manna.- "r .‘ m. V. V «53‘ ”r , . .53: .1: ¢. 7-,, -‘ - Ewaflnidt , amutflflr ‘ ‘, ri- - f ‘1‘ i '- .79.“ ’46?» a J," ' A.- ..‘JE? ’ .rlzi" “:2. - v» Wag-W wharf «3’- '3 «$5223 w-Fn‘nr-q "0‘1"" .,-.. :.'I-‘JZi-" " '4’ u: . n IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Lllils 3 1293 006296 LIBRARY Minot: State University This is to certify that the thesis entitled SUPPLEMENTAL REDUCED GLUTATHIONE, VITAMIN E AND SELENIUM FOR THE WEANLING PIG presented by Lora Lynn Foehr has been accepted towards fulfillment of the requirements for Master of Selenced' egree inAnJ'maLSnience J/mflf/fl Majorropfess Date 8-8-89 0-7 639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or botoro die or». DATE DUE DATE DUE DATE DUE MSU Is An Affirmative Action/Equal Opportunity Institution SUPPLEMENTAL REDUCED GLUTATHIONE, VITAMIN E AND SELENIUM FOR THE WEANLING PIG BY Lora Lynn Foehr A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1989 boBOYOl ABSTRACT SUPPLEMENTAL REDUCED GLUTATHIONE, VITAMIN E AND SELENIUM FOR THE WEANLING PIG By Lora Lynn Foehr Thirty-two 28-day old weanling pigs were used to determine the influence of supplemental reduced glutathione, vitamin E and selenium on their biological antioxidant status as indicated by plasma concentrations of selenium, a-tocopherol, glutathione peroxidase, aspartate aminotransferase, alanine aminotransferase, and the concentration of reduced glutathione in whole blood. Pigs were weighed and blood samples taken initially and weekly throughout a 4-week study. Plasma a-tocopherol concentration was elevated in pigs fed supplemental vitamin E. Plasma selenium, glutathione peroxidase and whole blood reduced glutathione were elevated in pigs receiving supplemental selenium. Plasma alanine aminotransferase was slightly lower in pigs fed supplemental reduced glutathione. There is no firm evidence that reduced glutathione is a limiting factor in the function of the glutathione peroxidase system of the weanling pig. ACKNOWLEDGEMENTS Upon completion of my Masters of Science Degree I wish to express my deepest thanks to Dr. E.R. Miller. Without his patience and understanding I would not have finished this part of my education. I would also like to thank the rest of my committee members, Dr. D.E. Ullrey, Dr. M.G. Hogberg and Dr. H.D. Stowe. I appreciate the time and advise each has given to me. I am indebted to Dr. P.K. Ku and Phyllis Whetter for their professional assistance and knowledge given in the laboratory. I also wish to thank my fellow graduate students who have given their time, support and friendships which have meant so very much to me. Lastly, but perhaps most importantly, I wish to express my most sincere appreciation and love to my parents who have always been my biggest supporters. Thank—youll TABLE OF CONTENTS LIST OF TABLES. . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . I. INTRODUCTION. . . . . . . . . . . II. REVIEW OF LITERATURE . . . . . . . A. Biological Need of Antioxidants 1. Definition. . . . . . . . 2. Free Radicals . . . . 3. Lipid Peroxidation. . . 4. Antioxidation . . . . . . B. Glutathione. . . . . . . . . . 1. Definition. . . . . . . . 2. 6-Glutamyl Cycle. . . . . 3. Redox Importance. . . . . 4. Metabolism. . . . . . . . C. Vitamin E. . . . . . . . . . . 1. Definition. . . . . . . . 2. General Functions . . . . 3. Antioxidation . . . . . . D. Selenium . . . . . . . . . . . 1. Definition. . . . . . . . 2. Metabolism. . . . . . . . 3. Functions . . . . . . . E. Summary. . . . . . . . . . . . III. MATERIALS AND METHODS . . . . . . A. Experimental Design. . . . . B. Sample Collection and Handling C. Analyses . . . . . . . . . . D. Statistical Analyses . . . . . IV. RESULTS AND DISCUSSION . A. Glutathione. B. Vitamin C. Selenium V. CONCLUSIONS . VI. E. LIST OF REFERENCES iv TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE 1. 2. 3. 10. 11. 12. 13. LIST OF TABLES FORMATION OF FREE RADICALS. . . . . . COMPOSITION OF DIETS. . . . . . . . . COMPOSITION OF VITAMIN-TRACE MINERAL PREMIX O O O O O O O O O C C O O O C CALCULATED ENERGY AND NUTRIENT ANALYSIS 0 O O O O O O O O O O O O O BODY WEIGHT OF PIGS AS INFLUENCED BY DIET AND TIME ON TRIAL . . . . . . FEED EFFICIENCIES . . . . . . . . . . PLASMA REDUCED GLUTATHIONE CONCENTRATIONS OF PIGS . . . . . . . PLASMA GLUTATHIONE PEROXIDASE ACTIVITY OF PIGS . . . . . . . . . . PLASMA ALPHA-TOCOPHEROL CONCENTRATION OF PIGS. . . . . . . . PLASMA SELENIUM CONCENTRATION OF PIGS O O O O C O O _ O O O O O O O PLASMA ASPARTATE AMINOTRANSFERASE ACTIVITY OF PIGS O O O O O O O O O O PLASMA ALANINE AMINOTRANSFERASE ACTIVITY OF PIGS. . . . . . . . . . ANALYSIS OF VARIANCE SIGNIFICANCE LEVELS O C C O O O C O C C O O O O O 44 45 46 55 56 69 7O 71 72 73 74 75 LIST OF FIGURES Figure 1. Possible damaging reactions of free radicals. O O O O O O O O O O O O O 0 Figure 2. The diatomic oxygen molecule . . .- Figure 3. Reduced glutathione. . . . . . . . . Figure 4. The gamma-Glutamyl cycle . . . . . . . Figure 5. Metabolic moduration of redox states in proteins . . . . . . . . . . . . . Figure 6. Action of glutathione reductase. . . . Figure 7. Levels of reduced glutathione in hamster oocytes O O O O O O O O O O O I O O 0 Figure 8. The interrelationships of GSH. . . . Figure 9. GSH metabolism and function. . . . . . Figure 10. Possible effects of GSH-GSSG disturbance. . . . . . . . . . . . . Figure 11. GSH metabolism. . . . . . . . . . . . Figure 12. Inter- and intraorgan metabolism and transport of GSH . . . . . . . . . . Figure 13. Mechanisms for antioxidative defense. Figure 14. Alpha-tocopherol (5,7,8-trimethyltocol) . . . . . . . Figure 15. Vitamin E reduction . . . . . . . . . Figure 16. Effects of supplemental GSH on plasma ALT . . . . . . . . . . . . . Figure 17. Effects of supplemental vitamin E on plasma vitamin E . . . . . . . . . . Figure 18. Effects of supplemental Se on plasma