ELSEVIER
Clinica Chimica Acta 244 (1996) 45-67
Phosphates precipitating from artificial urine and
fine structure of phosphate renal calculi
F. Grases*, O. S6hnel l, A.I. Vilacampa, J.G. March
University Illes Balears, Department of Chemistry, 07 071 Palma de Mallorca, Spain
Received 6 June 1995; revision received 1! September 1995; accepted 18 September 1995
Abstract
Phosphates precipitating from artificial urine in the pH range 6-8 were identified using Xray diffraction, chemical analysis and scanning electron microscopy. The influence of magnesium and citrate on phases precipitating from urine was established. From urine containing
a normal quantity of magnesium (around 70 ppm), brushite accompanied by hydroxyapatite
(HAP) precipitated at pH < 7.0 and struvite with HAP at pH > 7.0. HAP was formed exclusively from magnesium deficient urine at pH 7.0. Newberyite, octacalcium phosphate and
whitlockite were not identified. The chemical and phase composition and inner fine structure
of 14 phosphate calculi were studied. Three types of stones were distinguished based on their
magnesium content: (1) stones rich in magnesium composed of struvite, hydroxyapatite and
abundant organic matter, (ii) stones with low magnesium content constituted by calcium deficient hydroxyapatite, up to 5% of struvite, considerable amount of organic matter and occasionally brushite, and (iii) calculi without magnesium consisting of brushite, hydroxyapatite
and little organic matter. Conditions prevaling during stone-formation assessed for each type
of stone were confirmed by corresponding urinary biochemical data and corroborate the in
vitro studies of phosphates precipitation.
Keywords." Brushite; Hydroxyapatite; Struvite; Phosphate renal calculi; Magnesium; pH effect
1. Introduction
Six different phosphates, namely, hydroxyapatitc (HAP) Ca10(PO4)6(OH)2,
carbonate apatite CaI0(PO4,CO3,OH)6(OH)2, brushite (DCPD) CaHPO4.
* Corresponding author.
I Universityof Pardubice, Department of Inorganic Processes, wlm, Legii 565 532 10 Pardubice, Czech
Republic.
0009-8981/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved
SSDI 0009-8981 (95)06179-M
46
F. Grases et al. / Clinica Chimica Acta 244 (1996) 45-67
2H20, struvite (STR) MgNH4PO4.6H20, newberyite (NEW) MgHPO4.
3H20 and whitlockite (TCP)/3 Ca3(PO4)2, individually or in a mixture, were
detected as either major or minor components of human renal calculi [1,2].
Conditions under which those compounds are formed and their crystallising
behaviour were extensively studied. However, reported results are often controversial, due to widely different techniques, reaction conditions and initial
materials employed in the experiments.
The thermodynamically stable phase in contact with the aqueous CaPO4-Mg system is DCPD at pH values not exceeding approximately 5, and
HAP at pH > 5 [3,4]. Therefore, in such a system other calcium phosphates
finally transform, according to prevailing conditions, into one or other by a
sohition-mediated process. Conversion of DCPD to HAP seems to be a slow
process under physiological conditions [5,6] though even the opposite was
reported [7].
Inhibition of calcium phosphates crystallisation was also studied. Magnesium inhibits crystallisation of HAP [8-11] but exerts no influence on DCPD
[10], though some inhibitory effect on this compound is also reported [9].
Citrate exerts no effect on either crystal growth of DCPD [12] or formation
of amorphous TCP [13] but inhibits HAP crystallisation [14]. Proteoglycans
[15] and nucleoside phosphates [16] reduce the growth rate of HAP [13] and
gelatin prevents aggregation of amorphous TCP and promotes its transformation into the crystalline state [17].
Studies on phosphate precipitation from artificial or whole urine are rare.
Crystals of DCPD and HAP were detected in the voided urine [18]. Unfortunately, the composition of the urines was not reported and therefore this
finding merely signified that both phases could be spontaneously formed
under 'in vivo' conditions. Concentration of unspecified 'calcium phosphate
crystals' in voided urine abruptly increased when urinary pH was 6.0 or more
in comparison with urines ofpH less than 6.0 [18]. On aging, the whole urine
calcium oxalate monohydrate precipitated within the acidic range of pH,
TCP was formed between pH 6.0 and 7.0, HAP was detected at pH 7.0 and
STR appeared within the alkaline range [5]. During evaporation of the whole
urine, DCPD and HAP appeared at pH <5.5, formation of octacalcium
phosphate was suspected between pH 5.5 and 6.0 and STR at pH >7.5.
