THE EFFECTS OF VITAMIN E IN PLATELET FUNCTION Thesis for the Degree of M. S. MICHlGAN STATE UNIVERSITY I‘ERRY ALAN HOLMBERG 1975 (i. -$~}“5‘5€‘57?-3R"-?5'7" my? ' ‘ " Erna?) ._ A _ . - . : LEE 3 £31? 31’ Mia: Egan Starr: 1....Fniv0rsi E? It. ' W , .n“, r (mama , _.‘ H. i ..- " am‘omc By ‘52 . ‘ HUAB & SUNS' A . 800K Bwnm my. . LIBRAP‘ nlNDf “i W» C“: ‘ v- «9.. .w [Non N x ‘u ABSTRACT THE EFFECTS OF VITAMIN E IN PLATELET FUNCTION BY Jerry Alan Holmberg Two factorial experiments involving 14 young pigs were con- ducted to evaluate the effects of vitamin E on platelet function. The pigs were fed on 4 different diets: l) basal ration (deficient in vitamin E and selenium), 2) basal ration plus intramuscular (I.M.) injections of vitamin E, 3) basal ration plus I.M. injections of selenium, and 4) basal ration plus I.M. injections of vitamin E and selenium. Normal values for clot retraction (43—90%) and platelet factor three availability (24.4 :_7.4 sec.) were established for the pig. Platelet ultrastructural changes following hydrogen peroxide aggre- gation indicated that aggregation was followed by destruction of the platelet. Platelet factor three availability and hydrogen peroxide aggregation were two measurable parameters of platelet function which were altered in pigs fed a basal ration. The pig appeared to be a suitable experimental animal for determining the effects of vitamin E on platelet function. Jerry Alan Holmberg In addition, in viva experimentation on human platelets indi- cated that vitamin E neither inhibited nor augmented platelet aggregation. THE EFFECTS OF VITAMIN E IN PLATELET FUNCTION BY Jerry Alan Holmberg A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1975 DEDICATION To my wife, Rebecca Laura Holmberg, for her love and encouragement during my graduate study ii ACKNOWLEDGEMENTS I wish to express my deepest appreciation to Dr. Joan C. Mattson and Dr. C. K. Whitehair, my research advisors, for their guidance, patience and personal interest throughout the course of my investigation. I thank Mrs. Martha Thomas, my academic advisor, for her encouragement and guidance throughout my graduate study. I am grateful to Dr. G. Tishkoff for his willingness to serve as a member on my committee. I thank Donna Ladd and the Electron Microscopy Laboratory for the ultrastructural studies of platelets. iii INTRODUCTION . . . . . . LITERATURE REVIEW. . . . TABLE OF CONTENTS Lipid peroxidation of platelets . . . . . . Vitamin E as an anticoagulant . . . . . . . Role of vitamin E in hemorrhage . . . . . . Vitamin E, a cellular function. . . . . . . OBJECTIVES . . . . . . . MATERIALS AND METHODS. . Animals . . . . . Housing . . . . . Ration O I O I O 0 Experimental design Analyses. . . . . Vitamin E and its RESULTS. . . . . . . . . Normal values . . Platelet counts . Clot retraction . Platelet factor three availability. . . . . Aggregation . . . Enzymes . . . . . Red cell sensitivity. White blood counts. Tissue changes. . Vitamin E and its influence DISCUSSION . . . . . . . CONCLUSION . . . . . . . APPENDICES . . . . . . . LITERATURE CITED . . . . VITA iv on platelets in Page O‘U'IU'IU) 11 12 12 12 12 14 15 2O 21 21 24 28 28 28 28 31 31 31 32 34 4O 41 50 55 Table LIST OF TABLES Basal ration for experiments A and B. . . . Experimental design and pig numbers . . . . Normal values . . . . . . . . . . . . . . . Platelet counts averaged for each group . . Clot retraction averaged for each group . . Platelet factor three availability averaged each group. . . . . . . . . . . . . . . . . Page l3 14 21 25 29 30 LIST OF FIGURES Figure Page 1 Lipid peroxidation, free—radical chain reaction . . . 8 2 Hydrogen peroxide aggregation at 15 seconds . . . . . 23 3 Hydrogen peroxide aggregation at 35 seconds . . . . . 23 4 Bone marrow from a vitamin E-deficient pig. . . . . . 26 5 Multinucleated megakaryocyte from bone marrow of Vitamin E_defiCient pig 0 o o o o o o o o o o o o o o 26 6 Multinucleated megakaryocyte from bone marrow of Vitamin E-defiCient pig 0 o o o o .. o o o o o o o o o 27 7 Multinucleated normoblasts from bone marrow of vitamin E-deficient pig . . . . . . . . . . . . . . . 27 vi INTRODUCTION Platelets are a key component of hemostasis in that they plug the leaks in vessel walls and initiate the intrinsic clotting system (57). When the cellular layers of the vessel wall are ruptured and blood escapes through the interstitial spaces, hemo- stasis must arrest the bleeding before the animal hemorrhages to death (18). Inappropriate quantities of platelets or platelet dysfunction can lead to hemorrhage and death. Hypercoagulability and/or venous stasis, on the other hand, may result in the forma— tion of a thrombus. Vascular occlusion caused by thrombosis can restrict blood flow in an area and lead to focal necrosis (53). Occlusion of a coronary artery can lead to myocardial infarction and death (32). Increased lipid peroxidation, as occurs in vitamin E deficiencies (23,45,55), can alter platelet function to an extent to cause these pathologic lesions (39-41,50,13,l4,15). Since the middle of the twentieth century, vitamin E has been suggested for the treatment of cardiovascular disorders (37,38,42,46). It has been thought to have anticoagulant properties (21,56). Hemo- static disorders have been reported in vitamin E deficient animals, indicating that vitamin E is necessary for the maintenance of hemostasis (10,30,52). 2 In order to study the effects of vitamin E deficiency on platelet function, an animal model was needed that resembled man. The pig was chosen on the basis of its physiological similarities to man, its platelet activity and the volume of specimen needed (8). Since hemostasis is important in man and animal, research was undertaken to determine the role of vitamin E in platelet function, using the young pig as the experimental model. LITERATURE REVIEW The literature on platelet function is voluminous. This literature review pertains primarily to the effects of lipid peroxi- dation on platelet function. In addition, the role of vitamin E as an anticoagulant, some hemostatic disorders associated with vitamin E deficiency, and its possible role at the cellular level will be reviewed. Lipid peroxidation of platelets Lipid deterioration caused by peroxidation has occurred in the platelet (l3-lS,39—41,50). Aged platelets accumulate lipid peroxides and appear to lose their cellular defense mechanism against peroxidation (39). Lipid peroxides accumulate in stored platelets at 4C and this accumulation is even more marked at 20C. In the absence of vitamin E and its protective effect, this peroxi- dation may be enhanced. Lipids make up an integral part of the platelet and influence its function and maintenance. The lipid content of platelets is 2.98 per cent (19). Seventy-six per cent of the total lipids are phospholipids, 20 per cent are neutral lipids, and four per cent are lipoproteins (27). Platelets are unique in that they are capable of de novo fatty acid synthesis, utilizing the malonyl CoA pathway to maintain the phospholipids and membrane lipids (20,26). 