‘i W W I ? H I 1 r 4 i 4 r H .‘ 1 ‘ l | I i E‘éf'lTRATE ME) NETRWE TQXECBSES if? SHE? ‘E‘hesEs for the Ebegree cf NE. S. ifitiCHiGAN STAKE UE‘é-N‘ERSHY ROBERT LEE AMSTER 1970 (j ”WWWWW ''''''''' \\ RRRRRRR 3 1293000 imam, ; LIBRARY 1 Ivlichigan State I . . ' Umvetsuy ABSTRACT Nitrate and Nitrite Toxicosis in Sheep by Robert Lee Amster Sheep were fed lettuce which contained relatively high concentra- tions of nitrate to determine whether methemOglobin formation would occur. Sheep were administered large amounts (3.5 Kg. fresh weight) of lettuce after dehydration of the plant, powdering and then making a paste using distilled water. The oral administration of lettuce containing 3 grams of nitrate did not producesanoticeable rise in methemOglobin formation. Methem- oglobin concentrations caused by oral administration of NaNO3 and NaNO2 were used as criteria in evaluating the potential toxicity of nitrate or nitrite in lettuce. The oral administration of 600 mg. NaNO3/hg. and ”0 mg. NOQ/Kg. body weight resulted in a 35 to 55 Per cent conversion of hemoglobin to methemoglobin. The levels of nitrite (MO mg. NOQ/Kg. body weight) required to produce moderate to severe methemoglobinemia in sheep was considerably lower than previously reported. Since formation of methemoglobin is the major factor in the pathogenesis of nitrate tcxicosis and methemoglobin formation was variable in sheep administered NaNO3, methemOglobinization of blood in 313:2 was studied. Results in which NaN02 was used in vitro indicated that the variability of methemoglobin formation in sheep is probably due to factors in the rumen rather than in the blood. NITRATE AND NITRITE TOXICOSIS IN SHEEP By Robert Lee Amster A THESIS submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1970 Dedicated to My Family 11 ACKNOWLEDGEMENTS I wish 'to express my gratitude to those who have contributed significantly to the development of this work. Through their efforts and patience the completion of this project has been possible. A most heartwarming thanks to Dr. S. D. Sleight, my major professor, who has made this thesis possible from conception to delivery. I also WiSY11XD thank Dr. Langham who has borne the task of guiding me through my hours of histOpathology. - I wish to thank Dr. Ries and Paul Rogers of the Horticulture Department for their assistance. Special acknowledgement is made to my wife, Mary Frances, who assisted in the preparation of this thesis. Finally I would like to express my gratitude to the United States Air Force for making it possible for me to attend Michigan State University. iii INTRODUCTION . REVIEW OF LITERATURE MATERIAIS AND METHODS EXPERIMENTAL . RESULTS DISCUSSION . TABLE OF SUMMARY AND CONCLUSIONS REFERENCES . VITA . C ONTENTS iv 10 1h 17 33 37 39 ’42 Figures LIST OF FIGURES Page Mean methemogldbin values in sheep after oral administration of 150, 275, MOO and 600 mg. NaNO3/Kg0 bOdy I’eight O O 0 I O O O O O O O O O O O O 23 Mean methemoglobin values in sheep after oral administration of 20, 30 and U0 mg. Noe/Kg. bOdy weight 0 O O O O O O O O O O O O O O O O O O O O 27 In vitro methemoglobinization of sheep blood induced by NaNOo ho mg./lOO ml. blood. . . . . . . . 29 In vitro methemoglobinization of sheep blood induced by NaNOO 10 mg./lOO ml. blood. . . . . . . . 3o Tab 1 e l. 10. 11. LIST OF TABLES Methemoglobin and hemoglobin values of sheep administered powdered lettuce containing nitrite. . . . . . . . . . . Methemoglobin and hemoglobin values of sheep given 182.h Gm. of lettuce containing the equivalent of 3 Gm. of nitrate. O O O O O O O O O O I O O O O O O O O O O O O O Methemoglobin values in sheep after oral administration of 150 mg. NaNO3/Kg. body weight. . . . . . . . . . . . . Methemoglobin values in sheep after oral administration Of 275 mg. NaNO3/Kg. bOdy WCIght. o o o o o o o o o o o o Methemoglobin values in sheep after oral administration of ‘00 mg. NaNO3/Kg. body weight. . . . . . . . . . . . . Methemoglobin values in sheep after oral administration of 600 mg. NaNO3/Kg. body weight. . . . . . . . . . . . . Methemoglobin values in sheep after oral administration of 20 mg. N02/Kg. body weight. . . . . . . . . . . . . . Methemoglobin values in sheep after oral administration of 30 mg. NOE/Kg. body weight. . . . . . . . . . . . . . Methemoglobin values in sheep after oral administration of AO mg. NOE/Kg. body weight. . . . . . . . . . . . . . Methemoglobin values in sheep after oral administration of 275 mg. NaNO /Kg. body weight one half hour after oral administration f Ru—Bac. . . . . . . . . . . . . . . . . Nitrite values of lettuce after E. coli reduction and original nitrate value before reduction. . . . . . . . . vi 2O 21 21 22 25 32 INTRODUCTION Nitrite induced methemoglobinemia has'beer1 observed in man and animals for many years. With the increased use of nitrate and nitrite compounds in food preservatives and fertilizers, an increasing concern has occurred. The public health hazard is compounded by the accumulations of nitrate in plants. Nitrate in plants is potentially toxic to animals if reduced to nitrite since nitrite causes the conversion of hemOgIObin to methemOglobin. Methemoglobin is the ferric porphyrin complex of hemoglobin which is ferrous in its usual state. Methemogldbin is incapable of combining with molecular oxygen and thus cannot participate in the transport of oxygen to the tissues of the body. Although there are varied causes of circulating methemOglobin, this thesis will deal with methemoglobin caused by nitrate or nitrite toxicosis. It was important to include nitrate because nitrate in plants or food may be a target for bacterial reduction to nitrite. There are several types of bacteria capable of making this reduction including the coliform group which are common contaminants in food and water. With the increased use of fertilizers and herbicides, it has been found that the nitrate content in some plants may“ often be ex- tremely magi. Excessive nitrate in plants presents a double danger: in the diet of animals it can lead to poisoning, and plants stored in silos can liberate nitrOgenous gases that are highly toxic to all, animals, including man. The objective of this research was to determine if the amount of nitrate in commercial lettuce would elicit methemOglobin formation in sheep. A ruminant was chosen since nitrate reduction to nitrite by normal ruminal flora is known to occur. If methemoglobin formation occurred, plants of this nature have a potential toxicity for ruminants and possibly other animals including man. REVIEW OF LITERATURE Pathogenesis of Nitrate and_Nitrite Toxicosis Methemoglobin formation is the result of exposure to an agent capable of oxidizing the ferrous porphyrin complex of hemoglobin to the ferric porphyrin complex of methemoglobin. Cohen and