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Melamine Toxicity and the Kidney

Anthony Kai-ching Hau,*† Tze Hoi Kwan,† and Philip Kam-tao Li*

*Department of Medicine and Therapeutics, Prince of Wales Hospital, Chinese University of Hong Kong, and †Department of Medicine, Tuen Mun Hospital, Hong Kong

ABSTRACT The toxicity of caught the attention of physicians as a result of a recent gen in all organic nitrogenous com- spate of renal injury after exposure to melamine-tainted milk in China. Melamine is pounds, including melamine. In essence, an organic nitrogenous compound used in the production of plastics, dyes, fertil- nitrogen in the assay reacts with sulfuric izers, and fabrics. In the current incident, melamine was added to milk to elevate acid to form ammonium . Small falsely assay results for protein content. A variety of toxic effects from melamine, amounts of sodium hydroxide are then including nephrolithiasis, chronic kidney inflammation, and bladder carcinoma, all added, and the ammonium is con- have been studied in animals. We review here the epidemiology, clinical features, verted back to . Ammonia is re- and investigative findings concerning the only outbreak of melamine poisoning in acted with sulfuric acid again, and the re- humans. We also examine the renal toxicities of melamine and —a maining acid is quantified by adding by-product of its synthesis—and the associated risk factors on exposure and sodium carbonate with a methyl orange provide guidance on levels in foods. pH indicator to quantify the amount of ammonium salt (Figure 2). J Am Soc Nephrol 20: 245–250, 2009. doi: 10.1681/ASN.2008101065 The addition of 1 g of melamine to 1 L of milk falsely increases the protein con- tent by 0.4%. When melamine is dis- Melamine was an unknown substance to melamine foam, a polymeric cleanser. solved at the room temperature, 3.1 g of nephrologists until today. Although Other commercial products containing melamine can be dissolved in water with- studied as diuretics 50 yr ago,1 it never melamine include countertops, dry erase out forming precipitate, and protein came into clinical practice with the de- boards, fabrics, glues, housewares, and content will falsely increase by 1.2%. velopment of other potent and safer flame retardants. Melamine is also one of This can roughly lead to an overestima- means for naturesis. This compound be- the major components in pigment yellow tion of the protein content in liquid milk came headline news recently after the oc- 150, which is a colorant for inks and plas- by 30%. In case of milk powder, the currence of an outbreak of urinary stones tics. It is also a derivative of arsenical drugs, amount of melamine added can be in infants and children consuming mel- and Melarsoprol is one such drug used for greater because of its greater at amine-tainted milk in China. The neph- the treatment of African trypanosomiasis. higher temperature when adding warm rotoxic effects of melamine now warrant Beginning in 1958, melamine has been water. the attention of nephrologists, pediatri- used in fertilizers and is occasionally of- cians, urologists, and radiologists. fered as a nonprotein nitrogenous source for feeding cattle. Subsequently, however, METABOLISM IN ANIMALS it was shown to be an ineffective nonpro- WHAT IS MELAMINE? tein nitrogen source for animals because of Melamine is not metabolized by animals its slow hydrolysis in ruminants (Table 1). and is rapidly eliminated in the urine. Melamine is an organic base commercially More than 90% of ingested melamine is synthesized from with an intermedi- ate step producing cyanic acid (Table 1). WHY DID MELAMINE APPEAR IN Published online ahead of print. Publication date The reaction also results in the formation MILK? available at www.jasn.org. of other byproducts, including cyanuric Correspondence: Prof. Philip Kam-tao Li, Depart- acid, ammeline, and (Figure 1). Melamine was recently added to milk to ment of Medicine and Therapeutics, Prince of Wales Hospital, Chinese University of Hong Kong, Hong Melamine is 66% nitrogen by molecular elevate falsely its protein content by the Kong. Phone: 852-2632-3616; Fax: 852-2648-9864; weight. It is combined with formaldehyde Kjeldahl method (Figure 2). The first E-mail: [email protected] by industry to produce melamine resin, a step of the Kjeldahl method detects not Copyright ᮊ 2009 by the American Society of very durable thermosetting plastic, and only nitrogen in protein but also nitro- Nephrology

