Endogenous Digoxin-like Substance Research Articles

Development of a new ultra-high-performance liquid chromatography-tandem mass spectrometry method for the determination of digoxin and digitoxin in plasma: Comparison with a clinical immunoassay.

Cardiac glycosides digoxin and digitoxin are used in therapy for the treatment of congestive heart failure. Moreover, these compounds can be responsible for intoxication cases caused by fortuitous ingestion of leaves of Digitalis. Due to the narrow therapeutic range of these drugs, therapeutic drug monitoring is recommended in the clinical practice. In this context, immunoassays‐based methods are generally employed but digoxin‐ and digitoxin‐like compounds can interfere with the analysis. The aim of this study was to develop and validate an original UPLC–MS/MS method for the determination of digoxin and digitoxin in plasma. The method shows adequate sensitivity and selectivity with acceptable matrix effects and very good linearity, accuracy, precision, and recovery. A simple liquid–liquid extraction procedure was used for sample clean‐up. The method was applied for the analysis of n = 220 plasma samples collected in two different clinical chemistry laboratories and previously tested by the same immunoassay. The statistical comparison showed a relevant negative bias of the UPLC–MS/MS method versus the immunoassay. These results are consistent with an immunoassay overestimation of digoxin plasmatic levels due to cross‐reaction events with endogenous digoxin‐like substances.

ChemInform Abstract: Synthesis of Lysophosphatidylserine (I) with 19:4 Acyl Group, as a Novel Sodium Potassium ATPase Inhibitor, in Relation to DLIS-2, an Endogenous Digoxin-Like Substance.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

False-high blood salicylate levels in neonates with hyperbilirubinemia.

Drug assays may yield false-positive results caused by cross-reacting compounds. After finding a serum salicylate concentration of 81 microg/mL by using Trinder's colorimetric method, in a comatose child admitted to the authors' pediatric intensive care unit, in the absence of reported salicylate intake, the authors aimed to compare this situation with the phenomenon involving endogenous digoxin-like substances, which cross-react with the routine assay of digoxin. None of the participants in the study had been exposed to salicylate. Salicylate concentration was measured in all patients using Trinder's colorimetric method and in the second stage of the study also by AxSYM salicylate assay. Salicylate concentration using Trinder's method was 18 +/- 25 (4-81) microg/mL among nine seriously ill children in the pediatric intensive care unit, of whom two children with extensive burns had salicylate levels of 30 and 81 microg/mL, respectively. Salicylate concentrations were 107 +/- 24 (45-143) microg/mL and 60 +/- 25 (28-92) microg/mL, among 18 premature newborns and 18 term newborns, with hyperbilirubinemia, respectively. In the second stage, which involved 22 jaundiced term newborns and cord blood from 21 pregnant women, Trinder's method yielded elevated salicylate blood levels among the hyperbilirubinemic infants: 82 +/- 5 (73-89) microg/mL; however, the AxSYM assay yielded significantly lower blood levels: 2.5 +/- 3.4 (0-10.9) microg/mL (P < 0.0001). Among the pregnant women, salicylate cord blood levels were found to be low-within the limit error of the assay with both assay methods. In conclusion, when salicylate intoxication is suspected, particularly during the neonatal period, it is advisable to measure salicylate levels by immunoassay technology.

Pharmacology of inotropic agents in infants and children

Inotropic agents are drugs which increase the stroke work of the heart at a given pre-load and after-load. All of these agents work through a final common pathway involving the modulation of calcium interactions with various myocardial contractile proteins. The agents employed with pediatric patients include the cardial glycosides, catecholamine β-agonists and the selective phosphodiesterase III inhibitors. Digoxin is the prototypic cardiac glycoside which has a long history of safe and effective use in infants and children. Its utility in improving right ventricular dysfunction in patients with cor pulmonale leading to biventricular dysfunction makes it ideally suited to the pediatric population. Monitoring digoxin pharmacokinetics in infants is confounded by the presence of an endogenous digoxin-like substance. Nevertheless, the drug is well suited for subacute and chronic myocardial support. In contrast, the catecholamines are the drugs of choice for acute intervention. Their pharmacokinetics permit rapid dosing titration. In infants and children the greatest experience has been accrued with dopamine, a mixed α- and β-agonist but both epinephreine and norepinephrine are being used with increasing frequency as the need for drugs with increased potency and pressor activity becomes more common. The phosphodiesterase inhibitors amrinone and milrinone are the newest additions to our therapeutic armamentarium. In addition to their modest inotropic effects, amrinone and to a greater extent, milrinone offer significant pulmonary vasodilatation as part of their therapeutic package. These effects occur with little or any impact on myocardial oxygen consumpton while their lusitropic effects enhance relaxation in hypertrophied ventricular muscle. Of the two agents milrinone is probably preferred due to its greater therapeutic index and shorter elimination half-life. All of these agents remain important tools in the care of critically ill infants and children. The rational use of these drugs based upon their pharmacokinetic and pharmacodynamic properties is essential to achieve their optimal effects.