Hochstein (196M) found that hydrOgen peroxide was generated during the reaction between nitrite and hemoglobin. However, the role of hydrogen peroxide as essential intermediary product in the oxidation process has not been elucidated (Kiese, 1966). Winter (1962) reported that hydroxylamine was formed from nitrite in the blood and was itself capable of converting hemoglObin to methemoglobin. Bodansky (1951), emphasized that when a methemoglobin-forming compound is administered, methemOglobin accumulates in the blood at a rate characteristic of the compound. MethemOglobin attains a maximum concentration for a variable period of time and then begins to decrease. The shape of the curve is the result of three processes: (a) metabolism of the compound to other substances which do not oxidize hemoglobin, (b) formation of an intermediate compound which reacts directly with hemogldbin to form methemoglobin, and (c) reduction of methemoglObin to hemOglobin by the enzyme systems of the blood. These three processes are continuous and interdependent. Spicer (1950) noted differences in the reaction rate between nitrite and the hemoglobin of various species. Stolk and Smith (1966) found a greater methemOglobin reductase activity in red blood cells of the mouse and rabbit, whereas the cat, dOg and human red blood cells possessed lesser amounts of methemoglobin reductase activity. Methemoglobinemia leads to a hypoxia of all body tissues. Simon £3.21- (1958) regarded the hypoxic state in tissues and organs as the basis for the pathologic changes associated with nitrate and nitrite toxicosis. Nitrates in Ruminants Nitrates are normally converted to nitrite by bacterial or enzymatic action within the digestive tract of the ruminant. Nitrite is the first step in a normal process that converts the nitrate to nitrogen. Methemoglobin formation occurs when the amount of nitrate being ingested produces a greater amount of nitrite than can be utilized. Crawford gt 3i. (1958) found that it was difficult to specify the level of nitrate that may safely be fed to cattle. There is a range of levels from zero dose to maximum safe dose, or LDO within which no animals die. Tolerance of nitrate varies from animal to animal as well as from species to species. Therefore, it is impossible to establish the LD0 with absolute certainty unless every single animal under all possible circumstances is tested. Crawford 33 El‘ (1958) also emphasized that the danger to ruminants from nitrate depends on at least three major factors: (a) the concentration of nitrate in the forage, (b) the total quantity of nitrate consumed, and (c) the speed of intake of the toxic forage. Lewis (1951) noted that above a certain concentration of nitrate the rate of reduction of nitrite to ammonia becomes limiting, and consequently nitrite accumulates in the rumen. A bacterial homOgenate prepared from rumen liquor collected from a cow fed 30 Gm. NaNO3/lfiil(g. body weight reduced both nitrate and nitrite faster than a similar homogenate prepared from a control cow (Davison gt gl., 1962). Crawford gt El. (1960) stated that animals with stomach contents that are acidic (pig, dog, rat, man) do not normally harbor the intestinal bacteria that convert nitrate to nitrite. As a result, they are more unlikely to be poisoned by nitrate. However, in an abnormal situation, such as bacterial gastroenteritis, bacteria may be present that would convert the nitrate to nitrite. Methemoglobinemia in Man Caused by Nitrates or Nitrites Comly (l9h5) reported that many rural drinking water reservoirs for human consumption were contaminated with coliform organisms. He described cyanosis in infants caused by nitrates in well water which contained the coliform organisms. Water samples from 20 per cent of 91 dug wells in Iowa had nitrate levels above 65 parts per million (ppm). Comly suggested that well water used in infant feeding should have a nitrate content no higher'thanlo or, at the most, 20 ppm. According to the United States Public Health Service (1962) water containing AS ppm nitrate-nitrogen (NO3-N) has been considered hazardous for use in feeding infants. Hdlscher and Natzschka (l96h) reported cases of methemOglobin formation in infants fed spinach puree containing high levels of nitrite. The age of the child is one of the most important considerations in nitrate toxicity (Phillips, 1967). Keohane and Metcalf (1960) reported that hemoglobin from fetuses and the newborn is more sensitive to the action of methemOglobin forming agents such as nitrites. Sinha (1968) found blood of fetal guinea pigs more susceptible to methemOglobin formation than blood from adults. Ross (1963) reported that premature infants are more susceptible to methemoglobin formation than infants born at term. Metcalf (1961) reported that hemoglobin sensitivity to methemoglobin forming agents extends to pregnant women, patients with carcinomata and to patients with some other conditions. Using i2 21:39 studies, Metcalf, found that the nitrite conversion rates were significantly faster in children, pregnant women and adults with definite carcinomata. Keohane and Metcalf (1960: obtained little evidence as to the cause of the differing sensitivity of cells to nitrite in children and adults. There were four possibilities postulated. The difference may be in the plasma, in the cell membrane, in the hemOglobin itself or in some other intercellular constituent. Burden (1960) demonstrated that the higher gastric pH and greater relative fluid intake in infants facilitates the bacterial reduction of nitrate to nitrite. Sodium nitrate and sodium nitrite are incorporated in some fish and meat products at levels of 200 and 500 ppm, respectively, as preservatives (Fassett, 1966). Orgeron §t_§l. (1957) described several outbreaks of nitrite poisoning following the accidental incorporation of excessive amounts of nitrate and nitrite mixtures in meat products. Methemogldbinemia in Animals In a later report by Metcalf (1962) in work with rats, the reaction time to form methemoglobin of young rats as compared to that for older animals seemed to be more closely related to growth rate than to puberty. From this he concluded that a part of the diet may be involved. Of all the individual components of the diet tested, riboflavin alone, was capable of markedly slowing the reaction time of the young and pregnant rats. Riboflavin occurs in the body as a constituent of many enzymes and enzyme systems concerned with electron transfer. Metcalf suggested that a flavoprotein enzyme could delay the oxidation of the hemoglObin ferrous iron by nitrites, by acting as an electron acceptor until exhausted. Riboflavin Iwaquirements are especially great during rapid growth, pregnancy and debilitated conditions. Feeding trials by Crawford g3 g1. (1960) with