INFLUENCE OF DIETARY PROTEIN ON LIVER CONSTITUENTS OF TUBERCULOUS AND NON-TUBERCULOUS GUINEA PIGS Thesis for fhu Dogma of Ph. D. MICHIGAN STATE UNIVERSITY Roberta EIIen Bleiler 1958 0-169 This is to certify that the thesis entitled II‘IFL’LENCE OF DIETARY PROTEIN ON LIVER CONSTITUENTS OF TUBERCULOUS [AND NON-TUBERCULOUS GUINEA PIGS presented by Roberta Ellen Bleiler has been accepted towards fulfillment of the requirements for m— deqree in- Nu‘tI‘it ion 26:17,, {cg flcLaé/i/df/J (.411 % Major profess LIBRARY Michigan State University INFLUENCE OF DIETARY PROTEIN ON LIVER CONSTITUENTS OF TUBERCULOUS AND NON-TUBERCULOUS GUINEA PIGS BY ROBERTA ELLEN BLEILER AN ABSTRACT Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Foods and Nutrition 1958 Approved ”“9““62- 2‘44] “L" The amounts of fat, nitrogen, sulfur, solids, flavin adenine dinucleotide, flavin mononucleotide plus free riboflavin, and the activity of glutathione reductase were determined in the livers of tuberculous and non-tuberculous guinea pigs on different protein intakes. During 40- and 47-day experimental periods, purified diets containing 35 and 25 per cent protein were fed to 39 control guinea pigs and to 28 animals that had been inoculated withfig, tuberculosis HSVRv. The tuberculous animals consumed greater quantities of ration than did their respective controls, but failed to utilize the feed for weight gain as well as did the non-infected guinea pigs. The enlargement of the livers of the tuberculous guinea pigs indicated that possible structural.and func- tional alterations had occurred in the organ during the infection. The percentages of solids in the livers of the tuberculous animals were lower and the percentages of water higher than those for the respective controls. lbberculosis appeared to have little effect on the per- centages of fat, nitrogen, and sulfur in the livers of The amounts of fat, nitrogen, sulfur, solids, flavin adenine dinucleotide, flavin mononucleotide plus free riboflavin, and the activity of glutathione reductase were determined in the livers of tuberculous and non-tuberculous guinea pigs on different protein intakes. During 40- and 47-day experhmental periods, purified diets containing 35 and 25 per cent protein were fed to 39 control guinea pigs and to 28 animals that had been inoculated withfifi, tuberculosis H37Rv. The tuberculous animals consumed greater quantities of ration than did their respective controls, but failed to utilize the feed for weight gain as well as did the non-infected guinea pigs. The enlargement of the livers of the tuberculous guinea pigs indicated that possible structural and func- tional alterations had occurred in the organ during the infection. lhe percentages of solids in the livers of the tuberculous animals were lower and the percentages of water higher than those for the respective controls. Tuberculosis appeared to have little effect on the per- centages of fat, nitrogen, and sulfur in the livers of The amounts of fat, nitrogen, sulfur, solids, flavin adenine dinucleotide, flavin mononucleotide plus free riboflavin, and the activity of glutathione reductase were determined in the livers of tuberculous and non-tuberculous guinea pigs on different protein intakes. During 40- and 47-day experhmental periods, purified diets containing 55 and 25 per cent protein were fed to 39 control guinea pigs and to 28 animals that had been inoculated with g, tuberculosis HSVRV. The tuberculous animals consumed greater quantities of ration than did their respective controls, but failed to utilize the feed for weight gain as well as did the non-infected guinea pigs. The enlargement of the livers of the tuberculous guinea pigs indicated that possible structural and func- tional alterations had occurred in the organ during the infection. The percentages of solids in the livers of the tuberculous antmals were lower and the percentages of water higher than those for the respective controls. Tuberculosis appeared to have little effect on the per— centages of fat, nitrogen, and sulfur in the livers of the animals. limitation of dietary protein did not significantly alter the concentrations of these constitu- ents in the livers of the guinea pigs. Estimation of the activity of liver glutathione reductase suggested that mechanisms responsible for main- taining reduced glutathione in the livers of the guinea pigs were not disturbed either by the presence of tuber- culosis or by the restriction of dietary protein. A higher value for the percentage of flavin mononucleotide plus free riboflavin fraction in the liver was found for guinea pigs which received the lower protein ration than for those consuming the 55 per cent protein diet. The concentration of flavin adenine dinucleotide per gram.of liver tissue decreased during tuberculosis in the guinea pigs. It is possible that alterations occurred in the flavin adenine dinucleotide-linked enzyme systems of the livers of the tuberculous animals. The relationship between riboflavin retention and nitrogen storage in liver was not maintained during tuberculosis. .... 1F .5 1.1;} a. INFLUENCE OF DIETARY PROTEIN ON LIVER CONSTITUENTS OF TUBERCULOUS AND NON-TUBERCULOUS GUINEA PIGS By ROBERTA ELLEN BLEILER A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Foods and Nutrition 1958 g /<§' 1/5’ L/w/Iv’ Roberta Ellen Bleiler candidate for the degree of Doctor of PhilosOphy Dissertation: Influence of Dietary Protein on Liver Constituents of TUberculous and Non-tuberculous Guinea Pigs Outline of Studies Major subject: Nutrition Minor subject: Biochemistry, Biographical Items Born, October 5, 1950, Scranton, Pennsylvania Undergraduate Studies, The Pennsylvania State University, 1948-1952 Graduate Studies, University of Wisconsin, 1952-1954 Graduate Studies, Michigan State University, 1954-1958 Experience: Graduate Assistant, University of Wisconsin Graduate Assistant, Michigan State University ~ Instructor,‘Michigan State University Masher of Sigma Xi, Sigma Delta Epsilon, Iota Sigma Pi, Omicron Nu ACKNOWLEDGMENTS The author is deeply grateful to Dr. Tilina D. Brewer for her guidance and constant encouragement throughout this investigation. She is also particularly indebted to Dr. Walter N. Mack for his valuable assistance in the pathological aspects of this eXperiment. The author wishes to express her appreciation to Dr. James L. Fairley and to Dr. Carl A. Hoppert of the department of Biochemistry for their suggestions on analytical techniques; to Dr. Henrik J. Stafseth and to Dr. L. C. Ferguson for making available the facilities of the department of Microbiology; to Dr. William D. Baton for his assistance in developing the experimental design; and to Dr. Evelyn M. Jones for advice on special problems during this investigation. Acknowledgment is also made to Dr. Dena Cederquist and Dr. Margaret A. Ohlson, present and past heads of the department of Foods and Nutrition, respectively, for their support of this experimental work. . ‘1 Ill I'll. TABLE OF CONTENTS INTRODUC 11 ON . . C O O C O O O O I O O O O O O O O O 1 REVIEW OF LI mm m 0 O O O O O O O O O O O O O O O 2 Relationship between Protein Metabolism and the Liver Nutrition in Tuberculosis PRELDEINARYEXPERIMENT............... 19 Influence of Dietary Protein upon the Concen- trations of Riboflavin-containing Coenzymes in the Livers of Rats PRmEDURE O O O O l O O O O O O O O O O 0 O O O O O 24 General Experimental Plan Care of Animals Diets Sacrifice of Animals and Tissue Preparation Analytica1.Methods RESULTSANDDISCUSSION............... 36 Statistical Treatment of Results Observations at Autopsy Weight Gain and Food Intake Liver Fat and Solids Liver Weight and Body Weight Liver Nitrogen Liver Sulfur Glutathione Reduetase Activity in Liver Riboflavin-containing Coenzymes in Liver Interrelationships SMAH AND CONCLUSI CNS 0 O O O O O O O O O O O O O 87 LI ERA Tm c I m D O I O O O O O O O O O O O O O O O 90 APPENDIX 0 O O O O O O O O C O O O O O O O O O O O O 98 LIST OF TABLES Table 1 Changes in liver enzymes with dietary protein alterations, reported in literature . . . Table 2 Mean values for the nucleotides of riboflavin in the livers of rats fed eight per cent and 20 per cent protein . . . . . . . . Table 3 Experimental design . . . . . . . . . . . Table 4 Composition of the guinea pig diets . . . Table 5 Vitamin preparation for the guinea pig diets O O O O O O O O O O O O O O O O O O O O O 0 Table 6 Tuberculous and non-tuberculous animals Which survived the experimental periods on different protein intakes . . . . . . . . . . . . Table 7 Form.for the analysis of variance employed in the evaluation of the effects of separated housing units upon the experimental requ-ts O O C O O O O O O O O O O O O O O O O O 0 Table 8 Final form for the analysis of variance employed in evaluating the experimental results . Table 9 Average weight gain and food consumption for tuberculous and non-tuberculous guinea pigs on different protein intakes . . . . . . . . . . Table 10 Mean weights and percentages of fat in the livers of tuberculous and non-tuberculous guinea pigs on different protein intakes . . . . Table 11. Mean weights of solids and percentages of solids, fat-free solids, and water in the livers of tuberculous and.non-tuberculous guinea pigs on different protein intakes . . . . . . . . . . page 21 26 29 30 38 4O 41 44 51 54 page Table 12 Average body weights and liver weights for the guinea pigs . . . . . . . . . . . . . . . . 57 Table 15 iMean weights of nitrogen in the livers of tuberculous and non-tuberculous guinea pigs on different protein intakes . . . . . . . . . . . . . 65 Table 14 Average values for sulfur in the livers of tuberculous and non-tuberculous guinea pigs on different protein intakes; nitrogen-sulfur ratios 0 I O O O O O O O O O O O O O O O O O O O O 68 Table 15 Average rates for glutathione reductase activity in the livers of tuberculous and non-tuberculous guinea pigs on different protein intakes . . . . . . . . . . . . . . . . . . 72 Table 16 iMean values for riboflavin in the livers of tuberculous and non-tuberculous guinea pigs on different protein intakes . . . . . . . . . . . 74 Table 17 Ratio of riboflavin to nitrogen in the livers of tuberculous and non-tuberculous guinea pigs on different protein intakes . . . . . . . . . 78 LIST OF FIGURES page Figure 1 Comparison of the weight gains of Figure 2 guinea pigs per 100 grams of food consuned, by means of the studentized range 0 O O O O O O I O O O O O O O O O O 48 Relation between liver weight and body weight of tuberculous and non’tuberculous animals 0 o e e o e e e e 60 INTRODUCTION Among bacterial infections, tuberculosis is unique. The ability of tubercle bacilli to remain viable in a host for long periods of time without forcing an innnediate all-or-nothing struggle reflects the intractability of the disease. The efficiency of the host's defense mechanisms decreases due to the chronic nature of the infection. Known chemotherapeutic agents, at least in concentrations sufficiently low to prevent cell toxicity in certain body organs are tuberculostatic, rather than tuberculocidal. The host is not capable of destroying the attenuated parasites, rapidly. A healthy body can avoid mechanistic incompetency more easily than can one that is poorly nourished; moreover, tissue which was destroyed during the progress of the tuberculous infection must be regenerated. Herein lies the importance of nutrition with regard to both preventative and curative measures in tuberculosis. REVIEW OF LITERATURE Relationship between Protein Metabolism and the Liver The relationship between protein metabolien and the liver can be considered fran two approaches ; that is, the effect of the liver on‘protein metabolism or, conversely, ‘ that of protein metabolism on the composition and struc- ture of the liver. For example, the presence of hepatic disease, in spite of the ingestion of adequate amounts of nutritious foods, may lessen protein synthesis (Cannon, '50); on the other hand, protein malnutrition tends not only to produce a fatty liver, but also to promote patho- logical alterations of the liver, the central organ in protein metabolism (GyBrgy, '54). Injury to the hepatic parenchyma may occur when dietary patterns are altered adversely. Fatty infiltration of the liver was the first noticeable change in a choline deficiency which could be reversed with the feeding of methionine or other sulfur-containing amino acids (GyBrgy, '54). Hawk and Elvehjem ('55) have indicated that protein possesses a lipotrOpic action beyond that attributable to its content of sulfur-containing amino acids, particularly that of methionine. According to Himsworth.('47), every dietary component except carbo- hydrate has been indicated.as the cause of liver injury at one time or another. Diet-inducedumassive necrosis appears to result from multiple deficiencies, thile singular dietary limitations may caume cirrhosis of the liver (GyBrgy, '55). The recog- nition of dietary liver cirrhosis and dietary liver necrosis as separate and distinct entities does not pre- clude the possibility that fibrosis may succeed necrosis or that the death.of cells may occur during the cirrhotic process (Daft, '54: GyBrgy, '55). Furthermore, GyBrgy ('55) suggested.that preventative measures against cirrhosis and necrosis include all substances ihich.fa11 under’the terminology, lipotropic factors. Treatment of diseases of the liver by diets high in protein was based on the potentialities of protein in abetting regenerative proces- ses, in reducing fatty infiltration, and in regressing fibrosis. Siess and Stegmann ('50) suggested that, in a period of growth, the weight of the liver remains a constant fraction of the total body weight, but that a high level of dietary protein will increase the weight of the liver, slightly. According to Kosterlitz ('54), the entire cytOplasm of the liver cells may change withalterations in the dietary protein; the concentrations of protein within individual liver cells of rate increased with greater protein intake, but varied with the quality of the nutrient. Widdowson and McCance ('57) demonstrated that the amounts of nitrOgen present per 100 grams of liver in young rats depended upon the percentages of protein in the rations; when dietary protein was increased from 1.5 to 15.8 to 45 per cent, the nitrogen content per 100 grams of liver rose fran 2.4 to 5.0 to 5.5 grams, respectively. Protein deficiency in the diet has produced marked changes in the enzyme pattern and structure of the liver; these alterations were especially noticeable in young growing rats in comparison with adults (Srinivasan and Patwardhan, '55). Levy ('55) reviewed the influence of the nutritional state upon the activity of enzymes, and concluded that, on low protein diets, the majority of liver enzymes diminish in preportion to the amount of the nutrient lost from the liver. Knox 23 2.]: ('56) observed that when dietary protein was limited, the liver enzyme concentrations which did change were decreased uniformly; the concentra- tions of the catalysts increased when the diet contained extra protein or amino acids. Some changes in liver enzyme activities in relation to alterations in protein intake are given in Table l; the results agree generally with the observations of Levy (1223 gi23) and Knox.gt_§l, (123: citf). Cytochrome oxidase, however, demonstrated an in- creased concentration in the liver of rats when a protein-free diet was fed, according to Whinio 9§_§l, ('52). In 1951, Hepkins and Elliott preposed that a system capable of reducing oxidized glutathione existed in animal tissues. Meldrum and Tarr ('55) described an enzymatic reduction of oxidized glutathione in a coupled reaction with glucose-S-phosphate dehydrogenase (Zwischenferment) of blood cells or yeast in the presence of a triphospho- pyridine nucleotide preparation, and suggested that a glutathione reductase must have been present in order to explain the results. The enzyme was purified partially from.wheat germ and higher plants, and studied in Zwischen- ferment systems (Conn and Vennesland, '51; Anderson, g§_gl,, '52); materials which.contained glutathione reductase activity were extracted from.animal tissues by Hall and Lehninger ('52). The catalyzing ability of glutathione reductase in mammalian erythrocytes was about the same in Zwischenferment systems as in the presence of reduced triphosphOpyridine nucleotide, but the requiranent for a IHmuI fflwwd gmfixmfi emmoseou omoosemb onesero mm. «odousopaog one opossum oncogene commune eamflomm I am. macme=< I oncogene omenmaemaer equaeHw pm. ..MeI ue.Hm£poomomi camoneow. componnmomnoahmecamocope m. ..Hm He Hmnvsomom It emeoseemi oueowpmma cm. ..Me we Hmnvnemom, omeoseoo eueawwae mm. ..He we anpnomom omeonoop eemoaoem eoevomnm.onn eGAHeme .. e no 0 can commence omeesoeo comedy: mm. ..He woieacwea, oueosoop 44H omeesoeo o casewommhoJZWD mm. .nHw we owswmaii came osmoseoo ommmmwo mace onwadum mm. ..Me we odawms. came emeonoou 4 omscomonpbneu ewwfleQSu mm. ..ae.mm cacao; came emeosoou mm. maesmonIpse hpmn (I eeeeseoo I enmpmxo cammoese mm. .Me no edaama wade eawmi onmpfino enchanhn m. ..He..Ww admoaxaes emeosoep accessed mm. .Me we eacfima. oumoseem :1 05mm eueUHKo enfinpamn mm. ..He no ousdea emeosooo coaches“ no. .oseswon_uae hang oueeaoeo eeeoaxo osoanooahe .11. 