Se levels . . . . . . . . . . Figure 19. Effects of supplemental Se on plasma GSH-px. . . . . . . . . . . vi Figure 20. Effects of supplemental Se on whole blood GSH. . . . vii 66 I . INTRODUCTION The swine industry is becoming increasingly more competitive. It is economically vital for producers to raise pigs at the least possible cost. A source of expense is pig mortality, with death loss at weaning constituting a major portion. Physiological factors affect pig viability at weaning (Kornegay, 1980). Three changes occuring at weaning which affect the pigs physiological status are environmental, immune-competent and nutritional. At weaning pigs are removed from their dams, possibly intermixed with other litters, and placed in a new pen. Weaning necessitates that pigs adapt to entirely new physical surroundings. A weaned pig is also no longer receiving antibodies from it's dam and, in addition, it's own immune system is functioning only minimally. Weaning also introduces a different diet. Prior to weaning, milk has been the major feed consumed by the pig, and the gastrointestinal environment is developed to digest milk quite efficiently. Cereal-based diets require different enzymes for efficient digestion, and these are not yet in great concentration at weaning. These major changes at weaning introduce stresses to the pig. Newly weaned pigs characteristically respond to these stresses by going "ofif feed." _Nutrient deficiencies 2 are therefore more likely to develop during this period. Mahan (1986) suggested that serum tocopherol and selenium concentrations are significantly lower in the early post weaning period. Michel et al. (1969) and Trapp et al. (1970) diagnosed and characterized vitamin E selenium deficiency in swine herds. Some of the characteristic deficiency signs were sudden death of weanling pigs, liver necrosis, skeletal and cardiac muscle degeneration and edema. Researchers in Japan observed similar signs in yellowtail fish (Imada and Suga, 1986). Elevated serum enzymes, indicative of cellular damage, and hepatic disorder were characteristic of yellowtail fish with nutrient deficiency. In severely deficient fish, death resulted. Researchers at Kyowa Hakko, the parent company of Biokyowa, experimented with various compounds in an attempt to prevent the deficiency problem. Vitamin E was found to give some protection; however, another compound, reduced glutathione, was found to significantly lower certain serum enzyme activities and increase survival rate in yellowtail fish. The purpose of the following study was to determine the effects of supplementing reduced glutathione, vitamin E and selenium to the diet of the weaning pig. II. LITERATURE REVIEW A. Biological Need for Antioxidants 1. Definition Living organisms exist in a dynamic state. Within organisms the biochemical status is a continuum of anabolic and catabolic processes. In order for these processes to continue, energy is required. Highly regulated oxidative reactions are the ultimate source of energy for living organisms. The reduction of molecular oxygen to water is the final step in the process producing energy in the form of ATP. Intimately linked with biological oxidations is the process of electron flow and transfer. In addition to oxidation reactions being required to sustain life, they can generate products which are potentially harmful to the organism. Some of these products are free radicals (Table 1). 2. Free Radicals Molecules which contain an unpaired electron in their outer orbital are considered to be free radicals (Slater et al., 1987). These molecules may also be produced by the impact and/or absorption of radiation. Metabolism of xenobiotics can also generate free radical products. The metabolism of t-butyl hydroperoxide by erythrocytes produces 3 TABLE, 1. FORMATION OF FREE RADICALS E33333; """"""""""""""""""""""""""" (1) 'By the impact or absorption of radiation, or both: (a) high energy or ionizing radiation (b) ultra-violet radiation (c) visible light with photosensitizers (d) thermal degradation of organic material (2) By electron transfer ('redox') reactions: (a) catalysed by transition metal ions (b) catalysed by enzymes Modified from Slater et al. (1987) 5 t-butyl radicals (Trotta et al., 1983). The liver metabolizes carbon tetrachloride to trichloromethyl radical (Dianzani, 1987). Free radicals are usually very reactive, and are able to participate in numerous reactions such as: (l) electron donation, (2) electron acceptance, (3) hydrogen abstraction,(4) addition reactions, (5) self-annihilation reactions, and (6) disproportionations (Slater, 1984). A free radical's versatile and highly reactive nature make macromolecules such as proteins, lipids, carbohydrates and nucleotides susceptible to their action (Figure 1). Molecular oxygen is a free radical containing not one but two unpaired electrons in its outer orbital. (Figure 2). Oxygen is ubiquitous and required for aerobic life: however, it is also potentially toxic (Haugaard, 1968) Both electrons in oxygen's outer orbital have parallel spins, therefore, for oxygen to combine with another molecule in a two-electron reduction, there must be two corresponding electrons with antiparallel spins. This is in accordance with the Pauli principle. Consequently, oxygen is slow to react with non- radical molecules, and is more apt to undergo one-electron reduction reactions. In a review by Halliwell and Gutteridge (1984), it was stated that molecular oxygen can become more reactive if its spin restriction is removed. Singlet state oxygen has had the spin restriction removed by a rearrangement of the electrons. Some biological pigments have the ability to capture the energy necessary to raise oxygen to a 'singlet state'. Singlet oxygen is electrophilic and capable of reacting directly with surrounding molecules, mmoeoncaum_c becameogu .mevuuoca o» menace oeaeosoz mocasasee teguo mo mco_uucae ecu cu we seem .moeuumwu a an moueanczumwv oepmaau mausuotq agaveoumm co_uucac can ugzuuacum accesses :. «accuse .eovuau.xogea p.394 co>o=c=u :_euoce nemaogu:_ .mevouoga ou.oaesao ampponouoe u_a,— use mo.uv>.uuo oex~ee e. momeogu u_e._ use c.0aoca ea oepueva acupe>ou assoc. ou.c_:m_uu_a.ga e. dosage asexueo uemueoeourpowgu cu oucancaum_o :Aavowuua «exuceou ou.uoo_o:e so copuuaeummo m=o_uau:: mousse (zo sizes. ._.a m_.u_u.