Two principal categories of phosphate containing stones can be
distinguished: calcium phosphate stones and the so-called infection stones.
The former calculi are basically composed of HAP either pure or in a mixture
with calcium oxalate or DCPD, and the latter are comprised of STR [19].
A more detail classification shows that 9.5% of all stones are formed by
mainly hydroxyapatite (HAP), 2.1% by dicalcium phosphate dihydrate
(DCPD) and 14.6% by struvite (STR) mixed to various degree with HAP
[20].
F. Grases et al. / Clinica Chimica Acta 244 (1996) 45-67
47
Electron microscopic techniques facilitating observation of detailed stone
inner structure have seldom been employed in studying phosphate calculi
[21-24].
From this account of accumulated knowledge concerning phosphate
stones it transpires that insufficient information is available to clarify the formation and development mechanism of these calculi. A study of calcium
phosphate precipitation from artificial urine and the fine structure and composition of 14 phosphate calculi was carried out in order to contribute to
understanding the genesis of phosphate calculi.
2. Experimental
2.1. Studies on phosphate precipitation from artificial urine
Two series of experiments were performed. In the first set of experiments
solid phases precipitating from artificial urine at different initial pH values
were determined. The artificial urine was prepared by mixing together equal
volumes of two solutions, A and B respectively. These solutions were
prepared by dissolving analytical grade chemicals in distilled water. Solution
A contained 11.02 g/l Na2So4.10H20, 1.46 g/l MgSO4.7H20, 4.64 g/1 NH4CI,
12.13 g/1 KCI and 0.24 g/1 Ca 2+, and solution B 2.65 g/1 NaH2PO4.2H20,
18.82 g/l NaEHPO4.12H20, 13.05 g/1 NaCl, 1.0 g/1 Na3CrHsOT.2H20 and
0.05 g/1 C2042-.Ca 2+ and C2042- were added as standard solutions prepared
from CaCO3 dissolved in HC1 and oxalic acid neutralised by NaOH, respectively. The pH of the artificial urine, i.e. the mixture of A and B solution,
was 7.0. When experiments at different pH were performed, the pH of solution B was preadjusted to a required value by adding either HCI or NaOH
solution, as appropriate.
Both solutions A and B were first brought to 37°C in a water bath. Then
250 ml of solution A was placed in a beaker of 600 ml volume equipped with
four wall baffles and agitated by a double-bladed stirrer. The beaker was
situated in a constant temperature water bath kept at 37°C. Thereafter 250
ml of solution B was quickly poured into the beaker which was afterwards
covered with a lid to prevent evaporation. The resulting reaction mixture - artificial urine - - was sufficiently agitated throughout each experiment in
order to prevent sedimentation of formed solid phase.
In the second series of experiments the influence of urine components on
the composition of the phase precipitating at pH 7.0 was studied. Selected
components of urine were excluded when preparing solutions A and/or B.
After establishing solution temperature at 37°C, the ensuing experimental
procedure was identical to the first series; i.e. solutions were mixed together
in a beaker, beaker covered and agitated.
48
F. Grases et al./ Clinica Chimica Acta 244 (1996) 45-67
Samples were withdrawn from agitated reaction mixture at predetermined
intervals. A drop of suspension was placed on a microscopic cover glass and
left standing for a few minutes in order to allow sedimentation of crystals
present. Then, liquid was cautiously sucked off by a filter paper, the glass
was dried, sputtered with gold and observed in an Hitachi electron scanning
microscope (S.E.M.). The whole volume of reaction mixture was filtered
through a membrane filter with pore size of 0.45 t~m, the filter was dried and
thecollected precipitate subjected to X-ray diffraction and chemical analysis.
Chemical analysis was performed using atomic emission spectrometer with
inductively coupled plasma (Perkin Elmer 2000). Around 10 mg of
precipitate was precisely weighed and dissolved in 0.2 ml of diluted (1:1)
HC1. After further dilution with water to 100 ml this solution was analysed
for the content of Mg, Ca and P. Results expressed as mass of a respective
element in a unit volume of analysed solution, c(X), were converted into percentage of STR contained in the precipitate and the molar ratio Ca/P in the
solid remaining when STR was excluded.
Assuming that all determined magnesium was present in the form of
struvite and considering that in this mineral the molar ratio Mg/P = 1, then
the percentage, in moles, of struvite in a sample can be expressed as molar
%STR = (moles of P as STR/total moles of P) x 100.
