Endogenous Digoxin-like Immunoreactive Factors Research Articles

Inhibitory Effects of Digoxin on Testosterone Production in Rat Leydig Cells

Digoxin has beneficial effects on heart failure due to a direct positive inotropic action as an inhibitor of Na^+- K^+-ATPase and endogenous digoxin-like immunoreactive factors (DLIFs) found in mammalian blood also correlates to the same issue. We have demonstrated that digoxin inhibits rat testosterone secretion but the mechanisms remain unclear. In the present study, the direct effects of digoxin on the expression of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (P450scc) in rat Leydig cells were evaluated. Digoxin inhibited not only testosterone production after incubation of Leydig cells with or without human chorionic gonadotropin (hCG), forskolin, 8-Br-cAMP, but also pregnenolone production following stimulation of 25-hydroxy-cholesterol, suggesting that digoxin reduced adenylyl cyclase and P450scc activity. Digoxin significantly reduced the expression of StAR protein, but not that of P450scc. The stimulation of testosterone production induced by A23187 was decreased by digoxin indicating that digoxin might influence Ca^2+ utilization. The analog of digoxin, ouabain, did not affect testosterone production in rat Leydig cells, which indicated the drug specificity. These results suggested that, in rat Leydig cells, digoxin directly inhibited testosterone production partly by reducing P450scc activity, StAR protein expression, and Ca^2+ utilization. This report not only demonstrates the mechanism digoxin acts, but also implies a probable interaction between gonadal and adrenal axis linked by DLIFs (i.e. digoxin).

New Enzyme-Linked Immunosorbent Digoxin Assay on the ADVIA?? IMS??? 800i System Is Virtually Free from Interference of Endogenous Digoxin-like Immunoreactive Factors

Endogenous digoxin-like immunoreactive factors (DLIF) may crossreact with antidigoxin antibody and falsely elevate immunoassay results. Recently, a new enzyme-linked immunosorbent chemiluminescent assay for digoxin has been available for use on the ADVIA IMS (Integrated Modular System) 800i analyzer (Bayer Diagnostics). We studied potential interference of DLIF with this new digoxin assay. We analyzed 30 serum specimens from patients who have pathologic conditions that may increase serum DLIF concentrations. These patients were never exposed to digoxin or other agents that may lead to a measurable digoxin concentration. We also analyzed 10 specimens from neonates, 10 cord blood specimens, and 10 amniotic fluid specimens. Apparent digoxin concentrations were measured using the new enzyme-linked immunosorbent digoxin assay (IMS-Digoxin), a fluorescence polarization immunoassay (FPIA), and also a chemiluminescent immunoassay (CLIA, run on ACS:180(R) system from Bayer Diagnostics). We observed measurable apparent digoxin levels with the FPIA in 4 uremic patients (range 0.21-0.36 ng/mL, digoxin equivalent), 7 patients with liver disease (range 0.21-0.72 ng/mL), and 3 patients in the third trimester of pregnancy (0.22-0.66 ng/mL). We also observed measurable DLIF concentrations with the FPIA in 2 neonates (0.22 and 0.36 ng/mL), 5 cord blood specimens (range 0.21-1.18 ng/mL), and 5 amniotic fluid specimens (0.21-0.50 ng/mL). None of these DLIF-positive specimens showed any measurable digoxin concentration using the IMS-Digoxin or the CLIA assay. When serum specimens containing elevated concentrations of DLIF but no digoxin (as measured by FPIA) were supplemented with known concentrations of digoxin, we observed falsely elevated digoxin concentrations, as expected, only by the FPIA. In contrast, we observed a good agreement between the target and observed concentrations when the new IMS-Digoxin or the CLIA assay was used. We conclude that the IMS-Digoxin assay is free from interference of DLIF.