¢ mesa e>puueu¢ Possible damaging reactions of free radicals Figure 1. Adapted from Slater et al. (1987). O O O 6) CD 69 0 69 CD 69 M3<>oc>®<>® .2. 3 GD GD 0.2, ® ® .. ® @ 69 ~ 69 @ GD .. ® @ ® Figure 2. The diatomic oxygen molecule. Taken from Halliwell and Gutteridge (1984). @©©@@ @690 ©@ at “‘9 éfeeeee @690 or it may transfer its energy to another molecule causing it to become excited. Molecular oxygen's complete reduction requires the addition of four electrons. The intermediate reduction species in this process are known to be reactive and capable of damaging cellular processes. The first intermediate formed by the addition of a single electron is the superoxide radical (02-). According to Kanner (1987), activation of granulocytes, subcellular organelles and certain enzymes, the autoxidation of some molecules, and the oxidation of reduced metals are all reactions in which 02- may be produced. Most body tissues and fluids have a pH in the range of 6.4-7.5, therefore, most 02- remains in the unprotonated form. The protonated form (H02-) is a more powerful reducing and oxidizing agent than 02-. The superoxide radical acts mainly as a nucleophile, and is therefore able to reduce surrounding compounds. The destruction of 02- is usually through a dismutation reaction, where both protons and an enzyme catalyst are required. Superoxide dismutase (SOD) is the enzyme. The general dismutation reaction is as shown: SOD 02- + 02- + 2 H+ ------ > H202 + 02 An electron addition to superoxide produces the peroxide ion, (02-2),which has no unpaired electrons. Any 02-2 formed at‘a physiological pH will protonate to yield hydrogen peroxide (H202). There are also other reactions and enzyme systems which produce H202 as a product without having the 9 02-2 as an intermediate (such as D-amino acid oxidase in the peroxisome). Human granulocytes are yet another source of H202, which is produced during the phagocytic function (Root et al., 1975). Hydrogen peroxide, like 022', is not a radical. Usually H202 acts as a weak oxidizing agent, and is able to penetrate membranes fairly rapidly due to its nonpolar properties. Its precursor, 02'2, is not as capable of penetrating membranes. Another toxic product of intermediary oxygen metabolism is the hydroxyl radical (OH'). Homolytic fission of H202 will produce two OH‘ radicals. Metals such as iron (Fe) and copper (Cu) are also able to generate the OH' radical from H202, as shown below: (1) Fe2++ 3202 ---> Fe3+ + on- + on- (Fenton) Fe3+ (2) o - + H o ---> OH- + OH° + o (Haber Weiss) 2 2 2 2 (3) Cu+ + H20 ----> Cu2+ + on- + on: The reaction involving ferrous iron (Fe+2) has been termed the Fenton Reaction, and that involving ferric, the Haber- Weiss Reaction. The superoxide radical and H202 are also 3+ involvement thought to be capable of interacting without Fe to form water and the superoxide radical (Halliwell, 1985). The OH' radical is extremely reactive and undergoes three primary reactions: (1) hydrogen abstraction, (2) addition, and (3) electron transfer. Due to the highly reactive nature of the OH' it will affect molecules in the immediate vicinity of its production site. Oxygen intermediates have been associated with various 10 cellular injuries and diseases. In a series of papers by . Davies (Davies, 1987; Davies et al.,l987a,b; and Davies and Delsignore, 1987; and Davies et al., 1987) 'the disruption of various proteins by oxygen radicals was studied. These authors suggest, that proteins have an increased proteolytic susceptibility due to oxygen radicals (specifically OH°), causing an initial alteration of the primary protein structure. Alterations at the primary level lead to modifications of secondary and tertiary structures causing the peptide to unfold. Unfolding exposes previously "hidden" peptide bonds to protease hydrolysis. The hydroxyl radical was produced when oxyhemoglobin and methemoglobin were in the presence of H202 (Puppo and Halliwell, 1988) as well as other uncharacterized radical species. The authors suggest that H202 decomposed the heme moiety of methemoglobin causing the release of iron. The OH° was then produced via an iron-H202 interaction, as inthe Fenton reaction. Oxyhemoglobin, in the presence of H202, also produced a reactive species, however, it did not appear to be OH'. The ability of free hemoglobin to produce radicals in the presence of H202 may provide an explanation of hemoglobin toxicity. Nohl and Jordan (1987) performed an in vitro experiment incubating semiquinones with H202. They found that these radicals were capable of combining with H202 in a manner similar to the Haber-Weiss reaction. From this information, it appears that quinones normally involved in physiological processes may also play a role in catalyzing 11 the production of free radicals. Hydrogen peroxide is thought to act on the adenylate cyclase system of the B-adrenoceptor system, as well as to initiate lipid peroxidation. 8- adrenoceptor hyperstimulation and excessive radical formation have been linked with cardiotoxicity. Haenen et a1. (1988) incubated calf heart membranes with H202 to monitor its influence on the fi-adrenoceptor system. Hydrogen peroxide caused and increase in lipid peroxidation and a reduction in adenylate cyclase activity. Catecholamines, capable of autoxidation, may induce their myocardial toxicity through the production of H202. 