41 cosh deepen (a nude pehusaem cosh soaueameo Heap.pooomH awovcah. ameyoaml. noaemapaebaH aoauoase> hswpodo wnuhao Hospsoe sown huabfiuem oahmao sopflHime ewsene.~m seapeesdn oahsgm ensaeneoaa ad nonsense .eaoapenepae masochn hampedo_madw hpa>auec cahuae aopaa ad commune .n can—8”. ccfactor was evident when diphosphOpyridine nucleotide served as the hydrogen donor (Hancoer and Denstedt, '54). According to Backer ('55), fractionation of the acetone powders from beef liver enzyme with alcohol and isoelectric precipitation increased the glutathione reductase activity over that in the original extract; spectrOphotometrically, the concentrate demonstrated a more rapid reaction with triphosphopyridine nucleotide than with diphosphOpyridine nucleotide between pH 5.8 and 6.5, and showed a typical absorption spectrun of a protein. The relationship between dietary protein and glutathione reductase activity in the liver is unknown. Sulfur is a component of glutathione, a peptide functioning primarily in the maintenance of sulfhydryl groups in a reduced physiOIOgical state (Barron, '49; Bricas and Fromageot, '55). The sulfur-containing amino acids, which form an integral and essential part of the dietary protein, provide the majority of the body sulfur. Since a dietary deficiency of sulfur-containing amino acids had been suggested as a cause of necrosis of the liver, Linden and Work ('51) determined the amounts of glutathione present in necrotic livers. After nine days, a slight diminution in glutathione values was noted in the livers of albino rats fed a necrogenic yeast diet which supplied seven per cent protein, in comparison with the values obtained for the control animals. No further lowering of liver glutathione was observed until the experimental animals became ill suddenly and the livers showed symptans of necrosis; the glutathione values in the necrotic livers decreased to 20 per cent of those found for the livers of“ the control animals. Beck and Bianconi ('56) found that the non-protein sulfhydryl content of the livers of mice decreased markedly when the sulfur-containing amino acids of the diet were restricted. The content of sulfur within the tissues of the body is most closely associated with the tissue protein, speci- fically with nitrogen; the usual nitrogen to sulfur ratio of both food and body tissues lies between 14 and 16 (Shohl, '59). According to ‘Shohl (122. 941.), the average weight of the liver of an adult hunan being is 1.8 kilo- grams when the total body weight is 66.2 kilograms; the sulfur of this organ represents approximately 0.05 per cent (0.5 grams) of its weight. Little has been reported in the literature on the amounts of total sulfur in body tissues since 1950. Evidence has been accunulated to indicate that rela- tionships exist between protein metabolism and certain B-vitamins; riboflavin concentrations in tissues appear to depend not only upon the dietary content of the vitamin, but also upon the amount of protein in the diet. Sarett and Perlzweig ('45) studied protein and riboflavin inter- relationships in rats. 'mey reported that the riboflavin content per gram of liver appeared to bear a direct rela- tionship to the amount of nitrogen present per gram of tissue when the values for the high dietary protein groups were compared with those for the low protein animals, despite the fact that some of the high protein rats had low vitamin supplanentation. Although the total riboflavin present in the liver was slightly higher in both groups receiving the larger amounts of vitamins than in the livers of the respective low-vitamin animals, total nitrogen retention appeared to be the dominant factor with respect to the storage of riboflavin in the liver. The investi- gations of Czaczkes and Guggenheim ('46) demonstrated that the amount of riboflavin in the livers of rate increased when dietary casein was raised from 15 to 54 per cent. Riesen 91 El“ ('46) showed that greater amounts of nitrogen were retained in the livers of rats and that the riboflavin content increased from 14 to 25 to 29 micrograms per gram of ‘tissue as the casein content of the diet was augmented progressively from 8 to 18 to 50 per cent, respectively. 10 Riboflavin exists in the liver in three forms: free riboflavin which accounts for the smallest fraction; flavin adenine dinucleotide (FAD), the largest portion; and flavin mononucleotide (FMN) . FAD and FMN are co- enzymes; specifically, they are considered to be pros- thetic groups, since the apoenzyme and coenzyme moieties are firmly attached each to the other. The primary func- tion of the flavOproteins is in electron transport systans. In 1949, Bessey 23 51:, employing acid hydrolysis in a fluorometric procedure for the determination of esteri- fied riboflavin in animal tissues, reported that the FAD varied from nearly 90 per cent of the total riboflavin in the skeletal muscles to about '70 per cent in the kidney. During an imposed riboflavin deficiency, the concentrations of all three fractions decreased; the percentage decrease of the THIN appeared to be greater than that of either FAD or free riboflavin, but the investigators noted large variations between groups of animals. Free riboflavin in tissues is low, thus making measm‘ement difficult by this technique; generally, this fraction and FMN are expressed together. Trufanov ('46) found that the livers of rats fed a lower protein diet than the control animals for 50 days failed to synthesize a'normal quantity of FAD. ll Nutrition i2_gpberculosis Good nutrition appears to be one of the primary requisites in resistance to tuberculosis, according to the myriad reports concerned with the disease. Studies in countries involved in both.World war I and World war II adduced evidence ihich.strongly supported this theory, and furthenmore established an inverse relationship between economic level and the incidence of the disease (Faber, '58; Sneedin, '46; Rich, '51). Economic status influwnces food consumption, particularly that of protein; Faber (leg, Elfin) had associated the rise in the:mortality rate of tuberculosis in Denmark during werld war I uith.dietary protein deficiency. Cannon (Igg, 932,) attributed the increased susceptibility to infection displayed by starving persons to a depletion of protein reserves and to the inability to fabricate new supplies of antibody globulins because of an inadequate intake of essential amino acids; according to Rich (l_.g_c_. 91.3.), a dietary protein deficiency probably has an adverse effect even upon acquired resistance. In an attempt to establish.the protein requirement in tuberculosis, McCann ('22) maintained ten bed patients in nitrOgen equilibrium by feeding 60 to 90 grams of 12 protein daily. According to Brewer gt 2.1-.“ ('50), the retentions of nitrogen by six women with moderately advanced, active tuberculosis were similar to those of healthy college women when the protein intakes were can- parable; these patients were able to store some nitrOgen when the daily protein consumption was 80 grams. Tui gt _a__l_. ('54) reported that tuberculous patients do not require higher nitrogen intakes than normal subjects to achieve nitrogen equilibriun, and attributed the hypo- proteinemia in chronic tuberculosis to prolonged low food consunption rather than to physiological catabolism. With tuberculous girls in their adolescent years as subjects, thnston ('53) observed a correlation between adequate storage of nitrOgen and a decrease in the progress of the infection. In animal experimentation, Hornenann ('15) noted a lesser develoynent of tuberculous lesions in infected swine that received a high protein diet than in those fed a high carbohydrate or mixed diet. 'Ihe extensiveness of disease and mortality rate of tuberculous rats of the Wistar strain decreased as dietary protein increased fran 15 to 40 per cent (Koerner at 51., '49). In contrast, Hetcoff 9_t_ a}: ('49) studied three groups of infected rats fed diets which were (1) adequate, (2) protein 13 deficient, and (5) initially adequate, then severely deficient, respectively, and obtained results which favored the concept that dietary protein deficiency does not alter, significantly, the susceptibility of the rats nor the course of the disease. Following tuberculous injection, Swiss albino mice fed a diet of whole wheat and dried milk survived longer than those on a cornmeal, gelatin, and butter regime; the effects of a change from a good to an inadequate diet resulting in increased susceptibility to infection may reflect a non-specific physiological disturbance rather than a defined dietary deficiency, according to Dubos and Pierce ('48). Dubos gt gt. ('55) demonstrated that the susceptibility of mice to experimental bacterial infection can be increased consistently by various procedures de- signed to disturb animal metabolism. For instance, the fasting mouse manifested a decreased resistance within 56 hours after the beginning of the test period, and this alteration in susceptibility was further aggravated by feeding only glucose or an incomplete diet to the animal. Dubos ('55) hypothesized that metabolic disturbances which cause either a depletion of the glchgen reserves of the body or a reduction in the glycolytic activity in inflam- matory cells possibly with the accumulation of certain 14 polycarboxylic acids and ketones in the tissues can effect a decrease in resistance to tuberculous infection in mice. Furthermore, this investigator also reported that the resistance of mice appeared to be independent of the dietary protein level, within wide limits; whereas mice fed a diet very low in protein and high in carbohydrates proved markedly susceptible to tuberculosis, supplanting part of the carbohydrates with fat without deviating the protein content served to return their resistance to nomal. 'lhus, the evidence provided no indication that protein deficiency alone could accelerate the course of tubercu- losis. Dubos (loc. cit.) discussed diet and resistance to tuberculosis in man and concluded: It may be worth noting in this respect that investigators who have tried to correlate deficiency in nutritional factors with suscep- tibility to tuberculosis on the basis of epidemiological studies of the hunan disease have emphasized the importance of proteins of animal source. As deficiency in foods of SEE—LET origin is alm0st always accompanied by other dietary limitations, it seems possible that these, rather than low protein intake, are responsible for the increase in susceptibility to tuberculosis which has been observed in certain epidemiological situations, for example, during war time. According to Farber and Miller ('43) from observa- tions on 400 tuberculous patients, very 111 subjects often danonstrated multiple vitamin deficiencies which may 15 correlate in degree with the severity of the disease. Ascorbic acid metabolism appears to be disturbed during tuberculosis; it is generally conceded that the need for this vitamin by the tuberculous person is increased greatly.1 {the urinary levels of thiamine, riboflavin, and niacin were found to be lower in tuberculous subjects when canpared with the norm reported by Sartory gt gt. ('51), and the extent of diminution was preportional to the gravity of the infection, in agreanent with Farber and Miller (Egg. gi_1_;_.). On the other hand, Brewer gt g._l_._. (1.2g. gtt.) reported that the riboflavin metabolism of six women with moderately active, advanced tuberculosis was similar to that of college-age women, and that the urinary excretion of riboflavin was related directly to the dietary intake of the vitamin, but inversely to the retention of nitrogen. The values for pyridine nucleotides in erythro- cytes of tuberculous patients were less than those for non-tuberculous patients; the values for pyridine nucleo- tides in the red blood cells and livers of experimen- tally-infected guinea pigs fell below those for the control animals (Pfitidlfl, '54). When nicotinic acid amide and 1 Reviews include Perla and Mamorston ('57); Sweany gtgt. ('41); Shaw gtgt. ('50). 16 Coenzyme I were administered, orally, to infected mice, the spread of tuberculosis was suppressed; this effect was more marked when the treatment with niacin derivatives was instituted prior to-the inoculation of the mice (McKenzie gtgt., '48; Fitzpatrick, '55). line pathological characteristics of the general hosts, man, cattle, birds, and voles to the types of tubercle bacilli, hunan, bovine, avian, and murine, respectively, differ; the various strains can produce disease in the other hosts (Soltys gt gt” '52). the locations and nature of primary lesions in the hostsmay be dissimilar. In contrast to the pulmonary type of tuberculosis commt‘m in man and the rabbit, the lungs of guinea pigs were affected only in more advanced and nonspecific tuberculosis, while the spleen, liver, and lymphatic system danonstrated extensive necrosis; these symptoms were found also in rats (Willis, '25; Krause, '26; Wessels, '41; Iack, '56). Tuberculosis is a chronic disease with manifestations arising from areas involved in the infectious process, i.e., those which have tubercles, as well as from those without lesions. Chaudhuri and Martin ('54) measured the activity of succinic dehydrogenase in the tissues of guinea pigs which had been infected with virulent and nonvirulent strains of It. tuberculosis. Whether or not morphological 17 lesions were present in the tissues of the organs studied, the catalytic ability of the enzyme was altered in the spleen, kidney, and the liver in relation to that for the control animals. In man, hepatic functions were altered in pulm0nary tuberculosis (Steidl and Heise, '55; Hurst gt gt., '47). Gillman and Gilbert ('54) reported that, among Africans, tuberculosis contributed to widespread destruction of hepatic tissue even when the extent of infection of the liver was minimal. Ban ('55) found liver disease to be a prominent factor in 60 patients with active pulmcnary tuberculosis. Liver biOpsies revealed a gener- alized necrotic state of the organs; in some cases, the tissue was cirrhotic and particularly tended towards fatty degeneration. Proliferation of cells was noted also. Sarin gt gt. ('57) presented data on 100 tuberculous patients; pulmonary tuberculosis was diagnosed in 91 of these cases. or the patients who had pulm0nary tubercu- losis, 55 showed abdominal symptoms as anorexia, consti- pation, and diarrhea; 55 had enlarged livers. Derangement of liver function was found in 51 of the 100 cases of tuberculosis, and there appeared to be some correlation between hepatanegaly and malfunction of the liver. Fatty infiltration accompanied the cirrhotic changes which occurred in the livers of 27 patients. 'Ihe appearance 18 of fatty livers was attributed to the toxicity of the tuberculous infection, associated with malnutrition and metabolic disturbances. A preliminary experiment was designed in this laboratory to investigate the influence which protein consumption may have upon enzyme activity in the livers of rats. lhis was followed by a study of tuberculous and non-tuberculous guinea pigs ihich.consumed purified diets differing in casein content. The amounts of fat, nitrogen, sulfur, and.the activity of glutathione reductase in the livers of the guinea pigs were determined. lhe concen- tration of riboflavin, as flavin adenine dinucleotide and.flavin.mbnonucleotide plus free riboflavin was measured also. 19 PRELIMINARY WERIMENT Influence of Diets Protein u on the of Ergo Concentrations __ flavin-con ainIng Eoenzmes in tHe Livers o s Ihe riboflavin content of the liver appears to depend upon both dietary protein and dietary riboflavin. Sarett and Perlzweig (tgg. gtt.) reported that rats fed a diet higher in protein retained more riboflavin in the livers than did those which received lesser amounts of protein. Bessey gtgt. (tgg. gtt.) demonstrated that the tissue concentrations of flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and free riboflavin decreased when the amounts of riboflavin in the diet were low. In this laboratory, the concentrations of FAD and FMN plus free riboflavin were determined in the livers of rats fed diets containing different percentages of protein. mirty male, albino rats1 were distributed among three groups: Group I was given an eight per cent casein diet; Group II, the controls, received a diet containing AA 1 Sprague-Dawley, Madison, Wisconsin. Shipping weight, 100 grams. 