Cora J. Dillard

Measurement of fluorescent lipid peroxidation products in biological systems and tissues

Abstract A sensitive fluorometric assay for measurement of lipid peroxidation damage to biological preparations and tissues is described. The method consists of a chloroform-methanol extract of tissue followed by measurement of fluorescence characteristics of products derived from lipid peroxidation. Fluorescence analysis has been used successfully with rats and mice in vivo with stress induced during dietary and aging experiments and in peroxidation of subcellular organelles in vitro. Replicate extractions of fluorescent tissue showed a standard error of 2%. In a test of linearity with concentration, the amount of lipid-soluble fluorescent material in extracted samples was directly proportional to that added before extraction. Specific and general applications to in vivo and in vitro lipid peroxidation experiments are diseussed.

Fluorescent products of lipid peroxidation of mitochondria and microsomes

Liver microsomes and mitochondria and heart sarcosomes from rats fed diets with varying α-tocopherol concentrations and lipid contents were peroxidized over a 6 hr time period. Lipid peroxidation was measured by absorption of oxygen, production of thiobarbituric acid (TBA) reactants and by development of fluorescence. The spectral characteristics of the fluorescent compounds were the same for all peroxidizing systems; the excitation maximum was 360 nm and the emission maximum was 430 nm. As time of peroxidation increased, uptake of oxygen and production of fluorescent compounds increased. These two parameters as well as production of TBA reactants were dependent upon dietary antioxidant and all three had an inverse relationship with the amount of dietary α-tocopherol. The relationship between absorption of oxygen and development of fluorescent compounds was also dependent upon dietary polyunsaturated fats (PUFA). Subcellular particles from animals fed higher levels of PUFA produced more fluorescent products per mole of oxygen absorbed than did those from animals on a diet with lower PUFA content. TBA reacting products increased with time during the initial phase of peroxidation: in the microsomal systems their production stabilized or decreased by 4–6 hr of peroxidation. Using the synthetic 1-amino-3-iminopropene derivative of glycine as standard for quantitation of fluorescence, the molar ratios of oxygen absorbed per fluorescent compound produced were calculated. This ratio for subcellular particles isolated from rats fed diets with PUFA ratios similar to those in the average American human diet was 393∶1. The fluorescent compounds had the same spectral characteristics as the lipofuscin pigment that accumulates in animal tissues as a function of age, oxidative stress or antioxidant deficiency. The fluorescent molecular damage represented by that accumulated in human heart age pigment by 50 years of age was calculated to have been caused by approximately 0.6 μmole of free radicals per gram of heart tissue.

Whey Components: Millennia of Evolution Create Functionalities for Mammalian Nutrition: What We Know and What We May Be Overlooking

Referee: Dr. Phillip S. Tong, Associate Professor, Dairy Products Technology Center, California Polytechnic State University, San Luis Obispo, CA 93407 Nutrition is undergoing a revolution owing to the recognition that some foods contain trophic, health-promoting factors distinct from essential nutrients. In this revolution, whey is increasingly being viewed as more than a source of proteins with a particularly nutritious composition of essential amino acids. Milk evolved under continuous Darwinian selection pressure to nourish mammalian neonates. Evolutionary pressure appears to have led to the elaboration of a complex food that contains proteins, peptides, complex lipids, and oligosaccharides that act as growth factors, toxin-binding factors, antimicrobial peptides, prebiotics, and immune regulatory factors within the mammalian intestine. Importantly, these trophic macromolecules are not essential, although the health benefits that their biological activities within the intestine provide likely contributed to neonatal survival. Human and bovine milks contain many homologous components, and bovine whey may prove to be a source for molecules capable of providing biological activities to humans when consumed as food ingredients. To approach this potential, food and nutrition research must move beyond the description of food ingredients as delivering only essential nutrients and develop a mechanistic understanding of the interactions between dietary components and the metabolic and physiological properties of the intestine.