3. Lipid Peroxidation Lipid peroxidation, according to Pole and coworkers (1987), is a degradative process which is a consequence of the production and propagation of free radical reactions, primarily involving membrane polyunsaturated fatty acids (PUFAs). Girotti (1985) defined lipid peroxidation as the process of formation and breakdown of lipid hydroperoxides which are dioxygen adducts of unsaturated lipids. Lipid peroxidation has been associated with various cellular disorders, however; lipid peroxidation's role as a cause or effect of cell damage is uncertain. Some of the cellular disorders with which lipid peroxidation has been associated are: (1) membrane bilayer structural derangement and altered fluidity, (2) increased permeability of cytosolic constituents, (3) lysosomal enzyme release, (4) inactivation of intrinsic enzymes and transporters, (5) cross-linking of 12 , lipids and proteins, (6) polypeptide strand scission, (7) DNA damage and mutagenesis, and (8) depletion of NADPH due to antioxidant activity. Ivanov (1985) characterized some of the effects observed with lipid peroxidation as changes in the essential properties of membranes, such as permeability, viscocity, phase transitions, or as a change in lipid-protein interactions resulting in a potential loss of the enzyme activities of the membrane-bound and soluble systems. Haenen et al. (1988) found lipid peroxidation to occur in the calf heart membranes used in their experiment. Increased lipid peroxidation has been observed with some oxidatively stressful conditions and injuries. Davies et al. (1982) found a greater concentration of lipid peroxides in exercised rats than in sedentary controls. An earlier study by Brady and coworkers (1979) also showed an increase in lipid peroxidation products in exercised rats. Initiation of lipid peroxidation may or may not be enzymatically catalyzed. In a study using rat lysosomes, Fang et al. (1973) looked at how flavin enzymes affected membrane lipids. They observed increased lipid peroxidation of the membrane. They concluded that lysozyme enzymes generated 02-2, which became protonated to H202. Hydrogen peroxide, in the presence of iron, produced the free radical OH‘ in a Fenton reaction. The initiating event of lipid peroxidation is the interaction of a free radical with an unsaturated fatty acid causing a hydrogen to be abstracted from the lipid. The resulting alkyl radical (L') of the 13 fatty acid undergoes an intramolecular rearrangement forming a more stable conjugated diene. Molecular oxygen is able to add on to this alkyl radical forming the peroxyl radical (LOO'). The LOO‘ is then able to abstract a hydrogen from another PUFA, generating lipid hydroperoxide (LOOH) and another alkyl radical. It is obvious that the effects of a single free radical initiation reaction may be greatly amplified, especially in oxygen and unsaturated lipid environments. Several termination reactions of lipid peroxidation are known to occur, some of which are listed below: (1) 2 L' ------ > LL (2) 2 L00' ------ > LOOL + 02 (3) L' + LOO' ---> LOOL (4) RH + L‘ ----- > R' + LH (5) RH + LOO' ---> R‘ + LOOH 4. Antioxidation In order for organisms to survive they must have defense mechanisms to regulate the production and destruction of free radicals. Some molecules which are thought to have a role in antioxidation reactions are ascorbic acid, 8- carotene, a-tocopherol, and glutathione (Slater et al., 1987: Kanner et al., 1987). Usually, a good antioxidant must be an effective hydrogen or electron donor. Enzymes are also involved with antioxidation. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-px) are the three main antioxidant enzymes. Two forms of SOD exist, a cytosolic form containing copper and zinc, and a mitochondrial form containing manganese. SOD catalyzes 02—2 dismutation. As shown belowUICAT is involved in the destruction of H202, and is located within the peroxisomes. CAT H202 + H202 ----> 02 + 2 H20 Glutathione peroxidase, located both in the cytosol and mitochonddria, also catalyzes the destruction of H202, but is capable of destroying other peroxides as well. According to Simmons and Jamall (1988), GSH-px has a more important role than CAT in controlling peroxidation products. In its reduction reaction with peroxides, GSH-px utilizes reduced glutathione (GSH) as its electron donor substrate. Reed et al. (1986) found GSH-px activity to be critical in the prevention of damaging effects caused by cellular bioreduction reactions. B. Glutathione 1. Definition The substrate, glutathione (GSH), is widely distributed in animal and plant cells as well as in microorganisms. It is probably the most abundant low- molecular-weight thiol, as well as perhaps the most important nonprotein thiol in living systems. Reduced gluthathione is composed of three amino acids: glutamate, cysteine and glycine (Figure 3). Reduced gluthathione has two characteristic structural features: a sulfhydryl (SH) group on the cysteine moiety, and a 6-glutamyl linkage between the glutamate and cysteine residues (Meister and Tate, 1976). 15 Glutamic Acid NHZ HOOCCHCHZCHZCO - NH Glycine ‘ [_ [ascnzcaco f Nchzcmrrl Cysteine ' Figure 3. Reduced Glutathione. The glycine moiety protectsHESH from the degradative action of 6-glutamyl cyclotransferase, and the 6-glutamyl linkage of GSH renders it nonsusceptible to peptidases which cleave a- bonds. The 6-glutamyl bond may be degraded by the enzyme 6- glutamyl transpeptidase (6-GT). 6-GT appears to be mainly a membrane bound enzyme, and it hydrolyzes GSH into 6-glutamyl and cysteinyl-glycine. Once this occurs, peptidase enzymes are able to break the cysteine-glycine bond. 2. 