Takahiko Aoki

Involvement of cathepsins B and L in the post-mortem autolysis of mackerel muscle

Abstract The intracellular distributions of catheptic enzymes (cathepsins) in white muscle of mackerel were investigated using differential centrifugation. The enzymes included cathepsin B, cathepsin L, β- N -acetylglucosaminidase, and cathepsin D (a lysosomal marker), and alkaline phosphatase (a microsomal marker). The highest relative specific activities of each of the cathepsins and β- N -acet-ylglucosaminidase were in the lysosomal fraction, and the highest relative specific activity of alkaline phosphatase was in the microsomal fraction. The buoyant density of white muscle lysosomal particles ranged from 1.03 to 1.10 g ml −1 . In ageing muscle at 0°C, the activities of cathepsins B and L gradually decreased in the lysosomal fraction, while they increased in the supernatant fraction. Mackerel cathepsin L hydrolyzed myosin, troponin T, troponin I, and tropomyosin. Mackerel cathepsin B did not hydrolyze any of the myofibrils from mackerel white muscle. These results suggested that the main cause of post-mortem degradation of mackerel muscle tissue was due to the activity of cathepsin L.

High-performance liquid chromatographic method for the rapid and simultaneous determination of sulfamonomethoxine, miloxacin and oxolinic acid in serum and muscle of cultured fish

A rapid method for the simultaneous determination of sulfamonomethoxine (SMM), miloxacin (MLX) and oxolinic acid (OA) in serum and muscle of cultured fish by high-performance liquid chromatography has been developed. A Hisep shielded hydrophobic phase column (15 cm x 4.6 mm I.D.) and a mobile phase of 0.05 M citric acid-0.2 M disodium hydrogenphosphate buffer, pH 2.5 in 10 mM tetra-n-butyl ammonium bromide-acetonitrile (85:15) with ultraviolet detection at 265 nm were used. The recoveries of SMM, MLX and OA from serum and muscle samples were 72-101%. The detection limits of the three drugs were 0.05-0.1 microgram/ml or g of sample.

Pharmacokinetics of sulphamonomethoxine and sulphadimethoxine following oral administration to cultured rainbow trout (Oncorhynchus mykiss)

Abstract The pharmacokinetics and metabolites of sulphamonomethoxine (SMM) and sulphadimethoxine (SDM) were studied in rainbow trout after oral administration (300 and 200 mg/kg, respectively) at a water temperature of 15°C. Both sulphonamide levels were determined by high-performance liquid chromatography. The pharmacokinetics of SMM and SDM were evaluated by a one compartment model with first-order absorption. The absorption and elimination half-lives were 6.9 and 32.6 h for SMM and 5.0 and 24.5 h for SDM. The area under the serum concentration-time curve and mean residence time were 3081 (μg·h/ml) and 70.6 h for SMM and 2127 (μg·h/ml) and 45.8 h for SDM. N 4 -Acetylsulphamonomethoxine (AcSMM) and N 4 -acetylsulphadimethoxine (AcSDM) were detected in all tissues, and the concentrations of these metabolites were much higher in the internal organs, especially in bile, than in serum and muscle. The half-lives and elimination time of N 4 -acetyl metabolites were longer than those of parent sulphonamides. Glucuronide conjugates of SMM and AcSMM were detected in all tissues, and the glucuronide conjugates of SDM and AcSDM were detected in liver, kidney, and bile. Considerable amounts of conjugated AcSMM were observed in bile even at 32 days post-dosing.

Determination of oxytetracycline in blood serum by high-performance liquid chromatography with direct injection

A direct injection analysis by high-performance liquid chromatography has been developed for oxytetracycline in serum of animals and fish. A Hisep shielded hydrophobic phase column (15 cm x 4.6 mm I.D.) and a mobile phase of methanol-0.2 M oxalic acid (10:90, v/v, pH 7.0) with ultraviolet detection at 360 nm were used. The standard calibration curves in serum of chicken, hog, cattle and rainbow trout were linear over the range 0.1-20 micrograms/ml. The recoveries of oxytetracycline from all serum samples determined at two different concentrations (0.5 and 2.0 micrograms/ml) were 88-103%. The detection limit was 0.05 micrograms/ml for every serum sample.