Urinary Excretion of Free Dopamine and Digoxinlike Substances Correlates with Endogenous Secretion of Insulin in Normotensive Adults, but Not in Hypertensive Subjects

We investigated whether urinary excretion of free dopamine is related with the humoral factors which affect Na+, K+ ATPase activity in the kidneys. Subjects were 51 adults admitted in a hospital without renal insufficiency: they were divided into normotensive (n = 36, 60 +/- 3 years old, 122 +/- 3/73 +/- 2 mmHg) and hypertensive groups (n = 15, 65 +/- 5 years old, 157 +/- 6/91 +/- 2 mmHg). Urinary excretion of free dopamine was significantly and positively correlated with urinary excretion of C-peptide immunoreactivity of insulin (CPR) (r = 0.451, p = 0.014) in normotensive subjects, but not in hypertensive subjects (r = 0.155, p = 0.668). Urinary excretion of endogenous digoxinlike substances (EDLS) was also significantly and positively correlated with urinary CPR (r = 0.500, p = 0.006) in normotensive subjects, but not in hypertensive subjects (r = 0.275, p = 0.363). In normotensive subjects, urinary excretion of free dopamine and EDLS may be regulated at least in part by insulin secreted endogenously. In hypertensive subjects, however, this regulatory mechanism of the diuretic factors, such as insulin, EDLS and dopamine, is thought to be deranged, which might result in decompensation of a diuretic and antidiuretic balance leading to blood pressure elevation.

Plasma Endogenous Digoxin-Like Substance Levels are Dependent on Blood O2 in Man

1. Recently, we have demonstrated that hypoxic breathing is followed by an increase in plasma digoxin-like substance in normal men. 2. This study was undertaken in order to evaluate whether or not a low arterial O2 partial pressure is combined with an increase in plasma digoxin-like substance in chronic pathological conditions also. 3. Sixteen male patients (mean age 53.1 +/- 3.7 years) affected by chronic obstructive pulmonary disease of a mild stage were studied. They were further subdivided according to their arterial O2 partial pressure into 'mild hypoxic' (n = 8, mean age 52.5 +/- 2.7 years), with an arterial O2 partial pressure between 66 and 75 mmHg, and 'severe hypoxic' (n = 8, mean age 54.3 +/- 5.1 years), with an arterial O2 partial pressure < or = 65 mmHg, groups. Seven healthy men (mean age 48.5 +/- 4.8 years) voluntarily participated as the control group. 4. Plasma digoxin-like substance levels were significantly higher in 'severe hypoxic' patients (203.5 +/- 9.9 pg/ml) than in both 'mild hypoxic' patients (169.5 +/- 31.4 pg/ml, P < 0.02) and normal subjects (158.9 +/- 12.4 pg/ml, P < 0.0001) and were directly correlated with urinary Na+ excretion (severe hypoxic group, r = 0.756, P < 0.007; mild hypoxic group, r = 0.789, P < 0.02). Considering the two hypoxic groups together, plasma digoxin-like substance levels were negatively correlated with arterial O2 partial pressure (r = -0.740, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

血清中ジゴキシン濃度測定におけるＣＥＤＩＡ法の評価

Serum levels of digoxin and methyldigoxin were measured with the cloned enzyme donor immunoassay (CEDIA) method and the fluorescence polarization immunoassay (FPIA) method.In other samples, both of those cardiac glycosides were measured with FPIA method and radioimmunoassay (RIA) method.There was a significant correlation (r=0.835, n=85) between CEDIA and FPIA methods in serum levels of digoxin and an apparent that of methyldigoxin. But in some patients, especially in infants, the values of the CEDIA method were lower than those of FPIA method.In contrast, there was a higher significant correlation (r=0.939, n=63) between FPIA and RIA methods in serum levels of both cardiac glycosides.These results may indicate that sensitivities of these methods to the interfering substances (ex. endogenous digoxin-like substance) differ each other.

