Dansyl-labeled cholic acid as a tool to build speciation diagrams for the aggregation of bile acids

Article abstract of DOI:10.1021/jp308624h

Bile acids (BAs) are a family of natural steroids biosynthesized from cholesterol in the liver that tend to form aggregates in solution. A fluorescent derivative of cholic acid, namely 3α-Dns-ChA, was employed as a reporter to establish the speciation diagrams of the most abundant BAs that can be found mainly in three microenvironments, solution, and primary and secondary aggregates. The developed methodology is based on the analysis of the combined steady-state and time-resolved experiments performed on 3α-Dns-ChA whose emission behavior was found to be strongly medium dependent. In particular, speciation diagrams of sodium glycocholate (NaGCh), sodium taurocholate (NaTCh), sodium chenodeoxycholate (NaCDCh), sodium glycochenodeoxycholate (NaGCDCh), sodium deoxycholate (NaDCh), and sodium ursodeoxycholate (NaUDCh) were successfully built up.

Full text of DOI:10.1021/jp308624h

Article
pubs.acs.org/JPCB
Dansyl-Labeled Cholic Acid as a Tool To Build Speciation Diagrams
for the Aggregation of Bile Acids
Miguel Gomez-Mendoza, M. Luisa Marin,* and Miguel A. Miranda*
̀ ̀
Instituto Universitario Mixto de Tecnología Química (UPV-CSIC), Universitat Politecnica de Valencia, Avenida de los Naranjos s/n,
46022 Valencia, Spain
S
* Supporting Information
ABSTRACT: Bile acids (BAs) are a family of natural steroids
biosynthesized from cholesterol in the liver that tend to form
aggregates in solution. A fluorescent derivative of cholic acid,
namely 3α-Dns-ChA, was employed as a reporter to establish
the speciation diagrams of the most abundant BAs that can be
found mainly in three microenvironments, solution, and
primary and secondary aggregates. The developed method-
ology is based on the analysis of the combined steady-state and
time-resolved experiments performed on 3α-Dns-ChA whose
emission behavior was found to be strongly medium
dependent. In particular, speciation diagrams of sodium
glycocholate (NaGCh), sodium taurocholate (NaTCh), sodium chenodeoxycholate (NaCDCh), sodium glycochenodeox-
ycholate (NaGCDCh), sodium deoxycholate (NaDCh), and sodium ursodeoxycholate (NaUDCh) were successfully built up.
Chart 1. Chemical Structures of Primary and Secondary Bile
Salts
INTRODUCTION
■
Bile acids (BAs) are a family of natural steroids biosynthesized
from cholesterol in the liver,1 which play a key role in a number
of physiological functions and are the major final products of
cholesterol metabolism. Remarkably, they help lipid solubiliza-
tion by forming mixed micelles and stimulate the biliary
phospholipid secretion.2,3
The chemical structure of BAs includes an unusual cis fusion
between rings A and B, a different number of hydroxyl groups
on the α-face and a short chain ending in a carboxylic moiety.
All these characteristics make them amphiphilic entities, with a
hydrophilic α-face and a hydrophobic β-face, which results in a
tendency to aggregate in solution. Natural BAs are divided into
primary (cholic and chenodeoxycholic acid) directly biosynthe-
sized from cholesterol and frequently found conjugated to
glycine and taurine, and secondary (deoxycholic and lithocholic
acids) obtained from the corresponding primary BAs after
deconjugation of the amino acid and dehydroxylation at
position C-7. Ursodeoxycholic acid is the C-7 epimer of
chenodeoxycholic acid; it is found as a natural product in
humans, at low concentrations, but it is used as an effective
anticholestatic drug.1,4−7 The structures of the corresponding
bile salts (BSs) are shown in Chart 1.