Applying the analytical protocol described, and converting moles to mass,
the percentage in mass of STR in a sample can be expressed as
%STR = 127.4 c(Mg)/c(P)
and the molar ratio Ca/P
Ca/P = Ic(Ca)/40.08]/I Ic(P)/30.97] - [c(Mg)/24.3111
2.2. Renal calculi study
Stones classified as phosphate calculi were selected from our stone collection containing over 1000 specimens. Stones, often in the several pieces, were
fractured by a scalpel along different planes. One or several fragments of
every stone were mounted on a stub, sputtered with gold and observed by
S.E.M.
Remaining fragments were ground, the resulting powder was homogenised
and subjected to X-ray diffraction and chemical analysis as in the previous
section. Available urinary biochemical data of patients forming the stones
are listed in Table 1.
49
F. Grases et al. / Clinica Chimica Acta 244 (1996) 45-67
3. R ~
3.1. P h o s p h a t e s precipitating f r o m artificial urine
Experiments of the first series were performed at p H 6.0, 6.5, 7.0, 7.5 and
8.0. pH was constant throughout the experiments except those with initial
pH 8.0. In this case, p H during precipitation was gradually decreased and
reached a value around 7.6 after about 4 h.
The experiments were carried out for a maximum of 8 hours. In experiments performed at p H 6.0 and 6.5, a small amount of both individual and
intergrown crystals (Fig. 1), was formed in artificial urine after several hours.
A precipitate isolated after 8 h in both cases consisted of D C P D and H A P
(at pH 6.0, the presence of H A P was suspected). At p H 7.0, the reaction mixture became turbid within a few minutes after mixing solutions together and
15 min from the onset small spherulites of H A P appeared. After 1 h large
crystals of D C P D , occasionally surrounded by aggregated spherulites, were
detected. The size of both spherulites and crystals remained virtually unchanged during the experiment. The precipitate collected after 4 hours consisted of D C P D accompanied by HAP. At pH 7.5 small spherical particles
of amorphous matter were formed instantaneously on solution mixing. In 15
rain well developed crystals of STR were present. When initial p H was 8.0,
crystals of STR appeared immediately, even when adding the B solution.
Table 1
Urinary biochemical data of respective stone formers
No.
pH
DiuresisCa
Mg
P
Oxalate Creatinine
mg/l
Citrate Uric
acid
55.7
38.0
38.0
41.7
49.4
59.3
38.0
51.9
53.2
65.1
65.1
817
118
118
224
315
824
118
625
344
226
226
18.0
6.3
6.3
18.3
15.2
18.2
6.3
11.3
21.2
14.1
14.1
122
13
13
267
47
244
13
67
180
149
149
ml
1
3
4
5
7
8
10
11
12
13
14
6.42
6.88
6.88
5.15
6.72
6.42
6.88
7.17
6.26
6.06
6.06
1450
5100
5100
2650
1780
1755
5100
2200
2450
2030
2030
118
51
51
163
133
186
51
146
344
145
145
1014
310
310
733
600
401
310
560
1110
---
438
100
100
246
305
302
100
413
499
136
136
Stones nos. 13 and 14 and nos. 3, 4 and 10 were formed by the same person at different times.
50
F. Grases et al./ Clinica Chimica Acta 244 (1996) 45-67
STR crystals exhibited in all cases a typical surface structure (Fig. 2). In later
stages of this experiment spherical particles of apparently amorphous matter
precipitated. Precipitate present in the reaction mixture at pH 7.5 and 8.0
after 4 hours, was composed of STR and HAP.
The presence of 0.058 g of mucin in 250 ml of solution A at pH 7.0 and
8.0 was without any noticeable effect on the size of DCPD crystals formed
compared to precipitation carried out in the absence of mucin. However, the
precipitate prepared at pH 7.0 contained 13% of STR, contrary to precipitation without mucin when STR was completely absent at this pH.
In the second series of experiments performed at pH 7.0, spheres of HAP
precipitated from solutions containing both (i) Na2HPO4, NaH2PO4, NaCI
and KC1 in urine-like concentrations, and (ii) the same components with
added 500 and 1000 ppm of citrate (Fig. 3). A precipitate collected from the
reaction mixture after 4 h was pure HAP. In a solution (ii) with added magnesium in urine-like concentration (70 ppm of Mg in the form of MgSO4)
HAP spheres were detected after 15 min and abundant DCPD crystals appeared after 1 h of reaction. After (4 h) beginning the experiment the
precipitate was composed of pure DCPD as determined by X-ray diffraction.
A similar results was obtained in the presence of 30 ppm of magnesium.
However, only HAP precipitated in the form of typical spheres and also
loose flakes consisting of apparently crystalline matter with enmeshed
spheres from artificial urine containing 15 ppm of magnesium.
