Plasma pharmacokinetics of melamine and a blend of melamine and cyanuric acid in rainbow trout (Oncorhynchus mykiss)

Regulatory Toxicology and Pharmacology 61 (2011) 93–97

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Plasma pharmacokinetics of melamine and a blend of melamine and cyanuric acid in rainbow trout (Oncorhynchus mykiss) Min Xue a,b,⇑, Yuchang Qin a, Jia Wang a, Jing Qiu c, Xiufeng Wu a, Yinhua Zheng a, Qiuyan Wang a a

National Aquafeed Safety Assessment Center, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China Key Laboratory of Feed Biotechnology of Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China c Institute of Quality Standard and Testing Technology for Agro-products. Chinese Academy of Agricultural Sciences, Beijing, China b

a r t i c l e

i n f o

Article history: Received 15 February 2011 Available online 24 June 2011 Keywords: Oncorhynchus mykiss Pharmacokinetics Melamine Cyanuric acid Plasma

a b s t r a c t The objective of the study was to obtain pharmacokinetic parameters for melamine and blend of melamine (MEL) and cyanuric acid (CYA) in rainbow trout (Oncorhynchus mykiss). The single target dosage of MEL (20 mg/kg bw) and the blend of MEL and CYA (5 and 1.67 mg/kg bw, respectively) were designed and plasma samples were collected at 30 min, 1, 4, 8, 12, 20, 24, 36, 48, 72, 144 and 240 h sequentially. An optimized method for simultaneous determination of MEL and CYA in plasma and animal tissues by LC– MS/MS was used. The data were shown to best fit a non-compartment model with first order processes of linear characters for melamine, with half-life (t1/2) of 32.2–32.9 h, clearance (Clz/F) of 35.9–36.6 ml/h/kg, and volume of distribution (Vss) of 1.67–1.74 l/kg. Withdrawal of CYA was much more rapid than that of MEL with higher Clz/F (783.56 ml/h/kg) and shorter t1/2 (7.92 h). Tmax of MEL20 and MEL5 were 12 and 20 h, respectively, which showed that Tmax of MEL5 was delayed when MEL and CYA were given together. The results are quite different from those in mammals and showed much slower elimination of MEL and CYA from rainbow trout body. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction In 2007, there was a large outbreak of renal failure in cats and dogs in USA associated with ingestion of pet food which contained melamine and cyanuric acid (Brown et al., 2007). Furthermore, an increased incidence of kidney stones and renal failure in infants was reported in China, to be associated with milk powder contaminated with melamine. Melamine (MEL, 1,3,5-triazine-2,4,6-triamino-s-triazine) is a common chemical used for various of applications, such as laminates, plastics, coatings, glues or adhesives and kitchenware. Cyanuric acid (CYA, s-triazine-2,4,6-triol) is structurally related to melamine and used as a stabilizer in outdoor swimming pools and hot tubs to minimize the decomposition of hypochlorous acid by light (Allen et al., 1982). Both of MEL and CYA were reported as having low toxicity, with an oral LD50 in the rat of 3161 and 7700 mg/kg body weight, respectively (OECD, 1998, 1999), and no human data could be found on the oral toxicity of MEL or CYA separately until the issues of Chinese milk powder. In Europe, MEL is approved for use as a monomer and as an additive in plastics with a specific migration limit of 30 mg/kg food (EC, 2002) related to materials and articles ⇑ Corresponding author at: National Aquafeed Safety Assessment Center, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China. Fax: +86 10 82109753. E-mail address: [email protected] (M. Xue). 0273-2300/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2011.06.005

