(Hypertension. 2000;36:1059.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
Application of Supercritical Fluid Chromatography to Characterize a Labile Digitalis-Like Factor
From the Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah (S.W.G.); the Department of Analytical Chemistry, Uppsala University, Uppsala, Sweden (K.E.M.); and the Department of Radiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass (N.K.H.).
Correspondence to Dr Steven W. Graves, Department of Chemistry and Biochemistry, Brigham Young University, BNSN C-212, Provo, UT 84602. E-mail swgraves{at}chemdept.byu.edu
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Abstract |
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Abstract—A sodium pump inhibitor (digitalis-like factor), isolated from the peritoneal dialysate of volume-expanded, hypertensive patients with kidney failure who were treated with this dialysis modality, was further purified and characterized by means of supercritical fluid chromatography, a separation technique whose application to very-low-concentration biomolecules is new. Previous studies suggested that after high-performance liquid chromatography (HPLC) purification, this inhibitor was the only factor correlated with volume status and blood pressure in these patients. When this same HPLC fraction was furthered purified on 2-dimensional supercritical fluid chromatography, a single peak coeluted with [Na,K]ATPase inhibitory activity. When split specimens were used, there was a strict correlation between the peak area, measured by flame ionization detection, and activity (n=10, R=0.98, P=0.00001). Inhibitory activity after supercritical fluid chromatography was still correlated with the degree of volume expansion of donor patients (P=0.01). After HPLC purification, this volume-sensitive inhibitor was chemically labile. With further purification on supercritical fluid chromatography, the active peak was still labile with comparable half-life. Supercritical fluid chromatography coupled with flame ionization detection provided an estimate of the amount of the inhibitor present. Again using split specimens, we determined that the labile digitalis-like factor was
30-fold more effective than ouabain in
inhibiting renal [Na,K]ATPase activity and 
500 times more effective
than ouabain in causing vascular smooth muscle contraction. The data
suggest that we have purified to homogeneity a labile digitalis-like
factor that is readily distinguished from ouabain or bufalin, based on
chromatographic characteristics, chemical lability, and a
much lower effective concentration for its biological
activity.
Key Words: sodium pump • sodium • hypertension, sodium-dependent • ouabain
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Introduction |
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The recognition of the natural ligands of the opiate receptor1 2 raised the interesting possibility that other endogenous ligands exist, the action of which is mimicked by potent pharmacological agents. Among these, candidates have included the cardiac glycosides, which are known to interact with a highly conserved specific binding site on the active subunit of the sodium pump or [Na,K]ATPase. Inhibitors of the [Na,K]ATPase (termed digitalis-like factors, DLF) have not only been identified as present in plasma but have also been shown to increase after sodium loading.3 4 5 6 Elevations in plasma levels of this inhibitory activity have correlated well with the development of hypertension, both in experimental models and in patients.3 4 5 6 7 8 9
Over the years, a large number of candidates for the sodium pump inhibitor have been proposed.10 Recent attention has been focused primarily on a ouabain-like agent that was characterized from human plasma obtained from patients undergoing routine plasmapheresis.11 More recently, evidence for a bufogenin-like factor has also emerged.12 We have identified another candidate present in the peritoneal dialysate (PD) of volume-expanded patients with chronic kidney failure, which preliminary data suggest differs from ouabain, including the demonstration of its having chemical lability.13 14
To confirm the difference in the PD DLF and ouabain unambiguously and to evaluate the amount and representative activity of highly purified preparations of this additional candidate, we sought a separation method that provided robust separation capabilities, allowed for coupling to sensitive, quantitative detectors, and provided a less reactive environment during purification. The application of a novel biochemical separation technique, supercritical fluid chromatography (SFC, described in more detail in Methods below) has facilitated our characterization of this PD candidate in patients with chronic kidney failure and has allowed us to make assessments of the purity of this material, the amount present, and the concentrations at which this material is active in comparison to ouabain. This represents the central line of investigation in this report.