Endogenous digoxin-like substance as an important factor in the development of hypertension in children

In this study, endogenous digoxin-like substance within the plasma was measured by radio-immunoassay in 122 children with the systolic blood pressure equal to or greater than the 75th percentile (age = 12.3 ± 2.5 years) and 136 children with the systolic blood pressure at or less than the 50th percentiles (age = 11.9 ± 2.8 years) after a follow-up of one year. The levels of digoxin-like substance in the plasma of children with the higher blood pressure were much higher than those in the controls (32.7 ± 17.3 versus 22.7 ± 12.7 pg/ml, P<0.01) and were positively correlated to the blood pressure ( r=0.21, P<0.05 for systolic blood pressure; and r=0.41, P<0.01 for diastolic), and to the sodium content of the red cells ( r=0.36, P<0.001), plasma ( r=0.25, P<0.01) and 8-hour overnight urinary output ( r=0.32, P<0.05). The children in the group with higher blood pressure exhibited an exaggerated natriuresis after an oral saline-water load. We believe that a circulating inhibitor of sodium transport may play an important role in the early stage of the evolution of hypertension.

Generation of Monoclonal Anti-Digoxin Antibodies

Anti-digoxin monoclonal antibodies are a useful model for basic immunochemical studies, for investigation of endogenous digoxin-like substances and in immunoassay for cardiac glycosides. The complete phenotypic characterization is a requisite for the selection of antibodies with desired binding parameters for different purposes. Twenty-two high-affinity monoclonal antibodies specific for digoxin were obtained in two fusion experiments. Treatment of antigen-antibody complex with potassium thiocyanate (KSCN) and absorption ELISA were used for the selection of high-affinity antibodies at the earliest stages of hybridoma growth. The true affinity constants of selected antibodies were determined in ELISA. They had proven to vary between 10(-7) and 10(-10) M. The fine specificity of 21 anti-digoxin monoclonal antibodies was determined by cross-reactivity experiments with 25 structurally related compounds. Cardiac glycosides, digoxin metabolites, endogenous steroids and spironolactone were used in the elucidation of the antigenic recognition pattern of antibodies. The elucidation of the binding characteristics of anti-digoxin monoclonal antibodies makes possible the selection of antibodies possessing binding characteristics appropriate for a wide range of designations.

Effect of endogenous digoxin-like substances on the interpretation of high concentrations of digoxin in children

The objective of this study was to assess the effect of endogenous digoxin-like substances on the interpretation of excessive concentrations of digoxin in children. After the development of a high-pressure liquid chromatography (HPLC) method for digoxin in our laboratories, we analyzed sera of children in whom the fluorescence polarization immunoassay identified potentially toxic concentrations of the glycoside (greater than 3 nmol/L; 2.3 ng/ml). Sixteen of them were receiving long-term digoxin therapy, and one had an accidental overdose. The immunoassay yielded significantly higher concentrations (4.1 +/- 1.2 nmol/L; 3.2 +/- 0.9 ng/ml) than the HPLC method (3.3 +/- 1.6 nmol/L; 2.6 +/- 1.2 ng/ml; p less than 0.01). In five cases (30%) these differences were clinically significant because administration of digoxin had been discontinued in the presence of true digoxin concentrations within the therapeutic range and the lack of clinical toxic effects. These data suggest that therapeutic drug monitoring using immunoassays of digoxin may be too inaccurate to detect potential toxic effects, and that much more weight should be focused on clinical monitoring. The HPLC method for assay of digoxin is extremely meticulous and will not become clinically available; therefore the development of better immunoassays should be encouraged.

