Background
The saturation status of unconjugated bilirubin (UCB) is
relevant to understanding the pathophysiology of jaundice
and to interpreting experiments with UCB [ 1 ] . UCB, in
its diacid form (H
2 B), has a low solubility (S
o ) of 51 nM in water [ 2 ] . The total
solubility (S) at any pH is determined by S
o , pK
a
values, and pH [ 2 ] :
S = [H
2 B] + [HB -] + [B =] = S
o ·(1 + K
a 1 / [H +] + K
a 1 ·K
a 2 / [H +] 2) (
Eq. 1 )
Self-association of UCB dianion, B =, can also increase
S [ 2 ] .
Beginning in 1995, a series of papers reported the use
of 13C-NMR to study the ionization of a close analogue of
bilirubin-IXα, mesobilirubin-XIIIα (MBR), along with
several soluble reference acids, in buffered mixtures of (C
2H
3 )
2 SO and water [ 3 4 5 6 7 ] . Due to
the very low aqueous solubility of MBR, data were obtained
only at two high concentrations of (C 2H
3 )
2 SO (64 and 27 vol%). Its pK
a
values in "water" (actually in 1 vol% of ((C 2H
3 )
2 SO), obtained by an extrapolation
procedure based on the behavior of soluble reference acids,
were 4.2 and 4.9, far lower than values of 8.12 and 8.44
obtained by solvent partition between chloroform and
buffered aqueous solutions [ 2 ] .
These serious discrepancies led us to reexamine several
experimental aspects of the 13C-NMR studies and the
implications of their purported low pK
a
values for the solubility of UCB. Inaccuracies in the
measurements of buffer pH and of the pK
a
values of the reference 13C-carboxylic acids, related
to failure to correct for the strong effects of DMSO on the
pK
a
values of weak acids, have been previously noted [ 8 ]
and acknowledged [ 9 ] . In addition, as discussed later, a
thermodynamic theory about solubility in mixed solvents,
using the S
o values of 51 nM in water [ 2 ] and 10
mM in pure DMSO [ 10 ] , suggested that S
o of UCB would be only about 0.15 μM in
27 vol% DMSO and 2.2 μM in 64 vol% DMSO, which are well
below concentrations used in the MBR studies. This implies
serious supersaturation effects. In fact, turbidity was
reported for some of the systems used in the 13C-NMR papers
[ 4 ] , indicating the formation of coarse suspensions. By
definition, as stressed in the Conclusions section, pK
a
determinations are valid only for monomeric species
and require that solutions are below saturation at all pH
values studied. Even reversible aggregation of monomers in
undersaturated solutions is known to affect pK
a
estimates [ 2 ] . If supersaturation leads to the
formation of fine sols or coarse suspensions, the data are
unacceptable for determination of the pK
a
values of monomers.
In the present paper, we assess whether some of the
systems used in the 13C-NMR studies were supersaturated
with MBR. Our experimental work on sedimentation was done
with unconjugated bilirubin (UCB) and we used DMSO instead
of its deuterated analogue, (C 2H
3 )
2 SO. It is known that, when alkaline
aqueous UCB is acidified to neutral or low pH's, "Usually a
colloid suspension of bilirubin is formed and the solution
remains clear, as observed by the naked eye, thus inviting
an erroneous interpretation" [ 11 ] . Such systems, which
simulate true solutions, often show sedimentation on
centrifugation [ 12 13 14 15 16 ] . We here report that
sedimentation of UCB from apparently clear solutions can
likewise be extensive when UCB in DMSO is diluted with
aqueous buffers to final mole fractions (N) of DMSO =
0.025, 0.086 and 0.31, corresponding to 9, 27, and 64 vol%
of DMSO. A thermodynamic theory is used to examine the
effect of added DMSO on S
o . Our findings, consistent with our
partition-derived S
o , S and pK
a
values of UCB in aqueous buffers [ 2 ] , indicate that
the recently reported low pK
a
values of MBR in comparable (C 2H
3 )
2 SO -water systems [ 3 4 5 6 ] , were
determined above saturation.
