Clinical Chemistry 47: 95-101, 2001;
(Clinical Chemistry. 2001;47:95-101.)
© 2001 American Association for Clinical Chemistry, Inc.
Radioiodinated Tyrosyl-Ouabain and Measurement of a Circulating Ouabain-like Compound
Olli Vakkuri1,1,a,
Sighvatur S. Arnason2,
Päivi Joensuu3,
Jorma Jalonen3,
Olli Vuolteenaho1 and
Juhani Leppäluoto1
1
Department of Physiology, University of Oulu, PO Box 5000, 90401 Oulu, Finland.
2
Department of Physiology, University of Iceland,
Vatnsmyrarvegi 16, IS-101 Reykjavik, Iceland.
3
Department of Chemistry, University of Oulu, PO Box 333,
90571 Oulu, Finland.
a Author for correspondence. Fax 358-08-5375320;
olli.vakkuri{at}oulu.fi
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Abstract
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Background: Assays for endogenous ouabain, a cardiac glycoside
believed to be involved in blood pressure and volume regulation, are
characterized by laboratory-specific plasma values that are measured by
different assays. Because of this variability, our study
focused on the development of a new 125I-labeled ouabain
derivative for RIA of high sensitivity.
Methods: We generated rabbit antisera against a
ouabain-thyroglobulin conjugate. A tyrosylated ouabain
derivative for radioiodination was synthesized using periodate
and sodium cyanoborohydride reagents.
Results: Mass spectrometric analyses showed that the main product
of the tyrosylating reaction was tyrosyl-ouabain (molecular mass, 702
Da). This was radioiodinated with Chloramine-T and used as a
tracer in a RIA, which gave an assay detection limit of 5
pmol/L (4 ng/L), 2–100 times lower than that in the corresponding
3H-RIAs and 2–20 times lower than ouabain ELISAs, making
it possible to measure low plasma concentrations of immunoreactive
ouabain. Different amounts of SepPak C18-extracted plasma
samples displaced the 125I-labeled tyrosyl-ouabain tracer
at the same rate at which authentic ouabain was displaced.
Plasma immunoreactive ouabain coeluted with authentic ouabain in two
different HPLC conditions. Using the new RIA, we found plasma ouabain
concentrations, assayed as immunoreactive equivalents, of 10.0 ±
1.3 pmol/L in healthy women and 12.0 ± 0.9 pmol/L in
healthy men (mean ± SE; n = 10), as well as 41.2 ± 9.6
pmol/L in rats. The concentrations were 2–90 times lower than those
previously reported using different assay methods.
Conclusions: Our ouabain 125I-RIA enables reliable
measurements of low endogenous concentrations of a ouabain-like
compound for both physiological and clinical purposes.
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Introduction
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A sodium-potassium pump inhibitor has been isolated and chemically
characterized from human plasma (1)(2), bovine
hypothalamus (3)(4)(5), and bovine adrenals
(6)(7). Several studies, especially one using
nanogram-scale chemical derivatization (4), have indicated
that the compound isolated from human plasma and bovine hypothalamus is
an isomer of ouabain, rather than ouabain itself. However, in recent
studies using microgram amounts of purified material
(5)(7), molecular mass and proton nuclear
magnetic resonance (1H
NMR)1
spectroscopic measurements have demonstrated that the adrenal
and hypothalamic sodium pump inhibitors are indistinguishable
from authentic ouabain. Because cardiac glycosides, such as ouabain,
inhibit Na+,K+-ATPase
(8) and thus increase intracellular sodium, they might have
a physiological role (e.g., during pregnancy) as endogenous natriuretic
and vasoactive substances. Increased concentrations of
immunoreactive ouabain have been observed in hypertensive
patients, indicating that the measurement of an endogenous ouabain-like
compound might have clinical interest (9)(10),
especially because there are studies showing an endogenous, i.e.,
adrenocortical, origin of this compound (6)(11).