EFFECT OF DIETARY VITAMIN E ON EXPIRATION OF PENTANE AND ETHANE BY THE RAT

An analytical method for the measurement of hydrocarbon gases in the breath of rats is described. The method was used to follow the expiration in rat breath of in vivo formed scission products of hydroperoxides. The major products are pentane from the linoleic acid family and ethane from the linolenic acid family. Rats were fed 0, 11 or 40 i.u. vitamin E acetate/kg diet for 7 wk starting at age 21 days. Data obtained by gas chromatographic analysis of breath samples were analyzed by the Mann-Whitney nonparametricU-test. This statistical analysis showed that pentane evolved by the group of rats not supplemented with vitamin E was significantly higher during the period 1–7 wk than that evolved by either of the two supplemented groups of rats. Ethane from the nonsupplemented group was significantly higher than that from the group supplemented with 40 i.u. vitamin E/kg of diet by 5 wk, and significantly higher than both supplemented groups by 6 wk. By 7 wk, pentane production was tenfold greater in the nonsupplemented group than in either supplemented group, and ethane was about twofold greater. There was no significant difference between the groups supplemented with 11 and 40 i.u. vitamin E/kg diet for either ethane or pentane. This new technique, which measures scission products from in vivo lipid peroxidation, promises to be useful for application to many experimental areas where lipid peroxidation is expected or known to occur.

Composition, Structure and Absorption of Milk Lipids: A Source of Energy, Fat-Soluble Nutrients and Bioactive Molecules

Milkfat is a remarkable source of energy, fat-soluble nutrients and bioactive lipids for mammals. The composition and content of lipids in milkfat vary widely among mammalian species. Milkfat is not only a source of bioactive lipid components, it also serves as an important delivery medium for nutrients, including the fat-soluble vitamins. Bioactive lipids in milk include triacylglycerides, diacylglycerides, saturated and polyunsaturated fatty acids, and phospholipids. Beneficial activities of milk lipids include anticancer, antimicrobial, anti-inflammatory, and immunosuppression properties. The major mammalian milk that is consumed by humans as a food commodity is that from bovine whose milkfat composition is distinct due to their diet and the presence of a rumen. As a result of these factors bovine milkfat is lower in polyunsaturated fatty acids and higher in saturated fatty acids than human milk, and the consequences of these differences are still being researched. The physical properties of bovine milkfat that result from its composition including its plasticity, make it a highly desirable commodity (butter) and food ingredient. Among the 12 major milk fatty acids, only three (lauric, myristic, and palmitic) have been associated with raising total cholesterol levels in plasma, but their individual effects are variable—both towards raising low-density lipoproteins and raising the level of beneficial high-density lipoproteins. The cholesterol-modifying response of individuals to consuming saturated fats is also variable, and therefore the composition, functions and biological properties of milkfat will need to be re-evaluated as the food marketplace moves increasingly towards more personalized diets.

[41] Fluorescent damage products of lipid peroxidation

Publisher Summary Free-radical chain reactions, which take place during lipid peroxidation, lead to the formation of lipid hydroperoxides that decompose to many secondary products. Among the products, is a three-carbon dialdehyde, malonaldehyde. When ribonuclease reacts with peroxidizing polyunsaturated lipid and with malonaldehyde, it becomes inactivated and yellow fluorescent products are formed. The spectral properties of these chromophoric systems are nearly identical with those in chloroform–methanol extracts of age pigment (lipofuscin). Fluorescent product formation also occurs in peroxidizing subcellular particles, amino acid and phospholipid systems, peroxidizing arachidonic acid and DNA, and antioxidant-deficient mouse and rat tissues. A major portion of fluorescent molecular damage in biological tissues is found in the lipid-soluble phase of chloroform–methanol extracts. The simple method described is for the extraction and measurement of fluorescent molecular damage products of lipid peroxidation from animal tissues.

Fluorescent products from reaction of peroxidizing polyunsaturated fatty acids with phosphatidyl ethanolamine and phenylalanine

Fluorescent chromophores produced by reaction of peroxidizing arachidonic acid or methyl docosahexaenoate with synthetic dipalmityl phosphatidyl ethanolamine were lipid soluble, and those from reaction with phenylalanine were water soluble. In all reaction systems that contained polyunsaturated fatty acid and only one amine compound, the development of fluorescence was linearly related to oxygen absorption for 12–24 hr (p<0.001) and to the amount of thiobarbituric acid reacting materials until the rate of oxygen absorption decreased. The fluorochromes typically had maximum excitation at 360 nm and maximum emission at 430–440 nm, indicating that they were conjugated Schiff bases with the general structure R−N=C−C=C−N−R, where R represents the amino acid phenylalanine or the phospholipid phosphatidyl ethanolamine. The fluorochromes were similar to those extracted from isolated age pigments and tissues of animals that are aged, vitamin E-deficient, or stressed with highly unsaturated lipid diets.