The most widely accepted aggregation model postulates the
formation of primary aggregates by interaction of the β-faces of
few monomers (up to 10) at low BA concentrations, which
agglomerate as the concentration increases, giving rise to
secondary aggregates.8 However, how the number of hydroxyl
groups or the conjugation to glycine or taurine influences the
aggregation behavior of the different BAs is not yet fully
investigated. Moreover, the distribution of primary/secondary
aggregates over a broad concentration range, needed to build
the speciation diagram for every BA, has still to be addressed.
Several techniques have been employed to shed light on the
aggregation behavior of BAs, including NMR8,9 and photo-
physical techniques.10−13 Specifically, pyrene has been used as a
fluorescence probe to investigate the association of BSs in
aqueous solution.14−16 The ratio of the fluorescence intensities
of the vibronic bands of pyrene is strongly dependent on
solvent polarity, a property that has been exploited to
Received: August 30, 2012
Revised: October 21, 2012
Published: December 3, 2012
© 2012 American Chemical Society
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determine the critical micelle concentrations (cmcs).17,18 Due
to the fact that BS aggregates grow in size with increasing
concentration, the cmc range of BSs is broader than that of
other surfactants and hence difficult to be determined. Even
more complex is the problem of estimating the relative amounts
of BSs that can be found in the different aggregation stages. In a
preliminary communication,19 we have recently demonstrated
that derivatization of cholic acid by incorporating a dansyl
(Dns) fluorophore covalently attached to different positions of
the skeleton (3α, 3β, or 7α)20 gives rise to an appropriate
medium-dependent fluorescent reporter on the aggregation
behavior of sodium cholate, which can be successfully
employed to build the speciation diagram of this bile salt.
The concept is based on the enhancement of the emission
quantum yields and lifetimes, upon incorporation into the
aggregates. The unique properties of these ChA derivatives are
associated with their close structural similarity to BSs, which
allow them to occupy equivalent positions in the aggregates and
results in a nearly negligible influence on micellization.
with H2/N2 lamp (50/50, 1.5 ns pulse width) and a
stroboscopic detector. The concentration of 3α-Dns-ChA (20
μM) ensured that the absorbance of all solutions was below 0.1
at the isosbestic point used in each case as the excitation
wavelength (λexc = 327−360 nm for steady-state and λexc = 337
nm for time-resolved experiments, respectively). All the
photophysical measurements were performed under air, at
room temperature, in a quartz cell of 1.0 cm optical path length.
RESULTS AND DISCUSSION
■
In order to check the applicability of the methodology, the
UV−vis spectrum of 3α-Dns-ChA (20 μM) was recorded in the
presence of increasing concentrations of bile salts; this
produced no noticeable effect on the long-wavelength
absorption band (see Figure 1a for the case of NaGCh).
With this background, we wish now to report on the
extension of this straightforward methodology to build
speciation diagrams for sodium glycocholate (NaGCh), sodium
taurocholate (NaTCh), sodium chenodeoxycholate
(NaCDCh), sodium glycochenodeoxycholate (NaGCDCh),
sodium deoxycholate (NaDCh), and sodium ursodeoxycholate
(NaUDCh). For this purpose, the probe with a Dns unit bound
to C-3α of cholic acid, namely 3α-Dns-ChA (Chart 2), has
been selected as the fluorescent reporter.
Figure 1. Absorption spectra of 3α-Dns-ChA (20 μM) (a) upon
addition of increasing concentrations of NaGCh (0−100 mM), and
(b) in the presence of 30 mM NaCh (red), NaGCh (black), NaTCh
(green), NaCDCh (blue), NaGCDCh (turquoise), NaDCh (pink),
and NaUDCh (brown), in 0.2 M aqueous NaCl.
Chart 2. Chemical Structure of 3α-Dns-ChA
Likewise, addition of 30 mM NaCh, NaGCh, NaTCh,
NaCDCh, and NaUDCh did not have any influence on the
absorption spectrum of the probe in the 325−360 nm region
(see Figure 1b). Therefore, the excitation wavelength for the
subsequent emission experiments was selected in this region. In
the case of NaGCDCh and NaDCh, slight changes in the
absorption spectra were noticed; this made steady-state
emission measurements less reliable, but did not influence the
time-resolved results, which allowed us to build the speciation
diagrams. Unfortunately, the very scarce solubility of NaLCh in
aqueous medium prevented the performance of photophysical
experiments, and thus no further studies were carried out on
this BS.