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Toxicity can be classified as acute or tion of urinary changes relating to NH2 chronic. The most common toxicity is chronic exposure to melamine dates renal toxicity, which is also the area of back to 1953, when Hazleton Laborato- most concern to nephrologists. ries performed an experiment in dogs by feeding them 3% (30,000 ppm) mel- 8 N N Acute Toxicity in Animals amine by weight in food for 1 yr. Dis- Melamine has a low acute toxicity; the tinctive urinary changes were noted, in-

LD50, the lethal dose of a compound that cluding reduction in specific gravity, would result in death in 50% of the tested increases in urine output, melamine animals, for melamine in rats is 3.161 crystalluria, and proteinuria with micro- H2NNNH2 g/kg body wt.4 Acute dermal toxicity in scopic hematuria. The most commonly rabbits presents when the exposure is Ͼ1 reported chronic renal toxicity is stone g/kg body wt.5 formation. Uncertainty exists as to Direct contact results in skin irrita- whether melamine results in any chronic Figure 1. Chemical structure of melamine: tion and eye irritation, and inhalation damage to kidney other than aggressive There are six atoms of nitrogen in a molecule causes respiratory tract irritation. Oral in- stone formation. of melamine, constituting 66% of molecular gestion affects the digestive tract, present- weight. International Union of Pure and Ap- ing as nausea, vomiting, and diarrhea.6 Urinary Stone Formation plied Chemistry name: 1,3,5-triazine-2,4,6- Acute renal toxicity is best illustrated Most animal studies concerning sub- triamine; other names: cyanurotriamide, cya- by a sheep study done in 1953.7 When acute or chronic melamine exposure re- nurotriamine, or cyanuramide. sheep were fed a single 100-g oral dose of veal stone formation. Incidence ranges melamine, all of them died by the 11th from 5 to 100% depending on the dosage excreted within 24 h. The half-life of mel- day. When a daily dose of 25 to 50 g of of melamine, gender, and amount of wa- amine excretion in animal studies ranged melamine was given for 7 to 9 d, again all ter intake.4,9 The composition of stones from 2.7 to 4.04 h.2,3 The levels of mel- of the sheep died. They experienced is either a combination of melamine and amine in blood, liver, or plasma are sim- acute renal failure with elevation in uric acid or melamine in a matrix of pro- ilar.2 The volume of distribution of mel- blood urea nitrogen and creatinine fol- tein, uric acid, and phosphate.10 amine in pigs is 0.61 Ϯ 0.04 L/kg and is lowed by oliguria preceding death. Post- The incidence of stone formation in- not extensively distributed to most organ mortem examinations revealed crystals creases with daily exposure to melamine. tissues.3 This study in pigs also confirms in the kidney tubules, nephrosis, and The lowest possible daily dosage of mel- the excretion of melamine best fits a one- hemorrhagic cystitis. When the exposure amine that results in bladder stone for- compartment model in which melamine was reduced to 10 g/d melamine for 16 to mation is as low as 750 ppm for 13 wk.4 has a half-life of 4.04 Ϯ 0.37 h and a renal 31 d, two thirds of the sheep died. Again, The dose-response curve for the induc- clearance of 0.11 Ϯ 0.01 L/h per kg (ap- they experienced loss of appetite, oliguria, tion of urolithiasis in weanling rats is ex- proximately 27 ml/min).3 and elevation of blood urea nitrogen and tremely steep (Figure 3). This suggests creatinine before their death. Postmortem formation of calculi occurs in supersatu- analyses under these conditions also re- rated urine but not in urine that is under- TOXICITY OF MELAMINE vealed crystal deposition in the kidney. saturated.11 A study performed on B6C3F1 mice with exposure to 13 wk of Studies concerning the toxicity of mel- Chronic Toxicity in Animals melamine showed that male mice are amine taken orally in humans are nonex- Long-term exposure to melamine re- much more affected than females despite istent. Toxicity data mainly come from duces fertility and results in fetal toxicity similar body weights. Relative risk of studies in sheep, cat, dog, mice, and rat. in animal studies. The classical descrip- stone formation in males is twice as great as in females.4 Table 1. Summary of the chemical properties of Melamine2 A study of exposure to melamine for Property Description 36 wk in rats demonstrated the incidence of stone formation was reduced by in- Appearance at room temperature Whitish crystalline solid 9 Solubility Partially soluble in water creasing the amount of fluid intake. In 3.1 g/L in water at 20ЊC this study, one group of rats, the control 13.4 g/L in water at 50ЊC group, was fed diets containing 1% mel- 25 g/L in water at the boiling point amine. The other two groups of rats re- Insoluble in diethyl ether ceived feed containing 1% melamine to- Melting point 357ЊC gether with 5 or 10% of sodium chloride, Specific gravity 1.573 g/cm3 respectively. The increase in salt content Molecular weight 126.12 g/mol in the diet resulted in doubling and tri-