Results
Residual UCB in the supernatants after
centrifugation
Many systems initially appeared optically clear, but
well over 90% of initial UCB sedimented on centrifugation
of most samples below pH 7.2. Overall recoveries (UCB in
supernatant + UCB in redissolved precipitate) were
between 90 and 100%. Only residual [UCB] in supernatants
are reported.
Figs. 1A,1B,1Cplot the measured residual [UCB] in the
supernatant vs. the initial [UCB] at N
DMSO = 0.31, 0.086 and 0.025.
Deviations of their ratio below unity (dotted lines)
represent a decrease in [UCB], mainly from precipitation,
but possibly also from limited degradation. At N = 0.31
(Fig. 1A), [UCB] after sedimentation varied from 2.8 μM
at pH 5.86 to 166 μM at pH 8.38. At N = 0.086 (Fig. 1B),
[UCB] ranged from 0.3 to 2.4 μM at pH 4.50 and from 1.4
to 6.4 μM at pH 7.05 and most of the UCB sedimented. By
contrast, at pH 7.56 and 7.70 (phosphate), only minor
precipitation was observed. At N = 0.025 (Fig. 1C); [UCB]
ranged from 0.1 to 2.7 μM at all three pH values (4.15 to
7.18).
Sedimentation of bilirubin-albumin complexes
The supernatant from the original supersaturated
UCB-HSA system, when diluted with 1/8th vol. of buffer
and centrifuged again, showed more sedimentation and a
progressive rise in [UCB] as one moved down the column of
fluid. By contrast, after dilution with 1/8th volume of
DMSO to decrease UCB saturation, the supernatants
produced no further sedimentation and there were no
significant differences in [UCB] or protein
concentrations along the axis of the fluid column. Thus,
the UCB-HSA complex, which constitutes over 99.9% of the
UCB in this system [ 17 ] , did not sediment.
Discussion
Findings and their relation to thermodynamic
theory
Supersaturated aqueous systems of UCB, that are
optically clear before centrifugation, may exhibit
considerable variation in the extent of sedimentation [
12 13 14 18 ] . Although sedimentation is often
extensive, it is generally incomplete and may not be
observed at all. Our data in DMSO-water show the same
features, which are expected from the complex kinetics of
nucleation and growth of insoluble aggregates of UCB
diacid (H
2 B), leading to the formation of a
new solid phase [ 19 20 ] . Our centrifugation, 5 min at
14,000
g , was quite mild, and the short
20-minute period between preparation of UCB-DMSO-water
systems and centrifugation severely limited the
time-dependent growth to large aggregates. Lack of
sedimentation of the UCB-HSA complex (mol. wt. 68,000)
indicates that fine colloids composed of 100 UCB
molecules would be too small to sediment. Thus,
supersaturated systems lacking coarse, insoluble
aggregates may not show sedimentation, but any
sedimentation observed indicates their presence.
To evaluate the important effect of pH on
sedimentation efficiency, we calculated S in water using
chloroform-water partition data on UCB and the best
measure of S
o in chloroform, 0.88 mM [ 2 ] . S at
any pH, e.g. 62 nM at pH 7.4 and 0.32 μM at pH 8.5, can
be calculated from the fitted partition data, or,
equivalently, from Eq. 1, using the partition-derived S
o in water of 51 nM and pK
a
values of 8.12 and 8.44 [ 2 ] . In aqueous systems [
12 ] , the lowest [UCB] in water, below which no
sedimentation was observed at 100,000 ×
g for a few hours, was 100 nM at pH
7.4, modestly higher than our partition-derived S of 62
nM [ 2 ] . Even under such vigorous centrifugation, the
lowest [UCB] increased rapidly with increasing pH, to 17
μM (150 times S) at pH 8.05 and 34 μM (230 times S) at pH
8.2 [ 12 ] . This indicates increasing
charge-stabilization of fine, non-sedimenting colloids of
H
2 B by adsorbed UCB anions [ 12 19 20
] . In contrast, below pH 6.7, sedimentation of 10 μM UCB
was nearly complete [ 13 ] . This is compatible with a
dearth of stabilizing UCB anions at this pH, as expected
from the high pK
a
values of 8.12 and 8.44 [ 2 ] .
