Rf 535

Samples of the diets were analysed for crude protein (CP) by Kjeldahl-N × 6.25 (EC No 152/2009), crude fat using ASE® 350 Accelerated Solvent Extractor, dry matter (DM) by drying to constant weight at 104 °C (EC No 152/2009), ash by incineration at 550 °C (EC No 152/2009), acid detergent fiber (ADF) and neutral detergent fibre (NDF) using a fibre analyser system (Ankom200; ANKOM Technologies, Fairport, NY, USA) with filter bags (Ankom F58; ANKOM Technologies). Gross energy (GE) content was determined by a Parr 1281 Adiabatic Bomb Calorimeter (Parr Instruments, Moline, IL, USA) according to ISO (1998). Analysis of amino acids in the diets were carried out according to EC (2009) using Biochrom 30 Amino Acid Analyzer. Tryptophan in the diets was determined according to EC (2009) using high-performance liquid chromatography system (Dionex UltiMate 3000, Dionex Softron GmbH, Germering, Germany) and the fluorescent detector (Shimadzu RF-535; Shimadzu Corp., Kyoto, Japan).

MDA is the main lipid peroxidation marker. It is considered an oxidative stress index [31 ] and associated with cardiovascular diseases [32 (link)]. Thus, MDA levels were used to evaluate the cardiac lipid oxidation as follow:
Cardiac tissue (±150 mg) was homogenized (ULTRA-TURRAX® T25 basic IKA^® Werke Staufen/Germany) with 1.0 mL of cold phosphate buffered saline (PBS) pH 7.4, and centrifuged at 800 g at 4 °C for 10 min. Then, 100 µL from the supernatant was mixed with 700 μL of 1% orthophosphoric acid and 200 μL of thiobarbituric acid (42 mM). After this, the samples were kept at 100 °C for 60 min in a water bath, and immediately cooled on ice. In a 2 mL tube, 200 μL was mixed with 200 μL sodium hydroxide/methanol (1:12 v/v). After vortex, the samples were centrifuged for 3 min at 13,000 g. 200 μL from the supernatant was transferred to a glass vial and 50μL was injected into the column. The HPLC used was a Shimadzu LC-10AD system (Kyoto, Japan) with a C18 Luna column (5 μm, 150 × 4.60 mm, Phenomenex Inc., Torrance, CA, USA), and a Shimadzu RF-535 fluorescence detector (excitation 525 nm, emission 551 nm), and 0.5mL/min phosphate buffer flow (KH2PO4 1mM, pH 6.8) [25 (link)]. The MDA levels considered the peak area determination in the chromatograms relative to the standard curve of known concentrations.

The liquid culture medium and an equivalent volume of chloroform were dispensed into new microtubes and vortexed for 10 s. The chloroform layer was collected and transferred to a new microtube and then reduced to dryness in a draft chamber at 40 °C. The subsequent procedures followed the official method [28 ]. In this study, the limits of quantitation and detection were 2 μg·kg⁻¹ and 1 μg·kg⁻¹, respectively. An aliquot of trifluoroacetic acid (Wako Pure Chemical Industries) equal to 0.1 times the volume of the chloroform was added to the microtube to derivatize AFB₁ and AFG₁ with vortexing for 5 s. After incubation for more than 10 min, a mixture of acetonitrile in distilled water (10:90, v/v) was added to the microtube. The volume of acetonitrile/water was 0.9 times the volume of chloroform. An aliquot (20 µL) of the sample solution was injected into a high-performance liquid chromatography system (SCL-10A, Shimadzu, Kyoto, Japan) equipped with a fluorescence detector (λ_Ex = 365 nm and λ_Em = 455 nm; RF-535, Shimadzu). The mobile phase was a mixture of distilled water/methanol/acetonitrile (60:30:10, v/v/v) with a flow rate of 1 mL·min⁻¹. The column was an Inertsil ODS-3 (150 mm × 4.6 mm, particle size 5 μm; GL Sciences, Tokyo, Japan).

Culture medium and an equivalent volume of chloroform were dispensed into a new microtube, and vortex mixed for 10 s. The chloroform was collected and transferred to a new microtube, and then reduced to dryness in a draft chamber. An aliquot of trifluoroacetic acid (Wako Pure Chemical Industries) equal to 0.1 times of the volume of the chloroform was added to the microtube, followed by vortex mixing for 5 s. After incubation for more than 10 min, a solution of acetonitrile in distilled water (10:90, v/v) was added to the microtube, with the volume equal to 0.9 times the volume of chloroform. An aliquot (20 µL) of the sample solution was injected into a high performance liquid chromatography system (SCL-10A, Shimadzu, Kyoto, Japan) with a fluorescence detector (λ_Ex = 365 nm, λ_Em = 455 nm; RF-535, Shimadzu). The mobile phase was a mixture of distilled water:methanol:acetonitrile (60:30:10, v/v/v) at a flow rate of 1 mL/min.

Serum leucine concentration analysis was performed using a Shimadzu® high-pressure liquid chromatography (HPLC), model LC 10AD (Shimadzu Corporation, Tokyo, Japan), consisting of 2 continuous flow pumps for the mobile phase elution gradient. Samples of 10 μL of serum were added in 200 μL of methanol and placed in 1.5 mL tubes and centrifuged (Jouan Refrigerated Centrifuge, Model MR1812, Analytical Instruments Brokers LLC, Golden Valley, MN, USA) for 10 min at 1800 g. The supernatant was transferred to another 1.5 mL tube, and the methanol evaporated. After the total evaporation of the methanol, the dry serum was dissolved with 100 μL of the solution phase A. 10 μL of each sample were taken and placed in tubes suitable for analysis in the sampler (Sil 10A) with a capacity for 79 tubes. Then, 25 μL of the reaction product was injected into the C18 silica column, and the amino acids separated according to molecular weight and polarity and detected by the Shimadzu fluorescence detector model RF535, at 335 mm excitation and 455 mm emission [32 (link)].

Chromatographic separation of homotaurine and histidine was performed using a Shimadzu HPLC system consisting of two LC-20AD isocratic pumps, a DGU-14A degasser, an SIL-10AD autosampler and a fluorescence detector (RF-535, Shimadzu, Tokyo, Japan). The stationary phase was a reversed-phase LC-C₁₈ DB column (250 × 4.6 mm, 5.0 μm), Supelco (Bellefonte, PA, USA), thermostated at 30 °C. LC-solution^® software version 1.25 SP4 was used for hardware control and data manipulation. All separations were performed using a binary gradient elution program. Mobile phases A and B consisted of potassium phosphate buffer (pH = 7; 0.02 M) and methanol, respectively. The injection volume was set at 40 µL and the flow rate was set at 1 mL·min⁻¹. Homotaurine and histidine OPA derivatives were monitored at λ_ext = 340 and λ_em = 455 nm, respectively.

The identification and quantification of the tocopherol fractions were carried out by high-pressure liquid chromatography (HPLC) [59 ].The HPLC system consisted of a low-pressure quaternary pump HP-1050, a Rheodyne injection valve (20 mL loop), a thermostatic furnace, and a fluorescence detector RF-535 (Shimadzu, Kyoto, Japan). Separation was performed in a 250 × 4 mm particle size 5 μm LiChrospher Si-60 (Merck, Darmstadt, Germany) column. The column and detector were firstly separated and then detected with a UV detector.