Skip to main content
SpringerLink
Log in

Isolation of Human Small Intestinal Brush Border Membranes Using Polyethylene Glycol and Effect of Exposure to Various Oxidants In Vitro

  • Published:
Digestive Diseases and Sciences Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Caspery WF: Absorption: general aspects and transport mechanisms in the small intestine. In Structure and Function of the Small Intestine. WF Caspary (ed). Amsterdam, Excerpta Medica, 1987, pp 63-88

    Google Scholar 

  2. Wang X, Anderson R: Intestinal brush border function. Scand J. Gastroentrol 29:289-299, 1997

    Google Scholar 

  3. Welsh JD, Preiser H, Woodley JF, Crane GK: An enriched microvillus membrane preparation from frozen specimens of human small intestine. Gastroenterology 62:572-582, 1972

    Google Scholar 

  4. Schimtz J, Preisier H, Maestracci D, Ghosh BK, Cerdar JJ, Crane RK: Purification of the human intestinal brush border membrane. Biochim Biophys Acta 323:98-112, 1973

    Google Scholar 

  5. Hauser H, Howell K, Dawson RMC, Bowyer DE: Rabbit small intestinal brush border membrane preparation and lipid composition Biochim Biophys Acta 602:567-577, 1980

    Google Scholar 

  6. Ibrahim SA, Balasubramanian KA: Comparative study on brush border membranes prepared from rat and monkey small intestine by Ca2+ and Mg2+ precipitation. Comp Biochem Physiol 112B:65-69, 1995

    Google Scholar 

  7. Bjorkman DJ, Hagen CH, Trier JS: Structural features of absorptive cell and microvillus membrane preparations from rat small intestine. Gastroentrology 91:1401-1414, 1986

    Google Scholar 

  8. Proulx P: Structure-function relationships in intestinal brush border membranes. Biochim Biophys Acta 1071:255-271, 1991

    Google Scholar 

  9. Prabhu R, Balasubramanian KA: A novel method of preparation of small intestinal brush border membrane vesicles by polyethylene glycol precipitation. Anal Biochem 289:157-161, 2001

    Google Scholar 

  10. Otamiri T, Sjodahl R: Oxygen radicals: their role in selected gastrointestinal disorders. Dig Dis 9:133-141, 1991

    Google Scholar 

  11. Richter C, Gogvadze V, Laffranchi R, et al.: Oxidants in mitochondria: from physiology to disease. Biochim Biophys Acta 1271:67-74, 1995

    Google Scholar 

  12. Nalini S, Ibrahim SA, Balasubramanian KA: Effect of oxidant exposure on monkey intestinal brush-border membrane. Biochim Biophys Acta 1147:169-176, 1993

    Google Scholar 

  13. Lowry OH, Rosebrough NJ, Farr Al, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275, 1951

    Google Scholar 

  14. Thambidorai D, Bachhawat BK: Purification and properties of brain alkaline phosphatase J Neurochem 29:503-512, 1977

    Google Scholar 

  15. Dalhquist A: Assay of intestinal disaccharidases. Anal Biochem 22:99-107, 1968

    Google Scholar 

  16. Pfleiderer G: Particle-bound amino peptidase from pig kidney. Methods Enzymol 19:514-521, 1970

    Google Scholar 

  17. Indrani N, Hill PG: Partial purification and some properties of γ-glutamyl transpeptidase from human bile. Biochim Biophys Acta 483:57-62, 1977

    Google Scholar 

  18. Quigley JP, Gotterer SG: Distribution of (Na+-K+)-stimulated ATPase activity in rat intestinal mucosa. Biochim Biophys Acta 173:456-468, 1969

    Google Scholar 

  19. Hubscher G, West GR: Studies on the fractionation of mucosal homogenates from the small intestine. Nature 205:629-642, 1965

    Google Scholar 

  20. Dodgson KS, Spencer B: Studies on sulphatases. Biochem J 55:436-440, 1953

    Google Scholar 

  21. Djurdjic V, Mandic L: A spectrophotometric method of simultaneous determination of protein-bound hexoses and fucose with a mixture of L-cysteine and phelnol. Anal Biochem 188:222-227, 1990

    Google Scholar 

  22. Serifer A, Gerstenfeld S: Photometric determination fo sialic acid I serum and cerebrospinal fluid with thiobarbuteric acid method. Clin Chem Acta 7:467-475, 1962

    Google Scholar 

  23. Habeeb AFS: Reaction of protein sulfhydryl groups with Ellmans's reagent. Methods Enzymol 25:457-464, 1972

    Google Scholar 

  24. Ohkawn H, Ohishi, N, Yagi K: Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:351-358, 1979

    Google Scholar 

  25. Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purification. Can J Biochem Physisl 37:911-917, 1959

    Google Scholar 

  26. Chan HWS, Levett G: Autoxidation of methyl linoleate. Separation and analysis of isomeric mixtures of methyl linoleate hydroperoxides and methyl hydroxylinoleates. Lipids 12:99-104, 1972

