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CALCIUM RETENTIONS OF YOUNG ADULT WOMEN ON A CONTROLLED WEIGHT REDUCTION'DIET by BLAIR figmLIAMS A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of MASTER OF SCIENCE Department of Foods and Nutrition 1951 1 HESIS TABLE OF CONTENTS CHAPTER * INTRODUCTION . .'. . . . . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . . . . . . Factors affecting calcium retention . . . . . Dietary fat . . . . . . . . . . . . . . . . Dietary protein . . . . . . . . . . . . . . Additional factors influencing calcium ab- sorption . . . . . . . . 3 . . . . . . . The calcium.requirement of young adult women Calcium metabolism during weight reduction . EXPERIMENTAL PROCEDURE . . . . . . . . . . . . Subjects . . . . . . . . . . . . . . . . . . Experimental plan . . . . . . . . . . . . . . Collection and treatment of samples . . . . . Chemical methods . . . . . . . . . . . . . . RESULTS AND DISCUSSION . . . . . . . . . . . . The Weight reduction diet . . . . . . . . . . Calcium intakes and retentions of individual sub- Jects O O O 0 O O O O . O 0 O O 0 O O O O O O Intake and retention during self-selection Urinary calcium . . . . . . . . . . . . . . f3 5?: § i "4-! 33. 12 14 19 21 25 25 25 27 29 32 32 CHAPTER PAGE Fecal calcium . . . . . . . . . . . . . . . . . . 42 Post-reduction retention of calcium . . . . . . . 45 Group reSponse to weight reduction . . . . . . . . 45 SULMARYANDCONCLUSIONS............... 54 APPENDH O C O O O O O O O O O O O O O 0 O O O O O O 5 6 RWEMB CI TE 0 O O O O O O O O O O O O O O O O O 65 iv LIST OF TABLES TABLE _ PAGE 1. ‘Mean Daily Calcium.Intake and Retention of YOung College Women Reported in the Literature . . . 24 2. Age, Height and weight Schedule for Individual Subjects . . . . . . . . . . . . . . . . . . . 33 3. Comparison of Recommended Distribution of Fat, Protein and Carbohydrate With Distribution Provided in weight Reduction Diet . . . . . . . 35 4. Mean.Daily Calcium‘Metabolism of Individual Sub- Jects during Self-selection and during weight Reduction Periods . . . . . . . . . . . . . . . 57 5. TMean Daily Intake, Excretion, and Retention of Calcium.ror all Subjects during Self-selection and weight Reduction Periods . . . . . . . . . 47 6. Comparison of mean CalciumLRetentions from.the Present Study and weight Reduction Studies Reported in the Literature . . . . . . . . . . 51 TABLE APPENDIX PAGE 7. Recovery of Calcium.rrom Solutions Prepared by Wet and Dry.Ashing Procedures . . . . . . 57 8. A. Foods Included in.Menus during Seven.Day Period of Weight Reduction Diet . . . . . 58 B. Foods Added to weight Reduction Diet during weight Maintenance Period . . . . . 61 9. Individual Data on Average Daily Calcium Metab- olism of All Subjects during Self-Selection and during Weight Reduction Periods . . . . . 63 vi LIST OF FIGURES FIGURE PAGE I. The CalciumiRetentions of Subjects on Self- selected Diets and during weight Reduction . 38 II. Fecal Calcium as Related to Fecal Calories for All Subjects . . . . . . . . . . . . . . 43 III. The mean Calcium.Intake, Excretion, and Re- tention of Subjects on Self-selected Diet (Period 1) and during weight Reduction (Periods 2-6) . . . . . . . . . . . . . . 48 IV. Regression of CalciumuRetention on.Calcium.In- take for Young College WOmen on weight Reduc- tion Diets Plotted about Regression Line for 124 College wemen on.Selr-selected Diets (McKay,l942)................ 5O E11111“! I ACKNOWLEDGMENTS The author wishes to express her sincere gratitude to Dr. Wilma Brewer for her patient guidance and ready encour- agement throughout this study. ‘Appreciation is expressed to Dorothy Dunsing for her helpful cooperation in the lab- oratory, and to the young women who served as diet sub- jects. INTRODUCTION The problem of weight reduction is an.important one for the young woman, both from.an esthetic and physiologic standpoint. The physiological strain of excess body tissue has been.receiving increasing attention within the past few years and has resulted in a number of popular dietary re- gimes for weight reduction. Obesity is frequently the result of poor nutritional habits with an over balance of high calorie fats and carbo- hydrates in the diet to the exclusion of essential minerals and vitamins. .A weight reduction diet should not only cause reduction in excess fatty tissue, but should attempt to pro- mote good dietary habits by providing the necessary food nu- trients. This is particularly true of the young adult woman who should receive a diet which will be both adequate for completion of adolescent growth and in preparation for the child-bearing years. The deveIOpment.and refinement of methods in nutrition research and the accumulation of scientific information in this field have resulted in an appreciation of the complexi- ty of the interrelationships which exist between essential body nutrients. A.change in the quantity of one or two dietary essentials for any prolonged period may alter the 2 accepted pattern of food intake and result in an impairment of physiological processes; therefore, it is important to give consideration to interrelationships of dietary essen- tials. The possible interrelationship of calcium and fat me- tabolism has been of interest since the early studies of Bosworth (1918) who reported that calcium was excreted as calcium.soaps of the higher molecular fatty acids, and Holt (1920) who found that maximum calcium absorption was re- lated to a definite fat to calcium ratio. The purpose of the present study was to investigate the calcium.retention of young college women on a weight reduc- tion diet in which.the calories were supplied chiefly by protein and fat. The possible interrelationship of the di- etary constituents was also a matter of consideration. REVIEW OF LITERATURE The literature on calcium utilization in.human nutri- tion indicates that calcium.absorption is subject to consid- erable variation, not only between individuals, but also in reSpect to the same individual. The amount of calcium.which is available for the body depends, as with other food nutri- ents, upon the ability of the body to absorb it from the food ingested. The form of calcium present in the diet may be insoluble in the digestive tract; therefore absorption is limited. Further it has been shown that various factors may influence both the solubility of calcium in the intestinal tract, and the absorption of calcium from.the intestinal tract. The following review includes some of these factors ‘with particular reference to the literature which deals with dietary factors that influence calcium absorption and reten- tion. Factors Affecting Calcium.Retention Dietary fa . Many early studies on the interrelation- ship of dietary fat and calcium were concerned with infant feeding problems. Bosworth gt 2;, (1918) studied certain bottle-fed infants in respect to an apparent inability of the infants to digest and absorb fat. The modified cow's 4 milk formula regulated the intake of organic constituents so that the composition approximated that of breast milk; how- ever, the same adjustment was not made in respect to the in- organic constituents; thus the intake of calcium by these infants was much greater than those receiving breast milk. Observation of the stools of the breast-fed and bottle-fed infants revealed a greater excretion of calcium in the lat- ter. The increase in fecal calcium was characterized by an increase in calcium soaps and was attributed to the imbal- ance of calcium to fat in the formula. The greater excre- tion of calcium with the modified formula was cited as a method of protecting the body from an excess of this ele~ ment, but the point was made that in so doing nutritional disturbances might result. By decreasing the calcium.content of the modified formula it was found that the calcium.elim- inated as soap was decreased and the infants were able to utilize the fat present in a normal manner. Investigation of the stools of infants receiving the modified formula be- fore the calcium content was adjusted indicated the presence of high molecular fatty acids with a range in melting point