“ii? LUBQI‘LAVEN :‘QQETMiOigfiM G? .v'{ in COLLEEGE WSGg‘fiEN ON 13‘ ‘s‘r’ REMJCY‘EON mg? ' 1:9"? ‘mx .3 _‘ 5 «3-.» r: ’3 fans _ 1‘2 ‘.\:!J'§h\i£-x-\‘. A: A .3 EM ....2 a ,- I ’i ”3‘“ a, ‘ " h. L . Kf< Li, : f'g". *- .’ ~ 7., ‘Lw ‘: i... 3 - 1‘" ." s. “‘6 wfb K‘ll-c _ I This is to certify that the L thesis entitled ’ “ The Riboflavin Metabolism of -2 ' College Women on a. Weight Reduction Diet. ? presented by f Irene Hwei-lin Chang . has been accepted towards fulfillment of the requirements for Ldegree kw — Major professor ‘1'- .- an ._ ‘Ih‘.-J7' 1.- w-M-u-u- THE RI BOFLAV IN BETABOLI STE OF COLLEGE. WOEEN ON A 'L‘J'EI GET REDUCTION DIitll1 BY Irene Hwei-lin Qhang A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of IZAST ER OI‘ SCI EKCE Department of Food and Nutrition School of Home Economics 1951 THESlS ACKIIO LE DG EEK T The writer wishes to express her sincere thanks to Dr. Wilma D. Brewer, under whose con- stant supervision, and unfailing interest this investigation was undertaken and to whom the results are herewith dedicated. The writer also deeply appreciates the material and financial supports of the Food and Nutrition Department, School of Home Economics at Michigan State College,'which made it possible to complete this investigation. *********¥ *at#**** *t:#** **** ** t TABLE OF CONTENTS IIITRODTTCTIONOOCOO0.00.00.00.00.OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOIOOOO PLETIIE-ff OF LITE‘LATTRECOOIOOOOOOOOOOOOOO...OOOOOOOOOO...0.0.0.000... Riboflavin Metabolism.of College'Women....................... Influence of Diet High in Fat on Riboflavin metabolism....... lqethOdS Of Determination Of RibOflaVineeeeeeoeeoeeeeeeeeeeeoo 3} JFlr-EJ‘TITAL PllOCJiUU—REeeeeeeeeeeeeoeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeo Subjects..................................................... Experimental P1an............................................ flxperimental Diet............................................ Sampling of FoOd and Urine for Riboflavin Determinations..... Urinary Excretion of Riboflavin Following a Test Dose of RibOflflVineeeeeeoeeeeeoeeeoeoeeeeeoeeeeeoeeeeeeoeeeeeeeeeee Chemical PiethOdSOOOOOOOOOOOOOOOOOQQO...OOOOOOOOOOOOOOOOOOOOOO ElmULT MID DISCTJSSIOI‘I....O.......00......00.0..................... Riboflavin metabolism.of Overweight College women on Self- SeleCted DietSeeeeeeeooeeeoeeeeoeeeeeeeeeoeeeeeeeeeeoeeeeee Riboflavin Metabolism of Seven Overweight‘women on weight RedUCtion DietSOOOOeeeeoeeeeeeoeeeeeeeeeeeeeeeeeOeeeeeeeeeo Riboflavin.flbtabolism.of Three Subjects on Weight Reduction and Wbiflht Maintenanceooo.c..o.......o..................... The Variation of Riboflavin Content of Seven Day Diets....... STI‘E‘IARY MID COI\TCLYTSIOI\IOOO0.0.0.....0...O...OOOOOOOOOOOOOOOOOOOOOOO REFERETICES CITEI‘DCOOOOOOO...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. PACE 1 Coco»: 15 13 13 14 14 16 17 19 21 24 29 52 34 36 LIST OF TARLBS AND FIGURES TABLES PAGE I Typical Day's Diet for the Self-selected Diet Period and For the Visight RedUCtion Dieto.oo...o.o....o......o....... II Height, Weight and Age of the Nine Overweight College women. III The Intake and Excretion of Riboflavin by Seven Overweight College women on Self-selected Diets...................... IV The Average Intake and Excretion of Riboflavin By Overweight College‘women on Self-selected Diets and During Twelve lnlfeelfs Of Weight RedUCtionooeeeeeeeeeeeeeeeee.oeeeeeeeeeeee V The Average Intake and Excretion of Riboflavin of Three Sub- jects on Weight Reduction Diets and Weight maintenance DietSOOOOIOOOOOOOOOOOOOO0.00...OOOOOOOOOOOOOOIOOOOOO00.... VI The Riboflavin Content of Seven Day Composites of a Control DietOOOOOOOOOOOOOOOOOOOO0.000.....0OOOOOOOOOOOOOOOOOOOOOCO FIGURES l The Average Daily Intake and Urinary Excretion of Riboflavin of Seven Overweight College women on Self-selected Diets and During Twelve‘weeks on a Weight Reduction Diet........ 2 The Average Daily Intake and the Urinary Excretion of Ribo- flavin of Three Subjects for Ten'Weeks on a'Weight Reduc- ‘I tion Diet and Twelve'Ieeks on'fieight fiaintenance Diet..... 15 20 22 25 30 33 27 31 INTRODUCT ION INTRODUCTION Riboflavin has long been recognized as an essential nutrient in animal and human nutrition. The influence of riboflavin on growth was demonstrated among others by Bourquin and Sherman (1951), Clarke et a1 (1940) and MacLeod and Taylor (1944) who reported that the growth rate of animals was directly dependent upon the riboflavin intake. The slowing of the growth rate which resulted from a suboptimal intake of riboflavin was accompanied by a cor- responding retardation of general development. If shortage of riboflavin in the food supply was continued throughout the period of growth, the de- velopment of the individual remained permanently belOW'hlS native potenti- ality. Other studies which have demonstrated the physiological functions of riboflavin include those of Oden and Sebrell (1959), and Kruse et al (1940) who reported that riboflavin influenced the condition of the skin, mouth, and eyes, and Haas (1940) and Krebs (1935) who demonstrated that riboflavin took part in enzyme systems which regulated cellular oxidation. Riboflavin also may be related to some extent to fat metabolism. Tange (1941), fiannering (1941) and Czacjkes (1946) reported that increasing amounts of fat in the diet apparently increased the riboflavin requirement of rats. Obesity is considered to be a condition in.which an abnormally large amount of adipose tissue is present. Since riboflavin has been demonstrated to have a physiological function in cellular oxidation and since there has been some indication that riboflavin and fat metabolism may be interrelated, the possibility exists that riboflavin metabolisnlmay be disturbed in obesity. In the present study, the riboflavin metabolism of overweight college women on a weight reduction diet which was relatively high in fat and protein has been