20 20 per cent casein; the pair-fed animals, Group III, received the same diet as animals in Group II, but the amounts were restricted to the intakes of the respective pair-mate animals in Group I. 'Jhe basic diet was a characteristic, semi-synthetic ration for rats; cerelose was employed as a caloric equivalent to casein in the low protein diet. me diets supplied similar percentages of riboflavin. At the end of the 55-day experimental period, the animals were sacrificed. A modification of the fluoro- metric procedure of Bessey gtgt. (1.9.9.- gtt.) was used for the determination of riboflavin-containing coenzymes in the livers. 'me data for one animal are unavailable because of a laboratory accident. the mean values for the nucleo- tides of riboflavin in the livers of rats are given in 'Iable 2 and the individual data are presented in Appendix Table 1. line average amount of riboflavin, both as FAD and mm plus free riboflavin, which was stored in the livers of the animals that received eight per cent casein, was significantly less than the quantity found in the livers of either group of rats fed the higher protein ration (t-test; P5 0.01). 'lhere were no significant differences between the mean values for the riboflavin 21 naouonm Boa he.e-n.ev am.en-a.nmv on nonunsua e.>n n.e e.an oH om . "Houston HHH hm.oe-n.nv Am.an-n.emv n.en n.u o.um on on Houston HH he.e-m.mv A¢.nm-o.nav H.am n.n e.ua e m neurone non H .u\.um exam .w\.mm as e oabmauopan fidbmauondh ofionmo Hesse some 33 BE sausage heroes 98.8 22m encapsn :Houonn pace non om one pace non unwae con mama Ho nac>aa onv ma napsamonua no monapooacs: can you mosasb one: N odDMB 22 fractions in the livers of the two groups of animals that received the 20 per cent casein diet. then the low protein ration was fed, the mean values in micrograms per gram of liver were 17.6 and 5.5 for FAD and FIN plus free riboflavin, respectively. 'Ihe mean values for FAD and FIN plus free riboflavin in micrograms per gram of liver were 27.0 and 7.5, respectively, for the controls (Group II), and 51.9 and 6.5, respectively, for the animals in Group III which were pair-fed to the low protein rats. The animals in Group III, consuming ration in amounts equal to those of the pair-mate rats in Group I, received 245 grams of the 20 per cent casein diet per animal over the 55-day period; the corresponding controls consmied 400 grams of the 20 per cent casein ration per annal during the same length of time. Elhe data agree with those presented by Sarett and Perlzweig (tgt. gtt.); the livers of their rats fed high protein-high vitamin diets retained more riboflavin than did those of animals which consuned low protein-high vitamin rations. In the present study, when the protein content of the diet was decreased from 20 to eight per cent, the concentration of riboflavin in the livers of the rats diminished. ihe livers of the animals which received the 23 20 per cent casein ration in amounts similar to those given to the low protein rats ccntained.more riboflavin than did ‘the livers of the animals consuming eight per cent casein, even though dietary riboflavin was the same. ‘Jhe FAD and FMN plus free riboflavin appeared to decrease in proportion to the total riboflavin. It appeared of interest to investigate possible changes in riboflavin fractions in the livers of tubercu- lous guinea pigs when the protein in the purified ration was decreased from 55 to 25 per cent. 24 PROCEDURE General Mimental Plan lhe original experimental plan included 72 young adult guinea pigs,1 males, to be distributed equally among 12 study groups. Diets supplying two different quantities of protein were to be fed during two experimental periods to controls, to tuberculous animals, and to non-tuberculous guinea pigs, pair-fed with the infected animals. Because of the limitations in handling large numbers of animals, the guinea pigs were managed in three sets of 24 each, over a seven-month period. After the completion of the first phase of the study, it became apparent that the tuberculous animals consumed more feed, consistently, than did the non-tuberculous guinea pigs which were allowed the same amounts of rations as their pair-mates; thus, the non-tuberculous animals, pair-fed with the tuberculous guinea pigs, did not differ appreciably in food consumption from the control animals. No pair-fed animals were in- cluded in the second and the third sets; there were 40 1 Rockland Fams, New York. Shipping weight, 250 grams . 25 control animals and 52 tuberculous guinea pigs in the final experimental design, as shown in 'Iable 5. A 12-day adaptation period preceded the experimental periods. ‘Ihe guinea pigs were fed ground rabbit pellets1 and cabbage for two days; distilled water was given gt libitun throughout the study. In order to accustcm the animals to the consumption of a purified diet, a mixture of ground rabbit pellets and the 55 per cent protein ration which was used during the experimental periods was fed for ten days. By gradually increasing the percentage of the purified diet in the mixture and correspondingly decreasing that of the pellet derivative and the amount of cabbage, the guinea pigs appeared to adjust to the consunption of the purified diet, alone, by the end of the adaptation period. he animals were then distributed so that the average weight of each group approximated the others as nearly as possible. On the morning of the first experimental day, the animals which were to be in the tuberculous group were transferred to a special laboratory and injected between the dermal and the peritoneal layers of the right inguinal A A 1 nillctt Elevator a Coal Company, Millett, Michigan. 26 Iable 5 Experimental Design Group Animals no. Control 55 per cent protein 40 days 10 47 days 1 10 25 per cent protein 28 days 10 55 days 10 Tuberculous 55 per cent protein 40 days 8 47 days 1 8 25 per cent protein 28 days 8 55 days 8 l The experimental periods were either 40 or 47 days in length; the animals consuned the ration containing 55 per cent protein for 12 days and received the 25 per cent protein diet for the last 28 and 55 days, respectively. 27 region with 0.1 milligrams of tubercle bacillus H57Rv, mammalian strain, prepared in 0.85 per cent saline. lhe Michigan Department of Health donated the culture from which a transfer was made to modified Proskauer-Beck medium for prOpagation of the organism (Soltys gtgt., tgt. gtt.). The remaining animals served as controls. After 12 days of the experiment had elapsed, one-half of the tuberculous animals and one-half of the control guinea pigs were fed a 25 per cent protein diet, while the remaining animals continued to consume the 55 per cent protein ration. When 28 additional days md passed, one-half of the nunber of animals within each of these four groups was sacrificed, thus completing a 40-day experimental period. ihe other guinea pigs were continued for seven days and were sacrificed at the end of a 47-day experimental period. Care gt Animals 'Ihe tuberculous animals were housed in an air-condi- tioned, windowless room; the control animals were kept in a separate laboratory which had windows on two sides. Elhe temperatures in these rooms at several hours were recorded for a one-week period; these are given in Appendix {table ii. 28 lilo anMals were maintained individually in wire cages. An eight-ounce water bottle with a glass tip extending inward through the mesh was attached to the outside of each cage and a wide-mouthed feeding dish to the inside. Clean bottles were provided every other day and were rinsed well with distilled water on the alternate days; the glass tips were washed daily when a fresh supply of distilled water was given. lhe cages and collection trays were replaced at least once a week, the feed dishes as often as necessary. All equipnent was washed in hot, disinfectantl water and rinsed well with distilled water. more 'ihe purified diets for the guinea pigs in this study were modifications of those used by Reid and Briggs ('55) and Heinicke gt gt. ('55). 'ihe 25 per cent and the 55 per cent protein diets, fed gt libitun, differed only in the amounts of casein and cerelose in the mixture. 'Iable 4 and {table 5 present the composition of the diets. lhe amounts of ration consuned daily by each animal were recorded throughout the experiment. 'lhe guinea pigs were weighed twice weekly. l Phenol Coefficient 25. Table 4 Composition of the guinea pig diets 29 55 per cent 25 per c;nt Nutrient casein casein diet diet s-/ke- % s-/ke- % Vitamin-free caseinl 550.0 55.00 250.0 25.00 Roughage (Alpbacel)1 150.0 15.00 150.0 15.00 Corn oil 40.0, 4.00 40.0 4.00 Cerelose 581.5 58.15 481.5 48.15 Potassium acetate 25.0 2.50 25.0 2.50 Magnesium oxide 5.0 0.50 5.0 0.50 Salts .(P-H)1 40.0 4.00 40.0 4.00 Vitamin preparation 8.5 0.85 8.5 0.85 l Nutritional Biochemical Corporation, Cleveland. {table 5 1 Vitamin preparation for the guinea pig diets 30 Amount Nutrient in ration mg./kg. Choline chloride 5000.00 Ascorbic acid 5000.00 Inositol 2000.00 p-aminobenzoic acid 100.00 'Ihiamine hydrochloride 25.00 Riboflavin 50.00 Pyridoxine hydrochloride 25.00 Calcium pantothenate 80.00 Nicotinic acid 200.00 Biotin 1.00 Pteroylglutmmic acid 10.00 Vitamin 312 0.04 Alpha-tocopherol acetate 20.00 menadione 2.00 1 Vitamin A and Vitamin D were supplied weekly from Upjohn Super D Concentrate in 2000 and 200 unit amounts, respectively. 31 Sacgifice gt the Animals and Tissue Preparation The animals were sacrificed by a heart injection of chloroform on the morning following the last experiment day. As rapidly as possible, the livers were removed frcm the guinea pigs, blotted gently with filter paper to rid the tissue of superfluous blood, and weighed. During the autopsies of the tuberculous guinea pigs, smears were secured from organ and/or lymph node material and stained1 to permit examination for infiltration by tubercle bacilli. Each liver was hanogenized with distilled water of a known volune and tenperature; the weight of the water used was between one and two times that of the liver. For this procedure, a micro-Waring blendor was employed (Comar gt gt., '44; Home, '55); it was regulated by means of a voltage controller2 set at 60. After the h0m0genate was sampled for immediate determination of glutathione reduc- tase activity and per cent solids, the remaining material was (transferred into a small, brown- or green-glass container, the Opening of which was subsequently __i _L__ A_ A_ L AL _____ .1 ‘1; 1 Acid-fast staining by the modified Ziehl-Neelsen method (Conn, '27). 2 11115 voltage controller operates on a 115-volt input. Fisher Scientific Company, Pittsburgh. 32 sealed with parafilm and tightly capped. This hom0genate was frozen for use later in the following analyses: total sulfur, nitr0gen, and riboflavin-containing coenzyme groups. Analzti cal Methods Glutathione reductase activi A modification of the methods employed by Racker ('49, '50, and '55) to concentrate glutathione reductase from biological materials was used for the preparation of the enzyme from guinea pig liver. 'Ihe procedure was standardized to permit the calculation of relative enzymatic activity in the livers. Glutathione reductase was determined as the rate of decrease of the Optical density of a test system containing the extract, oxidized glutathione, reduced triphospho- pyridine nucleotide,1 and buffers. The test systan of 1.7 milliliters was prepared in 0.5-centimeter silica cells from reagents which were previously warmed to 57.50 C. Optical density was followed at 540 millimicrons in a 2 Beckmsn DU spectr0photaneter using a tungsten light source. 1 Acknowledgment is made to Sigma Chemical Cmnpany, St. Louis, for supplying reduced triphosphOpyridine nucleotide at a discount. 2 National Technical Laboratories, South Pasadena. 33 Satisfactory preportionality was dmonstrated between enzyme concentration and rate of oxidation of reduced triphosphOpyridine nucleotide in this type of test systan by Racker ('55). “line values used for expressing glutathione reductase activity were calculated by means of the following equation: R: x 5 80 8' A0.D. l 8 R is the rate for the enzyme in one gram of liver solids. A0.D. is the change in optical density. s is the number of lS-second intervals which elapsed during the change in optical density. 8 is the amount of liver extract expressed in milliliters in the test systan. w is the grams of wet liver in the aliquot of original hom0genate. lhe mathenatical factor, 5/25, arises from the dilution procedure employed (lining the extraction. Per cent solids Aliquots of the fresh liver homogenate were dried for 24 hours in an oven regulated to maintain 100° 0. Air was circulated through the oven by a motor-driven fan. 29.1; 9.921 Lei '1he.aliquot of hmogenate used for the determination of the per cent solids also served as the sample for the per cent fat. Purified ether was employed for the extrac- tion process in the Goldfisch fat apparatus.1 Lotgt sulfur The principal steps involved in the determination of total sulfur are two: (1) the conversion of all sulfur derivatives to sulfate; (2) the precipitation of bariun sulfate. In this laboratory, the liver sulfur derivatives were oxidized in the Parr oxycalorimeter;2 a turbidimetric method, patterned after the suggestions of Treon and Crutchfield ('42) for the quantitative measurement of sulfates, was used to complete the analysis. An aliquot of the thawed homogenate was weighed into a calorimeter capsule and dried. The material was ignited and the washings transferred to a beaker to be treated wim bromine water. 'me excess bromine was evaporated and the neutralized solution was made to a volume of 250 milli- liters. Barium chloride was added to the samples and the 1 laboratory Construction Company, Kansas City, Missouri. 2 Parr Instrunent Company, Inc., Molina, Illinois. 35 turbidity was expressed as optical density units in the Beckman DU spectrophotuneter:L at 540 millimicrons. The amount of sulfur present in the sample solution was obtained as sulfate directly from the standard sodiun sulfate curve; sulfur represents one-third of the sulfate value . Nitrogen The boric acid modification (Scales and Harrison, '20) of the Kjeldahl method was employed to measure the nitrogen content of the livers of the guinea pigs. Riboflavin-containng coenzymes The amounts of flavin adenine dinucleotide and those of flavin mononucleotide plus free riboflavin in the livers of the guinea pigs were determined by a modification of the fluorometric procedure proposed by Bessey gt gt. (tgg. gtt.) . The protein material in the livers was precipitated by trichloroacetic acid and removed by cold filtration. A Coleman 12A photofluorometer2 was used to measure the fluorescence of the deproteinized liver sample against an internal standard. 1 National Technical Laboratories, South Pasadena. 2 colenan Electric Company, Inc., Maywood, Illinois. 