dairy cattle on a typical ration, indicated that a rise in methemoglobin was not significant at levels of nitrate in forage up to 15 Gm. per h5 Kg. of body weight. A sharp increase in methemOglobin was not attained until levels greater than 25 Gm. of nitrate per MS Kg. of body weight were given. Death may not occur until amounts greater than 25 Gm. of nitrate per h5 Kg. of animal weight are consumed. It is impossible, however, to predict the exception to the rule. In their work with pregnant dairy cattle, Simon g3 gl. (1959) concluded that the concentrations of methemoglobin varied among animals within groups and in individual animals on different days. Setchell and Williams (1962) reported that sudden death in pregnant ewes occurred due to chronic ingestion of food containing 2 per cent potassium nitrate. However, the sheep had only a slight rise in methemOglobin and no determination was made as to the cause of death. Protection against the methemoglobinemia induced by orally administered sodium nitrate was obtained by drenching sheep twice daily with a sucrose solution or by the addition of corn to a forage ration (Emerick g3 g1., 1965). Nitrate in Plants The increased uptake of nitrate in plants is affected by limited moisture supply, low light intensity and low temperatures. This is probably because the growth of the plant is decreased to a greater degree than is the nitrogen uptake (Breniman gE_gl., 1961). Concentrations of nitrate high enough to be dangerous indicate that the conversion process has lagged behind the absorption process. The presence of nitrate in large quantities due to high nitrOgen fertilization is a ready source for plant uptake. Apparently the accumulation of nitrate is not harmful to the plant (Crawford EE.§l°: 1960). Marked differences are found in nitrate accumulation between plant Species as well as between different parts of the plant. The nitrate content is highest in the stems, intermediate in the leaves and lowest in seeds. Immature plants generally contain more nitrates than mature plants (Crawford 23.2l': 1960). Davison (1966) reported that the Species of plant is an important factor in suspected nitrate poisoning. Corn, oats and other small grains, sudan grass and other sorghums, and many common weeds may accumulate large quantities of nitrate. The utilization of nitrate by the plant is postulated to occur by nitrate reductasgg nitrite reductase the pathway of nitrate “‘ r nitrite ‘rt ammonia. Nitrate reductase appears to be the regulator of nitrates in this system (Hageman g3 gl., 1962). When fertilizers that supply nitrate are added to soils, the nitrate is rapidly absorbed by the growing corp (Crawford g3 g1., 1960). Ordinarily this nitrate is reduced quickly for incorporation into amino acids which are vital to the life processes of the plant. The field application of simazine [:2-chloro-h, 6-bis(ethylamino) -s- triazing] , a herbicide, increased the total nitrOgen level of tolerant plant Species (Ries _33 gl., 1963). In a later study by Ries gt g1. (1968) 0.23 Kg. of Simazine per acre caused more than an eightfold increase in nitrate content of dry rye grass forage. The protein content was increased in rye grass forage, rice foliage, alfalfa forage, peas, beans and oats. Simazine application to an established alfalfa stand increased the protein content but did not appreciably increase the nitrate level. MATERIAIS AND METHODS General Plan and Considerations The primary objective of this research was to determine if nitrate in lettuce was capable of causing methemoglobin formation lg XiXQ‘ The amount of nitrate present in lettuce was determined so that a dosage equivalent to this could be given as NaNO3. In another experiment an attempt was made to reduce the nitrate in lettuce to nitrite by means of Escherichia coli. In this manner it would be possible to determine if the reduction of nitrate to nitrite in the lettuce would cause more methemoglobin formation than that caused by unreduced nitrate in lettuce. It was necessary to determine the amount of methemoglobin formed after the administration of NaN03 and NaN02. These results were compared with those obtained after the administration of lettuce and this information made it possible to determine the percentage of nitrate in lettuce, or chemical, that would require reduction to nitrite to cause a methemoglob— inemia. Thus, 3 Gm. of nitrate present in lettuce could induce the same amount of methemoglobin formation as 3 Gm. of NaN03. It was desired to determine if a known amount of nitrate in lettuce reduced to nitrite could produce a methemoglobinemia comparable to an equivalent amount of NaNOQ. Preliminary tests with the sheep used in this study indicated that there was a variability among the sheep as to the amount of methemoglobin formed after being administered NaNO EXperiments using NaN02 lg vitro 3. were conducted to determine if the variability was due to individual blood characteristics or ruminal differences in the reduction of nitrate. The fact that the rumen bacteria are able to reduce nitrate to nitrite suggested a study on the effect of increasing the population of IO ll ruminal bacteria. An experiment was then designed to increase the number of bacteria present in the rumen in order to enhance the reduction of nitrate to nitrite by the normal ruminal flora. A known amount of NaN03 was used as the nitrate source in the altered ruminal flora experiment. Plasma nitrate and plasma nitrite values were determined during the studies so that their appearance and disappearance in the blood could be followed. Hemoglobin values were determined mainly as a means of obtaining methemogldbin values as per cent hemoglobin. Technigues, Chemicals ang_Care and Management of the Shggp Nine mature, crossbred sheep were used in the study. Fecal samples were checked for internal parasites and the sheep were treated with thiabendazole* at the beginning of the experiment. They were kept in a field lot with shelter from the rain and cold and hay was fed during the winter months. Nitrate levels were found to be insignificant in the hay, pasture and water. Ten milliliters of blood Obtained from the external Jugular vein were required for plasma nitrate, plasma nitrite, methemoglobin, and hemoglobin determinations. Heparinized** 10 ml. disposable syringes with 2.5h cm. disposable 20-gauge needles were used. MethemoglObin values were determined photometrically*** using the Evelyn and Malloy (1938) procedure. As soon as possible the blood *Equizole - Merck Chemical Division, Rahway, N. Y. **Heparin (ammonium salt) - Biological Research Inc., St. Louis, Mo. ***Coleman Model 6D Junior SpectrOphotometer, Coleman Instruments Inc., A2 Madison Street, Maywood, Ill. 12 (0.1 ml.) was mixed in M/60 phosphate buffer* in order