The Experimental Studies Of Urinary Stone In Infants And Children Induced By Melamine

Background Many children aged under3years old were diagnosed with urinary stones in the out-patient of urology and pediatrics in Lanzhou University Second Hospital since April2008. The primarily clinical manifestation of them was irritability, crying, nausea, vomiting, diarrhea, urine turbidity as well as upper respiratory tract infection. Some children presented with severe symptoms including gross hematuria and acute renal failure caused by oliguria and anuria. The urinary stones were confirmed in these children via examination of ultrasonic. At the same time, they had the feeding history of ingestion of infant formula produced by Sanlu Group in Hebei Province in China. Then, this emergency accident of urinary stones in children was promptly informed to the Health Department of Gansu Province by the department of urology in Lanzhou University Second Hospital. Following, the outbreaks of urinary stones in children were reported in other provinces in China, which triggered the strong attention to Ministry of Public Health of China. After investigation to the fed infant formulas produced by Sanlu Group in Hebei Province, it was found that melamine which forbided not to be used as food additives was illegally added to the infant formulas. According to the statistical data revealed by Chinese Center for Disease Control and Prevention, total294,000children who had the history of ingestion of melamine-contaminated infant formulas were accepted examination and treatment by end to Nov.27in2008, including51,900hospitalizations and6deaths. In order to further confirm the relationship between the ingestion of melamine and occurrence of urinary stone in children, samples of blood, urine and stones in hospitalized children with urinary stone induced by melamine in20hospitals of Gansu Province were collected in October2008and some related experimental studies to these samples also were conducted.Objectives To confirm the content of melamine in urine and stone samples of children with urinary stone induced by melamine to identify the relationship with melamine and occurrence of urinary stone in children and offer the etiological proof of urinary stone caused by melamine; To analyze the components of melamine-induced urinary stones and set up the Animal Models of renal stone in rats induced by melamine to theoretically extrapolate the mechanism of melamine-induced urinary stone formation in children in order to provide the experimental support and theoretical basis with the clinical prophylaxis and treatment with melamine-induced urinary stone in children.Methods Qualitative analysis for the components and types of crystallization of melamine-induced urinary stones using combination of chemical assay, infrared spectroscopy and X-ray powder diffraction methods; The fine morphology of crystals in melamine-induced urinary stones was observed by scanning electron microscopy and the content of melamine in stone samples was determined with high performance liquid chromatography; Quantitative analysis for the content of melamine in urine samples of children with melamine-induced urinary stones by high performance liquid chromatography to investigate the mechanism process of melamine in vivo; Animal Model of renal stone in rats induced by melamine was set up via administration of melamine or cyanuric acid alone or mixture of them to rats to identify the mechanism of renal crystals formation in rats caused by melamine and cyanuric acid; Based on the above results of experiment, the mechanism of melamine-induced urinary stone formation in children was theoretically extrapolated.Results Acorrding to the gross appearance of12stone samples, urinary stones in children induced by melamine was roughly classified to three types:brown color and sand-like stones, yellow-white color and granule-like stones and gray color and lump-like stones. The chemical components of9stone samples were qualitatively analyzed by the method of chemical assay including3uric acid stones,5ammonium urate stones and1mixture of ammonium urate, ammonium magnesium phosphate and carbonate apatite stone; The main components of crystals in9stone samples analyzed by infrared spectral indicated that1was composed of sodium urate monohydrate,2were composed of uricite,5were composed of uric acid dihydrate and ammonium urate and1was composed of ammonium magnesium phosphate hexahydrate, ammonium urate and carbonate apatite;6stone samples analyzed by X-ray powder diffraction method showed that the diffraction pattern of5stones were not matched with the diffraction pattern of known compounds and only1stone was matched with the diffraction pattern of ammonium magnesium phosphate hexahydrate, ammonium urate and carbonate apatite stone; Diversity of fine crystalline morphology in10stone samples was revealed by scanning electron microscopy observation, which had not only regular crystallization but irregular aggregations of crystals; The content of melamine in all of the12stone samples was determined by high performance liquid chromatography and found to be ranged from200to339000μg/g;40urine samples in children with melamine-induced urinary stone were detected and11of them were found that the content of melamine was above the limit of detection. The content of melamine in11urine samples was ranged from0.15to1.91μg/mL and the detection rate was27.5%;120male SD rats were divided into6groups according to different feeding patterns. The abnormalities of blood biochemistry, gross appearance and histopathological feature of kidney and liver were not found in rats after30days administration of the high dosage of melamine or cyanuric acid alone (100mg/kg/bw) compared to those in controls; The gross morphology of kidney presented with irregular color distribution and plaque on kidney surface. Renal swelling and white-yellowish color, needle point like granules which mostly distributed in collective system on the longitudinal section were observed in some rats after30days administration of the low dosage of melamine and cyanuric acid (10/10mg/kg/bw), and after3,15and30days administration of the middle (25/25mg/kg/bw) and high (50/50mg/kg/bw) dosage of melamine and cyanuric acid; The obvious elevation of blood urea nitrogen and blood creatinine were observed in rats after15and30days administration of the middle dosage of melamine and cyanuric acid and in rats after3,15and30days administration of the high dosage of melamine and cyanuric acid which had significant difference compared to those in controls (P<0.01); On renal pathological sections, crystals were found in renal tubules with inflammatory cell infiltration in some rats after30days administration of the low dosage of melamine and cyanuric acid, after15and30days administration of the middle dosage of melamine and cyanuric acid as well as after3,15and30days administration of the high dosage of melamine and cyanuric acid. The distribution of crystals was found in renal cortex, medulla and papillae.Conclusions (1) Melamine-induced urinary stones in children were classified to three types including brown color sand-like stones, yellow-white color granule-like stones and gray color lump-like stones according to the gross appearance of stone samples.(2) The main chemical components of melamine-induced urinary stone in children are uric acid dihydrate and ammonium urate; The structure of crystals in melamine-induced urinary stone is amorphous; The crystalline morphology of melamine-induced urinary stone is diverse.(3) Melamine was detected in11of40 urine samples in children with melamine-induced urinary stones (27.5%). However, the metabolic process of melamine in vivo of humans is not still accurately elucidated because of the random collection of urine samples in children with melamine-induced urinary stone.(4) The methods of determination of melamine in urine and stone samples in children with melamine-induced urinary stone using high performance liquid chromatography have lower cost and high sensitivity and accuracy, which are applicable for clinical research.(5) The administration of melamine or cyanuric acid in short-term can not induce the damages of toxicity to rats while the mixture of melamine and cyanuric acid administration can promote the formation of renal crystallization to induce the obstruction of renal tubules finally to lead to acute renal failure. The degree of renal injures in rats caused by melamine and cyanuric acid closely depends on the time and dosage of melamine and cyanuric acid administration.(6) The mechanism of urinary stone formation caused by melamine in children is different from that of in rats. We speculate that excessive amounts of melamine in the urine may combine with uric acid to develop melamine-urate precipitates to form uric acid stones; the high concentration of melamine in urine may impede the reabsorption of uric acid and interfere with the mechanism of ammonia in renal tubules resulting in hyperuricosuria and hyperuricoammonium. Finally, the direct consequence is the formation of uric acid and ammonium urate stones.