Fig. 1. Intergrowncrystals of DCPD formed at pH 6.5 in artificial urine.
F. Grases et al./ Clinica Chimica Acta 244 (1996) 45-67
51
The composition of precipitates prepared under different conditions is
given in Table 2. Therein %STR represents the percentage of struvite in calcium containing precipitate and the molar ratio Ca/P is the ratio of total calcium present to phosphorus non-associated with magnesium. Its value is
Fig. 2. (a) Crystals of STR surrounded by small spheres of apparently amorphous matter,
formed at pH 7.5 in artificial urine. (b) Fine structure of surface of STR crystal.
52
F. Grases et al./ Clinica Chiraica Acta 244 (1996) 45-67
Fig. 3. HAP spherulites formed at pH 7.0 in absence of Mg from artificial urine.
Table 2
Composition of precipitates formed in the short period experiments at 37°C under various
conditions
pH
medium
Chem. Analysis
% STR
Ca/P
X-ray
S.E.M.
DCPD
6.0
6.5
AU
AU
---
1.1
1.2
DCPD + X
DCPD + HAP
7.0
AU
--
I.I
DCPD
7.5
8.0
7.0
8.0
7.0
7.0
7.0
7.0
7.0
AU
AU
AU+muc
AU+muc
No Mg and Cit
No Mg, 500 ppm Cit
No Mg, 1000 ppm Cit
AU + 15 ppm Mg
AU + 30 ppm Mg
41.2
55.3
12.9
41.9
---2.7
2.7
1.7
1.7
1.3
1.4
1.6
1.5
1.7
1.6
1.4
STR + HAP
STR + HAP
DCPD
STR
HAP
HAP
HAP
HAP
DCPD
DCPD
+ HAP
DCPD+SP
STR + AM
STR + AM
DCPD + SP
STR + AM
HAP
HAP
HAP
HAP
DCPD+SP
SP spherulites of HAP; X, undentified compound (probably HAP); AU artificial urine, muc,
mucine; AM, small spherulites of apparently amorphous matter; Cit, citrate, S.E.M., scanning
electron microscopy.
F. Grases et al. / Clinica Chimica Acta 244 (1996) 45-67
53
given only to one decimal place since, due to the accuracy of the analytical
method (-4-2.5%), uncertainty in the ratio value is 4-0.05.
Crystallinity of DCPD and STR was well developed as signified by X-ray
diffraction patterns that were sharp and narrow. HAP, on the contrary, was
of poor crystallinity since only low and wide peaks appeared on high
background. At pH 6.0 a compound accompanying DCPD did not provide
a sufficient number of peaks for its unambiguous identification. However,
two clear peaks were identical with the most intensive peaks of HAP.
Therefore, this compound was regarded with high probability as HAP of a
poor crystallinity.
3.2. Phosphate renal calculi
Chemical and mineral composition of the phosphate calculi studied is
reported in Table 3. Predominant calcium phosphate constituting each stone
quoted in the fourth column is estimated based on the Ca/P molar ratio
realizing that it equals 1.67 for HAP, 1.5 for TCP, 1.4-1.5 for calcium deficient HAP [25-27] and 1 for DCPD. Composition is determined by X-ray diffraction accounts for crystalline components present in a quantity exceeding
the method detection limit, which is about 5 wt.% for most compounds and
Table 3
Composition of calcium phosphate calculi
Stone
Chemical analysis
X-ray diffraction S.E.M.
no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
%STR
Ca/P
Comp
18
27
5
6
5
60
6
45
49
5
7
0
0
0
1.3
1.6
1.4
1.4
1.4
1.6
1.6
1.5
1.4
1.3
1.5
l.l
0.9
0.9
-HAP
d-HAP
d-HAP
d-HAP
HAP
HAP
HAP
d-HAP
-d-HAP
DCPD
DCPD
DCPD
HAP, STR
HAP, STR
HAP(b)
HAP
HAP
STR,HAP
STR,HAP
STR,HAP
STR,HAP(b)
HAP
HAP
DCPD,HAP(b)
DCPD,HAP(b)
DCPD,HAP(b)
HAP, DCPD,CaOx,STR
STR,HAP, DCPD,O
HAP,ASP
HAP,ASP,O
HAP,ASP
STR,HAP,DCPD, O
STR,HAP,O
STR,HAP,O
STR,HAP,O
HAP,ASP
HAP,ASP,O
DCPD,HAP,O
DCPD, HAP,O
DCPD,HAP,O
O - - organic matter, HAP(b) - - very high background and low peaks of HAP, d-HAP - calcium deficient HAP, ASP - - aspidinic, S.E.M., scanning electron microscopy
54
F. Grases et al. / Clinica Chimica Acta 244 (1996) 45-67
in the case of HAP over 10 wt.%. Non-crystalline compounds, such as amorphous TCP, organic matrix, etc., cannot be detected. Mineral comlSosition
stated in the S.E.M. column is based on identification of observed particles
forming a calculus according to their appearance.