Our present data on residual [UCB] in DMSO-water
systems likewise show decreased sedimentation with
increasing pH (Fig. 1A,1B,1C). At each N
DMSO , the lowest [UCB] were at the
lowest pH values: 0.1 μM (N = 0.025, pH 4.15); 0.3 μM (N
= 0.086, pH 4.5); and 2.8 μM (N = 0.31, pH 5.9).
As in water, these are likely to be closest to the S
o values at each N. Indeed, they are
only moderately higher than the corresponding S
o values of 0.07 μM, 0.15 μM and 2.2
μM, respectively, calculated from Equation 2 using S
o values of 51 nM in water [ 2 ] and
10 mM in DMSO [ 10 ] .
log S
o,mixed = log S
o,water + (log S
o,DMSO - log S
o,water ) × N (
Eq. 2 )
Equation 2 is a thermodynamic relationship based on
assumptions of complete ideality of mixing [ 21 ] . In
general, a roughly linear variation of log S
o with N at low N is expected. For
example, data from 1-naphthoic acid in DMSO-water [ 22 ]
show that log S
o is a linear function of N up to N =
0.35. Such a relationship leads to a relatively small
effect of low N values on S
o . Thus, according to Equation 2, S
o increases by a factor of only 1.4 at
N = 0.025 and 2.9 at N = 0.086, but by a relatively
larger factor of 44 at N = 0.31. This would markedly
reduce the supersaturation factor ([UCB]/ S
o ), which is a measure of the
tendency of UCB to come out of solution at N = 0.31. This
explains in part the relatively high [UCB] at high pH at
N = 0.31 (Fig. 1A).
The pH effects on [UCB] at each N
DMSO are of interest also. The lowest
[UCB] at each pH registered relatively small increases
with significant increases in pH: for example from 0.1 μM
(pH 4.14) to 0.2 μM (pH 7.0) at N = 0.025; from 0.3 μM
(pH 4.5) to 1.4 μM (pH 7.1) at N = 0.086; and from 111 μM
(pH 7.1) to 166 μM (pH 8.4) at N = 0.31. These increases
are probably caused mainly by increasing
charge-stabilization of colloidal aggregates, as in
aqueous media [ 12 19 20 ] . If, instead, the relatively
small increases are ascribed entirely to increases in
true solubility (S) at the high pH (Eq. 1), the required
pK
a
values are about 7 at N = 0.025 and 0.086, and 8.5
at N = 0.31. The true pK
a
s of UCB in DMSO-water are thus probably
significantly higher.
We note that some variability in sedimentation results
from our short-term experiments, most evident at the low
residual [UCB] in Figs. 1Band 1C, in part magnified by
the log-log scale used. Some variability is expected,
however, because of the complexity of the kinetic
processes of nucleation, growth and flocculation that
precede sedimentation. In Fig. 1A, the difference between
acetate and Tris buffers is quite small (note the linear
scale), compatible with the 58% higher [H +] in the
acetate buffer. In Fig. 1B, the markedly lower
sedimentation from phosphate buffers at pH 7.6-7.7, as
compared to Tris buffer at pH 7.1, can be ascribed mainly
to the much higher pH values and ionic strength of the
phosphate systems. Another significant factor may be the
difference in charge between the buffer salts; phosphate
is anionic whereas Tris is cationic and zwitterionic. The
cationic species of Tris can, in principle, reduce the
negative charges on the surface of the colloidal H
2 B sufficiently to facilitate the
formation of coarser particles and, thus, increase
sedimentation.