from.50°-54° centigrade. To study the solubility of the calciumrfatty-acid complex pure butter was saponified with sodium ethylate. The solubility of this soap was found to be excellent in fresh ox bile, but upon the addition of cal- cium chloride, calcium soaps were precipitated. Further in- vestigation showed the non-volatile fraction of the fatty acids obtained from butter fat to be soluble in ox bile but heavily precipitated by calcium chloride. The volatile fat- ty acid fraction, on the other hand, remained in solution on the addition of calcium chloride. The soaps formed by the nonevolatile fatty acid fraction were similar to the type of soap found in the stools. Holt gt_gl. (1920b) found no relationship between a large intake of fat and excessive excretion of calcium; more- over, the calcium lost as soap was insignificant. In four~ teen of seventy-nine instances the calcium excreted by in- fants as soap did not exceed 10 per cent. When the form of calcium which was excreted in the stools was analysed, it was found that the calcium.found as soap was seldom more than three-tenths and never more than five-tenths of the total calcium.cxcreted. These investigators found that greatest calcium absorption took place when the fat intake was great- er than 3.0 grams per day with a fat: calcium oxide ratio of one gram of fat to 6.63-0.05 grams of calcium oxide. Tefler (1921) observed infants suffering from congeni- tal biliary atresia and concluded that an increase in the excretion of calcium soaps was evidently correlated with an increased concentration of fat in the diet and, moreover, that the form in which calcium and phosphorus was eliminated was dependent upon the concentration of the fatty acids in 6 the intestine. Tefler suggested that calcium phosphate was dissolved in the lower molecular fatty acids present in the intestinal tract with subsequent precipitation of calcium as calcium soaps by the high molecular fatty acids. These sec- ondary calcium.salts could not be absorbed. Boyd at 51. (1932) studied the relation of dietary fat to calcium utili- zation in white rats and found that the lower molecular fat- ty acids readily dissolved insoluble calcium.carbonate and calcium.phosphate, thus rendering them.available for absorp- tion. ‘However, in the presence of high molecular fatty acids, increased formation of insoluble calcium soaps was found. When pure calcium.soaps were fed in a synthetic food mixture which was otherWise free from.aalcium, the following order of utilization of calcium was found: lactate, 68.5 per cent, butyrate, 52.5 percent, oleate, 68.4 per cent, mixed soaps from.1ard, 46.7 per cent, palmitate, 59.4 per cent, and stearate, 30.0 per cent. When calcium.chloride was the source of calcium on a fat free diet, the calcium lost in the feces amounted to 34 per cent. When lard* was added to the diet, the calcium.lost in feces dropped to 22 per cent. It was calculated that 20.8 milligrams of calcium; were retained on the fat free diet, compared with 29.3 mil- ligrams when lard was included in the diet. The fat free * Lard was added to the amount of 10 per cent by weight; adjustment in the diet was made by a decrease in carbohydrate. diet containing calcium chloride caused a neutral or alka- line reaction in the lower intestine and cecum. The addi- tion of fat to the diet shifted the reaction of the upper half of the small intestine to greater acidity (pH 6.44 to 5.81). With this shift in acidity the amount of calcium re- tained by the experimental animals increased. When calcium lactate provided the source of calcium on a fat free diet, addition of fat caused little shift in intestinal reaction and increased the per cent absorptionof calcium.only slight- ly. The indications from this work were that moderate~ amounts of fat might benefit calcium absorption through the maintenance of a favorable intestinal acidity. The studies of Jones (1940) on the addition of lard to rachitOgenic di- ets of rats indicated that when.lard was included in the di- et in amounts of 5 to 25 per cent of the diet, on the basis of both weight and calories, definite antirachitic prepar- ties were observed which could not be accounted for in terms of the non-saponifiable portion. This work also suggested a beneficial effect of the addition of fat in the form of lard to calcium absorption. French (1942) studied weanling male rats and found that the utilization of calcium.decreased moderately and consist- ently as the fat content of the diet was increased. When the concentration of fat in the diet amounted to five per cent, a more acid condition existed in the intestinal tract 8 and the calcium.utilization paralleled the increased acid- ity. Average values for the pH of the jejunum contents were 5.81, 6.02, 6.18, and 6.65. These corresponded with the di- ets containing 5, 15, 28, 45 per cent fat reSpectively. The percentage of utilization for the four diets showed a pro- gressive decrease as the fat content of the diet was in- creased, resulting in the following values: 80.0 per cent, 77.7 per cent, 74.1 per cent and 46.8 per cent. A diet with a relationship of one gram of fat to 0.06 grams of calcium oxide was found to be most efficient for calcium absorption; as the ratio of fat to calcium.increased, the utilization of calcium was decreased. This work would seem.to confirm.that of Holt (1920) which reported the optimal effect of a certain calcium.to fat ratio on calcium.sbsorption. Bosworth 33,31, (1918) observed that the extent of soap formation, and the ultimate fecal excretion of calcium, were dependent upon the fatty acid of the soap formed. Calcium oleate being more readily soluble in bile than.either calcium palmitate or calcium stearate was absorbed from.the intestine in large quantities, even.with an excess of calcium ions. Chen, Mbrehouse and Deuel (1949) found that fecal calcium was increased when there was poor absorption of fats and fatty acids. They also found that calcium storage decreased under these circumstances even when the calciumlintake was high. The minimum.loss of calcium occurred when the dietary fats 9 had a low melting point; when the melting point exceeded 50° centigrade, there was marked loss of calcium which was pro- portional to the quantity of the fat fed. In an additional study of the relation of fat to fecal excretion of calcium, Calverley and Kennedy (1948) also found that fecal calcium.increased with an increase in the melting point of the dietary fat. The animals on a fat free diet excreted the least amount of fecal calcium. The free fatty acids calculated as soap in a study of the neutral fat and fatty acid in feces were found to be greatest when the diet included hydrOgenated cottonseed oil. With the short chain saturated fatty acids, or long chain.unsaturated acids, the increase in fecal fat was less marked. Mattil and Higgins (1945) studied the relationship of glyceride structure to fat digestibility and found that stearic acid was poorly digested, whether it was fed as mixed glyceride or as simple triglyceride. The reaction of stearic acid with calcium.ion resulted in fecal elimination of calcium» 1Hoagland and Snider (1945a) found no consistent relationship between the melting point of fats tested and the coefficient of digestibility; however, none of the fats mentioned in this study had a melting point above 509 centi- grade. The tendency was in the direction of decreased di- gestibility with increasing melting point at both 5 per cent and 15 per cent intake of fat by weight. In a later study lO (1943b) these same workers compared the digestibility of stearic, palmetic, myristic and lauric acids. The digesti- bility was found to be 21 per cent for stearic acid, 59.6 per cent for palmetic acid and 100 per cent for each of the other fatty acids. Whereas there seems to be little evidence to support the theory that calcium is detrimental to fat absorption as advanced by Bosworth (1918), evidence has accumulated which is in agreement with the observations of these investigators on the poor utilization of fatty acids of high molecular content (Hoagland and Snider, 1945b; Mattil and