investigated. if} .T 1 RIBOFLAVIN NETABOLISM OF COLLEGE WO‘ One of the earliest attempts to evaluate the riboflavin needs of women was reported by Strong (1941) who found that the twenty-four hour urinary riboflavin excretions for four young college women on unrestricted diets ranged from 500 to 800 micrograms on a daily intake of one to two milli- grams of riboflavin. Strong concluded that an intake of one to two milli- grams of riboflavin daily was no more than marginal and perhaps inadequate to supply the daily requirement. Sebrell (1941) studied the metabolism of ten women on a basal diet containing 0.5 milligrams of riboflavin per 2400 calories. After 89 to 232 days on this diet, six out of the ten subjects developed symptoms of riboflavin deficiencies which were cured by adding additional riboflavin to the diet. Sebrell concluded that the intake of 0.05 milligrams of ribo- flavin per kilogram of body weight or three milligrams per day exceeded the requirement of the adult women and that an intake of 0.025 milligram per kilogram was insufficient. The average daily urinary excretion of these subjects was 77 micrograms. Williams (1943) reported that when the intake of riboflavin was 0.8 milligrams per 1000 calories, there was no evidence of depletion of tissue stores, whereas an intake of 0.35 milligrams per 1000 calories produced depletion of the tissues. Williams concluded that an intake of 0.5 milli- gram per 1000 calories appeared to be the minimal daily requirement for riboflavin. This worker suggested that the riboflavin requirement of women was not less than 0.5 milligram and not more than 0.8 milligram per 1000 calories, or a total of 1.0 to 1.6 milligram per day, and considered that the recommendation of 2.2 milligrams per day which was made by the Food and Nutrition Board of the National Research Council (1943) for moderately active women provided a liberal margin of safety. Davis (1946) studied 12 young women between the ages of 19 and 32 years on diets in which the amount of riboflavin progressively increased for eight months, and estimated that the riboflavin requirement of young women was between 0.49 and 0.66 milligram per 1000 calories. The average daily urinary excretion of these subjects during the preliminary period on self-selected diet was 433 micrograms. Brewer et a1 (1946) compared data reported in the literature with observations on young college women and reported that dietary intakes of 1.3 to 1.5 nfilligrams riboflavin appeared to represent the upper limit of economical utilization of this vitamin when the caloric intake was from 2100 to 2300 calories per day. A similar estimate of the riboflavin requirement of adults was reported by Horwitt (1949) whose studies indicated that the daily requirement was between 1.1 and 1.6 milligrams daily. This estimation of the riboflavin requirement approximated the recommended intake of 1.5 milligrams riboflavin for women which was reported in the last revision of recommended daily allowances of the Food and Nutrition Board of the National Research Council in 1948. Harris (1949) found that the daily riboflavin excretion of young women on self-selected diets ranged from 463 to 1157 milligrams. A similar range was reported by Ingalls (1945) and Everson (1948). The use of the 24 hour urinary excretion of riboflavin to study the nutritional status of an individual has been questioned by Najjar (1941) and Keys (1945). These workers found that it was not possible to define a limit of twenty-four hour riboflavin excretion which was characteristic of riboflavin deficiency, since the urinary riboflavin varied with the immedi- ate intake and therefore did not indicate the state of the body stores of this vitamin. Additional factors which should be considered when the urinary excretion of riboflavin is used as an index to the metabolism of the vitamin include possible intestinal synthesis Najjar, 1944; Keys, 1945; Hathaway, 1946), tissue destruction (Sure and Ford, 1943), poor absorption and impaired kidney function (Seyle, 1943; mannering, Orsini and Elvehjem, 1944; ana, 1944), and interrelationships with other nutrients (Ferrebee, 1945 and Sure and Ford, 1942). The urinary excretion of riboflavin following a test dose has been considered closely related to the previous intake of this vitamin and an index to the nutritional status of the individual with respect to riboflavin by Najjar and Holt (1941), Williams (1943), Parsons (1944) and Copping (1945). The quantity of the test dose administered, the path of adminis- tration and the length of time of urinary collection after administration of the test dose have varied among workers. A test dose of one milligram of riboflavin in distilled water injected intravenously was used by Najjar and Holt (1941). These workers reported that individuals who had had an adequate dietary intake of riboflavin re- tained 32 to 72 per cent of the test dose at the end of a four hour period, while riboflavin-deficient individuals retained 81 to 93 per cent. In this study the excretion of test dose appeared to vary inversely with the weight of the subject. Najjar and Holt suggested that if the test dose had been based on the weight of the subject, the amount of variation observed for the group of normal subjects could be considerably reduced. Williams (1943) suggested that a test dose of one milligram.of sodium riboflavin injected subcutaneously would be a satisfactory test does for the adult whose minimal requirement varied between 1.5 and three milligrams of riboflavin. In studies with children, Oldham et a1 (1944) used a test dose of 75 micrograms per kilogram.body weight and found that the excretion of 20 per cent of the test dose in a four hour urine sample or the presence of nine milligrams of riboflavin in an one hour fasting urinary sample showed satisfactory nutritional status. Oldham indicated that the urinary ribo- flavin in the one hour fasting sample and four hour and 24 hour periods following the test dose could be used equally well in determining the nutri- tional status “nth respect to riboflavin and thiamine. Brewer (1946) also found that there was a significant relationship between the one hour fast- ing excretion and the twenty hour urinary excretion of riboflavin after a test dose of three milligrams which was given orally. 