Clinical Science and Molecular Medicine (1977) 53, 101-103.
SHORT COMMUNICATION
The crystalline salts of calcium bilirubinate
in human gallstones
D. J U N E SUTOR
AND
LYNETTE I. WILKIE
Department of Chemistry, University College London, London
(Received 21 January 1977; accepted 24 March 1977)
s-ary
1. The acid, neutral and ‘intermediate’salts of
calcium bilirubinate have been synthesized.
2. All are crystalline, and the intermediate
salt which has 14 molecules of bilirubin per
calcium atom probably has a crystal structure
containing molecules of both acid and neutral
salts.
3. The acid or intermediate salts have been
found in 14 out of 70 gallstones analysed
recently by X-ray diffraction.
Key words : bilirubinate, calcium bilirubinate,
gallstones, pigment.
Introduction
Pigment is present in most gallstones. Although
the term covers several compounds, their identity has not been conclusively established. We
have therefore investigated these substances,
starting with the crystalline material. What was
thought to be a crystalline pigment was found
in bovine and human gallstones by X-ray
diffraction (Epprecht, Rosenmund & Schinz,
1953; Bogren & Larsson, 1963; Sutor & Wooley,
1971) and was tentatively called calcium bilirubinate. However, Bogren & Larsson (1963)
showed that crystalline calcium bilirubinate
prepared by the method of Edwards, Adams &
Halpert (1958) was not the crystalline pigment
of gallstones. On the other hand, other workers
using infrared spectroscopy and the same synthetic material claimed that this calcium
Correspondence: Dr D. I. Sutor, Surgical Unit,
University College Hospital Medical School, The Rayne
Institute, 5 University Street, London WClE 6JJ.
bilirubinate is present in gallstones (Suzuki &
Toyoda, 1966).
We have now obtained two slightly different
X-ray-diffraction patterns for gallstone pigment,
both of which differ slightly from the somewhat
different single patterns given by Epprecht et al.
(1953) and Bogren & Larsson (1963). Moreover
the data published by these two groups do not
contain d spacings above 9.1 x 10-1 nm which
heIp to differentiate our two patterns. To
determine whether the crystalline pigment of
gallstones is calcium bilirubinate, this substance
was synthesized under different conditions,
examined chemically, crystallographically and
spectroscopically, and the X-ray-diffraction
photographs were compared with those from
gallstones.
Methods
Calcium bilirubinate was prepared by using
excess of calcium chloride to ensure that the
maximum amount of bilirubin reacted. Immediately before it was required, sodium bilirubinate
was prepared in the following way. Bilirubin
(85 pmol), supplied by BDH Ltd, was suspended
in 10 ml of deionized water and NaOH (1 molll)
added dropwise until all material dissolved. A
portion (10 ml) of triethanolamine solution
(0.29 mol/l) was adjusted to the required pH
with HCl (1 mol/l) and diluted to 25 ml. This
buffer was added to CaC1,,2Hz0 (3-5 -01)
and the freshly prepared sodium bilirubinate
was added with stirring. The gelatinous material
formed was collected by centrifugation, washed
several times with deionized water, methanol
and lastly chloroform. On drying in a vacuum
101
D. June Sutor and Lynette I. Wilkie
102
desiccator, a red-brown crystalline substance
was always obtained. The experiment was
carried out at initial buffer pH values of 6*3,6*7,
7.3, 7.8, 8.5 and 10.0, but addition of sodium
bilirubinate caused a rise in pH of up to 0.4 unit.
Experiments were performed with three times
the buffer concentration so that initial and final
pH values were the same. Calcium bilirubinate
was also synthesized by the method of Edwards
et al. (1958), in which sodium bilirubinate and
calcium chloride are mixed at pH 12.0.
All specimens were photographed by the
X-ray-powder method (Sutor & Wooley, 1971).
Routine elemental analyses for carbon, hydrogen, nitrogen and calcium were made on the
different crystalline salts obtained. The infrared
spectra of these salts and bilirubin were recorded
with a Perkin-Elmer spectrophotometer (model
no. 177), 1-2 mg of sample being mixed with
0-2 g of KBr and made into a pellet under
pressure.
Results
Three different crystalline calcium salts of
bilirubin were synthesized. Table 1 gives their
elemental analyses, pH range of formation in
the weaker buffer solution and X-ray-diffraction
pattern. The nature of the salt was established
from the calcium analysis, since the acid salt
contains 3.3% calcium and the neutral salt
6.4%. The material prepared in the range pH
6.3-7.4 (covering initial and final values) is the
acid salt, and the suggested formula is
(Ca3H3sN406)2Ca,H20.
However, the error in
the hydrogen measurement is k 2 atoms in the
above formula, thus the amount of water of
crystallization, if any, cannot be determined.
The salt stable above pH 7-8 is the neutral
salt, and, subject to the error in the number
of hydrogen atoms, the formula is
C33H34N406Ca,HZ0.
