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(Received for publication, July 3, 1995; and in revised form, January 3,
1996) From the
Digoxin-like immunoreactive factor (DLIF) from adrenal cortex is
an endogenous molecule with structural features remarkably similar to
those of digoxin, a plant-derived cardiac glycoside (Shaikh, I. M.,
Lau, B. W. C., Siegfried, B. A., and Valdes, R., Jr.(1991) J. Biol.
Chem. 266, 13672-13678). Two characteristic structural and
functional features of digoxin are a lactone ring and three digitoxose
sugars attached to a steroid nucleus. Digoxin is known to undergo
deglycosylation during metabolism in humans. We now demonstrate the
existence of several naturally occurring deglycosylated components of
DLIF in human serum. The components are identified as DLIF-genin,
DLIF-mono, and DLIF-bis, corresponding to the aglycone, and the
aglycone with one and two sugars, respectively. Similar components are
produced by acid-induced deglycosylation of DLIF isolated from bovine
adrenal cortex. The elution pattern and sequence of
DLIF-deglycosylation was identical to that of digoxin suggesting
identical sugar stoichiometry. However, analysis of these newly
discovered congeners by reverse-phase chromatography,
spectrophotometry, antibody reactivity, and kinetics of
deglycosylation, demonstrates that subtle structural and physical
differences do exist when compared to digoxin. DLIF was
chromatographically distinct from digoxin, and interestingly, the
mobility of the DLIF-genin was shifted toward increased polarity
relative to digoxigenin. DLIF and DLIF-bis, -mono, and -genin congeners
have absorbance maxima at 216 nm, whereas digoxin and its congeners
absorb at 220 nm. Reaction with specific antibodies directed at the
lactone portion of these molecules shows DLIF and its deglycosylated
congeners to be 10
Endogenous digoxin-like immunoreactive factors (DLIFs) (
Figure 1:
Structure of digoxin and
its deglycosylated congeners. Digoxin has a characteristic five-ring
structure (aglycone) to which are attached three sugar (digitoxoses) at
the C-3 position. The aglycone consists of a steroid nucleus with an
unsaturated lactone ring attached at the C-17 position. Note that
digitoxose residues are sequentially removed to form the bis, mono, and
genin derivatives.
The
In this
study we demonstrate the existence of several deglycosylated congeners
of DLIF in human serum and show that similar derivatives can be
produced in vitro by acid-induced deglycosylation of DLIF
isolated from bovine adrenal cortex. These congeners are analogous to
the formation of bis, mono, and genin components of digoxin by
sequential removal of the three sugars at the C-3 position of the
aglycone and suggest the likelihood of metabolism of DLIF in mammals.
We also describe a technique for purifying these newly discovered DLIF
congeners and investigate several physical properties indicating
structural differences between DLIF and digoxin at both the sugars and
the aglycone portions of these molecules.
Comparative chromatographic
mobility studies between DLIF (and its deglycosylated congeners) and
its counterparts digoxin (and its deglycosylated congeners) were
conducted. Pure DLIF and digoxin were mixed and co-injected on selected
an isocratic mode of 25% CH
Figure 2:
HPLC elution pattern of deglycosylated
DLIF and digoxin molecules. Panel A, standards of digoxin and
deglycosylated congeners, dig-bis, dig-mono, and digoxigenin; panel
B, pure adrenocortical DLIF (4.6 ng of digoxin equivalent (d.e.)) incubated without (open bar) and with (solid bars) 1% SSA for 60 s. Under these conditions, DLIF is
fractionated to components (DLIF-genin, DLIF-mono, DLIF-bis, and DLIF).
Note formation of two compounds less polar than digoxin eluting at
fractions 31 and 34 min, respectively. Panel C shows digoxin
(130 µmol/liter) treated with 1% SSA for 90 s and processed as
described in text.
Figure 3:
HPLC chromatograms of human serum. Panel A, digoxin-free serum taken from one subject and
incubated without (1 ml, open bar) and with (5 ml, solid
bars) 1% SSA for 60 s. Panel B, three normal human
digoxin-free sera (20 ml each) analyzed without acid treatment. d.e., digoxin equivalent.
