Saturation Supersaturation Nucleation Crystal

URINARY SATURATION AND UROLITHIASIS
Joe Bartges, DVM, PhD, DACVIM (SA IM), DACVN
Professor of Medicine and Nutrition
The Acree Endowed Chair of Small Animal Research
The University of Tennessee
Knoxville, TN 37996-4544
[email protected]
UROLITH FORMATION
Initiation and Growth of Uroliths
Urolith formation, dissolution, and prevention involve complex physical processes. Major factors include:
1) supersaturation resulting in crystal formation, 2) effects of inhibitors of crystallization and inhibitors of crystal
aggregation and growth, 3) crystalloid complexors, 4) effects of promoters of crystal aggregation and growth, and 5)
effects of non-crystalline matrix.(Finlayson 1978; Coe and Parks 1988; Brown and Purich 1992; Bartges, Osborne et
al. 1999)
Figure 1. Proposed sequence of events resulting in calcium oxalate urolith formation
Ca++
Ca++
Ca++
Oxalate=
Oxalate=
Ca++
Saturation
Supersaturation
Ca++
Oxalate=
Ca++
Ca++
Oxalate=
Oxalate=
Nucleation
Crystal aggregation and growth
Crystal retention
Stone formation
Urolith formation is associated with two complementary but separate phases: initiation and growth. It
appears that initiating events are not the same for all types of uroliths. In addition, factors that initiate urolith
formation may be different from those that allow it to grow. The initial step in formation of a urolith is formation of
a crystal nidus (or crystal embryo). This phase of initiation of a urolith formation, called nucleation, is dependent on
supersaturation of urine with calculogenic crystalloids. The degree of urine supersaturation may be influenced by the
magnitude of renal excretion of the crystalloid, urine pH, and/or crystallization inhibitors or promoters in urine.
Non-crystalline proteinaceous matrix substances may also play a role in nucleation in some instances.
Three theories have been proposed to explain initiation of lithogenesis.(Erwin 1976; Coe 1981; Smith
1990; Osborne, Bartges et al. 2000) Each theory emphasizes a single factor. The SUPERSATURATIONCRYSTALLIZATION THEORY incriminates excessive supersaturation of urine with urolith-forming crystalloids
as the primary event in lithogenesis. In this hypothesis, crystal nucleation is considered to be a physiochemical
process involving precipitation of crystalloids from a supersaturated solution. Urolith formation is thought to occur
independently of a) preformed matrix or b) crystallization inhibitors. The MATRIX NUCLEATION THEORY
incriminates preformed organic matrix (thought to be a mucoprotein with calcium-binding properties) as the primary
determinant in lithogenesis. It is based on the assumption that preformed organic matrix forms an initial nucleus that
subsequently permits urolith formation by precipitation of crystalloids. The role of organic matrix in lithogenesis has
not been defined with certainty; however, the similarity of the overall composition of matrix from human uroliths of
various mineral composition supports this hypothesis. The CRYSTALLIZATION-INHIBITION THEORY
proposes that reduction or absence of organic and inorganic inhibitors of crystallization are the primary determinant
of calcium oxalate and calcium phosphate lithogenesis. This theory is based on the fact that several lithogenic
substances in urine are maintained in solution at concentrations significantly higher that is possible in water (in other
words, driving forces for crystal precipitation of normally saturated urine are minimized by crystallization
inhibitors). Similarly, inhibitors are important in minimizing crystal growth and aggregation. These three theories
are not mutually exclusive. In fact, supersaturation of urine with the crystal's components is a prerequisite for each
theory of nucleation.
Further growth of the crystal nidus is dependent on the following: (1) its ability to remain in the lumen of
the excretory pathway of the urinary system; (2) the degree and duration of supersaturation of urine with crystalloids
identical or different from that in the nidus; and (3) physical characteristics of the crystal nidus. If they are
compatible with other crystalloid; some crystals may align themselves and grow on the surface of others. This is
called epitaxial growth. Epitaxy may represent a heterogeneous form of nucleation, and may account for mixed and
compound uroliths. For example, in man, the structural similarities of uric acid and calcium oxalate permit urolith
growth by epitaxis.
States of Urinary Saturation
An important driving force behind stone formation is saturation state of urine with lithogenic substances
(Figure 2). When a solution such as urine is saturated, it refers to the maximal amount of a substance, such as
calcium oxalate, that can be completed dissolved. This point is termed the thermodynamic solubility product.
When calcium oxalate is present in urine at a concentration less than the solubility point, the urine is
undersaturated with calcium oxalate and calcium oxalate completely dissociates and dissolves. When calcium
oxalate is present in urine at a concentration that is equal to the solubility point, the urine is saturated with calcium
oxalate and calcium oxalate begins to precipitate. When calcium oxalate is present in urine at a concentration above
the solubility point, the urine is supersaturated with calcium oxalate and calcium oxalate precipitates.
Urine contains ions and proteins that interact and/or complex with calcium and oxalic acid so as to allow
them to remain in solution. This explains why calcium and oxalic acid in urine do not normally precipitate to form
calcium oxalate crystals. Urine is normally supersaturated with respect to calcium and oxalic acid. But energy is
required to maintain this state of calcium and oxalic acid solubility, and, therefore, the urine must constantly
"struggle" to maintain calcium and oxalic acid in solution. Thus, urine is described as being metastable, implying
varying degrees of instability with respect to the potential for calcium oxalate crystals to form. In this metastable
state, new calcium oxalate crystals will not precipitate, but if already present, crystals can be maintained and even
grown in size. If the concentration of calcium and oxalic acid is increased, a threshold is eventually reached at
which urine cannot hold more calcium and oxalic acid in solution. The urine concentration at which this occurs is
the formation point of calcium oxalate. Above the thermodynamic formation product, urine is oversaturated and
unstable with respect to calcium and oxalic acid. Thus, calcium oxalate crystals will spontaneously precipitate,
grow in size, and aggregate together.