6-Glutamyl Cycle Gamma-glutamyl transpeptidase action is the first step in GSH breakdown. The biodegradation and biosynthesis of GSH is cyclical in nature and involves enzyme-catatyzed reactions. This "cycle," described by Meister (1981) and Meister and Tate (1976), is referred to as the S-Glutamyl cycle (Figure 4). GSH concentration determines the activity of 6-CC by nonallosteric feedback inhibition, therefore GSH acts as its own regulator. The intracellular concentration of GSH ranges from 1-50x10'4 M (Kosower and Kosower, 1976), and it may exist in heterogeneous pools (Tateishi et al., 1977). Reduced glutathione, through the action of GSH-px, becomes oxidized to yield oxidized glutathione (GSSG). Oxidized glutathione can be found in cells, but it is usually at a much lower concentration than GSH (6-200x10'6M). The ratio of GSH to GSSG has been used as an indicator of oxidant stress (McCoy et al., 1988). In a study utilizing exercised verses sedentary control rats, Lew and coworkers (1985) observed decreased GSH and increased GSSG in exercised rat 17 23 2.3.3. E +eo< 2.32.936 .ocsesgn. zoouk .>¢u>ouue. uzzzuk ~36. fiml 2 GSH + NADP+ GSH may also form mixed disulfides with cellular proteins. The structural integrity and maintenance of the functional processes of cell membranes and organelles are very important. According to Kosower and Kosower (1978), GSH, GSSG and SSS-protein are associated with numerous aspects of normal cellular structural maintenance and functional processes . 3. Redox Importance Olafsdottir and Reed (1987) observed that rat liver mitochondrial oxygen consumption leads to H202 production, and this in turn may cause lipid hydroperoxide development. Paller and Sikora (1988) estimated that from 1 to 10% of the oxygen in the respiratory chain is not entirely reduced. Reduced glutathione is used by GSH-px to reduce this oxygen, however, the concurrent oxidation of GSH has been shown to +2 permeability of the inner coincide with an increase in Ca mitochondrial membrane. GSH oxidation may therefore decrease a cell's ability to reduce important protein thiol groups involved with membrane function. Prasad et a1. (1986) suggested that any toxic process which increases the free 19 Muted mankks Mflflxfic Mannnmi Figure 5. Metabolic moduration of redox states- in proteins. Taken from Inoue et al. (1987). 20 - - G-s-Poeh dro enose Glucose 6 phosphate ’ 9 4 s-Phosphoglucoloctone nape? NADPH V Gluiolhione Reducmse A 500 Glutothione Peroxidase o —. H30; * , —: H20 2H corolose H20 I 0; Figure 6. Action of glutathione reductase. Taken from Kanner et al. (1987). ‘ 21 calcium concentration by inactivating either microsomal or +2 may lead to significant cellular mitochondrial pumps for Ca damage. Inactivation is usually via oxidation of protein sulfhydryls resulting in disulfide formation. GSH appears to reverse these effects (Erickson et al., 1987; Reed et al., 1987). Jones et al. (1983) also reported that thiols (GSH) 2+ sequestering system of the endoplasmic may protect the Ca reticulum. Comporti (1987) observed that certain xenobiotics exert their toxicity by causing decreases in GSH, resulting in the subsequent disulfide formation of vital sulfhydryls. Teaf and coworkers (1987) looked at male reproductive tissue in rats, and observed that protein alkylation, particularly of the cysteine-riCh protamines, resulted in toxic effects on spermatozoa. Disulfide crosslinks within protamines may be one reason spermatozoa have an extraordinary resistance to both chemical and mechanical insult. Reduced glutathione status appears to be involved in the resistance. Reduced glutathione status may also play an important role in the process of fertilization. It was shown, in a study by Perreault et al. (1987), that disulfide bonds in sperm have to be reduced in order for the nucleus to become reactivated. They concluded that mature hamster oocytes have more GSH than their immature precursors or those oocytes already fertilized (Figure 7). Mature oocytes have the capability to reduce the protamine disulfides via GSH, thereby initiating nuclear decondensing activity. Kuross and associates (1988) hypothesized that thiol oxidation was a potential explanation 22 HAMSTER OOCYTE GSH 2 G83 (pHolu/oooyto) 1.5 .. ..... \ - ................... l ''''''''''''''''''''''''''''''''''''' - . lI .................. . _, L ..................................... 1“. ‘.‘- ‘:‘ . . _ '7’ ...................................... , l ---------------------------------- . 9' 7 L ..................... . 1 :- -.._«—~ -_ » :-:-:.:-:-:-:-:-:-:-:-:-:-:.:-:-:-:~ ------------------ - ~ r . 1. , . i + ..................................... Hamster oocyte typ es r - Genuine! miolo um own-d Fertilizer! Figure 7. Levels of GSH in hamster oocytes. Adapted from Perreault et al. (1988). 23 , for some of the defects seen in sickle red blood cells (RBC). Garel et al. (1986) also worked with sickle RBC's, and they found GSH to be beneficial in reducing the severity of sickling symptoms. Reduced glutathione combined with hemoglobin to form a mixed disulfide; this resulted in increased oxygen affinity and a decrease in the incidence of sickled RBC's. Increasing the intracellular GSH concentration may have beneficial effects on cells. Mitchell (1988) suggested that the amount of intracellular GSH influences the cytotoxicity of cancer treatment modalities. Reduced glutathione has such functional diversity and assumes such a pivotal role in numerous bioreductive reactions that GSH modulation could obviously have an impact on the entire system (Figs. 8, 9 and 10). Arrick and coworkers (1982) observed an increased sensitivity of GSH-depleted tumor cells to oxidative cytolysis. Modulation of GSH is known to affect other processes as well (Figure 10). Fidelus et al. (1987) suggested that GSH plays an important role in lymphocyte activation and proliferation. Honda and Matsuo (1988) found that increasing the cellular CSH level in human diploid fibroblasts extended their in vitro life span. These cells are known to have a finite, replicative life span, and are used as a model system to study aging at the cellular level. Jensen and Meister (1983) found human lymphoid cells depleted of GSH to be more radiosensitive. In looking at the lens of 24 Y-Giutemyi u. Cysteine Trenspeptidase ‘1, . Drugs ‘ Ribosome Protein _ Xenobiotics . integrity Synthesis 02. GSH SOD Trsneterese ’ Mitochondrie Selenium Thloi Irensierese Pentose . " ‘ . , .