Isolation and characterization of a digoxin-like immunoreactive substance from human urine by affinity chromatography.
Many studies [5,6, 11] have found variations of digoxin-like immunoreactive substance (DLIS) concentrations in biological fluids (e.g., plasma and urine) after various stimuli, supporting the hypothesis that this substance is more than an aspecific interference occurring during RIA. Although different studies demonstrated that the inhibitor of [Na.sup.+]/[K.sup.+] ATPase might have the same biochemical structure and function of digoxin, and, in particular, that this substance cross-reacts with antidigoxin antibody, there is no conclusive evidence that the substances that cross-react with antidigoxin antibody are inhibitors of [Na.sup.+]/[K.sup.+] ATPase. The present study was carried out to investigate the nature of the substance that cross-reacts with antidigoxin antibody in human urine with an affinity chromatography method. After a further purification by HPLC, we studied the effect of this substance on [Na.sup.+]/[K.sup.+] ATPase activity by different methods.
Materials and Methods
Urine was obtained from healthy male volunteers who were not under treatment with digitalis preparations or other drugs. Every aliquot of urine (300 mL) was neutralized (pH 7.4) with 2 mol/L NaOH and then centrifuged at 2000g for 15 min at room temperature. The supernatant was filtered (filter type HA 0.45 [micro]m; Millipore, Molsheim, France) and sodium azide (150 mg) added. Processed urine was added to an affinity chromatography column at a flow rate of 1 mL/min.
Two grams of CNBr-activated Sepharose[R] 4B (Pharmacia Biotech, Uppsala, Sweden) were suspended and washed on a sintered glass filter with 1 mmol/L HCI. The ligand was an antibody (antiserum), Sigma product #D7782 (Sigma Chemical Co., St. Louis, MO), developed in rabbit with digoxin-bovine serum albumin as the immunogen. It was dialyzed overnight with 0.1 mol/L NaH[CO.sub.3], pH 8.3, containing 0.5 mol/L NaCl. The ligand and the swelled Sepharose were rotated end-over-end for 2 h at room temperature. The excess ligand was washed away with 0.1 mol/L NaH[CO.sub.3], pH 8.3, containing 0.5 mol/L NaCl, and the remaining active group was blocked with 1 mol/L ethanolamine, pH 9, for 2 h at room temperature. The gel was washed with three cycles of alternating pH (0.1 mol/L acetate buffer containing 0.5 mol/L NaCl, pH 4, and 0.1 mol/L Tris buffer containing 0.5 mol/L NaCl, pH 8).
Each urine sample was loaded and run overnight and then eluted with a 0.1 mol/L glycine buffer containing 0.1 mol/L NaCl, pH 2.8, and quickly neutralized with 2 mol/L NaOH. Concentrated eluant from more urine samples were further purified by HPLC steps.
The reversed-phase HPLCs (Varian model 5000,Varian 2050 UV/VIS variable detector; Varian Chromatography Systems, Walnut Creek, CA; [micro]Bondapack C18 column, 30 cm x 4.6 mm; Waters Corp., Milford, MA) were run with a linear gradient [H.sub.2]O:[CH.sub.3]CN from 65:35 to 35:65 in 20 min at a flow rate of 1 mL/min. The absorbance was monitored at 214 nm. Fractions were collected every 0.5 min and then dried and reconstituted in 0.01 mol/L phosphate buffer containing 0.15 mol/L NaCl and 1 g/L sodium azide, pH 7.4, for RIA. Active fractions from different batches were pooled and rechromatographed on the same column and eluted with an isocratic program of [H.sub.2]O:[CH.sub.3]CN 72:28.