36 RESULCIS AND DISCUSSION Statistical Treatment gt Results The statistical procedure used most frequently to aid in the interpretation of the results was analysis of variance. The experimental variables were: disease treatment; dietary protein; and length of experimental period. In the analysis, the disease treatment segregated the tuberculous guinea pigs from the non-tuberculous animals which were designated as the controls. The rations described by the term dietary protein were composed of purified materials containing either 55 or 25 per cent casein. The lengths of experimental periods were 40 and 47 days, since approximately six weeks are allowed to elapse for the developnent of tuberculous infection in guinea pigs used in diagnostic work. As was mentioned previously, the animals placed in the low protein group received the 55 per cent protein diet for the first 12 experimental days, and the 25 per cent protein ration for the last 28 and 55 days of the 40- and 47-day periods, 57 respectively. This plan was employed because it seemed doubtful that tuberculous guinea pigs could survive the experimental periods if fed only the low protein diet throughout the experiment . Five of the original 72 animals which began the ex- perimental periods failed to survive for the required lengths of time; Table 6 presents the nunbers of guinea pigs within each group for which data are available. The control animal, for which data are missing, died 10 days after the beginning of the experimental period; the cause of death appeared to be starvation. Aut0psies of the four tuberculous animals which survived approximately three weeks of the experiment showed that severe gastro- intestinal disturbances had occurred; it is not known whether failure to utilize the purified rations or the presence of disease was responsible for the abnormality. In order to avoid non-orthogonality in the statistical analyses, the averages of values obtained from determi- nations made for the surviving animals within each group were used for the unknown values. Since separate housing units were employed for the control and for the tuberculous animals, the possibility that confounding may have been introduced into the study Table 6 38 Tuberculous and non-tuberculous animals Which survived the expertmental periods on different protein intakes Animals Animals Group planned surviving for group in group no. no. Control 55 per cent protein 40 days 10 10 47 days 10 10 25 per cent protein1 28 days 10 9 55 days 10 10 Tuberculous 55 per cent protein 40 days 8 8 47 days 8 5 25 per cent protein1 28 days 8 7 55 days 8 8 1 All animals received the 55 per cent protein diet during the first 12 experimental days; the 25 per cent protein ration was fed for the last 28 and 55 days of the 40- and 47-day periods, respectively. 39 was explored. Two preliminary statistical analyses were completed, one from the data for the guinea pigs which received the 55 per cent protein diet and another fran the results for those consuning the lower protein ration; the form for the analysis of variance is shown in Table 7. The variability among the animals was estuated by the error mean square. The results indicated that the guinea pigs belonged to the same statistical p0pu1ation; that is, no significant effects due to housing facilities had been superimposed upon experimental values. Another analysis of variance danonstrated that the subdivision of the animals into three sets of 24 each for study over a seven-month period caused no significant differences among the data for the guinea pigs. It is doubtful, therefore, that the separation of the diseased animals from the controls and the distribution of the groups for study over seven months have affected the results beyond that which is usually attributed to experi- mental error. ‘Jhat the experiment may be considered as a statistical unit has been justified; the final form for the analysis of variance employed in the interpretation of the results is given in Table 8. Table 7 Form for the analysis of variance employed in the evaluation of the effects of separated housing units upon the experimental results 25 per cent protein diet 55 per cent protein diet df df Tbtal 55 Tbtal 55 Disease treatment 1 Disease treatment 1 Length.of period 1 ~Length of period 1 Interaction 1 Interaction l Error 52 Error 52 4O 'mble 8 Final form for the analysis of variance employed in evaluating the experimental results ‘21:. Total 71 Disease treatment (T) 1 Dietary protein (1’) 1 Length of period (D) 1 Interaction TP 1 Interaction TD 1 Interaction PD 1 Interaction TPD 1 Error 64 41 42 Observations gt Autogsx 'lhe guinea pigs which had been inoculated with it. tuberculosis H57Rv showed definite involvement of miliary tuberculosis: the enlarged lymph nodes contained caseous material; the spleens had amassed tubercles and almcst had doubled in size; the livers gave evidence of infil- tration by the bacilli and of enlargement. Slides made from the lymph node and/or splenic tissue of the tuber- culous animals danonstrated the presence of red rods, identified as tubercle bacilli. The disease was more widespread among the tuberculous animals surviving through the 47-day period than among the 40-day guinea pigs. Also, the extent of tuberculous lesions was greater among the animals that received the 25 per cent protein diet than among those which consumed the 55 per cent protein ration. For the guinea pigs receiving low dietary protein, symptoms of the infection frequently included fluid accumulation in the intestinal region; in some cases, far advanced tuberculosis was indi- cated by lung involvement. 0n the other hand, no distinct differences were observed for the control animals between the 25 and 55 per cent protein intakes or between the two 45 experimental periods; all of the control animals appeared healthy at autopsy. Weight Gain and Food Intake The mean gains in body weight and average food intakes of both the control and tuberculous animals are presented in Table 9; values for individual annals are presented in Appendix Table iii. Data for the guinea pigs which con- sumed the 25 per cent protein ration during 28 and 55 days of the 40- and 47-day periods, respectively, include the values from the first 12 experimental days on the 55 per cent protein diet. The average gain in weight of the animals which were given the 55 per cent protein ration was greater than that of the guinea pigs which received the 25 per cent protein diet. This was true of the tuberculous animals as well as the controls. The differences were found to be statis- tically significant by analysis of variance (P$0.0l). The tuberculous animals which received the 55 per cent protein diet for 40 and 47 days had average weight gains of 1661451 and 1721158 grams, respectively. 'Jhese weight A ~ ~ A¥ A ‘ 1 Nunbers following mean values represent standard deviations. .haopapeommoa .mmoaaom manure use too on» no shun mm was mm puma new you new new scheme awesome pace pom mm on» “sham ampoenaaeaxo ma pagan emu modest weapon oacpoaa some men on on» mopacooa mamaasm HH< n .nooapmabou meanness assurance ncsam> omefi msawoaaou uaonnsh m .naqadom rm madcapasn can now nonmaseaeo H e H 3 new fl mnfl me. w «3 m seen me n H me Sm Heme S 1. e3 m ease mm naoponn pace and mm m. H2 no Lu. mean mm H «S m n sees me e H3 one u." 33” me Home m seen on naopoam pace and mm 30H30h0959 e Mme meaflnom SHE 3 uses me e and n3 Hues as Home on shun mm macpoan pose pom mm a H mm as H 83 me Name 3 a uses 3 u H2 8 u... use as Home on case ow sacuonm unoo mom mm Hahn-.500 .w ooa\mw Hmaaom\m.w Hmaacm\m.w .on exupoa coon omens“ ofisw Hnamsaom macaw nod Sum unease 00cm unease noxmuoa seepage pnoaohhao no swam seofiam mooHSeaenspusoo com seafloononsp non noapmsznooo moon use oaaw unmask emancbd a manna 45 gains appeared to be similar to those of control animals which received the same diet. 'me consunption of the lower protein ration by the diseased animals resulted in smaller mean gains in weight than were observed for the respective controls; however, no real differences existed. The tuberculous animals which received the 55 per cent protein ration during the experimental periods consuned the largest amount of food. The control guinea pigs fed the lower protein diet after the first 12 experimental days consumed the smallest quantity of food of any of the groups of animals. Analysis of variance indicated that both the presence of infection and the consumption of the higher protein ration resulted in higher values for total food intake (rs. 0.01). 1110 gain in weight per 100 grams of food consuned by each of the guinea pigs was calculated as an index of food utilization; the mean values are given in Table 9. Tuber- culous animals which received the 55 per cent protein diet for 47 days gained 1721' 58 grams while consuning 1542': 95 grams of feed; this represented a mean weight gain of 15 grams per 100 grams of food. When the control animals received the same dietary treatment during the corresponding period of time, the average weight gain was 1811' 46 grams, 46 and the mean food intake, 1000295 79 grams; the calculated index of food utilization was 18 grams of weight gained per 100 grams of food consumed. The mean values thus determined as indices of food utilization indicated that the tuberculous animals required more food than did the controls to gain weight at a similar rate. The consunp- tion of the 25 per cent protein diet appeared to result in slightly smaller gains in weight per 100 grams of food during the respective experimental period than did the 55 per cent protein ration. The studentized range was used to test whether these differences were significant; the results of the analysis are shown in Figure 1. Each group of animals is repre- sented by a vertical line. The tip of the arrow head represents the average utilization value calculated from the grains of weight gained per 100 grams of food consmned by the animal. The cross-bar indicates the adjusted values obtained by subtraction of the correction factor derived from the standard error of the mean and the studentized range table at the five per cent level. When the bar of one group shows a value equal to or greater than that corresponding to the arrow tip of another group, the dif- ferences between the means of the two groups are significant. Figure l 47 manure .mSOHSononnp .oaopoum pace hon mm «bemaamm hmuiom .msodsoaonsp .Gaopoaa pace non mm mommaunm hunted .Hoouooc .odoposn used and mm mbwonmm hmouoe .Hoapooo .naoponm pace hem mm Momenmm hunter .ndoasoaonsa soaovoan home mom on “remain” hauled .naoasoaeasp shampoaa more son on normaumn humubm .Honpooo .naoponm some men mm “trauma hmnuov .Honuooe soaopoan some non an uoeoumn emoda consensuspu one no momma ho. .Uogmooo neck He macaw 00H son swam confism no noamw pnwuow one no nouuammsoo H enemas 48 svmhnm 05.7mm humane oemheo 26.3 03-90. 30.3 01.9.9... 0 1 m LI 4 11 .. 0. 1 1.. .m 00. \.m m [T 9.3.2. noon. + mud 22.0 4 FIO~M>> ¢ 1.. 11 .. m. a 4 1. .ON 49 The performance of the control animals was found to be superior to that of the canparable groups of tuberculous guinea pigs which received the same dietary treatment throughout a similar length of time. There were signifi- cant differences between the weight gains per unit of food intake for the animals given the 55 per cent protein ration and those which received the diet supplying 25 per cent protein when the experimental period was continued for 47 days following the inoculation of the test animals. However, this was not true for the corresponding groups onexperiment for 40 days. The group of animals with the poorest weight gain per 100 grams of food construed was the tuberculous animals which were fed the 25 per cent protein ration for the last 55 days of the 47-day experimental period. Although the mean gain in weight per 100 grams of food intake for these animals was not significantly different from that for diseased guinea pigs receiving the same ration for 28 of 40 days, the value was significantly less than for all other groups, either experimental or control. Symptoms of anorexia, which may accompany tuberculosis in hunan beings, were not observed in the tuberculous guinea pigs. Both the presence of tuberculosis and the 50 restriction of dietary protein apparently impaired the utilization of food for weight gain. The effect of these treatments appeared to be more pronounced over a longer period of time . Liver Fat and Solids ggt An examination of the mean values for fat in the livers of the guinea pigs indicated that the protein content of the diet had a greater influence upon the amount of fat which was deposited than did either the disease treatment or the length of the experimental period. These data are given in Table 10 and the individual values in Appendix Table iv. The control animals which were given the 55 per cent protein diet for 40 days were found to have a mean value of 0.465: 0.09 grams of fat in their livers; the livers of the respective tuberculous animals contained 0.52: 0.10 grams of fat. When the 25 per cent protein ration was fed to tuberculous animals during the last 28 days of the 40-day period, the amount of fat accumulated in the livers averaged 1.0531 0.56 grams. An analysis of variance indicated that no significant differences existed between the mean values. 51 .hao>auconmon .udoaaoa hmmubm use loo can no when we use mm puma emu non new no; pogo nfiopoam pace pom mm on» “when HmpoeanomKo NH pagan emu moaasm sesame odouoaa when son an on» mo>acooa namnaom HH< n .mnoapma>oo mandamus poomonmca metamb omofi moawoaaon naonnflz m .mamaaom no wnaoamsoa on» new Uopmasoamo H n.» 2.0.30.0 m seen no we 3.0.4.84 m seen me nuaoponm pace non mm m.» 3.9.25.0 m seen me on odoflmmd m ends 00 odopoaa unoo son mm moodnoacnsa 04. 30.4.8.0 0H seen no Te 0e.0.....:..0 0H seen we odouoam pace nod mm m.» «0.0.1320 on n sane 5.. in 80.230 0H secs 0... nacuoam unco pom an Hoflnco m w on unwael nopaa no pace hem unmaos anamnaod macaw use mommaoa suspend pooaokfidu no swam monflnw uneasenonspnsoo one odoasoncnap no nno>aa one an new no ncwmuooesca mom npnwaow odes OH OHQAH 52 The mean values for the percentages of fat in the livers were larger for the four groups of animals that received the lower protein diet than for the guinea pigs which consuned the 55 per cent protein ration. Am0ng the groups of animals to which the 55 per cent protein diet was fed, the livers of the controls on experiment for 47 days contained the greatest percentage of fat, i.e., 5.5; for the groups which received the diet supplying 25 per cent protein, the mean percentages for fat accunulation in the livers ranged from 5.5 to 4.8. These differences, however, were not significant. Harper gt gt. ('55) considered a fat deposit betwaen 2.9 and 4.5 per cent of the total weight of the liver of albino rats to be adequate and not excessive. Although the consumption of the 25 per cent protein ration under the conditions of the current study may have caused a slightly greater retention of fat per unit of liver tissue than did the 55 per cent protein ration, it does not appear that the diminution of lipotr0pic factors was sufficient to result in fatty livers. 55 Solids Table 11 and Appendix Table v include the mean values and individual data, respectively, for dry matter in (he livers of the guinea pigs. The total solid material determined in the livers of tuberculous animals which consumed the 55 per cent protein diet for 40 days averaged 6.61 0.6 grams, representing 25.2 per cent of the weight of the organ. The livers of the corresponding control guinea pigs contained 5.21:0.8 grams of solids, which was 26.5 per cent of the organ weight. The mean weight of dry matter in the livers of the control guinea pigs that received the 25 per cent protein diet for 28 of 40 days was 4.55:1.1 grams; this value was 24.9 per cent of the weight of the liver. It was found by analysis of variance that there was a significantly greater amount of solid material depos- ited in the livers of the tuberculous animals than in those of the controls (P$0.01); however, the percentage deposition was greater in the livers of the control animals than in those of the tuberculous guinea pigs (P $0.01). The total amount of dry material found in the livers of the animals was higher also when the ration supplied 55 per cent protein than when the 25 per cent 54 .haobdpooauoa .nmoanoa bruise one too on» no what mm use mm puma one sou new new oedema adepoam pace non mm on» “when HmaooaHAano NH pagan can modest pogo adepoam pace mom no on» mopaooon Madonna HH< m .nooapma>oe commoner commandos ucsam> uses wofisoaaou uncensz e .323 pass nod - 00H "manganese n .umh pace non u sudden p900 mom «newsflasamo m .namaflom moapaphsn on» ooh nonmasoamo H 0.2. mém 00m seduce m sees mm 0.2. 