0n the other hand, Berryman and French (1947) reported that the load test response was more significant than the fasting urinary excretion in the appraisal of nutri- tional status and Ingalls (1945) reported that the one hour fasting ex- cretion of riboflavin was a satisfactory measurement of riboflavin.nutri- tion when a group was studied but inadequate for evaluation of a single individual. Feder, Lewis and.Alden (1944) used a test dose of 0.016 milligram of riboflavin per kilogram of body weight given intramuscularly and reported that an excretion of less than 35 per cent of the test dose indicated a de- ficiency state and that the saturation dose seldom gave more information than could be obtained by analysis of a fasting hour specimen. The administration of several combinations of water'soluble'vitamins has been studied by Helnick (1945) who used a test dose of 10 milligrams of riboflavin and showed that extra urinary excretions of the water soluble vitamins were the same regardless of whether the vitamin was taken alone or in various Combinations. This fact also has been reported by Ingalls (1945) and Johnson and Robinson (1945). Johnson and Robinson (1945) observed that increased urinary excretion following an intravenous dose of riboflavin and ascorbic acid was no longer apparent after four hours and that the excretion of oral doses of these vitamins occurred in 12.6 and eight hours respectively. These workers sug- gested that the collection of urine for one hour after an intravenous dose and during the second hour after an oral dose might yield results'directly comparable with those obtained after collection periods of four to ten hours. Davis (1946) used a test dose of 0.02 ndlligram.of riboflavin per kilo- gram body weight and observed that the values for excretion per hour were more constant than the value for excretion per unit volume contrary to the finding of Hathaway (1946) and Feder (1944). INFLUENCE OF DIET HIGH IN FAT ON RIBOFLAVIN METABOLISH The literature concerning the effect of fat in the diet on riboflavin metabolism is controversial. Guerrant and Dutcher (1934) found that the fat content of the diet of albino rats did not bear any relation to the rats' requirement for vitamin G, and when restricted daily supplements of both vitamin B and G were fed to rats, greater growth rates were obtained for animals which received diets containing from 15 to 20 per cent of fat than for similar animals which received diets of IOW'fat content. Nhnnering, Lipton and Elvehjem (1941) fed diets containing 25 to 40 per cent of fat to growing rats and reported that the riboflavin require- ment increased when.the fat level in the diet was increased. In these experiments fat isocalorically replaced dextrin. Tango (1941) indicated that young rats which were fed a diet contain- ing 25 per cent fat over a period of seven weeks showed little difference in growth from.those fed a diet containing only five per cent fat when both diets were supplemented with 50 micrograms riboflavin. However when the riboflavin supplement was limited to less than 15 micrograms daily, the rats on the high fat diet showed impairment of growth after 12 days and a condition of the fur which resembled seborrhea. Additional intake of riboflavin cured these symptoms. Potter, Axelrod and Elvehjem (1942) reported that the isocaloric sub- stitution of lard for sucrose did not increase the riboflavin requirement of the growing dog and therefore considered that the riboflavin requirement of the dog was not related to the fat content of the diet. lhnnering, Orsini and Elvehjem (1944) reported that the growth of rats which received a diet containing seven per cent fat was superior to that of rats which received a diet containing 40 per cent fat. Further- more the high fat diet produced spastic paralysis and decreased the sur- vival time of the riboflavin deficient rats. These workers considered that the effect was due to the influence of fat on decreasing the intesti- nal synthesis of riboflavin. Czaczkes and Guggenheim (1946) reported that rats kept on high protein and high fat diets needed at least twice as much riboflavin as rats kept on the normal diet for the maintenance of the same concentration of ribo- flavin in the organs and urine; the high fat diets apparently diminished the number of viable bacteria in the flora. These workers confirmed the finding of lbnnering et a1 (1944) that fat decreased the intestinal synthe- sis of riboflavin. Reiser and Pearson (1949) extended studies of this nature to the avian species. Pearson reported that lard did not retard the rate of growth of chicks nor did a commercial hydrogenated vegetable fat. When 20 per cent cottonseed oil was used instead of lard in the diet, the chicks made less efficient gains than those chicks on the diets with no added fat. Here again, the author suggested that the unsaturated acids in the cottonseed oil interfered with the intestinal synthesis of riboflavin. These studies would appear to indicate that the riboflavin require- ment of animals may be increased by a diet which is relatively high in fat content. No studies of the interrelationship of fat and riboflavin in the metabolism of humans have been found in the available literature. 