Figure 4:
Chromatographic analysis of a mixture of
pure DLIF and its deglycosylated congeners (DLIF-bis, DLIF-mono,
DLIF-genin). Panel A, note identical retention times of the
DLIF congeners compared to the digoxin standards in Fig. 2A when the linear gradient (20-80% CH
Figure 5:
Spectrophotometric scans of purified DLIF,
its deglycosylated DLIF-genin, and the less polar component, DLIF-31.
Digoxin, digoxigenin, and cortisone were scanned for comparison.
Concentrations were: purified 24 µM DLIF and DLIF-genin
(estimated by molar absorptivity), 24 µM digoxin and
digoxigenin, 5 µM cortisone. Note the absorbance maxima of
216 nm (DLIF and DLIF-genin), 220 nm (digoxin and digoxigenin), and
that the absorbance at 250 nm is a characteristic feature of steroids.
The less polar species (F-31) of both digoxin (not shown) and DLIF have
a uv shift with an absorbance maximum at approximately 196
nm.
Figure 6:
Competitive displacement
(cross-reactivity) of
Figure 7:
Apparent molar fractional distribution and
time course of formation of DLIF and digoxin congeners after incubation
with acid. Conditions are as described under ``Experimental
Procedures.'' Panel A shows the relative production of
DLIF congeners from DLIF isolated from bovine adrenal cortex (starting
with 10 ng of digoxin equivalent DLIF) after treatment with 7.6% SSA
(7.6 g of SSA for each 100-ml homogenate). Panel B shows a
comparable fractional distribution profile for digoxin congeners
produced (starting with 50 ng of digoxin) under similar experimental
conditions. Note: time scales are different and that DLIF
deglycosylates more rapidly than digoxin.
Figure 8:
Chromatographic analysis of formation of
HPLC fraction 31 from digoxigenin. Panel A, digoxigenin (100
µmol/liter) treated with 1% SSA showing the formation of fraction
31. Panel B, one of the chromatograms used to construct the
above figure, representing the 2-h incubation of
digoxigenin.
Since the original discovery of DLIF in mammals (1, 2) little has been learned about the metabolism of
these endogenous steroid-like factors. We have recently identified the
existence of a naturally occurring reduced lactone ring form of
digoxin-like immunoreactive factor (dihydro-DLIF, analogous to that of
dihydrodigoxin) and showed that dihydrodigoxin is converted to a
digoxin-like immunoreactive compound by microsomal cytochrome p450
mediated activity(9) . In this study, we demonstrate the
existence of several naturally occurring deglycosylated species of DLIF
in human serum. We show that these derivatives can also be produced in vitro by acid-induced deglycosylation of DLIF isolated from
bovine adrenal cortex. The various DLIF species (DLIF-genin, DLIF-mono,
DLIF-bis, and DLIF, by analogy to the cardenolide counterparts, digoxin
and its deglycosylated congeners) correspond to the factor with no
sugars (aglycone), one sugar, two sugars, and three sugars (DLIF),
respectively. These findings provide a working model for characterizing
two important structural epitopes found on these endogenous factors and
also establish a basis for characterizing metabolic pathways of
synthesis of endogenous DLIF in mammals. We used several techniques,
each specific for probing structural differences on different portions
of the cardiac glycoside-like molecules. Reverse-phase HPLC is
sensitive to modifications affecting molecular polarity and solubility;
UV-spectral analysis is sensitive to structural features of or in close
proximity to the lactone ring; kinetics of deglycosylation of the
sugars detects differences in sugar stoichiometry and their binding to
the aglycone; and, immunoreactivity is sensitive to molecular
modifications near the C- and D-rings of the sterol and the
lactone-ring portion of DLIF or digoxin. By optimizing HPLC elution
conditions we demonstrate the ability to separate DLIF from digoxin and
their respective aglycones from each other, including two here-to-fore
unknown DLIF species. The HPLC elution pattern of deglycosylated
adrenocortical DLIF was identical to that of digoxin using the linear
elution gradient (Fig. 2, B and C). The DLIF
fractions eluted in fractions correspond to the standards of digoxin
and its deglycosylated congeners (Fig. 2A). A similar
chromatographic pattern was observed after acid-deglycosylation of DLIF
isolated from human serum (Fig. 3A) and, in fact, the