    Google Scholar 

  27. Sohal RS, Agarwal S, Dubey A, Orr WS: Protein oxidative damage is asociated with life expectancy of houseflies. Proc Natl Acad Sci USA 90:7255-7259, 1993

    Google Scholar 

  28. Zaltkis A, Zak B, Boyle AJ: A new method for the determination of serum cholesterol. J Lab Clin Med 41:486-492, 1953

    Google Scholar 

  29. Synder F, Stephen N: A simplified spectrophotometric determination of ester groups in lipids. Biochim Biophys Acta 34:244-245, 1959

    Google Scholar 

  30. Bartlett GR: Phosphorous assay in column chromatography. J Biol Chem 234:466-468, 1959

    Google Scholar 

  31. Tiruppathi C, Miyamoto Y, Ganapathi V, Leibach FH: Fatty acid-induced alterations in transport systems of the small intestinal brush-border membrane. Biol Pharmacol 37:1399-1405, 1988

    Google Scholar 

  32. Banhegi G, Lusini L, Puskas F, Rossi R, Fulceri R, Braun L, Mile V, Simplicio PD, Mandl A, Bendetti J: Preferential transport of glutathione versus glutathione disulfide in rat liver microsomal vesicles. J Biol Chem 274:12213-12216, 1999

    Google Scholar 

  33. Otamiri T, Lindahl M, Tagesson C: Phospholipase A2 inhibition prevents mucosal damage associated with small intestinal ischemia in rats. Gut 29:489-494, 1988

    Google Scholar 

  34. Dudeja PK, Harig JM, Ramaswamy K, Brasitus TA. Protein-lipid interactions in human small intestinal brush border membranes. Am J Physiol 257:G809-G817, 1989

    Google Scholar 

  35. Ohyashiki T, Ohtsuka T, Mohri T: A change in the lipid fluidity of the porcine intestinal brush-border membranes by lipid peroxidation. Studies using pyrene and fluorscent stearic and derivatives. Biochim Biophys Acta 861:311-318, 1986

    Google Scholar 

  36. Otamiri T: Oxygen radicals, lipid peroxidation, and neutrophill infilteration after small intestinal ischaemia and reperfusion. Surgery 105:593-597, 1989

    Google Scholar 

  37. Sanfey H, Bulkley GB, Cameron JL: Pathogenesis of acute pancreatitis. Role of oxygen free radicals. Surg Forum 34:222-224, 1983

    Google Scholar 

  38. Babbs CF: Free radicals and etiology of colon cancer. Free Radic Biol Med 8:191-200, 1990

    Google Scholar 

  39. Gonzalez-Crussi F, Hsueh W: Experimental model of ischaemic bowel necrosis: the role of platelet-activating factor and endotoxin. Am J Pathol 112:127-135, 1983

    Google Scholar 

  40. Smith SM, Grisham MB, Manci EA, et al: Gastric mucosal injury in the rat: role of iron and xanthine oxidase. Gastroenterology 92:950-956, 1987

    Google Scholar 

  41. Madesh M, Balasubramanian KA: Activation of liver mitochondrial phospholipase A2 by superoxide. Arch Biochem Biophys 346:189-192, 1997

    Google Scholar 

  42. Barker MO, Brim M: Mechanism of lipid peroxidation of erythrocytes of vitamin E deficient rats and in phospholipid model system. Arch Biochem Biophys 166:32-40, 1975

    Google Scholar 

  43. Smith MW, Collan Y, Kahng MW, Trump BF: Changes in mitochondrial lipids of rat kidney during ischemia. Biochim Biophys Acta 618:192, 1980

    Google Scholar 

  44. Lin JT, Xu JT, Lovelace C, Windhager EE, Heinz. Effect of Ca on Na-D-glucose cotransport across isolated renal brush border membranes. Am J Physiol 257:F126-F136, 1989

    Google Scholar 

  45. Pind S, Kuksis A: Isolation of purified brush border membranes from rat jejunum containing a Ca2+-independent phospholipase A2 activity. Biochim Biophys Acta 901:78-87, 1987

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Gastrointestinal Sciences, The Wellcome Trust Research Laboratory, Ida Scudder Road, Vellore-632 004, Tamil nadu, India

    Ramamoorthy Prabhu & K.A. Balasubramanian

  2. Department of Surgery, Christian Medical College, Ida Scudder Road, Vellore-632 004, Tamil nadu, India

    Benjamin Perakath

Authors
  1. Ramamoorthy Prabhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Benjamin Perakath
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K.A. Balasubramanian
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Prabhu, R., Perakath, B. & Balasubramanian, K. Isolation of Human Small Intestinal Brush Border Membranes Using Polyethylene Glycol and Effect of Exposure to Various Oxidants In Vitro . Dig Dis Sci 48, 995–1001 (2003). https://doi.org/10.1023/A:1023024301913

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1023024301913

Search

Navigation