intended to come into contact with foodstuffs. In 2008, US FDA established a tolerable daily intake (TDI) for MEL at 0.63 mg/kg body weight (bw) and this was subsequently revised to 0.063 mg/kg bw (USFDA, 2008). In 2009 and 2010, The WHO and the EFSA reported the TDI for MEL of 0.2 mg/kg bw, because it was certificated as much more toxic of MEL co-occurrence with CYA, which was confirmed as the key reason for renal failure of dogs, cats, pigs and fish (WHO, 2009; EFSA, 2010). Mast et al. (1983) reported that 90% of ingested MEL could be excreted by kidney of rat within 24 h. Fast elimination of plasma MEL were reported for several mammals, for example, the half-lives of MEL in plasma of pigs, rhesus monkey, male Fischer 344 rats and Sprague–Dawley rats were about 3–5 h (Mast et al., 1983; Baynes et al., 2008; Yang et al., 2009; Liu et al., 2010). However, half-lives of MEL in muscle of catfish and trout were 1.51 and 3.62 d, respectively, and which were delayed to 1.67 and 4.40 d if both MEL and CYA were given together (Reimschuessel et al., 2010). Compared to territorial animal, fish required much higher protein in diets because of theirs low carbohydrate utilization capability (Geurden et al., 2007; Kaushik and Seiliez, 2010). China is the largest producer and exporter of MEL in the world. MEL and its analogs were intentionally adulterated in protein sources for animal feed because of its high nitrogen content which give the appearance of normal protein levels when subjected to a test for protein levels that is based on nitrogen content. Qin et al. (2010) reported that MEL was found in many proteins sources, such as

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fishmeal, meat meal, corn and wheat gluten meal in China before 2009. Rainbow trout (Oncorhynchus mykiss) is a typical cold water and carnivorous species, which requires at least 35% of protein for normal growth (Wilson, 2002). Fish feeds were contaminated MEL and CYA both in USA and China during 2007 and 2008 (USFDA, 2007; Xin and Stone, 2008). A few studies had been conducted on renal effects and residue depletion in tissues of MEL and CYA in several fish species (Reimschuessel et al., 2008, 2010; Qin et al., 2010). However, there have no studies on plasma pharmacokinetic (PK) model of MEL and/or CYA in any aquatic animals. The current study was conducted to obtain plasma PK parameters of MEL and a blend of MEL and CYA with a single oral exposure in rainbow trout. 2. Materials and methods 2.1. Chemicals MEL (99%) and CYA (98%) were obtained from Sigma Aldrich (Shanghai, China). The stable-isotope-labeled internal standard was used in the LC–MS/MS determination. [15N3]–MEL and [13C3]–CYA (99% isotopic purity) were supplied by Cambridge Isotope Laboratories Inc. (Beijing, China). All other chemicals and solvents used in the analyses were reagent grade. 2.2. Animals Rainbow trout obtained from farm of Beijing Fisheries Institute were transferred into the tanks two weeks before the trial for acclimatization. During the acclimatization period, the fish were fed a commercial diet (440 g/kg crude protein, 160 g/kg crude lipid, produced by Uni-President Inc., Guangdong, China) to visual satiation. Prior to use, all feeds and ingredients were tested for MEL and CYA by liquid chromatography–triple quadrupole mass spectrometry (LC–MS/MS) (Qiu et al., 2009). No residues were detected above the limit of detection (LOD) of for MEL (0.05 lg/g) and CYA (0.1 lg/g). The feeding trial was carried out in the flowing water facilities of National Aquafeed Safety Assessment Center, Feed Research Institute, Chinese Academy of Agricultural Sciences (Beijing, China) using 20 fiberglass cylindro-conical tanks (capacity: 265 l; waterflow rate: 600 ml/min) at temperature of 16 ± 1 °C. Ten tanks with totally 200 fish were assigned to each treatment to ensure enough fish for sampling. All fish having a mean body weight of 107 ± 5 g were randomly allocated to each tank after 24 h starvation. Dissolved oxygen was maintained above 8 mg/l, pH was 7.5–8.5, ammonia <0.5 mg/l. The photoperiod was 12L:12D with the light period from 07:00 to 19:00. 2.3. Dosing and sampling Two extrusion diets were prepared with 2000 mg/kg MEL or a blend of 500 mg/kg MEL and 167 mg/kg CYA, respectively. The MEL and CYA blend was designed at 3:1 according to the ratio of scrap MEL in China (Xue, unpublished data). The diets were fed at 1% (80% of ad libitum) of fish body weight at a single target dosage of MEL (20 mg/kg bw) and the blend of MEL and CYA (5 and 1.67 mg/kg bw, respectively) to each tank. Test diets were carefully fed to fish to guarantee all diets were consumed by fish. Fifteen fish of each treatment (five from three different tanks of same group) were anesthetized with trichlorobutanol at 300 mg/l before each sampling. The blood was sampled at 30 min, 1, 4, 8, 12, 20, 24, 36, 48, 72, 144 and 240 h post-dose. Fish were dissected and to make sure feed eaten by fish and those without feed in intestine were abandoned during 30 min to 24 h sampling. Gastro-

