How does bovine serum albumin prevent the formation of kidney stone? A kinetics study

Article abstract of DOI:10.1021/jp057403b

To attain a better understanding of the crystallization of calcium oxalate crystals under the influence of the protein bovine serum albumin, we examined not only the nucleation kinetics but also the structural synergy between the biomineral and the biosubstrate. It follows that during the crystallization process of calcium oxalate crystals bovine serum albumin inhibits the nucleation of calcium oxalate by increasing the kink kinetics barrier. The results of scanning electron microscopy and X-ray diffraction show, however, that bovine serum albumin promotes the formation of calcium oxalate dihydrate. Apart from this, bovine serum albumin facilitates the ordered calcium oxalate crystal assembly by suppressing the supersaturation-driven interfacial structure mismatch. The physics questions behind the mentioned effects have been addressed from the kinetics point of view. This may explain why bovine serum albumin plays an important role in suppressing urine stone formation.

Full text of DOI:10.1021/jp057403b

J. Phys. Chem. B 2006, 110, 9085-9089
9085
How Does Bovine Serum Albumin Prevent the Formation of Kidney Stone? A Kinetics
Study
Junfeng Liu, Huaidong Jiang, and Xiang-Yang Liu*
Department of Physics, National UniVersity of Singapore, 2 Science DriVe 3, Singapore 117542
ReceiVed: December 20, 2005; In Final Form: February 27, 2006
To attain a better understanding of the crystallization of calcium oxalate crystals under the influence of the
protein bovine serum albumin, we examined not only the nucleation kinetics but also the structural synergy
between the biomineral and the biosubstrate. It follows that during the crystallization process of calcium
oxalate crystals bovine serum albumin inhibits the nucleation of calcium oxalate by increasing the kink kinetics
barrier. The results of scanning electron microscopy and X-ray diffraction show, however, that bovine serum
albumin promotes the formation of calcium oxalate dihydrate. Apart from this, bovine serum albumin facilitates
the ordered calcium oxalate crystal assembly by suppressing the supersaturation-driven interfacial structure
mismatch. The physics questions behind the mentioned effects have been addressed from the kinetics point
of view. This may explain why bovine serum albumin plays an important role in suppressing urine stone
formation.
Introduction
mismatch effect in the context of a new nucleation model.23,28-33
On the basis of this new nucleation model, some exciting results
Calcium oxalate monohydrate (COM) is the main inorganic
constituent of kidney stones.1-6 The cost of the treatment for
human kidney stone disease in the United States is estimated
to be more than 2.4 billion dollars per year.4,6 Thus, the study
of calcium oxalate crystal (CaOx) formation is of major
importance for human health. In particular, a fundamental
understanding of the crystallization is essential for the study of
micro-/nanostructure materials that have significant implications
for life sciences.3,4,6,7 Urinary proteins, such as the Tamm-
Horsfall protein,8-10 nephrocalcin,11-13 osteopontin,14-18 and
human serum albumin,3,7,14,19 are believed to have the potential
to influence the crystallization of calcium oxalate. Among them,
albumin seems to play a crucial role in kidney stone
formation.3,7,10,14,19-21 Some research work4,22,23 has suggested
that the proteins can influence crystal nucleation, growth, and
aggregation of calcium oxalate within the urinary system.
It was also believed that3,16,24-26 the modification of crystal
growth and crystal habit was achieved by the absorbance and
binding of the proteins to the surface of CaOx. These adsorbed
macromolecules can promote heterogeneous nucleation by
attracting calcium ions to their calcium-binding domains at the
surfaces.16,25 Nevertheless, there is no concrete data to support
those arguments. Apart from this, we notice that, to date,
although some aspects of nucleation, growth, and aggregation
of CaOx crystals in vitro have been studied including the effect
of some human proteins,7,27 no detailed studies on the crystal-
lization of calcium oxalate crystals have been reported to
elucidate the effect of these proteins. Evidently, an unambiguous
understanding of the effects of these proteins on the formation
of CaOx should be developed.
have been achieved in studying ice, calcium carbonate, and
hydroxyapatite,23,29,30 through a comparative analysis of the
effects of various selected additives (salts, proteins, and other
biopolymers). To obtain a better understanding of the CaOx
crystallization and the role of the protein (albumin) in the urine,
in this work, we employ the aforementioned model to examine
the nucleation of COM and the impact of bovine serum albumin
(BSA). In addition, we also examine how the protein (BSA)
influences the assembly of CaOx from the kinetics point of view.