Figure 2. States of urinary saturation(Bartges, Osborne et al. 1999)
Undersaturated Solution
An undersaturated solution is one that contains a sufficiently low concentration of a crystalloid to permit
dissolution of additional quantities of the crystalloid. Urine is undersaturated when the solute concentration (or
activity product) is below the solubility of the solute in question. Creation of urine which is undersaturated with
calculogenic crystalloids may permit varying degrees of urolith dissolution.
Saturated Solution
A saturated solution is one in equilibrium with the un-dissolved solute at a given temperature. The
solution contains so much dissolved substance, that no more can be dissolved at a given temperature. With respect to
urine, the saturation concentration is the concentration of a crystalloid in urine at which the urine can be mixed with
uroliths (or the solid phase) of that crystalloid without changing the concentration of the crystalloid in urine. The
saturation of salts in urine is influenced by several variables including pH, ionic strength, and temperature.
Supersaturated Solution
A supersaturated solution is one which is more saturated with a substance at a given temperature than
would be normally expected. In other words, it is any concentration greater than the saturation concentration.
Supersaturated urine contains a greater concentration of a crystalloid (cystine, phosphate, calcium, ammonium, etc.)
than the associated solvent (water) would be predicted to be able to normally hold in solution. Supersaturation can
vary in degree. At lower levels of supersaturation, urine is metastable. At higher levels of supersaturation, however,
urine becomes unstable with regard to its capacity to keep crystallogenic substances in solution. Factors that increase
the saturation of crystalloids in urine predispose to precipitation of crystals and thus urolith formation. Spontaneous
precipitation will occur if the concentration of the crystalloid is greater than the formation product of the crystalloid.
Metastable Region
The metastable region refers to the degree of supersaturation of a crystalloid that lies between the
solubility product and the formation product. Metastability applies to those solutions (such as urine) that have the
capacity to retain more of a compound in solution than would be predicted by knowledge of its true solubility in
water. The term "metastable" is appropriate because it implies a condition subject to change. A metastable solution
is thermodynamically unstable, but does not contain enough energy to initiate crystal formation. However, if crystals
are already present, they may grow. The region of metastability varies with the type of calculogenic crystalloid. It
has been estimated that the difference between the solubility product and the formation product of calcium oxalate in
urine is a multiple of about 8.5 to 10.0.
Oversaturated Solution
An over-saturated solution is one in which the degree of supersaturation of a crystalloid is greater than the
formation product. Recall that supersaturated urine exceeds the solubility product, but does not exceed the formation
product. Oversaturated urine is no longer metastable. Nucleation will take place in absence of heterogeneous factors.
It is thought that crystals observed by microscopic examination or urine sediment are caused by oversaturation of
urine.
Nucleation
Nucleation refers to the initial event in the formation (or precipitation) of uroliths and is characterized by
the appearance of submicroscopic molecular aggregates of crystalloids. Initially, the aggregates are approximately
100 molecules in size and represent potential crystal embryos (or a nidus). Crystals represent an orderly arrangement
of atoms in a periodic pattern or lattice. To become a urolith, crystal embryos must have a lattice arrangement that
allows continued growth. They must also be large enough to prevent dispersion back into the dissolved
phase.(Brown, Ackermann et al. 1994)
Nucleation has been classified as homogeneous (also called self nucleation or generalized nucleation) or
heterogeneous (also called localized nucleation). Homogeneous nucleation occurs spontaneously in highly
supersaturated urine in the absence of foreign substances. Therefore, the nidus is composed of identical crystalloids.
Heterogeneous nucleation is catalyzed by foreign material such as suture material, indwelling catheters, tissue
debris, crystal embryos of different composition, and so on (Figure 3). Urine contains many impurities that might
promote heterogeneous nucleation and initiate crystal formation at a concentration of crystalloids below the
formation concentration. These substances may be thought of as facilitators or potential facilitators of
crystallization. Any crystal type may be a potential nidus for nucleation of another crystal type. A greater degree of
supersaturation (that is, a higher formation product) is required for homogeneous nucleation than for heterogeneous
nucleation. Once nucleation has occurred, however, crystal growth can occur at any degree of supersaturation (even
at metastability).
Figure 3. Example of (a) homogeneous nucleation and (b and c) heterogeneous nucleation. (a) Ammonium urate
urolith removed from a 3-year-old, intact male, English bulldog. Note the laminations. (b) Compound urolith
removed from a 4-year-old, spayed female Miniature schnauzer. The outer layers are composed of infectioninduced struvite (S) around a calcium oxalate nidus (C). (c) Infection-induced struvite removed from a 2-year-old,
spayed female Miniature schnauzer. The urolith (S) formed around a piece of fibrous material (gauze sponge, G)
inadvertently left behind at a previous cystotomy for urolith removal.
