Endogenous digoxin-like factor: serum concentration and interrelations with the electrolyte picture in normal subjects

Endogenous Digitalis-Like Factor (DLF) is a putative hypothalamic Na+,K+-ATPase inhibitor that mediates natriuresis in response to intravascular volume expansion or sodium loading. The precise structure of this substance remains unknown; however, it cross-reacts with antibody to digoxin. Using a radioimmunoassay, we measured DLF concentrations in 26 normal subjects: mean value of this factor was 0.512 ng digoxin-equivalents/ml +/- 0.038 SEM; DLF correlated significantly with serum sodium levels (r = 0.59 - p less than 0.01) and daily urinary sodium excretion (r = 0.48 - p less than 0.05). Our results confirm that endogenous digitalis-like factor has a physiological role as regulator of natriuresis, in response to plasma sodium concentrations.     

Evidence for an adrenal contribution to plasma digitalis-like factors

Digitalis-like factors (DLF) were assayed on plasma collected serially using antibody to digoxin: (1) after intravenous ACTH (n = 3 patients) and (2) after 0.15 U/kg crystalline insulin, 50 micrograms gonadotropin-releasing hormone and 0.2 mg thyrotropin-releasing hormone (n = 7) as a combined pituitary function test. Patients had either hypothalamic-pituitary or ovarian problems. Mean values for DLF rose fourfold after ACTH, and by a factor of 2.3 with the combined pituitary function test. DLF rose at least 50% in all 10 subjects. In 3 dogs, adrenal vein values for DLF were on average more than double values in either adrenal artery or lower vena cava. These results suggest that certain DLFs have an adrenal origin.     

Digoxin-like immunoreactive factor in rat plasma: effect of sodium and calcium intake

Studies have been performed in rats to determine whether an endogenous material capable of binding to digoxin antibodies is present in the plasma. Such a material has been shown in other species and has been hypothesized to represent an endogenous ligand for the receptor on Na-K ATPase through which cardiac glycosides act. In rats consuming a normal rodent chow (1% calcium by weight) and drinking deionized water, endogenous binding of digoxin antibody in radioimmunoassay amounted to 23.1 +/- 4.6 fM digoxin equivalents/100 microliter of plasma (mean +/- SEM, n = 8). Since a hypothetical role for such an endogenous ligand is the regulation or renal sodium excretion by inhibition of renal Na-K ATPase, the effect of increased sodium intake on plasma levels of this digoxin-like immunoreactive factor (DLIF) was studied. Animals consuming the same chow, but drinking 0.5% NaCl solution in place of water for a 4 week period showed significantly greater DLIF in plasma which was measured at 109.2 +/- 20.3 fM digoxin equivalents/100 microliter of plasma (p less than 0.001). Because DLIF has been implicated in the pathogenesis of hypertension we also studied the effects of calcium intake on plasma levels of DLIF. In previous studies we have shown that rats allowed to drink 0.5% saline develop a moderate hypertension which can be reversed with calcium supplementation. In the present studies, 3 dietary calcium subgroups (0.01% Ca, 1.0% Ca and 4% Ca) were formed among animals drinking water or 0.5% saline for 4 weeks. No effect of low calcium intake on plasma DLIF was found either in water or saline drinkers. However, calcium supplementation produced a significant reduction in plasma DLIF in both water and saline drinking animals.     

Measurement of Plasma Digoxin Concentration by Radioimmunoassay

A rapid, sensitive, and precise method for measuring the plasma digoxin concentration has been developed with the radioimmunoassay technique. Seventy patients receiving digoxin were shown to have plasma digoxin concentrations between 0·4 and 5 ng./ml. Preliminary studies show that though there is a positive correlation between total daily dose and the plasma digoxin concentration, the relationship is not close, and a relatively wide range of plasma digoxin concentrations appear to be consistent with effective digitalization.     

Digoxin-like immunoreactive substance in patients with aneurysmal subarachnoid haemorrhage.

Digoxin was measured by radioimmunoassay in the plasma of 25 patients with aneurysmal subarachnoid haemorrhage who had not received digoxin treatment. After heating the plasma an endogenous substance cross reacting with antibodies to digoxin was identified in 18 cases. The presence of this substance was significantly related to the total amount of blood and to the presence of blood in the frontal interhemispheric fissure and could not be explained by hypertension or intake of water and sodium. A negative sodium balance and volume depletion occurred more often in patients who were positive for digoxin, but this relation did not reach statistical significance. It is concluded that a digoxin-like natriuretic factor is released in response to a subarachnoid haemorrhage, probably as a result of hypothalamic damage.     

