Melamine Deposits in Pet Food Preliminary Report

Eighty four commercially available pet food samples including cat food (38 solid, 17 semisolid) and dog food (20 solid, 9 semi-solid) were examined for melamine deposits by competitive enzyme linked Immunosorbant assay. Among solid cat food, 78.94% (n=30) were contaminated with 14.431.27 (range greater than 4-68 mg/kg) of melamine, while 82.35% samples of semi solid cat food (n=14) were found positive for melamine deposit with mean value 6.43 1.96 (range greater than 2- 13.14milligrams/kg). Likewise, 75% samples of solid dog food (n=15) were positive for melamine deposits with mean value 8.501.73 mg/kg (range greater than 4-49mg/kg). A total of 44.44% samples of semi solid dog food were positive for melamine with mean value 16.311.64 range of greater than 2-34.25milligrams/kg. From all positive samples, 53% samples of pet food were contaminated past the Codex Alimentarius Commission (i.e. 2.5mg/kg). Analytic data revealed existence of melamine deposits in imported pet food.

During passing few decades as a result of commercialization and rapid growth of creatures business, animal feed security problem has acquired great significance. Among creatures business, pet food business is booming exponentially in western world especially for their companionship with human beings. Pets are important not only for his or her loyal or playful characteristics but in addition look to offer their owners with major health benefit (Durrani et al., 2012). Upon wastes were generally raised pets, before /staying foods. Afterwards, for nutritionally balanced food condition, proper pet food business now broad selection of brands are offered in marketplace in accordance with age and species of pet and came into existence and being exported around the planet. Pet food is generally prepared through the use of different fixings i.e. cereals (corn, wheat gluten, corn gluten and rice protein) and meat portion. Sometime to elevate the obvious protein content, pet food's ingredients unique ly grains protein including corn gluten and wheat gluten is adulterated with cheaper non protein nitrogen sources i.e. urea, cyanuric acid, ammelide, ammeline and melamine and its analogues (Ingelfinge,2008;Lakhshmi, 2012).

Melamine or 1,3,5-triazine-2,4,6-triamine is an organic base with chemical formulae (C3H6N6). It's widely found in plastic (Skinner et al., 2010) and fertilizer sector due to its high nitrogen content (Gonzalez et al., 2009). It's shown that melamine includes 66 percent of nitrogen by mass. (Hau et al., 2009). Melamine was regarded as nontoxic because of the reason it is water soluble and economically (upto 90%) eliminated by kidneys within 24 h (Thompson et al., 2008). The possible toxicity of melamine was taken into account seriously after successive outbreaks of pet's renal failure and related deaths in Taiwan and United States during years 2004 and 2007 (Burns, 2007). The recalls in North Europe, America and South Africa came in response. The evaluation of suspected food and pathological studies of kidneys of dead creatures, it had been identified that melamine is inherent cause of these out breaks (Chen et al., 2009; Osborne et al., 2009; Nilubol et al., 2009; Park et al., 2011). Melamine in combination with cyanuric acid becomes more hazardous as compared to either melamine or cyanuric acid alone (Thompson et al., 2008; Yhee et al., 2009). It may precipitate in distal renal tubules and collecting ducts of kidney and chronic interstitial nephritis in animals and human beings (Dobson et al., 2008; Bhalla et al., 2009). From Europe, pet food is generally imported in Pakistan. As an emerging problem (melamine outbreaks), data viewing melamine deposits in pet food is missing link in Pakistan. In view of foregoing, a preliminary study was conducted to measure the deposit melamine in imported pet food advertised in Pakistan.

