Isolation of human small intestinal brush border membranes using polyethylene glycol and effect of exposure to various oxidants in vitro.
This study presents a method of brush border membrane (BBM) preparation from the human small intestine using polyethylene glycol (PEG) precipitation and also looks at the effect of in vitro oxidant exposure on structural and functional alterations in the membrane. Isolated BBM were relatively pure as judged by 10- to 14-fold enrichment of marker enzymes with less than 1% contamination by other subcellular organelles. These membranes showed uphill transport of glucose and lipid analysis showed a cholesterol-phospholipid (C/P) ratio of 1.19. Isolated BBM were found to be susceptible to superoxide generated by xanthine oxidase (XO), resulting in lipid and protein oxidation along with altered glucose uptake. Superoxide exposure also resulted in phospholipid alterations, especially generation of lyso phospholipids. These changes were prevented by inhibiting XO by allopurinol or scavenging superoxide by superoxide dismutase (SOD). Other oxidants studied did not have significant affect on these membranes. These studies suggest that PEG can be used for preparation of BBM from the human small intestine and these membranes undergo structural and functional alterations on exposure to superoxide.
Prabhu
Ramamoorthy
R
The Wellcome Trust Research Laboratory, Department of Gastrointestinal Sciences, Christian Medical College, Ida Scudder Road, Vellore-632 004, Tamil nadu, India.
Perakath
Benjamin
B
Balasubramanian
K A
KA
D000469:Alkaline Phosphatase / Q000494:pharmacology; D001692:Biological Transport / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000473:pathology; D046508:Culture Techniques; D006801:Humans; D007124:Immunoenzyme Techniques; D007421:Intestine, Small / Q000473:pathology*; D007931:Leucyl Aminopeptidase / Q000494:pharmacology; D015227:Lipid Peroxidation; D008563:Membrane Lipids / Q000378:metabolism; D008871:Microvilli / Q000187:drug effects / Q000473:pathology; D011092:Polyethylene Glycols; D012680:Sensitivity and Specificity; D018709:Statistics, Nonparametric; D013393:Sucrase / Q000494:pharmacology
D008563:Membrane Lipids; D011092:Polyethylene Glycols; D000469:Alkaline Phosphatase; D013393:Sucrase; D007931:Leucyl Aminopeptidase