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Methods |
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Clinical Protocol
Patients with kidney failure who are treated by chronic ambulatory peritoneal dialysis were recruited to a clinical protocol approved by the Committee on Human Research at the Brigham and Women’s Hospital.6 Each subject gave informed, signed consent before participation in the study. Patients were maintained on their current dialysis regimen; however, their sodium intake was increased and fluid intake was kept at 2.5 to 3.0 L per day as described in detail elsewhere.6 This brought about a gradual and sustained volume expansion, which has previously been shown to increase levels of a labile sodium pump inhibitor in their serum, and based on these previous studies, this same factor appeared in their PD.6
Production of Purified Labile Sodium Pump
Inhibitor
The first 2 L of PD of the day were submitted to a series of
rapid purification steps.6 Initially, 1 g of ascorbic
acid was added to each liter of dialysate because of evidence of
chemical lability. The PD was bubbled with argon while ultrafiltered
through a 1000-D exclusion membrane with a tangential flow
ultrafiltration device (Centrasette, Filtron) that filtered 1 L/10
min of dialysate. The ultrafiltrate was submitted to solid-phase
extraction by its being pumped through a preparative
C18 reverse-phase guard column (45 mmx11
cm, Dynamax, Rainin) at a rate of 20 mL/min. The active fraction was
eluted with 25 mL of methanol, dried, and redissolved in a small volume
of water (1.0 mL). This material was then loaded onto a semipreparative
C18 reverse phase column and eluted with an
ethanol–water–0.1% TFA gradient as described
elsewhere.6 This was followed by analytical
high-performance liquid chromatography (HPLC)
fractionation with a C18 reverse-phase column
eluted with a second ethanol–water–0.1% TFA gradient.6
The product of a single 2-L PD specimen was processed during the
morning of the collection day and used for further experimentation
within a few hours of its purification. The DLF used here was the only
candidate that was undetectable in PD when patients were euvolemic and
increased in proportion to the rise in body weight that followed the
liberalization of salt and fluid intake. Blood pressure and serum
activity of this factor rose in proportion to the rise in body weight
and in proportion to DLF levels in the simultaneously
acquired PD.6
Assessment of DLF Stability After HPLC Fractionation
PD was submitted to HPLC fractionation and DLF was collected
from the previously determined elution envelope and divided into 2
equal portions. Both halves were taken to complete dryness to remove
the organic solvents present in the HPLC mobile phase (ethanol,
0.01% trifluoroacetic acid). Immediately thereafter, half was
reconstituted in an aqueous assay buffer and was analyzed for
its [Na,K]ATPase inhibitory activity. The other half was
redissolved in distilled water and stored overnight frozen under argon
at -80°C. Storage was in silanized glass vials. The following day,
fractions were thawed, dried quickly (rotary evaporator), and brought
up in assay buffer, and the inhibitory activity was
measured. Other less polar cardenolides (strophanthidin or bufalin)
were also processed identically to assess resolubilization in assay
buffer and their stability.
Two-Dimensional SFC
SFC has features of both traditional gas
chromatography (GC) and HPLC. The columns used can be
either open tubular, similar to those used in GC, or packed, similar to
those used in HPLC. The mobile phase is typically
CO2 compressed until it is a supercritical fluid.
Advantages of SFC are that it typically provides a markedly greater
number of theoretical plates (the measure of separation efficiency)
than HPLC methods. Compared with GC, the supercritical fluid mobile
phase has greater solvating power than does its gaseous form, and the
method does not require molecules of interest to be volatile (requiring
high temperatures and often derivation). For these same reasons, it can
accommodate much larger molecules than GC. Other advantages include the
following: SFC is very clean; mobile phase contaminants are usually
trace quantities of other gases. The mobile phase is very free of
dissolved oxygen and is not particularly reactive and the mobile phase
is easily and rapidly removed. SFC allows for the quantitative delivery
of specimen to detectors such as flame ionization detection (FID),
which can provide quantification of resolved materials with a
sensitivity of 
0.1 ng. The major disadvantages of SFC are its limited
availability; highly polar molecules are not soluble in the mobile
phase, and current commercial systems do not allow for the quantitative
introduction of a specimen. Typically, commercial SFC moves a very
small fraction of a much larger specimen from a precolumn onto the
column. However, these limitations have been overcome through
instrumental modifications that more appropriately address
purifications of microscale and nanoscale quantities of
physiological molecules.15 More
sophisticated 2D systems (2D-SFC) allow for the interfacing of 2 SFC
columns having different column coatings or packings and thus provide
for orthogonal separation capabilities.