23 Ten Towed m sans mm nfioponm Home eon mm «.2. «Am mém 20.450 0 seen 5. mi. mém «.mm 0.0H00 m that 00 oaopoam pace pen on muoasonohsa has 2mm ném towed 0H when 3 H2. m.0m Tam 2me.5 0H shun mm mnaepoam pose Asa mm mi. due «no. newts 0H chap 5 was new mom madman 0H ease 0e :aopoan pace son an HOHHGOO R ¢.w .on MQMmoI no>HH Dumas: no>HH Ho wooo mom mooAMuumh he pace hem unwaca Hmasadn< muonm a nose: T unseen L 14 echoed“ afiepoam enmeshhdm so nwwm menasw encasemenaunooo mos macaroacnsp no onebda one om nous: mos .mmaaom coaunumh .mmfiaon H0 commonsense mos enaaon Mo mpnwaow one: HA canes 55 protein ration was fed; the differences between the means were found to be significant by analysis of variance (P $0.01). The differences between the mean percentages of solids accumulated in the livers of the animals con- suming either ration were not significant. jSince the weight of the solids in the liver probably depends to some degree upon the total weight of the liver, the comparisons of the mean percentages of solid material seem to be more indicative of the effects of the experi- mental factors upon the livers of the animals than do average weights of drywmatter.‘ The percentage of dry matter was less in each tuberculous group of animals than in the corresponding control animals. This was true also of the percentages of fat-free solids. By calculation, then, the percentages of water in the livers of the control guinea pigs were less than those in the livers of the respective tuberculous animals. These results suggest that a dilution effect may have occurred in the liver during tuberculosis. Itis possible that tuberculosis interferes with the structure within liver cells and thus may cause a decrease in the effective function of the organ. 56 Liver Weight and Bod: Weight The mean weights of the animals and of the livers are given in Table 12; Appendix.Table vi contains the individual data. The mean weights of the groups of guinea pigs that received the ration supplying 55 per cent protein were approximately the same; however, the mean weights of the livers of the tuberculous animals exceeded those for the respective ccntrols. When the 25 per cent protein diet was given to control animals for the last 28 days of the 40-day period, the mean weight of the livers was found to be 17.9 grams while that of the livers of the tuberculous guinea pigs was 22.5 grams; the mean body weight for the controls was approximately 25 grams greater than that for the tuber- culous animals. The livers of the tuberculous animals receiving the lower protein ration during the last 55 days of the 47-day period had.a:mean weight of 20.1 grams; the body weights averaged 405 grams. These data and the gross examinations of the animals upon sacrifice suggested that the presence of the tuber- culous infection caused enlargement of the liver. Since the individual body and liver weight values overlapped considerably, an attempt was made to evaluate the data statistically. 57 .hfle>apoommcn anneanoa hmmnbw mom uoe emu we when an mom wN puma on» son new me: asap oaopoam pooo hon am can “when HmuooafiAchc NH pmaaw one new oedema oaopoaa pace pom an on» uc>dcoou madness Had H Amme-eenv mos an.em-o.mav H.0m m sees no xmse-mmmv 00s is.mm-0.mev n.mm s seen we adopoam pace hon mN hose-aenv one x0.mm-m.0ev m.sm m H shes so homm-0mnv see x0.un-n.emv «.mm m shun 0e nacpoam pace men on macarononse xuemuennv one Am.em-e.mav 0.mH 0H sees no xoam-ummv 00¢ hm.mm-n.0 v e.ua a seen we unopoaa pose hem mN Aonn-0snv use Am.nmum.eav m.0m on H ease be imam-emnv use “n.0m-0.0av m.eH 0H sane 0e naopoam pace pom mn Hosance em em 00g assess to names: noted no names: sausage moose smug meadow on» new enemas! aobua one magmas: hoop ommaebm NH adage 58 A predicting equation, y I 0.04454 x, was calculated from the body and liver weight data of the control guinea pigs which were fed the 55 per cent protein ration; y represented the weight of the liver in grams and x the death weight of the animal. The theoretical weight of the liver of each animal was then calculated, according to the equation. The actual weights of the livers of the tuberculous animals on both protein rations differed significantly from the predicted weights (Chi Square; P50.05). The actual weights of the livers of the control guinea pigs which received the 25 per cent pro- tein ration were approximately the same as those predicted on the basis of body weight. Therefore, the liver and body weight data determined for both groups of control animals were used to calculate a second regression equation, y 8 0.04586 x, as shown in Figure 2. For the tuberculous guinea pigs, the actual weights of the livers deviated positively from those predicted by means of this regression equation, with one exception. It was thus established by a Chi Square test and a t—test (25.0.01), that the livers of the tuber- culous animals were enlarged with respect to body size, in comparison with the control animals. Figure 2 59 massage macadeaonspnoon mom maoggopsp no names: been. one unwaew no»: accrues. noaanem N 0.9me 60 “.me .2322... no F1052, Items 000 00... 00.. 00... 00m 00. b b b P p P O 34.2.24 woodbommmot o ‘ .0 352.24 somtzonc . -o. -n. ION X @QMVOAU u) 0 219m; . mm vOV 61 The data in Thble 12 show that the tuberculous animals receiving the 25 per cent protein ration weighed less than the corresponding controls, but the livers weighed more than those of the non-tuberculous guinea pigs. A failure to deposit body tissue or destruction of body tissue during tuberculosis may result in the appearance of heavier liver weights in relation to body weights. There appears to be no reason to doubt that hepatcmegaly occurred during tuberculosis; however, the wasting process of the disease may also be a factor in determining how enlarged the liver seems to be with respect to body size. ' Himsworth and Glynn ('45) studied dietary massive necrosis of the liver in rats. During acute necrosis, the livers were enlarged, and it was danonstrated that the injured organs were heavier in pr0portion to body weight than were those of the control animals. The inves- tigators noted that the increase in size and weight of the affected livers was due to edena, which appeared to be confined to the liver. Furthermore, approximately the same amounts of fat and a lesser concentration of protein were demonstrated to be present in the necrotic livers with respect to those in the livers of the control animals. 62 It was shown in the present study that the per- centages of water in the livers of the tuberculous animals were greater than those in the livers of the respective controls. A tendency for more of the solid material to be deposited as fat during the consumption of a lower protein ration was also indicated; however, the differ- ences between the mean percentages of fat in the livers of the animals consuning the 55 per cent protein diet and those receiving the 25 per cent protein ration were not significant. Ban (tgg. gi_t.) reported that certain tuber- culous patients were found to exhibit necrosis with pos- sible cirrhosis and fatty degeneration of the liver upon biopsy. GyBrgy ('55) described many causative agents in necrosis, but explained that a decrease in the consunption of lipotropic factors may result in fatty livers and, in some cases, cirrhosis of the liver. Changes attributable to degenerative processes in the liver may occur suddenly, according to Himsworth ('47). Liver cells proliferate readily; it is possible for the parenchymal cells to regenerate following necrosis, but the capacity for this process is dependent upon the type and severity of the injury. Himsworth (tgg. git.) also explained that atr0phy of the necrotic liver may succeed the stage characterized 63 by edema and may lead to postnecrotic cirrhosis. The data from the present study, shown in Table 12, indicate that the mean weights of the livers of the tuberculous animals on experiment for 47 days were less than those of the livers of the corresponding guinea pigs which were sacrificed upon the fortieth experimental day. The lack of histological evidence precludes the distinction of the type of liver injury that apparently occurred during the progress of the tuberculous infection in the guinea pigs. The results indicate that, during tuberculosis, the infectious process may be primarily responsible for injury to the hepatic parenchyma; the livers of the tuberculous animals were possibly necrotic and edemic. An extension of the experimental period may have resulted in atrophy of these livers. There were no indications that fatty infiltration into the livers of the tuberculous animals was greater than that into the livers of the control animals receiving the same quantity of protein. Moreover, the data did not suggest that the amount of fat in the livers of the tuberculous animals increased when the length of the experimental period was increased from 40 to 47 days. However, the infiltration of fat into portal spaces may be more marked in tubercu- losis when dietary protein is restricted. 64 Liver Nitrogen The average values for the retention of nitrogen in the livers of the guinea pigs are given in Table 15; the individual data are presented in Appendix Table vii. Analysis of variance showed that the tuberculous animals had significantly more nitrogen in their livers than did the respective controls (P $0.01), and that the consumption of the 55 per cent protein ration resulted in a greater deposition of total nitrogen in the livers than did the lower protein diet (£60.01). The control animals which.were fed 25 per cent protein for the last 28 days of the 40-day period had an average of 5573-3126 milligrams of nitrogen in the livers; the highest mean was 7522': 88 milligrams, for the tuberculous guinea pigs receiving 55 per cent protein for 40 days. When the nitrogen values were expressed as milli- grams per gram.of wet tissue, the livers of the control animals contained significantly greater amounts of nitrogen than did those of the tuberculous guinea pigs (P $0.01); per gram of wet tissue, dietary protein did not cause significant differences between mean nitrogen values for the livers. No significant differences were 65 .hHopHueomnon .mannom manure one now one he when mm one mN van on» no new as: poHu :Hopoam noon nod mN on» “when HmpsoaHaoaNo NH pmaHm on» oHnse sOHpsa aHopoaa pace sea on on» 00>Hcooe nHmnans HH4 n .noOHpmH>oo venomous poomenaoa nosHmb uses waHBoHHom nachnSZ N .nHeaHom moH>H>Asn on» new ecumHSono H «H M «ma 0 .m H0. an on H30 m were no Shana m.m “new 8 Memo m sees me asHopoaQ pace and mm m Heme HA H28 assumes m s seen 5 0H Hm: m.HHe.mm mm Hams m sans 0e nHoponm pose hem mm mfiOHnoaopss S Hm: m.» H0. .8 mm flab 0H sees an .2 H was 0. m He . He umaflmm 0H sans mm :nHepoam usoo hem mN 0H «use ed M 0. as 00520 0H .. seen 5 2 Meg s4 H m. an no H50 0H shes 0e oHopoam pace hem mm wfi HOHHGOO N N we N we on mmHHom Mo enanu now seem hem no nasm aom Henna HmHmaHa< macaw flowcthz nommpnfi sHopoea paeaoHMHU :0 mem mooHsm msoHsoaonsuuooo one nsoHsoaopsu mo nao>HH on» nH nowoapHG no mpnwdol use: 0H oHan 66 observed between mean values when liver nitrogen was expressed as milligrams per gram of solids. 'mat the analysis of variance showed that the tuberculous guinea pigs stored less nitrogen per gram of tissue than did the control animals may be attributable to the values obtained for the tuberculous animals on experiment for 40 days. ‘Ihese means were lower than those for the corresponding tuberculous guinea pigs on experiment for 47 days. When 35 per cent protein was given to tuberculous animals on experiment 40 and 47 days, the livers retained per gram of wet tissue 28.91’ 1.8 and 31.51'1.l milligrams of nitrogen, respectively. 'Ihe corresponding values for the tuberculous guinea pigs fed the 25 per cent protein ration were: 29.6123 milli- grams when the animals received the low protein diet for the last 28 days of the 40-day period; 31.0125 milli- grams, when the animals received the low protein diet for the last 55 days of the 47-day period. Himsworth and Glynn (log. 9.39) demonstrated that the concentrations of protein in the livers of rats fed necrogenic diets were less than those in the livers of the control animals; the animals that survived this acute dietary necrosis had livers which were chanically indistinguishable from those 67 of the unaffected animals. In the current experiment, the feeding of a 25 per cent protein diet for 28 and for 35 days apparently did not alter the liver nitrOgen values per gram of solids or per gram of wet tissue with respect to the values for the corresponding animals which received the higher protein ration. Nitrogen appeared to comprise a similar fraction of the dry matter in the livers of the experimental animals, regardless of the imposed treatment. he percentages of nitrogen deposited in the livers of the guinea pigs were not alteredby the decrease in dietary protein from 55 to 25 per cent. Ellie presence of tuberculosis appeared to have} influenced the percentage composition of the wet liver tissue, which possibly was a reflection of the increased moisture content of the organ. Liver Sulfur As shown in Table 14, the lowest mean value for sulfur per gram of dry material in the livers was 6.5i0.6 milligrams; this was determined for the tuberculous animals which received the 35 per cent protein ration for 40 days. The highest value, 7.5 milligrams for sulfur per gram of liver solids, was found for both the control and tubercu- lous animals consuming the 25 per cent protein diet during 68 .haobdpoommoa .nanaom hmoubv was uom on» no mhdu mm was mN umwa can now cow was noun :aouonm pace hog nN on» umhmm Haunoaanoaxo NH puma“ can you soapua :Hopoam unoo non no on» Uo>aooon massage HH< m .mnoapmfipem Unuoqum anemonaon mosaap qsoa wafisoaaou myopasz n .na manna 369% upon N .mamaaqa wnH>H>nnm on» non UopmHsono H Huméa 8 Meg 0 Hon mafia; m 236 an 1...}: mm «mom a. Hoe 90.4.2» m whee mm adopoam pace non mN 1mg; @2ng m H3 2095;. m a 2W8 3 flag; 8 Hm? can? manage m Page ow adopoam #200 men an muoafiononfia 125 am «in m «on «6.3.6 3 when an H333 8H H50 Swen 04“....» 3 3:6 mm dqaoponm pace Hem mN 195 ooafinao m Hon odflné 3 made 5 2.23 3 H50 m «S «toads S ”has 3 naopoam uqoo mom mn Honunoo .ms lama i mama .oc awash Naomonpan anhafim mwfiaou ho m\z Hence Hanna swam new amanaand macaw asuanm nodpwn ASMHSmuaowoauHa «moxwuna naeponm pqonohhfiu no nwdn meadow usoadononaunnoq one uncanononnu Mo mao>fia on» ca asHHSm non monas> omaao>¢ vH oanma 69 28 of 40 days; the standard deviations for this mean were 1.0 and 0.9, respectively. 'Ihe range for the means was small and no group average varied from the over-all mean of 7.1 milligrams by more than nine per cent; no significant differences between means for sulfur per gram of liver solids were found. 'me individual data are given in Appendix 'Iable viii. 'Ihe deposition of total sulfur in the livers er the guinea pigs was significantly higher when the percentage of protein in the ration was 55 than when 25 per cent protein was fed (13.40.05); significantly larger amounts of total sulfur were determined in the livers of the guinea pigs which had been inoculated with tubercle bacilli than in the livers of the control animals (Pf—0.01). 'lhe data in Table 14 show that when the values for the total nitrogen in the livers were high, values for total sulfur in the livers also were augmented. When the 55 per cent protein diet was fed to tuberculous animals for 40 days, the livers contained an average of 43:10 milligrams of sulfur and 7521' 88 milligrams of nitrOgen; the values for the corresponding controls were 57$ 9 and 6171’93 milligrams, respectively. The livers of the tuberculous guinea pigs which consumed the 25 per 70 cent protein ration for 28 days of the 40-day experi- mental period accunulated 401:7 milligrams of sulfur and 6581' 89 milligrams of nitrogen. The mean ratios of nitrogen to sulfur in the livers of the animals within the eight study groups ranged only from 16.431 to 17.9:1. ' The ratio of nitrogen to sulfur in organic matter approximates 16:1. Folin ('05) found that the ratios of nitrogen to sulfur in the urine of thirty human subjects decreased slightly when the protein in the diet was decreased. In severe wasting illnesses, the nitrogen to sulfur ratio in urine may increase markedly, since the excretion of nitrogen increases with degeneration of tissues. It was found in this laboratory1 that the urinary sulfur excreted by eight patients with terminal tuberculosis was not excessive when an excretion of one gram per day was considered as the norm. The relationship between nitrogen and sulfur in the livers of the guinea pigs apparently was not disturbed during tuberculosis or during restricted protein intake. 