3 4 In the absence of the protective mechanism of vitamin E, the accumulation of lipid peroxides can alter platelet aggregation. Platelet aggregation is dependent on the free sulfhydryl (SH) groups in the platelet membrane (43). Tateishi et al. (50) believed that it is the oxidation of the free membrane thiol (SH) by lipid peroxi- dation that alters its aggregation. The availability of platelet factor three (PF3), a phospholipoprotein which is thought to be located on the membrane (57), is greatly decreased after lipid peroxidation (50). Its activity is not detectable in the circulat— ing platelets but occurs after exposure to adenosine diphosphate (ADP) or aggregation. Platelet factor three is necessary for the initiation of the intrinsic clotting system (57). The decrease in activity after lipid peroxidation indicates either peroxidation of the membrane-bound phospholipoprotein or membrane change due to the oxidation of the free SH group. Okuma et al. (41) have reported that neither ADP nor epinephrine causes increased lipid peroxidation. They concluded that aggregation as such does not stimulate lipid peroxidation of the platelet. However, they did report an increase in lipid peroxides in aggrega- tion resulting from thrombin, glutathione (SH) inhibitors, polystyrene latex particles and platelet antibodies. Circulating peroxides, on the other hand, appear to enhance platelet aggregation. Peroxidized arachidonic acid enhances platelet aggregation in contrast to unperoxidized arachidonic acid (31). Platelet aggregation stimulated by lipid peroxides might contribute to thromboembolic phenomena. Polymorphonuclear leukocytes produce 5 peroxides (1,2) and can be part of a thrombus or line the periphery of it (7). Hydrogen peroxide has recently been used as an aggregating agent (l3~15,22). Higashi et al. (15) have suggested that peroxide caused permeability changes and/or membrane disruption which would release ADP and in turn cause platelet aggregation. Vitamin E as an anticoagulant Vitamin E has been thought to act as an anticoagulant and has been advocated for the treatment and prevention of thrombosis (38,42,46). Zierler et a1. (56) reported that administration of alpha tocopherol phosphate altered the clotting mechanism. Kay et al. (21) suggested that alpha tocopherol phosphate may be a normal circulating antithrombin. It was later reported that alpha tocopherol phosphate had antithrombin properties but that alpha tocopherol acetate did not. Monkhouse (33), in 1963, stated that the anti- thrombic activity was not due to the alpha tocopherol but to the phosphate ion. Role of vitamin E in hemorrhage Hemorrhagic lesions and sudden deaths have been reported in vitamin E deficiencies (10,30,52). The most prominent lesion reported has been the hemorrhagic necrotic liver and occasionally hemorrhagic lymph nodes. Grant (10), in an extensive study, was able to produce dietetic microangiopathies (MAP) in swine. The diets were high in unsaturated fat and, therefore, oxidatively unstable fatty acids, an inadequacy of tocopherol (vitamin E) and selenium. In dietetic MAP the red, hemorrhagic lesions of the heart gave it the appearance 6 of a "mulberry." These MAP lesions were located in the capillaries and small muscular vessels of the myocardium. The lesions contained a periodic acid-Schiff (PAS) positive material. There were also extracardiac MAP lesions in the lung, kidney, skeletal muscles, colon, stomach, abdominal wall, liver, uterus and the adrenals. Grant has suggested that the vascular changes, the hemolysis and the thrombocytopenia of MAP in swine resemble the pathologic changes seen in thrombotic thrombocytOpenic purpura (TTP) in man. A possible explanation for the hemorrhages and the vascular changes could be alterations in platelet function. Vitamin E, a cellular function The cellular function of vitamin E may include the stabiliza- tion of membranes, the breaking of free-radical chain reactions and participation in heme synthesis. It has been suggested by Lucy (23) that membrane phospholipids might form a stable complex with vitamin E in order to control membrane permeability and stability. The cis double bond of the arachidonyl acid forms a pocket in which the fourth and eighth carbons (C and C8) of tocopherol fit. This structural complex of 4 vitamin E in the biological membrane may act to inhibit oxidative destruction of polyunsaturated fatty acids (PUFA) in the cell and its membrane. Peroxidation of lipids has been seen in association with damage to cellular structures, proteins, enzymes and metabolic pathways (47—49,51). Cellular membranes may be broken by lipid peroxida- tion permitting their contents to leak out. In addition to the 7 membrane of the cell, the organelles may be affected. Tappel (48), who has done extensive work with antioxidants, has mentioned that lysosomal membranes may be damaged. This would allow the digestive enzymes to leak into the cell's cytoplasm. The mitochondria and the microsomes may also be damaged. The mitochondria contain approxi- mately 25 per cent unsaturated phospholipids and the microsomes 30—40 per cent. Vitamin E is the only known antioxidant in the mitochondria and the microsomes. Zalkin and Tappel (55) have stated that the membrane and the cytochromes are labile to lipid peroxida- tion. They concluded that it was this membrane change that resulted in the swelling of the mitochondria and altered metabolic activity. Schwartz (45) believed that vitamin E has a specific role in the mitochondria, that of preventing respiratory decline. The biochemistry of vitamin E enables it to act as an antioxi— dant in preventing free radical lipid peroxidation (48,49). Tappel, an authority on vitamin E, explained that if lipid peroxidation were permitted to proceed unchecked, it would advance in a free-radical chain reaction (Figure 1). When lipid peroxidation is initiated, a lipid free radical (R°) is generated. This lipid free radical combines with molecular oxygen (02) to form a lipid peroxyl radical (ROO-). The lipid peroxyl radical is now capable of continuing the peroxidative cycle by removing a hydrogen from another lipid source (RH), generating a lipid peroxide (ROOH) and another lipid free radical (R-). The lipid antioxidant, vitamin E, functions to supply hydrogen to the lipid peroxyl radical (ROO-) and break the chain reaction. The oxidized form of alpha tocopherol, alpha .cofluommu camno HMUAUMHImmHm .coflumpfixoumm pflmflq mmzoszoasmmzaoooe $1.35 m :> + zoom mlIISommEouE. $13,: I -m :> + .H musmflm .oom Tl:! O .m + IoomA mm + 9 tocopherquuinone, is the main product formed. It appears that one of the main functions of vitamin E is its reaction with free radical intermediates of lipid peroxidation and peroxides. In addition to its roles in membrane stabilization and control of lipid peroxidation, recent studies have indicated that vitamin E participates in heme synthesis (36,4). The first step in heme synthesis is the condensation of glycine and succinyl CoA within the mitochondria to form delta (5)-aminolevu1inic acid (ALA). Two mole- cules of é-ALA are condensed to form porphobilinogen outside the mitochondria. Both of these steps are controlled by the enzymes ALA synthetase and ALA dehydratase. Four molecules of porphobilinogen then combine through a series of reactions to form protoporphyrin IX. Protoporphyrin IX incorporates iron within the mitochondria to form heme. It is thought that vitamin E regulates the ALA synthetase and dehydratase at an inducer-repressor genetic site (36). Vitamin B may regulate heme synthesis somewhere between the translation and transcription of the inducer messenger ribonucleic acid (mRNA) by affecting the activity of the repressor. In the rat, vitamin E deficiencies result in a decrease in ALA