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croscopic hematuria with or without Protein + H2SO4 CO2 + (NH4)2SO4 + SO2 proteinuria. Melamine can be detected in the urine of the affected children by (NH4)2SO4 ++2NaOH Na2SO4 NH4OH biochemical test or the presence of fan- shaped crystals. Analyses of stone composition mainly 2NH4OH +H2SO4 (NH4)2SO4 + 2H2O demonstrated melamine and uric acid.15,16 Figure 2. Kjeldahl method, a universally used quantitative method for assessing protein Stones were radiolucent, and plain x-ray content in food. films failed to show their presence. Ultra- sonography or computed tomography scan can reveal the presence of stones CHANCE OF BLADDER STONE FORMATION and detect whether there is any obstruc- IN MALE F-344 RATS AFTER 13 WEEKS tion. In view of the large radiation dose of OF EXPOSURE TO MELAMINE computed tomography scans for chil- 1.0 dren, ultrasonography is the preferred first-line investigation. 0.8 Melamine stones in humans are char- acteristic. They usually occur bilaterally, 0.6 and multiple stones are often present.15 The acoustic shadow of stone can be ab- 0.4 sent. They are soft in nature and can be broken up easily. Most of the stones are Ͻ 0.2 usually 1 cm in diameter. Stones that are Ͻ4 mm in diameter can spontane-

Probability of stone formation ously pass to the bladder with adequate 0 0 5,000 10,000 15,000 20,000 hydration. When stones are Ͼ4mmor Exposure to melamine (ppm) show evidence of obstruction, medical treatment with hydration and follow-up Figure 3. Chance of bladder stone formation in male F-344 rats after 13 wk of exposure ultrasonography is the first-line attempt to melamine. Figure drawn using data from Melnick et al.4 at treatment. If conservative medical therapy fails, surgical drainage and re- pling the amount of fluid intake in an exposure to the tainted milk for ap- moval will be needed. Acute renal failure groups taking 5 and 10% sodium chlo- proximately 3 to 6 mo before the onset occurred in 2.5% of the cases.16 Mortality ride when compared with control. Inci- of stones. Later, the contamination was was recorded in four cases.17 dence of stone formation was reduced found in other brands of dairy product. from 37% in control group to 11% in the The highest content of melamine was group taking 5% sodium chloride and to in Sanlu milk powder, up to 2.563 g/kg RENAL PARENCHYMAL DAMAGE 5% in the group taking 10% sodium powder, whereas melamine in the chloride. other brands ranged from 0.090 to 619 Uncertainty exists as to whether mel- mg/kg.14 amine causes direct renal toxicity on The official data released by the Min- long-term exposure; however, in studies THE HUMAN OUTBREAK istry of Health of the People’s Republic of in which no chronic toxicity was ob- China on September 21, 2008, stated that served, the dosage of melamine was usu- The toxicity of melamine came to our at- a total of 52,857 children had received ally lower.18 Two studies specifically in- tention in Hong Kong because of a recent treatment for melamine-tainted milk13; vestigated the toxicity of melamine by outbreak of urinary stones in children 99.2% of the children were younger than looking at postmortem sections of kid- who consumed melamine-tainted milk 3 yr, although more children who were neys in animals. The first study from in China. On September 11, 2008, Xin- older than 3 yr were reported afterward. Melnick et al.4 observed a significant in- hua News Agency in China reported an Some of the children were asymptomat- crease in chronic inflammation in the outbreak of urinary stones in children ic; however, most symptoms included ir- kidneys of female rats exposed to mel- younger than 3 yr.12,13 The outbreak was ritability, dysuria, difficulty in urination, amine for 103 wk compared with con- initially linked to consumption of mel- renal colic, hematuria, or stone pas- trols (82% in rats fed a diet containing amine-tainted milk powder from the sage.15 Hypertension, edema, or oliguria melamine at 9000 ppm versus 34% in Sanlu Group, a leading dairy producer in also occurred in more severe cases. Uri- those on diets containing melamine at China. All of the children affected had nalyses of exposed children revealed mi- 4500 ppm versus 8% in control; P Յ