According to data available, plasma immunoreactive ouabain
concentrations are laboratory-specific (12), indicating that
reliable measurement is not without problems. Cardiac glycosides
conjugated to proteins are good immunogens, and several groups have
been able to establish enzyme and
3H-immunoassays for the measurement of ouabain
immunoreactivity in body fluids (13)(14)(15)(16)(17)(18). RIAs and ELISAs
are notoriously prone to nonspecific interference when used to measure
picomolar, i.e., low endogenous concentrations of biologically active
substances. This may be a reason for the different plasma ouabain
equivalents reported previously, varying from <5 pmol/L
(15) to 100–300 pmol/L
(14)(17)(18) to 
1000 pmol/L
(13) for human plasma, and from 
50 pmol/L (19)
to 200 pmol/L for rat plasma (18) in solid-phase-extracted
samples. It has even been reported that the immunoreactive ouabain in
human plasma is not authentic ouabain because in some HPLC analyses of
plasma extracts, no immunoreactivity was observed at the elution
position of authentic ouabain (15)(20). These
discrepant results indicate the need for further development of ouabain
assay methods suitable for physiological and clinical studies.
In the present study, we raised in rabbits high-affinity polyclonal
antisera against ouabain and synthesized a new
125I-labeled tyrosyl-ouabain derivative for use
as a tracer of high specific activity. The new RIA could be used, in
conjunction with solid-phase extraction, for routine measurement of an
endogenous ouabain-like compound present in biological samples.
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Materials and Methods
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materials
Ouabain (G-strophanthin) octahydrate, ouabagenin, dihydroouabain,
digoxin, digitoxin, digoxigenin, and other related steroids
(aldosterone, bufalin, cortisone, hydrocortisone, progesterone,
ß-estradiol, estrone, testosterone, and dehydroepiandrosterone),
bovine thyroglobulin, and sodium cyanoborohydride were obtained from
Sigma. Freund’s incomplete and complete adjuvants were
purchased from Difco Laboratories, L-tyrosine and
polyethylene glycol 6000 were from Fluka AG,
[3H]ouabain
([21,22-3H]ouabain) and
Na125I (IMS 300) were from Amersham Pharmacia
Biotech, and Sephadex G-50F was from Pharmacia Fine Chemicals.
NaIO4, Chloramine-T, and all other
chemicals were obtained from E. Merck AG.
preparation of ouabain antigen and antisera
Ouabain was coupled with bovine thyroglobulin as described by
Masugi et al. (13). Ouabain (29 mg) was allowed to react
with NaIO4 (11 mg) in distilled water overnight
at 4 °C. Ethanol (1 mL) was then added, and the reaction solution
was adjusted to pH 8 with 1 mol/L NaOH. Finally, bovine thyroglobulin
(20 mg dissolved in 1 mL of 0.05 mol/L sodium phosphate, pH 9) was
added dropwise to 1 mL of the reaction solution (14.5 mg of ouabain and
5.5 mg of NaIO4). After vortex-mixing and
incubation for 2 h, sodium cyanoborohydride (2.5 mg in 200 µL of
distilled water) was added and the reaction solution was applied to a
gel filtration column (Sephadex G-50F in 9 g/L NaCl) for
fractionation. The ouabain-thyroglobulin conjugate, eluting in the void
volume, was emulsified in Freund’s complete adjuvant and injected into
five rabbits (1 mg of conjugate per rabbit). Booster injections of 0.5
mg per rabbit, emulsified in Freund’s incomplete adjuvant, were given
at monthly intervals. As measured by the absorbance at 219 nm, the
hapten-carrier ratio of the conjugate was 160:1.
preparation of radioiodinated ouabain tracer
Ouabain was first coupled with L-tyrosine with a
method analogous to the one described above. After the reaction of
ouabain (3.1 mg) with NaIO4 (1.4 mg) in distilled
water (0.1 mL), ethanol was added to the reaction solution, and the pH
was adjusted to 8. L-Tyrosine (0.8 mg in 0.4 mL of 0.05
mol/L sodium phosphate, pH 9) was then added dropwise, followed by
incubation for 2 h. Sodium cyanoborohydride (0.25 mg in 0.2 mL of
distilled water) was added, and the reaction solution was fractionated
by reversed-phase HPLC [Vydac C18 column; 30-min
linear 5–50% methanol gradient in 0.5 mL/L trifluoroacetic
acid (TFA); flow-rate, 1.0 mL/min]. The main peak with
ultraviolet (UV) absorbance in the HPLC profile of the
tyrosylating solution (23 min) was used for radioiodination as follows:
Tyrosyl-ouabain (1 µg) was allowed to react with
Na125I (19 MBq) in the presence of Chloramine-T
(10 µg); after 30 s, sodium metabisulfite (10 µg) was added,
and the reaction solution was applied into the HPLC column (Vydac
C18) for reversed-phase fractionation (30-min
10–40% acetonitrile gradient in 1 mL/L TFA; flow-rate, 1.0
mL/min).