Our aim is to identify an effective approach to control the
formation of CaOx crystals and contribute to the treatment of
kidney stones.
Experimental Section
Stock solutions were prepared by dissolving reagent-grade
calcium chloride dihydrate (CaCl2‚2H2O, J.T. Baker), and
sodium oxalate (Na2C2O4, Merck) in deionized water. The pH
was adjusted to 6.0. A series of concentrations, ranging from
3.6 to 5.2 mM, of the two solutions was prepared by gradually
diluting the stock solutions. Both kinds of solutions were injected
through filters (200 nm) into a rectangular glass cell to examine
the kinetics of calcium oxalate formation from the reactants.
The nucleation kinetics was examined by using dynamic light-
scattering (DLS) employing a Brookhaven BI-200SM light-
scattering system with a He-Ne laser (632.8 nm) source.29 This
system can detect particles of down to 2 nm, which allows an
in situ measurement of the nucleation process and the size
increase of the nuclei. The induction time ts was measured by
monitoring the photon count rate recorded by the DLS equip-
ment against time. Subsequently, the effect of BSA (Sigma
Chemicals) on the nucleation and the aggregation of calcium
oxalate was also studied. Being synthesized by the liver and
the main protein of plasma, albumin is a well-characterized
protein3,7,10,14,19,21 having a molecular weight 6.9 × 104 Da, a
size of 11.6 × 2.7 × 2.7 nm3, an isoelectric point of 4.7-4.9,
Recently, the structural synergy between biominerals and
biosubstrates was examined. To that end particular emphasis
was placed on the templating effect of the substrate as well as
a newly identified supersaturation-driven interfacial structure
* Author to whom correspondence should be addressed. E-mail:
phyliuxy@nus.edu.sg.
10.1021/jp057403b CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/20/2006

9086 J. Phys. Chem. B, Vol. 110, No. 18, 2006
Liu et al.
respectively
[a(Ca2+)][a(C2O42-)]
Ksp(COM)
∆µ ) kT ln
(2)
Here Ksp(COM) is the solubility product at a given temperature,
and Ca2+, C2O42-, and COM denote, respectively, the calcium
ion, oxalate ion, and calcium oxalate. Through the use of eqs 1
and 2, the supersaturation, σ, can be calculated for different
solution conditions.
Taking into account the effect of the substrate on both the
nucleation barrier and the transport process, the nucleation
induction is given as28,31
Figure 1. XRD patterns of CaOx crystals obtained from a solution
with/without BSA. The crystal faces with open circles indicate the
presence of COM. The asterisks indicate the presence of COD
crystals: curve a, no additive; curve b, BSA present.
∆G/
J ) (Rs)2N0f(m)[f(m)]1/2B exp -
homof(m)
(3)
[
]
kT
with
and a good binding capacity for water and Ca2+. The experiment
f(m) ) ∆G/hetero/∆Gh/omo
(4)
was carried out at the temperature of 25 °C.
To confirm the crystal phases of the samples, X-ray diffrac-
tion (Philips, PW 1729) analyses were carried out. The X-ray
diffraction (XRD) patterns obtained were compared with those
of calcium oxalate crystals listed in the powder diffraction
database of the Joint Committee on Powder Diffraction Stan-
dards (JCPDS). To examine the morphology change of CaOx,
the microstructure of the calcium oxalate assembly obtained
under different conditions was observed using a scanning
electron microscope (JSM-6700F, JEOL) after coating with
platinum to increase the conductivity.
where Rs and N0 are the radius and the density of the substrates,
respectively, B is the kinetic constant, Ω is the volume of the
growth units, m relates to the interaction and (statistical)
interfacial structural match between the crystalline phase and
the foreign bodies, ∆Gh/omo is the homogeneous nucleation
barrier, and ∆G/hetero is the heterogeneous nucleation barrier.