10 LETHODS OF DETERMINE ION OF RIBOFLAVIN Riboflavin is determined both biologically and chemically. Biologi- cal methods for the determination of riboflavin have been based upon measurements of the effects of small quantities of the vitamin on the growth of rats or chicks or micro-organisms. In this connection, the bacteriological techniques of Snell and Strong (1937, 1939) and the rat growth procedure of Bourquin and Sherman (1931) have been quite extensively used. Chemical methods of determination of riboflavin have been based upon measurements of the light absorption of riboflavin or of the fluorescence exhibited by riboflavin in solution. Kuhn and Kaschars (1935) proposed a method based on measurement of the light absorption of the vitamin extract. The intensity of the color was measured either by a step photometer or by a polarograph. Kemmerer (1940) and Lingane (1941) have used this method to determine the amount of riboflavin in foodstuffs. The fluorometric method for determination of riboflavin has been the most commonly used method for determining the amount of riboflavin in urine, foodstuffs and other biological materials. This method is based upon the observation that riboflavin in solution possesses yellow green fluorescence as a result of excitation by light of certain wave lengths. The accuracy of this method depends upon the complete removal of certain interfering substances so that the fluorescence measured will be only that of riboflavin. Adsorption procedures have been suggested by many workers to remove the interfering pigments in urine and foodstuffs. Emmett (1917), Harayanan 11 and Drummond (1930) indicated that fuller earth absorbed the B vitamins. Neuwieler and Bierry (1936) adsorbed riboflavin in finally divided food- stuffs on fuller's earth. Ferrebee (1940) introduced two fuller's earth preparations, Florisil, and Supersorb, for the adsorption of riboflavin in urine. Other workers as Conner and Straub (1941) and Keys (1944) have employed this procedure to extract riboflavin from urine and foodstuffs. Some pigments in normal urine and foodstuffs have been found to interfere with the determination of riboflavin. Hodson and Norris (1939) used stannous chloride to reduce the interfering pigments, and Sure and Ford (1942) oxidized the interfering pigments with potassium.permanganate and destroyed the excess potassium.permanganate with hydrogen peroxide. A sample blank has been.used by many workers to eliminate the errors due to interfering substances which were not removed by adsorption and by oxidation. Najjar (1941) and Keys (1944) prepared blanks by exposure of the samples to ultra violet light or direct sunlight for one to two hours; this brought about photolytic destruction of riboflavin. Sure and Ford (1942) obtained sample blanks by adding sodium hydrosulfite to the sample to reduce the riboflavin to a non-fluorescent leuco form after the reading had been taken. Morell (1946) reported that urine contained not only apparent riboflavin but also precursors of apparent riboflavin which could be destroyed by stannous chloride reduction, by potassium.permanganate oxidation or during the process of adsorption on florisil. Morell used an internal blank to compensate for interfering substances. The use of acid hydrolysis and of enzymatic hydrolysis to liberate combined riboflavin in foodstuffs has been reported by some workers. 12 Conner and Straub (1941) recommended that samples should be incubated with clarase at a temperature of 45° C. for 24 hours. McLaren and Pearson (1944) extracted riboflavin from meat with a combination of papain and takadiastase enzymes at a pH of 4.0 for two hours. Klocke (1947) reported that enzymatic digestion with polidase -s for 16 hours at 45° c. was con- sidered adequate and practical for extraction of riboflavin from tissues. EKPB. TEN’I‘AL PROCEDURE 13 EEiPERIlETTAL PROCEDURE Subjects The subjects were overweight college women who acted as subjects for a research project in the Foods and Nutrition Department of the School of Home Economics at Michigan State College. This research was concerned with the utilization of calories and protein by overweight college women on a weight reduction diet. The subjects carried full college programs and en- gaged in the usual activities of the school. Physical examinations at the beginning of the study indicated that the subjects were healthy except for the condition of excess weight. The ages of the subjects varied from 18 to 23 years. Experimental Plan The study was begun in September, 1950 with two subjects, Sr. and Pe., who were given a weight reduction diet and continued on this diet, with the exception of the official school vacation period, until March 1951. In march, the subjects reached the desired weight for the body build and were given diets planned to maintain.their body weight. In January 1951, seven.subjects were added to the study. During the first two weeks, these subjects were permitted to eat without restriction from.diets typical of this region. All servings were weighed and served in the small apartment located in the Home Economics building. Food and urine collections were made during the second week of this period. After the preliminary period, the subjects were given a weight reduction diet and were continued on the diet until satisfactory loss of weight resulted or 14 until the end of the school year. One of these subjects'Wo reached the desired weight for her body build after 10 weeks of weight reduction and was given a diet planned to maintain a body weight. One subject, Se, left the experiment after four weeks on the weight reduction diet. Every third week on the weight reduction diet, seven day composites of food were analyzed for the riboflavin content and the riboflavin con- tent of the urine was determined. Expe rimental Diet The diet supplied approximately 1500 calories and 100 grams of protein daily. The calories of the diet were supplied chiefly by protein and fat. A typical day's diet for the preliminary period when.the subjects ate with- out restriction in.amounts of food and a typical day's diet during'weight reduction are given in Table I. Seven menus were planned for the weight reduction diet, and these menus were repeated each week. All