fluorescent microscopic observations confirmed that iron-free apdactoferrin was bound to bacterial cell surface but iron-replete lactofemn was not. Absence of E. coli cell surface binding of saturated lactoferrin as well as apolactoferrin was shown by immunofluorescent microscopy. These data point to the possibility that bacterial grqwth is influenced by the iron-chelating properties of lactoferrin at the bacterial cell surface. The amount of lactoferrin used in the incubation mixtures was not sufficient to bind all the available iron in the medium. These careful studies provide evidence for a direct

bacterial killing action of human lactoferrin perhaps exerted at the bacterial cell surface, and suggest that the pathogenecity and virulence of some bacterial species may be determinedin pa? by the ability of the organism to synthesize iron-binding enterochelins. 0 ~

1. J.J. Bullen, H.J. Rogers and L. Leigh: IronBinding Proteins in Milk and Resistance to Es-

cherichia Coli Infection in Infants. Brit. Med. J.: 1: 69-75, 1972 2. R.R. Arnold, M.F. Cole and J.R. McGhee: A Bactericidal Effect for Human Lactoferrin. Science 197: 263-265, 1977

LIPID PEROXIDATION IN MEMBRANE LIPIDS AND ACTION OF GLUTATHIONE PEROMDASE The selenium enzyme, glutathioneperoxidase, may act to prevent formation of peroxidized fatty acids, rather than to convert these compounds to hydroxy-fatty acids. Lipid peroxidation appears to be a continuing endogenous process in liver cells, even without the additionof agents whichpromoteperoxidation. Key Words: lipid peroxidation, polyunsaturated fatty acids, glutathione, glutathione peroxidase, malondialdehyde

The ceroid pigment which accumulates with aging appears to be a complex of peroxidized lipids and proteins.192 The reasons for accumulation of these pigments are not known, but lipid peroxidationhas been suggested as a major cause. Selenium and vitamin E are, of course, the two major dietary factors which are important in the inhibition of peroxidation.3~4 The enzyme glutathionine peroxidase, a selenium-containingenzyme, appears to be an important factor in controlling endogenous formation of peroxides from unsaturated fatty adds in cell membranes.s6 A lower activity of this enzyme in selenium deficiency could explain why some symptoms of selenium deficiency can be relieved by alpha-tocopherol and other antioxidants.' Purified preparations of glutathioneperoxidaseinvitro convert hydroperoxides of unsaturated fatty acids into hydroxy-fatty acids6 This reaction has been suggested as the probable mechanism for cellular action of this enzyme in preventing peroxidation. Otherwise, peroxidation of un-

saturated fatty acids, especially arachidonic and dmsahexaenoic acids, produces malondialdehyde.' This compound can cause protein damage by reacting with amino adds, especially the epsilon-amino group of lysine. Although formation of hydroxy-fatty acids has been suggested as the mechanism of action of glutathione peroxidase, no experiments have been done to measure the formation of hydroxy-fatty acids. Also, if these products are formed from peroxides, then formation of malondialdehyde should be reduced. This hypothesis has been tested by McCay and co-workers8 When rat liver microsomes were incubated in reaction systems promoting peroxidation, the polyunsaturated fatty acid content decreased, and considerable malondialdehyde appeared. These changeswere not prevented by addition of glutathione alone. Polyunsaturatedfatty acids still decreased and malondialdehyde appeared when glutathione peroxidase was added without addition of ,,glutathione. When both glutathione and glutathione peroxidase were added, there was little loss of polyunsaturated fatty acids, and little formation of malondialdehyde. Yet no NUTRITION REVIEWS IVOL. 36, NO. 1, I JANUARY 1978 23