mass spectrometric analyses of tyrosyl-ouabain
The main peak (23 min), as detected by UV absorbance, in the HPLC
separation of the tyrosylating reaction products of ouabain was further
purified in another HPLC using a Waters SymmetryShieldTM
RP8 column and a 30-min gradient from 0% to 30% methanol
in distilled water with a flow rate of 1.0 mL/min. The product (0.2–10
µg) was analyzed with a quadrupole time-of-flight tandem instrument
(Micromass Ltd) equipped with electrospray ionization and atmospheric
pressure chemical ionization (APCI) sources. The sample was
infused at a 1:1000 dilution in acetonitrile-water (50:50 by volume)
containing 2 mL/L formic acid into the electrospray source,
using a syringe pump (Harward Apparatus).
SepPak SOLID-PHASE EXTRACTION OF PLASMA SAMPLES
Human blood from healthy adults taking no medication was collected
from an antecubital vein into EDTA tubes at 9–10 h. Rat blood
was obtained by decapitation of adult Sprague–Dawley rats of both
sexes at 9–10 h. The blood samples were immediately
centrifuged, and the plasmas were stored at -20 °C. For ouabain
measurements, the plasma samples were extracted using SepPak
C18 cartridges (Waters) and an automated Gilson
Aspec system. Briefly, the cartridges were preconditioned with
2-propanol and 1 mL/L TFA. The 1-mL plasma samples were
acidified with 0.2 mL of 1 mol/L HCl containing 16 g/L glycine and
passed through the cartridges. After a 2-mL wash with 1 mL/L TFA,
ouabain was eluted with 3 mL of 400 mL/L acetonitrile in 1
mL/L TFA. After evaporation in a SpeedVac (Savant Instruments),
the extracts were reconstituted in 250 µL of RIA buffer.
ria procedure
The calibrators and samples were pipetted in duplicates
of 100 µL. The antiserum (a-ouabain-199-13-4-95) and tracer were
added simultaneously (total volume added, 100 µL). After an
overnight incubation, the bound and free fractions were separated by
double-antibody precipitation for 15 min at room temperature.
Precipitation was accelerated by the addition to the assay tubes of
polyethylene glycol 6000 in a final concentration of 57 g/L.
The precipitates were counted in a CliniGamma gamma counter (Wallac).
For 3H measurements, the precipitates were
resuspended in 250 µL of distilled water, transferred into counting
vials containing scintillation fluid, and counted in a Wallac beta
counter (16).
ria validation
Serial dilutions of SepPak extracts of human and rat plasma were
assayed against the ouabain calibrator. Plasma extracts were also
analyzed by reversed-phase HPLC, using a 4.6 x 150 mm Vydac
C18 218 TP column (Separations Group) and a
30-min linear gradient from 5% to 50% acetonitrile in 1 mL/L TFA with
a flow rate of 1.0 mL/min. The fractions were dried in a SpeedVac,
redissolved in RIA buffer, and subjected to the ouabain RIA. From a
selected chromatographic run, one-third of the peak fraction with
ouabain immunoreactivity was reanalyzed in another HPLC system with
different selectivity, using a 4.6 x 250 mm Vydac
C8 228TP pH-stable column and a 30-min
linear gradient from 0% to 30% acetonitrile in 1 mL/L TFA
with the flow rate of 1.0 mL/min. Blank runs, using water as the
sample, were carried out for both HPLC systems to control for
background immunoreactivity.
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Results
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antiserum titer
All five rabbits produced ouabain antisera against the
ouabain-thyroglobulin conjugate. After the first booster, the titers
were 1:3000–1:7000 with the tritiated tracer and
1:300 000–1:1 000 000 with the radioiodinated tracer. The
titer could be maintained or even increased for at least 1 year
with monthly boosters (Table 1
). The antiserum bleed selected for RIA use (see below) bound
30% of the 125I-labeled tyrosyl-ouabain tracer
(estimated specific activity, 2100 Ci/mmol) at a final dilution of
1:2 000 000 (Table 1
). By comparison, a dilution of 1:14 000 of the
same antiserum was required to bind 30% of
[3H]ouabain tracer (specific activity reported
by the manufacturer, 20 Ci/mmol).
radioiodinated ouabain tracer
The coupling of tyrosine to ouabain yielded several products
separable by HPLC. As seen in Fig. 1
, the main peak, as detected by UV absorbance, in the first HPLC
purification step eluted at 23 min, 5 and 15 min later than ouabain and
tyrosine, respectively. Radioiodination of the main peak and subsequent
purification by reversed-phase HPLC yielded a radioactive product that
showed high binding affinity to the ouabain antisera (Table 1
). The
125I-labeled tyrosyl tracer was relatively stable
and could be used in the RIA for 4 months when stored at 10 °C.