The factor f(m) describes the ability to lower the nucleation
barrier ∆G* due to the occurrence of foreign bodies and varies
from 1 to 0. It is important because it can determine the
heterogeneous nucleation barrier ∆G/hetero. As f(m) f 0,
∆G/hetero vanishes almost completely, implying that the grow-
ing crystals are well oriented and ordered with respect to the
structure of the substrate. While in the case of f(m) f 1, the
substrate exerts almost no influence on the nucleation, and the
nucleation is controlled by the kinetics of homogeneous
nucleation, which results in nuclei emerging disordered.23,29,31,34
The influence of foreign particles such as dust particles, proteins,
or even existing crystallites etc. on the nucleation barrier and
the association between the nucleating phase and the substrate
can be fully characterized by this factor.
Results and Discussion
According to the JCPDS database, calcium oxalate mono-
hydrate has monoclinic symmetry and belongs to the P21/c space
group with parameters a ) 6.29, b ) 14583, c ) 10.116, and
â ) 109.46, while calcium oxalate dihydrate (COD) has
tetragonal symmetry and belongs to the I4/m space group with
parameters a ) 12.35 and c ) 7.363. COM is thermodynami-
cally more stable than COD.
The crystal phases of the samples prepared in this study were
confirmed by XRD analyses. The XRD diffraction pattern
(Figure 1) shows that in the presence of BSA the product is a
mixture of calcium oxalate monohydrate and calcium oxalate
dihydrate. The peak intensity showed that in the product the
majority was COM and only a small portion was COD.
However, the COM crystal was the only product once the BSA
was removed. This indicates that BSA favors the formation of
COD. As COD is less likely to adhere to renal tubular cells
and is less injurious to cell membranes than COM is,4,16 we
concluded that BSA protects against stone formation.
To describe the kinetics of nucleation, one of the most
common ways is to measure the nucleation induction time (ts)
at different supersaturations. The nucleation rate J can be
defined23,28-33 as J = 1/(tsV), where V is the volume of the
system. Then from eq 3 then, we have
κf(m)
[ln(1 + σ)]2
ln ts )
- ln{V(Rs)2N0f′′(m)[f(m)]1/2B} (5)
where κ ) 16πγcf3Ω2/3(kT)3, which will remain constant under
a given condition.
In the following, we first focus on the nucleation kinetics of
COM, as the initial step of crystallization is nucleation. That is
a kinetic process by which embryos overcome the nucleation
barrier ∆G* to reach a critical radius rc and develop to become
crystals by a positive thermodynamic driving force ∆µ/kT. The
supersaturation σ provides the driving force and can be
given23,29,31 by
Since for the crystalline phase m and f(m) take on only certain
values corresponding to some crystallographically preferred
orientations, we expect a discrete set of intercepting straight
lines from the ln(ts) versus 1/[ln(1 + σ)]2 plot according to eq
5. These lines with different slopes, κf(m), indicate that
nucleation is governed by a sequence of progressive heteroge-
neous processes. Apart from supersaturation, the role of the
substrate is another important factor that influences the inter-
facial properties. Although the occurrence of a substrate will
promote nucleation by providing templating, it will also reduce
the effective collision of structural units to the surfaces of
clusters, which slows down the nucleation kinetics.23,31
ln(1 + σ) ) ∆µ/kT ) ln(a/ae)
(1)
Here k is the Boltzmann’s constant, T is temperature, a and ae
are the actual and equilibrium activities of a given ion,

BSA Prevents Kidney Stone Formation
J. Phys. Chem. B, Vol. 110, No. 18, 2006 9087
Figure 2. Plot of ln ts(sec) vs 1/[ln(1 + σ)}]2 for calcium oxalate
crystal nucleation under different conditions: curve 1, no additive; curve
2, with BSA at 0.5 mg/L; curve 3, with BSA at 1 mg/L.
To examine the influence of the supersaturation and the
templating effect of the substrate on the nucleation kinetics and
the subsequent structure correlation between calcium oxalate
crystallites, the ln(ts) versus 1/[ln(1 + σ)]2 plots were constructed
for calcium oxalate when BSA was fixed at a series of
concentrations, as presented in Figure 2. According to eq 5, for
a given system (κ, B ) constant), we can analyze the change
of the structure correlation between the substrate and the
crystalline phase in terms of the variation of the slope of these
plots. As shown in Figure 2, each of these depicted curves can
in principle be fit by two intersecting straight lines with different
slopes. This indicates that the nucleation is controlled by two
discrete values of f(m) within the corresponding regions. As
the supersaturation increases, f(m) increases sharply. This implies
that the structural match between the substrate and the nucleating
crystallites deteriorates as the supersaturation increases.