intestinal (GI) tracts and kidneys were inspected to inquire if the crystals were formed. Blood samples were collected from caudal vein of individual fish with a microhematocrit capillary tube with heparin as anticoagulant. Blood samples (2 ml of each fish) were centrifuged at 4000g for 10 min at 4 °C to obtain the plasma. All samples were frozen at 20 °C before determination. 2.4. Determination of melamine and cyanuric acid in plasma with LC– MS/MS An optimized method for simultaneous determination of MEL and CYA in plasma and animal tissues by LC–MS/MS was used in the present study (Qiu et al., 2009). An API 2000 LC–MS/MS System (AB Sciex Instruments) equipped with Turbo Electrospray Ionization (ESI) probe in the multiple reaction monitoring (MRM) scan mode was used to analyze samples. The MRM conditions of MEL and CYA is shown in Table 2. Liquid chromatography was carried out using an Agilent 1200 (Santa Clara, CA, USA) series HPLC equipped with a G1322 degasser, G1311A quatpump, G1316B column compartment, G1329A autosampler and a 20 ll sample loop (Wilmington, DE, USA). The separation was performed on a Waters Atlantis HILIC silica column with an HILIC silica guard column cartridge. The gradient elution used mobile phases of (A) acetonitrile/5 mM ammonium acetate/formic acid (95/5/0.1, v/ v/v) and (B) acetonitrile/5 mM ammonium acetate/formic acid (5/ 95/0.1, v/v) with a flow rate at 0.3 ml/min. The gradient program was that (A) ranged from 100% at 0 min to 40% at 3 min, held 2 min and then changed from 40% at 5.0 min to 100% at 5.1 min, finally held 6.9 min. The total run time was 12 min. The optimized LC–MS/MS parameters were as follows: Curtain gas (CUR) 30, Collision Gas (CAD) 3, Ion Source Temperature (Tem) 450 °C, Ion Source Gas 1 (GS1) 70 and Ion Source Gas 2 (GS2) 60 for both analytes, Ion Spray Voltage (IS) -4500 (CYA) and 2000 (MEL). The plasma (1.0 ml) was mixed with 9.0 ml of acetonitrile/water (70:30) by shaking for 20 min. After centrifugation at 5000 rpm for 5 min, the supernatant (500 ll) was mixed with 20 ll of 15N3-MEL (1.0 lg/ml), 40 ll of 13C3-CYA standard solution (1.0 lg/ml) and 440 ll of acetonitrile. The extraction solution was filtered through 0.22 lm PVDF syringe filter into an autosampler vial for analysis after centrifugation at 13,000 rpm for 10 min. A 10 ll-aliquot of each sample was then injected into LC–MS/MS system in replicates. The fortified samples were prepared by adding appropriate amount of MEL and CYA standard solutions to 1.0 ml of blank plasma and processing with above method. A series of calibration standards were directly prepared in acetonitrile. The optimized method using isotope-labeled analytes as internal standard was linear over a range of concentrations with 0.002–0.5 lg/ml for MEL and 0.004– 0.5 lg/ml for CYA. The mean recoveries of MEL and CYA in rainbow trout plasma are shown in Table 1. The limits of detection (LOD) for MEL and CYA, defined as concentration produced a signal-to-noise ratio of 3, respectively were 0.02 and 0.04 lg/ml of plasma. 2.5. Pharmacokinetic and statistical analysis PK analysis was performed using a non-compartmental modeling approach with the Winnonlin (Pharsight, Mountain View, CA, Table 1 Recovery of plasma for rainbow trout (n = 5). Analyte