Pharmacokinetics of nalidixic acid in cultured rainbow trout and amago salmon

Abstract The pharmacokinetics and tissue levels of the antibacterial agent, nalidixic acid, were determined after oral administration in cultured rainbow trout and amago salmon. Nalidixic acid levels were determined by a high performance liquid Chromatographic method. Serum levels of nalidixic acid were described using a one-compartment pharmacokinetics model. The theoretical time to peak serum concentration of drug ( t max ) was 16.6 h for rainbow trout and 17.6 h for amago salmon, and the concentration at t max was 11.4 gmg/ml and 12.3 gm/ml, respectively. Drug absorption was complete in approximately 48 h. The area under the serum concentration-time curve ( AUC) and the mean residence time calculated from AUC were similar for the two species. The biological half-life and elimination time in the tissues, with the exception of serum, were shorter in rainbow trout than in amago salmon. The glucuronic acid conjugate of nalidixic acid was detected in all tissues and in the bile of both species.

A cathepsin B-like enzyme from mackerel white muscle is a precursor of cathepsin B.

A cathepsin B-like enzyme from the white muscle of common mackerel Scomber japonicus was a cysteine protease that hydrolyzed Z-Arg-Arg-MCA, the substrate for cathepsin B. In a partial purified cathepsin B-like enzyme preparation at 4 degrees C left over time, a converted enzyme that hydrolyzes Z-Arg-Arg-MCA and Z-Phe-Arg-MCA appeared in the preparation. The converted enzyme was purified from the cathepsin B-like enzyme, characterized and was identified as mackerel cathepsin B. These results suggested that the mackerel cathepsin B-like enzyme was a precursor of cathepsin B. Mackerel cathepsin B formed in the purified cathepsin B-like enzyme preparation by adding of a small amount of the purified cathepsin B to the preparation. Therefore, mackerel cathepsin B-like enzyme was converted to the mature form of cathepsin B by autoactivation. The conversion of the cathepsin B-like enzyme (molecular mass 60 kDa) to cathepsin B (molecular mass 23 kDa) was detected by immunoblotting by using human anti-(cathepsin B) antibody. The intermediate forms of 40 kDa and 38 kDa were also detected during the conversion.

Pharmacokinetics and metabolism of sulphamonomethoxine in rainbow trout (Oncorhynchus mykiss) and yellowtail (Seriola quinqueradiata) following bolus intravascular administration

Abstract The present study examined the pharmacokinetics and metabolism of sulphamonomethoxine (SMM) in rainbow trout ( Oncorhynchus mykiss ) and yellowtail ( Seriola quinqueradiata ) after intravascular administration (100 mg kg −1 body weight). Rainbow trout and yellowtail were kept in tanks with running water at 15.0 ± 0.3 °C and running sea water at 21.3 ± 0.2 °C, respectively. Serum concentrations of SMM were determined using high performance liquid chromatography with direct injection. Serum concentrations of SMM in rainbow trout and yellowtail were best described by a two-compartment model. The calculated half-lives for the distribution phase and the elimination phase were 0.43 h and 30.9 h for rainbow trout, and 0.53 h and 5.8 h for yellowtail, respectively. The apparent volume of distribution ( V d ) was larger in rainbow trout ( V d = 0.83 1 kg −1 ) than in yellowtail ( V d = 0.56 1 kg −1 ). Total body clearance was calculated as 18.5 ml kg −1 h −1 in rainbow trout and 66.7 ml kg −1 h −1 in yellowtail. The behaviour of N 4 -acetyl metabolite, N 4 -acetylsulphamonomethoxine (AcSMM), in rainbow trout and yellowtail could be explained well by a one-compartment model. The formation rate constant of the metabolite and the elimination half-lives were 0.0694 h −1 and 15.4 h for rainbow trout, and 0.1896 h −1 and 8.1 h for yellowtail, respectively. Acetylation in rainbow trout and yellowtail was calculated to be 23% and 64%, respectively. The serum protein bindings in vivo of SMM and AcSMM were determined to be 6.4 ± 2.3% and 9.5 ± 3.6% for rainbow trout, and 5.8 ± 1.7% and 6.3 ± 2.3% for yellowtail, respectively.