In the following, we will discuss how BSA enhanced the
nucleation process of COM. First, let us review the general
nucleation process: The growth units (molecules or ions) in
the solution may, on collision, join into groups of two or more
particles to form embryos. To reach a thermodynamically stable
state, the embryos must overcome the nucleation energy barrier,
thus attaining the “growth” of embryos.29,31-33 During the
growth of an embryo, growth units should be transported from
the bulk solution to the surface of the embryo and become
incorporated into the kink sites. However, when foreign bodies
(impurities and/or protein molecules) are present, they become
incorporated on the nucleus surface. This not only suppresses
the nucleation barrier but also affects the transport of growth
units to the surfaces of the crystalline clusters.23,28,30 As shown
in Figure 3A, the foreign body causes a reduction in the
“effective surface” of the nucleus, that would normally be
available for the growth units to incorporate themselves into
the nucleus. This tends to slow the nucleation kinetics23,29-31
and increase the nucleation induction time.
Figure 3. (A) Schematic illustration of the effect of foreign particles
on the transport of structural units from the bulk to the nucleating site.
The presence of the substrate reduces the effective surface of the
nucleus. (B) Illustration of adsorption of BSA molecules at the kink
site. In the process of nucleation, the adsorption of additives at the
kink sites suppresses the approach of growth units to the embryo. (C)
In the process of nucleation, the adsorption of additives at the kink
Actually, during the embryo growth, the solvent and foreign
bodies that have been absorbed on both the kink sites and the
surfaces of the growth units need to be desolvated. Here, let us
define ∆G+kink to be the kink kinetics energy barrier, which
must be overcome to remove solvent molecules absorbed at the
kink sites, and (∆Gk+ink′)add to be the kink kinetics energy
barrier, which must be overcome to remove the absorbed foreign
bodies at the kink sites. Obviously, the adsorption of foreign
bodies on the surface of the crystal embryo, in particular at the
kink sites, will enhance the kink kinetics energy barrier by
site enhances the kink kinetics barrier by ∆(∆Gk+ink
)
) (∆G+kink′)add
add
- ∆G+kink
.
even terminated due to the increase of the kink kinetics energy
barrier.23,28,30
In our experiment, the presence of BSA inhibits crystal
nucleation by absorbing at the kink sites. Thus, it increases the
kink kinetics energy barrier and the induction time. According
to refs 30 and 31, in Figure 2, the vertical distance of the straight
lines along the y-axis equals ∆(∆Gk+ink
)
add/kT, from which we
can calculate the change of the kink kinetics energy barrier
∆(∆G+kink add. We calculated this equation for [Ca2+] )
∆(∆G+kink
)
add, which equals (∆G+ ′)add - ∆Gk+ink (Figures 3B
kink
and 3C). Consequently, the integration of growth units into
crystal nuclei at the kink site will be significantly slowed or
)

9088 J. Phys. Chem. B, Vol. 110, No. 18, 2006
Liu et al.
[C2O42-] ) 2.1 mM, in the presence of BSA at the concentra-
tions of 0.5 and 1 mg/L, and we found that the energy barrier
increase with reference to the case of homogeneous nucleation
(where BSA is absent) is 0.62kT and 1.07kT, respectively. When
[Ca2+] ) [C2O42-] ) 2.35 mM, the energy barrier increase from
homogeneous nucleation to nucleation in the presence of BSA
of 0.5 mg/L is 1.18kT. However, when [Ca2+] ) [C2O42-] )
2.45 mM, the energy barrier increase for an increase in the BSA
concentration from 0.5 to 1 mg/L is 1.18kT.
The supersaturation at which f(m) changes is defined as σ*.
A larger σ* implies that good crystalline alignment will be
achieved in a wider range of supersaturations.23 In Figure 2,
σ* for curve 1, 2, and 3 is 7.01 × 102, 8.6 × 102, and 9.4 ×
102, respectively, so that σ* exhibits a monotonic increase with
BSA concentration. This implies that the presence of BSA plays
a crucial role in suppressing the supersaturation-driven structure
mismatch effect occurring in the crystalline assembly over a
wide range of supersaturations. Since BSA can prolong the range
of supersaturation for a good structural match, we deduce from
the above nucleation kinetics discussion that a better calcium
oxalate crystallization assembly could be obtained in the
presence of BSA. This conclusion was confirmed by recording
the SEM micrographs shown in Figure 4.