foods eaten were weighed on a Hansen dietetics scale. After eight weeks on the weight reduction diet, one subject, So, was given reduced quantities of food in order to increase the weight losses for this subject. The reduction in the diet was approximately 10 per cent. However, on the lowered intake the rate of weight loss for the subject appeared to be not more than desirable, so she was returned to the same food intake as the rest of the group received. Sampling of Food and Urine for Riboflavin Determinations Aliquots of one-fifth of the serving of food were weighed on a trip balance and frozen after each meal. At the end of the seven day period, a TYPICAL DAY'S DIET FOR THE SELF-SELECTED DIET PERIOD TABLE I AND FOR THE WEIGHT REDUCTION DIET weight Reduction Diet 15 Self-selected Diet Food Amount Gm. Grape fruit section Orange juice 100 Egg, poached Eggs 100 Bread, whole wheat Butter 20 Nfilk Bread 20 Jelly Lamb patties 125 Coffee Pears, canned 100 Cream Lettuce 15 Bread Roast veal 125 Butter Peas 75 Cream of tomato soup Applesauce 100 Saltines 450 Lettuce Pears, canned Cookies Bread Butter Milk Steak, cubed Potatoes, boiled Green beans, buttered Lettuce Tomatoes Cottage cheese Bread Butter Milk Apple crisp Milk composite was made by blending the aliquots together in a Waring blender and making to a volume of 2000 milliliters. Aliquots were taken for ribo- flavin analysis. During the first three month period, the food intake for each week was determined; following this, only food samples of the self- selection period and balance periods were analyzed. Aliquots which could not be analyzed immediately after the composites were made were stored in brown bottles and frozen. Daily twenty-four hour urine collections were made during the balance period. The urine was collected in a brown glass bottle containing three milliliters of glacial acetic acid as a preservative and kept in the refrigerator during the period of collection. When the twenty-four hour collection was completed, the urine was transferred to a two liter glass stopped graduate cylinder and thoroughly mixed. The urine was then diluted to a volume corresponding to the nearest 500 milliliters with distilled water. The creatinine of the urine was determined daily as a check of the accuracy of the completed collection of the twenty-four hour period. One fifth of the daily sample was transferred to a three liter brown glass bottle and stored in the refrigerator. An analysis of riboflavin was made on composites of urine from the first three days, the second three days and the last day of the balance period. Urinary Excretion of Riboflavin Following a Test Dose of Riboflavin On the morning of the seventh day of the balance period, a test dose containing three milligrams of riboflavin was given orally. The urine excreted during the following 24 hours was collected in a brown glass bottle 17 containing three mdlliliters of glacial acetic acid as a preservative. Analysis of riboflavin was made. The percentage increment in riboflavin excretion of the 24 hours urine collected after the test dose over the average daily excretion of the six days preceding the test dose was calcu- lated to give the percentage excretion of riboflavin of the test dose. Chemical Methods Aliquots of food were acid hydrolyzed for one hour on a steam bath, then adjusted to pH 4.5 wdih.sodium.hydroxide using nitrazine paper as an indicator and incubated over night with Polidase S-enzyme (Klocke, 1947). The riboflavin content was determined by the Conner and Straub (1941) modification of the fluorometric method. This included adsorption of ribo- flavin on activated florisil, elution of the adsorbed riboflavin with 20 per cent pyridine-acetic acid solution and subsequent oxidation.with po- tassium.permanganate with the excess permanganate decolorized by treatment with hydrogen peroxide. The fluorescence of the riboflavin in solution 'was measured in a Coleman photofluorometer against a standard solution containing 0.1 milligram.of riboflavin per milliliter. The riboflavin content of urine was determined by the same procedure as modified by Keys (1944) and Demerre and Brown (1944). Corrections for interfering substances were made by using a separate blank for each sample. The sample blanks were obtained by photolytic destruction of riboflavin by exposure of urine aliquots under ultra violet light for two hours. After the second month of the experiment there was a failure of the mercury lamp and for the rest of the period, sample blanks were obtained by destruction 18 of riboflavin by sodium hydrosulfite which was added to the sample after the initial measurement of fluorescence was made (Assoc. of Vitamin Chemists, 1947). RESULT AND DISCUSSION 19 RESULT AND DISCUSSION The description of the subjects who participated in the weight re- duction study is given in Table II. The subjects ranged from 18 to 23 years old and were from seven to 51 per cent overweight. After ten weeks on the weight reduction diet, one subject, we, reached her desired weight and after 12 weeks two more subjects Sr and Po reached their desired weights also. The subjects were considered healthy; however, Go had received thyroid therapy for several years preceding the study. Obesity is considered to be a condition in which an abnormally large amount of adipose tissue is present. The condition of overweight is con- sidered to be the result of an intake of energy which exceeds the output and this disproportion results from an abnormal appetite (Hewburgh 1943). According to Newburgh a decrease of the calorie intake of obese individuals below their metabolic needs causes a burning of body tissues which results in loss of weight. The caloric intake of these overweight college women during the weight reduction diet was approximately 1500 calories whicn was lower than the 2000 calories recommended by the Food and Nutrition Board of the National Research Council in 