immunoreactive fragments were found naturally in human serum (3 of 22
subjects) without acid treatment (Fig. 3B). These
results are consistent with our recent hypothesis suggesting molecular
similarity between DLIF extracted from bovine adrenal cortex and that
from human serum(27) . Additional HPLC studies using mobile
phases clearly showed relative mobility shifts when comparing DLIF and
digoxin (Fig. 4B) and also for their respective
aglycones (Fig. 4C). Interestingly, the mobility of
DLIF-genin was shifted toward increased polarity relative to
digoxigenin when compared to the opposite relative mobilities of the
parent compounds. The genin compounds are aglycone sugar-free molecules
(see Fig. 1), hence, these findings are the first reported
observation indicating that structural differences between DLIF and
digoxin are in the aglycone portions of these compounds. Both the
chromatographic mobility shifts and the immunoreactive potency
differences observed for the two genin compounds of DLIF and digoxin ( Table 1and Fig. 6) suggests structural differences in/or
near the lactone ring for the aglycones. This is supported by the
spectral analysis showing a uv shift in the maximum absorbance for DLIF
(216 nm) compared to digoxin (220 nm) (Fig. 5). However, the
kinetics of acid-induced deglycosylation studied by monitoring the rate
of formation of different DLIF species showed that DLIF-deglycosylation
is considerably faster (approximately 8-fold) than that observed for
digoxin (see Fig. 7). This is an indication that the sugars on
DLIF are different than those on digoxin and thus in the strength of
the bond to each other in the linear linkage and/or to the
DLIF-aglycone at position C-3 (Fig. 1). Characterizing the
structural features of endogenous digoxin- or ouabain-like counterparts
of the cardenolides has been difficult because of their low
concentrations in tissues and difficulty in extracting sufficient
quantities for detailed structural analysis. For example, initial data
suggested that OLF (ouabain-like factor) was structurally identical to
ouabain using mass spectroscopy and other related
techniques(28, 29) . However, more recent studies
using an exciton-coupled circular dichroism (CD) technique show that
OLF and HIF (an isomer of ouabain from bovine hypothalamus) are
structurally different from ouabain(30) . The initial
suggestion of a structural similarity between OLF and ouabain was based
on the similarity in their chromatographic mobility, mass spectral
data, and their unit immunoreactivity with antibodies to ouabain.
However, in the case of DLIF we now have evidence of chromatographic,
spectral, and molar immunoreactivity differences which suggests that
DLIF is more structurally distinct from digoxin than OLF is to ouabain.
However, sufficient quantities of these compounds will still need to be
harvested to fully permit the identification of fine structure. Of
interest is the discovery of a new as yet undefined product of DLIF and
of digoxin (fraction 31, less polar by HPLC elution than DLIF or
digoxin) produced by acid-induced deglycosylation of these molecules
and also existing naturally in vivo in human serum. Our data
are consistent with the hypothesis that DLIF-31 (or digoxin-31) is
formed preferentially from the genin derivatives (Fig. 8). The
far uv shift to 196 nm observed for the DLIF-31 immunoreactive
component is, however, consistent with and characteristic of a
structural modification at or near the lactone ring. A similar uv shift
is observed after formation of dihydrodigoxin (196 nm) from digoxin
(220 nm) (31) or after modification by a double bond saturation
on the A- or B-ring of the steroid(32) . However, the presence
of a double bond in the A- or B-ring structures of the DLIF aglycone is
not suspected because that structural feature is usually associated
with a characteristic absorption band at 250 nm (see Fig. 5, e.g. cortisone). DLIF-31, therefore, is a new species and
since it is detected naturally in some human sera, may be a metabolic
product of DLIF. The 4-fold decrease in immunoreactivity of DLIF-31
compared to DLIF and relative to digoxin ( Fig. 6and Table 1) is again consistent with structural changes at or near
the lactone ring as is the spectral shift toward the uv(31) . Three principal routes of metabolism have been reported for digoxin