(a)
(b)
C
(c)
G
S
S
Inhibitors and Promoters of Crystal Formation
Urine is a complex solution containing a variety of substances that can inhibit or promote crystal formation
and growth.(Ryall 1997; Dussol and Berland 1998; Kavanagh, Jones et al. 1999; Shirane, Kurokawa et al. 1999;
Marangella, Bagnis et al. 2004; Guerra, Meschi et al. 2006; Kumar and Lieske 2006; Jaggi, Nakagawa et al. 2007;
Vella, Karydi et al. 2007) Inhibitors include molecules that reduce calcium oxalate and calcium phosphate
supersaturation. Some inhibitors (e.g., citrate, magnesium, pyrophosphate) form soluble salts with calcium, oxalic
acid or phosphate, and thereby reduce the quantity of calcium, oxalate or phosphate available for precipitation. Other
inhibitors (e.g. nephrocalcin, uropontin, glycosaminoglycans, Tamm-Horsfall glycoprotein, other inert ions)
interfere with the ability of calcium and oxalic acid to combine, and thereby minimize crystal formation and growth.
Also, glycosaminoglycans act as protectors by preventing adherence of crystals to the urinary tract mucosa.
METHODS FOR EVALUATING RISK OF UROLITH FORMATION
Various factors involved with urolith formation may be evaluated by several methods including: 1)
epidemiological studies performed at urolith centers and designed to identify risk and protective factors, 2)
measuring urine concentrations of calculogenic substances, 3) evaluating the influence of urine pH on crystal
formation, and 4) measuring the degree of undersaturation, supersaturation, and/or oversaturation of urine with
crystallogenic substances. Determination of urinary biochemical parameters and urinary saturation can only be done
in patients that are “stone free” because active urolith disease results in depletion of calculogenic compounds in
urine that alters results.(Laube, Pullmann et al. 2003)
An emphasis of urolithiasis research is evaluation of crystallization methods as urolith formation is
preceeded and advanced with crystal formation. “
Crystallization is a physical chemical process involving a change of state from solution to solid. The
supersaturation, which is a measure of the chemical energy available for this process, is a crucial factor and
governs all aspects of crystallization such as nucleation, growth, and aggregation. As the reaction proceeds, the
supersation will decline (unless replenished) and this in turn will impact upon the kinetic behavior of the
crystallization process. While the physical chemistry and kinetics are always important, the process of stone
formation takes place in a biological environment. – JP Kavanagh, 2006(Kavanagh 2006)
In addition to various techniques developed to evaluate crystallization, several “risk formulae” have been
proposed to evaluate propensity for urolith recurrence (primarily for calcium oxalate) in human patients,(Tiselius
1997) although debate exists as to the utility of these formulae.(Sutton 2006) These include: the urinary calcium-tomagnesium ratio, the urinary calcium-to-citrate ratio, saturation-inhibition index, 24-hour urine quotients [(calcium
x oxalate / magensium x creatinine) and (calcium x oxalate / magensium x creatinine x inhibitioin of calcium
oxalate crystal growth in dilute urine)], probability index, and the ion-activity product index.(Tiselius 1997)
Supersaturation
In solution chemistry, the difference in chemical potential of 2 states (Δµ) is dependent on the activities of
the crystallizing salt in the supersaturated solution (a) and in the solution when it has come to equilibrium (aeq):
Δµ = RTln(a/aeq)
where R is the universal gas constant and T is the absolute temperature (Kelvin). The activity of the crystallizing salt
is represented by the activity product (AP) for that salt where the activities of the ions comprising that salt are
multiplied. The term "activity" of a mineral is an index of the likelihood that the mineral will combine with other
substances in urine, and is determined by multiplying the concentration of the ion by the activity coefficient for
similarly charged molecules. For example, the activity of calcium is determined by multiplying the concentration of
calcium in solution (molarity) by the activity coefficient for a doubly charged molecule since calcium carries a “2+”
charge. The "activity" of a mineral is dependent on several factors including: 1) the urine concentration of that
mineral, 2) the urine concentrations of other substances such as sodium, potassium, calcium, etc., 3) the quantity and
functional state of non-mineral or non-measured mineral inhibitors and promoters of crystal formation, growth, and
aggregation, 4) urine pH, and 5) temperature of urine. Thus, a and aeq represent the AP’s for the salt in
supersaturated solution and the solution at equilibrium, respectively. Furthermore, the supersaturation ratio (S) =
a/aeq; therefore, Δµ/RT = ln(S).(Werness, Brown et al. 1985) For practical purposes, S is expressed as concentrations
(molarities) or activities (a). For example, for calcium oxalate:
S=
APcaox =
[Ca2+]x[Ox2-]
APcaoxeq
[Ca2+eq]x[Ox2-eq]
where “[ ]” represents the activities or concentrations of the ions, calcium (Ca2+) or oxalate (Ox2-). Usually, the
relative supersaturation (σ) is used rather than S, where σ = S–1.(Finlayson and Miller 1969; Finlayson 1978;
Werness, Brown et al. 1985; Brown, Ackermann et al. 1994; Kavanagh 2006)
Crystallization involves nucleation, growth, and aggregation. Nucleation may be heterogeneous (where a
foreign substance provides a nucleation catalyst for crystal formation) or homogeneous (where no foreign substance
is used). Supersaturation required for homogeneous nucleation is much higher than that required for heterogeneous
nucleation. There is still a supersaturation barrier that must be overcome before nucleation can occur (Figure 2).