The 2D-SFC apparatus used in these experiments included a
customized open-tubular specimen introduction system.15
More than 90% of specimen loaded into this precolumn was moved through
the SFC system. The first SFC dimension used a glyme column and a
thoroughly deactivated valving system that allowed for the
diversion of selected eluate regions from the first dimension,
quantitatively, to the second. The second dimension used a liquid
crystal column coupled to either a collection port or a flame
ionization detector with a detection limit of 0.1 ng.
N-eicosane and n-hexatricontane (typically 15 or
25 ng) were used as internal controls both to fix the migration
location of the inhibitor and, by comparison to the known
quantity of control, the amount of inhibitor
present.15 Initial studies that used collection
and assay of individual peaks defined the location of the labile DLF in
each dimension of the SFC chromatogram. A mass of 500 D was assumed for
the inhibitor, consistent with sizing
studies16 and other preliminary data (not shown), and used
to estimate concentration. Ouabain (30 ng) or bufalin (15 ng) were
applied to the SFC system, and the eluate was scanned for an FID
peak.
Assay of Inhibition of [Na,K]ATPase Activity
The assay monitored the influence of the labile sodium pump
inhibitor on [Na,K]ATPase ATP hydrolysis. To determine
the rate of hydrolysis, we used [32P]ATP
(Amersham) and canine renal [Na,K]ATPase (Sigma Chemical Co) in a
final volume of 130 µL of buffer (containing in mmol/L: Na 100,
K 5, Mg 3, EGTA 1, Tris 80, pH 7.5, 37°C) in the absence or presence
of labile DLF after correction for non-[Na,K]ATPase activity
(residual activity in the presence of
1x10-3 ouabain). The
reaction was started by adding 10 µL of 40 mmol/L
[
-32P]ATP (Amersham) and run for 30
minutes.17 Ouabain in graded concentrations was used for
comparison. DLF activity was expressed as the percent inhibition of
[Na,K]ATPase hydrolysis.
Vascular Smooth Muscle Contraction
The procedures used on animals complied with the guidelines of
the Committee on Animals of the Harvard Medical School and have been
described in detail previously.18 In brief, thoracic aorta
were harvested freshly from 15 New Zealand White rabbits. Strips of
tissue (2 mmx10 mm) were mounted on a pressure transducer
in a 4.0-mL muscle bath containing modified Krebs-Henseleit solution
(in mmol/L: NaCl 118.2, KCl 4.6, CaCl2 2.5,
KH2PO4 1.2,
MgSO4 1.2, NaHCO3 24.8, and
dextrose 10.0) and placed under 2.5 g tension at 37°C. The
chamber and solution were aerated with 95% oxygen/5% carbon dioxide.
Buffer solutions containing half of a specific preparation of the
labile sodium pump inhibitor replaced the equilibration
buffer, and changes in contractile force were recorded on a
polygraph recorder (Grass Instruments). The other half of the
labile DLF was used for quantitative assessment. Vascular smooth muscle
cell (VSM) response to graded ouabain concentrations was also measured
in duplicate.
Relation Between Clinical Volume Status and Post–SFC DLF
Activity
Weight gain during the clinical protocol was taken as a measure
of volume expansion. The increment in weight over baseline was
determined at the time of PD collection and divided into 3 response
ranges and compared with the inhibitory activity of labile
PD DLF from the same dialysate after both HPLC and SFC. DLF activity
was divided into 3 levels of inhibition.
Statistical Analysis
Data are expressed as mean±SEM. Comparisons of split DLF
specimens measured immediately and after being maintained frozen for
various periods >24 hours were made by paired t test.
Correlation between two parameters was assessed by
Pearson’s product moment correlation analysis. Comparison
of weight gain to labile DLF activity used a 3x3 matrix evaluated by
Spearman’s rank correlation test. Probability values <0.05 were
considered statistically significant.
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Results |
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Preparations of the labile DLF, representing the active HPLC fraction, were submitted to SFC. The only SFC peak that demonstrated consistent and significant inhibition occurred between 12.7 and 14.7 minutes of elution in the first dimension and at
18.3 minutes in the second dimension for these
experiments.15 A sample chromatogram is shown (Figure 1). Although this retention time was
highly characteristic of this factor for columns and conditions used
here, column or frit (needed for collection) replacement, even with
comparable pieces, can result in small variations in elution time.
