(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-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.
Endogenous digoxin-like immunoreactive factors (DLIFs) ()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) .
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
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) .
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% CHCN 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% CH
CN
mobile phase, respectively.
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% CHCN 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% CH
CN 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% CH
CN over 60 min clearly shows two distinct
chromatographic peaks.
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 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).
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 (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.