Metabolism and Elimination of Oxytetracycline in Hepatopancreas of Chinese Mitten Crab Eriocheir Sinensis

The kinetic profiles of Oxytetracycline (OTC) in hepatopancreas of Eriocheir sinensis was examined in this research. The results showed that after a single-dose of 40 mg/kg body weight OTC at (18±1°C) by intramuscular administration, the hepatopancreas concentration-time curve of OTC could be best described by a one-compartment model with first-order absorption and a time lag: C = 69.45×(e-0.005(t-0.0387)-e-8.195 (t0.0387)). The elimination half-life (t1/2β) was 130.71 h. It is suggested that a withdrawal time of 79.51d should be required before crabs are consumed safely.


INTRODUCTION
The Chinese mitten crab, Eriocheir sinensis (also known as river crab), is one of the most important aquatic products in China.With the enlargement of the cultivation scale, various diseases have emerged, which resulted in huge economic losses.Tremor Disease Agent (TDA) is one of the most hazardous pathogens.Based on our previous studies and molecular biology methods, TDA was thought to be a spiroplasm (Wang et al., 2003(Wang et al., , 2004a)).Oxytetracycline (OTC) was found to be very effective against TDA according to in vitro antimicrobial susceptibility test (Liang et al., 2009).OTC is the first fish antibiotic that approved by Food and Drug Administration (FDA) of USA, which has been widely used in control of aquatic animal diseases.Recently, much attention has been paid to safe medication in aquaculture, mainly focusing on fish and shrimp (Black et al., 1991;Chiayvareesajja et al., 2006;Poapolathep et al., 2008;Rigos et al., 2002).There is only a few work on pharmacokinetics of OTC in E. sinensis, which examined the drug concentrations in haemolymph and muscle (Feng et al., 2010(Feng et al., , 2011)).So far there is no report on OTC kinetics in hepatopancreas (the best edible parts of Eriocheir sinensis), which has the function of detoxification.In this study, the metabolism and elimination of OTC were investigated thoroughly.

Animals:
One hundred fifty healthy crabs (101.7±6.1g) were purchased from Hongqi Farm of Taizhou in Jiangsu province.E. sinensis were kept in circular tanks (4.0×3.0×3.0 m) at 18±1°C for two weeks before the experiment.
Drug delivery: Before giving the dose, crabs were surface disinfected with 75% alcohol.The drug was delivered through the arthrodial membrane in the hinge region between the fourth pereiopoda.The dosage was 40 mg/kg body weight, determined by former treatment experiment.
Samples were thawed at room temperature and broken up with electric homogenizer.5.000 g of samples were weight accurately and put into a 50 mL centrifuge tube with 10 mL 1% perchlorate solution.The tube was vortexed for 15~30 s, then centrifuged for 10 min at 10000 rpm/min.The supernatant was transferred.The procedure was repeated for three more times with 15, 15, 10 mL perchloric acid solution to extract OTC completely.All the supernatants were collected and filtered (0.45 µm nylon membrane).The samples were purified by a solid phase extraction procedure performed with AccuBOND ODS-C 18 (Agilent, 500 mg of 3 mL capacity).Columns were preconditioned with 5 mL of methanol, 2 mL of 5% EDTA and 5 mL ultrapure water.After this, columns were cleaned with 10 mL ultrapure and eluted with 20 mL 5% triethylamine methanol solution.The eluate was evaporated to dryness by rotary evaporator with water bath at 50°C, resuspended in 1 mL of mobile phase solvent.Before HPLC analysis, all the samples must be filtered (0.45 µm nylon membrane).
Chromatographic conditions: High performance liquid chromatography analysis was performed with an Agilent 1100 consisting of a quaternary pump (G1311A), an auto-injector (G1313A) and a variable wave-length detector (G1314A).Analytes (20 µL, sample volume) were separated on a ZORBAX SB-C18 250×4.6 mm i.d., 5 µm reverse phase silica column.The mobile phase was ACN-0.05 mol/L Na 2 HPO 4 containing 0.001 mol/L EDTA (pH2.5)(18:82, V/V), degassed and filtered before use.The column temperature was maintained at 37°C, the flow velocity, 1.0 mL/min; the detection wavelength is 355 nm, the flow rate was 1 mL/min and the UV detector was used at the wavelength of 355 nm.
Data analysis: 3P97 (Chinese pharmacological society mathematics professional committee compiled) was used to analyze kinetic profiles of OTC according to the residual sum of squares and the minimum Akaike's information criterion value.The elimination characteristics of the drug were calculated by using the last four time points.Least-square regression analysis was applied to estimate the elimination slope (β).The elimination half-lives were obtained from the equation: t 1/2 = ln2/β (Baggot, 1977).Optimization of dosage regimen was calculated with the formula: C min = Dose × e -βtd /V d(area), where C min is the minimum effective concentration and t d is the duration of the effect (Doi et al., 1998).
Withdrawal period was determined as the time post-dosing that OTC level fell below the maximum residue limit (MRL, 0.1 µg/g) and calculated with withdrawal-time calculation program WT1.4, which was adopted by the Committee for Veterinary Medicinal Products (CVMP) (Blanchflower et al., 1997).