1948 for women of moderate activity. A satisfactory weight loss of these subjects on this reduced calorie diet should be expected. The average weekly weight loss during this experimental period ranged from.0.4 to 1.4 kilogram.with an average of one kilogram.per week. It may be considered that these subjects lost weight at a satisfactory rate. However, the weights of some subjects highly exceeded their desirable HEIGHT, YaIGHT AND AGE OF THE NINE OVERWEIGHT COLLSGE W ”EH TABLE II 20 '— Subject Age Height Desirablel Weight at the Weight at the'Weight (Yr) (cm) Weight beginning of end of the change/ (kg) the Experiment Experiment week (kg) (ks) (ks/5k) G0 18 176.1 65.9 83.6 73.5 0.8 Ha 18 161.0 59.0 89.3 74.5 1.2 JO 19 168.9 68.1 83.4 72.5 0.9 39 22 176.5 72.7 78.5 73.2 1.4 SO 22 156.0 54.5 59.2 54.6 0.4 Va 18 166.0 65.9 84.6 70.9 1.1 W0 18 168.0 65.9 79.6 65.9 1.4 P6 22 155.0 54.5 60.3 51.5 0.7 Sr 23 166.2 61.3 73.4 63.8 0.8 1 Desirable weight for body build predicted from anthropometric measurements by Dr. Hargaret A. Ohlson. 21 weights at the beginning of the experiment; therefore at the end of the experiment two subjects Ida and Go still were 15 kilograms and eight kilo- grams overweight respectively and two other subjects Va and Jo were five kilograms overweight. Riboflavin Metabolism of Overweight College Women On Self-selected Diets The average intake of riboflavin of these subjects during the period in which the subjects selected their food from the diet table without re- strictions in amounts are given in Table III. The urinary exoretions of riboflavin during the self-selected diet period and the urinary excretion of riboflavin during the twenty-four hour period following the administra- tion of a test dose of three milligrams of riboflavin are also given. The percentage of test dose excreted represented the increnent in the urinary excretion of riboflavin during the twenty-four hour period following the test dose over the average daily excretion of riboflavin preceding the test dose. The average intake of riboflavin during the period of self- selected diet ranged from 1.58 to 2.06 milligrams per day, and the average for the seven subjects was 1.76 milligrams. The riboflavin intakes of all subjects exceeded the daily allowance of 1.5 milligrams per day which was recommended by the Food and Nutrition Board of the National Research Council (1948). I The average daily urinary excretion of the seven subjects ranged from 0.59 to 0.94 milligram per twenty-four hours, with an average daily excretion of 0.74 milligram. This range is slightly higher than the values of 0.5 to 0.8 milligrams per day which were reported by Strong (1941) for four average young women on unrestricted diets and within the range of 22 mm.o Hm.o sH.m mH.o mH.o e.s m.me .m .m H.me-m.em mm.a-ms.o H.em-H.mm em.o-en.o oo.m-mm.H H.ms-os docuuoesfi ownem m.mm no.0 s.~e es.o os.H N.es Neefi oweaoee e.sn NH.H H.om em.o eo.~ ~.as cmefi on e.em ms.o e.mn mm.o ms.H H.os OOmH on 0.0m ow.o m.ne mm.o cm.H m.ms mama om H.me mN.H H.se ea.o mm.H .o.es eOmH om ”.mm oo.H o.me Om.o mo.H o.o> oesfl oe N.em Na.o H.Nw no.0 no.m H.ms osHm em H.en No.H H.mm we.o ms.H m.ms soon on an: emv Ann em\.esv ooponoxm soon pose oxenaH Ase em\.env Ase ea\eswv Adm em nonv ewon emoa uovme M0 weaponoxm onan oMean oxdan poonnsm mo envenom nonpouoxm pgeouom madman: manna nfibeamonwm nfiopoam cauoaeo mwbefimonfim mo :ofipopoxm amenaab mHHHQ QMBomAmMImAmm zo zmfioa.momqqoo BmemBmeo zm>mw Mm szddmOmHm mo ZOHHmmoMm 92¢ mM¢EZH HMH HHH mqmumaa wszDD 924 memHQ omeomnmwlmqmm zo .fiHmQB mmmfldoo emem§4m>o Mm ZHbmqmomHm mo onHmmme QH< mm<9momHm n5 ZOHEmUNm 9,2 mfiE/HH magmas. mm; 31 .x m3 Mute tmxxs‘og x .EQR Q6xk3w§$k ximxmi Q to 33$: Q\ k0\. C0 33W); N\ “Q0 Kw?» wxuflwae) m. \Q kibbxcwmvk \Q QQX mCuXm. hkektb mot bee mktxtx mbe mmukmifi «ERR N MWQWh‘ Nuzvémfi >\\ V? klbxWi gun‘s Nx stem} Q\ «heme: N 5800f! 192 N/ 35‘00 .1 $3.1 50 N 0/1 33%.? JNJDa’Jd Ln N O b. 20xkODQMQ ktmvxwg “tum! Nx whom} Q\ “to?! .0. when? N \ L r L MHQQKMMK 96h \HMN I £2 t\\%&\\ \e “\QQMKURM \«ksktb \, max§\§\ £\\.§\\c .Nxmx \ h\\\\\\\\\\\\ 5300/! f? 330’ 9A! N/ NM Wjogla’ :10 NOUJHJXJ mW becz N/ 32 The Variation of Riboflavin Content of Seven Day Diets The riboflavin contents of the food samples collected during this experiment is shown in Table VI. The average amount of riboflavin in the diets used from September to December 1950 ranged from 2.10 to 2.34 nfllligrams riboflavin per day with an average of 2.16 milligrams per day. The average amount of riboflavin in the diets used from January to June 1951 ranged from 1.53 to 1.78 milligrams riboflavin per day with an aver- age of 1.64 milligrams riboflavin per day. Since the diets used in the above two periods were the same within each period, the small differences in riboflavin content of the seven day composites represent the variation which may be expected from week to week for a constant diet. This varia- tion includes the variability in food, the effect of cooking on the ribo- flavin content of foods, the sampling errors in preparing the food comp posites and the technical errors in riboflavin analysis. The diets used during the two periodS'were planned to provide the same intake of calorie and other nutrients. But at the beginning of January 1951, the diet had been modified slightly in selection of vegetables and.fruits; moreover, fewer fresh citrus fruits were used during the period froananuary to June 1951. These may have accounted for the lowered ribo- flavin intake during the months from.January to June 1951, than the months from September to December 1950. It is possible that the decrease also reflects a seasonal variation in riboflavin content of foods. TADLE VI THE RIBOFIAVII‘T COT‘TTEI'TT OF SEVEN DAY CO'POSITSS OF A CONTROL DIET 'Height Reduction Diet Riboflavin Content of Diet ( Ins/day) [September to December 1950 Sample 1 2.11 2 2.34 5 2.22 4 2.10 5 2.10 6 - 2.10 Average 2.1641: 0.10 Range 