Three types of phosphate calculi can be distinguished according to their
magnesium content, namely (i) magnesium rich uroliths, (ii) calculi containing low amount of Mg, and (iii) stones without magnesium.
3.2.1. Type I. Stones rich in magnesium, Nos. 1, 2, 6, 8 and 9. These stones
always contained STR as determined both by X-ray diffraction and S.E.M.
observation. STR constituted between 20 and 60% of the calculus mass containing phosphate, and the remaining part consisted of HAP and/or calcium
deficient HAP as indicated by the Ca/P ratio varying between 1.3 and 1.6.
These stones, classified as infection stones, did not exhibit any regular inner
fine structure (Fig. 4). Well developed STR crystals up to 100 #m in size were
disseminated throughout the calculus volume (Fig. 5). Individual spheres of
HAP, mostly accumulated in stone cavities, appeared in all studied infection
stones. These spheres of about 5 #m in diameter exhibited a smooth surface
containing no tiny crystals such as typically appear on the surface of artificially prepared spheres of HAP, and were occasionally aggregated into
Fig. 4. Fine structure of infection stones (Type I). No regular inner structure can be seen.
F. Grases et al. / Clinica Chimica Acta 244 (1996) 45-67
55
Fig. 5. Struvite crystals diseminated through the infection stones (Type I).
particles of about 50 #m that filled cavities. Typical plate-like crystals of
DCPD were occasionally associated with HAP spheres. DCPD crystals,
though observed in S.E.M., were not detected by X-ray diffraction. This indicates that the content of DCPD in calculus inorganic mass is lower than
5 wt%. All calculi contained a substantial amount of structureless organic
matter unevenly distributed throughout the calculus volume. Stone no. 1
contained crystalline layers of calcium oxalate situated near its outer surface.
These stones were soft, easily friable, and exhibited microporous texture
without occurrence of large caverns or a hollow centre.
3.2.2. Type II. Stones with low content of magnesium, Nos. 3, 4, 5, 7, 10
and 11. These calculi would contain about 5% of STR if all magnesium were
bound in this compound. However, neither X-ray diffraction nor S.E.M.
revealed its presence. In the case of X-ray diffraction such an amount of STR
is just around the detection limit and could therefore go undetected. Calcium
phosphate, forming the majority of the stone mass, exhibited the molar ratio
of Ca/P around 1.4 and thus can be either calcium deficient HAP or TCP.
Since X-ray diffractograms displayed several diffuse peaks of HAP on a high
background, this compound was considered as calcium deficient HAP of
poor crystallinity.
56
F. Grases et al. /Clinica Chimica Acta 244 (1996) 45-67
The inner fine structure of these calculi was characterised by an ample occurrence of layers of amorphous material, called 'aspidinic' layers after [23].
Aspidinic layers were structureless from the macroscopic viewpoint (Fig. 6),
but detailed inspection of broken surfaces revealed them to be composed of
small spheres, around 0.1 #m, of amorphous material cemented together
(Fig. 7). These stones contained a substantial amount of organic matter both
on their outer surface and inside the stone. Spheres of HAP of 5-10 #m in
size were composed of amorphous material with tiny crystals on their surface
(Fig. 8), or formed by layers of tiny crystals enveloping a cavity. HAP
spheres were largely accumulated in stone cavities as either individual entities
or agglomerates of about 50 #m in diameter. Calcium phosphate stones were,
as a rule, cavernous inside having always a hollow centre (Fig. 9). Sometimes
large spherical objects of over 100/zm in size were entrapped in calculus mass
(Fig. 10). These objects with an onion-like structure consisting of concentric
'aspidinic' layers were evidently formed independently and only later trapped
in the calculus. Any regular structure encompassing at least a part of calculus
was not detected. Regions formed by regular crystal growth were not
detected.
Calculus No. 7 was unique among this group of calculi from the viewpoint
of its composition. It contained STR, confirmed by chemical analysis, X-ray
diffraction and S.E.M., in a low quantity (6%) and the rest was formed by
Fig. 6. Aspidiniclayerspresent in the phosphate stones with low content of magnesium(Type
II).