synthetase activity in the bone marrow and a decrease of ALA dehydratase in the liver (4). The bone marrow in vitamin E deficiencies is usually hyperplastic with many red cell precursors, the majority being multinucleated (30,34,35). In addition, the megakaryocytes contain ten to twenty nuclei and have normal granular cytoplasm. There are two possible explanations for the anemia: destruction of red cells and decreased synthesis of heme. The red cell membranes in vitamin E deficiency are increasingly lO sensitive to hydrogen peroxide (l7). Jacobs et al. (17) explained this by postulating that the oxidation of the sulfhydryl groups (SH) in the membrane accompanied the hemolysis by hydrogen peroxide. There are other pathologic changes associated with vitamin E deficiency that appear to be related to excessive lipid peroxidation and membrane damage. These include a form of muscular dystrophy (36,25), cellular degeneration including liver necrosis, testicular degeneration, encephalomalacia, prematurity, fetal loss and malabsorp— tion syndromes (29). In summary, there appear to be two mechanisms by which peroxides affect platelet function: an external and an internal reaction. Tateishi et al. (50) indicated that accumulation of lipid peroxides within the platelet causes platelet dysfunction through membrane and organelle damage. External stimulation of platelets by lipid peroxides caused a hypercoagulative response (13-15,3l,22). It appears that vitamin E is needed to stabilize the platelet membrane and protect it from lipid peroxidation. OBJECTIVES The general objective of this research was to investigate platelet function in vitamin E-selenium deficiency, using the pig as the experimental animal. The specific objectives were: 1. To determine if a basal ration that induces a vitamin E- selenium deficiency would cause platelet dysfunction. 2. To determine if a basal ration that induces a vitamin E- selenium deficiency would cause enhanced platelet function. 3. To determine if a basal ration that induces a vitamin E- selenium deficiency would impair platelet production. 11 MATERIALS AND METHODS Animals Hampshire and Yorkshire—cross pigs were used as the experimental animals. In the first experiment, experiment A, eight litter mates and one non-litter mate were obtained from the Michigan State University Swine Research Farms. In the second experiment, experiment B, eight pigs from different litters, but the same boar, were obtained from the Richard Chaney Farm of Mason, Michigan. All pigs had iron injections prior to the experimentation. Housing All animals were housed in individual metal metabolism cages in a heated room. Ration The pigs were fed the ration given in Table l, twice daily. The basal ration used to induce a vitamin E—selenium deficiency was the same as that used previously by numerous workers and is described by Michel (30). The torula yeast was low in selenium and a protein source. The cod liver oil was used as the source of unsaturated fats to enhance the vitamin E—selenium deficiency. Water was available at all times in a separate bowl. 12 13 Table l. Basal ration for experiments A and B Torula yeast 32% Cod liver oil 5% Mineral mix* 6% Vitamin mix** 2% Cerelose 55% '1: Mineral mix: percentage of the total mineral mixture KCl 10.0 KI 0.002 FeSO4-H20 0.4 MgCO3 2.0 NaHCO3 25.0 CaHPO4-2H20 36.0 CaCO3 12.5 Cerelose 13.098 ** . . . Vitamin mix: Thiamin 0.3 gm Riboflavin 0.6 gm Nicotinic acid 4.0 gm Pantothenate 3.0 gm Pyridoxine HCl 0.2 gm PABA (para-aminobenzoic acid) 1.3 gm Ascorbic acid 80.0 gm Inositol 13.0 gm Choline 130.0 gm Folic acid 26.0 mg d-biotin 5.0 mg Cyanocobalamin 10.0 mg Menadione Na bisulfite 6.5 mg 14 Animals maintained on the basal ration plus vitamin E were given supplements of vitamin E as dl-alpha—toc0pherol acetate (Hoffman La Roche, Nutley, N.J.). In experiment A, 100 I.U. of vitamin E were injected I.M. weekly after blood specimens were collected. In experiment B, 200 I.U. of vitamin E were injected I.M. weekly. Animals maintained on the basal ration plus selenium were given supplements of 0.25 mg of selenium weekly. Animals fed the basal ration were not given supplements, and their ration was deficient in both vitamin E and selenium. Experimental design In experiment A, the eight pigs were divided into four groups (Table 2). In this experiment the platelet aggregation, platelet counts, platelet factor three availability, clot retraction, hemato- crit, white blood counts, erythrocyte osmotic fragilities, lactic dehydrogenase (LDH) and serum glutamate—oxalacetate transminase (SGOT) were determined in blood samples at selected intervals. Table 2. Experimental design and pig numbers Ration Experiment A Experiment B Basal 6,1 4,5,6,7,8 Basal plus vitamin E 8,4,9 3 Basal plus selenium 7,0 l Basal plus vitamin E, selenium 3,5 2 15 In experiment B, eight pigs were divided into a similar set of four groups, in order to examine the hydrogen peroxide red cell hemolysis and the hydrogen peroxide aggregation (Table 2). In addi- tion, all of the laboratory parameters monitored in experiment A were repeated in this experiment. Two of the eight pigs died from respira- tory infections before the first bleeding. One pig was fed from the basal diet group and one fed basal diet plus vitamin E and selenium. Blood specimens were collected from the anterior vena cava once a week for the period of the experimentation. The disodium salt of ethylenediaminetetraacetic acid (EDTA) was used as the anticoagulant in the hematologic studies (white cell counts, platelet counts, hematocrits and red cell fragilities). Sodium citrate was used as the anticoagulant in the platelet studies (aggregation, platelet factor three availability and platelet counts of the platelet rich plasma). Immediately after a specimen was drawn in a plastic syringe, 6% sodium citrate for pig platelets (12) and 3.8% sodium citrate for human platelets was added in a ratio of 1 part citrate to 9 parts blood. Tissue specimens were obtained at necropsy from three animals that died and two that were sacrificed at the end of the experimenta- tion and were examined by light microscopy. Analyses A. Platelet counts Platelets were counted manually using a phase contrast micro- scope on both anticoagulated whole blood (EDTA) and platelet rich plasma (PRP). A unipet capillary (Becton-Dickinson, Rutherford, N.J.) 16 was filled with the specimen (EDTA or PRP). This was then diluted in the unipet reservoir containing 1% ammonium oxalate. B. Clot retraction The clot retraction of whole blood was determined using a modification of the method described by Didishiem and Bunting as described by Bowie et al. (3). Four milliliters of whole blood was allowed to clot at room temperature and was then incubated at 37C for one hour. After an hour the clot was removed and described morpho— logically. The serum and cells remaining in the tube were centri- fuged. The volume of the sedimented red cells was then subtracted from the total volume to obtain a true serum volume. The clot retrac- tion was calculated as follows: Clot retraction (%) — (serum volume/blood value x plasmacrit) plasmacrit = 100 - hematocrit The normal value for man is 40—94% clot retraction. As the normal for pigs has not been established, the value for man was used. C. Packed cell volume Microhematocrits were determined by centrifugation. Capil- lary tubes were filled and sealed with clay. They were