Higgins, 1945), and the tendency of these fatty acids to bind dietary calcium in the form of insoluble soaps (westerlund, 1934; French, 1942; Boyd, 1952; Calverly, 1948; Kane, 1949). The relationship of calcium absorption to fat intake shows considerable variation with human subjects. The work of Holt (1920a, 1920b) with infants and young children dem- onstrated no close relationship of calcium absorption to fat intake; however, there was a definite ratio of calcium to fat which produced the best calcium absorption. Telfer (1921) found improved calcium.utilization with moderate amounts of fat in the form.of lard. Studies by Holt (1923) with children from two to six years of age supported the theory that fat has a beneficial effect on calcium absorp- tion. Five of the seven children studied showed marked re- ll duction in calcium absorption when the dietary fat was re- duced from approximately 30 to 5 per cent of the daily cal- orie intake. Mellon 93.31. (1930) found no evidence of an influence of fat intake on the calcium retentions of two young women on intakes of 105.6 grams of fat per day, and 5.8 grams per day. .A later study (Mallon.gt,gl.,ll933) gave similar fi ndings . The work of Kane g§_gl. (1949) using rats showed no difference in calcium absorption on diets containing one and twenty-one per cent of fat when young animals were the sub- ject of investigation but a decrease in absorption was evi- dent with increasing age. At 48 weeks female rats showed negative retentions of approximately the same magnitude on both diets; at 72 weeks the animals on the high fat intake showed a slightly greater negative balance than those ani- mals on the lower fat intake. At the age of 96 weeks there 'was marked difference between the calcium balances of ani- mals on the two diets; the animals which received a diet containing one per cent fat showed a balance of -l.1 milli- grams per day, while those with intake of twenty-one per cent fat showed a balance of -3.0 milligrams per day. These studies with experimental animals seem to paral- lel the findings of Holt (1920a, 1920b and 1923) for infants and young children, and Mellon (1930, 1933) for young women. As the maintenance of a positive calcium retention seems to l2 become more difficult'with increasing age in the human sub- ject, the relationship of fat to calcium.retention at dif- ferent ages gains in importance. Quetary;protein. MCCance g; 3;. (1942) using adult male subjects found that an increase in the amount of pro- tein in the diet markedly increased the absorption of cal- cium. 0n the basal diet, which contained 45-70 grams of protein,the average absorption of calcium was 32 milligrams per day, whereas on the protein supplemented diet the aver- age calcium absorbed was 94 milligrams per day. Hall and Lehmann (1944) theorized that the addition of protein to the diet, and therefore an increase in amino acid content of the intestine, should increase the solubility of calcium salts. These workers studied the effect of protein supplement using an increase in urinary calcium as an indication of increased calcium absorption. Fourteen of 18 subjects showed a higher urinary calcium.after the intake of peptone powders as against control powders which contained no form of protein or protein derivative. Two studies of calcium retention by young women (Kun- erth and Pittman, 1939; Pittman and Kunerth, 1939) also in- dicated that improved calcium.retention paralleled an in- crease in dietary protein. Three young women acted as sub- jects in each study of forty-five days including fifteen 3- day experimental periods. During the study of low protein 13 intake approximately 25 grams of protein per day were sup- plied. The second study provided a "medium? protein diet containing approximately 68 grams of protein per day. In each case meat accounted for 85 per cent of the protein al- lowance. Calcium intake was maintained at approximately the same intakein each study (mean per day, 0.458 grams and 0.459 grams respectively). In both studies all subjects were in negative calcium'balance. However, the beneficial effects of the higher protein intake were evident from the improved retention in the subjects during this period. In- dividual balances for the three subjects on the low protein intake were -0.054, -0.099, -.090 grams per day. These same subjects during the period of higher protein intake showed retentions of -0.025, -0.040, -0.039 grams per day respec- tively. The observation of Leverton and marsh (1942) on the me- tabolism of young women would indicate that the calcium in- take provided in these two diet studies was below that nec- essary for calcium equilibrium in this age group, and con- siderably lower than that given by Leverton and Marsh as the "mininal" requirement (0.83 grams per day). In eighteen studies of young women on self-chosen diets these investiga- tors found that 61 per cent were in negative balance when the intake of calcium was in the range of 0.400 to 0.599 grams per day. 14 Adolph g§_al. (1932) studied the calcium utilization of three Chinese adults, 18 to 27 years of age, on diets which provided protein in the form of milk and soybean curd. They found that an increase in protein, whether supplied by milk or by soybean curd, facilitated the establishment of calcium equilibrium. With a low protein intake all subjects were in negative balance. When the amount of protein was increased with the calcium intake remaining approximately the same, calcium retention improved in all cases. . Additional factors influenciggwcalcium.absorption. The intestinal output of calcium has been shown to be increased by an excess of phosphorus in the diet (Fearon, 1947); ab- sorption of the calcium on a low calciumzphosphorus ratio possibly is prevented by the formation of insolubleitrical- cium phosphate (Knapp, 1943). Tisdall 31;, g_1_. (1938) ob- served that rats fed a preliminary low calcium diet with subsequent increase in calcium.only, improved in retention of both calcium and phosphorus and concluded that the reten- tion of phosphorus seemed to be dependent upon the retention of calcium. Sherman (1948) concluded that the ratio of cal- cium to phosphorus would be satisfactory if an ample allow- ance for each nutrient were provided in the dietary. In the case of children Stearns(l93l) advocated a retention ratio ranging from 1.5 to 2 : l. The recommended dietary allow- ance for phosphorus for adults, as stated by the Food and 15 Nutrition Board of the National Research Council (1948), is approximately 1.5 times the intake of calcium; this ratio could be maintained readily if foods adequate in calcium were included in the diet. Other dietary factors which have been shown to decrease the absorption of calcium.from.the intestinal tract are phy- tic and oxalic acid (Fearon, 1947) and carbonate and sul- phate ions (Kleiner, 1948). Stewart and Percival (1928) in reviewing the literature on calcium metabolism.included the presence of excess potassium and magnesium.ions as detrimen- tal to calcium absorption whereas chloride ion.appeared to have a beneficial effect upon calcium absorption. .Aub g§_gl, (1937) found that additions of sodium chloride were effective in increasing calcium absorption only in massive doses. .Anchau (1930) studied the effect of increased bulk on output of fecal calcium and phosphorus in degs. With one exception the findings showed a greater calcium excretion with a high roughage diet. In the studies of Kunerth and Pittman (1939) previously mentioned it was found that the one subject who received a more bulky diet excreted a great- er amount of fecal calcium.than the other two subjects. However, this same subject in a later study (Pittman and Kunerth, 1939) also excreted a larger amount of fecal cal- cium on a diet containing approximately the same amount of 16 bulk as the others on the study. Aub 23_§l, (1937) found no relationship between calcium excretion and fecal volume. Brieter 23,31. (1942) in a study of the utilization of calcium from.carrots found that the addition of 700 grams of carrots per day resulted in an increased volume of fecal ma- terial and in most cases an increased intestinal activity. Five of the subjects studied showed poor utilization of cal- cium. The subject with good utilization of the calcium sup- plied by the carrot diet reported a decrease in fecal excre- tion. Deepite the increase in bulk produced by a diet pro- viding 93 per cent of the calcium intake through leaf let- tuce, mallon.