Intra-cellular electrolyte changes and levels of endogenous digoxin-like substance within the plasma in patients with congestive heart failure

In this study, the lymphocytes and erythrocytes from peripheral venous blood were used as the study model from which were measured the cellular contents of potassium, sodium, calcium and magnesium in 50 patients with chronic congestive heart failure and 39 control patients. Levels of endogenous digoxin-like substance in the plasma and activities of Na K ATPase in red cell membrane were monitored simultaneously. In the patients with heart failure, the intracellular contents of potassium and magnesium were decreased while those of sodium and calcium were increased significantly. The levels of endogenous digoxin-like substance were much higher in the plasma than those either in healthy controls or in patients with heart disease but without congestive failure (273.7 ± 35.5 vs 23.3 ± 2.2 and vs 32.9 ± 3.6 pg/ml, respectively, P < 0.001 for both). The activities of Na K-ATPase were much lower in the patients with heart failure than in the controls. Values for intracellular electrolytes were significantly correlated with the rising levels of digoxin-like substance in the plasma. Non-digitalis inotropic therapy was associated with the recovery of these alterations of heart function, with the levels of the digoxin-like substance decreasing and activity of Na K-ATPase going up. We conclude that endogenous digoxin-like substance might play a role in the imbalance of intra-cellular electrolytes seen in patients with congestive heart failure. Digoxin may exacerbate the loss of intracellular potassium.

Synthesis of lysophosphatidylserine with 19:4 acyl group, as a novel sodium-potassium atpase inhibitor, in relation to dlis-2, an endogenous digoxin-like substance

A lysophosphatidylserine with 19:4 acyl group proposed as a candidate of DLIS-2, an endogenous digoxin-like substance was chemically synthesized. The synthetic compound showed a significant activity of Na +,K +-ATPase inhibition, which has been accepted as a major indicator activity for the essential hypertension.

Digoxin-Like Immunoreactive Substance and Preterm Labor

An endogenous digoxin-like substance (DLIS) that may be related to preterm labor has been studied. Using a commercially available radioimmunoassay kit. DLIS levels were determined in 110 women, of whom 33 were in preterm labor, 27 were in labor at term, 26 had normally ongoing pregnancies before term and were not in labor, and finally 24 were at term but not in labor. Mean DLIS levels of women in preterm labor was found to be significantly high (greater than 0.2 ng/ml, p less than 0.05). Whether this finding points to a causative role for DLIS in preterm labor remains to be further investigated.

Endogenous Inhibition of Red Blood Cell Na, K-ATPase in Essential and Pregnancy-Induced Hypertension

Digoxin-like inhibitors of Na,K-ATPase have been implicated in the pathophysiology of essential (EH) and pregnancy-induced hypertension (PIH). A technique that enhances dissociation of digoxin from red blood cells (RBC) was used to displace endogenous digoxin-like substances from RBCs. RBC membranes were preincubated in Na and ATP (Release) or Na,K,Mg and ATP (Retention) prior to measuring ATPase activity. Groups studied were: 39 men with EH and 34 controls plus 10 women with PIH and 17 normotensive controls. All displayed similar increases in Na,K-ATPase activity (24.0 +/- 7.9%) following Release. Plasma digoxin immunoreactivity (DI) was measured in pregnant women, m = 0.25 +/- 0.07 ng/ml. No DI was detected in nonpregnant women, but RBCs from these women demonstrated the same increase in Na,K-ATPase activity after Release. The 24% increase in activity achieved by Na and ATP preincubation can be reversed by adding K and Mg to the Release suspension. However, after RBC-bound digoxin is displaced by Release preincubation, addition of K and Mg cannot promote renewed binding and pump inhibition. Thus, the observed endogenous inhibition is not due to displacement of a digoxin-like substance but probably is related to alteration of the enzyme-membrane interaction. Furthermore, even though pregnant women demonstrate DI, an inhibitory substance with digoxin-like binding could not be recognized using this technique.

Progesterone, some progesterone derivatives and urinary digoxin-like substances from pregnant women in radioimmuno- and 86Rb-uptake assays of digoxin.

Progesterone and some derivatives were tested in a radioimmunoassay (RIA) of digoxin and in a bioassay measuring the 86Rb-uptake into red blood cells as an index of Na+, K+-ATPase activity. The digitalis-like activity of the hormones was compared with that found in chromatographic fractions of material extracted from the urines of pregnant women at term. Progesterone at concentrations greater than 10(-6) M cross-reacted in the RIA, and at 10(-3) M it decreased 86Rb-uptake by 18%. The anaesthetic progesterone derivates 5 alpha-pregnane-3 alpha-ol-20-one and 5 alpha-pregnane-3,20-dione crossreacted to a lesser degree in the RIA and lacked effect in the bioassay. Similar results were obtained with pregnandiol-glucuronide, the major urinary metabolite of progesterone. In contrast, several fractions of the urinary material had significant effects in both assays. It is concluded that the digitalis-like activity of progesterone is not coupled to properties associated with its anaesthetic effects. Furthermore, although progesterone may account for a part of the endogenous digoxin-like substances in serum of neonates and pregnant women, neither progesterone proper nor pregnandiol-glucuronide explains the great amount of digoxin-like substances found in the urines.