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0.01). The observed chronic inflamma- A OH B tion in this study could not be attributed H H to stone formation, because no stones were detected in the urinary tract. The NNN male rats showed a slightly increased in- N N H H O cidence of chronic inflammation, but N N this was not statistically insignificant. HO N OH HH The second study from Ogasawara N N et al.9 demonstrated ischemic changes N in the renal cortex of rats (focal lesion HH of fibrosis, inflammatory cell infiltra- O N O tion, and renal tubular regeneration) after feeding on melamine for 36 wk. Ischemic changes occurred in 100% of H rats fed diets containing 3% melamine Figure 4. (A) Molecular structure of cyanuric acid, noted for its structural similarity with by food weight and occurred in only melamine. (B) Melamine can interact with the isomeric form of cyanuric acid to form 5% of rats when fed diets containing melamine cyanurate, explaining the increase in stone formation and toxicity. 1% melamine. spectively.20 No other adverse effects melamine and cyanuric acid is as high as Melamine Toxicity Combined with were noted. 74% in dogs and 61% in cats.19 Cyanuric Acid Melamine, however, can interact with A widely known epidemic of melamine the isomeric form of cyanuric acid to cre- poisoning is the infamous “pet food–in- ate crystals (Figure 4B). Melamine com- CARCINOGENICITY OF duced nephrotoxicity in North America” bined with cyanuric acid results in acute MELAMINE in 2007.19 In March 2007, numerous renal failure in cats within 48 h after in- cases of acute renal failure in dogs and gestion. The toxic dosage in their diet is There are no data concerning the carci- cats were associated with the ingestion of as low as 0.2% melamine and 0.2% cya- nogenicity of melamine in humans. The a variety of dog and cat pet foods. One of nuric acid in contrast to no evidence of carcinogenicity in animals was deter- the contaminants was melamine, which renal failure in groups taking either mel- mined from studies in rats and mice. Ex- was added for the same reason as in the amine or cyanuric acid alone up to 1% in posure produces urinary bladder and recent milk exposure in humans: To give their diet for 10 d.21 Urine analyses from ureteral transitional cell carcinomas in a falsely high protein content. In this pet affected cats revealed the presence of male rats but only urinary bladder hy- outbreak, not only was melamine amorphous or, in some cases, fan-shaped perplasia in male mice. Female rats or present, but also another toxic com- (Figure 5A) birefringent crystals. The mice did not have carcinoma, but transi- pound—cyanuric acid–was a contami- crystals were a combination of melamine tional cell papillomas were found in fe- nate, giving rise to a very high mortality and cyanuric acid.22 Cross-sections of male rats.4 The occurrence of urinary in these animals. the kidney demonstrated severe renal in- bladder tumors in male rats correlates Cyanuric acid (s-triazine-2,4,6-triol) terstitial edema and hemorrhage at the well with stone formation and exposure is structurally related to melamine (Fig- corticomedullary junction. Histopatho- to high dosages. A similar dosage-depen- ure 4A). It is used as a stabilizer in out- logic findings were limited to the kid- dent relationship was confirmed in an- door swimming pools and hot tubs to neys, and numerous crystals were found other study using male rats.9 The admin- minimize the decomposition of hypo- within the distal nephrons associated istration of sodium chloride to increase chlorous acid by light.20 How cyanuric with tubular epithelial necrosis and re- fluid intake and urinary output reduces acid got into the pet food is unknown. It generation (Figure 5B).22 In chronic the prevalence of stone and tumor occur- could have been added intentionally or cases, lymphoplasmacytic or granuloma- rence. remained as a contaminant during mel- tous tubulointerstitial inflammation and There is no evidence that melamine amine synthesis, because cyanuric acid is fibrosis were found and the associated undergoes biotransformation. Mutagen- a byproduct. crystals were larger.23,24 This toxicity was esis of melamine was not observed in Studies on the toxicity of cyanuric not limited to cats. Similar nephrotoxic- studies of exposure to Salmonella typhi- acid are limited, but it is likely to behave ity was observed when a combination of murium and Drosophila melano- as melamine as a result of its structural melamine and cyanuric acid was given to gaster.26,27 The urinary bladder tumors similarity. The subacute feeding of so- fish and pigs.25 The toxicity is size depen- seen in male rats exposed to high dosages dium cyanurate at 700 or 2200 mg/kg in dent, with cats affected more than dogs of melamine seem to be produced by a rats resulted in bladder calculi and some and small dogs affected more than large non–DNA-reactive mechanism involv- associated bladder epithelial changes, re- dogs.23,25 The mortality of combined ing epithelial hyperplasia secondary to

