Urolithiasis DOI 10.1007/s00240-013-0622-3
ORIGINAL PAPER
The true stone composition and abnormality of urinary metabolic lithogenic factors of rats fed diets containing melamine Xiaoming Cong • Xiaojian Gu • Yan Xu Xizhao Sun • Luming Shen
•
Received: 5 June 2013 / Accepted: 6 November 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract To better understand the toxicity of melamine to humans, the stone composition and urinary metabolic lithogenic factors of rats fed diets containing melamine including the infant’s melamine-induced stone composition were studied. Sixty 4-week-old male rats divided into three groups were, respectively, fed diets containing no melamine (control), 0.1 % melamine, and 1 % melamine for 4 weeks. At the end of experiment, the collected stones and 24-h urines from rats were, respectively, measured with compositions and metabolic lithogenic parameters. The stone from an infant who ingested melamine-adulterated formula was also included in compositional analysis. Across three groups, the stone was only detected in 1 % melamine group, with composition of almost melamine different from the affected infant’s stone composed of melamine and uric acid with a ratio of 1:2. Compared with control group, urine calcium and phosphate excretions were significantly increased in 1 % melamine group. Urine uric acid excretion was significantly increased but citrate excretion was significantly decreased in 0.1 % and 1 % melamine groups. Urine oxalate excretion and pH were indicated without any significant difference. In addition based on urine physicochemical characters, melamine–uric
X. Cong and X. Gu contributed equally to this work. X. Cong X. Gu Y. Xu Department of Urology, Jiangsu Province Hospital of TCM, Affiliated Hospital of Nanjing TCM University, Nanjing 210029, China X. Cong X. Sun (&) L. Shen Department of Urology, Affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, China e-mail: [email protected]
acid stone seems difficult to be formed in the rats due to their characters of urine high-pH and low-uric acid. These results demonstrated that (1) the stone composition of rats fed melamine was not and could not be as that of infants fed melamine-adulterated formula, two species had a different mechanism of melamine-induced stone formation; (2) the exposure of melamine could result in abnormalities of urine metabolic lithogenic factors to rats, perhaps as well as human beings. Keywords Melamine Rat Infant Stone Urinary metabolic lithogenic factors
Introduction In the summer of 2008, an outbreak of kidney stones and renal failure in infants caused by the ingestion of melamine-adulterated formula attracted a public concern to the toxicity of melamine for humans [1]. However, thus far the exact mechanism of stone formation in the affected infants remains unknown. In 2004 and 2007, two outbreaks of animal renal disease in some Southeast Asian countries and North America had been caused by both of melamine and cyanuric acid which formed crystals of melamine cyanurate in renal tubules and collecting ducts [2]. Unlike animal outbreaks, the Chinese melamine-adulterated formula contained only trace amount of cyanuric acid [2], and stones from the affected infants were composed of uric acid and melamine with a molar ratio ranged from 1.3:1 to 2.1:1 [3, 4]. In the studies of rats fed only melamine, Ogasawara et al. [5] found the formed stones with an equimolar ratio of melamine and uric acid, which was analogous to those of affected infants. However, Heck et al. [6] and recently Shen et al. [7] found the stone of rats fed melamine was
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composed primarily of melamine. It seems a re-analysis of stone composition of the rat fed melamine is necessary for us understanding how the human’s melamine-related stone was formed. A lot of studies showed that greater and longer exposure of melamine increased the formation of kidney stones in the melamine-adulterated formula event [8–10]. However, there were other factors that indicate the increase of melamine-related stone formation. Infants with preterm infancy and acid urine were demonstrated to more likely to suffer from melamine-related stones [9, 11]. Lam et al. [10] found that more than half of infants with melamine-related stones had predisposing urinary metabolic factors. Recently, Liu et al. [12] further reported a strong association between the exposure of low-dose melamine and calcium kidney stones in Taiwanese adults. In their study, patients with stones were detected with greater urinary melamine, as well as higher urinary calcium and uric acid excretion. These suggest that abnormalities of urinary metabolic lithogenic factors should be involved in the formation of melamine-related stones. However, whether abnormalities of these urinary metabolic lithogenic factors are caused by melamine exposure is unclear now. In this study, we will use a rat model fed melamine to analyze the stone composition and urinary metabolic lithogenic factors to help understand the toxicity of melamine to human beings.