Digitalis-like factor response to hyperinsulinemia accompanying a euglycemic hyperinsulinemic clamp or oral glucose tolerance test

Many studies of essential hypertension find evidence of insulin resistance in the same individuals, leading some to postulate a hypertensive role for insulin. However, the mechanisms by which insulin might exert a hypertensive effect are not fully resolved. An endogenous sodium pump inhibitor or digitalis-like factor (DLF) has been proposed as a hypertensive agent and its plasma concentrations are elevated in hypertension and in Type II diabetes, where insulin levels are elevated. Hence, we studied the effect of insulin on DLF using two approaches to achieve hyperinsulinemia. Normotensive men and women underwent a hyperinsulinemic, euglycemic clamp (40 mU/m2/min insulin, 40 mU = 1.6 x 10(-6) g) in which plasma insulin concentration was kept at high, but physiologic levels. Serum DLF (measured as inhibition of [Na,K]ATPase activity) and insulin levels were measured at baseline and every 30 min throughout the 2 hr clamp. Additionally, other subjects underwent an oral glucose tolerance test (OGTT) as a second means of increasing insulin levels. Insulin and DLF levels were measured prior to and hourly for 3 hours after receiving 100 gm of oral glucose. Serum DLF increased significantly during the clamp from a baseline of 4.6 +/- 0.81 to a peak of 8.7 +/- 1.2% inhibition (p=0.001). Comparison of the baseline and peak DLF levels with concomitant plasma insulin levels revealed a significant correlation (R=0.60, p=0.003). During the OGTT, DLF levels rose from a baseline of 2.4 +/- 1.0 to a peak level of 5.0 +/- 0.4%, p = 0.04. These results suggest that DLF, a factor that can cause vascular smooth muscle contraction and potentially influence blood pressure, is increased by hyperinsulinemia and provides a mechanism by which insulin may increase blood pressure.     

Behavior of the plasma level of digoxin in the rat in induced experimental hyperreninemia]

PRA, plasma and urine aldosterone levels and plasma digoxin were measured in rats in which digoxin had been administered under conditions of high PRA and high aldosterone levels experimentally induced by administering distilled water load and in rats in which digoxin had been administered without distilled water load. Results show that under conditions of high PRA and high aldosterone levels, plasma digoxin concentrations as measured 6 h after treatment were higher (45,3%) than in rats having received digoxin without water load. In assays carried out on rats sacrificed 12 h after digoxin treatment (with or without water load) all values approach basic levels again, thus suggesting that in rats too aldosterone might compete with digoxin at the level of tubular excretion.     

Influence of hypoxemia and respiratory acidosis on the plasma kinetics and tissue distribution of digoxin in the conscious dog

The aim of the present study was to investigate the influence of hypoxemia combined with respiratory acidosis on the kinetics of digoxin in conscious dogs. One group of three beagles was exposed to air and 7 days later to 10% O2, 10% CO2, and 80% N2. In a second group of three dogs, the order of exposure to the two atmospheric conditions was reversed. The dogs received 25 micrograms/kg digoxin and blood and urine samples were collected over the next 29 h. At the conclusion of the second treatment, the dogs were sacrificed to determine digoxin concentrations in the left ventricle, liver, renal cortex, and skeletal muscle. Digoxin total body clearance increased from 6.2 +/- 0.9 in control to 9.0 +/- 1.0 mL X min-1 X kg-1 in hypoxemic and hypercapnic dogs (p less than 0.05). The digoxin apparent volume of distribution at steady state (Vss) was increased in the dogs with hypoxemia and hypercapnia (11.63 +/- 1.11 vs. 8.62 +/- 0.41 L/kg in the controls, p less than 0.05). As a consequence the digoxin plasma half-life remained unchanged (18.6 +/- 1.5 h in hypoxemic and hypercapnic dogs versus 20.1 +/- 2.8 h in the controls). In dogs with hypoxemia and hypercapnia, the ratio of tissue to plasma digoxin concentrations tended to increase in the liver, in the renal cortex, and in the left ventricle and remained unchanged in the left hind leg muscle. In vitro studies showed that the digoxin total binding to erythrocyte membranes was slightly increased in the dogs with hypoxemia and hypercapnia, resulting from an increase in the apparent intrinsic association constant for digoxin (p less than 0.003). It is concluded that hypoxemia combined with respiratory acidosis changes digoxin disposition in the conscious dog and is the cause of a digoxin redistribution into the tissues.     