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amine. An additional safety factor was thus introduced to protect this most vul- nerable group; that is, children who are younger than 3 yr. Consequently, the lat- est TDI was further reduced to 0.32 mg/kg per d.31 Of course, in determining exposure, one has to take into account the average body weight of the target population and the amount of respective food eaten per day to calculate the toler- able exposure of melamine per person each day.

CONCLUSIONS

With the best available evidence in hu- man exposures and animal studies, we conclude several points regarding the toxicity of melamine: High-dosage melamine will result in urinary stones, crystalluria, and acute renal failure in both humans and animals; stone for- mation is likely enhanced by smaller body size, higher dosage of melamine, Figure 5. Histologic diagnosis of melamine-associated renal failure based on renal and smaller amounts of fluid intake; crystal characteristics. (A) Dilated distal cat tubule contains clusters of round green studies in animals show that males are melamine/cyanuric acid crystals with radiating spokes and concentric striations (arrow). Surrounding proximal tubules appear unaffected (hematoxylin and eosin; bar ϭ 45 ␮m). more affected than females; toxicity of (B) Dilated distal cat tubule contains fragmented or globular dense green melamine/ melamine is further aggravated by the cyanuric acid crystals (long arrows). Note attenuation of the lining epithelium with wide presence of other impurities associated separation of nuclei (short arrow) and mitotic figure (arrowhead) indicative of tubular with melamine synthesis, particularly epithelial necrosis and regeneration (hematoxylin and eosin; bar ϭ 45 ␮m). Reprinted cyanuric acid; tubular damage with ob- from Brown et al.,22 with permission from the American Association of Veterinary Labo- struction from crystals and chronic in- ratory Diagnosticians. flammation of kidney can occur; and toxicity may not be limited to stone the presence of melamine-containing (NOEL) of melamine in studies in ro- formation in animal studies if melamine bladder stones. These studies concluded dents is 63 mg/kg body wt per d. When is present in high dosages or in combina- that bladder tumors would not occur in this dosage is converted to a human tion with cyanuric acid. rodents unless exposed to dosages that safety limit, it is referred to as tolerable result in bladder stones. daily intake (TDI). TDI is usually defined Melamine has been classified as a as 1% of NOEL in view of the need for a DISCLOSURES group 3 carcinogenic risk by World tighter safety limit in humans; therefore, None. Health Organization,28 meaning that the TDI in humans may be equivalent to melamine is not classifiable as to its car- 0.63 mg/kg body wt per d, and this is why cinogenicity in humans, referring to the the recommendation from the Food and fact that the evidence of carcinogenicity Drug Administration gives a safety limit REFERENCES is inadequate in humans and inadequate of exposure to melamine and its struc- or limited in experimental animals. tural analogues to be Ͻ0.63 mg/kg per 1. Lipschitz WL, Hadidian Z: Amides, amines and related compounds as diuretics. J Phar- 29 d. The European Food Safety Author- macol Exp Ther 81: 84–94,1944 ity, however, recommends a daily expo- 2. Mast RW, Jeffcoat AR, Sadler BM, Kraska GUIDANCE ON LEVELS IN FOODS sure of melamine and its structural ana- RC, Friedman MA: Metabolism, disposition logues be Ͻ0.5 mg/kg per d.30 After and excretion of [14C]melamine in male Fi- Recommendations on the safety limits of reviewing the cases in China, children scher 344 rats. Food Chem Toxicol 21: 807– 810, 1983 melamine in food come from animal who are younger than 3 yr seem more 3. Baynes RE, Smith G, Mason SE, Barrett E, studies. The no observed effect level susceptible to the toxic effects of mel- Barlow BM, Riviere JE: Pharmacokinetics of

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