Implications for pK a values of mesobilirubin-XIIIα
(MBR)
In the recent 13C-NMR studies of the ionization of the
13C-COOH groups of MBR [ 3 4 5 6 ] , it was assumed that
the relevant physical properties of UCB and MBR, and of
(CH
3 )
2 SO (DMSO) and (C 2H
3 )
2 SO, are similar. Actually, as
expected from the replacement of two vinyl groups in UCB
with two ethyl groups in MBR, MBR is slightly more
soluble in organic solvents [ 23 ] and has a higher R
f on silica gel t.l.c. [ 24 ] ; MBR is
thus more hydrophobic and should be less soluble in water
than is UCB. Our low [UCB] in DMSO-water systems at
comparable N, therefore, indicate that many of the (C 2H
3 )
2 SO/buffer systems used in the
13C-NMR studies [ 3 4 5 6 ] were likely supersaturated
with MBR. In those studies, the MBR concentrations used
were stated to be 1 to 100 μM at N = 0.086 [ 3 ] ,
compared to our lowest [UCB] of 0.3 μM at pH 4.5 and 1.4
μM at pH 7.05. At this N, 9 of 11 MBR data points were
obtained at pH below 7.05 and 5 below pH 4.5 [ 4 ] , so
that even 1 μM MBR was likely to be supersaturated. At N
= 0.31, our lowest [UCB], 2.8 μM at pH 5.9, was close to
the lower limit of the 2 to 800 μM range of [MBR] used [
4 5 ] . Thus, many data points, obtained at pH values
down to 2 [ 4 5 ] , were probably from supersaturated
systems, despite being optically clear. As noted here and
elsewhere [ 12 13 14 15 16 18 ] , optical clarity gives
no assurance of the absence of supersaturation.
Actually, turbidity was reported in some of the
13C-NMR samples [ 4 ] , indicating that coarse, insoluble
aggregates of MBR were present. The claim that such
turbidity did not affect 13C-NMR measurements [ 3 5 6 ]
contrasts with evidence that even small multimers can
change NMR chemical shifts [ 25 26 ] . It should be noted
also that, at high concentrations of B =, extensive,
reversible self-association of B =can lead to apparently
stable supersaturation with no separation of an insoluble
phase [ 2 ] . For example, at pH 8.5 and a UCB
concentration of 20 μM (63 times S), the weight-average
aggregation number of UCB has been found to be 7.17 [ 18
] , corresponding to a molecular weight of 4,195. The
aggregation number remained fairly high, 4.2, in 60%
(w/v) ethanol [ 18 ] . The successful application of
equilibrium ultracentrifugation for that study [ 18 ]
suggests a complete absence of even small colloidal
species of UCB. Self-association of MBR dianions in (C 2H
3 )
2 SO-water mixtures cannot be ruled
out on
a priori grounds. It has been shown
that neglect of self-association of B =leads to an
artefactually low estimate of pK
a
values for UCB [ 2 ] .
In addition to the problems of insolubility,
supersaturation and self-aggregation of the MBR systems
in (C 2H
3 )
2 SO-water [ 3 4 5 6 ] , we had shown
previously that inaccuracies in the pH measurements
affected both the magnitude of ΔpK
a
(the change in pK
a
on adding (C 2H
3 )
2 SO to water), as well as the degree
of the variation of ΔpK
a
with N [ 8 ] . This is important for extrapolating
pK
a
values in (C 2H
3 )
2 SO-water to pure water (N = 0).
Indeed, remeasurement of one soluble acid raised its pK
a
by as much as 3 units at N = 0.31 [ 9 ] . Thus, the
inaccuracies in pH measurement produced serious errors in
reported pK
a
values of more than fifteen soluble acids used as
models for MBR, as well as for MBR itself [ 3 4 5 6 7 ]
.
Many methods, using appropriate pH measurements, have
been applied in the past to determine thermodynamic pK
a
values of soluble acids in non-aqueous or partially
aqueous media, including DMSO-water systems [ 27 28 ] .
Many other relevant references were given in our prior
paper [ 8 ] . In that paper, our pK
a
measurements on acetic acid in DMSO-water systems
were based on the potentiometric method, using properly
calibrated glass electrodes, which determine the activity
of H +, and on estimates of the activity coefficients of
the acetate ion. This method, which is well established
for aqueous solutions, yielded results in good agreement
with data from the literature that was based on a very
different method, measurements of electrical conductivity
[ 28 ] . In the 13C-NMR papers, therefore, it was not
justified, to assume that pH values do not change on
adding DMSO [ 3 4 5 6 7 ] , or to use uncalibrated pH
measurements for determination of the pK
a
values of soluble acids [ 9 ] .