1 Unpublished data, Department of Foods and Nutrition, Michigan State University. ’71 Glutathione Reductase activity i_n_ Liver The values calculated for the glutathione reductase activity in the livers of the guinea pigs were expressed as rates1 per gram of solids, per gram of fat-free solids, and per'gram.of nitrogen; the meme values for the eight study groups are given in Table 15. No significant differences were found.among these means by statistical analysis; the individual results varied greatly as can be seen in Appendix Table ix. The glutathione reductase activity in the livers of the tubercudoum annuals which received the 25 per cent protein diet for 55 days of the 47-day period, when expressed as the mean rate per gram of solids, was 0.82‘L’0.52; that of the respective control animals was 04610.17. For these two groups of guinea pigs, the average rates calculated per gran of fat-free solids and per gram of nitrogen were: tuberculous, 0.94:0.58 and 6.51537, respectively; control, 0.55:0.19 and 4311.8, respectively. There appears to be no evidence that glutathione reductase activity decreased during restricted protein 1 The basic equation for calculating glutathione redmtase activity is given on p. 55. 72 .hHObapoommoA .uUOHnoa hmonbm was uom emu no when on was NN unaH on» you new as: road aHovonm pace hem nN can “sham Hanuosanonxo NH umaHn es» you aoHpmn nHOponm paeo pen on on» Uo>HoeoA anand HH4 e .mnoapeHbom canvass» paomenaon mowmno>m quBOHHoH unobsnz n .mHuaHns qubabasn on» you copaneHmo N .mn .A no ao>Hw mH thproa onspQSan oQOHnampus wnHusHSono you QOHpqs o OHmmn can H r.» Hens 3.9.30.0 «adhmmd m sham on 9450...” 0n.0H8.0 mm.0Hme.0 m ease mm quouonm pace pom mN mémaé 5.0.320 m0.0Hm0.0 m same 5. m.m+a.e 0294.30 rm.0H0e.0 m ease 0e nHOpoam pqoo mom on nsOHsoaonps méhmé 3.9.30.0 S.0H0e.0 0H were me faunas mudflmné 00.0Hnn.0 0H ease mm quOponn pace non 0N fiaflmm madfieed me.0.flsn.0 0H mass 5 sense 00.0fl00.0 8.09.3.0 0H 298 0e :Hoponm pace non mm HORfiGOO .OQ MUHHon nomonuHa ooahnpwm uuHHom nHaEqu aroma no Edam hem noumm N noMmunH nHouonn unenohham no ume soanw usoHaoa°nnvuqoq was naoHSOnonsu no unopHH on» :H thH>Hpom ensuescen oGOHapsufiHm you money owshebd nH OHDMB 73 intake. The results presented both by Wainio 231 a}; (192. gig.) and by Knox 239;. (la. git.) indicated that, even during severe dietary protein limitation, not all enzymes danonstrated decreased activities. The mean values for glutathione reductase activity per gram of nitrOgen were slightly higher for the tuber- culous animals than for the respective control guinea pigs, except when 55 per cent protein was given for 40 days. Reduced glutathione is a hydrogen donor and may help protect ascorbic acid in the liver from oxidation. It is known that ascorbic acid metabolism is disturbed during tuberculosis . Under the experimental conditions imposed upon the guinea pigs, it appears that tuberculosis did not interfere with the mechanisms which function in maintaining liver glutathione in a reduced fonn. Riboflavin-containing Coenzymeg in Liver lean values for flavin adenine dinucleotide (FAD), for flavin mononucleotide (MN) plus free riboflavin, and for the suns of the two fractions designated total riboflavin in the livers of the guinea pigs are presented in Table 16. Individual data are given in Appendix Table x. Table 16 '74 Table Mean values for riboflavin in the livers of tuberculous and Liver riboflavin Group Animals1 ‘ Total FXN 32 FAD plus free 82 2 2 2 ug. 28° 2g. Control 55 per cent protein 40 days 10 585: 112 5201-112 651-21 47 days 3 10 6471: 117 5743104 75121 25 per cent protein 26 days 10 5512*: 125 4601’128 71:15 55 days 10 5481 96 471: 86 77:21 Tuberculous 55 per cent protein ' 40 days 8 6091': 81 5561' 85 75¢ 21 47 days 8 6222!: 65 542:1: 66 80115 25 per cent protein 26 days 3' s 558:9: 82 4761 78 8229217 55 days 8 5525:. 89 4521': 76 801' 27 1 Calculated from the surviving animals. 2 Numbers following mean values represent standard 3 All animals received the 55 per cent protein diet diet was fed for the last 28 and 55 days of the 40- 16 75 non—tuberculous gudnea pigs on different protein intakes Per gram of tissue Per gram of solids Total FMN Total FMN 32 FAD plus 82 FAD plus free B2 free 82 “2 2 2 2 2 2 EB- 28- BE- EB~ 28- ES- 29.e16.5 26.514.2 5.51’1.0 116116 100112 1614 62.214.1 28.616.6 6.611.0 127116 116116 141 29.916.6 25.616.7 4.612.o 120117 106117 1718 29.416.6 25.216.2 4.21l.0 108115 96116 1515 26.716.6 20.916.6 2.81o.7 94116 86116 1116 27.112.7 26.512.4 6.610.9 1091 7 951 7 1416 25.216.4 21.412.8 6.81l.l 1051 9 891 6 1615 26.716.7 22.7—16.5 4.01l.o 107116 91114 1616 deviations. for the first 12 experimental days; the 25 per cent protein and 47-day periods, respectively. '76 Effect 9‘1; dietary protein The consunption of the 25 per cent protein diet caused a decrease in the mean amounts of FAD and total riboflavin present in the livers of the guinea pigs in comparison with values determined for the animals fed the higher protein ration. An analysis of variance indicated that these differences were significant 0.4.0.01). There appears to have been no significant effect of dietary protein restriction upon the mean amounts of FAD and total riboflavin stored either per gram of liver tissue or per gram of solids. Limitation of dietary protein resulted in slightly higher mean values for FMN plus free riboflavin in the livers of the guinea pigs; the means for this frac- tion expressed per gram of wet tissue and per gram of solids in the livers of the guinea pigs consuming the 25 per cent protein diet were significantly higher than those found for the livers of the animals which received the 55 per cent protein ration ”$0.01). The control animals which received the 55 per cent protein diet for 40 days had an average of 58531112 micro- grams of riboflavin in the livers; the values for FAD and FMN plus free riboflavin were 5201112 and 65121 micro- grams, respectively. When the protein content of the diet '77 was limited to 25 per cent for 28 of 40 experimental days, the livers of the guinea pigs accunulated 5512‘: 125 micro- grams of riboflavin; there were 4601128 micrOgrams of FAD and 711’ 15 micrograms of PMN plus free riboflavin. For these two groups which.differed only in the percentage of protein in the ration, the mean values for FMN'plus free riboflavin per gram.of tissue were: 55 per cent protein, 5.5il.0 micrograms; 25 per cent protein, 4.5!.“ 2.0 micrograms. Fran the data given in Table 17, it can be seen that the livers of the animals which.received the 55 per cent protein diet contained more nitrogen as well as more riboflavin than did those of guinea pigs consuning the 25 per cent protein ration. Dietary protein appeared to have little effect upon the storage of nitrogen and total ribo- flavin either per gram of wet tissue or per gram of solids. When the milligrams of riboflavin were expressed per milli- gram.of nitrogen, it became evident that the retention of riboflavin in the livers of the guinea pigs was directly related to the storage of nitrogen. It would then appear that the tissue retention of riboflavin is related to protein consumption insofar as the storage of nitrogen is influenced by dietary protein. Sarett and Perlzweig 78 .hH0>Hpoommon .mmoHnom hsuube 0am 10¢ 0:» mo nhsu mm 0:0 mN pmaH may you new 00: chv :Heponm unoo neg 0N on» Nahum Hansonnnomwo NH unnfiu can non GOHpma nHouonm pace mom on on» Uo>Hooon 0HsaHn0 HH< m .9H oHpma aohh 0009 m .0H oHnma seem 0909 N .menaqm maH>H>n50 can you bopstoHso a 00.0 0H0 000 0 name 00 00.0 000 000 0 0000 00 quponm pace hon 0N 00.0 men 000 0 0 0000 be H0.0 00m 000 0 0000 00 nHoponm pace non mn nSOHsoaonna 00.0 000 000 0H 0000 00 00.0 000 H00 0H 0000 00 «GHOpoam pace non 0N H0.H 000 000 0a 0000 be 00.0 0H0 000 0H 0000 00 nHopoam pace son 00 Hoausoo .mfi\.wfi .ma .mfi .0: OHpsa semoaan NnH>0Huoan someepac-aa>sauonam Hesse Heaps massage dream H moannH nHouonm anonomuau co anm soast nsOHsvopapucon use mSoHaononap mo unobHH on» GH nomeana o» qH>sHmonHa he capum bH 0HD§B 79 (122, gig.) proposed the existence of a direct relationship between riboflavin retention in tissue and both nitrogen storage and protein consumption. The FAD content in the livers of the animals seemed to parallel that of total riboflavin. That the amount of FMN plus free riboflavin per gram of wet tissue and per gram.of solids in the livers of the guinea pigs increased when dietary protein was restricted is contrary to the data reported in the Preliminary Experiment for rats. When the dietary protein was decreased from 20 to 8 per cent without alteration in the quantity of ration fed to the rats, the FMN plus free riboflavin fraction expressed as:micrograms per gram of liver tissue decreased pr0portionately with total riboflavin and FAD. Bessey gt_al, (loc. cit.) found that a decrease in ribo- flavin consumption was reflected in a diminution of FMN plus free riboflavin values per gram of tissue in the livers of rats. The guinea pigs in the current study, ihich received the 25 per cent protein diet, had.alightly smaller food intakes than the respective guinea pigs on the higher protein ration; thus, they consumed less protein and smaller amounts of riboflavin than did the animals fed the 55 per cent protein ration. 80 Constituents in the livers of guinea pigs and of rats will not necessarily respond in a like manner to similar external stimuli. On the other hand, in the livers of the guinea pigs, the FAD fraction and that of MN plus free riboflavin did not appear to react similarly to imposed stress. Since the protein deficiency apparently was not severe, the MIN plus free riboflavin in the livers of the experimental animals receiving the 25 per cent protein ration may have reflected only a temporary alteration. Effect 93 disease treatment The riboflavin retention, either as FAD or as FMN plus free riboflavin, in the livers of the tuberculous guinea pigs was not significantly different from that in the livers of the control animals. However, per gram of wet tissue, the livers of the control animals which re- ceived the 55 per cent protein ration for 40 days contained 29.616.5 micrograms of riboflavin of which 26.514.2 micrograms were FAD, while the values for the corresponding tuberculous guinea pigs were 25.711545 and 20.91.". 5.5 micrograms, respectively. Tint the retentions of total riboflavin and FAD per gram of tissue were less during tuberculosis can be seen from the mean values given in 81 Table 16. These differences were significant (Psi-0.01) . This also was true for the total riboflavin and FAD values expressed per gram of solids ”$0.01). It can be seen in Table 17 that the livers of all of the control guinea pigs retained more riboflavin per milligram of nitrogen than was found in the livers of any tuberculous group; the differences between means were significant (P50.01). The control animals that received 55 per cent protein for 40 days stored 0.95 milligrams of riboflavin per milligram of nitrogen; for the corres- ponding tuberculous guinea pigs, the mean value was 0.81 milligrams of riboflavin per milligram of nitrogen. When the dietary protein was decreased to 25 per cent for the last 28 of 40 days, the livers of the control and tuber- culous animals contained 0.95 and 0.85 milligrams of ribo- flavin per milligram of nitrogen, respectively. The relationship between the retention of riboflavin and that of nitrogen in the liver appears to have been disturbed during tuberculosis. The tuberculous guinea pigs consuned more ration than did the corresponding con- trols receiving similar percentages of protein and ribo- flavin in the ration. The storage of riboflavin per milligram of nitrogen did not appear to be increased 82 {during tuberculosis when dietary protein was increased from 25 to 55 per cent. That the differences may be effects of simple dietary deficiency is not indicated by the data. Since it was the FAD»fraction of the riboflavin that appeared to decrease per gram of liver tissue and per gram of solids during tuberculosis, alterations may have occurred in physiological mechanisms of the liver which require FAD as a coenzyme. Consideration of the nitrogen data deter- :mined under these experimental conditions indicates that the apoenzyme:may not be as limiting a factor in FAD-linked enzyme systems as the coenzyme during tuberculosis in guinea pigs. Effect 9;; the length 9;: the experimental period The control and tuberculous guinea pigs which received the 55 per cent protein diet were found.to have slightly higher values for liver riboflavin after the 47-day experi- mental period than following the 40-day period. This was also true for the control animals fed 25 per cent dietary protein. When the diet supplied 25 per cent protein to tuberculous guinea pigs, the riboflavin in the livers decreased slightly from the fortieth to the forty-seventh experimental day. An analysis of variance showed that the small changes in the average riboflavin values in the livers of the guinea pigs due to the length of time on experiment were not significantly different. Interrelationship§_ The experimental data indicate that the restriction of protein in the diets of tuberculous gudnea pigs accele- rated the course of the disease, but did not affect, to a marked degree, the particular liver constituents under investigation in this study. The gross examination of the tuberculous animals suggested that the limitation of dietary protein propagated the infection at a more rapid.rate than was found when dietary protein was adequate. In general, the tuberculous guinea pigs on 25 per cent protein appeared to be more debilitated and gave evidence of'more widespread infection than did the corresponding tuberculous anhmals fed 55 per cent protein. The stress which.restriction of dietary protein hmposed Upon the tuberculous guinea pigs was most apparent in the influence on the utilization of food for weight gain. The weight gained by the tuberculous guinea pigs which received the high protein diet was approximately the 84 same as the increase recorded for the controls fed the same ration. The consunption of the low protein ration by diseased animals resulted in a lesser mean gain in weight than was observed for the respective controls. In the tuberculous animals there was a decreased ability to utilize food for weight gain; the restriction of protein in the ration seemed to exaggerate this stress. Limitation of food consunption which has been reported to be character- istic of hunan beings with far advanced pulmonary tuber- culosis was not apparent with the infected guinea pigs. The mean liver weights of the tuberculous animals were greater than those of the control guinea pigs; however, the percentages of solids in the livers were decreased during tuberculosis. Nitrogen and sulfur appeared to renain a constant fraction of liver solids regardless of disease treatment or dietary protein. The glutathione reductase activity of the liver probably was not disturbed during tuberculosis. Abnormal fat deposits were not produced in the livers of the tuberculous guinea pigs. During tuber- culosis, the concentrations of riboflavin, specifically as the FAD fraction, per gram of wet tissue and per gram of solids were markedly decreased; limitation of protein in the ration was reflected in an auwentation of FMN plus 85 free riboflavin per gram of wet tissue and per gram of solids. From these observations, it could be postulated that two Opposing stresses were placed upon the livers of the tuberculous animals which received the low protein diet. The data indicate, however, that limitation of dietary protein did not cause alterations in the riboflavin fractions in the livers of the tuberculous guinea pigs beyond those which resulted when similar restrictions in diet were imposed upon control animals. Thus the restriction of protein in the rations fed to tuberculous guinea pigs did not appear to exaggerate the stress placed upon the constituents of the livers by the presence of the infection. This does not preclude the fact that the consumption of a low protein ration by tuberculous guinea pigs may have caused stress in the livers of the animals, beyond that caused by the presence of tuberculosis alone. The homeostatic mechanisms may have been capable of counteracting the imposed dietary protein limitation so that.the various effects of the disease treatment were not magnified by inadequate diet. Dubos ('55) reported that experiments with mice pro- vided no indication that protein deficiency alggg_could accelerate the course of tuberculosis, and that the 86 resistance of mice appeared to be independent of the protein content of the diet, within wide limits. The resistance to tuberculosis was Judged by the cmnulative nunbers of mice surviving at the close of specified time intervals. The guinea pigs in the present experiment were sacrificed rather than permitted to survive until the imposed stresses effected the loss of life. That the limitation of dietary protein increased the progress of the infectious process in the guinea pigs can be implied only from observations of the physical conditions of the animals. 