Growth of crystals may occur through enlargement of existing crystals by direct incorporation of solution species
into the solid crystal lattice or by aggregation of crystals. The rate of crystal growth is described by second order
kinetics:
G = kσ2
where G is the growth rate and k is the rate constant.(Kavanagh 2006) Aggregation can also result in enlargement of
the crystal mass, and occurs through the net result of crystals colliding and either dispersing or consolidating, with
the outcome being depending on an efficiency factor. As consolidation is achieved by crystal bridges that fuse the
lattice structures of individual crystals, aggregation also is dependent on supersaturation.(David, Espitalier et al.
2001; Hounslow, Mumtaz et al. 2001)
There are several techniques used to evaluate urinary saturation. The most commonly used are
determination of relative supersaturation and activity product ratios.
Relative supersaturation
Determining the relative supersaturation (RSS) of a urolith-forming substance in a patient's urine is one
technique used to assess risk of urolith formation.(Pak, Hayashi et al. 1977; Brown and Purich 1992) RSS is
determined by measuring urine concentrations of several analytes including ammonium, calcium, chloride, citrate,
hydrogen (pH), magnesium, oxalate, phosphate, potassium, and sodium (and possibly cystine, sulfate, uric acid, and
other compounds), in urine. These values are then entered into a computer program (EQUIL or SUPERSAT), which
calculates the activity coefficients for the various ions and combines the relevant ion concentrations and activity
coefficients to produce the activity product (AP). For example, the AP of calcium oxalate is calculated as the
mathematical product of the activity of calcium and activity of oxalic acid. The AP for each urolith-forming
compound is divided by its known thermodynamic solubility product (SP) and the resultant RSS produced.
RSS = ion AP of the patient’s urine / ion SP
Relative supersaturation is related to the energy available for crystal nucleation and growth; however, RSS values
are limited by the fact that the thermodynamic solubility products used for these calculations have not been
measured in the patient's urine. This is of concern because it is probable that different macromolecules, including
inhibitors and promoters of crystal formation, growth, and aggregation, in the patient's urine have a pronounced
influence on free ion concentrations. By using calculations measured in urine from healthy human beings, RSS may
overestimate SP’s and AP’s of different minerals, and thus tend to underestimate the risk of urolith formation.
Another technical problem in evaluating dogs and cats is that the computer program used to calculate RSS involves
comparison of the pet’s urine values to standardized values based on the composition of human urine.
Activity product ratios
Activity product ratios (APR) also are designed to express the degree of supersaturation of solutions with
calculogenic minerals. APR's are obtained by calculating the ion AP in the patient's urine samples before and after
equilibrium with various seed crystals such as calcium oxalate.
APR = ion AP of patient’s urine before incubation with seed crystals
ion AP of patient’s urine after incubation with seed crystals
In determining the APR, the patient’s urine is incubated with preformed seed crystals composed of pure
urolith-forming mineral of interest (for example, calcium oxalate). Following incubation for 48-hours with the seed
crystals, the urine concentration of the same analytes are measured. The post-incubation concentrations of analytes
are then used to calculate a "post-incubation" AP. Dividing the "pre-incubation" AP by the "post-incubation" AP
gives the APR for that patient's urine sample.
An exact measurement of supersaturation is not obtained by determining APR, but the method provides
useful information about the relative increase or decrease of the ion AP in the patient's urine that result from seed
crystal growth or seed crystal dissolution. An APR less than one represents undersaturation of urine with the mineral
being evaluated An APR equal to one represents saturation of the patient's urine sample. An APR greater than one
indicates that the patient's urine sample is supersaturated.
APR’s can be calculated for any calculogenic mineral as long as pure seed crystals for that type of mineral
are available. Use of APR methodology will not eliminate errors associated with the effect of unknown factors such
as crystallization inhibitors or promoters on ion activities; however, since the same urine sample obtained from the
patient is analyzed before and then after equilibration with seed crystals (such as calcium oxalate), the same type of
error occurs in evaluation of both analyses and therefore the errors cancel. Whereas calculation of RSS can
overestimate supersaturation, saturation, and undersaturation, the APR method overestimates undersaturation,
underestimates supersaturation, and correctly measures saturation, provided that a sufficient amount of seed crystals
have been used. One limitation of APR determination is the assumption that urine has reached the SP for the salt
following 48 hours of incubation, which has been shown to be a false assumption in some cases.(Robertson, Jones et
al. 2002) Urine may not reach true equilibrium saturation level, particularly when coming from a supersaturated
level, presumably due to presence of various inhibitors of crystal growth that slow down the approach to
equilibrium. In this instance, when the true RSS is measured following 48 hours of seed incubation, the AP achieved
at that point may be 2-3 times higher than the thermodynamic solubility product. The APR calculated at this point,
therefore, systematically underestimates the actual level of supersaturation since the denominator (AP/SP) is too
large. The opposite may occur when the urine is undersaturated.
Other measures of urine saturation
There are other techniques for estimating urine saturation in addition to relative supersaturation and activity
product ratios. A newer method for evaluating risk of calcium oxalate urolith formation in human beings is use of
the Bonn Risk Index (BRI).(Laube, Schneider et al. 2000; Laube, Hergarten et al. 2001; Laube, Hergarten et al.