successive 24-h urines were collected with metabolic cages at the end of 4th week. Urine volume and pH were measured instantly. Urine sample was frozen at -20 °C for a biochemical analysis. At the end of 4th week rats were killed, the stones were collected and examined. Analysis of stone composition To compare the differences of melamine-related stone composition between rat and human, the stone received from a 10-month-old female infant who ingested Sanlu melamine-adulterated formula since birth in June 2008 was included. Stone compositions of the infant and rat were analyzed by the Fourier transform infrared spectroscopy (FTIR) and high-performance liquid chromatography (HPLC). In the analysis by FTIR, stone was firstly fragmented into powders, then compressed into a transparent wafer with potassium bromide (Sigma-Aldrich, MO, USA), at last examined by FTIR (Lambda Scientific Instrument Company, Tianjing, China). In the analysis by HPLC with a method of simultaneous determination of melamine, cyanuric acid, and uric acid [13], briefly, the dissolved stone sample was measured by HPLC system consisted of the Agilent 1,100 system and Agilent HC-C18 column (4.6 9 250 mm, 5 lm) (Agilent Technologies, CA, USA), with the eluted solution (10 mmol/l K2HPO4 ? 10 mmol/l KH2PO4, pH 7.50), flow rate of 0.8 ml/l, and detected wavelength of 254 nm. Measurement of blood and urine
Materials and methods Experimental animals Four-week-old male Sprague–Dawley rats were obtained from Animal facilities of Slac Laboratory Animal (Shanghai, China). Five animals each were housed in an individual plastic cage before the experiment under the temperature of 22 °C and a 12-h light/dark cycle condition. They were fed a basal pellet diet and tap water except as specifically noted below. All of experimental procedures were performed in accordance with NIH Guide for the Care and Use of Laboratory Animals. Experimental procedure Sixty rats were divided into three groups, and groups 1–3 were fed diets, respectively, containing 0, 0.1 and 1 % melamine (Sinopharm Chemical, Shanghai, China) for 4 weeks. Melamine diets were prepared by mixing powdered basal diet with melamine and then compressing them into pellets. Body weights, food consumption and water intake were recorded at the end of 4th week. Two
123
Blood biochemistry and urine calcium, phosphate, creatinine (Cr), and urine nitrogen (BUN) were conducted by a Hitachi 7600 biochemistry analyzer (Hitachi, Tokyo, Japan). Urine citrate and oxalate were measured by the Ion chromatography (IC) using a method [14]. The IC system consisted of the 792 Basic IC equipment, Metrosep ASUPP5-100 column (4 9 100 mm, 5 lm), and Metrosep ASUPP4/5 Guard (4 9 5 mm, 5 lm) (Metrohm, Herisau, Switzerland). All samples were eluted with a solution (17.5 mmol/l Na2CO3 ? 3.5 mmol/l NaHCO3) at a flow rate of 0.7 ml/l. The method of HPLC analyzing stone compositions was also used to measure urine melamine, uric acid, and cyanuric acid. Urine pH was measured by a PHS-25 type pH meter (INESA Scientific Instrument Company, Shanghai, China). Statistical analysis Statistical analysis was performed using SPSS 18.0. The Student’s t test was used to do comparisons between experimental groups. Differences were considered significant at P \ 0.05.
Urolithiasis Table 1 Body weight, food intake, water intake, and stone incidence Group
No.
Body weight (g)
Food intake (g/rat/ day)
Water intake (ml/rat/ day)
Control
20
446.12 ± 33.56
41.6
36.7
0
0.1 % melamine
20
444.5 ± 23.12
37.7
44.6
0
1% melamine
20
411.2 ± 42.16a
35.5
54.2
45
a
Stone incidence (%)
Significantly different from the control group value at P \ 0.05
Results During the total experimental period, there was no died rat in three groups. Compared with control group, body weights in 1 % melamine group were significantly lower (P \ 0.05). Across three groups, as the rats consumed higher melamine-contained diets, their water intakes were increased, but food intakes were on the contrary. Only nine rats (45 %) in 1 % melamine group suffered stones with faint yellow color and amorphous shape in the urinary
tract. There was no rat with detected stone in other two groups. The explicit data was shown in Table 1. The stones in nine rats were analyzed by FTIR and HPLC. By the FTIR, as the melamine standard shown in the data of Chen et al. [15], nine rats’ stones shared the same characteristic wave-number positions at 3,470, 3,420, 3,335, 3,215, 1653, 1,555, 1,470, 1,440, 1,030, and 815 cm-1 (Fig. 1a). By the HPLC, as shown in Fig. 1b, the stone was eluted as a distinct peak as melamine standard. The melamine contents in the nine rats’ stones were 98.6–99.5 %. Out of nine rats’ stones, three stones were detected of a trace of uric acid with contents 0.06–0.13 %, and no stone was detected with cyanuric acid. Figure 2 showed the melamine-related stone from the affected infant. This stone had the same characteristic wave-number position (Fig. 2a) with melamine–uric acid precipitate in our previous data [16]. In addition, as shown in Fig. 2b, this stone was eluted as two distinct peaks in agreement with these of uric acid and melamine standards. The content of melamine and uric acid in this stone was 23.6 and 63.7 % with a molar ratio of 1:2.0. The data of blood and urine parameters were shown in Table 2. There was no significant difference in blood BUN
Fig. 1 The analysis of a stone from rats fed diets containing 1 % melamine by FTIR (a), and HPLC (b). a, b, respectively, showed characteristic wave numbers and a distinct peak with retention time as the same as those of standard melamine
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Urolithiasis
Fig. 2 The analysis of stone from the infant with an ingestion of melamine-adulterated formula by FTIR (a) and HPLC (c). a Showed characteristic wave numbers as same as these of melamine–uric acid
precipitate (b), c showed two distinct peaks with retention times as the same as those of standard uric acid and melamine
and Cr as a functional index across three groups. Compared with control group, the urine volume of rats in 1 % melamine group was slightly increased but with a statistical significance (P \ 0.05). There was no significant value difference of urine pH across three groups. It is noted that urine pH of any rat in three groups was alkaline (7.0–8.0).