Cardiovascular investigations of an endogenous digoxin-like factor

A circulating factor with digoxin immunoreactivity has been demonstrated. Elevated levels of this substance appear to be present after volume expansion and salt loading, and in some forms of hypertension. The potentially causative role for this factor in hypertension can be demonstrated by the normalization of blood pressure after antidigoxin antibody infusions in low-renin and sodium-dependent hypertension. The possibility that renal excretory defects may be the initiating event to elevate endogenous digoxin is suggested by studies with normotensive humans and monkeys with renal disease. In the latter case cardiovascular deficits were noted that were analogous to those detected in renal hypertensive monkeys with elevated endogenous digoxin. Considered together, these results suggest the existence of a natriuretic and hypertensive substance that plays a role in body fluid homeostasis and blood pressure regulation.     

Plasma Digoxin Concentrations in Patients with Atrial Fibrillation

Plasma digoxin concentrations were measured by radioimmunoassay in 116 patients with atrial fibrillation on long-term oral treatment with the drug, and in 23 patients with digoxin toxicity. The mean concentrations were 1·4 ng./ml. and 3·1 ng./ml., respectively. Though an overlap occurred between the therapeutic and toxic ranges, toxicity is unlikely to occur below a level of 2 ng./ml. Plasma concentration showed a poor correlation with resting heart rate during atrial fibrillation. In patients with good renal function, however, a significant correlation was found between oral dose and plasma concentration. No evidence was obtained for increased sensitivity to therapeutic concentrations of the drug in elderly subjects, but the doses required to achieve these concentrations tended to be less than in younger patients.     

Mechanisms of digoxin-amiodarone interaction in the rat

Amiodarone and digoxin are often used in combination and clinical experience suggests that amiodarone may increase serum digoxin levels and toxicity. We have investigated the influence of amiodarone on digoxin pharmacokinetics and tissue distribution in the rat. Forty-nine rats were injected with 10 mg/kg amiodarone sc three times a day for 7 days, while 49 others were injected with saline only. On the eighth day, all the rats received 0.5 mg/kg digoxin ip; 4, 5, 6, 7, 8, 10, and 12 hr later, groups of 7 amiodarone-pretreated and control animals were sacrificed, and plasma, heart, liver, muscle, brain, and kidney digoxin concentrations measured by radioimmunoassay. Data were analyzed by two-way ANOVA, with group comparisons using the Waller-Duncan multiple comparison procedure. Digoxin levels were significantly higher in the plasma, heart, muscle, and kidney of the amiodarone-pretreated rats at most points of measurement (P less than 0.05) whereas liver digoxin levels were elevated at 8, 10, and 12 hr. Kidney/plasma, heart/plasma, muscle/plasma, and especially liver/plasma ratios in the control groups significantly exceeded the values found in the amiodarone-pretreated group at most time points. Concentrations of digoxin in brain were not changed. This suggests that the volume of distribution is significantly altered in the amiodarone-pretreated group. Amiodarone increases plasma digoxin levels in rats as it does in humans, but the mechanism is unclear.     

Endogenous digoxin-immunoactive factor in human subjects

Endogenous digoxin-like immunoactivity has been detected in the blood of adult patients in renal failure, newborn infants, and pregnant women in the third trimester. Blood levels of this activity increase in pregnant women as gestation progresses, and preliminary data suggest that the activity is increased in hypertensive pregnant women relative to normotensive pregnant women. Similar immunoactivity has also been detected in amniotic fluid and in the urine and serum of normal healthy subjects. The factors giving rise to this immunoactivity cross-react with antibodies used in many commercially available immunoassays for digoxin. The immunoactive factor isolated from human subjects is water soluble and exists tightly but reversibly bound to proteins in serum. The extent of this protein binding is altered in the clinical conditions studied relative to normal adults. This altered protein binding accounts for the detection of this factor by many of the commercially used immunoassays for digoxin. In this article I summarize recent findings related to detecting this activity in the blood of several clinical populations where the accurate measurement of digoxin may be compromised. I also summarize the preliminary isolation and characterization of the factor responsible for this immunoactivity.     

Renal Excretion of Digoxin in Swine and Goats

In experiments on swine and goats the renal excretion of digoxin was examined, and it was found that the renal clearance of non-protein-bound digoxin in swine was lower than creatinine clearance which expresses filtration clearance. Correlation analysis showed that the renal clearance of digoxin in swine was not significantly influenced by the concentration of non-protein-bound digoxin in plasma and the pH of the urine, while there was a significant positive correlation between the clearance and the urine flow rate (Table 4). On the other hand, the renal clearance of digoxin in goats was significantly influenced by the concentration of non-proteinbound digoxin in plasma and by urine pH (Table 4). From these results it is concluded that glomerular filtration and back-diffusion are involved in the renal handling of digoxin in both swine and goats. In addition active tubular secretion is also involved in the renal excretion of digoxin in goats.     

