New application of a subcellular fractionation method to kidney and testis for the determination of conjugated linoleic acid in selected cell organelles of healthy and cancerous human tissues

Abstract

To clarify the mechanism of the anticarcinogenic effect of conjugated linoleic acid (CLA), its intracellular distribution needs to be determined. Subcellular fractionation using centrifugation techniques is a method that is frequently used for isolation of cell organelles from different tissues. But as the size and density of the organelles differ, the method needs to be optimised for every type of tissue. The novelty of this study is the application of a subcellular fractionation method to human healthy and cancerous renal and testicular tissue. Separation of total tissue homogenate into nuclei, cytosol, and a mixture of mitochondria and plasma membranes was achieved by differential centrifugation. As mitochondria and plasma membranes seemed to be too similar in size and weight to be separated by differential centrifugation, discontinuous density-gradient centrifugation was carried out successfully. The purity of the subcellular fractions was checked by measuring the activity of marker enzymes. All fractions were highly enriched in their corresponding marker enzyme. However, the nuclear fractions of kidney and renal cell carcinoma were slightly contaminated with mitochondria and plasma membrane fractions of all tissues with lysosomes. The fraction designated the cytosolic fraction contained not only cytosol, but also microsomes and lysosomes. The CLA contents of the subcellular fractions were in the range 0.13-0.37% of total fatty acids and were lowest in the plasma membrane fractions of all types of tissue studied. C16:0, C18:0, C18:1 c9, C18:2 n-6, and C20:4 n-6 were found to be the major fatty acids in all the subcellular fractions studied. However, marked variations in fatty acid content between subcellular fractions and between types of tissue were detectable. Because of these differences between tissues, no general statement on characteristic fatty acid profiles of single subcellular fractions is possible.

Characterization of In Vitro Glucuronidation Clearance of a Range of Drugs in Human Kidney Microsomes: Comparison with Liver and Intestinal Glucuronidation and Impact of Albumin

“…Inclusion of BSA in HLM incubations increased CL int, u, UGT up to 7-fold, which is within the range of previously reported data (Kilford et al, 2009;Rowland et al, 2009;Manevski et al, 2011). In addition, the presence of BSA in the incubations led to increases in HKM and HIM CL int, u, UGT values, supporting reports that linoleic and arachidonic acids (the most potent UGT inhibitors of FFAs) (Tsoutsikos et al, 2004) are abundant in all three tissues investigated (Soydan et al, 1996;Hoffmann et al, 2005;Rowland et al, 2007). Inclusion of BSA (1% for acids and 2% for bases/neutral drugs) decreased the f u, inc for all drugs (Ͻ0.1 for four of seven drugs); consequently, depletion was very low for some of the drugs investigated.…”

“…Addition of BSA has resulted in up to 16-fold increases in glucuronidation intrinsic clearance (CL int, UGT ) obtained either by depletion or metabolite formation in human liver microsomes (HLM), resulting in improved prediction accuracy when in vitro-in vivo extrapolation (IVIVE) of such data was performed (Rowland et al, 2008(Rowland et al, , 2009Kilford et al, 2009;Manevski et al, 2011). Analogous to liver, arachidonic and linoleic acids represent two of the most abundant FFAs in renal and intestinal tissue (Soydan et al, 1996;Hoffmann et al, 2005;Rowland et al, 2007). However, to date there have been no studies assessing the effects of BSA on glucuronidation in human kidney (HKM) or intestinal (HIM) microsomes.…”

Anthocyanin inhibits high glucose-induced hepatic mtGPAT1 activation and prevents fatty acid synthesis through PKCζ

“…Cytosolic and membrane fractions were prepared as described previously ( 17 ). Mitochondrial fractions from the hepatocytes and livers of mice were prepared as described previously ( 17 ) and then solubilized in Tris buffer containing 1% digitonin, or 1% 3-[(3-cholamidopropyl) dimethyl-ammonio]1-propane sulfonate (CHAPS) at 4°C as reported previously ( 18 ).…”

Adaptor Protein LAPF Recruits Phosphorylated p53 to Lysosomes and Triggers Lysosomal Destabilization in Apoptosis

“…The purification of lysosomes from cells in a self-generated gradient of Optiprep (Axis-Shield) was performed according to manufacturer's instructions. Optiprep is based on a density gradient compound, Iodixanol, which has been used for isolation of lysosomes from other organelles (22). The purity of the isolated lysosomes was verified by biochemical analysis and Western blot analysis immediately after the isolation.…”

“…Several studies showed that p53 could trigger apoptosis in the absence of ongoing transcription and protein synthesis (4,5). Several transcription-deficient mutants of p53, like p53dl214 (contains amino acids 1-214) and p53Gln 22 / Ser 23 , have the capability of inducing apoptosis (6,7). In cell-free extracts, p53 could directly activate caspase-3 with the presence of mitochondria (8).…”