- Thioredoxin Shunt ion Deoxyribose Transpor . Synthesis Glucose Bio- Membrane DNA synthesis ”“9"” Synthesis .. 'v. Q . 1 \ / s, .. Leukotreines’ Prostagiandins Figure 8. The interrelationships of GSH. Taken from Mitchell (1988). 25 ”GSSG ’ I m\‘W)ie'A coenzwmc ’ FUNCTIONS IICS GSSG + ROH + H20 Jorgensen and Wegger (1979), as well as Jorgensen et al. 32 T-I.|l'll' I! ’IC'.‘IIII -zllI 0..vl.lll I'l"'|ll'. l-.illl|l‘l'|".llli'll|ab-. 3 . ......s. n .. ...s. . 33.; .o..:~. . .c 3.... 25.1.... - al-!!! O'ln.|ill. .16.: i. - III, £29.. 5:... >555: 2......- 3...... .fos..: .- o i o o "“oioc‘.-aulll“ 00000 0.8.2.3.}; 3.5.8.3! “000 ~ - - — _ - O -‘---—-—-—-—-—-- . 6.3.3:...21. .3... 3.3.2.3.»...3 38):: .58.»..33 3.2.5.... 8.3.21.3... (20 .o ..o...._....... _ _ . ..O . .3. . .5 ‘l.:-i..-.|ll.l ~09. ......us... 3...; .23: / \ .. 4. use... . .o: . 1A,!!! 6...“. ~... A” P s 3.61.30 ..-H .... . .o 66a .J .o ... «0 ‘Il €3.53... 0.3.25.0... Hechanisms for antioxidative defense. (1987). Taken from Das et al. Figure 13. 33 (1977), observed that pigs with higher GSH-px activity were less susceptible to disease than pigs having low GSH-px activity. Friendship and Wilson (1985) found a high degree of variability in the blood GSH-px levels among pig litters. They also found that GSH-px concentrations in one-day old pigs were not associated with body weight, but were, however, weakly correlated with piglet viability. Neonatal pigs with higher GSH-px concentrations may have a greater survivability rate. Mills (1959) suggested that GSH-px protects cells from the deleterous effects of H202. Flohe and Zimmermann (1970) found GSH-px to be an essential factor in preventing lipid peroxide accumulation and lysis of rat mitochondrial membranes in vitro. McCay (1976) proposed that GSH-px exerted its affect by preventing the initial attack on polyunsaturated membrane lipids by free radicals. When GSH becomes oxidized by GSH-px activity it may be regenerated through the reducing action of glutathione reductase (GR). GR is a riboflavin-containing flavin adenine dinucleotide enzyme. Pigs on a riboflavin-deficient diet exhibited a reduced amount of active GR within red blood cells (Brady et al., 1979). Glutathione reductase activity in muscle and liver was unaltered by riboflavin deficiency. Reduced glutathione within these cells was not found to be significantly affected. Paniker et al. (1970) also looked at GR in riboflavin deficient red blood cells. Their results were similar to Brady and colleagues (1979) in that GR did not seem to be a limiting factor in GSH production. 34 C. Vitamin E 1. Definition Vitamin E is a fat-soluble vitamin. Compounds which exhibit vitamin E activity have a 6-chromonal ring structure with an associated side chain. Side chain structure separates vitamin E into two main types, the tocols and the trienols. Tocols have phytol as a side chain whereas trienols contain three double bonds in the side chain. Methyl groups on the chromonol ring differentiate various tocol and trienol isomers. Alpha-Tocopherol (all rac) is the most active form of vitamin E, and it has methyl groups at carbons 5, 7 and 8 (Figure 14). Alpha-tocopherol is practically insoluble in water; however, it is highly soluble in organic solvents. Orally ingested tocopherol or its esters appear to be absorbed only at low levels (20-40%) (Machlin, 1984). The NRC (1988) recommends approximately 15 IU/kg feed for weanling swine to prevent deficiency signs. Vitamin E is absorbed as part of the lipid-bile micelle along with free fatty acids, monoglycerides, and other fat-soluble vitamins. From the intestine, chylomicrons are transported to the lymphatic capillaries. The vitamin E is then transported throughout the circulatory system. Erythrocytes also transport vitamin E, with all of it being located in the membrane. Most tissue vitamin E is associated with membranes. There is no primary tissue storage site for vitamin E; however adipose, liver and muscle tissue have significant concentrations (Bieri, 1987). Mobilization of vitamin E can be either rapid or slow depending upon the 35 “°\.,/‘\../‘\ c c )2 EH3 C EH3 ‘3 m3 \./ \o/l \c/W WV) Figure 14. Alpha-tocopherol (5,7,8-trimethyltocol). 36 storage site involved. 2. General Functions Vitamin E has been shown to have a role in immune function. Nikbin and coworkers (1986) looked at the splenocyte response in 3- and 24-month old mice supplemented with vitamin E for six weeks. They observed a mitogenic response of the splenocytes which may implicate vitamin E as being immuno-stimulatory. Prostaglandin (PGEZ) was also measured in this study due to its known inhibitory effect on lymphocyte proliferation. Prostaglandin levels were decreased in the 24-month old supplemented mice versus 24- month old controls. Vitamin E has also been found to decrease PGEZ synthesis, and this most likely explains the results observed. In a study by Peplowski et al. (1981) the effects of dietary vitamin E were examined in weanling swine antigenically challenged with sheep red blood cells. Vitamin E elevated hemagglutination titers which implicated it as an immuno-stimulant. 3. Antioxidation Vitamin E is thought to play a critical role in antioxidation functioning as a scavenger of reactive molecular species. Coquette et al. (1986) studied macrophages in vitro and showed that oxidative stress to the cells resulted in a 40% decrease in the intracellular tocopherol level. Though increasing the oxidative insult, these workers were unable to oxidize the tocopherol 37 Cy! C red Cyi c on 2 2 :X k 0- NAILXOX I a. C < z I m m C C XIX Vli E -OH VII E '- 0 X R02 R02 H Figure 15. Vitamin E reduction. Taken from Kanner et al. (1987). 