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Figure 1. HPLC separation of periodate/cyanoborohydride reaction
products of ouabain and tyrosine using a Vydac C18 column
and a 30-min linear gradient from 5% to 50% acetonitrile in 0.5
mL/L TFA.
Tyrosine, ouabain, and the main reaction product eluted as peaks at 8,
18, and 23 min, respectively, as measured by UV absorbance at 220 nm
(A220).
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mass spectrum of tyrosyl-ouabain
The APCI mass spectrum of the main HPLC peak of the coupling
reaction solution is shown in Fig. 2
. The mass of the suggested protonated molecule was 702 Da.
Furthermore, the exact mass measurements done using the electrospray
mode showed that the [M-H]+ ion of
tyrosyl-ouabain was 702.3510 Da, corresponding to the molecular formula
of
C37H52O12N.
Tandem mass spectrometric measurement revealed two main fragments at
m/z 439.2352
(C23H35O8)
and at m/z 264.1236
(C14H18O4N),
corresponding to cleavage of the glycosidic bond with a proton left on
either side. Fig. 3
shows the postulated reaction mechanism of ouabain and tyrosine
based on the schedule of Harris et al. (14). The schedule
leads to a tyrosyl-ouabain conjugate with a formula mass of 702 Da.
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Figure 2. APCI mass spectrum of the main coupling product of ouabain
and tyrosine purified in two HPLC systems.
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Figure 3. Postulated two-phase reaction mechanism in the coupling of
ouabain and tyrosine by periodate
(NaIO4) and sodium cyanoborohydride
(NaBH3CN) reagents.
Carbon, hydrogen, and oxygen losses are indicated in the
boxes.
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assay characteristics
As seen in Table 1
, the assay detection limit (defined at
5% displacement of tracer) for each antiserum was 1.8 fmol/tube
(1.3 pg/tube), which corresponded to a plasma concentration of 5
pmol/L (3 ng/L). The 50% displacement was achieved at 6–14 fmol
(4–10 pg). Fig. 4
demonstrates that the assay detection limit is 0.6 fmol/tube
(0.4 pg/tube) with the 125I-labeled tyrosyl
tracer and 44 fmol/tube (32 pg/tube) with the 3H
tracer, using the antiserum a-ouabain-199 selected for RIA use. The
displacement was linear between 3 and 50 fmol. The specificities of the
different antisera were tested with ouabain, digitalis, and related
compounds as well as with several steroids. The cross-reactivity data
of a-ouabain-199 are presented in Table 2
. Ouabagenin showed the highest cross-reactivity (52%),
followed by digoxin (1.7%) and digitoxin (0.6%), whereas the
cross-reactivity of all steroids was <0.001%. The intraassay CVs for
low (6.5 pmol/L), medium (37 pmol/L), and high (168 pmol/L) ouabain
concentrations were 8.5%, 3.7%, and 4.3%, respectively (n =
10). The interassay CVs for low (15 pmol/L), medium (42
pmol/L), and high (167 pmol/L) ouabain concentrations were
13%, 9.4% and 10%, respectively (n = 10). Nonspecific
binding was 1–2%.
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Table 2. Cross-reactivity of various analogs and other related
compounds of ouabain with ouabain antiserum
a-ouabain-199.1
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ria validation
Different amounts of plasma solid-phase extracts displaced
125I-labeled tyrosyl-ouabain tracer in parallel
with the authentic ouabain calibrator (Fig. 4
). Immunoreactive ouabain
in human and rat plasma extracts coeluted with authentic
ouabain in two different reversed-phase HPLC conditions (at 14
min in the first and 12 min in the second HPLC system; see Fig. 5
). The recovery of added ouabain in a HPLC test run was 89.6%.
