| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
DASRS, Aerospace Medicine Department and Chemistry & Technology Department, Aeroporto Pratica di Mare, 00040 Pomezia, Roma, Italy.
1
I Clinica Medica, Università di Roma "La Sapienza", Viale del Policlinico, 00161 Roma, Italy.
2
Department of Internal Medicine, Università
dell'Aquila, via S. Sisto n. 22, 67100 Aquila, Italy.
a Author for correspondence. Fax +39 6 9160 1079;
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Key Words: indexing terms: endogenous inhibitor • adenosine triphosphatase • radioimmunoassay
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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+/K+ 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+/K+ 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+/K+ ATPase activity by different methods.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
affinity chromatography
Two grams of CNBr-activated Sepharose® 4B
(Pharmacia Biotech, Uppsala, Sweden) were suspended and washed on a
sintered glass filter with 1 mmol/L HCl. 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 NaHCO3,
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 NaHCO3, 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.
hplc
The reversed-phase HPLCs (Varian model 5000,Varian 2050 UV/VIS
variable detector; Varian Chromatography Systems, Walnut Creek, CA;
µBondapack C18 column, 30 cm x 4.6 mm; Waters Corp., Milford,
MA) were run with a linear gradient
H2O:CH3CN 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 H2O:CH3CN 72:28.
ria
Cross-reactivity of the active fraction with antidigoxin antibody
was measured by a RIA kit from DPC (Coat-A-Count®
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 °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.
[3h]ouabain-binding inhibition
The ouabain-displacement activity of purified DLIS was measured by
competitive binding of [3H]ouabain with the active
fraction to canine kidney Na+/K+ ATPase
(Sigma). A medium containing 50 mmol/L Tris-HCl, 1 mmol/L EDTA, 2
mmol/L ATP, 2 mmol/L MgCl2, and 100 mmol/L NaCl, pH 7.4
(700 µL) was prepared to incubate with 100 µL of 2 x
10-9 mol/L [3H]ouabain (NEN-Dupont, Boston,
MA), 4 µg of purified Na+/K+ ATPase (100
µL), 0.01, 0.1, 1, 10, or 100 pmol of ouabain, digoxin (Sigma) (100
µL), 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 °C. The reaction was
quenched by addition of 3 mL of ice-cold medium.
86rb uptake assay
Sodium pump activity was estimated as ouabain-sensitive
86Rb uptake into human erythrocytes from healthy
donors as previously described (12).
Erythrocytes were washed and suspended to 50% cells in HEPES buffer pH 7.4 (20 mmol/L HEPES, 1 mmol/L CaCl2, 1 mmol/L MgSO4, 5 mmol/L NaH2PO4, 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 µL of
the same HEPES buffer, and incubated at 37 °C with 50 µL of
erythrocytes and 148 kBq (4 µCi)/mL 86Rb
(NEN-Dupont) in 100 µL for 90 min. The reaction was quenched with 750
µL of HEPES buffer (4 °C). Cells were separated from medium by the
addition of 250 µL 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 
-emission.
coupled enzyme assay
Na+/K+ ATPase activity was measured
as the decrease in absorbance at 340 nm due to NADH oxidation as
previously described by Haupert et al. (13). 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 µL of 20 mmol/L imidazole, 10 mmol/L
MgCl2, and 2 mmol/L H3PO4, pH 7.4
and incubated at 37 °C for 30 min with 10 µg of purified
Na+/K+ ATPase. Samples were cooled on ice and
put into the assay solution prewarmed to 37 °C. The assay solution
contained, in 1 mL, the following final concentrations (in mmol/L):
NaCl 56, KCl 25, MgCl2 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 °C.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A. 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.
|
The [3H]ouabain-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
86Rb+ into human erythrocytes. The
activity of Na+/K+ 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+/K+ ATPase measured as ouabain-sensitive
86Rb+ 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+/K+ 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 [3H]ouabain
displacement and a smaller degree of 86Rb+
uptake, or alternatively these data could mean that the coupled enzyme
assay is more effective to detect subtle differences in
enzyme-inhibiting properties.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The effect of purified DLIS on Na+/K+ ATPase was evaluated with three different techniques. The dose-dependent [3H]ouabain-binding inhibition shows that DLIS is able to interact at the specific glycoside-binding site on Na+/K+ ATPase. Inhibition of ouabain-sensitive 86Rb+ uptake into human erythrocytes demonstrates the specific inhibitory activity of DLIS on Na+/K+ 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+/K+ 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+/K+ 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. (10). However, the same author in another paper (14) 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+/K+ 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 (15) reported high concentrations of DLIS in rats after coarctation of the aorta and hypertension had been reversed by administration of antidigoxin antibodies. Goodlin (16) 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+/K+ ATPase and suggests its physiological significance.
|
Acknowledgments |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  M. A. El-Masri, B. J. Clark, H. M. Qazzaz, and R. Valdes Jr Human Adrenal Cells in Culture Produce Both Ouabain-like and Dihydroouabain-like Factors Clin. Chem., October 1, 2002; 48(10): 1720 - 1730. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Matthay, H. G. Folkesson, and C. Clerici Lung Epithelial Fluid Transport and the Resolution of Pulmonary Edema Physiol Rev, July 1, 2002; 82(3): 569 - 600. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, mean ± SE),
digoxin (
, mean ± SE), and DLIS (
, mean ± SE),
expressed as equivalents of authentic digoxin, in
[3H]ouabain displacement from dog kidney
Na+/K+ ATPase.