in mammals: sequential deglycosylation by the stepwise removal of sugar
molecules (with formation of bis-, mono-, and digoxigenin) resulting
primarily from acid hydrolysis in the stomach; conversion of digoxin to
polar metabolites like glucuronide and sulfate conjugates of
3-epi-digoxigenin(6, 33) ; and conversion of digoxin
to dihydrodigoxin by chemical reduction of the lactone ring occurring
within the gastrointestinal tract by bacterial action of Eubacterium lentum(34) . Extensive digoxin
biotransformation has been reported in some patients with polar
metabolites being predominant(35) . To date, however, little is
known about the metabolism of DLIF in mammals. Our data suggest that
the route of metabolism of DLIF may be similar to that of digoxin. The
recent discovery of a dihydro-DLIF species existing in the adrenal
establishes a basis for synthesis of DLIF in the adrenal
gland(9) . The DLIF deglycosylated congeners reported here
may explain some of the discrepancies reported in other studies
isolating digoxin-like compounds(36) . In fact, some of these
deglycosylated congeners (generated during isolation procedures and/or
those found naturally) may have been identified as different kinds of
digoxin-like immunoreactive compounds. DLIF is considered as one of the
most interesting and detrimental interferant to most commonly used
digoxin assays. The variable cross-reactivity and the different amounts
or ratios of the DLIF congeners in serum may explain the wide variation
in detection sensitivities for endogenous DLIF reported by digoxin
immunoassays(37) . Recognizing the presence of these newly
discovered congeners and understanding their relative immunoreactivity
with the antibodies used will provide a more accurate means of
therapeutic digoxin monitoring for digoxin(38) . DLIF is known
to exist bound tightly to a 22-36-kDa protein in
serum(25, 39) . The stoichiometry of
DLIF-glycosylation may also affect this protein binding and thus
influence the detection of these factors in serum when using different
digoxin immunoassays. This may well explain changes in the extent of
protein binding of DLIF observed in some clinical conditions, e.g. pregnancy induced hypertension (40) or renal
failure(2) . Digitalis cardiac glycoside molecules have two
important structural features linked to their ability to functionally
inhibit Na,K-ATPase: the lactone ring and the digitoxose sugar
residues. Studies of the molecular structure of the
Volume 271,
Number 15,
Issue of April 12, 1996 pp. 8731-8737
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
-fold less reactive than digoxin.
Kinetics of sugar removal suggests that DLIF is 8-fold more susceptible
to deglycosylation than is digoxin. Two less polar DLIF components
produced from the DLIF-genin have 
at 196 nm and are
4-fold less immunoreactive than DLIF. Our data suggest that subtle
structural differences exist between DLIF and digoxin at or near the
lactone ring as well as in the nature of the sugars. The presence of
deglycosylated congeners of DLIF in human serum, including the less
polar components, suggests in vivo deglycosylation of these
factors. This is the first demonstration of the existence of naturally
occurring deglycosylated derivatives of DLIF and establishes the
likelihood of active metabolism of DLIF in mammals.
)present in mammalian blood(1, 2) were
discovered in part as a consequence of their cross-reactivity with
antidigoxin antibodies. Considerable evidence suggests that endogenous
DLIF (3) and ouabain-like factors (OLF) (4) are
produced in the adrenal glands and may be linked to modulation of
Na,K-ATPase activity in a manner analogous to the family of cardiac
glycosides, digoxin or ouabain(5) . DLIF, a steroid-like
molecule (780 Da), has structural and molecular properties remarkably
similar to those of the cardiac glycoside digoxin(3) . These
properties include a characteristic five-ring structure (aglycone) to
which is attached three linearly linked sugar residues (digitoxoses, in
case of digoxin) extending from the aglycone C-3 position (Fig. 1). The aglycone portion of the glycoside consists of a
steroid skeleton with an unsaturated lactone ring attached at position
C-17. The drug digoxin undergoes removal of its digitoxose sugars
during metabolism, forming several deglycosylated species(6) .