2004) Supersaturation of urine with respect to a urolith-forming salt is a fundamental pre-requisite of salt
precipitation as supersaturation is the thermodynamic driving force behind the process; however, supersaturation
alone is not sufficient to induce pathologic salting-out. The BRI uses the ratio calculated from the urinary
concentration of ionized calcium and the amount of ammonium oxalate that is titrated to the urine in order to induce
a precipitation of calcium oxalate salts. A high BRI value indicates low risk of urolith formation (whereas a low
RSS indicates low risk) and a low BRI indicates a high risk of urolith formation (whereas a high RSS indicates high
risk). BRI has been shown to correlate with RSS.(Laube, Hergarten et al. 2001) For crystallization to occur in the
urinary tract, it is necessary that a correct combination of supersaturation and of factors that inhibit/promote the
nucleation process exists. To what extent this propensity for crystallization will actually relate to risk of urolith
formation also depends on factors that regulate steps leading from a crystal to a urolith. An advantage of BRI is that
fact that all urinary components contribute their effects in their native ratio to the determination. Thus, the BRI
includes an imbalance between promoters and inhibitors in the individual’s urine, if an imbalance is present;
however, the BRI is a non-specific method with respect to urinary constituents as only the concentration of ionized
calcium is measured. Additional urinary chemistry determination may be necessary to fully evaluate the metabolic
status of a patient. This technique has not been tested in animals, and the instrument (Urolizer, Raumedic Ag,
Munchberg, Germany) is not available in the United States.
Use of urinary saturation testing in dogs and cats
Limited studies utilizing urine saturation testing has been performed in veterinary medicine, particularly in
animals that have formed uroliths (Table 1). Despite the number of studies, very few have been performed in dogs or
cats that are urolith-formers and no studies exist that compares estimates of urinary saturation with recurrence rates
of uroliths. In dogs, calcium oxalate urolith formation typically occurs when urinary relative supersaturation for
calcium oxalate is greater than 10; the metastable zone lies between a relative supersaturation value for calcium
oxalate of 1 and 10 to 14.(Stevenson and Rutgers 2006) In cats, calcium oxalate urolith formation typically occurs
when urinary relative supersaturation for calcium oxalate is greater than 12; the metastable zone lies between a
relative supersaturation value for calcium oxalate of 1 and approximately 12.(Houston and Elliott 2008) Sterile
struvite urolith formation in cats typically occurs when urinary relative supersaturation for struvite is greater than
2.5; the metastable zone lies between a relative supersaturation value for struvite of 1 and approximately
2.5.(Houston and Elliott 2008)
Urinary supersaturation represents a risk for urolith formation, but as in human beings, there is overlap in
values between urolith-forming animals and healthy, non-urolith-forming animals.(Robertson, Peacock et al. 1968;
Kavanagh 2006); therefore, other factors are important. Use of urinary saturation studies can provide further
information as to mechanisms of urolith formation, screening of animals at risk for urolith formation, and
monitoring efficacy of urolith management.
CLINICAL APPLICATION TO DOGS AND CATS
So what does all of this mean? There are several issues to keep in mind:
- Urinary saturation is the most important, but not the only, driving force for crystallization and urolith
formation
- Several methods exist for estimating urinary saturation; however, none of them adequately describe what is
occurring naturally in the biological system (urinary tract)
- Determination of relative supersaturation and activity product ratios, while used to estimate urinary
saturation, give different results and information. Determination of relative supersaturation is a valuable
and reasonably reliable technique for estimating urinary saturation; however, it (a) is heavily influenced by
concentration of analytes measured, which, in turn, is influenced by urine volume, and (b) it does not
account for urinary constituents that are not measured including the influence of inhibitors. Because it is
influenced by urine volume, methods designed to increase urine volume (e.g. feeding canned foods,
administration of diuretics, and stimulating water consumption by increased levels of dietary sodium)
would be expected to lower the relative supersaturation; however, clinical studies in urolith-forming dogs
and cats are lacking.(Stevenson, Hynds et al. 2003; Lulich, Osborne et al. 2005; Hezel, Bartges et al. 2006;
Xu, Laflamme et al. 2006; Hezel, Bartges et al. 2007; Xu, Laflamme et al. 2009) Determining activity
product ratios do not give an exact estimation of the supersaturation; however, because a patient’s urine is
used pre- and post-incubation with seed crystals, this technique does account for unmeasured urinary
constituents and the influence of inhibitors.
- Medical dissolution of uroliths is accomplished by inducing a state of undersaturation of urine (below the
solubility product) with the minerals that formed the uroliths
- Medical prevention of uroliths is accomplished by induced a state of undersaturation of urine or at least a
state of saturation at the lower end of the metastability limit
- Despite use of estimates of urinary saturation, there are no published studies in urolith-forming dogs and
cats that validate their use as means to predict urolith recurrence. Until that time, these techniques are
useful for formulating diets, but await recurrence data for validation.
- Means to decrease urinary saturation include increasing urine volume (“dilution is the solution to
pollution”) thereby decreasing the concentrations of calculogenic substances and decreasing dietary intake
of calculogenic substances. Despite these measures, these measures do not guarantee prevention of urolith
recurrence in all patients demonstrating that urolith formation is a complex process and many questions
remain un-answered.
Table.1. Summary of studies utilizing relative supersaturation or activity product ratio estimates of urinary
saturation in dogs and cats. Data, when available, are presented as average (standard deviation).