F. Grases et al./ Clinica Chimica Acta 244 (1996) 45-67
57
Fig. 7. Small spheres that constitute the aspidinic layers (Type II).
Fig. 8. Spheres of hydroxyapatite observed in phosphate stones with low content of magnesium (Type II).
58
F. Grases et al. /Clinica Chimica Acta 244 (1996) 45-67
Fig. 9. Cross-section of a calcium phosphate stone with low content of magnesium (Type II).
Fig. 10. Large spherical objects consisting of concentric aspidinic layers entrapped in phosphate stones with low content of magnesium (Type II).
F. Grases et al. / Clinica Chimica Acta 244 (1996) 45-67
59
HAP. Its structure was partially chaotic since large STR crystals were randomly distributed in the calculus volume consisting of spheres of HAP and
organic matter. However, a kind of concentric lamination (Fig. 11) was
observed on the fracture plane. HAP spheres, often accumulated in specific
areas of a calculus, were not evenly distributed over the calculus volume.
Calculi of this type were soft, friable and rounded objects of smaller size than
infection stones.
3.2.3. Type IlL Calculi without Mg, Nos. 12, 13 and 14. These stones exhibited the Ca/P molar ratio around unity that is a characteristic value for
DCPD. Therefore, brushite represented the principal constituent of these
calculi and content of other phosphates, namely HAP, in these calculi cannot
exceed about 10 wt% of calculus inorganic mass. DCPD calculi were not
compact, but contained numerous cavities partially filled with spherical particles of HAP and organic matter. Organic matter was present in a noticeably
lesser quantity than in the previous groups of calculi. Plate-like brushite
crystals radially arranged in larger formations (Fig. 12) were mostly
separated by lateral micrscopic fissures (Fig. 13). Moreover, crystals were
often interrupted along their length, thus forming a sort of circular lamination evident in a macroscopic view. A closer inspection of this lamination
Fig. 11. Concentric laminations observed in a calcium phosphate stone with low content of
magnesium (Type II).
60
F. Grases et al. /Clinica Chimica Acta 244 (1996) 45-67
Fig. 12. Plate-like brushite crystals radially arranged observed in a calcium phosphate stone
without magnesium (Type III).
Fig. 13. Detail of brushite crystals exhibiting lateral microscopic fissures (Type III).
F. Grases et al. / Clinica Chimica Acta 244 (1996) 45-67
61
Fig. 14. Aggretages of well developed brushite crystals observed in a calcium phosphate stone
without magnesium (Type III).
revealed it to be composed of new crystals nucleated on top of an underlying
compact crystalline layer that later developed into a further crystalline layer.
Therefore, these arrays did not consist of individual columnar crystals extending over the total length of this formation, but represented an ordered configuration of numerous small DCPD plate-like crystals. Occurrence of
organic matter in these arrays was not apparent. Aggregates of well
developed crystals, assembled in calculus without any apparent order (Fig.
14) were also observed. The outer surface of these stones was largely formed
by tips of plate-like crystals.
Calculi of Type III are relatively small, rounded and generally completely
filled with solid without exhibiting a hollow centre. Available urinary biochemical data of the respective stone-former are included in Table 1.
4. Discussion
4.1. Studies on phosphate precipitation in artificial urine
From combined results of chemical analysis, X-ray diffraction and microscopic observations, identity of phases precipitating out from artificial urine
of different pH values at 37°C can be established.
Precipitates formed in artificial urine at pH 6.0, 6.5 and 7.0 are composed
of predominantly DCPD accompanied by about 10 wt.% of HAP. Small
62
F. Grases et al./ Clinica Chimica Acta 244 (1996) 45-67
spheres of HAP at pH 7.0 precipitate first and, later, developed crystals of
DCPD appear. STR and HAP precipitate at pH 7.5 and 8.0. Under these
conditions STR crystallises first and later HAP appears. STR comprises
about 50% of precipitate mass. Mucin apparently supports STR nucleation
since this compound is formed at a pH at which, without mucin, only DCPD
crystallises. HAP is formed at pH 7.0 either in absence or at low concentration of magnesium and when its concentration exceeds 30 ppm formation of
DCPD is favoured.
The observed effect of magnesium on calcium phosphate crystallisation
from artificial urine corresponds to the findings reported in the literature
established at 25°C in precipitating systems of different composition. This indicates the general inhibitory effect of Mg on HAP crystallisation. An inhibiting effect of magnesium on DCPD precipitation cannot be excluded
based on our results, but if it exists it is not significant.