to prevent reduction of methemogldbin (Sleight and Sinha, 1968). Hemoglobin values were determined by the cyanmethemoglobin method (Benjamin, 1961). Optical densities were recorded by the use of a spectrophotometer. To prepare protein-free plasma for nitrite and nitrate determinations the blood was centrifuged soon after collection and the plasma was removed. The procedure of Diven g3_g£. (1962) was followed. The nitrate determination had the following modification: instead of the addition of powdered zinc, a small amount of root nodule bacterioid was added. The determination of plasma nitrate and nitrite value was performed by using the color development procedure as described by Diven g3 El: (1962). For all experiments a fresh solution of sodium nitrate (NaN03)** and sodium nitrite (NaN02)*** was prepared. The dosages of NaNOé were determined as the amount of nitrite given. The core was removed from hO heads of lettuce, with a fresh leaf weight <3f 38 Kg.. The lettuce leaves were spread in a 60 C.drying room for ho hours. The dry weight of the leaves was 2.05 Kg., which was 5.h per cent of the fresh weight. The dry leaves were crushed and then ground in a Wiley Mill**** using a l mm.mesh screen, the final form being * A M/lS stock phosphate buffer (pH 6.6) was prepared by dissolving 9.0 Gm. of Na2HP0h°l2H20 and 5.7 Gm. of anhydrous KH2POh in water and diluting to 1 liter. Three volumes of water were added to 1 volume of the stock buffer to prepare M/60 buffer. **Sodium Nitrate Crystal (NaNO ) - "Baker Analyzed" Reagent, J. T. Baker Chemical Co., Phillipsburg, N. J. ***Sodium Nitrite Crystal (NaNOe) - "Baker Analyzed" Reagent, J. T. Baker Chemical Co., Phillipsburg, N. J. ****Wiley Mill - Mill, Laboratory, Wiley, Intermediate Model-A. 13 a course powder of lettuce. The lettuce was Obtained from Desert Products Inc., Holtville, California. Determinations of nitrate and nitrite in lettuce were made by using the Lowe and Hamilton procedure (1967). The nitrite determinations had the following modification: distilled water was added to the sample instead of bacterioids as nitrite was already present. Escherichia coli culture was used as a reducing bacteria to obtain nitrite from nitrate in lettuce. A culture was Obtained by inoculating a 1,000 m1. volume of trypticase soy broth* with a nonpathogenic E. coli, then incubating in a 37 C.culture room for 36 hours. The growth was exuberant as evidenced by the milky color of the broth when stirred. Three (186 Gm.) samples of powdered lettuce were placed into three 2,000xnl. Erlenmeyer ‘flasks. Distilled water was added to the powdered lettuce and E. coli mixture to form a slurry. The flasks were capped with gauze cotton and incubated at 37 C. After 18 hours of incubation 0.5 ml. of penicillin/streptomycin** was added to kill the E. coli. The reduced lettuce was refrigerated immediately and used within two hours. A sample of slurry containing approximately 2 Gm. lettuce (dry weight) was taken for nitrate and nitrite analysis. * trypticase soy broth - BBL, Division of BioQuest, Division of Becton Dickinson and Company, Cockeysville, Maryland 21030 P.D.C. - procaine penicillin G in dihydrostreptomycin solution, Jensen- Salsberry Laboratories, Division of Richardson-Merrell Inc., Kansas City, Mo. EXPERIMENTAL Determinations of Levels of MethemoglObinLHemoglobip, Plasma Nitrate and Nitrite Aftgr Adminigtgation of_Lgttuce Containing Known Quantities of Nitrate Two sheep, weighing approximately LO Kg. each, were randomly selected and each given 182.h Gm. of lettuce by stomach tube. The 182.h Gm. of lettuce represented 3 Gm. of nitrate. The powdered lettuce was suspended in 3 liters of distilled water and then pumped into the rumen. After the lettuce had been administered, blood samples were Obtained at 30 minutes, 1, 2 and 5 hours for methemoglObin, hemoglObin, plasma nitrate and nitrite determinations. A third sheep was given the same amount of lettuce by way of a large dose syringe. The same blood studies were performed. A blood sample was obtained prior to administration of the lettuce to the sheep. Determinations of Levels of MethemOglobin, Hemoglobin and Plasma Nitrite After Administration of Lettuce antaining Known Quantities of Nitrite Three sheep weighing approximately AO Kg. each, were randomly selected and given 182 Gm. of lettuce in which it had been attempted to reduce the nitrate to nitrite using an E. coli culture. The lettuce was administered to the sheep by way of a large dose syringe. A fourth animal was not given any lettuce but was used as a control animal. Blood samples were obtained before lettuce administration and at 30 minutes, 1, 1.5, 2, 3, A and 5 hours. The nitrite values are given in the footnote of Table 1. 1h 15 Determinations of Levels oprethemOglobin, HggoglObin, Plasma Nitrate and Nitrite After Oral Administratigg of NaNO3 Four experiments, 1, 2, 3 and h were conducted using four sheep each, randomly selected and weighed before each NaN03 administration. The NaNO3 was dissolved in distilled water to make a 10 per cent solution. The NaN03 dosages were given at the rate of 150, 275, hOO and 600 mg./Kg. body weight in experiments 1 through A respectively. The NaNO3 solution was given orally by using 50 m1. disposable syringes. The use of these syringes allowed drenching without loss of any solution. Blood samples were Obtained before NaN03 administration and at l, 2, 3, h, 5 and 6 hours for all studies and 8.5 hours for the sheep given H00 mg./Kg. body ‘weight, and 8, 10 and 12 hours for the sheep given 600 mg. NaNO3/Kg. body weight. Determinations of Levels of MethemOglobinL and Plasma Nitrite Afte: Oral Administration of NaNO2 Experiments 1, 2 and 3 were conducted using four sheep each, randomly selected and weighed before each NaNOQ administration. The NaNO2 dosages were given at the rate of 20, 30 and ho mg. NaN02/Kg. body weight in experiments 1 through 3 respectively. The NaNO2 solution was given orally by using 10 ml. disposable syringes. Blood samples were obtained before NaN02 administration and at 30 minutes, 1, 2, 3, h and 5 hours for the group given 20 mg. N02/Kg.; 30 minutes, 1, 2, 3, h, 5, 6 and 7 hours for the group given 30 mg. N02/Kg.; and 30 minutes, 1, 2, 3, h, 6 and 8 hours for the group given hO mg. NOE/Kg. body weight. 