§§,al, (1933) reported superior utilization when this diet was compared with a diet supplying approxi- mately the same quantity of calcium through whole pasteur- ized milk. The effect of cellulose upon mineral absorption in adults was studied by Morgan (1934) with two periods of nine days each differing only in the addition of 20 grams of re- . generated cellulose per day during the second period of a basically low roughage diet. The additional cellulose in- creased intestinal activity; however, six of eight subjects showed a positive calcium balance during the first period, while five of the eight subjects showed a negative balance during the second period ofLincreased bulk. The composite calcium deficit during this period was 12.9 per cent; about 17 12.5 per cent of the calcium was eliminated in the feces. Morgan concluded from this study that a high roughage diet had a definite limiting effect upon calcium.absorption. Mention should also be made of the influence of Vitamin D upon calcium absorption. .Although the mechanism by which the absorption is increased is not fully understood, it has been demonstrated that Vitamins D2 and D3 are most effective in promoting absorption.(Eddy, 1949). While a deficiency of Vitamin D is unlikely to occur in the adult, it may be a factor in border line cases of calcium deficiency (Ybumans, J. B., 1950). In addition to the influence of dietary pattern on cal- cium absorption McCance and Widdowson (1949) observed a sea- sonal variation. During July and August three of six sub- jects showed markedly greater absorption of calcium than in February and March, although the calcium intake was practi- cally constant during the different periods. Administration of 2,000 I. U} of Vitamin D in.March did not materially imp prove calcium absorption; this indicated that the seasonal change was not due to greater exposure to sunlight during the summer months. .A similar study of magnesium.and phos- phorus failed to show any seasonal rhythm. Sherman, Gil- lette, and Pope (1918) studied the calcium metabolism of healthy women and found no variation due to the menstrual cycle; however, a comparison of the retention values for one 18 of the subjects during successive months suggested a possible seasonal variation. During February, March and may this subject was in negative calcium balance; the average reten- tion in.February was -0.125 grams and in May the average re- tention was -0.05 grams. During June the subject showed a positive retention of 0.08 grams. Leverton.and Marsh (1942) reported the calcium.retentions of young women. In several cases Subjects were studied over a period of months. Exam- ination of these figures, and interpretation from informa- tion given regarding age at the time of each balance period, failed to suggest a seasonal rhythm. However, if seasonal variation is a true characteristic of calcium retention it must be taken into consideration in the evaluation of meta- bolic studies over a limited period of time. Fairbanks and Mitchell (1936) investigated the rela- tionship between calcium retention and the store of calcium in the body. In a preliminary period three paired groups of growing rats were fed diets providing 0.18 and 1.25 per cent 336 mg, 2336 mg), 0.32 and 1.25 per cent (610 mg, 2559 mg), 0.49 and 1.25 per cent (746 mg, 1916 mg) calcium.respective- ly. At the end of the preliminary period, three or four pairs from each group were sacrificed for analysis Of cal- cium to determine the body content. In all groups animals receiving the higher intake of calcium were found to have a greater store of body calcium. Retention studies during the 19 preliminary period were inversely proportional to the cal- cium intake. When the diet provided 1.25 per cent calcium, retention.was approximately 25 per cent while on the lower intakes (0.18, 0.32, 0.49 per cent) the percentage reten- tions were 106, 76, and 55 respectively. .After the comple- tion of the experimental period all animals were placed on a diet contributing 1.25 per cent calcium. Subsequent studies of the retention demonstrated that the degree of saturation of calcium stores in the preliminary period produced in- equalities in calcium.retention when a uniform high calcium diet was fed. Greater retention was found when the initial saturation of calcium was low. This would indicate the de- sirability of a period of adjustment to equalize calcium stores of experimental subjects, or information concerning previous calcium intake before interpreting data of calcium retention studies. The Calcium Requirement of Young Adult Women The periods of greatest calcium need are those associ- ated with the years of growth and during pregnancy and lac- tation. After adolescent growth is completed, there is in theadult a relatively low but continuous deposition and withdrawal of calcium going on in body tissues including bone. Over a reasonable period of time the withdrawal should be balanced by deposition so that no net loss of cal- cium occurs (Leitch, 1937). 20 Mitchell (1949) discussed the nutrient requirements for adult growth in reSpect to the nitrogen and phosphorus bal- ances of 23 male university students. Approximately one gram of nitrogen and 0.174 grams of phosphorus were retained daily from an adequate diet, presumably for adult growth in- cluding the skin, hair and nails and in the case of phos- phorus for delayed calcification of bones and teeth. The possibility of continuing saturation of bone after growth has apparently taken place was shown.in the investigations of Fairbanks and Mitchell (1936). MdKay 23 5;. (1942) also found in observations of young college students that reten- tion of calcium was continued after apparent growth was com- pleted when the calcium intake was adequate. Therefore it may be concluded that nutritional requirements of certain essentials continue in the adult over and above those re- quired for endogenous losses. Sherman (1920) estimated a daily requirement of 9.68 grams of calcium.per day for the theoretical 70 kilogram {man. Leitch (1937) analyzed the calcium.retentions of the literature by means of regression and suggested minimumlre- quirements for young adults as follows: Eighteen to twenty years, 0.9 grams per day; twenty to twenty-four, 0.7 grams per day. Leverton and Marsh (1942) concluded from.100 studies of young women that 0.83 grams per day represented the minimum 21 allowance. An intake of 1.08 grams per day was suggested as being "optimal" intake for the group studied. McKay 23731. (1942) in a study of the calcium, phosphorus, and nitrogen metabolism of 124 college women.representing four states found that mean daily intakes of 0.8 grams calciurlwere re- quired for equilibrium. Statistical analysis showed that place, age, and weight differences were not significantly related to calcium.retention for this group. Steggerda and Mitchell (1946a) reported that the average calcium require- ment for 43 adult subjects studied at the University of Il- linois was 0.65 grams of calcium.daily, with a coefficient of variation of 22.5 per cent. In a previous publication Steggerda and Mitchell (1946) suggested that 10 mg. of cal- cium per kilogram of body weight per day formed a good ap- proximation of the calcium.requirement of American college students and staff members on the accepted food pattern. The recommended dietary allowance of the Food and Nu- trition Board of the National Research Council (1948) for daily calcium intake of young women was 1.0 grams per day. Calcium‘Metabolism.During weight Reduction In the past decade studies of the metabolism.of young women during weight reduction have been reported from.two laboratories (Brown, 1946; Leverton, 1949, 1951). The in- vestigation of calcium metabolism has had an important part in each study. 