Our sedimentation data and their interpretation
indicate that significant additional uncertainties, not
important for the soluble acids investigated, exist for
the reported pK
a
values of the relatively insoluble MBR in (CD
3 )
2 SO-water (4.2 and 4.9 at N = 0.086
and 4.3 and 5.0 at N = 0.31), as well as their
extrapolation to obtain pK
a
values of 4.2 and 4.9 in water [ 9 ] . Indeed, if
these low aqueous pK
a
values, along with the experimental S values at pH
8.5 of 0.32 μM [ 2 ] , or 0.6 μM [ 17 ] , are applied to
Eq. 1, the calculated extremely low S
o values of UCB diacid of 4 or 8 × 10
-15M are seven orders of magnitude lower than the
experimental S
o , 5.1 × 10-8 M [ 2 ] . Applying the
S
o of 4 or 8 × 10 -15M to Eq. 2,
moreover, would indicate massive supersaturation (up to 8
to 10 orders of magnitude) of MBR at the concentrations
(1-800 μM) used in the 13C-NMR studies [ 3 4 5 6 ] .
Conclusions
The present sedimentation data for UCB in DMSO-water
demonstrate that the true solubilities of UCB, even at
fairly high pH values, are low at DMSO mole fractions up to
0.31. The results and related considerations are compatible
with similar results in purely aqueous solutions [ 12 13 14
] , and support both the estimated solubility (S
o ) of 5.1 × 10-8 M for uncharged UCB (H
2 B) in water, and the corresponding
high aqueous pK
a
values of 8.12 and 8.44, derived from our partition
studies [ 2 ] . These were performed in undersaturated
systems and took into account the self-association of B =.
Our experimental data indicate problems of insolubility,
supersaturation and self-aggregation of UCB in DMSO-water
mixtures with compositions similar to the MBR systems in (C
2H
3 )
2 SO -water [ 3 4 5 6 ] . In (C 2H
3 )
2 SO-water, DMSO-water [ 27 ] or any
other medium [ 8 ] , properly determined pK
a
values for the dissociation equilibria of a diacid H
2 A (H
2 A <--> HA -+ H +and HA
-<--> A =+ H +) must pertain to monomeric H
2 A, HA -and A =, the solute species
involved in the stated equilibria, and require unambiguous
determination of [H +] or pH. Unless pK
a
values are determined for monomeric systems, relative
concentrations of H
2 A, HA -and A =cannot be determined
from the pK
a
values and the pH. The 13C-NMR data, suggesting low pK
a
values for MBR in (C 2H
3 )
2 SO -water and water [ 3 4 5 6 9 ] ,
did not meet these essential requirements of proper pH
measurements [ 8 ] nor provide assurance that the MBR in
every system was below saturation and not self-associated [
2 ] . The issues raised are not trivial, since the pK
a
and S
o values of UCB are clinically relevant
to the effects of pH on the precipitation of calcium
bilirubinates in pigment gallstones and the neurotoxicity
caused by UCB diacid in severely jaundiced neonates [ 29 ]
.
Materials and Methods
Materials
UCB (Calbiochem) was purified by alkaline extraction
of a chloroform solution, recrystallized twice from
chloroform-methanol [ 30 ] , dried under Argon, stored in
vacuo in the dark and used within 6
weeks. DMSO was spectroscopic grade, 99.8% pure (UVASol,
Merck). Human serum albumin (HSA, lot 903635) was from
Calbiochem-Boehringer. All other chemicals were reagent
grade (Merck). Water used was deionized and distilled.
All flasks and tubes were Kimax glass, washed with 0.1 N
HCl and rinsed 4X with water and then dried before use.
Stock buffers, 1.0 M, were: Tris-HCl, pH 7.01; Na-
phosphate, pH 6.85, or 6.99; and Na-acetate, pH 4.01.
Stock UCB in DMSO (4 to 6 mM) and stock HSA, 613 μM in
0.1 M Tris-HCl buffer, pH 7.01, were prepared freshly for
each experiment.