Level (lg/ml)

Recovery (%)

SD

RSD (%)

MEL

0.04 0.4 4.0 0.08 0.8 8.0

90.9 99.7 87.2 91.6 79.7 96.2

4.2 5.7 2.9 4.1 5.6 8.6

4.6 5.7 3.4 4.5 7.0 8.9

CYA

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M. Xue et al. / Regulatory Toxicology and Pharmacology 61 (2011) 93–97 Table 2 Multiple reaction monitoring (MRM) scan mode for determination of MEL and CYA by LC–MS/MS. Compound

Retention time (min)

CYA

1.78

128.0

13

C2–CYA MEL

1.78 6.77

131.0 127.0

15

6.77

130.0

N3–MEL

Q3 mass (amu) *

42.0 85.0 43.0* 85.0* 68.0 87.0*

DP (V)

CE (V)

23 20 37 22 24 23

23 12 26 28 43 29

MRM used for quantification.

Table 3 The plasma pharmacokinetic parameters of melamine and blend of melamine and cyanuric acid following a single oral administration (mean ± SE*, n = 15). Parameters

Unit

MEL20

MEL5

CYA1.67

K t1/2 Clz/F AUC Tmax Cmax Vss MRT

h 1 h ml/h/kg h
lg/ml h lg/ml l/kg h

0.021 ± 0.00a 32.96 ± 1.51a 36.60 ± 1.83a 546.45 ± 10.32c 12.00 ± 0.50a 10.69 ± 0.87c 1.74 ± 0.02a 52.00 ± 0.23a

0.022 ± 0.00a 32.23 ± 2.13a 35.91 ± 1.98a 139.23 ± 5.38b 20.00 ± 0.53b 3.69 ± 0.02b 1.67 ± 0.01a 50.43 ± 1.38a

0.088 ± 0.001b 7.92 ± 0.35b 783.56 ± 12.12b 2.13 ± 0.04a 20.00 ± 0.15b 0.103 ± 0.01a 8.95 ± 0.08b 21.33 ± 1.26b

*

Values in the same row with no common superscripts are significantly different (P < 0.05).

14

MEL concentrations in plasma (µg/ml)

*

Q1 mass (amu)

12 10 8 6 4 2 0 0

USA). Parameters calculated using standard equations included elimination rate constant (K), elimination half life (t1/2), clearance (Clz/F), area under the curve (AUC), the maximal concentration (Cmax) and the time to maximal concentration (Tmax), steady state volume of distribution (Vss), and mean residence time (MRT). Because plasma CYA was lower than LOD for MEL-only group, three PK models were calculated and named as MEL20, MEL5 and CYA1.67, respectively. All data were subjected to one-way analysis of variance (ANOVA) followed by Duncan’s multiple-range test by STATISTICA 6.0 (Statsoft Inc., Tulsa, USA). Differences were regarded as significant when P < 0.05.

50

100

150

200

250

Time (h) Fig. 1. The mean concentration–time profile of plasma MEL and CYA following a single oral melamine administration of 20 mg/kg bw, in which CYA contents in plasma were low than LOD. The bars of standard errors were showed the coefficient of variation of data at each sampling point (n = 15).

783.56 ml/h/kg and shorter t1/2 of 7.92 h. Tmax of MEL20 and MEL5 were 12 and 20 h, respectively, which showed that deposition of MEL was delayed when MEL and CYA were given together.