22 Brown (1946) observed the calcium.retention of eight obese young women on a reduction diet which provided approx- imately 1200 calories per day with a distribution of 68 grams protein, 25-27 grams fat, and 125 grams of carbohy- drate. Calcium.retentions decreased in the period of weight reduction and the decrease continued in the post-reduction period. The mean coefficient of calcium absorption de- creased from 30 per cent in the preereduction period to 22 and 20 per cent in the weight loss and post-reduction peri- ods respectively. During the period of self-selected diet, at a mean daily intake of 1.253 grams, the average retention of calcium was 0.141 grams per day. The calcium intake dur- ing the period of low calorie intake was 1.096 grams per day and the calcium.retention was 0.079 grams. Post-reduction intake of calcium.for the subjects was 1.217 grams per day and the calcium.retention was -0.029 grams. Leverton and Rhodes (1949) studied the metabolism of fourteen young women during weight reduction. Seven of the group were maintained on a diet containing 60 grams of pro- tein and 43 grams of fat. The other seven subjects received a diet containing 105 grams of protein and 34 grams of fat. The diets provided 1150 to 1250 calories per day. The cal- cium.retention of the subjects on the low protein diet was 0.107 grams per day; this compared favorably with the value of 0.134 grams per day which was reported by Leverton (1942) 23 for young women on self-selected diets with a daily intake of 1.20 to 1.680 grams per day. The group receiving the high protein diet, with a calcium intake of 1.410 grams per day showed a mean.retention -0.074 grams. A further study (Leverton, 1951) of twelve young col- 1ege women utilized a low calorie diet of approximately 1200 calories per day with a customary distribution of food nu- trients. Mean daily calcium intake was 1.10 grams with a retention of -0.03 grams per day. Consideration of these studies would suggest that a weight reduction diet affects calcium.retention during the period of controlled diet and may also influence the utili- zation of calcium during the post-reduction.period. The di- ets cited provided a greater intake of calcium than the av- erage intake reported for young women on their own self- chosen diets by either Leverton (1942) or MbKay (1942). Table 1 summarizes the mean daily intake and retention of calcium for young women on self-chosen and weight reduc- tion diets reported in the literature. 24 Aammaveopnosoe oaaoaeo 00 H .eoapoeeem omo.o|. ooa.a NH new .25 em . “memdeofiete 33mm ”MWmmw .qogoseem 2.0.0.. 034 a. AmemHVqunosoe deepens .mam om..doaposeom aoa.o Hmm.fi a haemavmgoam eoeaonpeoo .eoapoeeon.pmom mmo.o.. pam.a m lmemavesonm canoaeo coma .qoaposeom meo.o mmo.a w Amemavesoam page demonouaaom Hea.o mmm.a m Amemavaeses page demoeouaaom omo.o Hem.o was Amemavaopnopoe page demoeo-aaem mao.o emm.o mm . ..3 #N mom .mam .pd .vm Hem .maw nopsmapmmqu aaoapaeeoo Hepaeaaaoasm soapqopom omepmH mwmommMm mmeeemmaHe ems zH mmemommm zmsoe mamaeoo ezpow so 23.5%.? 9,3 Baa/1H HSHSS H53 3% H £93. EXPERIMENTAL PROCEDURE Subjects Size overweight college women served as subjects for this study. The young women were subjects included in a me- tabolism study on weight reduction carried on by the Depart- ment of Foods and Nutrition of Michigan State College. The age range was from 18 to 23 years. The range of weight in excess of that considered desirable for each subject was from 10.15 pounds to 60.08 pounds.1 Experimental Plan The pattern of the dietary used differed from that ac- cepted customarily for this age group in the relationship of protein, fat, and carbohydrate. The total caloric intake was 1500 calories per day, except where noted. Preliminary to the period of weight loss, the subjects were maintained on a self-selected diet typical of the North Central region for a two week observation period. During the second week a balance study was conducted to establish calcium retention of the subjects on their usual food in- takes; thus each subject served as her own control. Follow- 1 Optimum weight determined from.Ba1dwinPWOod, height, weight tables for age, interpreted with reference to anthro- pometric measurements. 26 ing the preliminary period subjects were given the weight reduction diet. After two weeks on the diet a balance peri- od was maintained for a period of one week. Six balance periods were completed for sixLI subjects during the study which was continued from.January, 1951, to June, 1951. The balance periods were scheduled approximately every third week except during the vacation periods. The meals were prepared‘and served in the Home Econom- ics Department of the College under the supervision of trained personnel. The confinements of a study of this length were appreciated and every effort was made to main- tain a pleasant and companionable atmosphere. Subjects were kept on the controlled diet until satisfactory weight was reached, or until the termination of the school year. The subjects who reached their desired weight before termination of the study were given additional foods to study the diet- ary intake which would maintain the desired wieght. Throughout the study all subjects (except S. S.) re- ceived 312 I. U; Vitamin D per day. The amount of salt to be given for each subject was determined at the beginning of the study according to the taste preference of each individ- ual. .All foods were prepared without salt and weighed amounts were supplied at the table. There was no restric- tion on liquid intake but a record of all water, tea and coffee was kept in the individual record books of each sub- 27 ject. In view of the studies indicating the sometimes ap- preciable amount of calcium in water, a sample was analysed for calcium and the intake of calcium.from fluids was cal- culated for each subject for each balance period. Fifty per cent allowance was given for tea and coffee (Widdowson, 1943) . Collection and Treatment of Samples During the second week of the self selection period records of the weight of all servings were kept. .A weighed aliquot equal to one-fifth of the foods eaten was preserved by freezing. Similar aliquots of the weight loss diet were kept for all periods. Because of the volume of food col- lected daily aliquots were collected for two periods during the week. Liquids and solids were stored separately for ease in handling. ' At the completion of each week the frozen food compos- ites were thawed. The thawed material was transferred quan- titatively to a Waring food blender using wernlwater to wash out the container. This was blended for five minutes or un- til the mixture was homogeneous. The slurry was then trans- ferred quantitatively to a two-liter volumetric flask and made to volume with distilled water. After mixing by in- verting and rotating fifty times two samples were measured into 250 milliliter volumetric flasks. These samples were transferred quantitatively to weighed evaporating dishes 28 containing a glass stirring rod. Samples were placed in an oven at 40 degrees centigrade until dry (three to four days). Samples were stirred at intervals during drying to permit more even evaporation of moisture. .After they were dried to constant weight, the weight of the sample was determined. The dried sample was then scraped from.the sides of the dish and the entire contents ground in a mortar. A portion of the dried sample was transferred to a bottle and stored in a desiccator until analysed. Each slurry was analysed sepa- rately and the results added to give the total for the peri- od. Calcium.determinations were made on balance period samples only. During the balance periods daily fecal collections were made in waxed containers; carmine was used as a marker. At the end of each balance period the fecal collections were transferred to a blender and blended for five minutes. The sample was then transferred quantitatively to a two-liter volumetric flask and made to volume. After mixing fifty times three samples were measured into 100 ml. volumetric flasks and transferred quantitatively