuretic activity in the urine of salt loaded healthy subjects.