then centri- fuged (International Equipment Co., Needham Heights, Mass.) for four minutes. The percentage of packed red cells to volume was recorded. D. Platelet factor three availability Platelet factor three availability was determined using a modification of the procedures of Hardisty and Ingram (11). Two- tenths milliliter of an equal mixture of platelet rich plasma (PRP) l7 and platelet poor plasma (PPP) was mixed with 0.2 ml of Kaolin (J. T. Baker, Phillipsburg, N.J.). This was then incubated for 20 minutes with an occasional mixing. At the end of 20 minutes, the specimen was recalcified with 0.2 ml of 0.035 M CaCl2 and clot formation was timed in seconds. E. Platelet aggregation All labware used was plastic unless otherwise noted. The citrated specimen was centrifuged at 1360 rpm for 5 minutes to remove the red blood cells. The supernatant, citrated platelet rich plasma (PRP), was removed to another test tube until needed. Platelet aggregation studies were monitored using a commercial aggregometer (Chrono-Log Corp., Broomal, Pa.) with a strip chart recorder. Five-tenths (0.5 ml) milliliter of PRP was equilibrated in the aggregometer at 37C for a minimum of one minute. Then 20 lambda (0.02 ml) of the test aggregating agent was added to 0.5 ml of PRP; the concentration of the aggregating agent was then expressed as the final concentration. Three aggregating agents were used: (1) A final concentration of l x 10“4 M adenosine 5' diphosphate (ADP) was used as the stock solution. This stock solution was pre- pared by dissolving 11.8 mg of ADP disodium salt from equine muscle (Sigma, St. Louis, Mo.) in 10 ml of imadazole buffered saline (28). Various concentrations (1 x 10“5 M and l x 10-6 M) were made by dilu— tions from the stock solution. (2) Ristocetin (Abbott Laboratories, North Chicago, Ill.) was dissolved in saline to give a concentration of 104 mg/ml. This gave 18 a final concentration of 4 mg/ml. Various concentrations (1, 2 and 3 mg/ml) were made by dilutions of the stock solution. (3) A 1% solution of hydrogen peroxide was prepared by dilution from 30% hydrogen peroxide (Analytical Reagent, Mallinckrodt Chemical Works, St. Louis, Mo.). This gave a final concentration of 0.4%. F. Enzyme determination Lactic dehydrogenase (LDH) assays utilizing the reverse reaction of Wroblewski and LaDue (54) were performed. LDH-P Stat Packs (Calbiochem, La Jolla, Cal.) were used. Nicotinamide-adenine dinucleotide (reduced NADH) was used to activate the substrate, pyruvate. The change in the absorbance at 340 nanometers (nm) was determined for one minute. Serum glutamate-oxalacetate transaminase (SGOT) activities were determined using GOT Stat-Pack (Calbiochem, La Jolla, Cal.). The substrates employed were aspartic acid and alpha ketoglutarate, activated with NADH. The change in the absorbance at 340 nm was determined for 5 minutes. Lactic dehydrogenase isoenzymes were separated on cellulose acetate plates by electrophoresis at 180 volts for 25 minutes in high resolution buffer, pH 8.6 (Helena Laboratories, Beaumont, Tex.). The cellulose acetate plates were then stained with Helena LDH reagent (nitrozolium blue) for 30 minutes at 37C. After color development, the plates were fixed in 5% acetic acid for 5 minutes and then optically scanned on the Densicord (Photovolt Corp., New York, N.Y.). 19 G. Red cell sensitivity Red cell osmotic fragilities were determined in experiment A according to the method of Dacie as described by Lynch et al. (24). A series of graded strength buffered sodium chloride solutions was used which ranged in concentration from 0.1% to 0.8%. Five milli- liters of each concentration of sodium chloride were mixed with 0.02 ml of anticoagulated (EDTA) blood. The tubes were incubated for 30 minutes and centrifuged at 2000 rpm for 10 minutes. The 0.8% sodium chloride was used as the blank. The remaining tubes were measured against the blank at 540 nm in a spectrophotometer (Bausch and Lomb Spectronic 20, Rochester, N.Y.). The hydrogen peroxide hemolysis test used in experiment B utilized the modification of Rose and Gyorgy (44). A 5% suspension of washed red cells was made in 0.9% sodium chloride. Three tubes were prepared, each containing 0.25 ml of the 5% red cell suspension. To the first was added 0.25 ml of phosphate buffer. This served as the blank. To the second and third tubes were added 0.20 ml of phosphate buffer and 0.05 ml of 4 M hydrogen peroxide (Mallinckrodt, St. Louis, Mo.). All tubes were mixed, incubated for 15 minutes at 37C and then allowed to stand at room temperature for 2 hours and 45 minutes. At the end of three hours, 5 ml of saline-phosphate buffer was added to the blank and to the second tubes. Five milliliters of distilled water was added to tube three. All tubes were centrifuged for 10 minutes at 2000 rpm. Absorbances were determined for both tubes 2 and 3. The per cent hemolysis due to hydrogen peroxide sensitivity was determined by the following formula: 20 Absorbance saline-phosphate (tube 2) Absorbance water (tube 3) Per cent hemolysis = x 100 H. White blood cell counts In experiment A, white blood cell counts (WBC) were determined using an electronic particle counter (Coulter Counter, model B, Hialeah, Flor.). Coincidence corrections were made for all counts over 10,000 WBC/cu.mm. In experiment B, WBC counts were determined utilizing a unipet reservoir (Becton-Dickinson, Rutherford, N.J.). A 1:100 dilution was obtained using EDTA blood. Four square millimeters of a hema- cytometer with a chamber depth of 0.1 mm were counted and the WBC count was calculated as follows: WBC = cells counted (100/4 sq.mm x 0.1 mm depth) Vitamin E and its influence ongplatelets in man One human volunteer was used throughout the experiment. During this time no other medication was taken. Platelet aggregations were begun on the PRP after a week during which the volunteer consumed alpha tocopherol acetate (100 I.U. for 6 days and 400 I.U. for 7 days). In addition to the in viva experimentation, specimens were incubated in vitro with alpha tocopherol acetate (0.81 mg/ml). RESULTS Normal values Normal values (Table 3) for platelet factor three availability, platelet counts, LDH and SGOT were established from values obtained from the positive control (vitamin E and selenium supplemented) pigs. In both experiments for all pigs, the mean clot retraction and range were‘established. Table 3. Normal values Procedure X :_2 S.D. Platelet factor 3 (8) 24.4 sec. 7.4 Clot retraction (37) 71.9% 19.7 Range 43-90% Platelet counts (10) 624,050/cu.mm 258,974 LDH (8) 781 mU/ml 246 SGOT (9) 23 mU/ml 11 ( ) indicates number of analyses * standard deviation 21 22 A high concentration of ADP (1 x 10-4 M) was needed to sustain the primary wave of aggregation. Other concentrations were used (1 x 10—5 M and 1 x 10“6 M); however, a small initial wave was followed by dissasociation of the platelet aggregate. No secondary wave was observed in pig platelets. Ristocetin at final concentrations of l, 2, 3 and 4 mg/ml of PRP were used. A final concentration of 4 mg/ml was needed for pig platelets. Not only did this concentration cause platelet aggrega- tion but it also caused the conversion of fibrinogen to fibrin. Upon the addition of this concentration, there was a drop in light transmission from the ristocetin itself. This was followed by a rise in light transmission as a result of aggregation. Pig platelets produced a one wave tracing with this concentration of ristocetin. During experiment A, various concentrations (0.1% and 1%) of hydrogen peroxide were added to PRP and platelet changes observed. The 0.1% hydrogen peroxide (final concentration 0.004%) did not cause visible platelet aggregation. When 1% (final concentration 0.4%) hydrogen peroxide was added to equilibrated PRP in the aggregometer, there was a slight rise in the light transmission. This was followed by a sharp drop in light transmission, then an equally sharp rise in light transmission with much bubble formation. Transmission electron microscopy (TEM) was used to study the changes in platelets. These specimens were from pig 9 of experiment A (vitamin E supplemented), and are illustrated (Figures 2 and 3). It was observed that after the addition of 1% hydrogen peroxide, the light transmission increased 3%, plateaued, then increased another 9%. Transmission t x50,000. inser 2 Hydrogen peroxide aggregation at 35 .5 1. t a n O .1 t a 9 e r 9 9 a e d .1 x O r e P n m r d V. H x25,000. x17,500 Figure ‘3. Figure 2. seconds. seconds. {222.2 2 . .... 