In 24-h urinary metabolic lithogenic factors (Table 2), compared with control group, uric acid excretion was significantly increased and citrate excretion was significantly decreased in 0.1 % melamine group (P \ 0.05) and 1 % melamine group (P \ 0.001). Calcium and phosphate excretions were increased only in 1 % melamine group
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Urolithiasis Table 2 Renal functional, urinary metabolic factors, melamine, and cyanuric acid Group Serum BUN Serum Cr Urine volume (ml) Urine pH Urine calcium (mg/24 h) Urine phosphate (mg/24 h)
Control 8.2 ± 0.94
7.9 ± 1.12
8.3 ± 0.78
18.3 ± 2.82
24.7 ± 3.58
32.1 ± 9.30 7.33 ± 0.20
34.5 ± 8.11 7.53 ± 0.35
39.6 ± 9.20a 7.49 ± 0.35
2.0 ± 0.29
2.05 ± 0.26
2.39 ± 0.30a
19.49 ± 1.48
20.12 ± 1.79
20.85 ± 2.34a
0.86 ± 0.21
Urine oxalate (mg/24 h)
44.3 ± 6.02
Urine uric acid (mg/24 h)
2.46 ± 0.30
Urine cyanuric acid (lg/24 h)
1% melamine
18.61 ± 2.30
Urine citrate (mg/24 h)
Urine melamine (mg/24 h)
0.1 % melamine
none 8.13 ± 2.32
0.68 ± 0.29a 41.85 ± 5.94 2.69 ± 0.34a
0.43 ± 0.22b 45.6 ± 7.10 3.17 ± 0.27b
4.47 ± 0.50
36.25 ± 2.56
7.51 ± 1.72
7.92 ± 2.08
a
Significantly different from the control group value at P \ 0.05
b
Significantly different from the control group value at P \ 0.001
(P \ 0.05). There was no significant difference of oxalate excretion across three groups. Urine melamine excretion in 1 % melamine group was approximate eightfold than that in 0.1 % melamine group. Interestingly, there was the micro-dose of urine cyanuric acid detected in three groups but without a significant value difference.
Discussion By the stone compositional analysis, we showed the infant’s melamine-related stone was composed of uric acid and melamine with a molar ratio of 2.0:1, in agreement with the result of Sun et al. [3]. Zhang et al. [17] demonstrated melamine could be self-assembled and aggregated with uric acid to form lager clusters in water through hydrogen bonds. The predicted crystal structure was showed in the study of Dalal et al. [2]. With our previous study [16], we found the infrared spectrum of this infant’s stone was identical to that of melamine–uric acid precipitate not melamine–uric acid powders. It further demonstrated the infant’s melamine-related stone should be formed by melamine binding with uric acid through hydrogen bonds in urine to precipitate. In contrast, the formed stone in our rat model was composed of melamine and a trace of uric acid. This result is analogous to results of Hack et al. [6] and Shen et al. [7], but on the contrary with that of Ogasawara et al. [5]. To our knowledge, the
dissociation constant (pKa) of uric acid is 5.5. At a pH equal to the pKa, free uric acid and urate exist in equal proportions. At a pH of 6.5, 90 % uric acid would become urate and the remainder would continue to be free uric acid [18]. Unlike affected infants with the urine pH at 5.0–6.5 [10], the urine pH of Sprague–Dawley rats in our study was about 7.0–8.0. It means almost urine uric acid of our rats was urate ( 90 %), subsequently little free uric acid could complex with melamine free base through hydrogen bonds. Besides, urine uric acid level of rats should be significantly less than that of human because they had uricase which could convert uric acid to allantoin [19]. Therefore, we think it is very difficult to form melamine–uric acid stone for Sprague–Dawley rats, as well as for other strains of rats such as F344 and Wistar due to their alkaline urinary pH generally above 6.5 [20, 21]. After the event of melamine-adulterated formula, only four studies determined urine melamine level [10, 12, 22, 23], and three of them found the link between urine melamine level and urolithiasis risk [10, 12, 22]. To our knowledge, there is no study to investigate the effects of melamine on the urine metabolic lithogenic factors. Based on the results of urine biochemical analysis, we firstly showed the exposure of high-dose melamine could increase urine calcium, uric acid, phosphate excretions, decrease urine citrate excretion, and did not affect urine pH. It is usually thought melamine is primarily eliminated by urine excretion without metabolism [24, 25]. However, Xie et al. [26] found high-dose melamine could disrupt amino acid metabolism, and alter TCA and gut microflora structure. Perhaps the abnormalities of these urine factors showed in our study are related to the findings of Xie et al. As the study of Liu et al. [14], cyanuric acid was detected in urine of rat fed diets with or without melamine in our study. The uncorrelation between urine cyanuric acid and melamine excretions further suggests cyanuric acid should not be derived from melamine and involved in melamine-induced stone formation. In this study, we showed stone composition of rat fed melamine was not and could not be as that of infants fed melamine-adulterated formula. When using animal for risk assessment of melamine-induced stone formation, it is necessary to consider the difference of endogenous factors such as urine uric acid and pH. Since the exposure of melamine could cause abnormalities of urine metabolic lithogenic factors of rats, the melamine exposure may also cause it for human beings. Although the WHO and FDA lowered the melamine level of their recommended tolerable daily intake by tenfold [2], some studies indicated even low level of melamine exposure may risk urolithiasis formation both in infants and adults [12, 27, 28]. Considering melamine is a commonly used chemical in our daily life, and rat is usually used as a model to evaluate the toxicity of
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Urolithiasis
melamine on humans, we believe the present study is easy to understand the mechanism of infant’s melamine-induced urolithiasis, the limit of rat model used in the evaluation of toxicity of melamine to humans, and other possible toxicity of melamine to human beings.