4. Discussion 3. Results 3.1. Pharmacokinetic parameters of melamine after a single oral administration to rainbow trout Recoveries of the determination of MEL and CYA simultaneously were ranged from 79.7% to 99.7% with coefficient of variation ranging from 3.4% to 8.9%. A non-compartmental model was found to best model the data over one-compartment model. The concentration–time profiles of MEL and CYA in plasma after the single dose of MEL (20 mg/kg bw) and the blend of MEL (5 mg/kg bw) and CYA (1.67 mg/kg bw) are illustrated in Figs. 1 and 2 (Fig. 2a is for MEL5 and Fig. 2b is for CYA1.67, respectively). Plasma MEL and CYA were assimilated much more rapidly than elimination. The plasma PK parameters of MEL and blend of MEL and CYA following a single oral administration were shown in Table 3. PK model of MEL showed first order processes of linear (dose-independent) characters. Cmax of MEL20 and MEL5 were 10.69 and 3.69 lg/ml, respectively, and CYA1.67 was 0.103 lg/ml. Cmax of MEL5 and CYA1.67 was 35.8, which was much lower than the ratio of test diet. AUC were 546.45, 139.23 and 2.13 h
lg/ml for MEL20, MEL5 and CYA1.67, respectively. Cmax and AUC of MEL were increased with higher MEL dosage. Other parameters, including K, half-life (t1/2), Clz/F, Vss and MRT were at relatively stable values after dosing at 20 or 5 mg/kg bw levels. Withdrawal of CYA was much more rapid than that of MEL with much higher Clz/F at

Both of MEL and CYA are low molecular weight triazine with a pKb of 9.0 (NTP, 1983). Polar and small molecular weight amines have displayed similar PK model in rats (Smith et al., 1994). Although a wealth of data is available relating the toxicity of exposure to MEL or CYA individually in several species (WHO, 2009) and Center for Veterinary Medicine, US FDA reported some toxicological and metabolic data for combined exposure to MEL and CYA (Reimschuessel et al., 2005, 2008, 2010; Anderson et al., 2011), plasma PK models of fish were still not reported. In the present study, rainbow trout with a single oral dose of MEL or blend of MEL and CYA diet indicated quite different PK models from mammalian animals. The Vss of MEL in pigs (0.61 l/kg) is close to total body water and suggests that distribution of MEL may be limited to the extracellular fluid compartment and is not extensively distributed to most organ tissues, and compared the male Fischer 344 rats, lower Vss of pigs might be the reason for longer t1/2 (Baynes et al., 2008). However, in the present study, Vss of MEL in rainbow trout was similar to that in rats (1.8 l/kg), almost 3-fold greater in pigs, but even showed longer t1/2 (32.2–32.9 h) and slower clearance (35.9–36.6 ml/h/kg) than pigs. the half-lives of MEL in plasma of male Fischer 344 rats, pigs, rhesus monkey and Sprague–Dawley rats were about 2.71, 4.07, 4.41 and 4.9 h, respectively, which indicated that mammals cleared MEL much more rapidly than fish (Mast et al., 1983; Baynes et al., 2008; Yang et al., 2009; Liu et al., 2010). Similar to the results of elimination

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a

b

Fig. 2. The mean concentration–time profile of plasma MEL (a) and CYA (b) following a single oral melamine administration of a blend of MEL (5 mg/kg bw) and CYA (1.67 mg/kg bw). The bars of standard errors were showed the coefficient of variation of data at each sampling point (n = 15).

