Pediatr Nephrol (2007) 22:514–520 methods which would enable the detection of risk factors for urolithiasis. One of the risk factors is an increase in the crystallization of calcium oxalate (CaOx) in urine. Kavanagh and Laube recently published a review of methods used to assess the crystallization of CaOx in urine [6]. During the last few years, activity products (AP) of crystallization have usually been assessed by means of the APCaOx index or as the relative supersaturation (RS) of urinary calcium oxalate (RSCaOx) using the computer program EQUIL [7, 8]. Laube et al. showed that the ratio of calcium ions [Ca2+] to the amount of ammonium oxalate added to 200 ml of urine to induce crystallization [designated here as (Ox2−)] may be, at the moment of spontaneous crystallization of CaOx, an indicator of the risk of CaOx crystal formation [9]. The authors made this determination with a direct urine collection, without an initial processing. This ratio is known as the Bonn Risk Index (BRI): BRI=[Ca2+]/(Ox2−) 1/L [9, 10]. Due to its characteristics, this index is an accurate indicator of the individual state of balance between the quantities of the most important promoters and inhibitors of the crystallization process within urine [9, 10]. Measuring BRI is simple, cost-effective, and the results are repeatable. In patients with calculi formation, CaOx assessed using BRI is significantly higher when compared to healthy subjects [9]. A lack of published studies concerning BRI in pediatric patients led us to conduct the study in children and adolescents. The purpose of this study was to define the BRI value in healthy subjects aged 3–18 years, in relation to age and sex. Materials and methods The study was performed on a group of 1,050 healthy Caucasian children and adolescents (group I) aged 3– 18 years (mean ±SD: 10.51± 4.33), comprised of 525 boys and 525 girls. The study population was divided into 15 age groups, consisting of 70 children in each 1-year group (35 boys and 35 girls). The children reported no history of dietary restrictions. All participants met the criteria of the standard dietary energy and nutrient intakes recommended in Poland [11]. These children were free of infection at the time of the examination (serum C-reactive protein CRP <0.4 mg/dL, blood leukocyte count <10 × 109/L). Prior to inclusion, all participants were screened regarding serum levels of protein, albumin, calcium, phosphate, potassium, uric acid, creatinine and alkaline phosphatase as well as urine concentrations of citrate, oxalate, potassium, calcium and phosphate. Urinary dipstick testing (Bayer Diagnos- tics, Bridgend, UK) detecting nine parameters, including leukocytes, protein and blood, did not reveal any abnor- malities. Children with a family history of kidney stones were excluded from the study. Subjects with diseases 515 known to affect oxalate, calcium and phosphate metabo- lism and children treated with antibiotics were excluded. All children were screened using renal ultrasound exam- ination to exclude urolithiasis (Toshiba SSH-140A appa- ratus; probe Convex 3.75 MHz). Participants and their legal guardians gave informed consent, and the study was approved by the Ethical Committee of the Medical University of Bialystok. Bonn Risk Index The Bonn Risk Index was assessed using the method of Laube [12]. Each child had a 24-h urine collection into sterile containers, without additional preserving substances, which was stored at 4°C. The testing was always performed twice using the same urine collection from each subject. Two consecutive urine samples (each 200 mL) were incubated immediately after collection, at a temperature of 37°C and the calcium ion concentration was measured using calcium ion-selective electrodes of type Rapilab 855 (Bayer, Germany) and titrated with ammonium oxalate solution (40 mmol/L) at a rate of 0.75 mL/min. The onset of spontaneous crystallization was detected using an Eppendorff photometer (filter 585 nm) with a decrease in light transmission to 98% of the initial value. Each analysis was repeated twice. The BRI is presented as [Ca2+] mmol/L / (Ox2−) mmol = 1/L. Calibration and quality assurance procedures, based on the calibration curves, were con- ducted every day. Statistical analysis was performed using the program Statistica 6.0 PL. Mann-Whitney test was used for the analysis of two non-parametric independent variables, with P< 0.05 considered statistically significant. Assessment of the rank of two independent variables was conducted using Spearman correlation, with P< 0.05 considered statistically significant. For the purpose of plotting the curve of spontaneous crystallization (an association between the number of calcium ions and the amount of added ammonium oxalate leading to the spontaneous crystalliza- tion), we used the computer program with the range of values as a scatterplot with the option of adding curves. Results The characteristics of the study group are presented in Fig. 1. The anthropometric traits of participants, based on weight and height measurements and body mass index (BMI), were within the normal range in each subgroup, relative to the Polish references described by other authors [13]. Figure 2 shows the detailed results for the whole study group for BRI, defined as the ratio of [Ca2+] concentration