RESULTS STAND
HPLC assay: Under the conditions mentioned above, the retention time of OTC is about 5.344 min (Fig. 1  and 2).Though an impurity peak appeared in the sample, the target compound (OTC) was separated with good resolution (Fig. 2). Figure 3 showed that, the standard calibration curve for OTC was found to be  (L/kg) 0.460000 A: The zero time drug concentration intercepts of disposition and elimination; Ka, Ke: First order rate constants for drug absorption and elimination; T0: time lag; AUC0 -∞ : Area under the concentration from zero to infinity; MRT0 -∞ : Mean residence time from zero to infinity; CLS: The total body clearance; Vd/F: Apparent volume of distribution linear over the range 0.05~20 µg/mL, with correlation coefficients >0.999.

Kinetic analysis:
The hepatopancreas concentrationtime curve was presented in Fig. 2.There were two peaks in the curve.OTC concentration reached the first peak (102.02±10.92µg/mL) 2 h after dosing.In this stage, drug absorption is faster and then the concentration declined until 24 h.After that, the concentration started to rise again, the second peak (58.27±3.26µg/mL) appeared at the 48 h, then the concentration dropped slowly over time (Fig. 4).The concentration-time curve was best described by one compartment open model with time delay.The theoretical equation is: C = 69.45×(e -0.005(t-0.0387)-e -8.195(t-0.0387)The terminal elimination rate constant (β) is 0.005 h -1 .The corresponding elimination half-life (t 1/2β ) is 130.71 h.The main kinetic parameters could be found in Table 1.The dosage was optimized based on those parameters, 41.85 mg/kg body weight was recommended.

Withdrawal time calculation:
Referring to food safety standards of China, with the maximum residue limits (MRL, 0.1 mg/kg), the withdrawal time was calculated to be 79.51d (Fig. 5).

DISCUSSION
Perchloric acid was used as protein precipitant and the extractant for OTC from biosamples.Triethylamine was added to improve the chromatographic separation and avoid tailing phenomenon.EDTA functioned as chelator to remove any impure metal ions in chromatographic column, which could get better peak resolution (Oka et al., 2000).
The Limit of Detection (LOD) is 0.015 µg/mL, determined as 3 times of the baseline noise.The extraction recovery of OTC was 74.18 and 86.97% for samples spiked with 0.1 µg/mL and 0.2 µg/mL respectively.Coefficients of variation were 2.87 and 8.65% for intra-day and inter-day assays respectively (n = 6).
In former researches on liver tissue, twocompartment model was found to fit the experiment data (Wu et al., 2006;Zhang et al., 2008).While in this study, one-compartment model fitted better.Considering the differences of test conditions, sampling design, it is difficult to determine exactly which model is more appropriate.The non-compartmental model is not affected by the limitation of classical compartment model, which was applied for comparison between species.
The maximum OTC level was much higher than the values reported in Scylla serrata (32.12 µg/mL) (Chen et al., 2009) and Paralichthyidae olivaceus (13.78 µg/mL) (Ji et al., 2008).Tmax (2 h) was shorter than that observed in Scylla serrate (4 h) (Chen et al., 2009) and Paralichthyidae olivaceus (4 h) (Ji et al., 2008) respectively.The difference could be explained by factors such as medication administration route.In this study, the drug was delivered intramuscular injection.While oral administration was applied for the other two species, the first pass effect might play an important role in Tmax delay.
The Double Peak Phenomenon appearing in this study was also found in other researches, which suggested reabsorption of OTC in hepatopancreas (Guo et al., 2005;Li et al., 1997).The area under the concentration-time curve of terminal phase (the last four time points) contributed 67.61% to the area under the concentration from zero to infinity, which confirmed the existence of reabsorption in theory.The dose reabsorbed was unlikely to be from crustaceans exoskeleton, for OTC with strong capacity of chelating Ca 2+ /Mg 2+ couldn't release from exoskeleton within such a short period (2-14 d).It was also impossible to be explained by the slow release of OTC residues in the injection site, because of the open vascular system OTC would be absorbed completely soon after intramuscular injection.Warner (1977) considered that the main functions of the green gland (excretory organ) is not expelling waste from body but keeping ion balance and reabsorbing selectively.Hence the phenomenon of two absorption peaks in the hepatopancreas may be linked with reabsorption mechanism of green gland.
One purpose of this study was to optimize dosage regimen.Our former research showed that the minimal inhibitory concentrations of OTC are 0.04 µg/mL.According with one compartmental model selected, t d was calculated to be 719.38 h (nearly 30d).The optimized dosage is 41.85 mg/kg body weight, close to the dosage administrated (40 mg/kg body weight).
So far there are only two kinds of software's about withdrawal time calculation at home and abroad.The homemade one was only designed for two compartmental models with intravenous injection, which didn't apply to this study (Wang et al., 2009).The software WT-1.4 used here isn't subject to the limitations described above.18±1°C was chosen in this experiment, for simulating the water temperature conditions in late spring and early summer during clinical therapy.Considering the effect of water temperature on OTC elimination, too long or too short period of withdrawal time will affect the farmers negatively.The withdrawal time was converted into units of degree-day value (°C-day) for ease of reference.Thus under the conditions of this study (water temperature 18±1°C, single dose of 40 mg/kg body weight by intramuscular injection), the withdrawal period of OTC in hepatopancreas of E. sinensis is 1432°C-day.
Future research will focus on population pharmacokinetics characteristics of OTC after different administration ways (oral administration and bath), taking various temperature conditions and different physiology situation (health and sickness) into account, which will provide theoretic support and practical reference to production.