Dogs
Dogs
Health status*
Healthy Labrador
retrievers
Healthy Miniature
schnauzers
Healthy miniature
schnauzers, beagles,
labrador retrievers
Treatment group‡
Maintenance dry dog food
adult maintenance canned diet
Control diet + liquid potassium citrate
Control diet + potassium citrate tablet
Test and results#
RSScaox = 4.60 (1.66)
RSSbr = 0.47 (0.23)
RSScaox = 5.31 (1.62)
RSSbr = 1.22 (0.31)
RSScaox = 1.42 (0.63)
RSSmap = 2.59 (1.40)
RSScaox = 1.68 (0.83)
RSSmap = 3.55 (3.43
RSScaox = 1.24 (0.53)
RSSmap = 3.44 (2.63)
RSScaox = 4.02 (2.43)
RSScaox = 2.83 (2.25)
Dogs
Healthy beagles
Canned ltra-low protein, alkalinizing diet (0.24% DM)
Above diet + 1.2% NaCl DM
Dogs
Healthy Labrador
Retrievers
Maintenance dry dog food
Maintenance dry dog food + water
Maintenance dry dog food + 0.05 g NaCl/100kcal)
Maintenance dry dog food + 0.2 g NaCl/100 kcal
Maintenance dry dog food + 0.3 g NaCl/100 kca
Maintenance dry dog food
Maintenance dry dog food + water
Maintenance dry dog food + 0.05 g NaCl/100kcal)
Maintenance dry dog food + 0.2 g NaCl/100 kcal
Maintenance dry dog food + 0.3 g NaCl/100 kcal l
Low calcium (0.18), low oxalate (10)dry diet
Low calcium (0.18), medium oxalate (17.5) dry diet
Low calcium (0.18), high oxalate (25) drydiet
Moderate calcium (0.45), low oxalate (10) dry diet
Moderate calcium (0.45), moderate oxalate (17.5) dry diet
High calcium (0.75), low oxalate (10) dry diet
High calcium (0.75), high oxalate (25)dry diet
g/100kcal
Stone-formers
Non-stone-formers
Variable diets
RSScaox = 11 (6)
RSScaox = 9 (7)
RSScaox = 9 (4)
RSScaox = 5 (3)
RSScaox = 3 (3)
RSScaox = 14 (3)
RSScaox = 9 (5)
RSScaox = 15 (9)
RSScaox = 10 (6)
RSScaox RSS = 6 (3)
RSScaox = 2.5 (0.5)
RSScaox = 5.2 (4)
RSScaox = 4.3 (1)
RSScaox = 4 (2)
RSScaox = 4 (2)
RSScaox = 6 (5.5)
RSScaox = 5.8 (3)
Stone formers – baseline
1 month
12 months
Normal – baseline
1 month
Baseline = variable diets
1 and 12 mo = canned oxalate prevent diet
Adult Maintenance canned diet
RSScaox = 21.4 (15.8)
RSScaox = 7.8 (7.1)
RSScaox = 5.1 (2.9)
RSScaox = 4.1 (2.0)
RSScaox = 2.4 (1.4)
Healthy Miniature
schnauzers
Dogs
Healthy Cairn terriers
and Miniature
schnauzers
Dogs
Various
Dogs
Various
Dogs
Healthy beagles
Ultra-low protein, canned diet
Dogs
Healthy beagles
Ultra-low protein, canned diet with casein (10.4% DM)
Ultra-low protein, canned diet with casein (20.8% DM)
Dogs
Healthy beagles
Canned diet, casein-based (10.8% protein DM)
Dry diet, egg-based (9.2% protein DM)
Canned diet, chicken-based (11.1% protein DM)
Canned diet, chicken and liver-based (10.7% protein DM)
RSScaox = 21.4 (15.8)
RSScaox = 4.1 (2.0)
APRua = 0.05 (0.04)
APRnau = 0.04 (0.03)
APRau = 0.14 (0.07)
APRua = 0.005 (0.003)
APRnau = 0.004 (0.003)
APRau = 0.03 (0.03)
APRua = 0.005 (0.003)
APRnau = 0.005 (0.003)
APRau = 0.03 (o.009)
APRua = 0.02 (0.01)
APRnau = 0.03 (0.02)
APRau = 0.13 (0.10)
APRua = 0.007 (0.006)
APRnau = 0.015 (0.012)
APRau = 0.036 (0.028)
APRua = 0.033 (0.026)
APRnau = 0.50 (0.28)
APRau = 0.44 (0.33)
APRua = 0.007 (0.007)
APRnau = 0.042 (0.002)
APRau = 0.052 (0.046)
APRua = 0.008 (0.006)
APRnau = 0.064 (0.075
APRau = 0.15 (0.15)
Reference§
(Stevenson and
Markwell 2001)
EQUIL
(Stevenson,
Wrigglesworth et
al. 2000)
SUPERSAT¥
(Lulich, Osborne
et al. 2005)
EQUIL
(Stevenson,
Hynds et al.
2003)
SUPERSAT¥
(Stevenson,
Hynds et al.
2003)
SUPERSAT
(Stevenson,
Robertson et al.
2003)
SUPERSAT
(Stevenson,
Markwell et al.
2002; Stevenson,
Blackburn et al.
2004)
SUPERSAT
(Bartges,
Osborne et al.
1995)
EQUIL
(Bartges,
Osborne et al.
1995)
EQUIL
(Bartges,
Osborne et al.