11.
12.
13.
Conclusion The presented study demonstrated (1) the stone composition of rats fed melamine was not and could not be as that of infants fed melamine-adulterated formula, two species had a different mechanism of melamine-induced stone formation; (2) the exposure of melamine could result in abnormalities of urine metabolic lithogenic factors to rats, perhaps as well as human beings.
14.
15.
16. Acknowledgments Supported by the National Natural Science Foundation of China (grant 81070558) and Natural Science Foundation of Jiangsu Province (grant BK2010597). Conflict of interest
17.
No any conflict interest with others. 18. 19.
References 1. Ingelfinger JR (2008) Melamine and the global implications of food contamination. N Engl J Med 359:2745–2748 2. Dalal RP, Goldfarb DS (2011) Melamine-related kidney stones and renal toxicity. Nat Rev Nephrol 7:267–274 3. Sun N, Shen Y, Sun Q, Li XR, Jia LQ, Zhang GJ et al (2009) Diagnosis and treatment of melamine-associated urinary calculus complicated with acute renal failure in infants and young children. Chin Med J 122:245–251 4. Sun Q, Shen Y, Sun N, Zhang GJ, Chen Z, Fan JF et al (2010) Diagnosis, treatment and follow-up of 25 patients with melamineinduced kidney stones complicated by acute obstructive renal failure in Beijing Children’s Hospital. Eur J Pediatr 169:483–489 5. Ogasawara H, Imaida K, Ishiwata H, Toyoda K, Kawanishi T, Uneyama C et al (1995) Urinary bladder carcinogenesis induced by melamine in F344 male rats: correlation between carcinogenicity and urolith formation. Carcinogenesis 16:2773–2777 6. Heck HD, Tyl RW (1985) The induction of bladder stones by terephthalic acid, dimethyl terephthalate, and melamine (2,4,6triamino-s-triazine) and its relevance to risk assessment. Regul Toxicol Pharmacol 5:294–313 7. Shen Y, Sun N, Wang GN, He LJ, Jia LQ, Wang Y et al (2011) Pathological changes in rat model of urinary calculus induced by melamine. Zhonghua Er Ke Za Zhi (Chinese) 49:690–695 8. Wang IJ, Wu YN, Wu WC, Leonardi G, Sung YJ, Lin TJ et al (2009) The association of clinical findings and exposure profiles with melamine associated nephrolithiasis. Arch Dis Child 94:883–887 9. Guan N, Fan Q, Ding J, Zhao Y, Lu J, Ai Y et al (2009) Melamine-contaminated powdered formula and urolithiasis in young children. N Engl J Med 360:1067–1074 10. Lam CW, Lan L, Che X, Tam S, Wong SS, Chen Y et al (2009) Diagnosis and spectrum of melamine-related renal disease:
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plausible mechanism of stone formation in humans. Clin Chim Acta 402:150–155 Lu X, Wang J, Cao X, Li M, Xiao C, Yasui T et al (2011) Gender and urinary pH affect melamine-associated kidney stone formation risk. Urol Ann 3:71–74 Liu CC, Wu CF, Chen BH, Huang SP, Goggins W, Lee HH et al (2011) Low exposure to melamine increases the risk of urolithiasis in adults. Kidney Int 80:746–752 Li YY, Han HL, Lv RF, Xiao HZ, Yang SJ (2010) Simultaneous determination of melamine, cyanic acid and uric acid by HPLC. Modern Instruments and Medical Treatment (Chinese) 4: 30-32, Available at http://www.moderninstrs.org.cn/UploadFile/Article Page/20111415590100503.htm Zhang Z, Ou LL, Zeng GH, Yuan J, Li X, Wu KJ et al (2007) Simultaneous determination of oxalate and citrate acid in 24 hours urine by ion-chromatography. Chin J Urol (Chinese) 28:75–77 Chen WC, Wu SY, Liu HP, Chang CH, Chen HY, Tsai CH et al (2010) Identification of melamine/cyanuric acid-containing nephrolithiasis by infrared spectroscopy. J Clin Lab Anal 24:92–99 Cong X, Sun X, Ning B (2013) Could infrared spectroscopy identify melamine-related stone using melamine-contained mixture as a reference? J Clin Lab Anal 27:59–66 Zhang X, Chen T, Chen Q, Wang L, Wan LJ (2009) Selfassembly and aggregation of melamine and melamine-uric/ cyanuric acid investigated by STM and AFM on solid surfaces. Phys Chem Chem Phys 11:7708–7712 Ngo TC, Assimos DG (2007) Uric acid nephrolithiasis: recent progress and future directions. Rev Urol 9:17–27 Wu X, Wakamiya M, Vaishnav S, Geske R, Montgomery C, Jones P et al (1994) Hyperuricemia and urate nephropathy in urate oxidase-deficient mice. Proc Natl Acad Sci USA 91:742–746 Tannehill-Gregg SH, Dominick MA, Reisinger AJ, Moehlenkamp JD, Waites CR, Stock DA et al (2009) Strain-related differences in urine composition of male rats of potential relevance to urolithiasis. Toxicol Pathol 37:293–305 Arnold LL, Cano M, St John M, St John M, van Gemert M, Cohen SM et al (1999) Effects of dietary dimethylarsinic acid on the urine and urothelium of rats. Carcinogenesis 20:2171–2179 Wu CF, Liu CC, Chen BH, Huang SP, Lee HH, Chou YH et al (2010) Urinary melamine and adult urolithiasis in Taiwan. Clin