2.2.22... 22.... 2.2: ,2w2 .2 _. d2~22_2 H ..2wx~2 . . . . . 0... 2.222.... ..2... 2...... .... 2 ....: 22 : . .. ...... A . . _ ... 2,. ......w 2.2. 2..... 22...... .2... 2..... ._ ......22222222 ...... saw. 22.222.222.22. _ . , .. ... 2...... 2222.22.22.22 2... 2.2.2.222222222 . :2 222.222.2222.... g2—E 22.2 222.222.22.22... ._ .2. : _ .... ...22.2......222_ 2.. .. 22. ”2.2222,. . . . v2.22: 5+” 2222222.. 222—222 22 ..22 22 2222222 2 .22222 .22.... ._ .2 ...... . . . _ .2. .22.... . 2.2222222... .22.: . 222 ..... .. .. .... . . .. .... . .2222r..22.2222222222... 2222222 22222222252222... 2 .-- ..le|.. .D «02' u (I 24 electron microscopy revealed that the platelets were activated 15 seconds after the addition of hydrogen peroxide (Figure 2). At 35 seconds, TEM revealed (Figure 3) that the platelets were destroyed as evidenced by the presence of myelin figures. Another specimen was fixed for TEM 45 seconds after the addition of hydrogen peroxide; however, no specimen was recovered. Platelet rich plasma from pig 9 was then incubated with 0.81 mg/ml of alpha tocopherol acetate for 2 hours. The hydrogen peroxide aggregation tracing appeared the same as described in the previous paragraph. Polylysine (2 mg/ml) and epinephrine did not cause pig platelets to aggregate. Efforts to demonstrate platelet aggregation with my own preparation of bovine and pig collagen were unsuccessful; how- ever, it has been reported in the literature that collagen does aggregate pig platelets (6). Platelet counts Platelet counts were sporadically abnormal in all groups except specimens from pigs injected with selenium. The platelet counts never decreased below 12,000/cu.mm in any pig (Table 4). On bone marrow examination of a vitamin E deficient pig, mega- karyocyte production was not suppressed and the marrow appeared slightly hyperplastic. There was excessive multinucleation of the megakaryocytes. 25 Table 4. Platelet counts averaged for each group Basal + Basal + Basal + vita- Day Basal vitamin E selenium min E/selenium 3* 197,500 (1) 162,500 (2) 400,000 (2) 482,500 (2) 9** 532,488 (4) 508,400 (1) 756,450 (1) --- 10* 490,000 (1) 280,000 (2) 408,750 (2) 303,750 (2) 15** 561,250 (4) 902,500 (1) 977,500 (1) --- 17* 412,500 (2) 502,500 (2) 545,000 (2) 632,750 (2) 23** 466,667 (3) 760,000 (1) 482,500 (1) --- 25* 575,000 (1) 410,000 (2) 501,250 (2) 683,750 (2) 29** 471,875 (4) 430,000 (1) 772,500 (1) --- 32* --- 307,500 (2) 478,750 (2) 540,000 (2) 37** 367,500 (3) 550,000 (1) 495,000 (1) --- 40* --- 500,000 (2) 508,750 (2) 797,500 (2) ( ) indicates number of pigs * ** experiment A experiment B 26 ‘ ‘51.; 2. — 22‘. Figure 4. Bone marrow from a vitamin E-deficient pig. x45. Figure 5. Multinucleate: mega aryocyte marrow of vitamin E-deficient pig. x202.5. rom cone 27 \ ' "’ .41 Figure 6. Multinucleated megakaryocyte from bone marrow of vitamin E-deficient pig. x450. Figure 7. Multinucleated normoblasts from bone marrow of vitamin E-deficient pig. x450. 28 Clot retraction The clot retraction was rarely abnormal in any pig (Table 5). In both experiments the mean of all groups was 71.9% with a range of 43-90% retraction. The values are comparable to normal values in man with a range of 40—94% retraction (3). Platelet factor three availability Platelet factor three availability was sporadically abnormal in all groups throughout the entire experiment. The pigs injected with both vitamin E and selenium had a lower incidence of abnormal platelet factor three availability (Table 6). Aggregation A. ADP aggregation was sporadically abnormal in all groups throughout the experiments. B. Hydrogen peroxide aggregation was abnormal in all groups except the vitamin E supplemented group. C. Ristocetin caused platelet aggregation in all pigs; however, the conversion of fibrinogen to fibrin was sporadically inhibited in all groups. Enz es LDH: The LDH was slightly elevated in all pigs partially as a result of hemolysis of the specimens. The LDH elevation became increasingly marked as the pig approached the basal state. In experi- ment A, the LDH returned to normal after initiation of the supplemental injections (vitamin E and/or selenium). Electrophoresis revealed 29 Table 5. Clot retraction averaged for each group Basal + Basal + Basal + vita- Day Basal vitamin E selenium min E/selenium 3* 74.5% (1) 81% (2) 82.5% (2) 79% (2) 9** 68.5% (4) 43% (1) 72% (1) --- 10* 64.5% (1) 53.5% (2) 58% (2) 75% (2) 15** 77.25% (4) 75% (1) 70% (l) --- 17* 82.5% (2) 68.5% (2) 56.5% (2) 75% (2) 23** 71.5% (3) 90% (l) 66% (1) --- 25* 84% (l) 73% (2) 74% (2) 75% (2) 29** 74.75% (4) 77% (1) 51% (l) --- 32* --- 73% (2) 74.5% (2) 72.5% (2) 37** 60.3% (3) 77% (l) 77% (l) --- 40* --- 78.5% (2) 72% (2) 84% (2) ( ) indicates number of pigs * ** experiment A experiment B 30 Table 6. Platelet factor three availability averaged for each group Basal + Basal + Basal + vita— Day Basal vitamin E selenium min E/selenium 3* 23.3 (1) 24.7 (2) 24.4 (2) 22.8 (2) 9** 38.3 (4) 37.0 (1) 38.5 (1) --— 10* 16.6 (1) 30.5 (2) 25.5 (2) 33.3 (2) 15** 22.5 (4) 22.5 (1) 26.7 (1) --~ 17* 29.0 (2) 27.2 (2) 24.6 (2) 26.9 (2) 23** 54.5 (3) 28.0 (1) 26.2 (1) ~2- 25* 24.0 (1) 25.0 (2) 22.5 (2) 20.C (2) 29** 27.7 (4) 30.5 (1) 30.0 (1) --- 32* -—- 33.3 (2) 23.9 (2) 20.2 (2) 37** 42.8 (3) 24.0 (1) 33.6 (1) --- 40* --- 23.1 (2) 27.4 (2) 25.5 (2) ( ) indicates number of pigs ** experiment B * experiment A 31 that porcine LDH isoenzymes are composed of five fractions. The fifth fraction was elevated in relation to liver damage. SGOT: The SGOT was sporadically elevated in all pigs. Red cell sensitivity Osmotic fragilities were normal in all groups. Initial hemoly- sis appeared at 0.60-0.65% NaCl and was complete at 0.35—0.40% NaCl. Hydrogen peroxide hemolysis increased in all pigs fed a diet defic1ent in vitamin E. White blood counts White blood counts demonstrated no specific pattern throughout the experiments. Tissue changes Three pigs on the basal ration (deficient in vitamin E and selenium) died during the experiments. 