2.10-2.34 January to June 1951 Sample 1 1.53 2 ' 1.78 3 1.63 4 1.62 Average 1.64 i—O.10 Range 1053-1078 SUT 13' “.Y AID COITCLITSIO’i-I 34 Swissair AITD COTTCLUSI 01: The riboflavin metabolism of nine overweight college women, who acted as subjects for a research project in the Food and Futrition Depart- ment of the School of Home Economics at Hichigan State College was studied. This experiment was started in September 1950 with two subjects and later on in January 1951, seven more subjects were added to the study. The sub- jects were seven.to 51 per cent overweight and the age of thersubjects ranged from.18 to 23 years old. After ten weeks on the weight reduction diet one subject, and after 12 weeks on the weight reduction diet two more subjects reached their desirable weights and they were placed on a weight maintenance diet for a period of two weeks and 12 weeks respectively. One subject left the experiment after four weeks on the weight reduction diet. Five subjects were five to 15 kilograms overweight at the end of the experiment. The average weekly loss of weight during this period ranged from 0.4 to 1.4 kilograms per week with an average of one kilogram.per week per person. The weight reduction diet contained 1400 calories, 100 grams of . protein, 90 grams of fat and 1.61 to 1.78 milligrams of riboflavin per » day. The average daily urinary excretion of the seven subjects on the self- selected diets ranged from 0.59 to 0.94 milligram.per day with an average of 0.74 milligram per day. The average per cent of daily intake of riboflavin excreted ranged from 32.1 to 56.1 per cent with an average of 42.7 per cent. The per cent of three milligrams test dose given orally excreted in 35 24 hours ranged from 24.1 to 43.1 per cent with an average of 32.6 per cent. The average daily urinary excretion of the same seven subjects after two, four, ten and twelve weeks on the weight reduction diet were 0.69, 0.89, 0.65, 0.72 milligram riboflavin per day. The average per cent of daily riboflavin intake excreted were 45.3, 49.8, 40.4 and 44.3 per cent respectively, and the per cent of test dose excreted in 24 hours were 39.1, 24.5, 35.7 and 36.1 per cent respectively. The riboflavin metabolism of three subjects on weight maintenance diets appeared to be similar to that of the subjects on the weight re- duction diets. The riboflavin metabolism.of the overweight college women on a re— duction diet was similar to that of healthy women. The relatively high fat content of the reduction diet did not appear to interfer with the riboflavin metabolism of the nine overweight college women. Under the conditions of this experiment an intake of 1.6 milligrams of riboflavin per day appeared to be adequate for these subjects. REESRI‘NCES CITED 36 RSFSRETCES CITED Association of Vitamin Chemist, 1947. Axelrod, A. E., T. D. Spies and C. A. Elvehjem 1941 A study of urinary riboflavin excretion in man. J. Clin. Invest. vol. 20, p. 229. Berryman, G. H., C. E. French, H. R. Baldwin, S. L. Bell and C. R. Henderson 1947 The relation of load test response and fasting ex- cretion levels to tissue content of thiamine and riboflavin. Am. J. Physiol., vol. 149, p. 254. Brewer,'W. D., T. Porter, 3. Ingalls and M. A. Ohlson 1946 The urinary excretion of riboflavin by college women. . Nutrition, vol. 32, P0 5830 Bourquin, A. and H. C. Sherman 1931 Quantitative determination of vitamin G (B2). J. Am. chem. Soc. vol. 53, p. 3501. Clarke, 2. F., M. Lechycka and C. A. Cook 1940 The biological assay of riboflavin. J. Nutrition 20, p. 133. Conner, R. T. and G. J. Straub 1941 Combined determination of riboflavin and thiamine in food products. Ind. Eng. Chem. (Anal. Ed.), v01. 13, p0 3850 Copping, A. V. 1945 Some aspects of riboflavin nutrition in man. Nutrition Absts. and Revs., vol. 14, p. 433. Czaczkes, J. W. and K. Guggenheim 1946 The influence of diet on the ribo— flavin metabolism.of the rat. J. Biol. Chem., vol. 162, p. 267. Davis, M. V., H. G. 01dham.and L. J. Roberts 1946 Riboflavin excretions of young women on diets containing'varying levels of the B vitamins. J. Nutrition, vol. 32, p. 143. Defierre, L. J. and'W. S. Brown 1944 Effect of various lighting condi- tions on riboflavin solutions. Arch. of Biochem., v01. 5, p. 181. Emmett, A. D. and L. H. “cKim 1917 The value of the yeast vitamin fraction as a supplement to a rice diet. J. Biol. Chem., vol. 32, p0 4090 Everson, G., E. 0., H.'Wa1ker and W} J. Caulfield 1948 Availability of riboflavin of ice cream, peas, and almonds judged by urinary excretion of the vitamin by women subjects. J. Nutrition, vol. 35, p. 209. Feder, V. H., G. T. Lewis and H. S. Alden 1944 Studies on the urinary excretion of riboflavin. J. Nutrition; vol. 27, p. 347. 37 Ferrebee, J. W. 1940 The urinary excretion of riboflavin, fluorometric methods of its estimation. J. Clin. Invest., vol. 19, p. 251. Ferrcbee, J. W. and N. Weissman 1943 Riboflavin and Thiamine inter- relationships in rats and in man. J. Nutrition, vol. 26, p. 459. Food and Nutrition Board, National Research Council 1943 Recommended dietary allowances. National Research Council Reprint and Circular Series no. 115. Food and'Nutrition Board, National Research Council 1948 Recommended dietary allowances. National Research Council Reprint and Circular Series no. 129. Guerrant, N. B. and R. A. Dutcher 1934 Some effects of the composition of the diet on the vitamin B and vitamin G requirement of the growing rat. J. Nutrition, v01. 8, p0 3970 Haas, E., B. L., Horecker, and T. R. Hogness 1940 The enzymatic reduction of cytochrome C, cytochrome 0 reductase. J. Biol. Chem., vol. 136, p0 7470 Harris, J. N. and F. I. Secular 1949 Riboflavin metabolism.of young women on self-selected diets. J. Nutrition, vol. 38, p. 435. Hathaway, ML L. and D. E. Lobb 1946 A comparison of riboflavin synthesis and excretion in human subjects on synthetic and natural diets. J. Nutrition, vol. 32, p. 9. Hodson, A. F. and L. C. Norris 1939 A fluorometric method for determining the riboflavin content of foodstuffs. J. Biol. Chems, vol. 131, p. 621. Horwitt, i. K., 0. W} Hills, 0. C. Harvey, E. Liebert and D. L. Steinberg 1949 Effects of dietary depletion of riboflavin. J. Nutrition, V01. 