Effects of dietary vitamin E, selenium, and polyunsaturated fats on in vivo lipid peroxidation in the rat as measured by pentane production

Starting at 21 days of age, groups of six rats each were fed a basal Torula yeast diet supplemented with 0,4% L-methionine and varying amounts of vitamin E as dl-alpha tocopherol acetate, selenium as sodium selenite, and with either 10% stripped corn oil, stripped lard, or coconut oil. By 7 wk, pentane production by rats fed a corn oil diet deficient in both vitamin E and selenium was twice that by rats fed 0.1 or 1 mg of selenium per kg of the same basal diet. Blood glutathione peroxidase activity after 7 wk was proportional to the logarithm of dietary selenium. Groups of rats fed the vitamin E- and selenium-deficient diets with lard or coconut oil had one-half the pentane production of rats fed the vitamin E- and selenium-deficient corn oil diets. The plasma level of linoleic plus arachidonic acid was 1.8 times greater on a wt % basis in rats fed corn oil than in rats fed lard or coconut oil as the fat source. Pentane production by rats fed 40 i.u. dl-alpha tocopherol acetate per kg of the selenium-deficient corn oil diet was one-sixth of that by rats fed the same diet without vitamin E; the plasma of the rats fed the vitamin E-supplemented corn oil diet had a level of vitamin E that was about six times greater than that of the rats fed the vitamin E-deficient corn oil diet.

Bioactive components in milk.

Milk is the only biomaterial that evolved under the Darwinian selective pressure to nourish growing mammals. The purpose of this article is to review the scientific research that is using new techniques of integrating biological sequence, structure and function, to understand the innovative biology underlying the products of that evolutionary pressure. As it emerges that milk is actively communicating between the maternal mammary epithelia and the infants gastrointestinal system, actively directing and educating the immune, metabolic and microflora systems within the infant, enhancing nutrient absorption and delivery, and conferring multiple means of protection, nutritionists are gaining a host of new molecular targets towards which to build scientific strategies for future foods and clinical applications. As new components and functions are being discovered in milk by using traditional methods and modern genomic tools, the complexities of demonstrating in vivo, and particularly in humans, the functional mechanisms behind milks newly observed physiological benefits are becoming the next challenge of this rapidly growing field.

Effect of antioxidants on lipid peroxidation in iron-loaded rats

Indirect evidence has suggested that lipid peroxidation is associated with iron overload in vivo. As a measure of lipid peroxidation, pentane expired in the breath of rats loaded with an accumulated dose of either 100 mg or 186–200 mg of iron injected intraperitoneally as iron dextran was measured over a 7 to 8 week period, and the effect on pentane production of feeding antioxidant-supplemented diets was determined. By the seventh week of feeding the diets, rats fed 0.3% L-ascorbic acid produced 17% less (P=0.03) pentane than did rats fed the basal antioxidant-deficient diet, whereas rats fed 0.004% dl-α-tocopherol acetate produced 92% less (P<0.001). After being fed the basal diet for 7 weeks, iron-loaded rats produced 76±9 pmol pentane/100 g body wt/min. When synthetic antioxidants were added to the diet at a concentration of 0.25%, the order of effectiveness in decreasing pentane production after 1 week was: N,N′-diphenyl-p-phenylenediamine > ethoxyquin > butylated hydroxyanisole > butylated hydroxytoluene > propyl gallate ∼ no antioxidant. After removal of either ethoxyquin or N,N′-diphenyl-p-phenylenediamine from the diets for 1 week, pentane production increased to a high level. The total amount of lipid soluble fluorophores in individual spleens of rats fed N,N′-diphenyl-p-phenylenediamine, ethoxyquin, dl-α-tocopherol acetate, ascorbic acid and no antioxidant were correlated significantly with the corresponding total integrated amount of pentane produced by the individual rats over the 7 to 8 week period. This study has provided some of the most direct evidence to date that lipid peroxidation is associated with iron overload in vivo.