to weighed evaporating dishes supplied with stirring rOds. Samples were dried par- tially over a steam bath. ~Drying was completed under.infra red lamps. When.drying was complete (constant weight reached), the weight of the sample was determined. The 29 dried sample was scraped, ground, and stored in bottles in a desiccator until analyzed. TWenty-four hour urine collections were made during balance periods. After determination of the volume of the sample in a glass stOppered mixing cylinder the urine was diluted with distilled water to a volume easily divisible by five. The sample was mixed by inverting the cylinder twenty times and one-fifth of the daily sample transferred to a three-liter brown bottle and star ed in the refrigerator. The procedure was repeated daily until a composite for the balance period was obtained. Chemical Me tho ds weighed portions of the dried samples of food and feces were prepared for calcium determinations by dry ashing in a muffle furnace at.a temperature of approximately 490° centi- grade (A.0.A.C., 1940). The temperature was maintained for approximately 48 hours. The furnace was cooled and the cru- cibles removed to a desiccator. The crucibles were reweighed and the weight of the ash determined. Completeness of the ashing process was checked by reheating for 12 hours and again determining the weight of the ash. After the first two periods of the controlled diet, reheating was discontinued. Completeness of ashing was accepted on the basis of the per- centage ash obtained. The ash was taken up with hydrochloric 30 acid (1-e4), transferred quantitatively to a 100 milliliter volumetric flask, and made to volume. Urine samples were prepared for determination of cal- cium by a process of wet ashing. An aliquot of fifty milli- liters of the urine composite for each balance period was transferred by pipette tc»a 150 milliliter erlenmeyer flask. Five milliliter portions of concentrated sulphuric acid, 1 were added concentrated nitric acid and hydrogen peroxide slowly in the order mentioned. Samples were kept on a hot plate between medium and fast heat until clear and color- less. During ashing additions of nitric acid were made when the contents became charred due to carbonization of organic material, or when the contents tended to become dry. Sev- eral additions were found necessary for satisfactory ashing. Complete ashing required one to two weeks. When ashing*was completed, the ash was transferred quantitatively to a 100 milliliter volumetric flask and made to volume. In some cases a precipitate formed during ashing so that hydrochloric acid (1-+4) was used to transfer the ash. Solutions of both dry and wet ash were analyzed for calcium using a modification of MOCrudden's method (1911). Aliquots of five milliliters each of the solutions of ashed 1 Supersol. 31 faces and urine, and aliquots of ten milliliters of the so- lutions of food were taken for analysis. Brom cresol purple was used in adjusting the pH (Kramer, 1926). Care was necessary in adjusting the pH to avoid interference with magnesium, phosphorus and iron. Concentrated ammonia was added until the sample turned purple. Sufficient concentrated hydrochloric acid was then added to return the color to yellow. This pH has been found satisfactory for calcium determination in this laboratory. After satisfactory adjustment of pH, 10 ml. each of 2.5 per cent oxalic acid and 20 per cent sodium acetate (12 per cent anhydrous) were added slowly in order given to precipitate the calcium.as calcium oxalate. The following day the sample was filtered through Jena glass filters, the precipi- tate washed with dilute ammonia to remove traces of oxalic acid, dissolved in approximately normal sulphuric acid and titrated between 70° and 900 centigrade with approximately 0.01 normal potassium permanganate. Standard sodium oxalate was used to determine the normality of the potassium perman- ganate.at frequent intervals. Experimental procedures were checked by recovery of known quantities of calcium.and are recorded in Table 7 (Ap- pendix). RESULTS AND DISCUSSION The body weights of the subjects at the beginning of the experiment and during weight reduction are presented in Table 2. These data indicate that the subjects lost weight satisfactorily on the experimental diet. Two of the six young women (Aw, SS) reached desired weight within the peri- od of study. The remaining subjects (MJ, EH, FG, VA) showed steady weight losses, although they did not complete the recommended reduction before the end of the experimental period. The weight Reduction Diet The distribution of calories in the weight reduction diet differed from.that ordinarily accepted. The general pattern of the high fat, high protein diet presented by Pen- nington (1949) was modified to include a wider variety of foods. The diet reported by Pennington was low in calcium since milk was not included. Omission of milk from the diet makes it difficult to provide the necessary dietary require- ment of calcium. The weight reduction diet used in this ex- periment included one pint of milk per day. The recommended distribution of protein, fat and carbo- 33 e.aa e.mu m.aa m.mm m.mw mod we as >.¢ m.em a.e m.em «.mm and mm mm m.oa m.ma a.m m.mm o.mm ems we on m.» m.ea m.ea «.3m n.mw was me as H.ma e.mm m.ma m.mm m.m> ems we as e.aa o.ma m.~m a.mm e.mw Hos we me .me. .wa .mu .3. .wu .Eo made.» mmoq Harm museum eonamon .53ng Em 3m was 303.6 pawns; meomhmpm eaan>Ho2H mom assessom emeHma oza emeHmm .mee N Hagan. 34 hydrate in the normal diet as compared with the distribution provided in the diet study is presented in Table 3. The Food and Nutrition Board of the National Research Council (1948) recommended 75 grams of protein for women 16-20 years of age. This would provide 300 calories from protein in the diet. It was further recommended that the fat in the diet should provide at least 20-25 per cent of the total calories. 0n the basis of 1500 calories this would amount to approximately 350 calories from dietary fat. The remaining 850 calories would be derived from carbohy- drate. The weight reduction diet used in this study con- tained approximately 90 grams of protein and 80 grams of fat. Thus the diet provided about 360 calories from protein and 720 calories from fat. The remaining 420 calories would be supplied by about 80 grams of carbohydrate. Typical menus for one week during weight reduction are given in Table 8 (Appendix). The calculated calcium.and phosphorus intake per day (Bureau of Human Nutrition and Home Economics, 1945) were 0.66 and 1.1 grams per day re- spectively, giving a calcium : phoSphorus ratio of 1 : 1.6. The calculated value for calcium (0.66) was 13 per cent low- er than 0.76 grams per day found by analysis of food compos- ites for the corresponding weight loss period; however, va- riation between calculated and analyzed values has been 35 TABLE 3 COMPARISON'OF RECOMMENDED DISTRIBUTION OF FAT, PROTEIN AND CARBOHYDRATE WITH DISTRIBUTION PROVIDED IN WEIGHT REDUCTION DIET _-;_ l Recommended Allowance Fat Protein hygiggg Total Percent of calories 25.0 21.4 53.5 99.99 Total calories 350.0 300.0 850.0 1500.0 Present study Percent of calories 51.4 25.7 22.8 99.99 Total calories 720.0 360.0 420.0 1500.0 36. noted to be particularly characteristic of calcium.(Bassett, 1931; Hummel, 1942; Widdowson, 1943). Calcium Intakes and Retentions of Individual Subjects Intake and retention during self-selection. The cal- cium metabolism of individual subjects for the period of self-selection, weight reduction and maintenance (subject AW) is given in Table 4. Individual values for all subjects in all periods are given in Table 9 (Appendix). Individual retention values for calcium retentions during all periods are shown graphically in Figure 1.. The mean.daily intakes during the period of self- selected diets ranged from 0.93 grams for MJ to 1.16 grams for SS. The retention values for these subjects were 0.06 and 0.05 grams per day, reSpectively. The greatest reten- tion value was that for VA (0.27 grams per day) on an.intake of 1.11 grams per day. The remaining three subjects (EH, FG, Aw), on intakes of 1.06, 1.07, and 0.98 grams per day, showed corresponding retentions of 0.10, 0.14, and 0.04 grams per day. When these values are compared with.va1ues reported by McKay (1942) for young college women on self-selected diets, it is apparent that the young women subjects of the weight reduction study were retaining calcium in greater quantities than those on comparable intake ranges in the MbKay study. Thirteen subjects reported by mcKay on a mean daily intake .moOHnmm.ooaoooumn sumac; Mo mmmmm P mu loo.01.