No ouabain immunoreactivity was detected in the blank samples.
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Figure 5. HPLC characterization of ouabain immunoreactivity of human
[A and B (as inset)] and
rat (C) plasma extracts.
SepPak-extracted plasma samples (6–12 mL) were chromatographed
in a reversed-phase HPLC using a Vydac C18 column and a
30-min 5–50% acetonitrile gradient in 1 mL/L TFA (A
and C) or a Vydac pH-stable column and a 30-min 0–30%
acetonitrile gradient in 0.1 mol/L NH4HCO3
(B). (- - - -), detection limit of the ouabain RIA.
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plasma concentrations of immunoreactive ouabain in healthy humans
and rats
The recovery of ouabain added to human plasma samples (1 mL) from
solid-phase extraction with SepPak C18 cartridges
was 102.8% ± 2.5% (mean ± SE; n = 5) for a low dose (34
fmol added) and 93.6% ± 1.3% for a high dose (103 fmol added). In
accordance with these results, the recovery in acidic extraction
conditions with respect to neutral conditions (HCl and TFA replaced
with distilled water) was 97.5% ± 2.1% (mean ± SE; n =
6). Plasma samples (1 mL) from healthy adult women and men and from
adult rats of both sexes were extracted by the solid-phase cartridges
and measured in the ouabain RIA. The plasma concentrations were
10.0 ± 1.3 pmol/L (mean ± SE) in women 22–45 years of age
(n = 10), 12.0 ± 0.9 pmol/L in men 23–55 years of age
(n = 10), and 41.2 ± 9.6 pmol/L in rats.
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Discussion
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In the present study, we developed a
125I-RIA for ouabain measurements with a
detection limit almost 100-fold lower than that of our corresponding
3H-RIA. The key components in our method are the
new 125I-labeled tyrosyl-ouabain tracer and the
high-affinity ouabain antiserum. The work was based on our previous
results involving the synthesis and use of a radioiodinated tracer in
melatonin RIA (21)(22). We adopted a
modification of the method of Masugi et al. (13) to make the
ouabain-thyroglobulin conjugate, which was used as an immunogen, and to
make the ouabain-tyrosine conjugate, which was used to prepare
radioiodinated tracer for the RIA. In both cases, the insertion
probably takes place in the sugar moiety of ouabain. This is supported
by the cross-reactivity profile of the ouabain antiserum
(a-ouabain-199) raised against the ouabain-thyroglobulin conjugate,
which demonstrated the highest cross-reactivity (52%) to the aglycone
ouabagenin. The steroid nucleus with its hydroxyl groups and apparently
also the lactone ring thus form the antigenic determinant. Previously,
Takemura et al. (23) used periodate to cleave the sugar ring
of digoxin. However, they used 2-hydroxy-3-methylbenzoyl-hydrazide,
instead of sodium cyanoborohydride, in the preparation of a derivative
for radioiodination. This derivatization did not eliminate the biologic
or immunologic activity of digoxin. In some digoxin assays,
radioiodinated tracers have also been prepared by coupling digoxigenin
to tyrosine via succination without periodation
(24).
We characterized the structure of the synthesized putative
tyrosyl-ouabain by mass spectrometry. The product was first
purified to homogeneity by reversed-phase HPLC. APCI analysis gave a
molecular ion mass of 702 Da. Interestingly, this is consistent with
the reaction mechanism proposed by Harris et al. (14) for
ouabain and bovine serum albumin. In this proposed two-phase reaction
mechanism, the rhamnose moiety of ouabain is first opened by
periodation, losing a carbon atom, a water molecule, and two
hydrogen atoms. Subsequently, in the coupling phase, the side
chain of the tyrosine molecule is attached to the opened rhamnose
moiety of ouabain when two more oxygen atoms are lost. The mechanism
would provide a molecular mass of exactly 702 Da to the
product, tyrosyl-ouabain.
Several studies have shown that human plasma contains a cardiac
glycoside, i.e., a ouabain-digitalis-like factor, the structure of
which is indistinguishable from plant-derived ouabain as judged from
various HPLC, mass spectrometric, and RIA analyses
(1)(2)(13)(14)(17)(18)(25)(26)(27). However, a mass spectrometric
analysis by Mathews et al. (27) raised the possibility of
slight differences in hydroxylation positions of the steroid nucleus of
the human plasma compound. Initially, this review was reinforced by
studies using nanoscale fluorescent naphthoylate derivatives of the
human and bovine compounds (3)(4). However,
recent NMR studies (5) showed that the bovine compounds
formed complexes with borate under isolation conditions and NMR
processes carried out in borosilicate glassware
(4)(5). Therefore, although not confirmed by
direct observation, it was inferred that the bovine hypothalamic
compound as isolated would have been indistinguishable from ouabain.