The SEM micrographs confirm the conclusions obtained from
the nucleation kinetics discussion. Figure 4A is the SEM picture
of COM obtained from a solution at low concentration ([Ca2+
]
) [C2O42-] ) 0.2 mM) without additives. However, in the
presence of BSA, no crystals were found even when all of the
other conditions, such as supersaturation, pH, and temperature,
were kept unchanged. Thus we arrived at the same conclusion
as in the previous discussion obtained by the nucleation kinetics
results, namely, that BSA inhibits the nucleation and the crystal
growth of CaOx. Figure 4B is the SEM picture of randomly
oriented COM crystals obtained from a solution at a high
concentration ([Ca2+] ) [C2O42-] ) 0.35 mM) without BSA.
It shows a distorted morphology35 as the crystals are highly
aggregated. Meanwhile, the samples obtained in the same
conditions as those in Figure 4B, except for the presence of
BSA, are shown in Figure 4C. The image shows a good structure
match and the coincident formation of COD and COM. This
proves that the presence of BSA can prolong the range of
supersaturation for a good structural match and achieve a better
crystal assembly. Also the conclusion from the XRD analysis
that the presence of BSA favors the formation of COD is
confirmed.
As urinary stone formation is a time-restricted process,
increasing the induction time may allow the crystals to remain
small enough to be excreted out freely.4 Moreover, COD and
COM crystals differ in their ability to adhere to the wall of the
kidney and/or renal tubule. As a consequence COD can be
expelled out by the urine more easily than COM can, and COM
is retained in the urinary system and induces the formation of
urine stones.3,4,16 Therefore, COD is less harmful to the human
body than COM, and the presence of BSA could reduce the
danger of urine stone formation. This process is illustrated by
Figure 5.
Figure 4. (A) SEM picture of COM twin crystals obtained from a
solution at a low concentration ([Ca2+] ) [C2O42-] ) 0.2 mM) without
additives: scale bar, 5 µm. (B) SEM micrograph showing COM
crystallites obtained from a solution at a high concentration ([Ca2+] )
[C2O42-] ) 0.35 mM) without additives: scale bar, 5 µm. (C) SEM
micrograph showing COM and COD crystallites obtained from a
solution at a high concentration ([Ca2+] ) [C2O42-] ) 0.35 mM), where
BSA is used as an additive. Because of the templating effect of BSA,
the crystallites show good structural synergy: scale bar, 5 µm.
Conclusions
In our investigation, a new nucleation model is employed in
studying the nucleation of COM under the influence of BSA.
The results show that the BSA protein not only inhibits the
nucleation of COM by increasing the kink kinetics barrier but
also promotes the structure synergy match between the protein
and the substrates and favors the formation of COD. This study

BSA Prevents Kidney Stone Formation
J. Phys. Chem. B, Vol. 110, No. 18, 2006 9089
(10) Ryall, R.; Harnett, R. M.; Hibberd, C. M.; Edyvane, K. A.; Marshall,
V. R. Urol. Res. 1991, 19, 181.
(11) Nakagawa, Y.; Ahmed, M.; Hall, S. L.; Deganello, S.; Coe, F. L.
J. Clin. InVest. 1987, 79, 1782.
(12) Worcester, E. M.; Sebastian, J. L.; Hiatt, J. G.; Beshensky, A. M.;
Sadowski, J. A. Calcif. Tissue Int. 1993, 53, 242.
(13) Tang, Y.; Grover, P.; Moritz, R.; Simpson, R.; Ryall, R. Br. J.
Urol. 1995, 76, 425.
(14) Baumann, J. M.; Affolter, B.; Caprez, U.; Henze, U. Eur. Urol.
2003, 43, 421.
(15) Konya, E.; Umekawa, T.; Iguchi, M.; Kurita, T. Eur. Urol. 2003,
43, 564.
(16) Ryall, R. L.; Chauvet, M. C.; Grover, P. K. BJU Int. 2005, 96,
654.
(17) Reinhold, F. P.; Hultenby, K.; Oldberg, Å.; Heinegård, D. Proc.
Natl Acad. Sci. U.S.A. 1990, 87, 4473.