Partial Characterization of Endogenous Digoxinlike Substance in Human Urine

Partial Characterization of Endogenous Digoxinlike Substance in Human Urine

Endogenous digoxin-like substance(s) associated with uneventful and high-risk pregnancies.

We assessed the existence of endogenous digoxin-like substance(s) (EDLS) in mother-neonate pairs using a routine radioimmunoassay for digoxin. None of those studied had been treated with cardiac glycosides during or before pregnancy. In uneventful pregnancies, cord EDLS levels (0.31 +/- 0.02 ng/ml mean +/- SEM) were significantly higher (p less than 0.001) than both antepartum and postpartum maternal levels (0.14 +/- 0.02 and 0.17 +/- 0.02 mg/ml, respectively). This observation was in contrast with findings in high-risk pregnancies. In general, EDLS levels in the high-risk group were significantly higher than in normal pregnancies (cord 0.94 +/- 0.38 ng/ml; antepartum 1.63 +/- 0.54 ng/ml; postpartum 0.89 +/- 0.73 ng/ml). In the high-risk group there was a remarkably wide range of maternal and cord EDLS concentrations. The present studies suggest that following pregnancies of high risk for a variety of reasons, EDLS determination may be commonly high in the perinatal period and may affect the determination of 'true' digoxin. Consequently, digoxin dosing based on monitoring drug concentration may be futile.

Partial Characterization of Endogenous Digoxinlike Substance in Human Urine

Urinary samples were collected from individuals not taking cardiac glycosides. Aliquots of 30 ml were passed through preparative octadecylsilane-bonded phase columns and eluted in fractions by stepwise increasing concentrations of acetonitrile. Eluted fractions were analysed for their contents of endogenous digoxinlike substance (EDLS) by radioimmunoassay of digoxin and by a bioassay of cardiac glycosides, which measures the uptake of rubidium (86Rb) by erythrocytes as an index of Na+, K+-ATPase activity. In both assays, digoxinlike activity was found in several fractions, but the highest values were consistently measured in the fractions eluted with 40% acetonitrile. Greater amounts of EDLS were recovered from the urine of pregnant women than from the urine of men and nonpregnant women.

Discrepancies Between the Calculated Digoxin Clearance and the Serum Digoxin Level in Elderly Patients-Reply

—Dr Reider's comments on our recent article in theArchives 1 raise a fundamental question of the reliability of digoxin radioimmunoassay (RIA). Discrepancies in the measurements made with different kits are well recognized. 2,3 Analytical recovery studies have shown that RIA consistently overestimates serum digoxin levels. 2 Whether this is due to the endogenous digoxinlike substance (endoxin) has not been conclusively demonstrated. In our study, the calculated digoxin level underestimated the serum levels in five patients and overestimated it in two (patients 5 and 14). This observation indicates that the presence of endoxin cannot be the sole explanation for the discrepancy between the measured and calculated serum digoxin levels. Digoxin concentration measurements done by commercially available RIA methods may not be optimal. 2,3 However, the purpose of our study was not to establish the reliability of digoxin RIA, but to determine the predictability of immunoassayable serum digoxin concentrations measured by

Discrepancies Between the Calculated Digoxin Clearance and the Serum Digoxin Level in Elderly Patients

To the Editor. —Regarding the article by Mooradian and Wynn in the April issue of theArchives,1about discrepancies between the calculated digoxin clearance and the serum digoxin level in elderly patients, it should be no surprise that the serum digoxin levels by radioimmunoassay (RIA) were higher than expected. In the study by Erickson and Green,2analysis of human plasma supplemented by known amounts of digoxin... [by] RIA methods consistently yielded results >100%. This tendency of the RIA to produce falsely elevated levels may be related to the endogenous digoxinlike substance called endoxin,3which has been shown to be present in patients with a variety of clinical states, including renal impairment4and hypertension,5as well as in normal individuals.6Thus, the dismissal of estimates of digoxin clearance based on serum levels of digoxin by RIA (which, at least potentially, are falsely high) may