Then, 3α-Dns-ChA was subjected to steady-state emission
measurements in the presence of increasing concentrations of
NaCh, NaGCh, NaTCh, NaCDCh, and NaUDCh (see Figure
2a for the case of NaGCh). In general, addition of the BSs
resulted in a marked enhancement of the emission intensities
(Figure 2b), accompanied by a significant hypsochromic shift of
the maximum (Figure 2c), as a result of the progressive
incorporation of the fluorophore to the hydrophobic micro-
environment provided by the BSs.
Furthermore, time-resolved measurements were performed
for NaCh, NaGCh, NaTCh, NaCDCh, and UDCh, as well as
for NaGCDCh and NaDCh. Thus, the lifetime of 3α-Dns-ChA
was determined upon addition of increasing concentrations of
BS. The fluorescence decays of 3α-Dns-ChA in the presence of
NaGCh are shown in Figure 3a as an example.
EXPERIMENTAL METHODS
■
Chemicals. The BA salts NaCh, NaGCh, NaTCh,
NaCDCh, NaGCDCh, and NaDCh as well as lithocholic acid
(LChA), ursodeoxycholic acid (UDChA), sodium chloride,
sodium hydrogen carbonate, and dimethyl sulfoxide (DMSO),
were purchased from Sigma Aldrich and used without further
purification. The free acids LChA and UDChA were basified
with the stoichiometric amount of NaHCO3 to obtain the
corresponding salts NaLCh and NaUDCh. Millipore water was
used for sample preparation. The fluorescent reporter 3α-Dns-
ChA was synthesized as previously described.20
Instrumentation. Absorption spectra were registered on a
Cary 300 UV−vis spectrophotometer (UV0811M209, Varian).
Fluorescence spectra were recorded with a LP S-220B (Photon
Technology International) equipped with a 75 W Xe lamp. All
the spectra were corrected with baseline control experiments to
subtract the solvent Raman emission. Fluorescence lifetime
measurements were obtained on a PTI (TM-2/2003) equipped
In all cases, a significant increase of the average lifetime was
observed as the concentration of BS increased (Figure 3b). It
was also found that, at moderate BS concentrations, in addition
to the known lifetime in aqueous solution (4.8 ns),20 a second
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that is more than twice the value found in solution. Formation
of the secondary aggregates is also associated with a lifetime
enhancement, which is especially remarkable in the case of
NaCDCh and its conjugate derivative NaGCDCh.
Interestingly, when the changes in the relative fluorescence
intensities (I/I0) or in the average lifetimes (τ/τ0) of 3α-Dns-
ChA versus BS concentration were plotted together (see Figure
4 for NaGCh), it became evident that the two methodologies
reveal exactly the same trend, which can be taken as a proof for
the dynamic nature of the observed emission changes.
Figure 2. (a) Emission spectra of 3α-Dns-ChA (20 μM) in 0.2 M
NaCl upon addition of increasing concentrations of NaGCh (λexc
=
327 nm) in the range 0−100 mM. (b) Changes in the relative emission
intensities (I/I0). (c) Changes in the emission λmax vs BS
concentration for NaCh (red), NaGCh (black), NaTCh (green),
NaCDCh (blue), and NaUDCh (brown) in 0.2 M aqueous NaCl.
Figure 4. Changes in the relative emission intensities (I/I0) (black
squares) or lifetimes (τ/τ0) (green triangles) vs NaGCh concentration
in 0.2 M aqueous NaCl.
After the emission behavior of 3α-Dns-ChA as a function of
added BS concentration was examined, a further effort was
made to build the speciation diagrams for all BAs. For this
purpose, the percentage of each component in 0.2 M aqueous
NaCl (% i) within the different microenvironments, i.e.,
solution (% S), primary aggregates (% I), and secondary
aggregates (% II), was determined using eqs 1 and 2.