(18) Brown, L. F.; Berse, B.; Van de Water, L.; Papadopoulos-Sergiou,
A.; Perruzzi, C. A.; Manseau, E. J.; Dvorak, H. F.; Senger, D. R. Mol.
Biol. Cell 1992, 3, 1169.
Figure 5. Scheme showing a renal tubule, in which supersaturated
urine with CaOx is flowing. The arrow indicates the flow direction of
the urine. In the urine, after the nucleation and growth of CaOx, most
of the COM is bonded to the renal tubule, while most of the COD is
propelled out.
also predicts the potential application of the BSA protein to
the cure of urine stone disease.
(19) Nancollas, G. H.; Wu, W. J. Cryst. Growth 2000, 211, 137.
(20) Jung, T.; Kim, W.-S.; Choi, C. K. Mater. Sci. Eng., C 2004, 24,
31.
(21) Wassell, D. T. H.; Hall, R. C.; Embery, G. Biomaterials 1995, 16,
697.
(22) Combes, C.; Rey, C. Biomaterials 2002, 23, 2817.
(23) Jiang, H.; Liu, X.-Y. J. Biol. Chem. 2004, 279, 41286.
(24) Jauregui-Zuniga, D.; Reyes-Grajeda, J. P.; Moreno, A. Plant Sci.
2005, 168, 1163.
Acknowledgment. The authors appreciate Janaky Narayanan
for her help with this work.
References and Notes
(1) Nancollas, G. H.; Gardner, G. L. J. Cryst. Growth 1974, 21, 267.
(2) Girija, E. K.; Latha, S. C.; Kalkura, S. N.; Subramanian, C.;
Ramasamy, P. Mater. Chem. Phys. 1998, 52, 253.
(3) Cerini, C.; Geider, S.; Dussol, B.; Hennequin, C.; Daudon, M.;
Veesler, S.; Nitsche, S.; Boistelle, R.; Berthe´ze`ne, P.; Dupuy, P.; Vazi, A.;
Berland, Y.; Dagorn, J.-C.; Verdier, J.-M. Kidney Int. 1999, 55, 1776.
(4) Khan, S. R. Calcium Oxalate in Biological Systems; CRC Press:
Boca Raton, FL, 1995.
(25) Asokan, D.; Kalaiselvi, P.; Farooq, S. M.; Varalakshmi, P. Urol.
Res. 2004, 32, 357.
(26) Jung, T.; Sheng, X.; Choi, C. K.; Kim, W.-S.; Wesson, J. A.; Ward,
M. D. Langmuir 2004, 20, 8587.
(27) El-Shall, H.; Jeon, J.-h.; Abdel-Aal, E. A.; Khan, S.; Gower, L.;
Rabinovich, Y. Cryst. Res. Technol. 2004, 39, 222.
(28) Liu, X. Y. J. Chem. Phys. 2000, 112, 9949.
(29) Liu, X. Y.; Lim, S. W. J. Am. Chem. Soc. 2003, 125, 888.
(30) Du, N.; Liu, X. Y.; Hew, C. L. J. Biol. Chem. 2003, 278, 36000.
(31) Liu, X. Y. J. Phys. Chem. B 2001, 105, 11550.
(32) Liu, X. Y. J. Cryst. Growth 2002, 237, 1806.
(33) Liu, X. Y. Surf. ReV. Lett. 2001, 8, 415.
(34) El-Shall, H.; Jeon, J.-h.; Abdel-Aal, E. A.; Khan, S.; Gower, L.;
Rabinovich, Y. Cryst. Res. Technol. 2004, 39, 214.
(35) Walton, R. C.; Kavanagh, J. P.; Heywood, B. R. J. Struct. Biol.
2003, 143, 14.
(5) Francesch, V. R.; Nakata, P. A. Annu. ReV. Plant Biol. 2005, 56,
41.
(6) Joseph, K. C.; Parekh, B. B.; Joshi, M. J. Curr. Sci. 2005, 88, 1232.
(7) Grover, P. K.; Moritz, R. L.; Simpson, R. J.; Ryall, R. L. Eur. J.
Biochem. 1998, 253, 637.
(8) Grover, P. K.; Ryall, R. L.; Marshall, V. R. Clin. Chim. Acta 1990,
190, 129.
(9) Yoshioka, T.; Koide, T.; Utsunomiya, M.; Itatani, H.; Oka, T.;
Sonoda, T. Br. J. Urol. 1989, 64, 463.