This is the salt prepared
by the method of Edwards et al. (1958). The salt
formed within the range pH 7-3-74 contains If
molecules of bilirubin per calcium atom. X-raydiffraction photographs show that this material
is not a mixture of the acid and neutral salts in
any fixed proportion. It is therefore likely to be
a mixed crystal, i.e. its crystal structure contains
molecules of both acid and neutral salts. We
have called it the intermediate salt.
When calcium bilirubinate was synthesized in
the stronger buffer solution, the acid salt was
obtained up to pH 7.7. At pH = 8.0 the intermediate salt formed, but the point at which the
neutral salt crystallized was not determined.
The identity of the salts was confirmed by
their infrared spectra. The peak at 1700 cm-'
in the bilirubin spectrum represents the carbonyl
band for carboxylic acids. When these groups
TABLE
1 . Data for the three crystalline salts of calcium bilirubinate
Interplanar ( d )spacings and their relative intensity ( I ) are given together with the carbon: hydrogen:nitrogen atomic
proportions, the percentage calcium and the pH range in which each salt is formed in the weaker buffer solution.
Relative intensities are indicated by: s = strong, m = medium, w = weak, d = diffuse, v = very.
Acid salt
Intermediate salt
I
d (1o-l nm)
d (10-l nm)
I
Neutral salt
d (10-l nm)
I
~
Diffraction
pattern
vs d
15.9
10.1
8-34
6.39
5.69
4.74
4.41
3.68
3.33
C:H:N
(numbers of atoms)
Ca (%I
Approximate
pH range
vs d
14.9
vs d
13.2
S
S
8.48
S
8-7
S
5.65
483
432
3.68
3.34
m
s d
5.3
4.16
4-04
3.61
3-22
wd
m
m
m
md
W
m
md
W
W
md
W
W
md
33:36:4
3.6
33:35:4
4.8
33:36:4
6-2
6.3-7.4
7.3-7.8
> 7.8
Calcium bilirubinate in gallstones
are ionized, as in salts, this peak vanishes
(Bellamy, 1975). Thus this band will be less
marked in the acid salt and missing in the
neutral salt. These changes were observed in our
acid and neutral salts, and the small shoulder
given by the intermediate salt suggests it has
un-ionized carboxyl groups but fewer than the
acid salt.
The X-ray-diffraction patterns of the acid and
intermediate salts correspond to our two
patterns obtained from gallstones. A recent
study of the crystallinecompositionof gallstones
from 70 unselected patients from University
College Hospital, London, showed the acid salt
was present in eight stones and the intermediate
salt in another six. The crystalline constituents
accompanyingcalcium bilirubinate were usually
calcium phosphate and/or calcium carbonate.
The remaining 56 stones and possibly some of
the others contained pigment@ not detectable
by X-ray diffraction,either because the pigment
was present in too small an amount or, more
likely, because it was amorphous.
Discussion
It has been established that only the acid and
intermediate salts of calcium bilirubinate can
crystallize in gallstones in quantities detectable
by X-ray diffraction. The salt formed when
crystallization occurs depends on the pH and
composition of the solution and probably other
factors as well. Thus, from our results, it is
impossible to define a pH range in which each
salt crystallizes from bile.
As already mentioned, some infrared spectro-
103
scopists reported the presence in gallstones of a
calcium bilirubinate, which we showed was the
neutral salt. Non-detection by X-ray diffraction
might result from the small amount present or
its non-crystallinity. However, none of the
spectroscopists mentions material corresponding to the acid and intermediate salts, which
occurred in 20% of our gallstones. Therefore a
re-examination of the infrared data with
standards of known composition is necessary,
to determine if neutral calcium bilirubinate is a
gallstone constituent.
Acknowledgments
We thank the Medical Research Council for
financial support and Mr M. Jackson for the
calcium determinations.
References
BELLAMY,
L.J. (1975) The Infrared Spectra of Complex
Molecules, 3rd edn, pp. 183-202. Chapman and Hall,
London.
BOGREN,
H. & LARSSON,
K. (1963) On the pigment in
biliary calculi. Scandinavian Journal of Clinical and
Laboratory Investigation, 15,56%572.
EDWARDS,J.D.,JR, ADAMS.W.D. & HALPERT,B.
(1958) Infrared spectrums of human gallstones.
American Journal of Clinical Pathology, 29, 236-238.
W., ROSENMUND,
H. & Scanrz, H.R. (1953)
EPPRECHT,
Chemische, mineralogische und Rontgen-Feinstrukturuntersuchungen an Gallensteinen des Menschen
und des Rindes. Rdntgenfortschritte, 79, 1-19.
SUTOR,D.J. & WOOLEY,
S.E.(1971) A statistical survey
of the composition of gallstones in eight countries.
Gut, 12, 55-64.
SUZWKI,
N. & TOYODA,
M. (1966) On infrared absorption spectra of bilirubin and calcium bilirubinate.
Tohoku Journal of Experimental Medicine, 88, 353360.