Furthermore, another recent study based on mass spectrometric and
1H NMR analyses showed that the compound isolated
from the bovine adrenal glands was also indistinguishable from ouabain
(7). Thus, it now seems that the endogenous bovine adrenal
and hypothalamic ouabain-like material is ouabain (circulating compound
remains to be resolved).
Circulating ouabain or ouabain-like activity has been measured by many
techniques, including bioassays, radioreceptor assays using membrane
fractions from several tissues (28), and ouabain and digoxin
immunoassays. The bioassay methods, which are based on
Na+,K+-ATPase inhibition
(ATP hydrolysis by the sodium pump or the inhibition of the in-trans
location activity of the
Na+,K+-ATPase) are
ouabain-specific and have been used to verify the authenticity of
isolated endogenous compounds. These methods are not ideal for routine
measurements because they are cumbersome and have lower sensitivities
than immunoassays.
The available ouabain immunoassays have recently been compared in
reviews by Semra et al. (16) and by us (12).
Although the best enzyme immunoassays and RIAs are believed to
possess detection limits (10–30 pmol/L) suitable for
physiological studies, widely varying plasma concentrations, ranging
from 0.04 to 1.1 nmol/L, have been reported for healthy adults in
different studies (13)(14)(15)(16)(17)(18). Moreover, no circulating ouabain
immunoreactivity was detected in some studies
(15)(20). These laboratory-specific results
might be related to differences in antiserum specificity and
sensitivity; the use of different tracers, generally
[3H]ouabain and enzyme-linked ouabain analogs;
the use of different sample volumes, causing variable matrix effects;
and/or the presence of potential interfering factor(s) in plasma,
leading to overestimation of concentrations
(17)(28). Because of the laboratory-specific
differences, a reference interval of normal
concentrations cannot be reliably inferred, especially when
sufficient validation has not been carried out in all studies regarding
the substance measured in the assay. Thus, at present, ouabain
equivalents are preferred over ouabain, meaning that from a
methodological perspective, plasma measurements are lacking a
laboratory routine.
Therefore, it was necessary for us to carry out several validation
tests giving special attention to the identity of plasma
immunoreactivity measured in our RIA. We found that the extraction of
plasma samples is essential for valid measurements. The SepPak
C18 reversed-phase extraction method provided
necessary purification and a nearly complete recovery of ouabain added
to human plasma and rat samples. Validation of quantification of plasma
immunoreactive ouabain was demonstrated by the parallel dilution of
plasma extracts with the ouabain calibrator. Furthermore, additional
support for the analytical validity of the measurements was obtained by
the finding that immunoreactive ouabain in human plasma extracts
coeluted with authentic ouabain in two reversed-phase HPLC systems with
different selectivities. Moreover, our assay detected the previously
isolated ouabain-like compound from human plasma (endogenous ouabain
discovered by Hamlyn) with high cross-reactivity
(12).
The results obtained with our new sensitive RIA support previous
findings that ouabain itself or ouabain-like compound is present in
normal human plasma (1)(2)(13)(14)(18)(26)(27). In the
present study, we were able to detect immunoreactive ouabain both
before and, unlike in the study of Lewis et al. (15),
after HPLC fractionation. According to our plasma measurements,
however, immunoreactive ouabain concentrations are low in healthy
adults, 1.5- to 3-fold lower than those reported in the most sensitive
enzyme immunoassay-based and 3H-RIA
studies (19)(29)(30).
In conclusion, we have synthesized and characterized by mass
spectrometry a tyrosyl-ouabain conjugate suitable for radioiodination.
The new molecule made possible development of a ouabain
125I-RIA with superior sensitivity. Coupled with
solid-phase extraction of plasma samples, the assay reliably detects
immunoreactive ouabain concentrations of 5 pmol/L. In our HPLC and
other validation tests, this immunoreactive ouabain behaved like
authentic ouabain. Thus, our RIA allows measurement of the proposed low
endogenous ouabain equivalents in both physiological and
pathophysiological situations. The new method provides an analytical
tool with which it is possible to establish the potential homeostatic
and clinical significance of endogenous ouabain.