0n necropsy, each had some- what different morphologic changes. In gross examination of pig 8 (experiment A), the major abnor- mality seen was a markedly patchy, necrotic liver. All organs and muscles were pale. The gastrointestinal tract was normal and no hemorrhagic lesions were present in the heart, striated muscles or parenchymal organs. Microscopically, the liver demonstrated evidence of marked necrosis, lobular collapse and mononuclear cell infiltration. Some small vessels of the heart contained platelet aggregates. The bone marrow was hypercellular and contained abundant, excessively multinucleated megakaryocytes in which signs of platelet production 32 were evident. In addition, many normoblasts were multinucleated. The kidneys appeared congested. LDH was increased in pig 6 (experiment A) by the second week. It remained elevated until the pig died during the fourth week of the experiment. At necropsy the muscles were pale but no hemor- rhages or petechiae were noted. There were no gross abnormalities of the liver. The most striking lesion was an ulcer approximately 3 cm in diameter at the esophageal-gastric junction. The intestines contained coffee ground-like material. Microscopically the liver appeared normal except for scattered mononuclear infiltration in the sinusoids. Occasionally, there were some polymorphonuclear cells. In the heart there were platelet aggregates in venous spaces. The kidneys were congested but otherwise appeared normal. Pig 6 (experiment B) died suddenly and unexpectedly. Necropsy was performed shortly after death. Grossly, the major abnormalities seen were a hemorrhagic liver and focal petechiae in the cortex of the kidney. There was no ulceration of the gastrointestinal tract. There were no hemorrhagic lesions in the heart or striated muscles. Microscopic focal hemorrhages were seen in the liver; the kidneys had marked vascular congestion and stasis. No microthrombi were found in the heart, striated muscles, liver, or kidneys. Vitamin E and its influence on platelets in man Baseline aggregation studies of human platelets demonstrated that upon the addition of 1% hydrogen peroxide (0.04% final concen- tration), the light transmission rose slightly (2%) for approximately 10 seconds. This was followed by a precipitous drop in transmission 33 of 25% for approximately 25 seconds. There was then an equally sharp rise of 20-25% above the initial baseline. Bubble formation accompanied these changes. This was the same pattern that occurred in pigs fed the basal ration supplemented with vitamin E and selenium. Vitamin E was ingested, 100 I.U. for 6 days and then 400 I.U. for the following 7 days. The aggregation tracings appeared the same as the baseline except that the final phase of the tracings rose only to the baseline. Alpha tocopherol acetate was incubated in vitra with specimens from the week when the daily ingestion was 100 I.U. The tracings were the same as the in viva tracings. In comparison to the baseline ADP aggregation (5 x 10.6 M) and ristocetin (1 mg/ml) aggregation, consumption of 100 or 400 I.U. alpha tocopherol acetate did not alter platelet aggregation. In vitra incubation Wlth vitamin E neither inhibited nor augmented the aggregation response to ADP or ristocetin. DISCUSSION The young pig appeared to be a suitable experimental animal for research on platelet physiology and function. It consumed the basal ration and tolerated the torula yeast diet well. Deficiency occurred in some groups within 10 days. The hematologic and circulatory systems are relatively similar to those in man (8). Pig platelets are similar in appearance to human platelets. However, they require higher concentrations of aggregating agents for stimulation. The size of the pigs allowed the collection of an adequate volume of blood for the analyses. The pig was occasionally difficult to bleed, especially when less than three weeks of age. Hemolysis and an increase in tissue thrombo— plastin from traumatic punctures presented problems in some blood specimens. New parameters of pig platelets were evaluated. Reports on values for clot retraction and platelet factor three availability normals were not found in the literature. Clot retraction in the pig (43-90%) is comparable to human normals (40-94%) (3). Platelet factor three availability (24.4 sec. :_7.4) is also comparable to man (less than 30 sec.) (11). 34 35 Platelet precursors in a vitamin E-deficient pig indicated that there was an interference in megakaryocyte division. The multi- nucleation seen in many normoblasts was another indication that vitamin E deficiency interferes with cellular division. Even though the megakaryocyte from the vitamin E—deficient pig had alterations in cellular division, the marginal budding of platelets appeared normal. The quantitative platelet counts were adequate in all groups indicating that the deficiency does not decrease the quantity of circulating platelets. The platelet function of clot retraction was rarely abnormal. This suggested that the function of retraction was not impaired in the deficiency. The release of the phospholipid, platelet factor three, was abnormal in all groups but to a lesser extent in those pigs injected with vitamin E. Platelet factor three is a phospho- lipoprotein, thought to be located on the membrane of the platelet (57), which may undergo peroxidation in the absence of the antioxi- dant. Therefore, it appears that vitamin E is necessary in the stabilization of platelet factor three and its availability. It is postulated that a deficiency of vitamin E might enhance platelet aggregation and produce a state of hypercoagulation. This could explain the production of microthrombi described by Grant in vitamin E-deficient pigs (10). If vitamin E deficiency can enhance aggregation, one might suspect that vitamin E supplementation would be capable of inhibiting aggregation. Indeed, this has been postu— lated by Schute, who uses vitamin E therapeutically in coronary patients (46). There was no evidence that dietary supplementation 36 of vitamin E inhibits ADP aggregation. Furthermore, in none of the pigs fed the basal diet was there any evidence of enhanced aggre- gation. In contrast, subnormal ADP aggregation was observed in each group. The poor response of platelets to ADP stimulation could reflect previous stimulation by ADP released from hemolyzed red cells or hydrogen peroxide or lipid peroxides produced in the absence of vitamin E protection. Ristocetin is a relatively new tool as an aggregating agent. It is used to evaluate the interaction between factor VIII and the platelet membrane. Ristocetin was used in these experiments to determine if excessive lipid peroxidation altered the membrane-factor VIII interaction. In man aggregation is initiated at a low concen- tration (0.5-1.6 mg/ml); the conversion of fibrinogen to fibrin is seen only at a higher concentration (greater than 1.6 mg/ml) in both PRP and platelet poor plasma (PPP) (16). In these experiments, 4 mg/ml of ristocetin was needed to aggregate platelets. The plate- lets would form large aggregates, then ball up in a fibrin clump. In every group there were some atypical ristocetin curves. Visibly, the platelets in these specimens would form large aggregates but would fail to ball up in the fibrin clump. This would indicate that the platelets were functional and adequate to interact with factor VIII at the membrane. The problem appeared to be the level of fibrino- gen available for conversion to fibrin. Liver injury due to vitamin E deficiency might decrease the production of fibrinogen. It is postulated that, if hydrogen peroxide caused in vitra aggregation, then in viva circulating peroxides could also cause 37 intravascular aggregates. The hydrogen