Constituents of Urinary Calculi, some Metabolic Screening, and Detection of Polymorphism in SLC3A1 gene in Sudanese Children with Cystine Calculi

Abstract

The objectives of this study were to insight the light in urinary calculi constituents in Sudanese children, evaluation of some metabolic risk factors and detection of polymorphism in exon 8 at SLC3A1 gene in patients with cystine stones. One hundred and seventy five urinary calculi from pediatric patients which were removed surgically in Soba Teaching Hospital at Khartoum state Sudan were analyzed during the period October 2005- May 2009. The patient’s age was between 3 months- 16 years with mean of (4.96± 4 years). The stones were analyzed by semi quantitative chemical method. Using a kit supplied by DiaSys Diagnosistic systems GmbH. (Germany). The stone was powdered and the standard both was analyzed for calcium, oxalate, ammonium, phosphate, magnesium, uric acid, and cystine. One hundred and thirty blood samples (80 patients, 50 controls) were collected from patients to screen some metabolic risk factors, serum calcium, serum phosphate, serum uric acid, and serum creatinine. Fifty samples, of 24 hours urine collection (30 patients, 20 controls) were collected to determine the excretion of urine calcium, phosphate and uric acid. It was analyzed by auto analyzer Cobs Integra 400 Plus. Ten blood samples were collected in EDTA container from patients with cystine stones, DNA was extracted by phenol chloroform isoamyle alcohol method, then the exon 8 of SLC3A1 gene were amplified by using thermo cycler machine (X Bioer, China), and the exon 8 was sequenced by Genetic auto analyzer AB3130 ( Applied Biosystem). Urinary calculi were more predominant in male than female ratio 2.8:1 .The stones were located in the upper urinary tract in 75%, lower urinary tract in 25% and 1.1% in both upper and lower tract. On presentation 66.8% had flank pain 27.4% had hematuria, 49.1% had urinary tract infection (UTI), and 6.2% had renal failure, 20% had positive family history of stones formation. The age group 2-6 years was the most frequent of stones onset for all types of stones except cystine stone in which the age group was found to be less than two years. 20% had recurrent stones formation. Calcium oxalate stone were the commonest constituents (55.4%), followed by ammonium urate (48.6%), Struvite (15.9%), calcium phosphate (11.4%), uric acid stones (8%), and cystine stones (5.7%). The components of the upper urinary tract calculi were calcium oxalate (40%), ammonium urate (35.4%) and calcium phosphate (21%), whereas the main components of the lower urinary tract calculi were ammonium acid urate (23%), struvite (9.1%), and calcium oxalate (10.4%). There was significant association between upper urinary tract and calcium phosphate stones, cystine stones P< 0.028, 0.054 respectively, and lower urinary tract with Struvite stones P<0.0001. Cystine stones were more common in patients with positive family history P< 0.000. This study showed that there was no difference between the mean of total serum calcium, phosphate, serum uric acid, and serum creatinine in patients compared to control group, the means of total calcium for patients were (9.6± 0.85 mg/dL) , and control (9.5± 0.7 mg/dL), P<0.56, mean of serum phosphate for patients (4.3± 0.4 mg/dL), and control (4.2± 0.4 mg/dL), P<0.345, mean of serum uric acid in patients were (3.5± 1.1 mg/dL), control (3.5± 1 mg/dL) P< 0.979, and serum creatinine in patients were (0.46± 0.24 mg/dL), control (0.4± 0.1 mg/dL), P< 0.089. There was a reduction in urine volume per 24 hours in patients with renal stones (675± 200 mL/day) compared to control group (898± 350 mL/day), P<0.011.There was increase in urine calcium excretion mg/kilogram/day mean (1.98 ± 0.8) mg/kg/day compared to control (1.4± 0.5)mg/Kg/day P< 0.009, urine uric acid excretion in patients were (1.0± 0.5) mg/Kg/day, and control (0.6 ± 0.4) mg/Kg/day P<0.013, and no significant variation between the mean of urine phosphate excretion in patients (5± 1.9) mg/Kg/day, and control (5.2 ± 1.2) mg/kg/day P< 0.627. The mutation which was found in exon 8 at SLC3A1 gene was M467K, due to substitution of one base thymine (T) to adenine (A), which affects the transport function of cystine and dibasic amino acids in the kidney and small intestine.