1995)
EQUIL
Dogs
Healthy beagles
Ultra-low protein, canned diet + allopurinol (15 mg/kg PO q12h
Week 4
Week 8
Dogs
Healthy Beagles and
Labrador Retrievers
Maintenance dry dog food
Cats
Healthy DSH cats
Maintenance canned cat food
Cats
Healthy
Whiskas low pH canned
Waltham feline pH control canned
Cats
Healthy
RC Veterinary cats young adult dry
PD Feline c/d dry
Hill’s hariball control dry
Eukanuba low pH/O dry
Cats
Various – stone
formers
Diet on which formed stone
Canned oxalate preventative diet
Cats
Healthy
Adult Maintenance Canned Diet with 0.4% Na
Adult Maintenance Canned Diet with 0.8% Na
Adult Maintenance Canned Diet with 1.2% Na
Cats
Healthy DSH
Adult maintenance, dry diet
Diet with hydrochlorothiazide (1 mg/kg PO q12h)
Cats
Healthy DSH
Adult maintenance, dry diet
Diet with prednisolone (2.2 mg/kg PO q24h)
APRua = 0.01 (0.006)
APRnau = 0.02 (0.013)
APRau = 0.32 (0.27)
APRxan = 0 (0)
APRua = 0.003 (0.003)
APRnau = 0.004 (0.002)
APRau = 0.03 (0.02)
APRxan = 0.26 (0.09)
APRua = 0.005 (0.003)
APRnau = 0.009 (0.004)
APRau = 0.088 (0.051)
APRxan = 0.27 (0.12)
RSScaox = 1.21 (0.03) S
RSScaox = 1.52 (0.03) E
RSSmap = 1.48 (0.25) S
RSSmap = 6.61 (1.17)E
RSScaox = 0.97 (0.03) S
RSScaox = 1.14 (0.03) E
RSSmap = 1.35 (0.15) S
RSSmap = 5.74 (0.58) E
RSSmap = 0.16 (0.14)
RSScaox = 0.37 (0.24)
RSSmap = 0.58 (0.18)
RSScaox = 0.45 (0.16)
APRcaox = 1.11 (0.19)
APRmap = 0.72 (0.28)
APRcaox = 1.20 (0.23)
APRmap = 0.32 (0.06)
APRcaox = 1.21 (0.23)
APRmap = 0.66 (0.34)
APRcaox = 1.25 (0.22)
APRmap = 0.91 (0.2)
RSScaox = 14.3 (8.4)
APRcaox = 3.86 (1.59)
RSScaox = 5.9 (1.9)
APRcaox = 2.01 (0.59
RSScaox = 4.04 ((2.04)
APRcaox = 6.30 (13.69)
RSSmap = 0.06 (0.04)
APRmap = 1.26 (0.51)
RSScaox = 2.97 (2.04)
APRcaox = 4.76 (3.69)
RSSmap = 0.06 (0.04)
APRmap = 1.13 (0.51)
RSScaox = 2.52 (2.04)
APRcaox = 4.20 (3.69)
RSSmap = 0.1 (0.04)
APRmap = 0.79 (0.51)
RSScom = 3.48 (1.12)
RSScod = 1.49 (0.46)
RSSmap = 3.82 (2.30)
RSScom = 1.12 (0.70)
RSScod = 0.48 (0.30)
RSSmap = 1.35 (0.05)
RSScom = 0.36 (0.33)
RSScod = 0.47 (0.38)
RSSmap = 0.38 (0.32)
RSScom = 0.62 (0.42)
RSScod = 0.49 (0.40)
RSSmap = 1.59 (0.88)
(Bartges,
Osborne et al.
1994)
EQUIL
(Robertson,
Jones et al. 2002)
SUPERSAT
AND EQUIL
(Markwell,
Smith et al.
1999)
EQUIL
(Devois, Biourge
et al. 2000)
EQUIL
(Lulich, Osborne
et al. 2004)
EQUIL
(Xu, Laflamme
et al. 2006)
EQUIL
(Hezel, Bartges
et al. 2007)
EQUIL
(Geyer, Bartges
et al. 2007)
EQUIL
Cats
Healthy DSH
Commercial adult maintenance, dry foods: A
B
C
D
E
F
G
H
I
Cats
Healthy
Purified adult maintenance diet
Purified diet with 0.45% MgCl
Purified diet with 0.45% MgOxide
Adult maintenance canned diet
Adult struvite preventative canned diet
RSScaox = 2.96 (0.68)
RSSmap = 19.12 (5.42)
RSScaox = 5.66 (0.91)
RSSmap = 4.08 (1.36)
RSScaox = 5.40 (0.91)
RSSmap = 3.22 (1.23)
RSScaox = 6.52 (1.9)
RSSmap = 2.85 (1.43)
RSScaox = 2.88 (1.69)
RSSmap = 2.98 (2.22)
RSScaox = 1.30 (0.52)
RSSmap = 1.63 (1.14)
RSScaox = 3.68 (2.09)
RSSmap = 0.85 (0.69)
RSScaox = 3.47 (1.59)
RSSmap = 12.18
RSScaox = 2.32 (1.15)
RSSmap = 0.75 (0.37)
pSAPcalc = 9.18 (0.83)
pSAPEQUIL = 10.76 (0.65)
pSAPcalc = 11.40 (0.24)
pSAPEQUIL = 12.10 (0.24)
pSAPcalc = 7.80 (0.91)
pSAPEQUIL = 10.21 (0.60)
pSAPcalc = 10.78 (0.38)
pSAPEQUIL = 11.61 (0.28)
pSAPcalc = 10.11 (0.66)
pSAPEQUIL = 11.32 (0.38)
pSAP = 9.07 (0.34)
pSAP = 9.91 (0.34)
Cats
Healthy
Dry diet with 29% protein DM corn gluten and fish meal
Dry diet with 55% protein DM corn gluten and fish meal
Cats
Healthy
Dry diet with 32.6% protein as meat meal
Dry diet with 32.5% protein as corn gluten meal
pSAP = 10.17 (0.34)
pSAP = 10.11 (0.34)
Cats
Healthy
Cats
Healthy
Adult dry diet with 39% protein DM as meat meal
Adult dry diet with 39% protein DM as chicken meal