38 completely. A redox cycle may exist to maintain tocopherol in its active form (Figure 15). Yamamoto and coworkers (1986) looked at vitamin E- deficient and sufficient red blood cells exposed to an oxidative stress. A greater percentage of vitamin E- deficient cells were seen to undergo hemolysis. Hemolysis appears to occur when oxidative reactions overwhelm the cell's ability to counterbalance them. Vitamin E, in those cells having a sufficient concentration, was utilized to scavenge peroxy radicals (A02‘) and lipid peroxy radicals (LO') which were the primary oxidation products. Lipid and protein oxidation, though significantly less, still occurred in cells with 'sufficient' vitamin E. Meydani and associates (1988) studied the effects of vitamin E and selenium deficiency in brain tissue. Chronic vitamin E deficiency signs in neurological disorders include axonal swelling and dystrophy, demyelination of nerves, increased oxidative enzyme activity, and neuronal pigment accumulation. Brain tissue is particularly susceptible to the activity of free radicals due to its high rate of oxygen consumption and high phospholipid content with PUFAs. Results of the Meydani study showed that brain regions respond to dietary vitamin E uniquely. Regions with greater a-tocopherol concentration had decreased lipid peroxidation. Amemiya (1987) investigated muscular changes in response to vitamin E deficiency,and observed that vitamin E deficiency caused both endothelial and mitochondrial degeneration. The degeneration may be a result of peroxidation due to insufficient vitamin E 39 protection. D. Selenium 1. Definition Selenium (Se) is a metalloid, and shows a close relation in its chemical properties to sulfur. Se is an essential trace element for animals (Schwartz and Foltz, 1957: Thompson and Scott, 1969). The NRC (1988) suggests that the dietary Se requirement of swine is between 0.1 and 0.3 ppm, and presently the U.S. Food and Drug Administration allows the addition of 0.3 ppm Se to all swine diets. Meyer et al.(1981) indicated that weanling swine may require an even greater amount of Se early in the post-weaning period. Se may exist in various organic and inorganic forms, and the bioavailabilities of each form differ (Ku et al., 1972, 1973). Rats given Se as either selenite, selenocysteine or selenomethionine exhibited no significant differences in GSH-px activity; however, the tissue Se content tended to be greater with selenomethionine supplementation (Deagen et al., 1987). Glutathione peroxidase is considered an accurate diagnostic marker in determining Se status (Rotruck et al., 1973). Hassan (1986) studied the efficacy of various Se forms on the prevention of exudative diathesis (ED) in chicks. Sodium selenite was most efficient in preventing ED, and increasing plasma GSH-px activity compared to wheat, barley and fish meal Se. Cardiac muscle Se concentrations were elevated more by organic Se from feed ingredients. Wheat and barley Se appears to be in 40 the form of selenomethionine, but the form in fish meal remains uncertain. 2. Metabolism Selenium is absorbed mainly in the duodenum. Absorbed Se is immediately taken up by red blood cells and then becomes translocated into the plasma. 'Plasma proteins then bind Se and deliver it throughout the circulation. Lee et al. (1969) showed that in order for plasma proteins to bind Se, the Se must have been within an erythrocyte. Red blood cells themselves may be carriers of Se, but it is only as a component of the enzyme GSH-px (Rotruck et al., 1973). Tissues retain Se at differing concentrations. The kidney has the highest concentration followed by the liver. Skeletal muscle has the highest absolute amount of Se (Ullrey, 1983). Both the dietary amount and form affect tissue Se status. 3.Functions Se appears to play a role in the modulation of immune responses (Kiremidjian-Schumacher and Stotzky, 1987). Petrie et al. (1986) found in vitro lymphocyte proliferation could be affected by Se. These researchers suggested that Se may have a role in regulating lymphocyte response to antigen challenges. In swine, a syndrome termed vitamin E and selenium deficiency (VESD) by Hakkarainen and coworkers (1978) was protected against by 5 mg DL-a-tocopheryl acetate and 135 ug Se/kg of feed (as sodium selenite). Vitamin E selenium deficiency is described as a myopathy characterized 41 by muscle degeneration, weakness, and the sudden death of pigs. Exudative diathesis may also occur in swine, which results in a pooling of fluids in the muscle and subcutaneous regions. Bengtsson et al. (1978a) found VESD signs in Se- deficient pigs supplemented with vitamin E. Alpha-Tocopherol was shown to delay the onset of VESD signs, but some Se is required for prevention. In a second study by Bengtsson and coworkers (1978b) when a-tocopherol was deficient and Se was adequate in pig diets, pigs also developed VESD. Selenium most likely exerts its protective effects through the enzyme GSH-px of which it is a part. Hafeman and coworkers (1974) found rats supplemented with Se had higher plasma and red blood cell GSH-px activity than deficient controls. Control animals had lost most GSH-px activity (activity < 1% of weaning amount) by day 24 postweaning. Death usually occurred by day 28 in these rats, and hepatic necrosis was diagnostically determined in all cases. Glutathione peroxidase does appear to plateau at a certain Se supplemental level (Hafeman et al., 1974; Meyer et al., 1981). Noguchi et al. (1973) studied the effects of Se on ED in chickens. Glutathione peroxidase activity in the red blood cells of Se-deficient chicks was high at the onset