Substances and procedures

Eighty four commercially available pet food samples including cat food (38 solid, 17 semi- solids) and dog food (20 solid, 9 semisolid) were gathered from departmental shops of local marketplace within an interval of June to August 2011. The source of pet food samples were from UK, EU and Korea. Samples were kept at 4C till additional investigation. Direct competitive ELISA kit (AgraQuant(R) Romer, Singapore) was used for the determination of melamine deposits. For evaluation, 2g of homogenized pet food samples were combined with methanol: water (1:9; v/v) and vortexed vigorously for 2 min. The sample extracts were subsequently filtered by using 0.45m syringe filter (MiniSart(R) Germany). Filtrate was diluted with diluent solution. Dilution factor for solid and semi solid food were 100x and 200 x as per directions given by ELISA kits manufacturing companies. Optical density (OD) values were recorded by using BioTek(R) ELISA Reader Elx808 (BioTek(R), USA).The OD data was computed by using BioTek(R) Gen5 applications (BioTek(R), USA) for melamine quantification. Limit of detections for semi- dry and solid pet foods were 2-50mg/kg and 4- 100mg/kg respectively. Through the use of linear regression equation of anticipated versus observed melamine calibration curve was drawn. Restorations for spiked pet food samples i.e. semisolid and solid ranging from 0 to 3000ppb, showed good linearity i.e., R2 0.993 (solid) and 0.996 (semisolid) respectively (Fig. 1). For descriptive statistics, using SPSS16 Software examined data.

Results and discussion

A total of 75% pet food samples (n=63) were found positive for melamine ranging from greater than 2- 68mg/kg. Comprehensive results of all accumulated samples including cat and dog food samples are summarized in Table I. Data was farther computed for frequency distribution of melamine in dogs and cat foods. Maximum percent of positive samples were discovered in the number of greater than 5-10mg/kg and greater than 20- 40mg/kg respectively Melamine will not show organized toxicity independently, but capable to complex with other materials for example endogenous uric acid or cyanuric acid to form crystal in the tubules which cause

Table I. Melamine contaminants (MeanSE) in accumulated pet food samples.

Kidney damage (Puschner et al., 2007; Park et al., 2011). Pathological studies of kidney of the impacted/ expired dogs and cats identified crystals of calcium oxalate and ammonium urate in the distal tubules and yellowish discoloration of interior element of medulla with deposit of yellow green crystals in pelvic tubes (Skinner et al., 2010; Cocchi et al., 2010).In fact, concurrent administration of melamine and cyanuric acids results in severe toxicity due to the formation of insoluble merchandise i.e. melamine cyanourate at low pH i.e. 5.8. (Cianciolo et al., 2008; Bhalla et al., 2009). The general range of pee pH of cats and dogs is 5.5-7.0 depending upon the nature of diet. It is almost always more inclined towards values that are acidic. Thus, low urinary pH is a leading risk factor for crystals formation in kidneys (Cocchi et al., 2010). Maximum degree (68mg/kg) found in current study demonstrated a steady dietary exposure to melamine could cause rocks formation and raises the prevalence of urinary bladder in pets especially male cats (Park et al., 2011; Puschner et al., 2007). In current study, all samples were of imported brands as well as the countries of origin were assumed to be leaders for declaring strict legislations (i.e. EU and UK for melamine in babies and pet foods).Yet, these remaining amounts might be due to plastic packing materials leaching. Ludn and Petersen (2006) reported that constant melamine migration may happen through the duration of the merchandise if it is subjected to hot acidic foods. Additionally, pH of present accumulated solid pet food samples were 5.8- 6.4 whereas, pH of semisolid was 5.95- 6.8. Samples set during warm climate (i.e. June to August, 2011) might be the reason for the melamine existence in pet food samples. During these month temperature ranged between 44 to 48C of Pakistan. Thus, melamine may be leached out from plastic packings to pet food as a result of improper storage, transfer and direct exposure to heat/ sun (Bradely et al., 2005; Luden and Petersen, 2006).

Data found much over the legislative limits and of current study revealed the existence of melamine deposits. Findings of current study might be used as preliminary info by keeping in view of other melamine associated chemical compounds especially cyanuric acid to plan biological trials.