Chim Acta 411:184–189 Cheng WC, Chen SK, Lin TJ, Wang IJ, Kao YM, Shih DY et al (2009) Determination of urine melamine by validated isotopic ultra-performance liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 23:1776–1782 Mast RW, Jeffcoat AR, Sadler BM, Kraska RC, Firedman MA et al (1983) Metabolism, disposition and excretion of [14C]melamine in male Fischer 344 rats. Food Chem Toxicol 21:807–810 Liu G, Li S, Jia J, Yu C, He J, Zhu J (2010) Pharmacokinetic study of melamine in rhesus monkey after a single oral administration of a tolerable daily intake dose. Regul Toxicol Pharmacol 56:193–196 Xie G, Zheng X, Qi X, Cao YM, Chi Y, Su M et al (2010) Metabonomic evaluation of melamine-induced acute renal toxicity in rats. J Proteome Res 9:125–133 Chen B, Liu X, Li S, Zhou Y, Jiang Q (2009) Melamine exposure assessment in children with nephrolithiasis. Pediatr Nephrol 24:2065–2067 Li G, Jiao S, Yin X, Deng Y, Pang X, Wang Y (2010) The risk of melamine-induced nephrolithiasis in young children starts at a lower intake level than recommended by the WHO. Pediatr Nephrol 25:135–141
ORIGINAL PAPER
The true stone composition and abnormality of urinary metabolic lithogenic factors of rats fed diets containing melamine Xiaoming Cong • Xiaojian Gu • Yan Xu Xizhao Sun • Luming Shen
•
Received: 5 June 2013 / Accepted: 6 November 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract To better understand the toxicity of melamine to humans, the stone composition and urinary metabolic lithogenic factors of rats fed diets containing melamine including the infant’s melamine-induced stone composition were studied. Sixty 4-week-old male rats divided into three groups were, respectively, fed diets containing no melamine (control), 0.1 % melamine, and 1 % melamine for 4 weeks. At the end of experiment, the collected stones and 24-h urines from rats were, respectively, measured with compositions and metabolic lithogenic parameters. The stone from an infant who ingested melamine-adulterated formula was also included in compositional analysis. Across three groups, the stone was only detected in 1 % melamine group, with composition of almost melamine different from the affected infant’s stone composed of melamine and uric acid with a ratio of 1:2. Compared with control group, urine calcium and phosphate excretions were significantly increased in 1 % melamine group. Urine uric acid excretion was significantly increased but citrate excretion was significantly decreased in 0.1 % and 1 % melamine groups. Urine oxalate excretion and pH were indicated without any significant difference. In addition based on urine physicochemical characters, melamine–uric
X. Cong and X. Gu contributed equally to this work. X. Cong X. Gu Y. Xu Department of Urology, Jiangsu Province Hospital of TCM, Affiliated Hospital of Nanjing TCM University, Nanjing 210029, China X. Cong X. Sun (&) L. Shen Department of Urology, Affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, China e-mail: [email protected]
acid stone seems difficult to be formed in the rats due to their characters of urine high-pH and low-uric acid. These results demonstrated that (1) the stone composition of rats fed melamine was not and could not be as that of infants fed melamine-adulterated formula, two species had a different mechanism of melamine-induced stone formation; (2) the exposure of melamine could result in abnormalities of urine metabolic lithogenic factors to rats, perhaps as well as human beings. Keywords Melamine Rat Infant Stone Urinary metabolic lithogenic factors
Introduction In the summer of 2008, an outbreak of kidney stones and renal failure in infants caused by the ingestion of melamine-adulterated formula attracted a public concern to the toxicity of melamine for humans [1]. However, thus far the exact mechanism of stone formation in the affected infants remains unknown. In 2004 and 2007, two outbreaks of animal renal disease in some Southeast Asian countries and North America had been caused by both of melamine and cyanuric acid which formed crystals of melamine cyanurate in renal tubules and collecting ducts [2]. Unlike animal outbreaks, the Chinese melamine-adulterated formula contained only trace amount of cyanuric acid [2], and stones from the affected infants were composed of uric acid and melamine with a molar ratio ranged from 1.3:1 to 2.1:1 [3, 4]. In the studies of rats fed only melamine, Ogasawara et al. [5] found the formed stones with an equimolar ratio of melamine and uric acid, which was analogous to those of affected infants. However, Heck et al. [6] and recently Shen et al. [7] found the stone of rats fed melamine was
123
Urolithiasis
