IN CALCIUM OXALATE CRYSTAL MORPHOLOGY AS A FUNCTION OF SUPERSATURATION
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MARCOS A. VIEIRA
of Nephrology, Clinic of Research in Nephrolithiasis, Evangelic University
Hospital and Catholic University of Paraná (PUC - PR), Curitiba,
To study the changes in calcium oxalate crystal morphology induced by
different levels of supersaturation (SS) in human urine.
Materials and Methods: Twenty-four hours
urine samples from 5 normal men were collected. Each specimen was centrifuged
and filtered. About 200 mL of each sample was dialyzed overnight. Aliquots
of 2 mL of urine was then added to a 24-wells tissue culture plate and
checked for crystal absence. Calcium oxalate crystals were precipitated
from each sample by adding sodium oxalate and calcium chloride in sufficient
quantities to induce spontaneous crystallization. Finally, each plate
hole was examined with an inverted polarized microscope (X500 magnification).
Initial SS of each sample relative to calcium oxalate was calculated using
an iterative computer program.
Results: Crystal formation was connecte
to relative calcium oxalate (CaOx) SS. At SS of 10, small crystals of
similar shape were formed, mainly CaOx dihydrate morphology. At SS of
30, there was an enormous increase in the number of crystals, that kept
the same size. SS greater than 50 produced larger crystals with different
shapes and multiple crystalline aggregates. Urine was able to tolerate,
i.e., to avoid crystal formation, until SS ratios of approximately 10.
Conclusions: Relative CaOx SS and the concentration
ratio of calcium to oxalate are important determinanting factors of crystal
morphology. Non-dialyzable urinary proteins can act as inhibitors and
influence the structure of formed crystals. Additional studies from patients
with kidney stones are needed in order to establish whether crystal size
and habit distribution are different from crystals in normal urine.
words: urolithiasis; crystallization; calcium oxalate
Int Braz J Urol. 2004; 30: 205-9
the process of water conservation, kidneys supersaturate urine (1). Supersaturation
(SS) in relation calcium oxalate and phosphate salts is the driving force
for crystallization in solutions like urine, which means that it will
contain crystals that are formed spontaneously. If inhibitors of crystal
formation were not able to act and control their size, the final result
will be nephrolithiasis and/or nephrocalcinosis (2,3).
The understanding of crystalluria requires
some knowledge of crystal nucleation, growth and aggregation, all of which
depend greatly on solution concentration. Both the monohydrate and dihydrate
species of calcium oxalate (CaOx) crystals are present in kidney stones
(4). It has been proposed that crystalluria may be predictive of a nephrolithogenic
tendency (5). Also, crystalluria with oxalate crystal volume measurement
is a non-invasive, easily performed investigation, and can give feedback
on the efficacy of urolithiasis therapy (6).
The aim of this study was to examine the
changes in calcium oxalate crystal morphology induced by different levels
of SS in human urine.
hours urine samples from 5 normal subjects was collected without preservative.
This urine was screened to exclude bacteria, protein and glucose before
being pooled. Each specimen were centrifuged for 15 min at 8,000x g and
passed through Millipore filters with a pore size of 0.22-mm (Millipore
Corp., Bedford, Massachusetts). About 200 mL of each sample was dialyzed
for 24 hours against 5 liters of distilled water at a temperature of 4°C,
with 2 changes of water. The dialysis membranes had 3-kd molecular weight
Stock solutions of calcium chloride (2 mm)
and sodium oxalate (0.5, 1 and 4 mm) were used. All solutions were prepared
with reagent-grade chemicals, and double-distilled water.
Aliquots of 2 mL of dialyzed urine was added
to a 24-wells tissue culture plate at 37°C and checked for crystal
absence. After that, calcium oxalate crystals were precipitated from each
urine sample by adding 0.5 mL of sodium oxalate and 0.5 mL of calcium
chloride. Oxalate ion was added as the last component, after mixing the
other 2 components. One well had no oxalate added to serve as control.
The plate was incubated in a shaking water
bath at 37°C for 1 hour. Finally, the content of each plate well was
examined for crystal identification with an inverted polarized microscope
(X500 magnification) and optical photomicrographs of any crystals present
were taken. The size of individual crystals was estimated using a graduated
scale in ocular lens.
The SS of each sample in relation to calcium
oxalate at the point of precipitation was calculated by an interactive
computer program (Equil 1.3, University of Gainsville, Fl, USA).
SS increased due to the increase of oxalate concentration. Using dialyzed
urine and calcium chloride at a fixed concentration of 2 mm, the addition
of 0.5, 1 and 4 mm sodium oxalate resulted in SS of 10, 30 and 50, respectively.