-mm.oll Amw.ouma.oa Aao.anmm.ov “ea.o-mo.o. mo.o:. mw.o am.o ma.o soaposoon names; em.o HH.H m .o mo.o noeeeoaee-eeon «a lma.o+.-ma.ouv 1mm.ouah.ov Ame.oumm.ov .ma.onmo.o. . eoo.on. Hm.o am.o ma.o noaeeeeoe senses no.0 ma.a a .o ea.o noaeeoaee-aaon mm Amo.o+ nom.o;. lam.oue>.ol 1m».onme.o. “mm.oumm.o. No.0). mw.o m.o mm.o soaposcen powers ¢H.o uo.a m.o ww.o ooHpoeHomuuHmm on Ama.o+ -Nm.o(V hem.o->e.o. 1mm.o-mm.ol lam.oumm.o. mo.o.. mm.o mm.o am.o moaeoeeee enmao: oo.o .o mm.o H~.o noaeeoeoouceoo as ¢H.OI. wa.m mm.o mm.o ooomsepaams Amo.o+ umo.oul Amm.m- p.01 1N>.osmm.o. 1mm.ouwm.ov 0.0. o .o em.o om.o eoeeooeoe posses o.o mm.o a .o mm.o doaeeoaomuuaeo 34 lma.o+ uee.om. 1am.oume.o. 1m~.oume.o. elmm.ouma.ol mo.o.. em.o mm.o mm.o noaeeeeen cameos oa.o mo.a m .o em.o eoaeooaeonaaoe me o\msw e\msw e\msw c\mew amomh hamsanp noaeeoeom esoeoH eoaeom eoeneem oofipenonm [III I) mQOHmHm ZOHBDDQmm BmUHMS QZHmDD Q24 ZOHBomqmmlhAHm UZHmDQ meombmbm AdDDHPHQZH mo EmHAom¢Bm2 EDHOH [:80 \ ' -' .-/ ‘ .1 7 I I ‘-. I , 09 V \ I \. I ‘ ‘3‘ b \.. I I \ I ‘ I .' / \ :0, \ " I ‘ c .\ / 0.8 ’ , I \ . 1 ‘ * ~. " 70 ”S‘ . .- . ‘ J \ ,’I W ” ‘ \ l \ I 4 ." ‘. I .' ’1 ‘. .' .' I I I \ ’ I 0 7» V I ' K I x1 ‘ I: \ \ 1' Z ’1 ) \ [I ‘ .A ' \ I f'" a I " 3‘ I \‘ \ I; \‘ '. .I. I’I \ ~ “ I l- 06 \ I, Z I' . .. a"' . q p “ .' ' .‘ \‘ II ‘\\\-. £1 8% 71.", \ \ '0‘, ‘60 8 . \v" ' \‘I I . 0.5 ' I! ‘ ) ‘5‘ 0'! v '. \\ 1'! \ . ' \ ' ' 0.4» < V, I » \\., ‘. -.-’ ‘50 \‘v'.’ \ . 0'3) . » V EH AW MJ. LZ 7 7 .1 490 I ‘2 ..I'. 4 I I > » \, f » I'- .\ I I; \ \. '° : \ no). « I \ .-' --4 I, ! '-\ Inc "I "\ ’ f \ l -. \‘1 g : I '- : \ 0.9 ' \‘\. r :I” r \ ! °-\ 2‘, A .I' '\ I = e I \ I ’I ‘ . I . g \ If" 08 ’ “. ‘ \ ’4 y. I ‘ I’ 4 . ' I '. 4 7 o m . \‘. I .’ / ‘ :l \ . \ O. -' \s / \ I’ '- ' " I ‘ ., 1’.--'\“+" '. :' \ I A \ 0.7 t ‘1 ° I " 3 I r » . ' I '. Q ‘\ II V \ . - I I ‘\ \ ~ \. :I - e \ : , . .,/-'°-“‘ ‘.\ ‘1 \.. 3 1‘ . I I \‘ ‘1‘” 0.5. h“ '1' . I ‘. x 4' . .\\ V‘ [I \“ [xxx .50 5 .\~ «I: V, \\ 1” ‘\ 1’ “i (I. 7 °' -' ’ \ \ I 0.5 \I I " x”: 0" III 04 > 4 > b a 50 ¢ 3 Q3) , —"— CALORI [s . ( "" CALCIUU U J n 2 3 4 s e n 2 3 4 5 e I 2 3 4 s e f, 53. VA. EC. In! It. 0 I E T P E R I O D S FIG.I[ FECAL CALCIUM AS RELATED TO FECAL CALORIES FOR ALL SUBJECTS 44 drawn for fecal calories; the increase in fecal calories was reflected in most cases with an increase in calcium.ex- cretion. The relationship was most marked for subjects.AW, MJ, and VA. The situation for AW must be interpreted dir- ferently from that of M3 or‘VA. The former subject showed a tendency to a reverse relationship from period three to period five with a sharp increase in both calcium and fecal calories in period six. As this period represented main- tenance for the subject, it cannot be considered comparable to the other periods. The sharp change in direction from. rqpid decline to increase in both calcium excretion and fecal calories for ME and VA.is particularly interesting in view of the marked difference in retention shown by these two subjects during the same periods. The mean retention values of calcium for periods five and six were -0.13 and -0.15 grams per day respectively, while the retention value for M3 during period five was -O.22 and for period six -0.32 grams per day. Retentions for‘VA were -0.28 and -0.32 grams per day reSpectively for these two periods. This sharp decrease in calcium.retention, in view of the sharp increase infecal calories, might be interpreted as due to an interference with calcium absorption by some or- ganic, calcium binding material subsequently eliminated in the feces. 45 Post-reductign retention of calcium. The two subjects reaching desired weight, SS and AW, indicate different re- actions to the post-reduction regime. During the third re- duction period SS was given a diet providing 80 per cent of the regular weight reduction diet. The final period repre- sents a return to the full weight loss diet; hence this pe- riod of maintenance provided the same food distribution as the weight loss periods. The daily retention for this pe- riod was at equilibrium, which would seem to reflect re- sponse to the variation in intake, or perhaps a final ad- justment to the dietary distribution of food. The response of AW is interesting because this subject was given.the regular weight maintenance diet, which increased the cal- cium intake for this subject to 1.17 grams per day. De- spite this increase, which provided a greater intake than during the preliminary period, the retention.remained nega- tive with a value of -O.l4. This retention was an improve- ment over the previous period and might indicate that the ‘period of adjustment to the new intake required time. Group Response to Weight Reduction Individual variation of small experimental groups tends to limit the value of interpretations of group averages. However, as mass metabolism studies, comparable to the weight reduction study reported, are impractical, it is of value to observe the group trend in relation to the diet re- 46 gime. Group averages are also of value in comparing studies from different laboratories. The mean daily intake, excretion, and retention of calcium.for all subjects are given in Table 5 and are shown graphically in Figure III. The mean.retention:for the period of self-selection on a mean intake of 1.05 grams of calcium per day was 0.11 grams per day. During the first two periods of weight re- duction when the mean intake of calcium was 0.86 grams per day, the average retention was 0.02 grams per day. This is similar to the value for calcium.retention of 0.034 which Twas reported by MdKay (1946) for subjects on an intake of 0.85 grams per day. The retentions for the remaining weight reduction periods were -0.01, -0.13 and -0.15 grams per day respectively. The initial decrease in average calcium.retention could be accounted for by the decrease in intake following the change from self-selected dietary to the controlled weight reduction diet. If this were the only factor influ~ encing calcium retention, the retention might be expected to stabilize at the new value. In the present study a slight downward trend continued in.period four, with a sharp drOp in period five. Period six showed continuing decrease in calcium retention, although less marked than in the previous period. The continued decline in.retention 47 .aOHpeapee euwenepm p .noapeseon sumac; mcoaueq wqfisaason “oofincm aoapoewemnuaom e 3.0.“ 5.0 9.0“ 3.0 8.0..“ 0.0.0 3.0“ 0m.0 m 3.0“ 3.0 5.0“ $0 00.0.... 8.0 no.0“. 320 m 5.0..“ 8.0 3.0..” $0 8.3 3.0 no.0“ :0 s 9.0“ «0.0 00.0”. 3.0 00.0“ 3.0 3.0..“ 3.0 m 3.3 00.0 3.0“ 3.0 mode 3.0 8.0... 00.0 m 00.0....30 3.33.0 3.38.0 0000.30; «A ..E em mom .3m ..3 ¢m “cm .maw ..3 am non .maw ..3 am neg .maw aoflampom seems snags 333 eowmmm soapenon MQOHmmm ZOHBODDmm BmGHMB Q24.ZOHBomHmthHMm mszDn maombmbm Had mom SDHoqdo ho ZOHBZHBmm 924 .ZOHBmmUNH .MMflezH MAHma Z¢m2 m an: . U o . s I ...... 4 III!