composed primarily of melamine. It seems a re-analysis of stone composition of the rat fed melamine is necessary for us understanding how the human’s melamine-related stone was formed. A lot of studies showed that greater and longer exposure of melamine increased the formation of kidney stones in the melamine-adulterated formula event [8–10]. However, there were other factors that indicate the increase of melamine-related stone formation. Infants with preterm infancy and acid urine were demonstrated to more likely to suffer from melamine-related stones [9, 11]. Lam et al. [10] found that more than half of infants with melamine-related stones had predisposing urinary metabolic factors. Recently, Liu et al. [12] further reported a strong association between the exposure of low-dose melamine and calcium kidney stones in Taiwanese adults. In their study, patients with stones were detected with greater urinary melamine, as well as higher urinary calcium and uric acid excretion. These suggest that abnormalities of urinary metabolic lithogenic factors should be involved in the formation of melamine-related stones. However, whether abnormalities of these urinary metabolic lithogenic factors are caused by melamine exposure is unclear now. In this study, we will use a rat model fed melamine to analyze the stone composition and urinary metabolic lithogenic factors to help understand the toxicity of melamine to human beings.
successive 24-h urines were collected with metabolic cages at the end of 4th week. Urine volume and pH were measured instantly. Urine sample was frozen at -20 °C for a biochemical analysis. At the end of 4th week rats were killed, the stones were collected and examined. Analysis of stone composition To compare the differences of melamine-related stone composition between rat and human, the stone received from a 10-month-old female infant who ingested Sanlu melamine-adulterated formula since birth in June 2008 was included. Stone compositions of the infant and rat were analyzed by the Fourier transform infrared spectroscopy (FTIR) and high-performance liquid chromatography (HPLC). In the analysis by FTIR, stone was firstly fragmented into powders, then compressed into a transparent wafer with potassium bromide (Sigma-Aldrich, MO, USA), at last examined by FTIR (Lambda Scientific Instrument Company, Tianjing, China). In the analysis by HPLC with a method of simultaneous determination of melamine, cyanuric acid, and uric acid [13], briefly, the dissolved stone sample was measured by HPLC system consisted of the Agilent 1,100 system and Agilent HC-C18 column (4.6 9 250 mm, 5 lm) (Agilent Technologies, CA, USA), with the eluted solution (10 mmol/l K2HPO4 ? 10 mmol/l KH2PO4, pH 7.50), flow rate of 0.8 ml/l, and detected wavelength of 254 nm. Measurement of blood and urine
Materials and methods Experimental animals Four-week-old male Sprague–Dawley rats were obtained from Animal facilities of Slac Laboratory Animal (Shanghai, China). Five animals each were housed in an individual plastic cage before the experiment under the temperature of 22 °C and a 12-h light/dark cycle condition. They were fed a basal pellet diet and tap water except as specifically noted below. All of experimental procedures were performed in accordance with NIH Guide for the Care and Use of Laboratory Animals. Experimental procedure Sixty rats were divided into three groups, and groups 1–3 were fed diets, respectively, containing 0, 0.1 and 1 % melamine (Sinopharm Chemical, Shanghai, China) for 4 weeks. Melamine diets were prepared by mixing powdered basal diet with melamine and then compressing them into pellets. Body weights, food consumption and water intake were recorded at the end of 4th week. Two
123
Blood biochemistry and urine calcium, phosphate, creatinine (Cr), and urine nitrogen (BUN) were conducted by a Hitachi 7600 biochemistry analyzer (Hitachi, Tokyo, Japan). Urine citrate and oxalate were measured by the Ion chromatography (IC) using a method [14]. The IC system consisted of the 792 Basic IC equipment, Metrosep ASUPP5-100 column (4 9 100 mm, 5 lm), and Metrosep ASUPP4/5 Guard (4 9 5 mm, 5 lm) (Metrohm, Herisau, Switzerland). All samples were eluted with a solution (17.5 mmol/l Na2CO3 ? 3.5 mmol/l NaHCO3) at a flow rate of 0.7 ml/l. The method of HPLC analyzing stone compositions was also used to measure urine melamine, uric acid, and cyanuric acid. Urine pH was measured by a PHS-25 type pH meter (INESA Scientific Instrument Company, Shanghai, China). Statistical analysis Statistical analysis was performed using SPSS 18.0. The Student’s t test was used to do comparisons between experimental groups. Differences were considered significant at P \ 0.05.