Figure 3. (a) Time-resolved fluorescence of 3α-Dns-ChA (20 μM) in
0.2 M aqueous NaCl upon addition of increasing concentrations of
NaGCh (λexc = 337 nm). (b) Changes in the relative lifetime (τ/τ0) vs
BA concentration for NaCh (red), NaGCh (black), NaTCh (green),
NaCDCh (blue), NaGCDCh (turquoise), NaDCh (pink), and
NaUDCh (brown) in 0.2 M aqueous NaCl.
(%S)ϕS + (%I)ϕI + (%II)ϕII
100ϕS
I
I0
=
(1)
100Aϕ
i
i
%i =
AS
ϕS
AI
ϕI
AII
ϕII
+
+
(2)
lifetime value (τI) had to be introduced to get a good fitting,
which was taken as an indication of the formation of the
primary aggregates. Moreover, at higher BS concentrations, a
third lifetime (τII) was needed for a satisfactory fitting, in
agreement with formation of the secondary aggregates. Table 1
shows the values found for the lifetimes of 3α-Dns-ChA (τI and
τII) when incorporated into every studied BS. According to
these data, incorporation of the probe into the primary
aggregates provokes an increase of its fluorescence lifetime
where I/I0 is the emission enhancement at a given BS
concentration, ϕS, ϕI, and ϕII are the corresponding
fluorescence quantum yields, and AS, AI, and AII are the pre-
exponential factors of the decay fittings in the time-resolved
experiments.
The value of ϕS determined for 3α-Dns-ChA was 0.05; the
values of ϕII were initially estimated assuming that, when the
plateau is reached, at high BS concentration, secondary
aggregates are the overwhelmingly predominating species.
Primary aggregates are expected to predominate in the
intermediate BA concentration range. Therefore, initial ϕI
values were selected by assuming the simplification that
primary aggregates are the only existing species at intermediate
concentration. In the case of NaGCDCh and NaDCh, for
which the steady-state experiments were less reliable, the values
of ϕI and ϕII were taken from the plot of the relative lifetimes
versus BS concentration. This is based on the parallelism
between the steady-state and the time-resolved behaviors,
which was already demonstrated for the other BAs (see Figure
4). The first estimated quantum yield values were fed into eqs 1
and 2 and subsequently refined within 0.02 until the
Table 1. Fluorescence Lifetimes of 3α-Dns-ChA in Primary
(I) and Secondary (II) Aggregates
τI (ns)
τII (ns)
NaCh
10.6
12.1
11.6
8.5
12.8
16.5
13.2
15.5
17.2
12.3
14.9
NaGCh
NaTCh
NaCDCh
NaGCDCh
NaDCh
NaUDCh
9.5
9.5
9.0
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simulated results were superimposable to the experimental
ones. The final quantum yield values are shown in Table 2.
concentration for the primary aggregates is reached at 25−30
mM while for NaCh it is reached at ca. 15 mM (see Figure 6).
Table 2. Fluorescence Quantum Yields of 3α-Dns-ChA in
Primary (I) and Secondary (II) Aggregates Determined by
Applying Eqs 1 and 2
a
a
ϕI
ϕII
NaCh
0.14
0.13
0.12
0.13
0.13
0.11
0.10
0.19
0.19
0.14
0.18
0.20
0.18
0.16
NaGCh
NaTCh
NaCDCh
NaGCDCh
NaDCh
NaUDCh
Figure 6. Composition between the BA concentrations where the
percentage of primary aggregates is maximized for NaCh (red),
NaGCh (black), NaTCh (green), NaCDCh (blue), NaGCDCh
(turquoise), NaDCh (pink), and NaUDCh (brown) in 0.2 M aqueous
NaCl.
a
Errors were lower than 5% of the stated values.