39, P. 3570 Ingalls, R. L. 1945 Urinary excretion of riboflavin of college women on a self chosen diet and the response to a saturation dose of riboflavin and thiamine. Thesis, M.S.C. Johnson, R. E., L. A. Contreras, F. C. Consolazio and P. F. Robinson 1945 A comparison of intravenous and oral vitamin tolerance tests. Am. J. Physiol., vol. 144, p. 58. Kemmerer, A. R. 1940 Report on riboflavin. J. Assoc. Off. Agri. Chem., vol. 23, p. 346. Keys, Am, A, F. Henschel, 0. Mickelsen, J. H; Brozek, and J. H. Crawford 1944 Physiological and biochemical functions in normal young men on a diet restricted in riboflavin. J. Nutrition, Vol. 27, p. 165. 38 Keys, A., A. F. Henschel, H. L. Taylor, 0. Hickelsen and J. M. Brozek 1945 Experimental studies on.man.with a restricted intake of the B vitamins Am. J. Physiol., vol. 144, p. 5. Klocke, J. F., P. I. Tack, M. A. Ohlson, R. Nitchals, E. Leffler and N. S. Henry 1947 Nutritive value of fish from.Michigan water, II. Thiamin of lake herring, carp, common sucker, burfiot and smelt. Food Res., vol. 12, p. 36. Krebs, H. A. 1935 Hetabolism.of Amino acids III. Determination of Amino acids Biochem. J., vol. 29, p. 1620. Kruse, H. D., V. P. Sydenstricker,'W. H. sebrell and H. H. Cleckley 1940 Ocular manifestations of ariboflavinosis. U. 3. Pub. Health Repts., v01. 55, p0 1570 Lingane, J. J. and 0. L. Davis 1941 Polarographic determination of ribo- flavin (vitamin B2) and other vitamin B factors. J. Biol. Chem., vol. 137, p0 5670 lbeleod, G. and C. M. Taylor 1944 Rose's Foundations of Nutrition, 4th Ed. annering, G. J., M..A. Lipton and C. A. Elvehjem. 1941 relation of dietary fat to riboflavin requirement of growing rats. Pro. Soc. EXP. B1010 M9d., V01. 46’ p0 1000 Lbnnering, G. J., D. Orsini and C. A. Elvehjem 1944 Effect of the comp position of the diet on the riboflavin requirement of the rat. J. Nutrition, vol. 28, p. 141. MCLaren, B. A., S. Cover and P. B. Pearson 1944 A simplified fluoro- metric method for riboflavin in meat. Arch. Biochem., vol. 4, p. l. Melnick, D., M. Hockberg, and B. L. Oser 1945 Physiological availability of the vitamins. I. The Human Bio-assay technic. J. Nutrition, vol. 30, p. 67. Herell, D. B. and E. C. Slater 1946 The Fluorometric determination of riboflavin in urine. Biochem. J., vol. 40, p. 652. Najjar, V. A. 1941 The fluorometric determination of riboflavin in urine and other biological fluids. J. Biol. Chem., vol. 141, p. 355. Najjar, V. A. and L. E. Holt 1941 A riboflavin excretion test as a measure of riboflavin deficiency in man. Bull. Johns Hopkins Hosp. v01. 69, p0 4760 39 Najjar, V. A., G. A. Johns, G. C. Medairy, G. Fleischmann and L. E. Holt Jr. 1944 The biosynthesis of riboflavin in man. J. Am. Med. Assn., vol. 126, p. 357. Newburgh, L. H. 1942 Obesity Arch. Inter. Hed., vol. 70, p. 1033. Narayanan, B. T., and J. C. Drumond 1930 The concentration of vitamin 82. Biochenu J., vol. 24, p. 19. Oden, J. W}, L. H. Oden, Jr. and W. H. Sebrell 1939 Report of three cases of ariboflavinosis. U. S. Pub. Health Repts., vol. 54, p. 790. Oldham, H. F., J. S. Kleiger and H. H. Arismendi 1944 A study of the ribo- flavin and thiamine requirements of children of pro-school age. J. Nutrition, vol. 27, p. 435. Parsons, H. T. 1944 Further studies on human requirements for riboflavin. Fed. RrOCo, V01. 3, p. 162. Peterson, Y. J., D. E. Brady, and A. 0. Shaw 1943 Fluorometric determina- tion of riboflavin in pork products. Ind. Eng. Chem. (Anal. Ed.), vol. 15, p. 634. Potter, R. L., A. E. Axelrod and C. A. Elvehjem 1942 The riboflavin re— quirement of the dog. J. Nutrition, vol. 24, p. 449. Reiser, R. and P. B. Pearson 1949 The influence of high levels of fat with sub-optimm levels of riboflavin on the growth of chicks. J. Nutrition, vol. 38, p. 247. Sebrell, W} H., R. E. Butler, J. G. Wooley and H. Isbell 1941 Human ribo- flavin requirement estimated by urinary excretion of subjects on con- trolled intake. U. S. Pub. Health Repts., vol. 56, p. 510. Selye, H. 1943 The role played by the gastrointestinal tract in the ab- sorption and excretion of riboflavin. J. Nutrition, vol. 25, p. 137. Snell, E. E., F. M. Strong and W. H. Peterson 1937 Growth factors for bacteria VI Fractionation and properties of an accessory factor for lactic acid bacteria. Biochem. J., vol. 31, p. 1789. Snell, E. E. and F. M. Strong 1939 .A Microbiological Assay for riboflavin. Ind. Eng. Chem. (Anal. Ed.), vol. 11, p. 346. Strong, F. M., R. E. Feeney, B. Moore and H. T. Pearsons 1941 The ribo- flavin content of blood and urine. J. Biol. Chem., vol. 137, p. 363. 40 Sure, B. and Z. W. Ford, Jr., 1942 Vitamin interrelationships II. ‘ Thiamine and riboflavin interrelationships in metabolism. J. Biol. Chem., vol. 146, p. 241. Sure, B. and Z. W} Ford, Jr. 1943 Influence of increasing doses of thiamine and riboflavin on efficiency of their utilization. J. Nutrition, V01. 26, p0 6590 Ume, Tango 1947 Relation between fat and riboflavin requirement of grow~ ing rats. Chem. Abstract, vol. 41, p. 4550. (From J. Agr. Chem. Soc. Japan, vol. 17, p. 1050, 1941). Unna, K., H. 0. Singher, C. J. Kensler, H. C. Taylor and G. P. Rhoads 1944 Effect of dietary protein on liver riboflavin levels and on in- activation ofEBtradiol by liver. Pro. Soc. Exp. Biol. Ved., vol. 55, p0 2540 ' Williams, R. 0., H. L. Mason, P. L. Cusick and R. H. fiilder 1943 Observa- tions on induced riboflavin deficiency and the riboflavin requirement of man. J. Nutrition, vol. 25, p. 361. ; "Fm.” new?!” ' ‘ l l - i .‘ . a a- ’ ‘1"? 7" :J ”1‘1""? I ‘3 V M473}: " "'- r V - " ROOM {SSE WU" r; 1‘12 (1“ v."_ f {Tu .I- _ f. ' " _ .u' . , kwi . 433‘ r 1 ‘ A? ‘5; ‘i‘ ‘81" ’ «("h- ’3’ Ly‘hgr" “Ybfifla‘éfia J § ." ““ ‘ ' .‘JM— .\ -..x.-u .- 3‘