of ED and remained high throughout the experimental period. Glutathione peroxidase in the livers of these chicks fell to approximately 50%, and in plasma the level was nearly zero. Even though plasma GSH-px was almost zero by day five, signs of ED did not appear until day eight. Glutathione peroxidase may be the first line of defense against peroxidation of the 42 unsaturated lipids of the capillary plasma membrane. In an experiment involving rat erythrocytes by Rotruck and coworkers (1972) glucose addition was capable of preventing oxidative damage in Se-sufficient RBC's. Glucose most likely provides the NADPH necessary for the reduction of GSSG by GR. Combs et al. (1975), in an article on the mechanisms of action of Se in the protection of membranes, relates the biological activity of Se to that of GSH-px. As a component of GSH-px, Se is involved in the protection against damaging effects of both lipid peroxides and hydrogen peroxide. E. Summary It. is critical for living organisms to ‘maintain antioxidant defense mechanisms in adequate condition. Reduced glutathione, vitamin E, and Se all have an association and function.‘with.Ibiological antioxidation, and. therefore (are likely to be interrelated in their activity. III. MATAERIALS AND METHODS A. Experimental Design A split-block, repeat-measure with 2 x 2 x 2 factorial in the whole plot (over 5 weeks in the sub-plots) design was constructed to involve two levels of supplemental Se (0.0 or 0.3 ppm), two levels of supplemental vitamin E (0.0 or 50 IU/kg), and two levels of supplemental GSH (0.0 or 20 ppm). Selenium was supplemented as sodium selenite, vitamin E as dl-a-tocopheryl acetate, and GSH in its reduced form (Biokyowa). Supplements were added to the control diet at the expense of corn starch. Diet compositions are shown in Table 2. The composition of the vitamin-trace mineral premix used in all diets is listed in Table 3. Table 4 shows the calculated nutrient and energy density of the control weanling starter diet. Thirty-two pigs, weaned at 4 weeks of age, were blocked into treatments from 4 litters (one pig/litter) of eight pigs. Blocking was also according to sex and initial weight. Pigs were housed in the north, enviromentally- controlled nursery trailer at the MSU Swine Research and Teaching Center. Pens were 1.2 x 2.4 meters in dimension. The pens had a two gutter, gravity flow system, and the gutters were covered by woggn wire. Separating the gutters 44 mcopguauspm pounce; u ow. o mcvcwaucoo stmtao .cppppuvcma mx\ns mm use .ocwuaguwsowpsm mx\ms CHH .mcwpuxuaguaago—go mx\ms cafl m:_=.oucouu .xpsosa pagmcps «out» :Fsauv> ea copuwmoqsoo to; m «each womb .mcpmxp was m:F:_aucoua r.---.‘t'----""l'-’"t--"'---"‘""-"-"|'-"'""."|t|l“-.."t""'"r"'.. so No. No. as . as so. so. so macaque Pogogaouos-~ga_m._u ms. mN. co co mN. NH. as so ou.:mpmm 53.nom m.o m.o m.o m.c as co so so . w=o_gsnsapo uauzuoa mN. mN. mN. mN. mN mN mN. mN. a u8N ampomg=< m o m.o m. c m o m o m a m.c m.o xcsata zp> m o m. o m. o N o m c m.o m.o N.o . uxcsata mu.topguogumg means? A m.o N,o . N.c mic m. o N.o N.o moo seam o.N c.N, o.N o.N o N o.~ o.N a.” «uncontau so.u~uu m.N a,” m.N m.N m N m.N m.“ m.H mangamoga anu_ausu.o=cz mN mN mN mN mN mN mN mN Aavev Paws cooasom m. cN m oN m cN m cN NN HN NN NN grog um__mgm u==ONu as. .mo es. NH. as. ac. es... ma. . . gurus” grog .-.a,L;,,n, ,m\_ ,m N, - N ,|,Nm .+»N N mucm.cuga=_ , . . . . mumwa . . . . .hmua mzh mo zouhmmomzou .N u4m paucmsmpaaamum “Amuvcopmm s=+o6m ma amp; mxxms m.ov savcopom Poucwsmpaasmumm ”AuuNC mxxas owv ocorguauapm oceans;.paucmsopaaawnw.u=auzmaa .mx .ucmsumogu boa mm_a a we agave: mongw>m amozmaamzu m< mom; moahzumwz >aom .m m4mhs>NNa< amh~>~hu< mm<¢mmmz<¢hczuz< uh< a .'.r.-"-'.|"-.'-'---"-'0-'0'----‘E"0'-"-'--I|"'-"'-"""'l"“"‘-'|"'O'T' ¢N.N ON.N NN.N Ne.” NN.N NN.N ca." ao.N ea raa: NN-N aN.N NN.N. oa.N em.N NN.N NN.N NN.N an r max NN.N ao.N NN.N .ea.N oN.N NN.N NN.N NN.N eN ram: N¢.N NN.N Na.“ NN.N Hm.N aa.~ aa.~ N4.N . N Nam: aN.N oa.~ NN.N Na.” NN.N as.N oe.~ aN.N aarrrer uu+am+m- lama.w a+a am+a N am zma .-Feracoa .arra . .w . a mucmmuaarh xuwuavo ww_. .w_ _ .u :6 as_» 0“Ell-0.05.}‘ty00‘50‘t0‘lml‘05500050-00000505---0-50-0--.I-rL-"t-"nv'0‘500555500500 .muua mom>h~>ubu< mm<¢mmmzm mz Fc. mz cc. mz mz mz a x am x mac az mz mz mz mz mz mz a x am az mz mz mz az mz az a x emu mz mz mz mz az az mz am x mmo mz .cc. az mz cc. Pcc. mz am mz mz mz mz az az .cc. a az az mz cc. az mz az mac mz mz az ac. mz mz az a x am x :ao x 3 az mz .c. mz az mz «2 m x mm x 3 mz mz mz mz az az mz a x nae x z mz mz mz mz az az so. am x emu xnz mz mz az az az az Pccc. a x axaaz mz .ccc. az mz cc. .ccc. az am x axaaz mz mz mz cc. az az cc. mac x axaaz unmaaz xalmao ema sea mac am a auoauua ammuommm: .mflm>mn NUZ ho mHm>A¢24 .mp qudfi V. CONCLUSIONS 1) Glutathione supplementation of the diet of weanling pigs lowered plasma ALT activity slightly (P<0.05) in weeks 3 and 4 of the 4 week trial period. Glutathione supplementation had no significant influence on whole blood GSH, plasma GSH— px, plasma a-tocopherol, plasma Se, or plasma AST. 2) Vitamin E supplementation significantly elevated plasma a-tocopherol in weeks 2, 3 and 4 (P<0.01). Whole blood GSH, plasma GSH-px, Se, ALT or AST were not significantly affected by dietary vitamin E. 3) Selenium supplementation significantly elevated plasma Se levels in weeks 2, 3 and 4 (P<0.01). Supplemental Se also significantly elevated plasma GSH-px activity in weeks 2, 3 and 4 (P<0.01). Selenium elevated whole blood GSH slightly (P<0.06) Alanine aminotransferase and AST were not affected by supplemental Se. 76 LIST OF REFERENCES Amemiya, T. 1987. Differences in muscular changes in rats with vitamin E and with selenium deficiency. Internat. J. Vit. Nutr. Res. 57:139. Anderson, M.E., R.J. Bridges and A. Meister. 1980. 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