Inclusion of internal control materials ensured that the same peak was
studied in every run. Ouabain (up to 30 ng) injected onto the precolumn
(at least 6 experiments) was too polar to be appreciably soluble in the
supercritical fluid carbon dioxide and consequently could not be found.
Under the same SFC conditions, bufalin eluted after the labile DLF at
21.5 minutes in the first dimension and was not carried into the second
dimension.
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DLF activity, measured as inhibition of [Na,K]ATPase hydrolysis, decreased with time after partial purification on HPLC, as noted previously.6 In this study, those observations were confirmed with the use of additional specimens. The HPLC prepared labile DLF was divided in 2 equal portions and half assayed immediately after removal of HPLC solvents. Half was dried, dissolved in water, and stored frozen at -80°C under argon for 1 to 7 days and then assayed immediately after thawing. DLF activity was lost with a half-life of 25.3±9.1 hour (n=6). Strophanthidin and bufalin, both cardiac glycosides with comparable or reduced water solubility, showed no loss of activity when processed similarly (data not shown).
After the volume-sensitive, labile DLF had been isolated by HPLC and subsequently submitted to SFC FID, the single peak was still labile, showing a 58% reduction in amount (area under curve) when split specimens were analyzed, half on the first day of study and half 24 hours later (Figure 2, n=13; t=0: 408±80 U of area versus t=24 hours: 170±36 U of area, P=0.004) after being frozen (<-80°C) under argon in aqueous solution. The mean of the half-lives calculated for each pair was 34.7±13.0 hours, which was not different than the half-life of the HPLC-purified DLF (Figure 3, P=0.56).
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The initial amount of inhibitor estimated by FID from individual purifications was variable but probably represented the degree of volume expansion. In other experiments, [Na,K]ATPase inhibitory activity purified from a single collection of dialysate was significantly related to the degree of volume expansion in the same patient at the time PD was obtained (Table, P<0.01).
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The labile DLF, purified by SFC, produced a concentration-dependent inhibition of [Na,K]ATPase hydrolysis, consistent with previous reports.3 4 5 6 The amount of labile DLF in half a specimen could be closely estimated from its FID response in comparison to known quantities of internal controls. The other half of the labile DLF specimen was submitted to biological assay. Assuming a molecular weight range similar to that of strophanthidin or ouabain, the labile DLF produced inhibitions of canine renal [Na,K]ATPase activity equivalent (in timing and magnitude) to those of ouabain but at concentrations 30 times lower. Fifty percent inhibition occurred at a concentration estimated as 1.7x10-8 mol/L for the labile inhibitor compared with 5.4x10-7 mol/L for ouabain. There was a strict relation between the amount of material after 2D SFC estimated by FID of half the specimen and the inhibition produced by the other half (n=10, R=0.98, P=0.00001, Figure 4).
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Other specimens of the labile DLF were split, half assayed against VSM
and half assessed by SFC FID for quantity. Graded concentrations of
ouabain were applied to other strips of aorta for comparison. The
labile DLF and ouabain produced concentration-dependent increases in
contraction of rabbit aortic rings (Figure 5). The response threshold for the labile
DLF was 5x10-11 mol/L
as compared with
1x10-7 mol/L for
ouabain. One gram of contraction was achieved at a concentration of
1x10-9 mol/L for the
labile DLF and 5x10-7
mol/L for ouabain.
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) were applied to rabbit aorta; slowly developing contractile response was measured at its maximum. Several purified preparations of labile DLF (•) were divided, half used to determine amount of DLF present by means of FID and half added to tissue chambers containing rabbit aorta. FID allowed for close determination of amount of DLF, which in turn was used to estimate DLF concentration in muscle bath in conjunction with resultant VSM response. |
Discussion |
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Supercritical fluid chromatography provides a powerful purification technique that has greater separation efficiency than liquid chromatography and uses a mobile phase that is readily removed by decompression of the liquefied gas, allowing it to be coupled easily to sensitive and quantitative ionization techniques such as FID or mass spectrometry. The mobile phase, because it is a gas, is very clean, which is highly desirable when working with trace quantities of an unknown. In this particular application, HPLC-purified specimens of the labile DLF were applied to SFC, and, as described in previous work,6 15 gave rise to a single region of DLF activity, which coincided with one peak completely resolved from other peaks present in the SFC eluate. When we divided HPLC-purified DLF specimens in half, the area of the peak obtained from half the specimen, measured by its ionization response, was strictly correlated with the inhibitory activity of the other half. The close relation between a single region of activity and the area of one peak suggests strongly that the DLF obtained from peritoneal dialysate was represented by this single peak that had been purified to homogeneity.