The metabolic products of digoxin are important because they interact
in a very structure-specific manner with sodium pump isoforms (7) as well as with highly specific antibodies raised against
digoxin(8) . However, to date no study has identified the
presence of naturally occurring metabolic products of DLIF. With
improved chromatographic techniques we recently identified the presence
of a DLIF with a chemically reduced lactone ring, dihydro-DLIF,
analogous to the metabolic product dihydrodigoxin (9) .
subunit of the sodium pump is the only known functional receptor
for digitalis compounds. The stoichiometry of the sugars at the C-3
position on digoxin (8) or of steroids (e.g. progesterone) (10) affect their binding to this receptor.
The individual isoforms of Na,K-ATPase have been shown to possess
differing affinities for binding of digitalis, ouabain, and their
respective deglycosylated derivatives(11, 12) . The
importance of characterizing the molecular and physical properties of
DLIF is further underscored by the linkage observed between increased
DLIF concentrations in blood and clinical or pathological conditions
associated with altered ion-transport homeostasis such as: renal
failure(13, 14) , hepatic failure(15) ,
pregnancy(16) , neonatal development(17, 18) ,
diabetes- and exercise-induced stress(19, 20) , and
hypertension(21, 22, 23) . The source of
digitalis-like factors in a hormone-secreting tissue such as the
adrenal, presence in blood, and the distribution of likely receptors in
target tissues suggest the likely possibility of a new hormonal axis
linking the adrenal cortex, endogenous digitalis- or ouabain-like
factors, and sodium pump activity(5, 24) .
Chemicals and Reagents
All chemicals used were
reagent grade. 5-Sulfosalicylic acid (SSA), calcium carbonate
(CaCO), and bovine serum albumin were obtained from Sigma.
Digoxin was purchased from Sigma and its related congeners
(digoxigenin, mono- and bis-digitoxosides) were donated by Burroughs
Wellcome Co. (Research Triangle Park, NC). Pure DLIF (quantitated as
dig-equivalent concentration) isolated either by the current or our
previous procedures (3) was used as a standard to observe the
formation of DLIF-deglycosylated congeners. The most common steroids in
the adrenal cortex (cortisone, corticosterone, progesterone, and
cortisol) were obtained from Sigma and used as standards to define
their chromatographic mobility and cross-reactivity. Acetonitrile
(CHCN) was HPLC grade and obtained from Aldrich.Equipment and Materials
We used a Polytron PT-3000
(Brinkmann, Westbury, NY) for homogenizing adrenocortical tissue and an
Orion pH/SE meter model 710A (Orion, Cambridge, MA) for pH
measurements. The Sep-Pak reverse-phase C-18 cartridges were obtained
from Waters Associates. The HPLC fractions were dried in a Savant
SpeedVac Plus SC210A (Savant, Farmingdale, NY) connected to a
Refrigerated Condensation Trap RT 400 and a Virtis Freeze Mobile 12
Lyophilizer (Virtis, Gardiner, NY). A Beckman JA-2 centrifuge (Beckman)
was used for centrifugation in the isolation procedures.Tissue Preparation
Bovine adrenal glands were
obtained from Pel-Freeze Biologicals (Rogers, AR) and kept at -80
°C until dissected. Tissues were maintained at 4 °C during the
isolation procedure (dissection, extraction, and centrifugation), while
other steps were performed at 22-25 °C. Briefly, after
isolating the glands from any surrounding connective tissues and fats,
the cortex was separated from the medulla based on the physical
appearance of the outer cortical ring. Each gland weighed approximately
15 g and contained twice as much cortex as medulla. The cortical tissue
was sliced (3-mm thick sections), chopped, and homogenized in 2 ml of
dH
O/g of cortex. The homogenate was then centrifuged three
times at 34,000 
g for 30 min at 4 °C in which the
pellet from each cycle was resuspended in dHO (1 ml/g
cortex). The pooled supernatants were stored at -80 °C until
further use.Protein Precipitation
The thawed extracts of
adrenal cortical tissue were re-centrifuged at 34,000 g for 30 min at 4 °C to remove any remaining cellular debris.