16 Methemggldbinization of Sheep Blood Induced by NaNO9 (in vitro) Five milliliters of heparinized blood was added to test tubes (15 by 125 mm.) then placed in a 37 C. water bath. A l per cent solution of the NaNO3 was added to the blood so that the tubes contained the equivalent of 10 mg. and ho mg. per 100 ml. of blood. During sampling, the tubes were slowly agitated in the water bath in order to keep the red blood cells in suspension. During sampling, the tubes were slowly inverted several times to also facilitate red blood cell suspension. Hemoglobin and methemoglobin were determined at l, 10, 25, A5, 60 and 90 minutes when hO mg. of NaNO2/100 ml. of blood was used and 0, 20, AD, 60 minutes, 1.5, 2, 2.5, 3, h, 5, 6, 7 and 10 hours when 10 mg. NaN02/100 ml. of blood was used. The samples of blood to which 10 mg. of NaN02 was added were allowed to remain in the water bath for a total period of 10 hours for reduction to hemoglobin. Determinations of Levels_of Methemoglobin, Plasma Nitrate and Nitrite After Oral Administration of NaNO3 FollowiggfiRu-Bag_Administration To introduce additional bacteria to the ruminal contents four sheep were given BE'EEE* one half hour before the oral administration of 275 mg. NaN03/Kg. body weight. The dosage of Rg-ng for each sheep was 1% teaspoons in 10 ml. of distilled water given as a drench. Blood samples were obtained before NaN03 administration and at l, 2, 3, h, 5, 6 and 7 hours after administering the NaN03. * Rg-Bac - Rg-Bac with enzymes powder, Haver-Lockhart Laboratories, Kansas City, Mo. RESULTS In this thesis all methemoglobin values will be expressed as the percentage of hemoglobin oxidized to methemoglobin. Results of Nitrate Determigations in Lettuce The average value of nitrate in lettuce was l6,h26 micrograms per (kn. (dry weight) of lettuce. With this figure it was calculated that 182.h Chn of lettuce (dry weight) would be equivalent to 3 Gm. of nitrate. ‘Dgterminations of Levels of Methemoglobin; Hemoglobin, Plasma_Nitrate and Nitrite After Administration of Lettuce Contaigigg Known Quantities of Nitrite and Nitrate The three sheep that were administered lettuce containing approximately 50 mg. of nitrite and the control sheep not given lettuce did not have an appreciable rise in methemoglobin (Table 1). There was a smaller amount of nitrite present in the lettuce than had been expected. The three sheep that were administered lettuce containing the equivalent of 3 Gm. of nitrate did not have an increase in methemoglobin (Table 2). The nitrate in the lettuce was not toxic even in the large quantity represented by 182 Gm. dry lettuce (3,370 Gm. fresh weight). All hemoglobin values remained within the normal range. Determinations of Levels of Methemoglobin, HemOglObin, Plasma Nitrate and Plasma Nitrite After Oral Administration of NaNO3 In the first experiment the dosage was 150 mg. of NaNO3/Kg. body weight. Blood samples collected at zero time, 30 minutes and hourly 17 18 Table 1. Methemoglobin and hemoglobin values of sheep administered powdered lettuce containing nitrite. Sheep 19 Sheep 32 Sheep A9 Sheep 172 uh Kg. * 37 Kg. * ul Kg. * us Kg. * Time Hgb. % Hgb. % Hgb. % Hgb. % Gm./ MetHb. Gm./ MetHb. Gm./ MetHb. Gm./ MetHb. 100 ml. 100 ml. 100 m1. 100 m1. 0 12.2 3.1 11.3 3.3 13.0 3.2 12.1 1.1 30 min. 11.3 3.3 11.2 3.1 12.3 3.8 12.8 . 1.0 1 hr. 10.9 3.8 11.0 h.7 11.9 h.l 12.6 1.2 1.5 hr. 10.9 3.8 10.8 5.2 11.3 h.6 12.u 0.7 2 hr. 10.6 3.2 10.6 h.u 11.9 3.9 12.h 1.9 3 hr. 10.3 1.8 10.9 3.u 11.9 2.0 12.3 0.0 h hr. 10.9 0.8 11.2 0.h 12.0 1.1 12.3 0.0 5 hr. 10.9 0.8 10.9 0.0 12.0 2.0 12.h O.b * Sheep 19 1.10 mg. NOE/Kg. body wt. Sheep 32 1.66 mg. NOE/Kg. body wt. Sheep ho 1.07 mg. NOE/Kg. body wt. Sheep 172 Control, was not administered lettuce 19 through the sixth hour did not contain any distinct change in methemoglobin (Table 3). The highest value of nearly 5 per cent methemoglobin is not appreciably greater than the normal range of O to 3.5 per cent for this group of sheep. The second study, in which the dosage was 275 mg. NaNO3/Kg. body weight, revealed a low increase in circulating methemoglobin in one animal (Table A). The methemoglobin value for the one sheep reached a maximum of 12.3 per cent, the remaining three Sheep were within the normal range of l to 3.5 per cent. In the third experiment the dosage was increased to AOO mg. NaNO3/ Kg. body weight. A moderate rise of 5 to 2h per cent methemoglobin was attained (Table 5). The fourth study in which the dosage was increased to 600 mg. NaNOB/Kg. body weight there was an appreciable increase in methemoglobin to 19 to A6 per cent (Table 6). In these h-experiments there was arl indication that the greater the methemoglobin values the greater the length of time required to attain maximum circulating methemOglobin(Figure 1). In the comparison of the means for these h studies, 19 to 31 per cent methemoglobin values were attained 6 to 8 hours after the administration of 600 mg. NaNO3/Kg. body weight. A peak of 10 per cent methemoglobin was attained h hours after the administration of AOO mg. NaNO3/Kg. body weight. Determinations of Levels of Methemoglobin, annglasma_NiE£ite After Oral Administration of NaN02 In the first study the dosage was 20 mg. NO2/Kg. body weight. The highest methemoglobin concentration (7.25 to 8.25 per cent) was present at one hour and the value then steadily decreased (Table 7). 2O Table 2. Methemoglobin and hemoglobin values of sheep given l82.h Gm. of lettuce containing the equivalent of 3 Gm. of nitrate.* Sheep 35 Sheep 173 Sheep 19 39 Kg. h3 Kg. ho Kg. Time Hgb. % Hgb. % Hgb. % Gm./ MetHb. Gm./ MetHb. Gm./ MetHb. 100 ml. 100 ml. 100 ml. 0 12.6 .21. 12.2 .13 10.5 .00 30 min. 12.8 .21 11.0 .21 11.2 ..00 1 hr. 11.h .00 11.5 .00 10.5 .00 2 hr. 12.6 .08 11.0 .13 ll.h .00 5 hr. 11.2 .00 11.0 .00 10.5 .00 *Approximately 75 mg. NO3/Kg. body weight. Table 3. Methemoglobin values in sheep after oral administration of 150 mg. NaNo3/Kg. body weight. Animal Methemoglobin (%) at Intervals No. Wt. 0 min. 30 min. 1 hr. 2 hr. 3 hr. h hr. 5 hr. 6 hr. 19 37 Kg. 2.h 3.7 2.0 2.7 2.5 0.0 0.0 3.6 .165 h9 Kg. 3.1 2.h 0.0 2.0 1.1 1.0 1.1 2.5 172 37 Kg. 0.0 2.9 0.0 3.2 1.0 3.1 3.1 1.0 173 hR Kg. 0.0 0.0 0.0 h.8 3.9 3.0 2.6 2.6 Mean 1.h 2.2 0.5 3.2 2.1 1.8 1.7 2.h s. D. 1.6 1.6 1.0 1.0 1.h 1.5 1.h 1.0 21 Table h. Methemoglobin values in sheep after oral administration of 275 mg. NaNO3/Kg. body weight. Animal WW.“ No. Wt. 0 min. 1 hr. 2 hr. 3 hr. u hr. 5 hr. 6 hr. 19 3h Kg. 1.3 2.0 1.9 1.0 2.0 0.0 1.1 32 30 Kg. 1.0 1.0 1.u 3.0 3.0 h.o 1.1 165 50 Kg. 0.0 1.5 1.1 0.0 0.0 0.0 0.0 172 37 Kg. 2.3 6.6 12.3 11.2 10.6 2.8 0.5 Mean 1.2 2.8 h.2 3.8 3.9 1.7 0.7 s. D. 0.9 2.6 5.6 5.1 h.6 2.0 0.5 Table 5. Methemoglobin values in sheep after oral administration of hOO mg. NaNO3 body weight. Animal Methemoglobin ($7 at Intervals No. Wt. 0 hr. 1 hr. 2 hr. 3 hr. h hr. 5 hr. 6 hr. 8.5 hr:__ 167 h5 Kg. 0.5 h.2 1h.1 1h.1 7.9 6.5 0.0 u.0 175 h5 Kg. 1.1 5.” 