Preparation of UCB-DMSO-buffer systems
Test systems (4.0 mL) of UCB were prepared in
duplicate: to 0.4 mL of the stock buffer were added
successively the appropriate volumes of water, DMSO and,
finally, up to 150 μL of stock UCB/DMSO solution. To
minimize UCB oxidation, all tubes and solutions were
deoxygenated with Argon and kept in the dark [ 31 ] . To
determine the [UCB] in the UCB/DMSO stock, 6.0 μL, was
added to 3.0 mL of the HSA stock. The absorbance,
A , at 460 nm was read against a
blank containing 2.0 mL of HSA stock plus 4.0 μL DMSO,
and the [UCB] calculated using the extinction
coefficient, ε, 47,000 M -1.cm -1 [ 18 ] . The pH
measurement of each final DMSO/aqueous buffer system
included an electrode calibration using the strong acid,
HClO
4 [ 8 ] . The 0.1 M phosphate buffer
in N = 0.31 DMSO was centrifuged because of partial
insolubility [ 8 ] and only the supernatant was used.
Centrifugation and analysis of residual UCB in
supernatants
Fifteen min after mixing, samples were assessed
visually for turbidity or precipitation. After
Vortex-mixing, duplicate 1.8 mL aliquots were transferred
to polypropylene tubes and centrifuged for 5 min at 25°C
and 14,000
g (Mikroliter centrifuge, Hettich,
Tuttlingen, Germany). The supernatants were assayed
spectrophotometrically within 20 min. The precipitates
were washed once with 1.8 mL of water, again centrifuged,
the water aspirated, and the packed precipitate dissolved
in DMSO for spectrophotometry. Absorbance (
A ) was measured in triplicate at
458 nm on each sample, diluted, when necessary, with DMSO
or DMSO/buffer mixture to
A of 0.2 to 0.8; a comparable
medium without UCB was used as a blank. In all systems,
including the redissolved UCB sediments, we applied the
extinction coefficient of 0.0634 μM -1cm -1at the peak
wavelength of 458 nm for UCB in pure DMSO [ 18 ] .
Preliminary calibration studies. of the effects of DMSO
concentration and buffer composition on
A had confirmed this value for pure
UCB in pure DMSO and in DMSO/buffer systems containing 64
vol% DMSO. In the systems containing 27 and 9 vol% DMSO,
the spectrum developed a plateau between 458 and 450 nm,
but
A at 458 nm remained within ± 10%
of the value expected from applying the extinction
coefficient of 0.0634 μM -1cm -1to the measured quantity
of UCB dissolved in each system. The variability is in
part due to degradation, discussed above, and in part due
to the low absorbances at [UCB] below the saturation
limit in some buffer/DMSO systems. For these reasons, no
corrections were made for these minor differences in
A .
Sedimentation of bilirubin-albumin complexes
To determine if UCB bound to HSA would sediment, we
prepared a system containing HSA, 300 μM, and UCB 170 μM,
in 0.1 M Tris-HCl buffer, pH 7.01. Microcentrifugation
for 5 min yielded a small amount of precipitated UCB.
Three aliquots of the clear supernatant were diluted with
1/8th volume of DMSO and a fourth aliquot diluted with
1/8th volume of buffer. The diluted samples (in
duplicate) were then microcentrifuged for another 10 min
and 25 μL samples taken from the top, middle and bottom
of the fluid column in each tube, using a Hamilton
syringe. Protein concentrations were determined with the
Bio-Rad bicinchonic acid method, which is unaffected by
bilirubin. After dilution with 1.8 mL DMSO, triplicate
A readings were taken at 458
nm.
Abbreviations
UCB, unconjugated bilirubin; H
2 B, UCB diacid; B =, UCB dianion; DMSO,
dimethylsulfoxide; MBR, mesobilirubin XIIIα; N = mole
fraction of DMSO in DMSO-aqueous buffer systems; NMR,
nuclear magnetic resonance; S, solubility of UCB or MBR at
a given pH; S
o , solubility of UCB diacid.
Authors' note
An abstract of this work has been published
(Gastroenterology 2000; 118:A1477)
Authors' contributions
All three authors collaborated in the conception, design
and writing of this study. The work was performed by JDO
while he was a visiting professor at the Academic Medical
Center in Amsterdam, the Netherlands. All authors read and
approved the final manuscript.
