Liquid chromatograph (Shimadzu Co.) and 5-cholestane as an internal normal. Liver protein content was determined according to the system of Lowry et al. (1951) utilizing bovine serum albumin as a normal. Fecal bile acid content was measured based on the method of Bruusgaard et al. (1977). The fatty acid composition of liver lipids have been analyzed having a fused silica capillary column (Omegawax 250, Supelco Co., Ltd.) immediately after methylation by sodium methoxide utilizing a GC-14B gas-liquid chromatograph (Shimadzu Co.). Analysis of liver enzyme activity Every single liver was homogenized in ten volumes of 3 mM Tris?HCl buffer (pH 7.four) containing 0.25 M sucrose and 1 mM EDTA-2Na. The homogenate was centrifuged at 500 g for 10 min at four , and the supernatant was obtained (fraction 1). The supernatant was recentrifuged at 9,000 g for ten minTable three Fatty acid composition of soybean oil and fish oil Fatty acid Dietary lipids (wt ) Soybean oil 14:0 15:0 16:0 16:1 n-7 16:1 n-9 17:0 17:1 18:0 18:1 n-7 18:1 n-9 18:two n-6 18:3 n-3 18:three n-6 20:1 n-9 20:four n-6 20:five n-3 22:five n-3 22:5 n-6 22:6 n-3 Other people N.D. not detected N.D N.D 10.6 N.D N.D N.D N.D three.eight 1.three 21.8 53.7 five.eight N.D N.D N.D N.D N.D N.D N.D 3.0 Fish oil 3.4 0.7 16.1 0.8 5.3 1.1 0.5 four.six two.3 12.7 1.4 0.six 1.eight 1.2 2.1 11.1 1.9 1.9 29.1 1.J Meals Sci Technol (March pril 2013) 50(two):266?at 4 to sediment the mitochondria (fraction two). The supernatant was ultracentrifuged at 105,000 g for 60 min at 4 , and the supernatant was obtained (fraction three). Acyl-CoA oxidase (ACOX, EC 1.3.3.six) activity of fraction 1 was measured as described previously (Ide et al. 1987). Carnitine palmitoyltransferase-2 (CPT-2, EC 2.three.1.21) activity inside the mitochondrial fraction (fraction 2) was measured as described by Markwell et al. (1973). Fatty acid synthase (FAS, EC 2.three.1.85) (Kelley et al. 1986) and glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) (Kelley and Kletzien 1984) activities in fraction 3 had been measured spectrophotometrically.Methyl 2-amino-3-hydroxybenzoate In stock The protein content of every single fraction was determined according to the technique of Lowry et al.578729-05-2 uses (1951) as described previously.PMID:26760947 Analysis of mRNA expression Total RNA was extracted in the livers working with an RNeasy Mini Kit (Qiagen, Tokyo, Japan). cDNA was synthesized from total RNA making use of a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems Japan Ltd., Tokyo, Japan). Real-time quantitative polymerase chain reaction (PCR) analysis was performed employing an automated sequence detection method (ABI Prism 7000, Applied Biosystems). 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), cholesterol 7-hydroxylase (CYP7A1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression levels have been measured using TaqMan Gene Expression Assays (Applied Biosystems). PCR primers (HMGR: Rn00565598_m1; CYP7A1: Rn00564065_m1; GAPDH: Rn99999916_s1) were bought from Applied Biosystems. mRNA expression levels of ATP-binding cassette (ABC) A1 (ABCA1), ABCG5, ABCG8, low density lipoprotein receptor (LDLR), stearoyl-Coenzyme A desaturase (SCD)-1 scavenger receptor class B sort 1 (SR-B1), and GAPDH were measured utilizing SYBR Green PCR Master Mix (Applied Biosystems). PCR primers had been as follows: forward: 5CCCGGCGGAGTAGAAAGG-3 and reverse: 5-AGGGC GATGCAAACAAAGAC-3 for ABCA1; forward: 5CCTCAAGGGCTCCGAGAACT-3 and reverse: 5ACCACACTGCCCCATAAGCT-3 for ABCG5; forward: 5-GCCATGGACCTGAACTCACA-3 and reverse: 5GCTGATGCCAATGACGATGA-3 for ABCG8; forward: 5-CCGTGGCTTTTTCTTCTCTCA-3 and reverse: 5GCATTCGGAACAGTGCAACA-3; fo.