Comparable Effects of DIGIBIND and DigiFab in Thirteen Digoxin Immunoassays

Digoxin is widely prescribed for the treatment of cardiac conditions (1). Because of its narrow therapeutic range, digoxin-related toxicity resulting from acute or chronic overdose is common. Metabolites of digoxin as well as related compounds, including digitoxin, tanshinones, bufandienolide, and oleander, can contribute to or independently produce digoxin toxicity (2)(3). Digoxin toxicity can be rapidly and safely reversed by administration of anti-digoxin immune fragments (Fab) such as DIGIBIND®, which has been available in the US since 1986. Therapeutic Fab products act by binding digoxin with high affinity (109–1010 L/mol), favoring movement of digoxin out of tissue and thus promoting elimination. Factors that impact dosing with Fab products include known or suspected digoxin load, patient weight and history, and renal function (4)(5)(6)(7). Monitoring the free digoxin concentration after Fab administration may help ensure appropriate dosing, prevent deadly recrudescent toxicity, and determine when digoxin therapy should be resumed (8)(9)(10). Monitoring free digoxin in serum is challenged by the positive interference that has been extensively described with DIGIBIND, which interferes with immunoassays by competing with assay capture antibodies. The degree of interference depends on incubation times, washing steps, and the affinity of capture antibody for bound vs free digoxin (11)(12)(13). Consequently, monitoring of free digoxin in ultrafiltrates is a popular strategy for managing DIGIBIND-treated patients. Although ultrafiltration eliminates interference produced by large molecules, such as endogenous digoxin-like immunoreactive factors (DLIFs) and DIGIBIND, it does not eliminate interferences produced by small molecules known to interfere with digoxin immunoassays, such as spironolactone (14). In addition, ultrafiltration methods are not standardized, may require matrix-specific calibration, add expense and manual manipulation, and lengthen turnaround time (8)(15)(16). DigiFabTM is a …

Interference From Digitoxin-like Immunoreactive Factors Reduced in a New Monoclonal Chemiluminescent Digitoxin Assay

Endogenous digoxin-like immunoreactive factors (DLIF) can interfere with some digoxin immunoassays. We looked for similar interference, called digitoxin-like immunoreactive factors (DTLIF) in two digitoxin immunoassays: A new chemiluminescent assay (CLIA), processed on the automated random access immunoassay system ACS:180, and a fluorescent polarization assay (FPIA), processed on the semiautomated TDx batch analyzer. One hundred thirty-seven samples of sera were tested from nondigitalized pregnant women, patients with liver or kidney diseases, and cord blood. The CLIA digitoxin assay uses a murine monoclonal antibody and requires no sample pretreatment; the FPIA digitoxin assay uses a polyclonal rabbit antibody and requires sample precipitation. Both assays have a similar dynamic range and sensitivity and give comparable results with commercial controls and external quality control survey samples. Although the CLIA detected no digitoxin in any sample tested, the FPIA showed apparent digitoxin concentrations of more than 2.0 ng/ml for 100% and 44% among cord blood and liver disease specimens, respectively. The highest DTLIF concentration was found in serum from a patient with liver disease (18.1 ng/ml). When spiked with 32 ng/ml digitoxin, six of the samples containing DTLIF generated FPIA digitoxin values of 6% to 27.5% more than the expected digitoxin levels. Two specimens with no detectable DTLIF activity were run as controls, and when spiked with digitoxin, showed target digitoxin concentrations in the FPIA. The CLIA recovered near the target digitoxin values (32 ng/ml) in all spiked samples. It was concluded that the polyclonal FPIA digitoxin assay may give discordant digitoxin concentrations in some patient groups because of interference from digitoxin-like immunoreactive factors. The CLIA digitoxin assay is not affected by DTLIF interference.