Urine was able to tolerate, i.e., to avoid crystal formation, until SS
ratios of approximately 10.
Crystal formation seems to be related to
calcium oxalate SS. At SS of 10, small crystals of similar shape were
formed, mainly CaOx of dihydrate morphology (Figure-1). Their size could
be estimate to vary between 15 to 20 mm.
Using sodium oxalate in 1 mm to produce
a relative SS of 30 caused an enormous increase in the number of crystals,
most of then keeping the same size and morphology observed with an SS
of 10 (Figure-2). These octahedral crystals also have the characteristic
morphology of CaOx dihydrate crystals.
SS greater than 50 produced larger crystals
(Figure-3) with different shapes and multiple crystalline aggregates (Figure-4).
Some of these crystals suffered a peptization process and generated fragments
of 50 to 100 mm of diameter. We should also observe that, when the concentration
ratio of calcium to oxalate was kept above one, the crystals formed had
a typical CaOx dihydrate morphology.
of calcium oxalate are often found in urine (7). Even though crystalluria
by itself could be considered as a harmless phenomenon, in some patients
it could imply an existing or preexisting urinary SS and an increased
risk of nephrolithiasis (8). A pathologic calcification like a Randall’s
plaque can also be generated by intense deposition of crystals inside
the urinary tract (9).
In urine, measured CaOx SS rarely exceeds
30. This suggests that nucleation inside the urinary tract is heterogeneous.
In this scenario, clusters of crystal-salt ions can be generated by a
central nucleus of cell debris, for example. Higher CaOx SS, probably
around 80 is apparently necessary to create homogeneous nucleation (10).
This kind of nucleation is characterized by a decrease in the average
crystal size caused by intense precipitation and peptization of crystals.
In this work we were able to demonstrate
that different urinary CaOx SS modulates structure and crystalline habit
in human urine. Using this model, we could verify that relative SS and
the concentration ratio of calcium to oxalate are important factors in
the determination of crystal morphology. The urine blocked formation of
crystals until CaOx SS reached 10. This effect is probably due to several
non-dialyzable macromolecular inhibitors present in it, like nephrocalcin,
glycosaminoglycans, Tamm-Horsfall/uromodulin, and osteopontin for example
(2-3,11-12). When the relative SS varied between 10 and 30, there was
an interesting relationship among these inhibitors of crystallization
and the physicochemical burden imposed by high concentration of calcium
and oxalate. We can attribute the increase of crystal number to SS and
the preservation of crystal structure to urinary proteins, as previously
demonstrated by Wesson et al. (4). When the CaOx SS was higher than 50,
there was almost no space for inhibitors of crystallization to act and
the crystals aggregated and peptized almost immediately.
Very similar conclusions to this study were
obtained by Burns & Finlayson (13). In their work, the crystals were
examined optically and with X-ray diffraction and their morphology was
found to be closely related to relative SS. Nevertheless, they used simple
buffer solutions, rather than urine, and even the authors pointed that
doubtfully their results could apply to a complex solution as urine.
CaOx SS and the concentration ratio of calcium to oxalate are important
determinants of crystal morphology, mainly in the range of SS of 10 to
30. Non-dialyzable urinary proteins can act as inhibitors and influence
the structure of formed crystals. Additional studies from patients with
kidney stones are needed to establish whether crystal size and habit distribution
are different from crystals in normal urine.
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Received: January 23, 2004
Accepted after revision: May 10, 2004
Dr. Mauricio Carvalho
Kidney Institute, Clinic of Research in Nephrolithiasis
Rua Augusto Stelfeld, 2034,
Curitiba, Pr, 80730-150, Brazil
Fax: + 55 41 222-6029
this manuscript, the authors assess changes in calcium oxalate crystal
morphology with various changes in urine supersaturation. Using human
urine in an in vitro system, the authors found various changes in crystal
morphology as the human urine supersaturation was increased from 10 to
greater than 50. The authors conclude that the relative calcium oxalate
supersaturation and the concentration ratio of calcium to oxalate are
important determinants of crystal morphology. Overall, this is an interesting
concept, but one that is not particularly new. Moreover, the authors comment
that urinary proteins such as glycosaminoglycans, Tamm-Horsfall proteins
and osteopontin may act as inhibitors of calcium oxalate crystallization.
However, based on the present findings, they provide no evidence that
these proteins were present in the urine samples and their observations
with regard to the inhibitory characteristics of these proteins are speculative
and based on previous publications.
Comprehensive Kidney Stone Center
Duke University Medical Center
Durham, North Caroline, USA