Protein was precipitated by incubating the supernatant with 1% SSA (1 g
of sulfosalicylic acid/100 ml supernatant) at room temperature for 60 s
followed immediately by adding an excess amount of CaCO until the pH increased and stabilized at 5.2 (the pH of the
normal adrenal cortex starting homogenate). This extract was then
centrifuged at 80,000 g for 10 min at 4 °C
followed by vacuum filtration of the supernatant using two layers of
Whatman #1 filter paper.Solid Phase Extraction (C-18 Cartridge
Column)
C-18 Waters reversed-phase Sep-Pak solid-phase
extraction column (Vac 3 cc) was primed with 1 volume of
CHCN and rinsed with 2 volumes of dHO. The
protein-free supernatant was passed through the column twice at a rate
of 1 ml/min which was then washed with 2-3 volumes (typically 10
ml) of dHO and eluted with 20 ml of 60% CHCN.
To remove the CHCN, the eluates were evaporated to dryness,
reconstituted in HO, and filtered through 0.22-µm
Whatman PVDF in preparation for HPLC. Reconstitution was made to
achieve average yields of immunoreactivity of approximately 10,000
pg/ml by the RIA.HPLC Reverse-phase Chromatography
We used a C-18
reverse-phase micro-Bondapak column (3.9 300 mm, 10-micron
particle size) connected with a Waters 600E System Controller and a
Waters 966 Photodiode Array Detector to monitor the HPLC output eluent
for both UV absorbance and spectral analyses. The eluate was
fractionated on HPLC using a linear 20 to 80% CHCN in
HO elution gradient. Unless otherwise indicated, typically,
1-ml (1 min) fractions were collected during each chromatographic run
over 40 min using a Waters Fraction Collector from Millipore Corp.
(Milford, MA). The time delay between detector and the fraction
collector was typically 18 s. All fractions were evaporated to dryness,
reconstituted in 1 ml of HO, and measured by
dig-radioimmunoassay. Steroids co-elute with the fractions of interest
(at 15, 19, 23, and 25 min representing DLIF-genin, DLIF-mono,
DLIF-bis, and DLIF, respectively), were separated by re-injected the
fractions individually on selected isocratic mobile phase modes (18,
25, 30, and 39% CHCN, respectively) for 20 min.
Radioimmunoassay measurements showed a single chromatographic peak for
each species with immunoreactivity eluted at fraction 6.00 min for
DLIF, 8.30 min for DLIF-bis, 12.30 min for DLIF-mono, and 16.30 min for
DLIF-genin. The final yield of DLIF and its related deglycosylated
congeners were measured by uv spectra and their digoxin equivalent
molar absorptivity were determined.CN mobile phase for 60 min.
Similarly, the genin, the mono- and the bis- derivatives of the two
parents DLIF and digoxin, were run on 15, 20, and 25% CHCN
mobile phase, respectively.Purification of DLIF from Human Serum
Normal
dig-free fresh-frozen human serum obtained from the American Red Cross,
Louisville, KY, was treated with SSA (final concentration 1% SSA),
vortexed, and centrifuged for 2 min at 9,500 g.
Immediately after centrifugation, we applied the entire supernatant to
a small primed C-18 reversed-phase Sep-Pak solid-phase extraction
cartridge. The column was then washed with 3 ml of HO and
eluted with 2 ml of 100% CHCN. For serum samples not
treated with SSA, we applied the whole 1-ml sample directly to the
Sep-Pak cartridge. In both cases, to remove the CHCN, we
evaporated the eluates to dryness, dissolved the residue in 1 ml of
dHO, and filtered the solution through a Whatman
0.22-µm polyvinylidine difluoride filter in preparation for HPLC.Molar Absorptivity and Concentration of DLIF
The
molar absorptivity of digoxin was determined experimentally by
injecting known weighed amounts of digoxin on HPLC and the measured
peak area was monitored. At their respective maximum absorbance
wavelengths, we assumed comparable molar absorptivity between digoxin
and DLIF. Measured amounts of DLIF and its related congeners (by
dig-radioimmunoassay equivalence) were injected on HPLC and the
apparent molar-immunoreactive concentration of DLIF (or the respective
congeners) was determined. From the above data the percentage
cross-reactivity of DLIF and its congeners (competitive displacement of 
I-digoxin from polyclonal digoxin-specific antibodies)
were obtained.Protein Assay
Protein concentrations were measured
at different steps of the purification procedure using a Pierce BCA
protein assay reagent (Rockford, IL). Absorbances were measured at 562
nm on a Beckman DU-64 Spectrophotometer (Beckman) and protein
concentration was estimated using bovine serum albumin as standard.Digoxin-like Immunoreactivity
DLIF was measured
using competitive displacement of I-digoxin of polyclonal
digoxin-specific antibodies relative to digoxin standards as described
elsewhere(25) . The standards consisted of digoxin dissolved in
human serum and ranged in concentration from 25 to 2,000 pg/ml. The
zero standard had no digoxin. The polyclonal antisera and components
for the assay were obtained from DuPont NEN (Wilmington, DE). All
radioimmunoassay measurements were done in duplicate.