peroxide aggregation tracing reflects a complex and poorly understood series of events. Hydrogen peroxide produces an initial small increase (H) followed by a pre- cipitous drop in light transmission. This is followed by an equally sharp return of the curve to at least the baseline (Figure 2). These three components of the curve are designated as phases 1, 2 and 3 of hydrogen peroxide aggregation. Ultrastructural studies show the first phase to represent platelet aggregation. The sharp decline (phase 2) in light transmission is due to platelet disruption with release of platelet content. It is in this phase that platelet disruption is discernible as myelin figures. The cause of the final rise in light transmission is unclear as recovery of platelets for TEM was impossible. It was postulated that the excessive liberation of oxygen, as evidenced by bubble formation, in combination with the total platelet destruction seen on electron microscopy, caused entrap— ment in platelet debris. The bubbles apparently carried the debris to the surface leaving the specimen more transmissible to light. Evaluation of hydrogen peroxide aggregation was based on the first phase of the tracing (H). A rise in the first phase indicated aggregation. All groups of pigs except those injected with vitamin E demonstrated inhibition of this small wave of aggregation. Tissue enzymes (LDH and SGOT) were used to monitor vitamin E deficiency. Normal values were established for these experiments. SGOT appeared to be the best indicator of tissue changes. The LDH results were often elevated. This could have been due to hemolysis. Lack of information in the literature hampered adequate correlation 38 between isoenzymes and specific organ damage. I was able to demon- strate that pig LDH can be separated into five fractions by electro- phoresis. An increase in the fifth isoenzyme appeared to be associated with liver damage. This would be comparable to similar changes seen in man. Red cell sensitivity, monitored by osmotic fragility, was not a good indicator in increased lipid peroxidation. The results did indicate that porcine red cells have an increased fragility ranging from initial hemolysis at 0.60-0.65% NaCl to complete hemolysis at 0.35-0.40% NaCl. The hydrogen peroxide hemolysis test, which was used in experiment B, was the better test in monitoring lipid peroxidation. Increased peroxide sensitivity was seen in pigs in all groups except those that were injected with vitamin E. This confirms previous reports in the literature (44). Alpha tocopherol acetate and, to a greater extent, alpha tocopherol nicotinate have been reported to inhibit hydrogen peroxide and ADP aggregation (22) . It was thought that if vitamin E can inhibit aggregation in vitra it might have similar effects in viva. This could have great clinical significance in the treatment and prevention of thrombosis. Neither oral consumption nor in vitra incubation with alpha tocopherol acetate had any effect on platelet response to hydrogen peroxide, ristocetin and ADP. It is concluded that vitamin E does not alter the platelet release to ADP. This is in keeping with the recent reports by the American Heart Association (9) on the effect of vitamin E on ADP and epinephrine aggregation. 39 Although no conclusive evidence was obtained in this research to link platelet dysfunction to the hemorrhagic lesions reported in pigs, there was evidence that vitamin E deficiency caused alteration in two measurable parameters of platelet function, platelet factor three availability and hydrogen peroxide aggregation. This informa- tion can now be the basis for more sophisticated research into the interaction between platelets and vitamin E. Specifically, the effects of prostaglandin and lipid peroxides can be evaluated using these two sensitive tests. Some exciting areas of platelet research today concern the intraplatelet synthesis of prostaglandin and its intermediates such as LASS (labile aggregating stimulating substance). These substances are peroxides and their synthesis is blocked by aspirin. It would be worthwhile to determine if vitamin E promotes or inhibits prostaglandin in the platelet. CONCLUSION Two factorial experiments involving 14 young pigs were con— ducted to evaluate the effects of vitamin E on platelet function. The pigs were fed on four different diets: l) basal ration (deficient in vitamin E and selenium), 2) basal ration plus intra- muscular (I.M.) injections of vitamin E, 3) basal ration plus I.M. injections of selenium, and 4) basal ration plus I.M. injections of vitamin E and selenium. Normal values for clot retraction (43-90%) and platelet factor three availability (24.4 :_7.4 sec.) were established for the pig. Platelet ultrastructural changes following hydrogen peroxide aggre- gation indicated that aggregation was followed by destruction of the platelet. Platelet factor three availability and hydrogen peroxide aggregation were two measurable parameters of platelet function which were altered in pigs fed a basal ration. The pig appeared to be a suitable experimental animal for determining the effects of vitamin E on platelet function. In addition, in viva experimentation on human platelets indi- cated that vitamin E neither inhibited nor augmented platelet aggregation. 4O APPENDICES APPENDIX A PREPARATION OF REAGENTS APPENDIX A PREPARATION OF REAGENTS Platelet factor three availability 1. Kaolin. A suspension of 4 gm of kaolin (J. T. Baker, Phillipsburg, N.J.) in 160 m1 of 0.85% NaCl. 2. 0.035 M calcium chloride. 3.9 gm of anhydrous CaCl2 (Mallinckrodt, St. Louis, Mo.) was dissolved in distilled water and diluted to a liter. Osmotic fragility of RBC l. Buffered salt stock solution (10%). 180 gm of NaCl, 27.31 gm of dibasic sodium phosphate (NazHPO4) and 4.86 gm of monobasic sodium phosphate (NaH PO ~2H20) were dissolved in dis- tilled water and diluted to 2 liters. Hydrogen peroxide hemolysis test l. 0.9% sodium chloride. 9.0 gm of analytical grade (NaCl) was dissolved in distilled water and diluted to 1 liter. 2. 4 Molar hydrogen peroxide. Mix 1 part of 30% hydrogen peroxide and 1.5 parts of phosphate buffer. 3. Fifteenth Molar monobasic potassium phosphate (KH P04). 2 9.078 gm of monobasic potassium phosphate was dissolved in distilled water and diluted to 1 liter. 41 42 4. Fifteenth Molar dibasic sodium phosphate (Na HPO4). 11.876 2 gm of dibasic sodium phosphate was dissolved in distilled water and diluted to a liter. 5. 0.05 M phosphate buffer, pH 7.4. 61.2 ml of 1/15 M NaZHPO4 and 14.6 ml of 1/15 M KH2P04 were mixed with 24.2 ml dis- tilled water. 6. Saline-phosphate buffer. Equal parts of the stock saline and the phosphate buffer solutions were mixed. APPENDIX B LABORATORY DATA FROM PIGS FED BASAL RATION, EXPERIMENT A 43 mnom owam oowv .¢Bm5 .« ucmswummxo .coflumu Amman vow mmwm Eoum sumo xuoumuonmq .m xHozmmm¢ v... ....... m z 82 Au. .82.... 2..: 8sz 88.25 ...... ...... a... .20 ....... m 2 82.3 ......» 8.2.3... 83: Sm ...: 3-2: ....... z 8...: £2 228.83 in .28 I: ..o ..... m 2 88.8 82 88.3... 82.32 ...: ...: on-.. Ems. NON: .29. 2mm 82. B: 2.2 5.2 .22: <28 2.5.: 3. 88 a 2.. xmflfimmwx xxxx.www xxxxxxxx xxxxuuuu unuuuuuu uuuunuux xuuuunnx uxmflflflkx2mmflflflflflw .KKKKKKKt‘KKKKK .822 ....... m .2 8a.... 2.3 88...: 8.2.8... .5. ...... 3-3 .8 ....... m : ---- 2....." 68.8. 2.8 TB .8 m s Nan 8.2.228 .--- ..-: 2: 8A 822. N3. 822 U “I. E a .22: <28 5.8.: 3. 