8'7 SLMMARY AND C ONCLUSIONS The concentrations of certain liver constituents were. determined for 28 tuberculous and 59 non-tuberculous guinea pigs which were fed 55 and 25 per cent protein during 40- and 47-day experimental periods. Autopsies of the tuberculous guinea pigs revealed that the consump- tion of the lower protein ration resulted in more advanced and diffuse tuberculous infection, as judged by gross symptoms. The amounts of ration, containing either 25 or 55 per cent protein, that were consumed by guinea pigs inocu- lated with tubercle bacilli, exceeded those consumed by the respective non-tuberculous controls. However, the utilization of feed, which was represented as grams of weight gained per 100 grams of food intake, was signifi- cantly less by the injected animals than by the controls. The percentages of solids in the livers of the tuber- culous animals were less and the percentages of water higher than those values determined for the respective controls; the percentages of fat were similar. Comparison 88 of the relationships existing between body weights and liver weights of tuberculous animals and of their controls demonstrated that enlargement of the liver occurred during tuberculosis. This incongruity in liver size with respect to body weight appeared to reflect the accunulation of excessive amounts of fluid in the livers with possible structural and functional alterations. These data indicated that the liver injury manifested by the tuberculous guinea pigs may have been necrosis, without fatty infiltration. The ‘ratio of nitrogen to sulfur in the livers of the guinea pigs was not altered during tuberculosis or by restriction of dietary protein. Nitrogen cmnprised a similar fraction of liver solids for all guinea pigs; this was true also of sulfur. Limitation of dietary protein and the presence of tuberculosis infection probably did not cause disturbances in mechanisms responsible for maintaining reduced gluta- thione in the livers of the animals. The decrease in total riboflavin and the FAD content of the livers of guinea pigs fed a diet supplying only 25 per cent protein was not observed when the values were expressed per gram of tissue and per gram of solids; the 89 FMN plus free riboflavin fraction increased with dietary restriction. The amount of riboflavin present in the livers of the guinea pigs per’milligram of tissue was less for the tuber- culous than for the control animals. The FAD fraction of riboflavin decreased per gram.of tissue and per gram of solids during tuberculosis. It is possible that altera- tions occurred in FAD-linked enzyme systems of the livers of the tuberculous animals. When the experimental period of 40 days was lengthened to 47 days, no significant changes in the concentrations of liver constituents were noted beyond those found for the shorter time interval. 90 LITERATURE CITED Anderson, D. G., H. A. Stafford, E. E. Conn, and B. Vennesland 1952, The distribution in higher plants of triphosphOpyridine nucleotide-linked enzyme systems capable of reducing glutathione. Plant Physiol. 223675 Awapara, J. 1955, Effect of protein depletion on the transaminating activities of some rat organs. J. Biol. Chem. 200:557 Ban, B. 1955, Hepatic damage in chronic pulmonary tuber- culosis. Am. Rev. Tuberc. 12571 Barron, E. S. G. 1949, Cellular metabolism and growth. "The Chemistry and Physiology of Growth” ed. A. K. Parpart. Princeton University Press, Princeton, New Jersey, p. 151 Beck, L. V. and.AtiM. Bianconi 1956, Resistance of mouse tissue sulfhydryl to alterations by changes in dietary intake of sulfur amino acids. J. Nutrition 695197 Benne, E. J. 1955, Personal communication Bessey, O. A., O. H. Lowry, and R. H. Love 1949, The fluorometric measurement of the nucleotides of riboflavin and their concentration in tissues. J. Biol. Chem. 1§Q;755 Brewer, W. D., H. L. Tbbey, De Hwei Peng Kan, M. A. Ohlson, and C. J. Stringer 1950, Riboflavin, nitrogen, and thiamine metabolism of women with active tuberculosis. J. Am. Dietetic Assn. g§;861 Bricas, E. and C. Fromageot 1955, Naturally occurring peptides. Adv. Prot. Chem. §;12 Cannon, P. R. 1950, "Recent Advances in Nutrition". University of Kansas Press, Lawrence, Kansas,.p. 74 91 Chaudhuri, S. N. and S. P. Martin 1955, Effect of infection with M. tuberculosis and of tuberculin shock on the succinic demdrogenase activity of guinea pig tissues. J. Exp. Med. 98:99 Comer, C. L., E. J. Miller, M. N. Richard, and E. J. Benne 1944, Adaptation of a Waring Blendor for continuous emulsification. Ind. Eng. Chem. l_§_:717 Conn, E. E. and B. Vennesland 1951, Glutathione reductase of wheat germ. J. Biol. Chem. 192:17 Conn, H. J. 1927, "An Elementary laboratory Guide in General Bacteriology". The Williams and Wilkins Company, p. 145 Czaczkes, J. W. and K. Guggenheim 1946, The influence of diet on the riboflavin metabolism of the rat. J. Biol. Chem. 162:267 Daft, F. S. 1954, Experimental differentiation between liver necrosis and liver cirrhosis and some dietary factors affecting their development. Ann. N. Y. Acad. Science 53615 Dubos, R. J. and C. Pierce 1948, The effect of diet on experimental tuberculosis of mice. Am. Rev. Tuberc. 57:287 Dubos, R. J., J. M. Smith, and R. W. Schaedler 1955, Metabolic disturbances and infection. Proc. Royal Soc. Med. 48:911 Dubos, R. J. 1955, Effect of metabolic factors on the susceptibility of albino mice to experimental tuberculosis. J. Exp. Med. 101359 Faber, K. 1958, Tuberculosis and nutrition. Acta Tuberc. Scandinav. l_2_:287 Farber, J. E. and D. K. Miller 1945, Nutritional studies in tuberculosis. II. Niacin (nicotinic acid) and riboflavin deficiency. Am. Rev. Tuberc. 483412 Fitzpatrick, F. K. 1955, Nicotinamide in murine tubercu- losis. Proc. Soc. Exp. Biol. Med. 88:54 92 Folin, 0. 1905, Approximately complete analyses of thirty "normal urines. . J. Physiol. 18:45 Francoeur, M. and O. F. Denstedt 1954, Metabolism of mammalian erythrocytes. VII. The glutathione reductase of the mammalian erythrocytes. Can. J. Biochem. and Physiol. 883665 Gillman, J. and C. Gilbert 1954, Aspects of nutritional liver disease - human and experimental. Ann. N. Y. Acad. Science 513757 Gydrgy, P. 1954, On some aspects of protein nutrition. ‘ Am, J. Clin. Nutrition 83251 GyBrgy, P. 1955, Nutrition and liver injury. Borden's Rev. Nutrition Research.XVI Harper, A. E., M. E. Winje, D. A. Benton, and C. A. Elvehjem 1955, Effect of amino acid supplements on growth and fat deposition in the livers of rats fed polished rice. J. Nutrition 883187 Hawk, E. A. and C. A. Elvehjen 1955, The effects of vitamins B12 and B1 f on growth, kidney hemorrhage, and liver fat in rats fed purified diets. J. Nutrition 483495 Heinicke, H. R., A. E. Harper, and C. A. Elvdhjem 1955, Protein and.amino acids requirements of the guinea pig. I. Effect of carbohydrate, protein level and amino acid supplenentation. J. Nutrition §_'_7_:485 Himsworth, H. P. and L. E. Glynn 1945, The gross chemical changes in the liver in dietetic necrosis. Biochem. J. 593267 ‘Hhmsworth, H. P. 1947, "The Liver and its Diseases". Harvard.Uhiversity Press, Cambridge, Massachusetts, pp. 204 Hopkins, F. G. and K. A. C. Elliott 1951, The relation of glutathione to cell respiration with special reference to hepatic tissue. Proc. Royal Soc. London, Series B 198358 93 Hornemann, 0. 1915, Experimentelle beitrfl e zur Frage *der Beziehungen von Infektion und Ern ung. II. Mitteilung. Biochem. 2. 813475 Hurst, A., HQ‘M. Maier, and S. A. Lough 1947, Studies of hepatic function in pulmonary tuberculosis. Am. J. Med, Sciences 2143451 Johnston, J. A. 1955, "Nutritional Studies in Adolescent Girls and their Relation to Tuberculosis." Charles C. Thomas, Springfield, Illinois, pp. 520 Knox, W. E., V. H. Auerbach, and E. C. C. Lin 1956, Metabolic adaptations. Physiol. Rev. 883164 Koerner, Th A., H. R. Getz, and E. R. Long 1949, Experi- mental studies on nutrition in tuberculosis. The role of protein in resistance to tuberculosis. Proc. Soc. Exp. Biol. Med. 283154 Kosterlitz, H. W. 1954, The liver and protein metabolism. "Liver Injury" ed. F. W. Hoffbauer. Corliss, Macy & Company, Inc., New York, pp. 195 Krause, A. K. 1926, Studies on tuberculous infection. XV. Summary, analysis and applications of the studies of tuberculous infection. Am. Rev. Thberc. 143271 lack, C. H. 1956, Chronic tuberculous infection in experi- mental animals. Am. Rev. Tuberc. 183578 Lévy, M. 1985, Influence des etats nutritionnels sur les activites enzymatiques. Ann. Nutrition Aliment. Z3167 Lévy, n. and J. Legrand 1953, La rele de la choline dans différents systémes enzymatiques. Arch. Sci. Physiol. 2365, cited in Nutrition Abs. Rev. 883572 Linden, O. and E. Work 1951, Glutathione and experimental liver necrosis. "Liver Disease" ed. 3. Sherlock. The Blakiston Company, Philadelphia McCann, W. S. 1922, The protein requirement in tubercu- losis. Arch. Int.‘Med. 88355 94 ‘McKenzie, D., L. Malone, S. Kushner, J. J. Oleson, and 'Y. SubbaRow 1948, The effect of nicotinic acid amide on experimental tuberculosis of White mice. J. Lab. Clin.‘Med. 8831249 Meikleham, 7., I. C. wells, D. A. Richert, and W. W. Westerfeld 1951, Liver esterase and xanthine oxidase during protein depletion. J. Biol. Chem. 1923651 Meldrun, N. U. and H. L. A. Thrr 1955, The reduction of glutathione by the Warburg-Christian system. Biochan. J. 88le8 Metcoff, J., D. Darling, D. Wilson, A. Lapi, and F. J. Stare 1949, Nutritional status and infection response. II. Electrophoretic, circulating plasma protein, hematologic, hematOpoietic, and pathologic responses to mycobacterium tuberculosis (H57RV) infection in the protein-deficient rat. J. Lab. Clin.Med. 883555 P331313, J. 1954, The amount of pyridine nucleotides (Coenzymes I and II) in blood in experimental tuber- culosis before and during isoniazid treatment. Am. Rev. Tuberc. 283455 Perla, D. and.J. Marmorston 1957, Role of vitamin C in resistance. Arch. Path. 883545 and 685 Racker, E. 1949, Aldehyde dehydrogenase, a diphospho- pyridine nucleotide-linked enzyme. J. Biol. Chan. 1773885 Hacker, E. 1950, Crystalline alcdhol dehydrogenase from bakers' yeast. J. Biol. Chen. 1843515 Hacker, E. 1955, Glutathione reductase from bakers' yeast and beef liver. J. Biol. Chem. 2173855 Rall, T. W. and A. L. Lehninger 1952, Glutathione reduc- tase of animal tissues. J. Biol. Chem. 1943119 Reid, M. E. and.C. M. Briggs 1955, Development of a seni-synthetic diet for young guinea pigs. J. Nutrition 883541 95 Rich, A. R. 1951, "The Pathogenesis of Tuberculosis". Second edition. Charles C. Thomas, Springfield, Illinois, pp. 1028 Richert, D. A. and w. w. Westerfeld 1952, The effect of diet on tissue choline oxidase. J. Biol. Chem. 1993829 Riesen, W. H., B. S. Schweigert, and C. A. Elvehjem 1946, The effect of the level of casein, cystine, and methionine intake on riboflavin retention and protein utilization by the rat. Arch. Biochan. 103587 Rosenthal, 0., C. S. Rogers, H. M. Vera, and C. G. Ferguson 1950, Arginase, adenosinepyrcphosphatase and rhodanase levels in the livers of rats. J. Biol. Chan. 1853669 Rosenthal, 0., J. C. Fahl, and H. M. Vars 1952, Response of alkaline phosphatase of rat liver to protein deple- tion and inanition. J. Biol. Chan. 1943299 Sarett, H. P. and W. A. Perlzweig 1945, The effect of protein and B-vitamin levels of the diet upon the tissue content and balance of riboflavin and nicotinic acid in rats. J. Nutrition 883175 Sarin, L. R., K. C. Samuel, and R. K. Bhargava 1957, Hepatic derangement in pu1m0nary tuberculosis. Am. Rev. Tuberc. 283410 Sartory, R., I. Meyer, M. F. Foussereau, and I. Touiller 1951, Le taux des vitamines 81, 82, et PP en strepto— mycino-therapie dans lee tuberculoses pulm0naires. Rev. de la tuberc. 163207. Abstracted by Leites, V. 1952, Vitamins in pfifinonary tuberculosis. Am. Rev. Tuberc. Abs. 88382 Scales, F. M. and A. P. Harrison 1920, Boric acid modifi- cation of the Kjeldahl method for crOp and soil analysis. Ind. Eng. Chem. 883550 Shaw, C. R., F. Beck, H. Pilcher, and J. Parker 1950, A study of the relation of nutritional status to pu1m0nary tuberculosis. Am. Rev. Tuberc. 88358 96 Shohl, A. T. 1959, "Mineral Metabolism". Reinhold Publishing Corporation, New York, pp. 584 Siess, M. and H. Stegmann 1950, Arch. Path. Anat. Physiol. (Virchow's) 5183554 in Mann, F. C. and F. D. Mann 1955, Liver. Ann. Rev. Physiol. 883475 Sneeden, V. D. 1946, Observations on human malnutrition. Am. J. Clin. Path. 883580 Soltys, m. A., c. A. St. Hill, and I. Ansell 1952, "Tubercle bacillus and Laboratory Methods in Tuber- culosis”. E. 8c S. Livingstone Ltd., London, pp. 212 Srinivasan, P. R. and V. N. Patwardhan 1955, The effect of protein deficiency on (a) some liver constituents and (b) enzymes in liver, pancreas, and blood plasma in albino rats. Indian J. Med. Research 8831 Steidl, J. and F. H. Heise 1955, Studies on liver function in advanced pulmonary tuberculosis. Am. J. Med. Sciences 1863651 Sweany, H. C., C. L. Clancy, M. H. Redford, and V. Hunter 1941, Body economy of vitamin C in health and disease with special studies in tuberculosis. J. Am. Med. Assoc. 1883469 T‘reon, J. F. and W. E. Crutchfield, Jr. 1942, Rapid turbidimetric method for detemination of sulfates. Ind. Eng. Chem, Anal. Ed. 1.83119 Trufanov, A. V. 1946, Fate of riboflavin during protein deficiency. Biokhim. 81355, cited in Chem. Abs. 4035815 Tui, C., N. H. Kuo, and L. Schmidt 1954, The protein status in u1m0nary tuberculosis. Am. J. Clin. Nutrition __3252 Wainio, W. W., B. Eichel, H. J. Eichel, P. Person, F. L. Estes, and J. B. Allison 1955, Oxidative enzymes of the liver in protein depletion. J. Nutrition 883465 97 Wessels, G. C. 1941, Tuberculosis in the rat. II. Gross organ changes and tuberculin sensitivity in rats infected with tubercle bacilli. Am. Rev. Tuberc. 883449 Widdowson, E. M. and R. A. McCance 1957, Effect of a low-protein diet on the chemical composition of the bodies and tissues of young rats. Brit. J. Nutrition 883198 Willis, H. S. 1925, Studies on tuberculous infection. I. The early dissemination of tubercle bacilli after intracutaneous inoculation of guinea pigs of first infection. Am. Rev. Thberc. 