Acid Hydrolysis of Adrenocortical DLIF
The
retention times of a mixture of pure digoxin and several deglycosylated
digoxin congeners as standards are shown in (Fig. 2, panel
A). A similar HPLC pattern (elution time and order) was observed
when digoxin was treated with SSA (Fig. 2, panel C).
Incubation of bovine adrenocortical DLIF with SSA (Fig. 2, panel B) resulted in immunoreactive fractions with identical
retention times as those of the digoxin standards or the digoxin
treated with the same acid. Based on the known sequential removal of
sugars by acid hydrolysis (26) and the similarity in the
retention times observed for the new DLIF fractions and those of the
digoxin congeners (digoxigenin, mono-, and bis-digitoxosides), we named
the DLIF fractions eluting at 15, 19, 23, and 25 min as DLIF-genin,
DLIF-bis, DLIF-mono, and DLIF, respectively. In addition, note the
additional immunoreactive DLIFs appearing in fractions corresponding to
less polar products of digoxin.
Acid Hydrolysis of Human Serum DLIF
DLIF was
extracted from human serum (subject not taking digoxin) as described
under ``Experimental Procedures.'' The initial HPLC data
shows a small peak of endogenous DLIF as seen in Fig. 3A (open bar) with a retention time (25 min) similar to that
of digoxin or of adrenocortical DLIF. When similar serum was treated
with SSA (final concentration 1%) for 60 s, the major part of the
25-min fraction was converted to products with retention times similar
to those after acid exposure of digoxin or of DLIF from bovine adrenal
cortex, including the less polar product at 31 min (Fig. 3A,
solid bar). Fig. 3B shows HPLC analyses of serum
drawn from three human subjects (out of 22 human subjects not taking
digoxin) but processed without acid treatment. The HPLC elution pattern
shows evidence of several naturally occurring DLIF species consistent
with the deglycosylated congeners of DLIF. This may indicate in
vivo metabolism of DLIF by the cleavage of the sugar moieties
attached at the C-3 position of the steroid nucleus (deglycosylation
pathway). The remaining 19 subjects showed only one DLIF component at
fraction 25, suggesting possible inter-individual differences in
metabolism of DLIF.
Chromatographic Mobility Study
Physical properties
related to molecular polarity and solubility can be distinguished by
HPLC analysis. Fig. 4, panel A, demonstrates the
baseline resolving capacity of the HPLC system used to separate various
deglycosylated DLIF congeners. After DLIF and its related
deglycosylated congeners were purified and separated by HPLC
chromatography, they were mixed and re-chromatographed on HPLC using
the gradient described in Fig. 4, panel A. Absorbance
at 216 nm is used to monitor elution of the various species and
demonstrate the capability of isolating the individual deglycosylated
congeners of DLIF by HPLC. Pure DLIF and digoxin were mixed and
co-injected on HPLC using an isocratic 25% CHCN mobile
phase. The two molecules separated by a minimum of 2-3 min and
absorbance was monitored at 220 or 216 nm (Fig. 4, panel
B). Similarly, the genin compounds (aglycone without sugar
molecules) of both parent compounds were clearly separated by
1.5-2.0 min on an isocratic 15% CHCN mobile phase (Fig. 4, panel C). Interestingly, the elution order of
the genin compounds (DLIF-genin and dig-genin) were reversed from the
parent compounds (DLIF and dig). Similar separation results to the
parent molecules (DLIF and digoxin) were obtained when DLIF-bis and
DLIF-mono were mixed with their counterparts (dig-bis and dig-mono) on
selected various isocratic mobile phase (25 and 20% CHCN
mobile phase, respectively) (data not shown).