88 .2 2.. APPENDIX C LABORATORY DATA FROM PIGS FED BASAL RATION, EXPERIMENT B 44 AmHm>HoEmn n 82 .m ucwfifinwmxm .coflumu Hmmmn 0mm mmflm Scum mumc >uoumuonmq . 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2:38:me .------ ------ --- --- ----- ---- ------ ---- ---- ---- ---- ---- ---- 88-88 -----. ------ ------ --- ------ ---- ---- ---- ----- ------ ---- ---- ----- 88-88 888.. .8 v.8 t .882 .8 8 88.88 88 82.8 888.88 8.88 888.882. 888.888 2.88 2.88 88-88 - 88.8.8 88. f8: 8.88 -:.- 2.88 888 8.88 8888 88 8 z 8 888: 88 8888 888.88 8.88 888.888 888.888 888 888 88-88 888.8 .28 8 2. 8. 8888 88 .888 888.88 8.88 888.88.. 888.888 888 888 88-88 8:888: 88888 888: 88< 288 888 8888 :88 88: 828 888 888 8888 8888.: 88 8888 8 888 Vggxxxxxxxx xxxxxxxx xxxxxxxx xxxxxxxxnxxxxxxxx xxxxxxxx xxxxxxxx xxxxxxxx xxxxxxxx xxxwvnxnxfigxxx wyxxxxxx xxxxxx 8888 88-88 8888 88 8 a 8 .---- 88 8888 888.88 8.88 888.888 888.888 888 888 88-88 8888 8888 a 2 8 88.88 .8 88 .L8888 888.88 8.88 888.888 888.888 888 888 88-88 ---- 88 8 z 8 88.88 88 8888 888.88 8.88 888.888 888.888 888 888 8-88 8888 ----- a a 8 888: 88 8888 888.88 8.88 888.888 888.888 888 888 88-88 8888 88 8888 = 8 8888 88 8888 888.88 8.88 888.888 888.888 288 888 88-88 88.888... 8882. 8888. 898 288 882. 888 8.2 88... 88 8888 82... 28.2.8.8 88.8.: 82. 8888 2. 888 885828 APPENDIX F LABORATORY DATA FROM PIGS FED BASAL RATION PLUS INJECTIONS OF VITAMIN E, EXPERIMENT A 47 282882080: u 88 .¢ quE2Hmmxm .m C2Emu2> mo mcowuomnC2 892m cowumu 268mg 0mm mm2m Scum mumc xuoumnonmq .m xHozmmmd 8888 .888 ...... .888. 2 888 888.82 888.8 8.28 888.882 888.882 888 888 8-82_ 8888 2.8 -8 2 . 88 888.82 8.88 888.888 888.882 888 888 88-8 8588.. 2.8228 808.: .2288. 2288 R228 .288 E2 on: 88.8 8.2.. .2228 «Em 82.8. m 82. 2020 8 2828 qunxnxnxnxuxnx188-212-5225. 8:38:82: 53:33:32 83:32:: 5:853:22 8:258:22 88:28-22: 8:82.332: 5:325:38 xnxnxnxnx-x-xnxu. 2:21:58: 2:22-2:58 28:2: 8288 20 -r- m z 88 888 oc2882 8.88 cco.omm ooo.mom 888 828 8-22 8888 a. .888 ----- 2.88.8 z 88 888 888.88 s. 8.88 888.888 888.888 888 888 88-82 8888 2.8 ----- 8 2 88 888 888.82 8.88 888.888 888.888 888 888 88-82 8888 20 ---- m z 88 888 oom.8~ 8.88 ooo.82m oo8.888 828 828 82-02 8888 888 ...... 8 z 28 888 888.82 8.82 888.888 888.888 888 888 8-82 0888 20 --- m 2 mm -- oom.- 8.88 oom.NwN coo.oo~ 888 888 om-m 88.888... 8882. 8888. 82.8 28.8 882. .288 88... 88: 8... 8888 82.8 8882-. 88.8.2 82. 8828 8 82.. mhmAM8KAA 0838009 nxax-xnxnxnxux-x Ix-xnxnxflx nxuxuxxx-u-x :- ux- 22: xxx-xsx-x-xx 188-2-242-2an 8212-2-2-2-2-2-2 5228:2159 12-88-1252 2153395 52530252812252; 85023 8888 20 ------ m z 88 ~88 cc8.n2 o.o~ oom.8~8 888.888 888 888 8-22 owo8 xo --- m 2 on 888 ocw.2~ 8.28 oom.NM8 ooo.mm8 888 888 mN-O2 2828 .2. 8.8 ----.- ... 8 z 88 288 888.8 8.88 888.888 888.888 888 888 88-82 8888 88 V.8 .2. 8 z 88 888 888.22 8.88 888.888 888.888 888 888 88-82 8-O2 om-m 828282 8.2882. 8882 8228 2888 8222. 2.88 2.8-. 8222 8.8 ...-88 82... 2.82.8. 88.8... 82. 8828 8 8... m 2.5.4.2.; APPENDIX E LABORATORY DATA FROM PIGS FED BASAL RATION PLUS INJECTIONS OF SELENIUM, EXPERIMENT B 46 AmHm>2osmn n «V .m quEHuwmxm .85228288 80 mcofluomnGH 892m GOHUMH 28883 8mm 802m scum mumn muoumuonmn .m xHszmmd 8888 ------------ 88-82_ 8888 a 228 r a x. 8 88 .8. 828 888.88 8.88 8.888 8.888 888 888 82-82.. 8888 ... .288 #323 2.. 8 88.28 88 w. 888 888.82 8.88 .888 8.888 888 828 82-82 ...... 228 8 8 82.88 .8. 88 w 888 888.82 8.88 88.888 88.888 888 888 8-82 8888 828 a 8 88.22 88 888 888.88 8.88 .888 8.888 888 888 88-22 8888 88 8 88.8 28 888 882.82 8.88 8.888.2 88.888 888 888 88-22 2628: 88822. . 888: 898 E8 8822 888 :22 88: 88 8828 82: <88 28.: 88 2828 2 828 282.2 2:.zw4wm 48 8888>20805 u 88 .m GHEmuH> mo mcofluowhcfl 8888 . 88 8 .8- 88 8. 8: x. 8 ----- 88 8882 882.82 8.88 8888 88 8888 : .2888 88.8 8.. 88 ... 8882 888.88 8.88 ----- .88 8888 I 8888 88.8 88 8882 888.88 8.88 8888 88 88 8288 z .8. 8 8888 88 82. 8282 882.88 8.88 2 8888 88 8 : 8 82.8 88 8822 888.88 8.88 828282 88828 888: 888 288 888 8888 :82 882 888 888 8888888888888888888888888888888888888888888888888888888888888888888888888088888888 m Z_2<._._> .m unmeflummxm 858m coaumu Hmmmn 6mm 808m Eonm mumw muoumuonmq 888.888 888.888 888.888 888.888 888.888 888.888 88.888.2 888.888 888.828 888.888 888 8888 92-52-59) 922-585 .0 xHozmmm¢ _ 88-82 888 888 82-82. 888. 888 82-82% 888 888. 8-82_ 888 888 88-22. 888 888. 88-22 HHHU.= 0“ HOHU m 0:. iii-53328. APPENDIX H LABORATORY DATA FROM PIGS FED BASAL RATION PLUS INJECTIONS OF BOTH VITAMIN E AND SELENIUM, EXPERIMENT A 49 m :HEmuH> anon mo mcofluummcfi msam :oHumu Hammn wmm moan Scum mumu wuoumuonmq .m xHszmmm 8mm xo ....... m z a E 8}: 93 833 2563 2m 3m 0-: E: vs ...... m z 2 RN 8%: 0.3 25.23 2569.. 3». . E mg: 32 v3 ...... m z 3 N8 83: Ni 83% 838 g *2 NE: 28 xo ...... m z 3 mg 85.2 mgm 83: 256% $8 an 3-3 33 Son ...... m z a 02 85.2 EN 89g 838 “an 30 I: 88 . xo ...... m z .8 81: .NAN 8m.~$ 8.38 .3 2w om-m :55: 962 NON: an? 2mm and .88 :3 um: n: Sm 55 SB 38.: 3. 88m o: xxxxxxxxafimmmmfimmw.:wxxxxmw.mxmmmmmw.mmxmwxmwflwxwmlmmw.mmxmxwxw.gmxmflxxx. 333000006JOSOOOOOOJOOODOOOG303330003433333. :8 ... xo ...... ¢ m z 3 3.0 83.... SN 8am; 89m: 3 3w 0-: :3 {Son ..... m z .8 N8 8}: 98 255$ 25.23 *3 *2 8-2 28 V3 .m z 3 «mm Sam ”.8 25.23 832 am 2K 2: 22 go ..... m z 2 32 coma». mg 805m 83$ *3 w: I: 3% ~89 83 z Sc St: 58.: N.:. 832 83: *3 E I: 3?. H89 m z 8 25.2 v.3 255% 8:3 «.3 :5 85 :33: 8m? 83 a3 2mm RE .88 a: 82 E SE “an LITERATURE CITED LITERATURE CITED Baehner, R. L., Gilman, N., and Karnovsky, M. L.: Respiration and Glucose Oxidation in Human and Guinea Pig Leukocytes: Comparative Studies. J. Clin. Invest., 32; 692, 1970. Baehner, R. L., Nathan, D. G., and Castle, W. B.: Oxidant injury of Caucasian Glucose-6-Phosphate Dehydrogenase- Deficient Red Blood Cells by Phagocytosing Leukocytes During Infection. J. Clin. Invest., E9; 2466, 1971. Bowie, E. J. W., Thompson, J. H., Didisheim, P., and Owen, C. A.: Test of Platelets and Blood Vessels, in_Mby0 Clinic Laboratory Manual of’Hemostasis, pp. 29-33. w. B. Saunders, Philadelphia, 1971. Caasi, P. J., Hauswirth, J. W., and Nair, P. P.: Biosynthesis of Heme in Vitamin E Deficiency. Ann. N.Y. Acad. Sci., 203: 93-102, 1972. Call, F. L., II, Lucas, C. T., and Williams, W. J.: Phospho- lipid Synthesis in Human Platelets. J. Clin. Invest., fig; 13a, 1969. Cazenave, J. 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L., Grob, D., and Lilienthal, J.: On the Anti- thrombic and Antiproteolytic Activity of Alpha Tocopherol Phosphate. Am. J. Physiol., 153: 127-132, 1948. Zucker, M. B.: Platelet Function, in Hematology: edited by W. J. Williams, E. Beutler, A. J. Erslev, and R. W. Rundles, pp. 1014-1022. McGraw-Hill, New York, 1972. VITA The author was born in Brooklyn, New York, on March 1, 1950. He attended Bob Jones University in South Carolina and graduated from Saginaw Valley College, where he received a B.S. degree in Biology, after completion of an internship in Medical Technology in 1972. He interned at St. Luke's Hospital in Saginaw, Michigan, where he remained on the staff until June 1973. He was admitted to the graduate program in Clinical Laboratory Science at Michigan State University in September 1973. During his graduate studies he was a graduate assistant in the undergraduate pathology teaching laboratories. The author is married to Rebecca Laura Scharrer. They are presently anticipating the blossom of their love. 55 MICHIGAN STATE UNIVERSITY LIBRARIES 0 3015 4560 3 1293