kinetics of triazine in fish muscle or mammal’s plasma, CYA t1/2 was shorter than that of MEL in rainbow trout plasma (Allen et al., 1982; Barbee et al., 1984; Reimschuessel et al., 2010). This might be related to the difference in metabolism between mammal and fish. Fish are cold-blooded animals and live in much lower temperature environment than mammals, and the optimal water temperature for rainbow trout culture is below 21 °C (Evans and Claiborne, 2006). Temperature is one of most important factor to affect animal metabolic rates, and in fish, residue half-lives of drugs in edible tissues is frequently longer than those in plasma (Reimschuessel et al., 2005). Warm water species, such as catfish, showed significantly higher K and shorter t1/2 than trout both for MEL and CYA in fillet, and the t1/2 for MEL was between 1 and 2 d in catfish and between 3 and 4 d in trout. CYA t1/2 was less than 1 d in catfish and between 1 and 2 d in trout. Besides, the t1/2 increased if both chemical were given together (Reimschuessel et al., 2010). Except for excretion, kidney and gill were the organs for fish osmotic pressure adjustment. Freshwater teleosts excrete plenty of watery urine, which comprised trace amount of ions, such as divalent ions, ammonia, amino acid and a very small quantity of urea. Eckert and Randall (1988) reported that 75–93% of low molecular weight nitrogen waste of freshwater fish species will be excreted by gills. However, gills of freshwater fish are unlikely to be an excretory organ for the macromolecular compounds, such as MEL and CYA. Even urea, a much smaller compound, is excreted by gills in fish only with specific transporters (Wilkie, 2002). It was not reported whether PK models of MEL were dose-independent or dosedependent. In the present study, PK models of MEL showed first order processes of linear (dose-independent) characters generally after given different dosage of MEL, although some parameters might be affected by co-administration of MEL and CYA. For example, Tmax and of MEL5 was delayed to 20 h when co-administered

with MEL and CYA, which might be due to crystal formation in the GI tract and renal system. Reimschuessel et al. (2008) reported that renal crystals were induced in fish (rainbow trout, Atlantic salmon, channel catfish and tilapia) kidney, intestine and even in mucosa and submucosa of stomach via co-administration of MEL and CYA at a very high dose of 400 mg/kg bw of each compound for 3 d. However, crystals were not found in tissues of fish administered melamine alone, and only 1 rainbow trout and 1 salmon had renal crystals in groups that fed CYA alone. Thereafter, Reimschuessel et al. (2010) found the crystal formation within the kidney of catfish and trout after a single dose of MEL and CYA at 20 mg/kg bw synchronously on day 1–day 7. Only 1 and 2 catfish given only MEL or CYA developed renal crystals. However, in the present study, we did not found obvious stones in GI tract or kidney during whole period of the study inspected by naked eye and even by optical microscope. This might due to the lower dose of co-administration of MEL and CYA and the different ratio of MEL and CYA. Before the experiment, we inquired and determined the compositions of the scrap MEL from different factories, and those scrap MEL with nitrogen level of 32.8–52.7%, MEL and CYA at ratio of 10:1 to 3:1 were the main protein adulteration sources into animal feed. All the limited information about risks assessment of co-administration of MEL and CYA were based on the ratio of 1:1, which might be an important factor to form the obvious crystals combined by MEL and CYA. The ratio of 3:1 of MEL and CYA at present levels might not induce obvious crystal in fish GI and kidney, but enough to affect the PK model of plasma. The present study and almost all kinetics studies of MEL were based on a single oral dose, while in a real world the contamination of feeds would result in multiple doses. From 2010 to now, several articles reported the data of continuous, low-dose oral administration of MEL in lambs, pigs, laying hens, laying ducks and catfish (Lv et al., 2010; Li et al., 2010; Bai et al., 2010; Gao et al., 2010; Anderson et al., 2011). The published data and the results of present study are important for public health officials to assess risks imposed by such contamination incidents.

Conflict of interest statement The authors declare that there are no conflicts of interest. This work was supported by Special Fund for Establishment of Maximum Residue Limit (MRL) of Melamine in Feed (Ministry of Agricultural of China).

Acknowledgments We wish to thank Prof. Ying Xie and Miss Hexiang Zhou of Peking University for data analysis, and thank Dr. Gen He of Laboratory of Molecular and Cellular Neuroscience, the Rockefeller University, USA for language improvement.

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