Urolithiasis Table 1 Body weight, food intake, water intake, and stone incidence Group
No.
Body weight (g)
Food intake (g/rat/ day)
Water intake (ml/rat/ day)
Control
20
446.12 ± 33.56
41.6
36.7
0
0.1 % melamine
20
444.5 ± 23.12
37.7
44.6
0
1% melamine
20
411.2 ± 42.16a
35.5
54.2
45
a
Stone incidence (%)
Significantly different from the control group value at P \ 0.05
Results During the total experimental period, there was no died rat in three groups. Compared with control group, body weights in 1 % melamine group were significantly lower (P \ 0.05). Across three groups, as the rats consumed higher melamine-contained diets, their water intakes were increased, but food intakes were on the contrary. Only nine rats (45 %) in 1 % melamine group suffered stones with faint yellow color and amorphous shape in the urinary
tract. There was no rat with detected stone in other two groups. The explicit data was shown in Table 1. The stones in nine rats were analyzed by FTIR and HPLC. By the FTIR, as the melamine standard shown in the data of Chen et al. [15], nine rats’ stones shared the same characteristic wave-number positions at 3,470, 3,420, 3,335, 3,215, 1653, 1,555, 1,470, 1,440, 1,030, and 815 cm-1 (Fig. 1a). By the HPLC, as shown in Fig. 1b, the stone was eluted as a distinct peak as melamine standard. The melamine contents in the nine rats’ stones were 98.6–99.5 %. Out of nine rats’ stones, three stones were detected of a trace of uric acid with contents 0.06–0.13 %, and no stone was detected with cyanuric acid. Figure 2 showed the melamine-related stone from the affected infant. This stone had the same characteristic wave-number position (Fig. 2a) with melamine–uric acid precipitate in our previous data [16]. In addition, as shown in Fig. 2b, this stone was eluted as two distinct peaks in agreement with these of uric acid and melamine standards. The content of melamine and uric acid in this stone was 23.6 and 63.7 % with a molar ratio of 1:2.0. The data of blood and urine parameters were shown in Table 2. There was no significant difference in blood BUN
Fig. 1 The analysis of a stone from rats fed diets containing 1 % melamine by FTIR (a), and HPLC (b). a, b, respectively, showed characteristic wave numbers and a distinct peak with retention time as the same as those of standard melamine
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Fig. 2 The analysis of stone from the infant with an ingestion of melamine-adulterated formula by FTIR (a) and HPLC (c). a Showed characteristic wave numbers as same as these of melamine–uric acid
precipitate (b), c showed two distinct peaks with retention times as the same as those of standard uric acid and melamine
and Cr as a functional index across three groups. Compared with control group, the urine volume of rats in 1 % melamine group was slightly increased but with a statistical significance (P \ 0.05). There was no significant value difference of urine pH across three groups. It is noted that urine pH of any rat in three groups was alkaline (7.0–8.0).
In 24-h urinary metabolic lithogenic factors (Table 2), compared with control group, uric acid excretion was significantly increased and citrate excretion was significantly decreased in 0.1 % melamine group (P \ 0.05) and 1 % melamine group (P \ 0.001). Calcium and phosphate excretions were increased only in 1 % melamine group
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Urolithiasis Table 2 Renal functional, urinary metabolic factors, melamine, and cyanuric acid Group Serum BUN Serum Cr Urine volume (ml) Urine pH Urine calcium (mg/24 h) Urine phosphate (mg/24 h)
Control 8.2 ± 0.94
7.9 ± 1.12
8.3 ± 0.78
18.3 ± 2.82
24.7 ± 3.58
32.1 ± 9.30 7.33 ± 0.20
34.5 ± 8.11 7.53 ± 0.35
39.6 ± 9.20a 7.49 ± 0.35
2.0 ± 0.29
2.05 ± 0.26
2.39 ± 0.30a
19.49 ± 1.48
20.12 ± 1.79
20.85 ± 2.34a
0.86 ± 0.21
Urine oxalate (mg/24 h)
44.3 ± 6.02
Urine uric acid (mg/24 h)
2.46 ± 0.30
Urine cyanuric acid (lg/24 h)
1% melamine
18.61 ± 2.30
Urine citrate (mg/24 h)
Urine melamine (mg/24 h)
0.1 % melamine
none 8.13 ± 2.32
0.68 ± 0.29a 41.85 ± 5.94 2.69 ± 0.34a
0.43 ± 0.22b 45.6 ± 7.10 3.17 ± 0.27b
4.47 ± 0.50
36.25 ± 2.56
7.51 ± 1.72
7.92 ± 2.08
a
Significantly different from the control group value at P \ 0.05
b
Significantly different from the control group value at P \ 0.001
(P \ 0.05). There was no significant difference of oxalate excretion across three groups. Urine melamine excretion in 1 % melamine group was approximate eightfold than that in 0.1 % melamine group. Interestingly, there was the micro-dose of urine cyanuric acid detected in three groups but without a significant value difference.