Similar conclusions can be reached regarding the influence of citrate on
HAP erystallisation. The inhibitory effect reported in [14] cannot be important, otherwise HAP would have not appeared in artificial urine containing
500 and 1000 ppm of citrate. Thus, citrate evidently exerts either little or no
influence on crystallisation of HAP.
Based on reported results, formation of calculi composed of DCPD and
a minor quantity of HAP is expected at pH 7.0 or less from urine containing
a typical quantity of magnesium. However, under similar conditions from
magnesium deficient urine, i.e. urine containing 30 or less ppm of magnesium, stones containing mostly HAP and no or little DCPD should be formed. Stones containing a major amount of STR should appear from urine of
pH exceeding 7.0. At pH around 7.0 struvite can occur as a minor component. HAP can accompany STR in all cases. Hence HAP will be the most
wide-spread calcium phosphate precipitating from urine. Other phosphates,
namely NEW, octacalcium phosphate and TCP, can only exceptionally
occur in renal calculi. Also, monomineral stones composed exclusively of
DCPD, STR or HAP, can be seldom encountered since HAP precipitates out
of urine under most physiological conditions.
4.2. Studies on phosphate renal calculi
The findings previously reported correspond closely to composition of the
renal phosphate uroliths studied. Thus, three main types of phosphate
stones, distinguished by their magnesium content, are characterised by different phase composition, inner structure and external features. Therefore,
different formation mechanisms of these types can be expected.
4.2.1. Type I. Stones rich in magnesium. These large calculi filling one or
several caliceal spaces are soft, easily friable and without caverns or a hollow
F. Grases et al./ Clinica Chimica Acta 244 (1996) 45-67
63
centre. Uroliths of Type I consist of a random mixture of well developed
and/or partially dissolved struvite crystals, hydroxyapatite spheres and a
large amount of organic matter. STR and HAP crystallize directly from urine
at pH > 7.0, as described in the previous section, and a similar situation must
prevail in the stone-forming period. Unfortunately, urinary biochemical data
reported in Table 1 were determined when infection was virtually over and
hence do not convey any information on conditions prevailing during the
stone-forming period. Urinary pH over 7.0 can only be established during severe renal infection when bacteria present in the kidney produce ammonia,
changing urinary pH into the alkaline region. However, urinary pH can temporarily drop below 7.0 due to pharmacological treatment and/or infection
cycles under such conditions. Then, STR crystals in contact with urine start
to dissolve. Thus, occurrence of partially dissolved STR crystals signifies
changes in urinary pH during the stone-forming period. Bacterial attack on
the urothelium producing abundance of organic matter and bacterial detritus
are responsible for a high amount of organic matrix in calculi. Clinically,
calculi of Type I correspond to infection stones since conditions requisite for
their formation can develop only under severe kidney infection.
Absence of any organized inner structure predominating even over limited
regions of the Type I calculi interiors, suggests their sedimentary origin. A
similar disorganised inner structure is displayed by detritic rock formed by
sedimentation [28]. Stratification, pronounced in these rocks, is missing in
these calculi owing to largely uniform composition of solid suspended in
urine over the stone-forming period. Mechanical properties of the Type I
stones, i,e. softness and friability, corroborates their sedimentary origin. Sedimentary formations, not subjected to subsequent consolidation by pressure
or through development of crystalline bridges, are never hard, compact and
difficult to pulverize, contrary to concretions formed by crystal growth.
The following formation mechanism of the Type I calculi can be deduced:
crystals of inorganic constituents of calculus, mainly STR and HAP,
nucleate and grow in urine inside the kidney. Particles reaching a sufficient
size within the residence time in the upper urinary tract are not carried away
by liquid flow into the bladder but remain in the kidney and sediment due
to gravity. Similarly, organic particles of adequate size sediment independently. With changing kidney position due to body movement, sedimentation successively proceeds in various directions. Therefore, a crust
covering the calix inner surface that represents the infection stone outer surface is formed first. As this shell becomes filled with further sediment, a free
space in the centre serving as a urine outlet gradually shrinks. At a certain
moment this free space virtually vanishes. Urine from affected papilla, i.e.
papilla whose adjacent caliceal space is filled with sediment, can still for
64
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some time 'diffuse' through this sediment. However, when the concentration
gets dense enough, it becomes practically impenetrable to liquid and further
production of urine by nephrons connected to impaired papilla practically
ceases.