Cross-reactivity of the active fraction with antidigoxin antibody was measured by a RIA kit from DPC (Coat-A-Count[R] Digoxin; Diagnostic Products Corp., Los Angeles, CA). To prepare the calibration curve (from 0.150 to 10.2 nmol/L) we dissolved pure digoxin (Sigma) in absolute ethanol and prepared dilutions with 0.01 mol/L phosphate buffer containing 0.15 mol/L NaCl and 1 g/L sodium azide, pH 7.4. The test was performed with a solid phase RIA in which 125I-labeled digoxin competed, for 1 h at 37 [degrees]C, with cross-reacting substance, in the chromatographic fractions, or digoxin, in the calibrators, for antibody sites. After decanting the contents, the tubes were counted for 1 min in a gamma counter.
The ouabain-displacement activity of purified DLIS was measured by competitive binding of [[sup.3]H]ouabain with the active fraction to canine kidney [Na.sup.+]/[K.sup.+] ATPase (Sigma). A medium containing 50 mmol/L Tris-HCI, 1 mmol/L EDTA, 2 mmol/L ATP, 2 mmol/L Mg[Cl.sub.2], and 100 mmol/L NaCl, pH 7.4 (700 [micro]L) was prepared to incubate with 100 [micro]L of 2 x [10.sup.-9] mol/L [[sup.3]H]ouabain (NEN-Dupont, Boston, MA), 4 [micro]g of purified [Na.sup.+]/[K.sup.+] ATPase (100 [micro]L), 0.01, 0.1, 1, 10, or 100 pmol of ouabain, digoxin (Sigma) (100 [micro]L), or the same amount of digoxin equivalent of DLIS, or dilutions of a different chromatographic peak that elicited no DLIS activity. Incubation was performed for 60 min at 37 [degrees]C. The reaction was quenched by addition of 3 mL of ice-cold medium.
[sup.86]RB UPTAKE ASSAY
Sodium pump activity was estimated as ouabain-sensitive [sup.86]Rb uptake into human erythrocytes from healthy donors as previously described .
Erythrocytes were washed and suspended to 50% cells in HEPES buffer pH 7.4 (20 mmol/L HEPES, 1 mmol/L Ca[Cl.sub.2], 1 mmol/L Mg[SO.sub.4], 5 mmol/L Na[H.sub.2]P[O.sub.4], 138 mmol/L NaCl, 11 mmol/L glucose).
Aliquots of DLIS (0.01, 0.1, 1, and 2 pmol digoxin equivalents) or same amount of digoxin or ouabain were dried, reconstituted with 50 [micro]L of the same HEPES buffer, and incubated at 37 [degrees]C with 50 [micro]L of erythrocytes and 148 kBq (4 [micro]Ci)/mL [sup.86]Rb (NEN-Dupont) in 100 [micro]L for 90 min. The reaction was quenched with 750 [micro]L of HEPES buffer (4 [degrees]C). Cells were separated from medium by the addition of 250 [micro]L of 1:1 silicon:phthalate and spun in a microfuge (10 000g) for 2 min. The unbound counts were removed by aspiration and the cell pellets counted for [gamma]-emission.
COUPLED ENZYME ASSAY
[Na.sup.+]/[K.sup.+] ATPase activity was measured as the decrease in absorbance at 340 nm due to NADH oxidation as previously described by Haupert et al. . Aliquots of DLIS (0.01, 0.02, 0.04, 0.08, 0.16, 0.32, and 0.64 pmol of digoxin equivalent) or same amount of digoxin or ouabain were dried and reconstituted with 50 [micro]L of 20 mmol/L imidazole, 10 mmol/L Mg[Cl.sub.2], and 2 mmol/L [H.sub.3]P[O.sub.4], pH 7.4 and incubated at 37 [degrees]C for 30 min with 10 [micro]g of purified [Na.sup.+]/[K.sup.+] ATPase. Samples were cooled on ice and put into the assay solution prewarmed to 37 [degrees]C. The assay solution contained, in 1 mL, the following final concentrations (in mmol/L): NaCl 56, KCl 25, Mg[Cl.sub.2] 6, phosphoenolpyruvate 1.4, dithiothreitol 1, HEPES-triethylamine 20, disodium ATP 3, EGTA 0.1, NADH 0.26, pyruvate kinase 10 mg/L, and lactic dehydrogenase 10 mg/L. The absorbance was recorded (Lambda 3; Perkin-Elmer, Uberlingen, Germany) every 20 s after 30 s of incubation at 37 [degrees]C.