The [Na,K]ATPase inhibitor that we had previously identified as present in peritoneal dialysate and that fulfilled criteria for its being a digitalis-like factor6 13 14 17 18 was unstable, even after bubbling argon through aqueous solutions of the factor and freezing them until assay. This lability, while precluding stockpiling of this factor, provided a unique chemical characteristic of this compound that readily distinguished it from other cardiac glycosides. Consequently, when the HPLC-purified, labile DLF was applied to SFC, we not only were able to monitor [Na,K]ATPase inhibitory activity but also chemical lability. The one region of [Na,K]ATPase inhibitory activity consistently found with SFC separation was also labile, with a half-life comparable to that seen for the labile DLF after HPLC, suggesting strongly that it is the same material that had been previously studied and characterized. Moreover, even with the additional SFC purification, the DLF inhibitory activity of this one peak was greater when from the dialysate of a patient having greater volume expansion. This further confirmed that the SFC-purified DLF material was indeed the volume-sensitive DLF previously isolated by HPLC and characterized as part of clinical studies.
The ability to couple SFC to FID allowed for a measure of the
amount of material represented by the DLF peak. By assuming
a molecular weight in the range of 500,16 the
concentration of the labile DLF could be estimated. By then using a
strategy of dividing specimens, determining the amount (and hence
concentration) of half the material, and comparing that with the
inhibition of [Na,K]ATPase or its bioactivity against VSM, the
concentration-activity profile of the labile DLF could be
characterized, at least in part. Against canine renal [Na,K]ATPase,
the labile DLF was 30 times more active than ouabain for a given
concentration over the concentration range we were able to study.
Against rabbit aorta, the labile DLF was 500 to 1000 times more
effective than ouabain in producing VSM contraction over the available
concentration range. The demonstration that the labile DLF itself
produced different levels of [Na,K]ATPase inhibition in different
tissues at comparable concentrations has been studied previously and
appears to be due to the sodium pump isoform composition of the two
tissues and to an apparent preferential effect of the labile DLF for
rabbit 
2 isoform compared with its effects on
canine 1.14 In addition, the
labile DLF demonstrated enhanced inhibition when compared with
comparable concentrations of ouabain for both tissues, especially so
against rabbit aorta. This probably is caused by the labile DLF having
somewhat greater affinity for the 1 (dog
kidney) but a markedly greater affinity for the
2 isoform (abundant in rabbit vascular smooth
muscle) compared with ouabain.
Both ouabain and bufalin were applied to SFC. Ouabain was insufficiently soluble in the supercritical CO2 to allow detection, consistent with its being more polar than the labile DLF. Bufalin was readily soluble and provided a consistent and stable FID peak, the location of which was several minutes later in the first SFC chromatogram.
In summary SFC provided a useful tool in the purification of the labile DLF from residual impurities present even after multiple HPLC purifications. It also easily distinguished the labile DLF from other cardenolides or bufadienolides, specifically ouabain and bufalin. The ability to couple SFC with FID allowed us to characterize the labile DLF concentration-activity profile and demonstrate that this factor has a profound contractile effect on rabbit VSM, with detectable changes achieved at subnanomolar concentrations, a concentration-response remarkably different than that of ouabain. One final but future consideration in the use of SFC is that it can be coupled to a mass spectrometer and allow for important further characterization of this inhibitor of [Na,K]ATPase despite its inherent instability.
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Acknowledgments |
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The clinical portions of these studies were performed in the General Clinical Research Center at Brigham and Women’s Hospital, supported by a National Institutes of Health grant (2-M01-RR-02635).
Received March 14, 2000; first decision April 25, 2000; accepted June 13, 2000.
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