Discussion By the stone compositional analysis, we showed the infant’s melamine-related stone was composed of uric acid and melamine with a molar ratio of 2.0:1, in agreement with the result of Sun et al. [3]. Zhang et al. [17] demonstrated melamine could be self-assembled and aggregated with uric acid to form lager clusters in water through hydrogen bonds. The predicted crystal structure was showed in the study of Dalal et al. [2]. With our previous study [16], we found the infrared spectrum of this infant’s stone was identical to that of melamine–uric acid precipitate not melamine–uric acid powders. It further demonstrated the infant’s melamine-related stone should be formed by melamine binding with uric acid through hydrogen bonds in urine to precipitate. In contrast, the formed stone in our rat model was composed of melamine and a trace of uric acid. This result is analogous to results of Hack et al. [6] and Shen et al. [7], but on the contrary with that of Ogasawara et al. [5]. To our knowledge, the
dissociation constant (pKa) of uric acid is 5.5. At a pH equal to the pKa, free uric acid and urate exist in equal proportions. At a pH of 6.5, 90 % uric acid would become urate and the remainder would continue to be free uric acid [18]. Unlike affected infants with the urine pH at 5.0–6.5 [10], the urine pH of Sprague–Dawley rats in our study was about 7.0–8.0. It means almost urine uric acid of our rats was urate ( 90 %), subsequently little free uric acid could complex with melamine free base through hydrogen bonds. Besides, urine uric acid level of rats should be significantly less than that of human because they had uricase which could convert uric acid to allantoin [19]. Therefore, we think it is very difficult to form melamine–uric acid stone for Sprague–Dawley rats, as well as for other strains of rats such as F344 and Wistar due to their alkaline urinary pH generally above 6.5 [20, 21]. After the event of melamine-adulterated formula, only four studies determined urine melamine level [10, 12, 22, 23], and three of them found the link between urine melamine level and urolithiasis risk [10, 12, 22]. To our knowledge, there is no study to investigate the effects of melamine on the urine metabolic lithogenic factors. Based on the results of urine biochemical analysis, we firstly showed the exposure of high-dose melamine could increase urine calcium, uric acid, phosphate excretions, decrease urine citrate excretion, and did not affect urine pH. It is usually thought melamine is primarily eliminated by urine excretion without metabolism [24, 25]. However, Xie et al. [26] found high-dose melamine could disrupt amino acid metabolism, and alter TCA and gut microflora structure. Perhaps the abnormalities of these urine factors showed in our study are related to the findings of Xie et al. As the study of Liu et al. [14], cyanuric acid was detected in urine of rat fed diets with or without melamine in our study. The uncorrelation between urine cyanuric acid and melamine excretions further suggests cyanuric acid should not be derived from melamine and involved in melamine-induced stone formation. In this study, we showed stone composition of rat fed melamine was not and could not be as that of infants fed melamine-adulterated formula. When using animal for risk assessment of melamine-induced stone formation, it is necessary to consider the difference of endogenous factors such as urine uric acid and pH. Since the exposure of melamine could cause abnormalities of urine metabolic lithogenic factors of rats, the melamine exposure may also cause it for human beings. Although the WHO and FDA lowered the melamine level of their recommended tolerable daily intake by tenfold [2], some studies indicated even low level of melamine exposure may risk urolithiasis formation both in infants and adults [12, 27, 28]. Considering melamine is a commonly used chemical in our daily life, and rat is usually used as a model to evaluate the toxicity of
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melamine on humans, we believe the present study is easy to understand the mechanism of infant’s melamine-induced urolithiasis, the limit of rat model used in the evaluation of toxicity of melamine to humans, and other possible toxicity of melamine to human beings.
11.
12.
13.
Conclusion The presented study demonstrated (1) the stone composition of rats fed melamine was not and could not be as that of infants fed melamine-adulterated formula, two species had a different mechanism of melamine-induced stone formation; (2) the exposure of melamine could result in abnormalities of urine metabolic lithogenic factors to rats, perhaps as well as human beings.
14.
15.
16. Acknowledgments Supported by the National Natural Science Foundation of China (grant 81070558) and Natural Science Foundation of Jiangsu Province (grant BK2010597). Conflict of interest
17.
No any conflict interest with others. 18. 19.
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