4.2.2. Type II. Stones with low magnesium content. These calculi, composed
of calcium deficient HAP, small amount of STR and considerable amount
of organic matter, represent rounded objects generally smaller than infection
stones. Urinary pH around 7 and a low concentration of magnesium is expected to prevail during the stone-forming period, since, as explained above,
only under such conditions does HAP unaccompanied by other phases originate. Urinary biochemical data of patients forming Type II stones given in
Table 1 support this conclusion. Urinary pH fluctuates around 7.0 and magnesium content is lower than in urines of patients forming calculi of other
types.
The inner structure of Type II calculi is characterized by a wide occurrence
of aspidinic layers, HAP spheres, large spheric objects entrapped in calculus
mass and a hollow centre. An absence of any ordered structure indicates the
sedimentary origin of these stones. Mechanical properties exhibited by the
Type II stones are consistent with their sedimentary origin. However, the
calculi's limited size, rounded shape and hollow centers point to their formation in confined space with poor urodynamic efficacy, rather than in the kidney calix. A round-bottomed cavity in the kidney with an opening small in
comparison to the cavity inner diameter in the kidney [8] is assumed to represent an appropriate place for these stones to form. The cavity is for most of
the time filled with liquid. If a low-speed fluid stream flows past its opening,
the liquid in the cavity starts to rotate around the geometrical center of the
cavity forming a single vortex [28]. A flow pattern established due to this
vortex tends to distribute small particles suspended in this liquid over the
cavity perimeter.
Thus, small particles of calcium deficient HAP originating in urine are distributed in a form of a layer over the cavity wall or inner surface of developing calculus. This layer is assumed to be later transformed through ageing
into aspidinic structure. HAP larger spheres and large spherical objects
enmeshed in calculus mass formed independently outside a cavity are only
subsequently entrapped in the cavity. Since these particles are too large to
be carried by a slowly moving liquid in the cavity they sediment and become
incorporated in the stone. Large particles of organic origin behave similarly.
When Type II calculi are either completely filling the cavity volume or are
accidentally blocking the narrow cavity opening they cannot develop any
further. In the latter case, development of the calculus has not been finished
and it contains little or no solid in the centre, which remains filled with liq-
F. Grases et al./ Clinica Chimica Acta 244 (1996) 45-67
65
uid. Such a calculus, when dried, exhibits a hollow centre, a feature invariably found in all samples studied.
Clinically, Type II calculi must correspond to patients with low urinary
magnesium content and urinary pH values around 7.0, whose kidneys have
confined spaces with poor urodynamic efficacy. Consequently, in such cases
an increase in the magnesium excretion and a decrease in the urinary pH,
that can be accomplished through adequate dietary recommendations, could
solve the problem.
4.2.3. Type III. Stones without magnesium. These calculi are composed of
brushite accompanied by a small amount of HAP and organic matter. As
shown above, brushite with a small amount of HAP precipitates from urinelike liquors containing a normal quantity of magnesium, around 60 mg/l, at
pH < 7.0. Similar conditions are expected to prevail during the stoneforming period. This conclusion is supported by the urine composition of patients producing these stones (Table 1), exhibiting pH around 6.0 and a normal quantity of magnesium. The predominant component of these stones,
brushite, appears as larger organised formations evidently originating
through regular crystal growth. That is, each crystal when nucleated grows
by depositing growth units from supersaturated urine. These formation,
composed of closely attached individual plate-like crystals, cannot be formed
by sedimentation of individual crystals formed independently in bulk urine
since in this way no organised inner structure can arise, as discussed above.
That is, brushite crystals are nucleated on the kidney wall or on some equivalent surface and grow by depositing building units from supersaturated
urine. HAP spheres, formed in the urine bulk, sediment among neighbouring
brushite formations. Thus, the formation mechanism of brushite stones
represents a combination of regular crystal growth of brushite and sedimentation of HAP spheres formed in the urine bulk. The calculi shape, size and
absence of a hollow centre suggest their formation in the kidney cavities with
poor urodynamic efficacy exhibiting relatively large openings that can only
rarely be blocked by a developing stone.
Under exceptional conditions one can imagine a small stone of Type I
originating in the kidney cavity with wide opening or a calculus of Type III
developing in a cavity with a narrow opening. In this case, however, stone
of Type III can contain a hollow centre.
Clinically, Type III calculi must be assigned to patients with urinary pH
values over 6.0 and under 7.0 with kidney cavities with poor urodynamic efficacy. Therefore, in such cases a decrease in the urinary pH below 6.0, and
an increase in the urinary crystallization inhibitory capacity could solve or
minimize the problem.
66
F. Grases et al./ Clinica Chiraica Acta 244 (1996) 45-67
Acknowledgements
Financial support of D.G.I.C.Y.T. Espafia (grant no. PB 92-0249) is
gratefully acknowledged.
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