Pharmacokinetics of sodium nifurstyrenate in cultured yellowtail after oral administration

Abstract The pharmacokinetics and tissue levels of sodium nifurstyrenate (NFS) were studied in cultured yellowtail after oral administration at a dose of 100 mg/kg. NFS levels were determined by our high performance liquid chromatographic (HPLC) method. The recoveries from spiked serum and tissues were 97, 90, 97, 90 and 98% for serum, muscle, liver, kidney and bile, respectively. The detection limit of the method was 0.05 μ g/ml for serum, muscle, liver and kidney, and 0.1 μ g/ml for bile. Serum levels of NFS could be evaluated by a one-compartment model with first-order absorption. The theoretical time to peak serum concentration of drug ( t max ) was 1.9 h, and the concentration at t max was 6.98 μ g/ml. The area under the serum concentration-time curve (AUC) and the mean residence time calculated from AUC were 31.6 μ g/ml and 3.0 h. The elimination half-life ( T 1 2β ) was 1.4, 2.4, 29.7 and 1.5 h for serum, muscle, liver and kidney, respectively. The elimination time ( E t ) was calculated as 16.2, 9.5, 101 and 15.0 h for serum, muscle, liver and kidney, respectively, from their elimination curves. The T 1 2β and E t of NFS in bile could not be calculated because the elimination phase could not be recognized until 96 h.

Pharmacokinetics of sulphamonomethoxine in cultured yellowtail after oral administration

Abstract The pharmacokinetics of sulphamonomethoxine (SMM) and N4-acetyl-sulphamonomethoxine (AcSMM), N4-acetyl metabolite, were studied in cultured yellowtail. SMM and AcSMM levels were determined simultaneously by high performance liquid chromatography. The pharmacokinetics of SMM could be evaluated by a one-compartment model with first order absorption. Drug absorption was complete in approximately 3 h. The area under the serum concentration-time curve (AUC) and the mean residence time calculated from AUC were 932 (μg h/ml) and 8·0 h, respectively. The elimination half-life of SMM in serum, muscle, liver and kidney were 4·5, 5·1, 69 and 3·1 h, respectively. Residual SMM remained much longer in bile, and the elimination phase could not be recognized. AcSMM was detected in all tissues, and the elimination half-life of AcSMM was longer in liver (43 h) and bile (82 h) than in other tissues. The glucuronide conjugates of SMM and AcSMM were detected in all tissues. SMM, AcSMM and their glucuronidated metabolites were observed in bile even at 20 days post dosing.

Effect of bleeding on the quality of amberjack Seriola dumerili and red sea bream Pagrus major muscle tissues during iced storage.

The effect of bleeding on white muscle quality in amberjack and red sea bream was evaluated by measuring ATP-related compounds, volatile basic nitrogen (VBN), and the trimethylamine (TMA) content. The freshness was evaluated by the K value, and the degree of spoilage was elucidated by VBN and TMA. ATP was rapidly converted to ADP and AMP in the muscle and IMP was the main product of ATP degradation during iced storage. For both species, the IMP content was higher in the muscles of fish that were bled than in those unbled during iced storage. Conversely, the K value and the levels of hypoxanthine (Hx) and VBN were higher in the muscle tissue of unbled fish than in the bled tissue of both species. Similarly, the TMA content was higher in the unbled muscle tissues of both species after a week of storage.