Er and feces have been analyzed using a SE-30 column (Shinwa Chemical Industries LTD., Kyoto, Japan) applying a GC-14B gas-liquid chromatograph (Shimadzu Co.) and 5-cholestane as an internal normal. Liver protein content material was determined in accordance with the strategy of Lowry et al. (1951) utilizing bovine serum albumin as a typical. Fecal bile acid content material 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.) just after methylation by sodium methoxide utilizing a GC-14B gas-liquid chromatograph (Shimadzu Co.). Analysis of liver enzyme activity Each liver was homogenized in 10 volumes of 3 mM TrisHCl buffer (pH 7.4) containing 0.25 M sucrose and 1 mM EDTA-2Na. The homogenate was centrifuged at 500 g for 10 min at four , along with the supernatant was obtained (fraction 1).X-GAL supplier The supernatant was recentrifuged at 9,000 g for 10 minTable 3 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 22:5 n-3 22:five n-6 22:six n-3 Other people N.D. not detected N.D N.D ten.6 N.D N.D N.D N.D 3.eight 1.3 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.four 0.7 16.1 0.8 5.3 1.1 0.five 4.six 2.three 12.7 1.4 0.six 1.eight 1.2 two.1 11.1 1.9 1.9 29.1 1.J Meals Sci Technol (March pril 2013) 50(two):266at four to sediment the mitochondria (fraction 2). The supernatant was ultracentrifuged at 105,000 g for 60 min at 4 , and also the supernatant was obtained (fraction 3). Acyl-CoA oxidase (ACOX, EC 1.3.three.6) activity of fraction 1 was measured as described previously (Ide et al. 1987). Carnitine palmitoyltransferase-2 (CPT-2, EC 2.3.1.21) activity in the mitochondrial fraction (fraction 2) was measured as described by Markwell et al. (1973). Fatty acid synthase (FAS, EC two.three.1.85) (Kelley et al. 1986) and glucose-6-phosphate dehydrogenase (G6PDH, EC 1.Disodium 5′-inosinate Autophagy 1.PMID:22943596 1.49) (Kelley and Kletzien 1984) activities in fraction 3 had been measured spectrophotometrically. The protein content material of each and every fraction was determined based on the strategy of Lowry et al. (1951) as described previously. Evaluation of mRNA expression Total RNA was extracted from the livers employing 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 utilizing an automated sequence detection program (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 were measured utilizing TaqMan Gene Expression Assays (Applied Biosystems). PCR primers (HMGR: Rn00565598_m1; CYP7A1: Rn00564065_m1; GAPDH: Rn99999916_s1) had been 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 variety 1 (SR-B1), and GAPDH were measured making use of 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.
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