Adult dry diet with 39% protein DM as corn gluten meal
Dry diet (72% protein, 7.5% fat, 14% NFE, 0.4% fiber DM)
Dry diet (52% protein, 9% fat, 32% NFE, 0.8% fiber DM)
Dry diet (52% protein, 14% fat, 25% NFE, 3.3% fiber DM)
Diets as above, but daily intake normalized for protein
Dry diet (72% protein, 7.5% fat, 14% NFE, 0.4% fiber DM)
Dry diet (52% protein, 9% fat, 32% NFE, 0.8% fiber DM)
Dry diet (52% protein, 14% fat, 25% NFE, 3.3% fiber DM)
Adult dry diet with 29% protein DM
Adult dry diet with 55% protein DM
Adult dry diet with 29% protein DM
Same diet with 1.5% ammonium chloride
Same diet with 0.75% sodium chloride
Adult maintenance dry diet
Diet with 1% d,l-methionine
Diet with 2% d,l-methionine
Experimental diet with 27% protein DM
Experimental diet with 1.5% ammonium chloride
Adult maintenance canned diet
pSAP = 9.27 (0.31)
pSAP = 9.20 (0.31)
pSAP = 9.61 (0.52)
pSAP = 9.45 (0.38)
pSAP = 9.04 (0.55)
pSAP = 9.29 (0.42)
Cats
Healthy
Cats
Healthy
Cats
Healthy
Diet + 0.1 mg/BWkg takushya
Diet + 0.5 mg/BWkg choreito
Cats
Healthy
Adult maintenance canned diet
Same diet with 0.25 gm/BWkg chorieto
Same diet with 0.5 gm/BWkg chorieto
Same diet with 1 gm/BWkg chorieto
Same diet with 2 gm/BWkg chorieto
Same diet with 4 gm/BWkg chorieto
pSAP = 9.48 (0.39)
pSAP = 9.05 (0.45)
pSAP = 8.99 (0.39)
pSAP = 9.08 (0.68)
pSAP = 9.71 (0.63)
pSAP = 8.81 (0.45)
pSAP = 9.00 (0.73)
pSAP = 10.56 (0.66)
pSAP = 9.71 (1.13)
pSAP = 9.46 (1.13)
pSAP = 10.61 (1.13)
pSAP = 8.43 (3.04)
pSAP = 9.65 (3.04)
RSSmap = 5.70 (4.74)
pSAPmap = 9.52 (0.44)
RSSmap = 3.47 (2.02)
pSAPmap = 9.76 (0.45)
RSSmap = 2.53 (2.56)
pSAPmap = 9.92 (0.44)
pSAP = 8.5 (0.3)
pSAP = 8.9 (0.3)
pSAP = 9.2 (0.6)
pSAP = 9.2 (0.6)
pSAP = 9.3 (0.4)
pSAP = 9.4 (0.2)
(Smith,
Stevenson et al.
1998)
EQUIL
(Buffington,
Rogers et al.
1990)
Hand calculated
or EQUIL
(Funaba,
Hashimoto et al.
1996)
Hand calculated
(Funaba,
Matsumoto et al.
2002)
Hand calculated
(Funaba, Oka et
al. 2005)
Hand calculated
(Funaba,
Uchiyama et al.
2004)
Hand calculated
(Funaba, Yamate
et al. 2003)
Hand calculated
(Funaba, Yamate
et al. 2001)
Hand calculated
(Buffington,
Blaisdell et al.
1997)
EQUIL
(Buffington,
Blaisdell et al.
1992)
Cats
Healthy
Adult maintenance dry struvite preventative diet
Adult maintenance canned struvite preventative diet
Adult maintenance dry high fiber diet
Adult maintenance canned high fiber diet
APRmap = 0.47 (0.31)
APRcaox = 2.51 (1.15)
APRmap = 0.68 (0.29)
APRcaox = 1.43 (1.27)
APRmap = 0.84 (0.49)
APRcaox = 2.21 (0.90)
APRmap = 1.98 (0.96)
APRcaox = 0.52 (0.30)
(Bartges, Tarver
et al. 1998)
* Healthy – non-urolith-forming animals, DSH = domestic short-hair
‡ NFE = nitrogen free extract
# RSScaox = relative supersaturation for calcium oxalate, APRcaox = activity product ratio for calcium oxalate,
RSSmap = relative supersaturation for struvite (magnesium ammonium phosphate), APRmap = activity product
ratio for struvite, RSScom = relative supersaturation for calcium oxalate monohydrate, RSScod = relative
supersaturation for calcium oxalate dihydrate, RSSbr = relative supersaturation for brushite, APRua = activity
product ratio for uric acid, APRnau = activity product ratio for sodium urate, APRau = activity product ratio for
ammonium urate, APRxan = activity product ratio for xanthine, pSAP = negative logarithm of struvite activity
product where pSAP is negatively related to struvite crystal formation.
§ EQUIL = EQUIL program (various versions), College of Medicine, University of Florida, SUPERSAT =
SUPERSAT program, Dr. W.G. Robertson.
¥ Values are approximate based on figure in manuscript; results were not included in table in text
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