6.1 17.3 22.9 23.8 22.7 8.0 35 3h Kg. 3.7 1.3 5.6 1.h 1.h 2.8 1.h u.2 119 36 Kg. 0.9 h.9 3.2 2.3 2.8 .2.3 l.h 6.h Mean 1.6 h.0 7.3 8.8 10.3 8.9 6.h 5.7 S. D. 1.5 1.9 h.7 8.1 10.0 10.2 10.9 1.9 22 Table 6. Methemoglobin values in sheep after oral administration of 600 mg. NaK03/Kg. body weight. Methemoglobin (as % hemoglobin) Hour Animal Animal Animal Animal Mean Standard No. 35 No. h9 No.167 No.175 Deviation 39 Kg. 35 Kg. 50 Kg. A5 Kg. 0 2.9 0.5 0.0 0.0 0.9 1.5 1 3.2 3.7 3.6 5.7 h.1 1.3 2 5.h 7.2 15.5 12.h 10.1 h.6 3 7.0 10.7 22.7 12.7 13.3 6.7 h 10.5 9.u 27.h 16.8 16.0 8.3 5 10.7 9.3 37.3 20.h 19.u 12.9 6 19.6 1h.9 u6.h 23.2 26.0 lu.0 8 3h.7 18.8 no.5 29.h 30.9 9.2 10 3u.7 13.5 22.7 30.6 25.6 8.9 12 17.3 8.2 7.9 15.0 12.1 h.8 A8 0.5 2.8 1.0 1.h 1.u 1.0 23 35w 30‘ .g 600 mg. é em 25‘ o e (D :1 ea m 20‘ e“; a "4 £3 15— r—i u) o e 2 g; 10. hOO mg. 51 ,/--..‘.____._‘275 mg. 50 mg. ‘\ ‘ O ’ ‘ 7 r 7 v I I l I I :‘/——-1 2 3 u 5 6 '7 8 9 10 12 M8 Hours Figure 1. Mean methemoglObin values in sheep after oral administra- tion of 150, 275, too and 600 mg. NaNO3/ Kg. body weight. 2h In the second study the dosage was 30 mg. NQZ/Kg. body weight. Three sheep had maximum methemoglObin values of 1h to 2k per cent while a fourth sheep had a considerably higher methemoglObin value of ho per cent (Table 8). In this study the maximum methemoglobin values were attained at approximately two hours and had decreased by the fourth hour. In the third study the dosage was no mg. Roz/Kg. body weight. Individual variation manifested itself in that three sheep had maximum methemOgIObin values of 6h to 82 per cent and the fourth sheep had a considerably lower methemoglObin value of 17 per cent (Table 9). In this study the maximum methemoglObin values of 17 to 82 per cent were attained at approximately two hours and had decreased significantly by the eighth hour. During the study the sheep that had the 82 per cent methemoglObin value became weak one hour after NaNOé administration. Two hours after NaNOQ administration this sheep was down and became semicomatose for one and one half hour. After being down for two and one half hours this sheep got up and walked about, but wObbly. Six hours after NaNOé administration this sheep appeared normal. During the semicomatose period the sclera had the characteristic chocolate color as seen in nitrite toxicity. The comparison of the mean values for these :3 experiments indicates that the 30 to ho mg. nitrite dosages produced a maximum circulating methemoglObin value one hour later than that of the 20 mg. nitrite dose (Figure 2). In these 3 studies the hemoglObin values remained within the normal range. 25 Table 7. Methemoglobin values in sheep after oral administration of 20 mg. NO2/Kg. body weight. Animal __ Methemoblobin (%) at Intervals No. Wt. 0 min. 30 min. 1 hr. 2 hr. 3 hr. 5h hr. 5 hr. 19 35 Kg. h.0 6.5 7.7 u.u 2.1 2.3 2.3 172 38 Kg. 1.9 7.5 7.5 3.6 1.8 1.8 0.7 167 A5 Kg. 2.0 u.3 7.2 2.3 0.h 1.3 0.0 32 30 Kg. 0.8 2.8 8.3 h.o 3.5 0.h 0.8 Mean 2.2 5.3 7.7 3.6 2.0 1.5 1.0 s. D. 1.h 2.1 0.5 0.9 1.7 0.9 1.0 Table 8. Methemoglobin values in sheep after oral administration of 30 mg. NOZ/Kg. body weight. Methemoglobin (as % hemoglObin) Hour Animal Animal Animal Animal Mean Standard No. 19 No. 32 No. 35 No.167 Deviation 3A Kg. 29 Ks- 3h Ks. h5 Kg. 0 h.3 6.0 2.8 7.3 5.1 1.9 0.5 16.9 8.2 20.6 9.8 13.6 5.9 1 23.7 13.7 33.6 15.0 21.5 9.0 2 20.0 12.7 no.2 20.7 23.u 11.8 3 11.9 h.8 h0.u 8.0 16.3 8.9 h 7.6 5.0 20.2 h.3 9.3 7.h 5 3.1 3.1 13.5 2.2 5.5 5.u 6 0.5 N.D. 10.3 N.D. 5.3 h.0 7 0.9 N.D. h.9 N.D. 2.9 1.7 26 Table 9. MethemOglobin values in sheep after oral administration of MO mg. Noe/Kg. body weight. Animal Methemogldbin (%) at Intervals No. Wt. 0 hr. 0.5 hr. 1 hr. 2 hr. 3 hr. u hr. 6 hr. 8 hr. M9 36 Kg. 3.5 hu.2 6u.8 81.6 77.9 71.0 55.8 31.5 165 h8 Ks. 5.0 h0.7 55.9 6h.u 57.0 su.u 28.9 6.h 173 M9 Kg. 3.8 13.3 16.9 11.9 7.1 u.3 2.3 5.u 175 A5 Kg. 2.7 no.8 56.0 68.8 62.1 60.5 32.8 7.6 Mean 3.8 3h.3 A8.u 56.7 51.3 50.5 30.0 12.7 s. D. 1.0 11.8 21.h 30.7 30.6 30.3 21.9 12.5 27 60. 1401 “0 ms- Methemogldbin (as % hemoglObin) ID Lu sheep 167. 3O 30 7 h) U1 1 m 0 4 H U1 0 Methemogldbin (as % hemoglObin) 10‘J 5 i2; Hours Figure A. la vitro methemoglobinization of sheep blood induced by NaNO 10 mg./100 ml. blood. X~ --axsheep 35,~ --sheep A9, o- ~2~—~—«>sheep 173, r —————— a sheep 175. 31 Determinations of Nitrite in Lettuce After E. coli Reduction Analysis of the lettuce after E. 3213 reduction revealed that there was not an appreciable amount of nitrite present. The mean nitrite value of 270 mg./100 Gm. dry weight represents approximately 1.7 per cent reduction of the nitrate originally present (Table 11). Plasma Nitrate and Nitrite Results for All Blood Studies Results were negative for all determinations for plasma nitrate and plasma nitrite during this experiment. 31 Determinations of Nitrite in Lettuce After E. coli Reduction Analysis of the lettuce after E. 3211_reduction revealed that there was not an appreciable amount of nitrite present. The mean nitrite value of 270 mg./lOO Gm. dry weight represents approximately 1.7 per cent reduction of the nitrate originally present (Table 11). Plasm§_Nitrate and Nitrite Results for All Blood Studies Results were negative for all determinations for plasma nitrate and plasma nitrite during this experiment. 32 Table 10. MethemOglobin values in sheep after oral administration of 275 mg. NaNO /Kg. body weight one half hour aft administration f RB-Bac. er oral Animal Methemoglobin (3) at Intervals No. Wt. 0 hr. 1 hr. 2 hr. 3 hr. h hr. 5 hr. 6 hr. 7 hr. 19 39 Kg. 0.0 5.9 8.h 11.3 7.h 1.9 1.9 1.0 32 33 Kg. 1.3 2.h 3.8 2.h h.8 6.8 5.8 u.7 165 50 Kg. 1,u 1.h 1.0 1.0 1.5 2.6 1.5 2.0 172 39 Kg. 1.3 5.h 12.3 15.7 5.8 5.8 0.0 0.“ Mean 1.0 3.8 6.u 7.6 u.9 h.3 2.3 2.3 s. D. 0.2 2.2 5.0 5.7 2.5 2.u 2.5 148 Table 11. Nitrite values of lettuce after E. coli reduction and original nitrate value before reduction. Lettuce Administered Dosage Lettuce Nitrate Nitrite to Sheep mg. NOQ/Kg. Dry Microgm. microgm. Body Wt. Weight Per Gm. Per Gm. No. Wt. Gm. Dry Wt. Dry Wt. 19 AA Kg. 1.10 186 l6,h26 259 32 37 Kg. 1.66 186 16,u26 325 A9 hl Kg. 1.07 186 16,h26 236 DISCUSSION One of the manifestations of nitrite toxicity is the oxidation of hemoglObin to methemoglObin. The resulting methemOglobinemia is the major factor in the pathogenesis of nitrite toxicosis. A public health problem exists due to current agricultural practices of heavy nitrate fertilization. The important factor is not so much that there is a lot of nitrate present but that plants are often subjected to conditions that allows the increased uptake of the nitrate. These conditions have been recognized for many years but it has been only recently established that the application of herbicides increases the uptake of nitrate in certain plant species. It was also determined that some commercially grown lettuce has a high nitrate content (21,000 to 2h,000 ppm). The nitrate in lettuce fed to sheep did not produce a rise in the circulating methemoglobin. Continued normal methemoglobin values were probably the result of (l) a relative small amount of nitrate being reduced to