With the determined percentage of each species (% i) it was
possible to build the speciation diagrams of each BA as shown
in Figure 5. Significant differences can be found among the
seven studied analogues. For instance, in the case of NaCh and
the corresponding glycine and taurine derivatives (Figure 5a−
c), it is clear that conjugation results in more hydrophilic BAs
that extend their presence in solution until 40 mM. In
accordance with this increased solubility, the maximum
In addition, the lack of a hydroxyl group at position C-12 has
also a marked influence on the distribution of the three species,
as shown for NaCDCh and NaGCDCh (Figure 5d,e). Their
presence in the bulk solution is scarce above 8 mM, and the
primary aggregates are predominating at around 5 mM (Figure
6). Again, the increased solubility due to conjugation with the
Figure 5. Percentage of BA in solution (black squares), in primary (red circles) and secondary aggregates (green triangles) for (a) NaCh; (b)
NaGCh; (c) NaTCh; (d) NaCDCh; (e) NaGCDCh; (f) NaDCh; and (g) NaUDCh.
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amino acid moves the three curves to higher concentrations,
although in this case the effect is less remarkable than in the
NaCh/NaGCh pair. As expected, very similar speciation
diagrams were found for NaCDCh (Figure 5d) and NaDCh
(Figure 5f), since both contain two hydroxyl groups in the α-
face; changing the hydroxyl group at C-7 to the β-face of the
skeleton as in NaUDCh results in a higher solubility (Figure
5g), and therefore the maximum percentages of primary
aggregates appear at higher concentration (Figure 6). This is
interesting in connection with the pharmacological activity of
NaUDCh, which has been attributed in part to the fact that its
administration to patients renders the BA composition of bile
less hydrophobic, reducing the concentration of NaCDCh/
NaGCDCh and NaDCh/NaGDCh and hence the toxic
potential.21,22 Overall, the observed trends are in reasonable
agreement with the cmc values determined by independent
methods, such as that based on the relative intensities of the
vibronic bands in the pyrene fluorescence spectrum.16
However, speciation diagrams are much more informative
than just cmcs, and the fact that a single Dns-labeled BA can be
used to probe aggregation in a full series of analogues
constitutes a general validation of the methodology.
(8) Small, D. M.; Penkett, S. A.; Chapman, D. Biochim. Biophys. Acta
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CONCLUSIONS
■
In summary, the usefulness of 3α-Dns-ChA as a fluorescent
reporter to establish the distribution of the most abundant bile
acids in the main three microenvironments, solution, and
primary and secondary aggregates, has been demonstrated. The
developed methodology is based on the combined analysis of
steady-state and time-resolved experiments, which reveal a
strongly medium-dependent emission behavior. The obtained
information can be advantageously used to build the speciation
diagrams, where clear differences associated with the degree of
hydroxylation and/or conjugation have been found.
(20) Rohacova, J.; Marin, M. L.; Martinez-Romero, A.; O’Connor, J.
E.; Gomez-Lechon, M. J.; Donato, M. T.; Castell, J. V.; Miranda, M. A.
Org. Biomol. Chem. 2009, 7, 4973−4980.
(21) Paumgartner, G.; Beuers, U. Clin. Liver Dis. 2004, 8, 67−81.
(22) Moroi, Y.; Okabe, M. Colloids Surf. A−Physicochem. Eng. Asp.
2000, 169, 75−84.
ASSOCIATED CONTENT
* Supporting Information
Absorption spectra, steady-state spectra, time-resolved decays,
and comparison of experiments of the bile acids. This material
is available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: marmarin@qim.upv.es (M.L.M.); mmiranda@qim.
upv.es (M.A.M.). Fax: +34963879444. Tel: +34963877807.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We gratefully acknowledge financial support from the General-
itat Valenciana (GV/2009/104 and Prometeo 2008/090), the
■
Spanish Government (Red RETICS de Investigacion
́
de
Reacciones Adversas a Alergenos y Farmacos (RIRAAF),
́
CTQ2009-13699, and Predoctoral FPU fellowship AP2008-
03295 for M.G.-M.).
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