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Acknowledgments
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We thank Tuula Lumijärvi and Micromass Ltd application
laboratory for expert assistance and support. Sighvatur S. Arnason was
supported by the Icelandic University Research Fund.
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Footnotes
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1 Nonstandard abbreviations: NMR, nuclear magnetic resonance; TFA, trifluoroacetic acid; UV, ultraviolet; and APCI, atmospheric pressure chemical ionization. 
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References
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|---|
-
Hamlyn JM, Blaustein MP, Bova S, DuCharme DW, Harris DW, Mandel F, et al. Identification and characterization of a ouabain-like compound from human plasma. Proc Natl Acad Sci U S A 1991;88:6259-6263.[Abstract/Free Full Text]
-
Ludens JH, Clark MA, DuCharme DW, Harris DW, Lutzke BS, Mandel F, et al. Purification of an endogenous digitalis-like factor from human plasma for structural analyses. Hypertension 1991;17:923-929.[Abstract/Free Full Text]
-
Tymiak AA, Norman JA, Bolgar M, DiDonato GC, Lee H, Parker WL, et al. Physicochemical characterization of a ouabain isomer isolated from bovine hypothalamus. Proc Natl Acad Sci U S A 1993;90:8189-8193.[Abstract/Free Full Text]
-
Zhao N, Lo LC, Berova N, Nakanishi K, Tymiak A, Ludens JH, Haupert GT. Na-K ATPase inhibitors from bovine hypothalamus and human plasma are different from ouabain: nanogram scale CD structural analysis. Biochemistry 1995;34:9893-9896.[Medline]
[Order article via Infotrieve]
-
Kawamura A, Guo J, Itagaki Y, Bell C, Wang Y, Haupert GT
Jr, et al. On the structure of endogenous ouabain. Proc Natl Acad Sci
U S A 1999;96:6654–9..
-
Laredo J, Hamilton BP, Hamlyn JM. Ouabain is secreted by bovine adrenocortical cells. Endocrinology 1994;135:794-797.[Abstract]
-
Schneider R, Wray V, Nimtz M, Lehmann WD, Kirch U, Antolovic R, Schoner W. Bovine adrenals contain in addition to ouabain, a second inhibitor of the sodium pump. J Biol Chem 1998;273:784-792.[Abstract/Free Full Text]
-
Goto A, Yamada K, Yagi N, Yoshioka M, Sugimoto T. Physiology and pharmacology of endogenous digitalis-like factors. Pharmacol Rev 1992;44:377-399.[Web of Science][Medline]
[Order article via Infotrieve]
-
Hamlyn JM, Lu ZR, Manunta P, Ludens JH, Kimura K, Shah JR, et al. Observations of the nature, biosynthesis, secretion and significance of endogenous ouabain. Clin Exp Hypertens 1998;20:523-533.
-
Doris PA, Bargrov AY. Endogenous sodium pump inhibitors and blood pressure regulation: an update on recent progress. Proc Soc Exp Bio Med 1998;218:156-167.[Medline]
[Order article via Infotrieve]
-
Perrin A, Brasmes B, Chambaz EM, Defayne G. Bovine adrenocortical cells in culture synthesize an ouabain-like compound. Mol Cell Endocrinol 1997;126:7-15.[Web of Science][Medline]
[Order article via Infotrieve]
-
Vakkuri O, Arnason SS, Pouta A, Vuolteenaho O, Leppäluoto J. Radioimmunoassay of plasma ouabain in healthy and pregnant subjects. J Endocrinol 2000;165:669-677.[Abstract]
-
Masugi F, Ogihara T, Hasegawa T, Tomii A, Nagano M, Higashimori K, et al. Circulating factor with ouabain-like immunoreactivity in patients with primary aldosteronism. Biochem Biophys Res Commun 1986;135:41-45.[Web of Science][Medline]
[Order article via Infotrieve]
-
Harris DW, Clark MA, Fisher JD, Hamlyn JM, Kolbasa KP, Ludens JH, DuCharme DW. Development of an immunoassay for endogenous digitalis-like factor. Hypertension 1991;17:936-943.[Abstract/Free Full Text]
-