Positive material for digoxin RIA, as eluted during the first HPLC step, is shown in Fig. 1A. The retention time of ouabain and digoxin is shown (2.7 and 6.5 min) in the same Figure. We found a single peak with DLIS properties in human urine, and its retention time was identical to that of digoxin. Results of the second step of chromatography of this material are shown in Fig. 1B. A single major peak of activity was recovered at 17.5 min. Also, in this second elution program, DLIS had the same retention time of authentic digoxin, different from ouabain. The chromatographic data allow us to say that our isolation procedure, and in particular the antiserum to digoxin we used, was able to isolate one compound that coelutes with digoxin. The fact that the compound coelutes with digoxin does not prove that it is digoxin.
[FIGURE 1 OMITTED]
The [[sup.3]H]ouabam-binding inhibition showed a dose-response curve for DLIS (Fig. 2), whereas different chromatographic fractions treated with the same steps did not (data not shown). The dose-response curve of DLIS ran parallel to those of cold ouabain and digoxin.
Figure 3 shows the effect of DLIS on the ouabain-sensitive uptake of [Rb.sup.+] into human erythrocytes. The activity of [Na.sup.+]/[K.sup.+] ATPase was inhibited by DLIS in a dose-dependent manner while the enzyme was in its physiological site, the cytoplasmic membrane. Ouabain shows the highest inhibition of [Na.sup.+]/[K.sup.+] ATPase measured as ouabain-sensitive 86[Rb.sup.+] uptake. In comparison, DLIS seems to have a higher ability to inhibit the pump than authentic digoxin. Consistent with these findings, we observed a dose-dependent inhibition of enzyme activity also when [Na.sup.+]/[K.sup.+] ATPase was isolated and purified, and the activity was measured, as consumption of ATP, by coupled enzyme assay (Fig. 4). DLIS seems to be more effective than ouabain and digoxin in inhibiting Na'/K+ ATPase when the enzyme is isolated from its physiological environment. However, this higher activity could depend on an inadequate method of measuring DLIS. For example, the RIA method could not measure this substance 1 to 1 with digoxin, in this case indicating that much more of this factor may be required to produce the same degree of [[sup.3]H]ouabain displacement and a smaller degree of 86[Rb.sup.+] uptake, or alternatively these data could mean that the coupled enzyme assay is more effective to detect subtle differences in enzyme-inhibiting properties.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
Numerous endogenous digitalis-like substances have been purified or identified [1-3,10] from biological fluids or tissue. Depending on the study, the substance has been defined as ouabain-like or digoxin-like. No conclusive progress has been made in this field, and it is still not known whether the substance that cross-reacts with antidigoxin antibody is the endogenous inhibitor of [Na.sup.+]/[K.sup.+] ATPase or whether it only represents an interference during RIA. In the current study we answered this question. As the first step, we used an affinity chromatography column made with an antiserum to digoxin. This kind of approach allowed us to isolate and elute cross-reacting substances. Further steps in HPLC discriminate, with a RIA, specific reactive substances among eluted materials. Our results show that only one peak is significantly positive with RIA. However, we cannot infer that other DLISs retained by or measured by other digoxin antisera can be found. This urinary DLIS apparently shares chromatographic characteristics with authentic digoxin in two different elution programs. On the contrary, its retention time was considerably different from ouabain. These data, together with the cross-reactivity with antidigoxin antibody, suggest, but do not prove, that the DLIS we isolated is structurally similar to digoxin or is digoxin itself. In fact, in absence of a complete physicochemical characterization, our data cannot exclude the possibility that the compound we isolated with our procedure is not digoxin or a strictly related molecule.
