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Relative Supersaturation (RSS) is a physicochemical method to assess the risk of CaOx or MAP deposits forming in the urinary tract of dogs and cats. This method is one of the most widely used methods in humans to predict mineral crystallization in urine. In 1969, Dr WG Robertson developed an original method for calculating urine supersaturation in human: Supersat™. . In collaboration with the Waltham Center for Pet Nutrition (WCPN), this software was validated in both dogs and cats. Dr Robertson and the R&D team of Royal Canin have upgraded the software to make it easier to use and to take into account the composition of urine of dogs and cats fed present day petfood.
Measuring the crystallisation of urine is a useful way of developing and monitoring therapeutic interventions for patients with urolithiasis. The risk of a given salt or acid precipitating, and the rate and extent to which crystallisation will proceed, are largely dependant upon their level of supersaturation in the urine . RSS is the algorithm used by most workers in the field of human urolithiasis to measure urinary supersaturation. This method has recently been validated for both dogs and cats .
Briefly, the formation, growth, and dissolution of urinary crystals depends on the concentrations of the minerals that make up the crystal (i.e. for CaOx, calcium and oxalate, and for struvite, magnesium, ammonium and phosphate) that are free to react with each other. The concentration of a solute is not synonymous with its activity, and activity can only be determined by correction for the effect of ionic strength and formation of complexes with extraneous ions.. Indeed in the case of CaOx, in urine, calcium ions will interact with phosphate, sulfate, and citrate ions, whereas oxalate ions will interact with magnesium, sodium, and potassium ions. Therefore CaOx crystals will only form if enough calcium and oxalate ions are free to interact with each other. Comparable to CaOx, struvite crystals will only form if enough magnesium, ammonium and phosphate ions are free to interact.
It is possible to calculate the free fractions of calcium and oxalate; the product of concentrations is called the activity product. The RSS for a given salt is defined as the ratio of the activity product divided by the thermodynamic solubility product for that salt. The thermodynamic solubility product is the maximum amount of a given salt that can be dissolved in a solvent (i.e. water), for a given temperature (i.e. 37°C), and a given pH (i.e. 6.0).
A RSS < 1 means that the urine is undersaturated (the activity product, calculated from concentrations of soluble ion complexes in the urine, is below the solubility product at standardised temperature and pH); crystals will not form, but will dissolve.
A RSS > 1 means that the urine is supersaturated (the activity product, calculated from concentrations of soluble ion complexes in the urine, is above the solubility product at standardised temperature and pH); crystals might form, and will not dissolve. Estimating RSS requires numerous and complex calculations. Software such as Supersat™ has been designed to calculate RSS for CaOx and struvite, from the urinary pH and concentrations of ten solutes (calcium, magnesium, sodium, potassium, ammonium, phosphate, citrate, sulfate, oxalate, uric acid) [1,2].
In a complex solution like urine, it is possible to have a RSS for CaOx or struvite above 1 without spontaneous precipitation of crystals . This is due to electrical fields (caused by ionic bonds) induced by the numerous ions in solution, and the presence of inhibitors of crystallisation. Both prevent the free fractions of minerals from interacting and forming crystals. This level of supersaturation is referred to as metastable supersaturation (the activity product calculated from concentrations of soluble ion complexes in the urine is higher than the solubility product at standardised temperature and pH, but lower than the formation product at standardised temperature and pH). At this level of saturation, crystals will not form spontaneously, but might occur in the presence of a nucleus. In the zone of metastable supersaturation, crystals (and thus stones) will not dissolve.
At higher urinary mineral concentrations, crystals will form spontaneously within minutes to hours. This is labile supersaturation (the activity product calculated from concentrations of soluble ion complexes in the urine is higher than the solubility product at standardised temperature and pH, and higher than the formation product at standardised temperature and pH). The limit between metastable and labile supersaturation is called the formation product. Kinetic precipitation studies in urine have shown that the formation product is 2.5 for struvite and 12 for CaOx.
The method used to study the influence of diet on urinary CaOx and struvite RSS in dogs and cats has been described earlier . The measurement of RSS consists of (1) collecting urine from animals fed the diet that is checked for compliance, (2) analysing pH and concentrations of the ions involved in the formation soluble ion complexes, and (3) calculating the ratio of the activity product to the solubility product.
It is important that the length of the urine collection period and volume of pooled urine from this period is adequate to obtain a representative and repetitive measure. Briefly, groups of 6 to 8 healthy adult cats or small dogs (< 10 kg body weight) are housed in accommodation designed for urine collection. They are adapted to the diet for 9 days, then all urine is collected over the subsequent 5 days. The animals are fed and water is available ad libitum. For individual animals, the various urine samples produced over the course of the study are pooled and stored at 4°C in a bottle containing 1 mL of chlorhexidine 20% (Hibitane®). At the end of the urine collection period the pooled sample is well-mixed and the total volume, specific gravity, and urine pH are recorded. An aliquot is taken, titrated to pH 2 with HCl 37%, and kept at -20°C pending analysis. We validated the use of chlorhexidine, as well as storage for up to 4 years at -20°C; neither affected struvite or CaOx RSS. We also found that following a 7-day dietary adaptation period, CaOx and struvite RSS values remained constant over time.
Urinary calcium, magnesium, sodium, potassium, ammonium, phosphate, citrate, sulfate, oxalate, and uric acid concentrations are determined by ionic chromatography (Dionex, Sunnyvale, CA, USA). The analysis of ions involved by ion-exchange chromatography is estimated to have a Reproducibility Relative Standard Deviation (RSDR) < 5% for mineral ions and < 10% for organic ions. Cations are eluted with methanesulfonic acid and anions with potassium hydroxide.
The ratio between the activity product and solubility product must be calculated taking the potential interactions between ions into account, which is done by a computer based program. Today 2 software programs exist, the original method Supersat™ and Equil-2. Both softwares were validated for dog and cat urine  and showed significant differences between Equil-2 and Supersat™ for struvite and CaOx RSS in dogs and cats. Both softwares calculated reasonably accurate RSS values for CaOx though Equil-2 gave overestimated results, whereas only Supersat™ provided an accurate measure of struvite RSS. The discrepancies found can be due to differences in the values of many stability constants used to calculate the mutual complexing of the various cations and anions, and differences between the values of the solubility products used in the 2 programs. Overall, the RSS for CaOx and struvite may be accurately calculated using the Supersat™ software.