Lewis LK, Yandle TG, Lewis JG, Richards AM, Pidgeon GB, Kaaja RJ, Nicholls MG. Ouabain is not detectable in human plasma. Hypertension 1994;24:549-555.[Abstract/Free Full Text]
-
Semra YK, Butt AN, Swaminathan R. Effect of salt intake on excretion of endogenous ouabain-like substance, measured by RIA. Clin Chem 1996;42:1949-1954.[Abstract/Free Full Text]
-
Harwood S, Little JA, Gallacher G, Perrett D, Edwards R, Dawnay A. Development of enzyme immunoassay for endogenous ouabain-like compound in human plasma. Clin Chem 1997;43:715-722.[Abstract/Free Full Text]
-
Ferrandi M, Manunta P, Balzan S, Hamlyn JM, Bianchi G, Ferrari P. Ouabain-like factor quantification in mammalian tissues and plasma: comparison of two independent assays. Hypertension 1997;30:886-896.[Abstract/Free Full Text]
-
Gomez-Sanchez EP, Foeking MF, Sellers D, Blankenship MS, Gomez-Sanchez CE. Is the circulating ouabain-like compound ouabain?. Am J Hypertens 1994;7:647-650.[Web of Science][Medline]
[Order article via Infotrieve]
-
Doris PA, Jenkins LA, Stocco DM. Is ouabain an authentic endogenous mammalian substance derived from the adrenal?. Hypertension 1994;23:632-638.[Abstract/Free Full Text]
-
Vakkuri O, Leppäluoto J, Vuolteenaho O. Development and validation of a melatonin radioimmunoassay using radioiodinated melatonin as tracer. Acta Endocrinol 1984;106:152-157.
-
Vakkuri O, Lämsä E, Rahkamaa E, Ruotsalainen H, Leppäluoto J. Iodinated melatonin: preparation and characterization of the molecular structure by mass and 1H NMR spectroscopy. Anal Biochem 1984;142:284-289.[Web of Science][Medline]
[Order article via Infotrieve]
-
Takemura Y, Fujibayashi Y, Dote N, Taniuchi H, Iijima N, Konishi J, Yokoyama A. Development of glycoside-bound radiopharmaceuticals: novel radioiodination method of digoxin. Biol Pharm Bull 1994;17:97-101.[Web of Science][Medline]
[Order article via Infotrieve]
-
Barbieri U, Gandolfi C. Digoxin radioimmunoassay: a comparison of the assay performances using tritiated and 125I radioiodinated tracers. Clin Chim Acta 1977;77:257-267.[Web of Science][Medline]
[Order article via Infotrieve]
-
Bernini G, Paci A, Sgro M, Moretti A, Salvetti A. Endogenous digitalis-like factor and ouabain immunoreactivity in adrenalectomized patients and normal subjects after acute and prolonged salt loading. Am J Hypertens 1998;11:1-7.[Web of Science][Medline]
[Order article via Infotrieve]
-
Butt AN, Semra YK, Lane SJ, Lee T, Swaminathan R. Endogenous ouabain secretion in man is not regulated by ACTH. J Steroid Biochem Mol Biol 1998;66:151-157.[Web of Science][Medline]
[Order article via Infotrieve]
-
Mathews WR, DuCharme DW, Hamlyn JM, Harris DW, Mandel F, Clark MA, Ludens JH. Mass spectral characterization of an endogenous digitalis-like factor from human plasma. Hypertension 1991;17:930-935.[Abstract/Free Full Text]
-
Paci A, Ledda A, Ciarimboli G, Pascal B, Bernini GP. Commercial enzyme immunoassay reagent pack for ouabain compared with human placenta radioreceptor assay. Clin Chem 1996;42:648-650.[Free Full Text]
-
Naruse K, Naruse M, Tanabe A, Yoshimoto T, Watanabe Y, Kurimoto F, et al. Does plasma immunoreactive ouabain originate from the adrenal gland?. Hypertension 1994;23(Suppl 1):I102-I105.
-
Balzan S, Montali U, DiBartolo V, Ghione S. Further evidence for an endogenous digitalis-like compound in newborn and adult plasma detected by antiouabain antiserum. Life Sci 1997;60:893-898.[Web of Science][Medline]
[Order article via Infotrieve]
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Abstract
Introduction
), as well as a displacement curve of
125I-labeled tyrosyl-ouabain with different amounts of
human plasma extracts (
).