883427 APPENDIX Table No . i ii iii iv vi vii viii ix LIST OF TABLES Page Individual determinations of the nucleotides of riboflavin in the livers of rats . . . . . . . . . . . . . i Temperatures in the animal rouns recorded over a weekly period . . . . . ii Individual weight gain and food con- sumption of tuberculous and non-tubercu- lous guinea pigs on different protein intakes O O O O O O O O O O O O O O O I 111 Individual weights and percentages of fat in the livers of tuberculous and non-tuberculous guinea pigs on different protein intakes . . . . . . . . . . . . v Weights of solids and percentages of solids and fat-free solids in the livers of tuberculous and non-tuberculous guinea pigs on different protein intakes . . . vii Body weights and liver weights for the guinea pigs . . . . . . . . . . . . . . ix Individual weights for nitrogen in the livers of tuberculous and non-tuberculous guinea pigs on different protein intakes xi Individual weights for sulfur in the livers of tuberculous and non-tuberculous guinea pigs on different protein intakes xiii Rates for glutathione reductase activity in the livers of tuberculous and non-tuberculous guinea pigs on different protein intakes . . . . . . . . . . . . xv Riboflavin in the livers of tuberculous and non-tuberculous guinea pigs on different protein intakes . . . . . . . xvii Table 1 Individual detenminations of the nucleotides of riboflavin in the livers of rats FMN Group Dietary FAD plus free Total casein riboflavin riboflavin % gay/g- ast- EJ8- I Low protein 8 16.6 2.8 19.4 18.5 5.4 21.9 19.9 5.7 25.6 19.1 5.0 22.1 lost lost lost 15.8 4.6 18.4 14.4 5.8 18.2 14.0 4.0 18.0 20.4 5.4 25.8 22.1 5.0 25.1 II Control 20 26.1 8.0 54.1 27.2 10.7 57.9 26.7 5.7 52.4 21.8 8.8 50.6 27.6 7.1 54.7 28.5 5.5 55.8 26.5 6.2 52.7 29.8 6.7 56.5 25.0 7.7 52.7 50.6 6.6 57.2 III Control: 20 50.1 7.8 57.9 ”11“de 3201 900 41-1 to low 51.1 5.5 56.6 protein 50.7 5.8 56.5 51.2 5.2 56.4 50.6 6.0 56.6 57.2 8.8 46.0 51.5 6.0 57.5 54.8 6.2 41.0 29.6 5.1 54.7 Table 11 Temperatures in the animal roams recorded over a weekly period 11 Housing for Housing for Date Hour tuberculous Hour control animals animals °F. °F. 2/25/57 8 AM 68 1 PM 72 12 N 80 5 m 74 6 m 82 10 PM 77 2/26/57 8 AM 72 6 AM 75 12 N 72 12 N 78 5 PM 74 6 PM 74 2/27/57 8 AM 70 6 AM 68 1 PM 74 12 N 74 5 m '74 6 PM 77 2/28/57 8 AM 66 6 AM 65 11 AM 66 12 N 75 6 PM 70 6 PM 78 5/1/57 8 AM 70 6 AM 68 1 m 66 12 N 76 5 m 70 6 PM 78 5/2/57 9 AM 70 6 AM 67 1 m 69 12 N 68 5 PM 76 6 m 68 5/5/57 8 AM 68 6 AM 62 12 N 68 111 Table iii ea aeoa oea eon ea eee noa eon ea eena era 6 ea aee «ea eaa ea «baa oea sen om eee eea baa ea eeea era 5 ea eae mea oaa ea eeea eea 8 cm eooa sea eaa e meea aoa eea ea aeoa «ea eoa ea eeea eee sea em neoa awe boa ea eeea eea eea om nee eea eoa ea neea eea eea we eeaa wee eoa ease 88 ea oee mea eon e wee ea aon ea pee eea ewe em aee eea eaa oa eeea eea nee am aee eea eaa e enna oe Nee om one eea eaa ea eeea eee aen ea nee eaa aaa ea eeoa nea «ma om see mea eoa ea aaea eam nea em aoe eae eoa ea eeaa aea mea me «me ene eoa ea eeoa eea aea am wee eea aoa whee oe dampenn pace non on wooa\ w m w wooam w w w soapsn campsa namm soaueu excusa qwmw -aaaee eeee sewaee aaesaea -aaaes eeee paeaee aeeaea doom II .11 wwom meoagononpb Honpsoo meMeuQa naeponm psoaemnau so mwaa wedadm enoaSOAeQSpneos one nsoaseaopSH no soauQESmnoo 800M use saew unmaes aespa>apsH HHH manna iv .eouspaeeese asonw nee mosae> seas “meaeeaa even n .haobapoedmea .oUOHAeaehepuew use now on» me when no use mm puma on» non cow was pcae caepoam name you mm can “sham aspneaanemxe NH penam emu pom soapsa naopoam name pom ma can pebaoooa massage Had m ODQDPCD ¢H bH omna Coma nwOH Nona mow ¢NOH mow mood mnOH mmmH mNOH me fimm web 00m wa .asoaw on» ea uawaase wnababhse can now mneoa on» one mesaeb esp manoaw can 86am wsameas maeEHGm on» psomenaea maeppoa aaesm a mad mm Hm boa nNH mbH omH mmm mmm «mm mm mm Hm bNOH ¢®OH mom mam war owed 0mm mwo How NDOH Ham Ham mnm who mmb bop mbm Cow 0H0 00b mad mOH and and mm HON mm mm Had omm mod #0 on Nod HON and mm an add wma ease en 6888 we seem adepoam hem mm Table iv e.m 08.0 eon 0.m 8n.0 eon m.e 88.0 e m.n 08.0 eaa e.m ee.0 8mm 0.8 e8.0 8aa m.e 88.0 e a.e ne.0 eaa m.e 88.0 a e.a ea.a eaa 8.m 88.0 mma 8.m me.0 eoa 0.8 ea.a 8ma m.m 0e.0 80a e.a ee.0 ema m.m ne.0 00a m.e me.0 ema e.m 88.0 eoa e880 8e e.m ee.0 men e.a m0.0 aoe e.m me.0 eme 0.m 08.0 eaa 0.m 0e.0 eme 0.m 00.0 eaa e.a ae.0 mmn a.m 88.0 maa e.a 08.0 ame 0.m 08.0 aaa 0.m ee.0 «ma a.m ee.0 «0a e.a me.0 nma e.m ee.0 noa e.a me.0 mma m.m ee.0 m0a 0.m 0e.0 ama m.m ne.0 aoa 8880 08 aaoponn goo pea .m .m 5 en pamae: se>aa pamae: mm>aa 0e pace pea eewaoe, aaesaee the pace gem .Awamaes aeaaee In 888.:‘ amen msoHsoaeebs Honmqov moxqua qweponm psoaokhau so swam eoaasm adoasoanSpnaoq was esoHSOHonsp Mo maobaa on» Ga new 80 memeusoenen use magmas? amswa>adsa 8a eaeea 53500018090 £800.89 haoubv 0:0 :08 one no 0.000 we use mm puma 0:» no.“ can 003 #36 530.3 pace pea mm 003 “when asuneaaaemxo ma p03.“ can 90.... £0309 530.3 .8080 .009 an one 025000." 30590 .338 N .Bponw 003 sa 305:0 msa>a>§0 on» no.“ 0.0.005 one 0.3 0250b 05 “macaw 23 Son.“ weaned: wage—”80 2.3 98800.50." 090.30..” aamfim a 0.0 80.0 000 0.0 a0.0 000 a.0 00.0 0mm 0.0 08.0 0a a.0 00.0 . 800 0.0 80.a 8a 0.0 80.0 000 0.0 a0.0 0a 0.0 08.0 000 8.0 a0.0 0a 0.8 08.0 00 0.8 08.0 0 0.8 a0.a 7. 80 0.8 00.a 8 8.0 80.0 00 0.8 00.0 0 e.a 08.0 00 0.0 00.0 0 0800 00 0.0 08.0 mom 0.0 00.0 aom 0.m 08.0 800 0.8 00.0 8a 8.0 00.0 000 a.0 a8.0 0a 0.0 88.0 mmm 8.8 a0.a ma 0.8 00.a e 0.0 80.a aa 8.8 e0.a 80 8.0 00.0 8 0.0 00.0 00 0.a 80.0 0 8.0 08.a mm 0.a 80.0 m 0.0 m0.a am a.8 a8.0 0 0800 0m sHouOHQ anew hem mm vii Table v a.mm a.m won m.mm 0.m non 0.8m 8.0 o 8.8m m.¢ maa 8.0m 0.m 8mm H.0m m.¢ baa 0.8m 8.0 a 8.80 0.0 0aa 0.8m 8.m 0 0.8m 0.8 maa 8.0m 0.8 mma m.om a.m moa 0.0m m.w bma 0.0m a.o boa 0.8m 0.m 0ma 0.0m 8.0 00a 8.00 0.0 0ma 0.80 0.0 00a 0880 88 0.00 w.m Non 0.0m 0.8 Hon 0.0m 0.m ¢mn b.8m m.¢ 8HH 0.00 0.0 0m0 0.0m 0.0 0aa m.mm 0.m man 8.0m «.8 maa 0.0m m.m awn 0.8m 0.m aaa H.0m 0.m 8ma m.mm n.m 80a «.8m m.8 mma 0.mm 0.m noa m.om 8.8 mma 8.0m a.8 moa a.0m 0.0 ama 0.0m e.8 a0a 0880 08 samponm pace non an am am pawaes ae>aa pander nebaa «0 0000 mma newaea aaesaee 00 eeee gem pamaee. assaea mmHHom 0wMHnm unedsonehpa Hpausoo noxepna saoponm psenokhau so awam mosasw 050HSoaenSpnsos ps0 0500508095» mo 0a0>aa esp ea meaaoe eenMupmm use 08aa00 mo memepseeaom 0:0 meaaom Mo enamaes 8 eanea viii .haepapoeauea .ueoanoa h08388 use :08 on» he whee mm 0:0 mm puma an» new com 003 peap caepoaa pace nod mm on» “whee aeuaefianoaxe ma puma“ 0gp no“ soapea saopona pace sea 00 can eebaeeea 0a08Hs0 aa< m .asonm on» ma madaasm wsa>a>950 can no“ 0:008 one one mesamp can “macaw on» Sony msaumaaneamaasm on» psomonmon weeppea Haeam a 0.80 8.8 000 a.8m 0.8 000 8.00 0.8 000 ‘ 0.00 , 0.8 0a a.8m 0.0 800 0.00 0.0 8a 8.00 0.0 000 0.00 0.8 0a 0.00 8.8 000 0.80 0.0 0a 0.80 0.8 00 0.8m 0.0 0 0.00 a.0 80 0.80 8.8 8 0.80 8.8 00 0.00 0.0 0 8.00 8.0 00 a.e0 8.0 0 8880 00 0.80 a.8 000 0.00 0.0 aom a.0m 0.8 800 0.00 a.0 8a 0.00 a.0 000 8.80 0.0 0a 8.80 0.0 000 0.00 0.0 0a a.8m 8.0 e 0.00 0.8 aa m.a0 0.0 80 8.00 0.0 8 0.00 8.8 00 8.80 0.0 0 0.00 8.0 00 8.80 a.0 0 0.00 0.0 a0 e.80 0.8 0 0800 00 saepoan i seem non mm Table vi 1:: 008 0.00 000 008 0.88 000 0080080 0080080 o 080 0.88 088 888 . 0.08 800 088 0.08 888 0080080 0080080 p 088 0.00 088 0080080 0080080 0 008 0.08 088 800 0.08 008 088 8.08 008 008 0.00 808 080 0.00 808 088 0.80 008 008 8.80 008 088 0.00 008 000 0.00 008 0800 88 088 0.08 000 000 0.08 800 808 0.80 800 808 0.00 888 088 8.80 000 008 0.08 088 080 0.80 000 008 8.08 088 000 0.80 800 888 0.00 888 000 0.80 808 008 8.08 808 088 0.00 008 808 0.08 008 008 0.00 008 080 0.00 008 008 0.80 808 008 0.08 808 0800 08 GHOponn pnoo you an .0 .0 .0 .0 000800 00080: 00080: 00080: 800808 00808 800808 800808 00888 800808 nfioafiononpa denunco mw8m moadaw onp pom 0p£m8os 80>88 0:0 0pn0803 800m H> manna NH 90888 on» pom £08808 nfioponm pcoo pom mm on» Uob80oom mamsaqd 88¢ 888 000 880 808 880 008 000 008 now $08 8H8 waamnaa New o>¢ «an .haopfipoomnon 00008808 hwdnbw 0:0 :08 can 80 0800 an dam mm #008 on» you 00% 003 8080 naouonm ammo 80m mm map 80800 prnoaanomxo N.mH 0.am 0.0m 0.5H 0.mH .m.nm O.©H a.8m N.mm b.wm n.nm mn8008E 0.mH $.md w.HN b.mm wmm bmm 0mm mum bm mm mm 800 mmm mmm o ¢N mm mm Hm i ..v w®¢ mmw mmw now $00 080 A 089 mma fion ¢b¢ O¢¢ 0N8 bow nmw Can 08* won 08¢ 80» mcumuda m.bH $.bH m.mH m.bH $.0H m¢.HN N.mH $.bH b.0H m.HN ¢.®H m.mH m.HH m.mm b.Hm 0.mH 0.m 0.0m w.om mn8008a .51 8&9 wow wow ma EH wH ma H H 0800 mm 0800 00 auoponm 9:00 non mm Table v11 xi n08 0.00 000 000 . _ 808 0.00 088 won 008 0.80 088 o 008 0.00 800 088 808 0.00 000 800 008 0.00 800 888 008 0.80 088 p 008 0000 .800 088 008 0.80 088 0 N88 «.80 .080 088 088 8.00 088 008 088 8.00 080 008 808 0.00 000 808 008 8.00 808 808 008 8.00 000 0N8 008 0.80 000 008 088 8.80 000 008 008 8.80 008 008 0800 88 mm 0.00 mum «on 008 0.00 008 800 008 8.80 080 800 008 0.00 800 888 088 8.00 8mm man 008 n.8n 000 088 008 0.00 000 mmn 8N8 0.00 080 N88 008 0.00 080 800 088 0.00 800 888 008 8.80 000 808 888 8.00 000 808 088 0.00 880 008 008 0.00 800 008 088 0.00 800 008 088 0.00 080 008 888 0.00 008 808 008 8.00 000 808 0800 08 0808080 pqoo 800 mm .08 .08 .08 .03 .03 .05 008800 050080 008800 05008» no 803 no 80909 8088s8 mo 803 no 80pofi 88038a 8080 new 808m 8mm. $080 80% B080 80% :0woppmz ccmopu8z 05085080935 H0880000 _ 0080pq8 0800080 800808880 :0 0080 005850 05085opon5pnaoc 0C0 05085080959 «0 080>88 can :8 20008985 808 0930803 80508>80q8 88> 08908 x11 .h80p8u00000n .0008noa h00u>¢ 0G0 n0¢ 00v Mo 0h00 00 000 mm p008 on» you 00% 00: p080 GaouOhQ 0:00 pom mm 030 Mnhmv 800noaanomxo m8 00980 030 now £08009 c80ponm 0:00 000 00 on» 00>8000h 0808H20 88¢ a 008 0N8 008 008 008 >m8 0N8 00 008 808 >88 ¢m8 >08 N08 0N8 ¢N8 .95090 000 08 0800830 wn8>8>h50 03¢ now 0:008 00v 090 00580> 8.80 N.00 >.0N 0.m0 0.m0 0.m0 $.00 $.0N 0.00 0.00 0.mm 0.0m 8.00 0.80 m.00 0.>N 000 000 000 000 080 0>> 000 080 000 880 0>0 000 0N0 m>0 00> ¢w> 0mm >Nm . 000 0mm >N 0m mm 008 008 0N8 008 N0 008 00 008 008 >08 8N8 >88 008 m08 008 008 008 008 >N8 0N8 N.00 «.00 0.00 0.00 0.>N 0.mm 0.0m $.om 0.00 m.80 0.00 8.00 0.00 0.mm >.80 0.00 0.m0 0.00 0.80 $.80 000 0>0 800 08> vm¢ 880 00¢ 080 N00 000 Now 000 >00 0¢0 000 «00 >00 000 000 >00 00m on» “@5090 03» Song 058008EH08058G0 on» psomonmon 0n09p08 88080 8 0h00 00 0000 00 anew 58oponn non 0m xiii Table V111 00 >.0 000 00 0.0 000 00 0.> o 00 0.0 088 00 0.> >00 00 0.0 >88 00 0.> n 00 0.0 088 00 0.> 0 00 0.0 088 00 0.0 008 00 0.0 008 00 >.> >08 «0 0.0 >08 00 0.0 008 00 0.0 008 00 >.0 008 >0 0.> 008 0000 >0 00 0.0 000 00 0.> 800 00 0.0 000 00 0.0 088 >0 0.0 000 00 0.0 088 00 0.0 000 00 8.0 088 00 >.0 800 80 0.> 888 00 0.0 008 00 0.0 008 00 8.> 008 80 0.0 008 00 8.> 008 00 0.> 008 00 0.0 808 >0 0.> 808 0000 00 n8oponm 0500 mom 00 .wfi .w5 .w5 .ma . 0000800 no 008800 no H0000 0000 000 800000 0080000 0000 000 803000 A5m85mx‘ n5mH5m 050HMMAonPb 8mmu000 00809q8 n80ponm p90900080 no 0089 000850 05085onop5pnson 0cm 0508500005» 00 000>88 00p :8 950850 00% 0p£m803 80508>8008 888> 08909 xiv .h80>8poogmon .mcoahon hwUn>¢ can n0¢ on» Mo mhmo mm and mm 9008 0:» gap 609 mm: 0086 q8oponm name you mm on» Muhmu Hmpnoaanomxo NH meHM ofip you nO8pwn GHOponm paoo hog mm can co>aooon mawaacw 884 m .Qfioaw on» :8 namaaqw wqfi>8>950 059 now names 080 mosamp may “macaw anuAaonm wn8008a mawaaaw on» anemonmon mnmppoa Hausa 8 v 00 _ >.> mom m0 «.0 000 0.> mma 00 o.> 08 0.0 >00 00 8.> >8 m.> 000 on «.0 08 o.> 000 80 0.0 08 m.> 00 00 0.0 m 0.0 >m 00 o.> > >.0 mm 00 0.0 0 0.0 mm 00 >.0 0 nhmu 00 00 0.0 mom 00 0.> 800 0.0 000 08 0.0 08 0.0 000 m0 >.0 08 m.> «mm 00 0.0 m8 0.> o 00 0.0 88 8.0 «m 08 0.0. 0 m.> 0m 00 0.0 0 m.> mm 00 «.0 m 0.> 8m 00 0.> 0 ”>00 00 mn80ponm anon non mm Table 1:: XV 0.0 00.0 00.0 won e.a mn.o 00.0 000 8.0 00.0 00.0 0 m.8 00.0 00.0 m88 0.0 00.0 00.0 >00 a.0 om.o 00.0 >88 8.0 00.0 00.0 n 0.0 00.0 00.0 088 8.0 00.0 00.0 0 0.0 00.0 00.0 088 0.0 00.0 00.0 mm8 0.0 00.0 00.0 m08 0.0 m0.o 00.0 008 a.0 88.0 08.0 >08 0.0 08.8 mm.o 008 0.0 00.0 00.0 008 0.8 00.0 00.0 008 0.0 >0.0 00.0 008 0000 >0 m.¢ 80.0 00.0 «on 0.08 00.0 80.8 800 8.0 00.0 00.0 000 0.0 00.0 mn.o 088 0.0 00.0 mm.o 000 0.0 00.0 00.0 088 0.0 mo.o 00.0 000 0.0 00.0 >m.o 088 0.0 00.0 00.0 800 0.0 mm.o 00.0 888 0.0 00.0 00.0 008 m.8 00.0 00.0 008 0.m 00.8 00.0 008 0.0 mo.o 00.0 008 0.8 08.0 08.0 008 8.8 08.0 08.0 008 0.8 00.0 08.0 808 0.0 00.0 00.0 808 0000 00 nHoponn 9:00 009 mm 008800 008800 ammonu8c 000 n 0 008H00_ 8058a< n0w0098n can u a 00880l .8wa8q< ho swam 00 opmn m ho 800m 00 opwn .0p8p8p00 ommpofiwmm, 0 .5808900 omeQ—bom _0 nsoHdononbE, Honmqobx noxprH a8oponm pnmnouh8u :0 anQ mon8dm 0308sononfiuuno: 0C0 05085000n59 mo mno>88 on» :8 8008>8000 ommpofivon 0:08nudnn8m you nopdm N8 o8nma xvi .h80>8poommoh .000800Q hmonbfi cam now on» mo 0h00 an 0:0 mm 9008 on» you 60% 003 H086 :8maonm ammo 00m mm 0nu «mhdu 8wpn088homxo N8 #0080 0ma hog :08pmn :80pOAm 9:00 009 mm 03» 60>80000 08058G0 88¢.n .gsoaw 00» n8 08088c0.mn8>8>050 on» non 0:003 000 00:80> on» “macaw 0gp Scan wc8008a_08038q0 0gp ua0000a00 0009908 880am m .00 0w0m no 00>8w 08 00808000 000005000 0n080u0ps8w wd8008no800 000 00800500 08009 one 8 __ 0.0 00.0 00.0 _ 000 0.8 00.0 00.0 800 0.0 00.8 >8.8 000 0.0 0>.0 00.0 08 o.> 00.8 >0.0 >00 0.8 00.0 00.0 >8 0.08 00.0 00.8 000 0.0 00.0 00.0 . 08 0.0 00.0 >0.0 000 0.0 00.0 00.0 08 0.0 00.0 00.0 00 o.> 0>.0 >0.0 0 8.0 00.0 00.0 >0 0.0 00.0 00.0 > 0.0 00.0 00.0 00 0.0 0>.0 00.0 0 0.0 >0.0 00.0 00 0.0 00.0 00.0 0 0000 00 0.8 08.0 08.0 000 0 .0.0 >0.0 >>.0 800 0.0 00.8 00.0 000 0.0 88.0 00.0 08 0.0 0>.0 00.0 000 0.8 80.0 08.0 08 8.0 00.0 00.0 000 0.0 00.0 00.0 08 0.0 80.0 00.0 o 0.8 00.0 08.0 88 >.0 00.8 0>.0 00 0.0 00.0 00.0 0 0.0 08.0 08.0 00 >.0 00.0 00.0 0 0.8 00.0 00.0 00 8.> 00.0 00.0 0 0.8 >0.0 00.0 80 >.0 00.0 00.0 0 0000 00 nn8ouoam 0000 90m mm , xvii Table 1: >0 >00 000 000 00 000 000 000 00 000 000 o 00 000 800 088 00 000 000 >00 0> >00 00> >88 00 000 000 n 00 000 000 088 00 000 000 0 00 000 >00 088 00 000 000 008 0> >80 800 008 0> 000 000 >08 00 00> 000 >08 >0 080 000 008 >> >00 00> 008 008 000 >0> 008 008 000 0>> 008 0000 >0 0> 0>0 000 000 0> 000 0>0 800 0> 000 0>0 000 00 000 000 088 0> 000 0>0 000 00 800 000 088 >0 000 000 000 00 000 000 088 00 080 00> 800 00 800 000 888 00 0>0 000 008 00 000 000 008 00 000 000 008 00 >0> 00> 008 00 08> 0>> 008 008 000 00> 008 00 0>0 000 808 00 0>0 000 808 0000 00 a80uonm 0a 0% cm 0% 0a ca ““00 9H0“ on mm 009% m «M 009M m 0:80 00m 0 8805800 0080 000.% . 0 803800 Ills?! 8000.8. 085 LEE EOHfichomWF Homflop Ap0>88 non 05090090850 00M0pa8 :80ponm paohohhfiu so 0w8m 00n85w 050850h09§p|qon 6cm 0508:000930 mo 000>88 0n» :8 s8>08hopam x 08000 xviii .h80p8000m00n .000890m h0©ab0 000 :00 on» no 0h00 mm 020 mm 0008 00¢ non 00% 00B 0080 aaopOAQ 9:00 non mm on» 80h0u 80psoaan0qxo N8 #0080 030 new :o8p0n 0809099 9000 00m 00 0gp 00>8000n 0803800 884 m 00580> 0gp “macaw 0n» Scam mn8008E 080BHG0 0n» paononmon 0009008 88080 Pb 80¢ 00 mwm No man no 8b¢ $0 m8n om mom mp mma N08 mm¢ wm 0&0 am Now 8w mmm mm mbw nb mma >08 80¢ 008 80m mm fiwm mom #80 >00 mam mmm mmm 00¢ 0&0 Q80 0mm 0mm mma mn¢ mom 00b bmo .gfionw 00D 08 080aan0 mn8babhdm on» non 0000aflon0 80¢ nm¢ 00¢ mam $00 8bm omv 08¢ con man mn¢ 00¢ 0mm ham nmm woo ©m8 wmm omw 06$ ‘ N80 00m «mm mmo 0mm haw 08m bflfi man how wm¢ 8mm man N80 m¢© mam bbm now 080 800 . 8 0000 00 0h00 mm anom adoponn 009 mm xix Table x (continued) 0.8 0.0m 0.0N 000 0.0 0.00 0.00 non 0.m 0.0N 8.0m o 0.0 >.0N >.mm 088 0.0 o.>m 0.00 ban 0.0 0.00 0.00 >88 0.m 0.0N 8.>N n 0.0 0.mm 0.0N 088 0.0 0.0N 8.0m 0 0.0 0.mm 0.00 088 0.0 0.0m N.on 0N8 0.m >.0m 0.00 008 0.m 0.08 N.NN >m8 0.0 0.00 0.00 >08 0.m 0.mm n.0m 0N8 N.n 0.0m N.mm 008 0.0 0.00 0.00 008 0.0 0.00 0.00 008 0000 00 0.0 8.0m 0.mm Non 0.0 0.08 b.0N 800 0.0 8.0m n.0m 0mm 0.m 8.0m 0.mm 088 0.0 8.0m 0.0m 0N0 N.N 0.mm 0.80 088 0.8 0.NN 0.0m NNn 0.0 N.0N 8.80 N88 0.N 0.08 8.08 8N0 0.m 0.mm 8.0m 888 0.m N.mm 0.0m 0N8 0.m 0.0N 0.mm 008 0.m 0.08 0.08 0N8 N.N 0.00 m.0n 008 0.m 8.0m 0.0N NN8 8.0 b.0m 0.mm N08 0.0 0.08 0.00 808 8.0 0.00 0.00 808 0000 00 n80ponm uaoo 90% mm .mm .mm .mm .mm .mm .mm mm 00mm N mm 00AM N 0080 000 0 8800000 0:80 000 0 8800000 anr .80909 ame .kuos mSO850n0nfifi 8oaunoo‘ p0>H8 00: mo swam p09 mswpmopofiev 00x :8 :80pOA pc0h0nu80 no 008g woadsw MSOHSOHopfiptflog Uflw mfiogOhmpdn. MO WHOPHH Gnu. 8H.“ Gdbwflfiopdm A005n8pnoov M 08909 GOGDOCOL‘OO OSCDCDOGJCOCOG) .h8ob8po09000 .muoanom hduub0 0n0 :00 000 no 0000 on 0G0 0N 9008 0:9 000 000 00: 9080 n8oaonm pa0o mom 0N 0nu 80000 809n08800QN0 N8 00089 05v 000 c08900 n80uomm 0:00 000 on 0gp 00>80o0n 08088q0 880 N .90000 050 G8 08088G0 mn8>8>0§0 on» non 0:008 000 00580» 0:» “@5000 0£p_fionw wa8mmdfi 08088G0 0n» pn000090n 0000908 88050 8 0.0 0 0.00 0.00 000 i 0.0 0.00 0.00 000 .0 0.00 0.00 000 0.0 0.00 0.00 08 .0 0.00 0.00 000 0.0 8.00 0.00 08 .0 8.08 0.08 000 0.0 0.08 0.00 08 .0 0.00 0.80 000 8.0 0.00 0.00 08 .0 8.00 8.00 00 0.0 0.00 0.00 0 .0 0.80 0.00 00 0.0 0.00 0.00 0 .0 0.00 8.00 00 0.0 0.00 0.00 0 .0 0.00 0.00 00 0.0 0.00 0.00 0 0000 00 0.0 0.00 8.00 mom 8.0 0.00 0.00 800 .0 0.08 0.00 000 0.0 0.00 0.00 08 .0 0.08 8.00 000 0.0 0.00 0.00 08 .0 0.00 0.00 000 0.0 0.00 0.00 08 .0 0.80 0.00 0 0.0 0.00 0.00 88 .0 8.00 8.00 00 0.0 0.00 0.00 0 .0 0.80 0.00 00 0.0 0.00 0.00 0 .0 0.00 0.00 00 0.0 0.80 0.00 0 .0 0.08 0.80 80 0.0 0.00 0.00 0 0000 00 Nn8oponn unoo 00m 0N Table 3: (continued) 0 888 000 08 008 000 00 008 o 88 008 088 008 088 000 08 008 088 00 008 p 08 088 088 00 008 0 00 008 088 00 088 008 08 088 008 00 00 008 08 808 008 008 088 008 08 008 008 00 008 008 08 008 008 0000 00 08 00 000 08 00 800 808 088 000 0 088 088 00 008 000 0 088 088 00 00 000 08 008 088 00 00 800 08 008 888 00 00 008 08 008 0O8 00 00 008 0 808 008 00 008 008 08 008 008 00 00 808 m8 0O8 808 0000 00 a8ouonm 9:00 mom on .mm .mm .mm .mm mm 0000 N 000 00 800800 0080H 0 8800800 H0000 .000 I 80090 05085 monnqoo 000 8008800 00>“ 00 500 L 0 mEdLUCLO.nEM moxdpGH n80p00m 9n0000080 so 0089 00m85m 05°85ono95punon 0G0 0508500095» mo 000>88 09p n8 q8>08mo98m 8005c89noov N 08905 xxii .080585005005 00008505 005300 550 now 05» Mo 0000 on 050 mm 5008 055 50% 505 003 5080 5805055 5500 505 mm on» “0000 8055058505N0 «8 50585 055 505 508505 5805055 5500 505 mm 05» 00580005 0808850 885 m .5505w 05» 58 0808850 w58>8>550 055 505 05005.050 00580> 05v “m5o5w 05v 5050 m58mm8a 0808850 05» 550005505 0505508 8808m 8 m08 ow N08 mO8 $88 588 088 mm 08 ON mm 98 08 88 M8 v88 mn8 m88 n88 or 088 008 mm 008 908 mm8 #08 ¢N8 mm 0&8 mm8 mm8 mn8 n08 Om8 mom 0000 00 0000 mm «5805055 5500 505 mm mom USE ONLY m mmmuwwm