Vitros Digoxin Immunoassay Evaluated for Interference by Digoxin-like Immunoreactive Factors

Serum digoxin concentrations are routinely monitored because of the narrow therapeutic range of digoxin. Endogenous digoxin-like immunoreactive factors (DLIFs) may cause a false analytic increase in the measured digoxin concentration of patients undergoing digoxin treatment (1)(2). DLIF interference is most commonly found in the sera of neonates, and cord blood and neonatal sera frequently produce false positive results for digoxin (3)(4). Here we examine the newly introduced Vitros digoxin immunoassay (DGXN, Johnson & Johnson Clinical Diagnostics) performed on the Vitros 250 analyzer and compare it to a method (OnLine, Roche Diagnostic Systems) substantially free from DLIF interference (5) and to a method (Digoxin II, Abbott Laboratories) where DLIF interference is known to be present (1). These particular methods are not unique with regard to interference by DLIFs, and methods vary considerably in the impact of DLIF interferences (1)(2)(6)(7). Serum samples used in this study were aliquots of samples that had been sent to St. Louis Children’s Hospital or Barnes-Jewish laboratories (BJC Health System) for the routine measurement of serum digoxin or aliquots of samples received for other routine chemistry analyses. Samples were from newborns (<2 weeks of age) and from adults. The study followed a protocol approved by the Human Studies Committee of Washington University. All samples were analyzed immediately or stored at −20 °C until use, with no more than three freeze-thaw cycles preceding analysis. The Vitros DGXN digoxin assay is an enzymatic heterogeneous, competitive immunoassay that uses dry slide technology. Digoxin-peroxidase conjugate and sample digoxin compete for a limited number of binding sites on the immobilized antibody reagent. After the unbound conjugate is eluted and …

Digoxinlike Immunoreactive Factor Isolated From Human Pleural Fluids is Structurally Similar to Digoxin

AbstractTo further define the chemical structure of human endogenous digoxinlike immunoreactive factors (DLIF) we used human pleural effusions as a source of the substance. Digoxinlike immunoreactive factor activity was detected by radioimmunoassay in the pleural fluid of each of four patients; average concentration was 0.35 ng/mL. The chemical profile of DLIF was determined by initial extraction and concentration of DLIF by ion exchange chromatography followed by reverse phase-high-pressure liquid chromatography (RP-HPLC) separation and purification. Using high-pressure liquid chromatography cochromatography of DLIF, together with several radioactively marked glycosides, we observed a single peak of DLIF activity that was chro- matographically identical to digoxin. The present study further supports the recent finding that DLIF is related structurally to the cardiac glycosides, and for the first time it has been proven that DLIF is present in pleural fluids

Purification and characterization of endogenous digoxin-like immunoreactive factors in chicken blood

Studies have been performed to determine whether an endogenous material capable of binding to digoxin antibodies is present in the chicken plasma. In the blood of 12 chickens without feed control, endogenous digoxin-like immunoreactive factors (DLIF) binding of digoxin antibodies in enzyme immunoassays amounted to 866 302 pg digoxin equivalents/mL of plasma (mean SEM). Immunoreactivity of DLIF increased to 1848 331pg/mL with a double value of control after boiling and acid pretreating the plasma. The major purification steps employed in this report were gel filtration column chromatography, high performance liquid chromatography (HPLC) and isoelectric focusing (IEF). Using HPLC for the separation, at least 10 chicken DLIFs with different molecular weight (MW) have been found. The MW of the smallest is 300 daltons (Da) while the largest is 100 kDa. The value of the isoelectric point of the most abundant type of DLIF from untreated chicken plasma is 6.3 as determined by IEF. The partially purified DLIF inhibits Na +, K +-ATPase from a porcine cerebral cortex as well as three human red blood cell membrane preparations in a doseresponse fashion.

058 Endogenous digoxin-like immunoreactive factors (DLIF): Impact on the results of two homogeneous immunoassays for digoxin

058 Endogenous digoxin-like immunoreactive factors (DLIF): Impact on the results of two homogeneous immunoassays for digoxin