[FIGURE 4 OMITTED]
The effect of purified DLIS on [Na.sup.+]/[K.sup.+] ATPase was evaluated with three different techniques. The dose-dependent [[sup.3]H]ouabain-binding inhibition shows that DLIS is able to interact at the specific glycoside-binding site on [Na.sup.+]/[K.sup.+] ATPase. Inhibition of ouabain-sensitive 86 [Rb.sup.+] uptake into human erythrocytes demonstrates the specific inhibitory activity of DLIS on [Na.sup.+]/[K.sup.+] ATPase, when the pump is embedded in its physiologic milieu. Finally, coupled enzyme assay shows the direct inhibitory action of DLIS on isolated and purified [Na.sup.+]/[K.sup.+] ATPase. Altogether, our results demonstrate that radioimmunoreactivity for digoxin in biological fluids is not a simple interference with substances of an unprecise nature, but is essentially due to the presence of the substance we isolated and that this substance is an effective inhibitor of [Na.sup.+]/[K.sup.+] ATPase.
The procedure of isolation exclusively based on the cross-reactivity with antidigoxin antibody is original and assures the correct approach to the aim of the present study. Our data confirm, with different methodologies and a different point of view, the findings of Goto et al. . However, the same author in another paper  studied two different digitalis-like factors in response to chronic alterations in dietary salt intake. The less polar compound was a DLIS and did not change after high sodium intake. These observations suggest that digoxin-like immunoreactivity may be different from natriuretic hormone. In contrast, our results indicate that DLIS is an inhibitor of [Na.sup.+]/[K.sup.+] ATPase and suggest some physiological importance. Furthermore, the role of DLIS in human diseases seems to be predicted mainly from isolated reports. Huang and Smith  reported high concentrations of DLIS in rats after coarctation of the aorta and hypertension had been reversed by administration of antidigoxin antibodies. Goodlin  reported a case of a pregnant woman with severe toxemia with hypertension unresponsive to therapy and very high serum digoxin concentration. Also in this case, the intravenous administration of the Fab fragment of antidigoxin antibodies (Digibind) determined a significant reduction in her blood pressure.
At present we cannot assert that this substance is produced in the human body or represents contamination from food, in particular, vegetables. Our previous findings [4, 6], as well as other reports [3, 5], show that plasma and urine concentrations of DLIS are modifiable with physiological or pathological changes of sodium intake or respiratory oxygen concentrations, suggesting a pathophysiological role of DLIS and its endogenous source.
However, the compound we isolated in the present study could not be the substance measured in previous studies, even if in our own studies [4, 6] we used exactly the same RIA procedure and antibody.
In conclusion, this study demonstrates that cross-reactivity with digoxin antibody is mainly due to a single substance that shares chromatographic characteristics with authentic digoxin. Furthermore, the study adds evidence that this substance is able to inhibit [Na.sup.+]/[K.sup.+] ATPase and suggests its physiological significance.
We thank Garner T. Haupert Jr., who taught us some of the techniques used in the present study, for helpful discussions. We also gratefully acknowledge the language assistance of Paul Codley.
Received October 21, 1996; revised April 14, 1997; accepted April 29, 1997.
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Claudio De Angelis,* Massimiliano Riscazzi, Riccardo Salvini, Alfonso Piccoli, (1) Claudio Ferri, (1) And Anna Santucci (2)
DASRS, Aerospace Medicine Department and Chemistry & Technology Department, Aeroporto Pratica di Mare, 00040 Pomezia, Roma, Italy.
(1) I Clinica Medica, Universita di Roma "La Sapienza", Viale del Policfinico, 00161 Roma, Italy.
(2) Department of Internal Medicine, Universita dell'Aquila, via S. Sisto n.
22, 67100 Aquila, Italy.
* Author for correspondence. Fax +39 6 9160 1079; e-mail email@example.com.
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|Title Annotation:||Drug Monitoring and Toxicology|
|Author:||De Angelis, Claudio; Riscazzi, Massimiliano; Salvini, Riccardo; Piccoli, Alfonso; Ferri, Claudio; Sa|
|Date:||Aug 1, 1997|
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