CN over 35
min) was used. The injected amounts of DLIF and its congeners measured
as digoxin equivalent by radioimmunoassay were: DLIF, 2.5 ng; DLIF-bis,
1.5 ng; DLIF-mono, 1.5 ng; DLIF-genin, 2.0 ng. Panel B represents a mixture of pure amounts of DLIF (2.4 ng) and digoxin
(10 µg) co-injected and run on 25% CHCN over 60 min.
Note the separation of the two molecules. Panel C,
co-injection of DLIF-genin (2 ng) with its counterpart digoxigenin (20
µg) on 15% CHCN over 60 min clearly shows two distinct
chromatographic peaks.
Spectrophotometric Analysis of DLIF Congeners
Fig. 5shows the absorbance spectra of the DLIF,
DLIF-genin, digoxin, and digoxigenin. The results show that DLIF
congeners (like the parent DLIF) have the same characteristic
absorbance maxima at 216 nm compared to that of digoxin and its
congeners at 220 nm (see Table 1). The absorbance spectra of
digoxin, digoxigenin, and cortisone are included for comparison. The
absorbance at 250 nm is a characteristic feature of steroids. The less
polar species (digoxin- and DLIF-31) has a uv shift with an absorbance
maximum at approximately 196 nm. This suggests some structural
modification at or near the lactone ring on the fraction 31 compound.
Molar Immunoreactivity of DLIF and Its
Congeners
We investigated the relative molar immunoreactivity
based on displacement of I-labeled digoxin by purified
DLIF and its deglycosylated congeners from the antiserum to digoxin. We
applied the assumption of comparable molar absorptivities of DLIF (at
216 nm) and digoxin (at 220 nm) to calculate the picomole of actual
DLIF congeners per pmol of digoxin equivalent immunoreactivity. Note
that deglycosylated DLIF derivatives all show a similar immunoreactive
potency (approximately 1000-fold less than digoxin) identical to their
parent compound, DLIF. On the other hand, DLIF-31 is approximately
4,000-fold less immunoreactive than is digoxin ( Fig. 6and Table 1).
I-digoxin from polyclonal
digoxin-specific antiserum by DLIF and its congeners. Digoxin as a
standard is included for comparison. The concentration of DLIF and its
congeners were determined by multiplying the digoxin equivalent of DLIF
molar concentration with a factor obtained by absorbance (see text, Fig. 5, and Table 1).
Kinetics of DLIF Species Formation
To study the
kinetics of formation of the individual DLIF congeners, we compared the
time course for production of both DLIF and digoxin congeners by acid
hydrolysis. Fig. 7shows the production of the deglycosylated
congeners as a result of incubating DLIF or digoxin with 7.6% SSA. The
reaction was terminated (by neutralization) at zero time (no acid) plus
9 time points over the course of 24 h. After each neutralization at the
times indicated the contents of the reaction were processed through
Sep-Pak and then fractionated by HPLC chromatography. Having identified
the HPLC fractions above (Fig. 2), we constructed a fractional
molar distribution versus time graph (Fig. 7). This
graph provides the relative amount of each species present in the
reaction mixture at each time point. Note that acid hydrolysis of both
digoxin and DLIF produces a less polar immunoreactive product (F-31).
To confirm that digoxigenin (and DLIF-genin by analogy) was being
converted to fraction 31, we incubated digoxigenin with 1% SSA at 22
°C and monitored the formation of fraction 31 by HPLC. Fig. 8shows the disappearance of digoxigenin (monitored by
absorbance at 220 nm) and the simultaneous appearance of fraction 31
(monitored at 196 nm).
(catalytic)
subunit of the sodium pump (Na,K-ATPase) has revealed the involvement
of specific regions of the polypeptide in binding the sugar portion of
the cardiac glycosides (41, 42, 43, 44) . Our data taken
together are consistent with subtle but important structural
differences existing between DLIF and digoxin. Differences are evident
on all three major portions of the molecule: sugars, lactone ring, and
sterol nucleus.
)CN, acetonitrile; OLF, ouabain-like
factors.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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