nitrite, (2) the released nitrite was converted to ammonia by the rumen bacteria or (3) the methemoglobin produced was reduced to hemoglObin as fast as it was being formed. The second hypothesis is supported by the Observations of lewis (1951) that nitrite was readily reduced to ammonia but that it did have a point at which nitrite began to accumulate in the rumen and was then absorbed into the blood. The third hypothesis is supported by the Observations of Kitchen (personal communication) in that sheep blood readily reduces methemOglobin to hemogldbin. The method for giving the sheep large amounts of plant 33 3h material (as compared to fresh weight) proved successful. In this experiment an equivalent of 3.5 Kg. fresh weight lettuce was given as a paste made from the 182 Gm. of powdered lettuce. The nitrate given at 150 and 275 mg. NaNO3/Kg. body weight did not produce a distinct rise in the circulating methemoglObin. The ADD to 600 mg. NaN03/Kg. body weight levels of nitrate did produce a moderate rise in circulating methemoglObin. This is substantiated by lewis (1951), that with less than 0.25 mole of nitrate (l9 Gm. NaNO3) a linear relation- ship exists between the amount of nitrate administered and the extent of methemoglobinemia. The greater quantities of nitrate result in a disproportionately large amount of circulating methemoglObin. This he attributed to the reaction of nitrite being reduced to form ammonia if the dosage is below 0.25 moles of nitrate. After the dosage exceeds 0.25 mole of nitrate the nitrite that is not converted to ammonia is absorbed into the blood stream and causes methemoglObinemia. This, Iewis, termed as the "limiting factor". The nitrite studies were more clear cut as methemOglobin values were not as erratic as those in the nitrate studies. Nitrite given in the amount of 20 to 30 mg. N02/Kg. body weight produced a mild to moderate methemoglObinemia. In the experiment in which 30 mg. NOQ/Kg. body weight was administered there was 1 sheep that had a twofold higher methemoglObin value than the others. This can only be attributed to individual variation. Another individual variation was seen in the sheep given ho mg. Noe/Kg. body weight, as 1 sheep had a moderate methemoglObinemia and the other 3 had a severe methemoglObinemia. The levels of nitrite-N (ho mg. Nob/Kg.‘body weight) required to produce moderate to severe methemoglObinemia in sheep was considerably lower than 35 that reported in 1951 by Lewis (115 mg. nitrite-N/Kg. body wt.) and in 1967 by Sinclair and JOnes (60 mg. nitrite-N/Kg. body wt.). The study of methemoglobinization of sheep blood by nitrite 33 11:39 indicated that the variation in methemoglObin concentration seen in 13 yixg studies is due to variations in the rumen. The 13 11:39 methemoglobin formation in blood samples from h sheep were all in close approximation to one another. This close approximation also occurred in the reduction of methemoglObin to hemoglobin., There was only a minimal reduction of nitrate to nitrite in the lettuce treated with E. 3911. The final analysis of the nitrite present in the lettuce indicated that only a 1.7 per cent reduction of the nitrate had occurred (Table 11). This resulted in a small dose of nitrite to the sheep which explains the lack of rise in methemoglObin. In working with the E. 9913 reduction method there was a concern about the nitrate being reduced to ammonia by the nitrite reductase activity found with these bacteria. A better procedure for reducing nitrate to nitrite without the presence of nitrite reductase may be to use bacterioid nitrate reductase. In this procedure a greater nitrate reduction to nitrite would result without loss of nitrite. The addition of bacteria to the sheep rumen before giving NaNO3 may have enhanced the reduction of nitrate to nitrite as shown in Table 10. However, in this experiment the Observed difference was mainly due to 1 sheep. Apparently enough excess nitrite may be formed in a shorter period of time so that the limiting factor (Lewis, 1951) can be lowered. That is, the greater than normal amount of bacteria present in the rumen allows for a greater reduction of nitrate to nitrite in a given amount of time. 36 From this work one should not conclude that there is no potential toxicity in plants containing large amounts of nitrate. From a public health stand point further work in this area should be conducted. Suggested studies may include working with simple stomached animals, such as swine, with gastrointestinal infections. The addition of bacteria to the normal intestinal or rumen flora to enhance nitrate reduction may increase the toxicity. Additional work with herbicides in which higher levels of nitrate in plants are observed may necessitate further work to assess potential toxicity for animals and man. SUMMARY AND CONCLUSIONS This work was designed to determine if lettuce which contained relatively high concentrations of nitrate would elicit a methemOglOb- inemia in sheep. Basic to the accompanying characterization of the effect was a series of studies on the extent of methemoglObin formation caused by NaNOQ 32.12352 and ig_zi!2, and by NaNO3 lg gigg. Determi- nations of hemOglobin, methemoglObin formation, plasma nitrate and plasma nitrite were made. Hemoglobin values were also determined for each blood sample. From these experiments the following conclusions were made: 1. In the lettuce administered, the levels of nitrate (l6,h62 micrograms N03/Gm. dry wt.) did not produce a methemoglObinemia in sheep. Approximately 186 Gm. of powdered lettuce is equal to 3,370 Gm. of fresh lettuce. Thus, even in large quantities the nitrate in lettuce was not toxic. 2. ‘12 11339 E. 3911 reduction of nitrate to nitrite is not easily controlled. Possibly a better method would be to use nitrate reductase. Ifintherinvestigation along this line could reveal better methods. 3. Sodium nitrate (600 mg./Kg. body wt.) or sodium nitrite (ho mg. N02/Kg. body wt.) orally administered results 111 mean methemoglObin concentrations corresponding to approximately 35 to 55 per cent conversion of the total hemoglObin, respectively. A. After a moderate 13 vitro methemoglObinization (30 per cent), the blood enzyme system was capable of reducing methemoglObin to 95 per cent hemoglobin 7.5 hours after peak methemoglObinization. 37 38 5. In spite of some relatively high levels of methemoglObin, the hemoglObin values remained within their normal ranges in nitrate and nitrite treated sheep. 6. The level of nitrite (ho mg. N02/Kg. body wt.) required to produce moderate to severe methemoglObinemia in sheep was considerably lower than previously reported (approximately 125 to 150 mg./Kg. body wt.). 7. The limiting factor of nitrate being reduced to ammonia before nitrite attains excess and becomes absorbed into the blood stream may possibly be lowered by the addition of bacteria to the rumen of sheep. 