The present study investigates in a experimental system in vitro the relationship between the non-enzymatic (ascorbate-Fe2+) and enzymatic (NADPH) lipid peroxidation in rat liver microsomes and nuclei. Chemiluminescence was measured as cpm/mg protein during 180 min at 37 degrees C. Approximately 50-55% of the fatty acids located in rat liver microsomes and nuclei are polyunsaturated with a prevalence of C18:2 n6 and C20:4 n6. The values of total light emission during the non-enzymatic and enzymatic lipid peroxidation were highest in microsomes than in nuclei. A significant decrease of C20:4 n6 and C22:6 n3 in rat liver microsomes and nuclei was observed during the lipid ascorbate-Fe2+-dependent peroxidation, whereas a significant decrease of C20:4 n6 in rat liver microsomes was observed during enzymatic lipid peroxidation. Over the time course studies, analysis of chemiluminescence in microsomes and nuclei demonstrated that the lipid peroxidation in the presence of ascorbate-Fe2+ reach a maximum at 50 and 30 min, respectively, whereas in the presence of NADPH it reachs a maximum at 20 min in both organelles. In liver microsomes and nuclei the peroxidizability index (pi) which indicates the degree of vulnerability to degradation of a selected membrane showed statistically significant differences between control versus ascorbate-Fe2+ when microsomes or nuclei were compared. Our results indicate that non-enzymatic (ascorbate-Fe2+) and enzymatic (NADPH) lipid peroxidation are operative in rat liver microsomes and nuclei but the sensitivities of both organelles to lipid peroxidation evidenced by chemiluminescence was greater in the presence of ascorbate-Fe2+ when compared with NADPH.
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http://dx.doi.org/10.1023/b:mcbi.0000044302.59193.00 | DOI Listing |
Methods Enzymol
October 2024
Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, Freiburg, Germany; CIBSS Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany; BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany. Electronic address:
In addition to fluorescence microscopy, the subcellular fractionation of eukaryotic cells remains one of the central methods for the basic characterization of proteins. Here we describe an optimized procedure for the subcellular fractionation of yeast cells, specifically for mitochondrial studies. Major recommendations are to separate the fractions immediately after each centrifugation step, to carefully discard a significant part of the supernatant fractions which is in the direct vicinity to the pellets and, in addition, to perform an extra homogenization step of the post nuclear supernatant fraction.
View Article and Find Full Text PDFJ Lipid Res
May 2024
Depts of Pediatrics and Biochemistry & Molecular Biology, Atlantic Research Centre, Dalhousie University, Halifax, Nova Scotia, Canada. Electronic address:
Intestinal epithelial cells convert excess fatty acids into triglyceride (TAG) for storage in cytoplasmic lipid droplets and secretion in chylomicrons. Nuclear lipid droplets (nLDs) are present in intestinal cells but their origin and relationship to cytoplasmic TAG synthesis and secretion is unknown. nLDs and related lipid-associated promyelocytic leukemia structures (LAPS) were abundant in oleate-treated Caco2 but less frequent in other human colorectal cancer cell lines and mouse intestinal organoids.
View Article and Find Full Text PDFSci Rep
November 2023
Laboratory of Organelle Pathophysiology, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Japan.
Yes-associated protein (YAP) is a transcriptional coactivator that is essential for the malignancy of various cancers. We have previously shown that YAP activity is positively regulated by phosphatidylserine (PS) in recycling endosomes (REs). However, the mechanism by which YAP is activated by PS in REs remains unknown.
View Article and Find Full Text PDFMolecules
June 2023
Modern Research Center for Traditional Chinese Medicine, Beijing Research Institute of Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China.
As a first-line agent for cholestasis treatment in a clinic, ursodeoxycholic acid rectifies the perturbed bile acids (BAs) submetabolome in a holistic manner. Considering the endogenous distribution of ursodeoxycholic acid and extensive occurrences of isomeric metabolites, it is challenging to point out whether a given bile acid species is impacted by ursodeoxycholic acid in a direct or indirect manner, thus hindering the therapeutic mechanism clarification. Here, an in-depth exploration of the metabolism pattern of ursodeoxycholic acid was attempted.
View Article and Find Full Text PDFAnal Chem
July 2023
Key Laboratory of Drug Quality Control and Pharmacovigilance (Ministry of Education), State Key Laboratory of Natural Medicine, China Pharmaceutical University, Nanjing 210009, P. R. China.
Subcellular compartmentalization ensures orderly and efficient intracellular metabolic activities in eukaryotic life. Investigation of the subcellular metabolome could provide in-depth insight into cellular biological activities. However, the sensitive measurement of multi-subcellular metabolic profiles is still a significant challenge.
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