Isolation of digoxin-like immunoreactive factors from mammalian adrenal cortex

Endogenous digoxin-like immunoreactive factors (DLIF) are present in serum and tissues of humans and animals. To date, a tissue source for these factors has not been rigorously defined nor have these factors been isolated to identifiable homogeneity. In this study, we define the distribution of DLIF in mammalian tissues, demonstrate the adrenal cortex to be the principal source of this factor in bovine, and isolate DLIF to chromatographic homogeneity using high performance liquid chromatography (HPLC). DLIF concentrations in tissue extracts from rats measured as follows: adrenal glands, 44.3; serum, 6.3; liver, 5.2; kidney, 1.2; heart, brain, or lungs, less than 1.4 ng of digoxin-equivalent per g of protein. Human tissues showed similar results. In dogs, the ratio of the DLIF concentration in lumbar vein serum to that in infrarenal inferior vena cava serum was 3.3 +/- 0.4 (mean +/- S.E., n = 4). Bovine adrenal cortex contained 7 times more DLIF per g of tissue than the adrenal medulla. 70 +/- 4% (n = 7) of the total bovine cortical DLIF activity (6,159 pg of digoxin-equivalent) applied to a reverse phase HPLC column eluted as one definitive fraction. 60% of the digoxin-like immunoreactivity extracted from bovine serum also co-eluted with DLIF from adrenal. None of the 14 steroid molecules or 7 cardiac glycoside congeners co-eluted with the major DLIF activity. Our data indicate that 947 pmol of DLIF is equivalent to 1 pmol of digoxin-equivalent immunoreactivity. Preliminary mass spectral analysis suggests that purified DLIF has a molecular mass of 780 daltons comprised of one 390-dalton aglycone component plus several sugar moieties. This study establishes a definitive link between DLIF in serum and the adrenal cortex as a source tissue. We also demonstrate a method for purifying DLIF to chromatographic homogeneity with an extraction capacity of 1.2 nmol of DLIF per g of adrenal cortex.

Criteria for identifying endogenous compounds as digoxin-like immunoreactive factors in humans

Endogenous digoxin-like immunoreactive factors (DLIF) are factors in plasma that interact with anti-digoxin antibodies. In this report we propose specific empirical criteria that must be satisfied by any group of endogenous compounds purported to account for DLIF activity in human plasma. These criteria include immunoreactive potency relative to existing physiologic concentrations as well as the biochemical and protein binding properties of these compounds. Recent studies have identified several congeners of fatty acids and phospholipids, hydrocortisone, and dehydroepiandrosterone-sulfate as compounds likely to account for DLIF activity in plasma. Using the above criteria we demonstrate that the highest reported plasma concentrations of these compounds combined account for < 25% of DLIF reported in healthy adult subjects, < 11% in newborns, < 27% in pregnant women, and < 39% in patients with renal failure. Human serum albumin at a concentration of 40 g/l completely abolished any detectable interaction of these compounds with both anti-digoxin antibodies or canine kidney Na/K-ATPase. The immunoreactive and physical properties of these compounds are also not consistent with those reported for DLIF. We conclude that these compounds do not account for the plasma DLIF concentrations measured in human subjects nor are they likely to play a role as specific endogenous regulators of Na/K-ATPase.

Excretion of endogenous digoxin-like immunoreactive factors in human urine is a function of urine flow rate.

We studied the effect of varying water and salt intake on the renal excretion of endogenous digoxin-like immunoreactive factors (DLIF). DLIF were measured in human urine and serum by competitive displacement of 125I-labeled digoxin from anti-digoxin antibodies. Diuresis was selectively induced in normal healthy humans by acute water ingestion, and natriuresis was preferentially induced by acute saline ingestion. We found the amount of endogenous immunoreactivity excreted in urine to be correlated with urine flow rate but not with urinary sodium excretion. Urinary excretion of DLIF, normalized to creatinine, was 3.6-fold greater at a urine flow rate of 5.5 mL/min than at 0.5 mL/min. On the other hand, saline intake increased urine flow rate 1.9-fold and increased sodium excretion threefold, but did not affect urinary excretion of DLIF. Fractional excretion of DLIF was linearly related to fractional excretion of water. This study demonstrates that normalization of DLIF values to urinary creatinine does not make DLIF excretion independent of urine flow rate and underscores the need for information on urine flow rate when DLIF measurements in urine are being interpreted.