8. Sheep can be administered large amounts (2.5 to 3.5 KB.) Of plant material after dehydration of the plant, powdering and then making a paste from the powder. In sheep the most successful means of administering the paste was by a large (12 oz.) dose syringe. REFERENCES Benjamin, M. M.: Outline of Veterinary Clinical Pathology, 2nd. ed. The Iowa State University Press, Ames, Iowa, 1961. Bodansky, Oscar: Methemoglobinemia and methemoglobin-producing compounds. Pharmacol. Revs., Vol. 3, No. 2, 1951. Breniman, G. W., Neumann, A. L., Smith, G. S., Zimmerman, J. E.: Factors affecting the nitrate content of forages. J. Animal Sci.,20:926 (Abst.) 1961. Burden, E. H. w. J.: The toxicology of nitrates and nitrites with particular reference to the potability of water supplies. The Analyst, 86zh20, 1961. Cohen, 0., and Hochstein, P.: Generation of hydrogen peroxide by hemolytic agents. Biochemistry, 3:895, 196A. Comly, H. H.: Cyanosis in infants caused by nitrates in well water. J.A.M.A., 129:112-115, (Sept.3) l9u5. Crawford, R. F., Kennedy, W. K., and Wright, M. J.: Nitrate in forage crops and silage. Bulletin 37, New York State College of Agriculture, 1960. Davison, K. L., Hansel, W., Wright, M. J., and McEntee, K.: Adaptation to high nitrate intake by cattle. Proceeding for 1962 of the Cornell Nutrition Conference for Feed Manufactures. Davison, K. L.: Sublethal nitrate poisoning - is it really a problem? Feed Age, 23-27, (March) 1966. Diven, R. H., Pistor, W. J., Reed, R. B., Trautman, R. J., and Watts, R. E.: The determination of serum or plasma nitrate and nitrite. Am. J. Vet. Res., 23zh97-h99, 1962. Emerick, R. J., Embry, L. B., Surley, R. W.: Rate of formation and reduction of nitrite-induced methemoglobin 12 vitro and 12 vivo as influenced by diet of sheep and age of swine. Journal of Animal Sci., 2A:221-228, (Feb.) 1965. Evelyn, K. A., and Malloy, H. T.: Microdeterminations of oxyhemOglobin, methemoglobin, and sulfhemOglobin in a single sample of blood. J. Biol. Chem., 126:655-662, 1938. Fassett, E. W.: Toxicants occurring naturally in foods. Nitrates and nitrites. National Academy of Science - National Research Council Publication, l35h:250-256, 1966. 39 DO Hageman, R. W., Cresswell, C. F., and Hewitt, E. J.: Reduction of nitrate, nitrite, and hydroxylamine to ammonia by enzymes extracted from higher plants. Nature, 193:2h7, 1962. Hblscher, P. M. and Natzschka, J.: MethaemOglobinaemia in young infants due to nitrite in spinach. Germ. Med. Mon., 9:325-327, 196A. Keohane, K. W. and Metcalf, W. K.: An investigation of the sensitivity of juvenile and adult erythrocytes to methaemoglobinization. Physics in Medicine and Biology, 5:27-35, 1960. Kiese, M.: The biochemical production of ferrihemoglObin—forming derivatives from aromatic amines, and mechanisms of ferrihemo- globin formation. Pharmacol. Rev.,'l8:283-290, 1966. Lewis, D.: The metabolism of nitrate and nitrite in the sheep. I. The reduction of nitrate in the rumen of the sheep. Biochem. J., h8:175-180, l9h8. Metcalf, W. K.: A biochemical change in the blood in pregnancy and malignant disease. Phys. Med. Biol., 5:259-268, 1961. Metcalf, W. K.: The sensitivity of intracorpuscular hemOglobin to oxidation by nitrite ions. 1. The effect of growth, starvation and diet. Phys. Med. Biol., 6zh27-h35, 1962. Orgeron, J. D., Martin, J. D., Caraway, C. T., Martine, R. M., and Hauser, G. H.: MethemOglobinemia from eating meat with high nitrite content. U. 8. Public Health Rept., 72:189, 1957. Phillips, W. E. J.: Changes in the nitrate and nitrite contents of fresh and processed spinach during storage. J. Agr. Food Chem., Vol. 16, No. 1, 88-91, (Jan.-Feb.) 1968. - Ries, S. K. and Cast, A.: The effect of Simazine on nitrogenous components of corn. Weeds, 13:272-27h, 1965. Ries, S. K.: The increase in protein content and yield of Simazine— treated crops in Michigan and Costa Rica. Bio Science, Vol. 18, NO. 3, 205-208, 19€)80 Ross, J. D.: Deficient Activity of DPNH-dependent methemoglobin idaphorase in cord blood erythrocytes. Blood, 21:51, 1963. Setchell, B. P. and Williams, A. J.: Plasma nitrate and nitrite concentration in chronic and acute nitrate poisoning in sheep. AUSt. V813. Jo , 38:58‘62, 1962. Simon, J., Sund, J. M., Wright, M. J., Winter, A., and Douglas, F. D.: Pathological changes associated with the lowland abortion syndrome in Wisconsin. J.A.V.M.A., 132:16h-l69, 1958. hl Simon, J., Sund, J. M., Douglas, F. D., Wright, M. J., and Kowalczyk, T.: The effect of nitrate or nitrite when placed in the rumen of pregnant dairy cattle. J.A.V.M.A., 135:311-3hl, 1959. Sinha, D. P.: Pathogenesis of abortion in acute nitrite toxicosis in guinea pigs. Ph.D. Thesis, Michigan State University, 1968. Sleight, S. D. and Sinha, D. P.: Prevention of methemoglObin reduction in blood samples. J.A.V.M.A., 152:1521-1525, 1968. Spicer, S. 8.: Species differences in susceptibility to methemoglobin formation. J. Pharmacol. Exp. Therap., 99:185-19h, 1966. Stolk, J. M. and Smith, R. P.: Species differences in methemoglObin reductase activity. Biochem. Pharmacol., 15:3A2-351, 1966. United States Public Health Service Drinking Water Standards: U. S. Public Health Service Publication, A7, 1962. Winter, A. J.: Studies on nitrate metabolism in cattle. Am. J. Vet. Res., 23:500-505, 1962. VITA RObert Lee Amster was born in Madison County, Kentucky on October 12, 193A. He graduated from Lafayette High School, Lexington, Kentucky in 1952. In 1955, after service in the United States Navy, he enrolled at the University of Kentucky and in September 1958 transferred to Auburn University, Auburn, Alabama and there received his degree of Doctor of Veterinary Medicine in 1962. From 1963 to 1966 the author was employed by Florida Department of Agriculture Diagnostic Laboratory in Kissimmee, Florida. While there he co-authored two papers, "Studies of the Lipoprotein Methods Used for Detecting Altered Serum LipOpoteins in Equine Infectious Anemia" and "Altered Serum LipOproteins in Equine Infectious Anemia: Comparisons of Values Among Normal Horses and Horses Infected with Babesia caballi". In October 1966, the author received a commission as Captain in the United States Air Force, and has served active duty at Wilford Hall Hospital, Lackland Air Force Base, Texas and at Battelle Memorial Institute Pacific Northwest Laboratory, Richland, Washington. After completing schooling at Michigan State University, he will continue his studies at the Armed Forces Institute of Pathology, Washington D. C. Dr. Amster is married to the former Mary Frances Hall and is the father of three children; Robert Koerner, Deborah Gale, and Brenda Leigh. A2 HICHIGRN STATE UNIV. 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