Statistical survey of “saturation analysis” calibration curve data for prednisolone,prednisone and digoxin

An extensive survey of radioimmunoassay calibration data for prednisolone, prednisone and digoxin indicated that the common practice of preparing calibration curves with individual subject's pre-dose plasma or serum, and using this to estimate unknown concentrations for the same subject, is not supported by statistical considerations. Preparation of calibration plots from pooled data is better because this introduces less bias in estimated concentrations. Such a method also saves a great deal of time, since it is not necessary to repeat the calibration procedure each time “unknowns” are being assayed. The data suggest that there is no optimum calibration plot for all radioimmunoassays. Rather, each antibody-drug combination should be investigated thoroughly to determine the best calibration plot for the particular combination. We found that the best calibration plots are; the logistic-logarithmic plot for prednisolone; nonlinear least squares fit to a polyexponential equation for prednisone; and a weighted least squares regression of normalized % bound $\text{versus}$ concentration for digoxin. The error in the radioimmunoassay is usually concentration-dependent, and, in certain regions of the standard curve, is larger than the literature indicates, since, frequently, the error has been gauged from % bound values, but should be gauged from inversely-estimated concentrations.     

Identification and estimation of endogenous digoxin in biological fluids and tissues by TLC and HPLC.

A procedure for estimation of digoxin in biological samples after adding a known quantity of digoxin followed by extraction, separation by TLC and HPLC is described. The identity of digoxin thus extracted from rat brain has been established by reaction with digoxin antibody and by its inhibition of Na(+)-K+ ATPase activity. The method could be a better substitute to the routine radioimmunoassay as interfering substances are removed by TLC and HPLC.     

CANADIAN MEDICAL NEWS

Using the radioimmunoassay technique for measuring serum digoxin, it was found that patients who were given 0.25 mg. digoxin orally per day had a mean serum level of 0.83 ± 0.06 ng. per ml. In patients given 0.5 mg. daily the mean level was 1.30 ± 0.14 ng. A higher 24-hour urinary excretion of digoxin was associated with the higher serum levels in the latter group. Individuals who exhibited electrocardiographic evidence of digoxin toxicity had a mean serum level of 2.81 ± 0.21 ng. The majority of patients with high serum levels had evidence of impaired renal function, and it is in this clinical situation that knowledge of serum digoxin levels is likely to be most helpful in determining dose schedules.The method is specific, sensitive and reproducible. Repeated measurements on the same patient on maintenance therapy showed little variation. To obtain dependable serum levels blood should be drawn at least five hours after oral, and three hours after intravenous administration.     

Seasonal kidney and plasma renin concentration in Testudo hermanni Gmelin

Seasonal plasma and kidney renin concentration was studied by radioimmunoassay of angiotensin I in the terrestrial chelonian Testudo hermanni Gmelin under several methodological conditions (different substrates, pH, temperature). Evidence has been found for seasonal variations of the renin levels in relation to the physiological phase of the animals: active animals demonstrated high renal and plasma renin concentration, while lower values were obtained in hibernating animals.     

Serum digoxin in patients with thyroid disease.

Serum digoxin concentrations were measured by radioimmunoassay in 17 hyperthyroid and 16 hypothyroid patients after a seven-day course of oral digoxin. The significantly higher levels of serum digoxin in patients with hypothyroidism and lower levels in those with hyperthyroidism were closely related to the measured changes of glomerular filtration rate and digoxin serum half time in these two groups. Differences in serum digoxin concentration contribute to the altered sensitivity to digoxin shown by patients with thyroid disease.     

Radioimmunoassay of N'-nitrosonornicotine.

Antibodies specific for the precarcinogen N′-nitrosonornicotine (NNN) were obtained in rabbits immunized with a N′-nitroso-5′-carboxynornicotine-human serum albumin conjugate. The NNN derivative was synthesized by reduction and nitrosation of 2′,3′-dehydronornicotine and covalently conjugated to the macromolecule with the use of a carbodiimide. A radioimmunoassay was developed which could detect as little as 0.2 ng of NNN. Analyses of plasma supplemented with NNN indicated that it was possible to detect 2 ng NNN/ml sample. Nicotine, cotinine, pyridine, and pyrrolidine derivatives are ineffective inhibitors of the antigen-antibody reaction. N′-Nitrosoanabasine inhibits effectively, but this derivative has been reported not to occur in tobacco. The radioimmunoassay has been used to monitor nitrosation of nornicotine and anabasine under chemical conditions and to determine the rate constants for the reactions.