Improved interassay correlation of digoxin results in patients with and without renal failure by elimination of digoxin-like immunoreactive factors.

Use of immunoassays that do not detect endogenous digoxin-like immunoreactive factors (DLIF) in serum significantly improves the between-assay correlation of digoxin results for patients. We investigated five different immunoassay methods (Abbott, Clinical Assays, Corning, Du Pont, and Syva), measuring digoxin by all five assays in sera from 38 patients in renal failure and in 40 patients with normal renal function, all taking digoxin. The mean standard error of the estimate (Sy X x) of digoxin results (compared for all five assays) were significantly lower for patients with normal renal function than for patients in renal failure (0.148 vs 0.293 microgram/L, P less than 0.001). Assays previously shown (Clin Chem 1987;33:401) to be the least sensitive to DLIF (Syva and Corning) gave the lowest mean scatter about the regression (Sy X x = 0.192 microgram/L, renal failure; 0.114 microgram/L, normal renal function) for all 10 assay correlations. Evidently, discrepancies between digoxin values as measured by different immunoassay kits for patients with renal disease can be attributed to DLIF. Moreover, because inaccurate digoxin results attributed to DLIF may not be limited exclusively to groups of patients with known increased concentrations of DLIF, the possibility of "latent" DLIF interference may be a problem in many other human subjects.

Endogenous digoxin-like immunoreactive factors eliminated from serum samples by hydrophobic silica-gel extraction and enzyme immunoassay.

Elimination of endogenous digoxin-like immunoreactive factors (DLIF) that interfere with accurate measurement of digoxin requires use of a highly specific anti-digoxin antibody, or that DLIF be separated from digoxin before immunoassay. Several commercial digoxin-assay kits include a step for separating serum proteins and other substances from digoxin before immunoassay. We tested six different immunoassay methods (some having pretreatment steps) for their ability to detect DLIF in serum from patients in renal failure, pregnant women, and neonates, all of whom were not taking digoxin. Extracting digoxin on a column of derivatized silicagel eliminated detectable DLIF from serum as measured by enzyme immunoassay (EMIT; Syva Co.), but recovery of added digoxin was quantitative. In contrast, protein precipitation with 5-sulfosalicylic acid left significant amounts of DLIF in samples, most probably because the procedure (TDx assay; Abbott Labs.) disrupted protein-DLIF binding. A glass-bead radioimmunoassay (Immophase; Corning Medical) had the most digoxin-specific antisera. By preparative silica-gel-chromatography of serum we could eliminate or significantly minimize inaccurate digoxin measurements attributable to endogenous DLIF.

Endogenous digoxin-like immunoreactive factors: impact on digoxin measurements and potential physiological implications.

Various laboratories have reported endogenous digoxin-like immunoreactive factor(s) (DLIF) in blood from patients in renal failure or liver failure, from newborn infants, and from third-trimester pregnant women. Similar immunoreactivity has been detected in amniotic fluid, in cord blood, and in urine and serum from normal subjects. The factor(s) giving rise to this immunoreactivity cross react with antibodies used in many currently available immunoassays for digoxin, sometimes causing apparent digoxin concentrations exceeding the therapeutic range obtained for exogenous digoxin, with consequent errors in measurement and in subsequent clinical interpretation of digoxin results. Here, I summarize findings in our laboratory and those of others. DLIF evidently exist in three states in serum: tightly protein-bound, weakly protein-bound, and unbound (free). In normal subjects, greater than 90% of the total DLIF in serum is tightly but reversibly bound to serum proteins and is not readily detectable by direct measurement of digoxin in serum with conventional immunoassays. However, there seems to be a redistribution of the more weakly bound and unbound components in patients with renal failure, pregnant women, and newborns. The increased values detected in these groups are ascribable to increased amounts of weakly bound and unbound DLIF rather than to increased total DLIF. Carrier proteins may play a prominent role in the transport of these factors in blood. I discuss the potential physiological and pharmacological implications of detecting endogenous immunoreactive factors that cross react with antibodies to drugs.