hydroxy-fatty acids were found in the fatty acids isolated from phospholipids. These data indicate that glutathione peroxidase did not prevent malondialdehyde formation by conversion of peroxides to hydroxy-fatty acids, but rather must have prevented peroxide formation from fatty acids. McCay and co-workers also studied lipid peroxide formation and membrane fatty acid alterations in isolated rat liver Since isolatedcells are independentof bloodflow and metabolic contributions of other tissues, their cellular environment can be more closely controlled than is possible with animals. Liver cells from fasted rats were incubated in bicarbonate buffer. Malondialdehyde formation was again used as the index of peroxidation. When increased peroxide formation was needed, carbon tetrachloride was added. Previous work showed that the treatment of liver membranes with carbon tetrachloride produced small amounts of conjugated diene fatty acids, together with decreases in polyunsaturated fatty acids.10 As expected, more malondialdehyde was formed when carbon tetrachloride was added, and there was also loss of fatty acids and protein from microsomal membranes in these cells. Cells of rats previously treated with phenobarbital also formed larger amounts of malondialdehyde. Phenobarbital increases formation of the endoplasmic reticulum and cons& quentiy the amount of fatty acids available for peroxidation, since lipids of the endoplasmic reticulum are rich in unsaturated fatty acids. A most significant observation was the fact that definite amounts of malondialdehydewere formed in cells of normal rats fasted for 24 hours. This occurred without the addition of carbon tetrachloride or pretreatment with phenobarbital. The amount formed by cells from the fasted rats was greater than reported for cells from fed rats,ll perhaps because the glutathione level is lower in fasted rats.12 The higher level in fed rats may protect against peroxidation. These results emphasize that peroxide formation is a normal occurrence in animal cells. Therefore this observation provides a basisfor accumulationof peroxidizedlipids and ceroid pigments as animals age. This isolated 24 NUTRITION REVIEWS I VOL. 36, NO. 1 JANUARY 1978

cell system should be useful for study of conditions which might protect against peroxidation. The isolated hepatocytes constitute a more physiological system than isolated liver membranes. Observations made with these cells can be expected to be similar to what occurs in intact animals. 0

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3. 4.

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N. Haugaard: Cellular Mechanisms of Oxygen Toxicity. Physid. Rev. 48: 311-373, 1968 M. Hasan and P. Glees: Genesis and Possible Dissolution of Neuronal Lipofuscin. Gerontologia 18: 217-236, 1972 Seleniumand Human Health.Nutrition Reviews 34: 347-348, 1976 Selenium: An Essential Element for Glutathione Peroxidase. Nutrition Reviews 31: 289-291, 1973 L. Flohd and R. Zimmerman: The Role of GSH Peroxidase in Protecting the Membrane of Rat Liver Mitochondria. Biochim. Biophys. Acta

223: 210-213, 1970 6. B.O. Christophersen: The Inhibitory Effect of Reduced Glutathione on the Lipid Peroxida-

tion of the Microsomal Fraction and Mitochondria. Biochem. J. 106: 515522, 1968 7. Malondialdehyde and Liver DNA. Nutrition Reviews 27: 316-318, 1969 8. P.B. McCay, D.D. Gibson, K-L. Fong and K.R. Hornbrook: Effect of Glutathione Peroxidase Activity on Lipid Peroxidationin Biological Membranes. Biochim. Biophys. Acta 431: 459-468, 1976 9. C.C. Weddle, K.R. Hornbrook and P.B. McCay: Lipid Peroxidation and Alteration of Mem-

brane Lipids in Isolated Hepatocytes Exposed to Carbon Tetrachloride. J. Biol. Chem. 251: 4973-4978, 1976 10. B.K. Tam and P.B. McCay: Reduced Triphos-

phopyridine Nucleotide Oxidase-Catalyzed Alteration of Membrane Phospholipids. Ill. Transient Formation of Phospholipid Peroxides. J. Bid. Chem. 245: 22952300, 1970 11. J. Hogberg, P. Molddus, 8. Arborgh, P.J. OMen and S. Orrenius: The Consequences of Lipid Peroxidation in Isolated Hepatucytes Europ. J. Biochem. 59: 457-462, 1975 12. N. Tateishi, T. Higashi, S. Shya, A. Naruseand Y. Sakamoto: Studies on the Regulation of Glutathione Level in Rat Liver. J. Biochem. 75: 93-103, 1974