- 8 8 """ “CALCIUM INTAKE m-FICAL CALCIUM ---UIINAIIY CALCIUM ---.ITINTION 8 V AND II‘I’INTION A8 CIAMS I , mrnou , CALCIUM IMTAKC p a A 8 DIET PERIODS FIGS!!! TI-IE MEAN CALCIUM INTAKE, EXCRETION, AM) RETENTION OF SUBJECTS ON SELF- SELECTED DIETIItmoonAm DURING WEIGHT REDUCTION (names 2 -0) A 49 .might indicate some alteration in endogenous calcium metab- olism. The values for urinary excretion would reflect an- dogenous changes, but with the exception.of period two, in ‘which the mean urinary excretion was 0.26 grams per day, the mean.values were constant and in keeping with the mean value for the period of self-selection. Increased percent- age excretion of calcium in the feces might represent imp ‘pairment of absorption. The values for fecal excretion are more consistent with the decreased retention values than are the urinary excretion values. The individual values for calcium.retentions of sub- jects on the diet used in this study, and the calcium re- tention values from other published weight reduction stud- ies, Brown (1946), Leverton (1949, 1951), are shown in.Fig- ure IV plotted about the regression line drawn from the predicting equation for calcium retention.against calcium intake reported by MdKay (1942),1 from.observations of 124 normal young women of college age. The mean calcium reten- tion values during weight reduction for these same studies are given in Table 6. The regression line for the data from the present weight reduction.study was determined.2 Comparison of the 1 y o.ogo+o.2392 (ac—0.941) 2 y 0.019+(-o.0773x) II 50 0.4' OBROWN I940 .LEVERTON I943 I oLEVERTON I940 11 . 03> ~LEVERTON I95| * "PRESENT STUDY 0 “-0.2? ‘ . 9 ¢ ,, .. I s a- E * I I I In ~ ‘ u -‘ - A C fl 3: - A U I: “ 0 % I; 1 fl / - ° 1 ‘ n = a .1 :0." ‘ ‘ 3 D U s A ‘ ° ° n a < 0 g. . 3 2 9 . P i 2 U I... O LIJ C I 3 o 3 ( U # x - - # ' 0.3 0.1 cc 0.. IO II .2 L3 III III CALCIUM INTAKE As CRAMs PER 24 HRS. no.1! REGRESSION OF CALCIUM RETENTION ON CALCIUM INTAKE FOR YOUNG COLLEGE WOMEN ON WEIGHT REDUCTION OIETs PLOTTED ABOUT REGRESSION LINE FOR I24 COLLECE WOMEN ON SELF-SELECTED DIETS (MCKAY, I942) 51 no.0I no.0 NS” m , 55.5 “3000.3 no.0- Ha.a em NH hamma.eopnopoq ¢>0.0I 0H¢.H mm s aflopoum swam .HH ~0H.0 Hmm.a mm s eaoponm_sog .H “sema.aopee>og m>0.0 mm0.H mm m Amemavazonm .30 mom .maw hen non .maw muse mews. fin. .mmmmm. .03“. 2...... IIIIIII rllnlI'I fimbagaHH ME... 2H Qmamommm mmHPDBm ZOHBDDQmm emcHBs n24 MQPHw Emmmmm E Scmh mZOHBszmm EDHQHdo Zfimfi ho ZomHmdnmzoo m mamas 52 slope of the line of the present study (b = -0.0773) with the slope of the line from.the MdKay study (b - 0.2392) was made using the "t" test1 (Snedecor, 1946); there was not a significant difference. This indicated that the relation- ship between calcium intake and retentions for the subjects of the study was similar to that found for the subjects of average height and weight studied by MOKay (1942). Indication that calcium.retentions of subjects of this study decreased steadily during weight reduction was pre- sented in both the individual data (Figure I) and by the group trend (Figure III). .A decrease in ability to retain calcium.during weight reduction has been observed in each of the other studies mentioned (Brown, 1946; Leverton, 1949, 1951), and suggests some change in calcium metabolism caused by weight reduction. The studies discussed repre- sent considerable variation in diet regime. Brown (1946) and Leverton (1951) provided low calorie diets with a gen- erally accepted food distribution; Leverton (1949) used a moderate protein intake for one group, and a high protein intake for a second group during the same study; the pres- ent study provided a high protein, high fat diet. Thus the decrease in calcium.retention seemed to occur irrespective of the diet employed, which might support the hypothesis 53 that the decrease in retention may be due to metabolic dis- turbance during weight reduction. Although the subjects in the study reported by Brown (1946) were in equilibrium on a self-selected diet, it is of interest to note that the tendency toward negative cal- cium.retention of the weight reduction period continued in- to the period of post-reduction. The study of Brown showed a lower retention during this period than during weight re- duction. On an average calcium intake of 1.096 grams per day, during weight reduction the average retention of eight subjects was 0.079 grams. During the post-reduction period with an intake of 1.211 grams of calcium.per day the reten- tion decreased to -0.029 grams per day. Subject AW of the study reported here was given a post-reduction diet of ac- cepted food distribution. .Although the retention was imr proved during this period, -0.14 grams per day compared with -0.21 grams for the preceding period on weight reduc- tion, the subject did not attain calcium equilibrium during the three weeks that she was on the weight maintenance diet. Since Brown also found negative balance during the post- reduction period, it would appear that further study in calcium.retention during weight loss and during adjustment to weight maintenance is needed. SUMMARY.AND CONCLUSIONS The effect of weight reduction on the calcium reten- tion of six young women has been studied. The subjects were part of a group of overweight women of college age taking part in a weight reduction study carried on by the Nutrition Department of Michigan.State College. Observa- tions of the calcium.retentions of these six subjects were made at regular intervals from.January to June, 1951. .A period of self-selected diet preceded the weight reduction periods. The diet used during weight reduction provided ap- proximately 80 grams of fat and 90 grams of protein per day; the intake of carbohydrate was adjusted to keep the total calorie intake at approximately 1500 calories per day. During the course of weight reduction a decrease in calciunIretention was apparent for the group as a whole. Individual subjects showed wide variation. All subjects were in positive calcium balance during the period of self- selection on an average intake of 1.05 grams of calcium per day With.a range in retention of 0.04 to 0.27 grams per day. During weight reduction, with an average calcium in- take of 0.83 grams per day, the range in retention was 0.19 b to -0.32 grams per day. Calcium retentions averaged -0.05 . 55 grams per day, a value which was not significantly differ- ent from that of MdKay (1942) for college women. Decreased calcium intake during weight reduction peri- ods did not seem to give an entirely satisfactory explana- tion for the decrease in calcium retention. Urinary cal- cium.values during weight reduction remained close to the pro-reduction average for the group, suggesting little evi- dence of decreased retention which might be due to unsatis- factory calcium to phosphorus ratio in the body, or to for- mation of ketones due to incomplete fat oxidation. Fecal calcium increased during successive weight reduction peri- ods, indicating decreased absorption of calcium from the intestinal tract. The possibility of a relationship be- tween this increase in fecal calcium and fat digestion is discussed. Data have been discussed for individual subjects and averages for the subjects considered for indications of group trends. findings of the study have been discussed in relation to the studies on weight reduction from other lab- oratories (Brown, 1946; Leverton, 1949, 1951). APPENDIX 57 H.~m emmH0.0 mmma0.0 wmmao.0 Hem00.0 oneness aeaoaeo p.00a sm>m0.0 m¢em0.0 me>m0.0 emm00.0 oneness aeaoaeo eoosIfiw “.5 m.em «H0000.0 m0000.0 m0000.0 eH0000.0 avenues assoaeo m.mm eam00.0 pam00.0 0e000.0 mmm00.0 openopneo asaoaeo onanDIInme no...» hnepooom 550.30 200000 0.33di 00004 oamaem OH 00004 . SHOHUO .maw ESHOHGO SSHOHOO * mac aaspo4 copsasoaeo .mac .maw asaoaeo no such magnmoomm oszmd. Mg 82. E 20% mmmdmmmm mZOHBbAOm 20mm “ah—Hugo ho Mmmboomm a aumda .A.FOODS INCLUDED IN MENUS DURING SEVEN DAY PERIOD TABLE 8 OF WEIGHT REDUCTION DIET II First Day Meal Food Weight 811130 Breakfast Grapefruit and orange juice 100 E88 100 Bread 20 Buttsr (total for day) 10 Milk 150 Lunch Pork chop 125 Broccoli 100 Milk 150 Dinner Hamburg 125 Creamed potato 100 Milk 150 Fresh grapefruit sections 100 Second Day Breakfast Orange juice 100 E88 100 Bread 20 Butter (tota1.for day) 20 Milk 150 Lunch Lamb patties 100 Lettuce 15 Pears (drained) 100 Milk 150 1.Approximate.weight of two eggs. Eggs scrambled in 5 gm. portion of butter from.day's allowance. 2 Whole milk. 59 . TABLE 8 (continued) Wm Meal Food Weight Dinner Roast veal 125 Peas 75 Applesauce 100 Milk 150 Third Day Breakfast Orange juice 100 E83 100 Bread 20 Butter (total for day) 20 Milk 150 Lunch Meat loaf 125 Cottage cheese - 20 Fresh tomato 0 Lettuce 0 Milk 150 Dinner Roast beef 125 Boiled potato 80 Sweet cherries 80 ‘Milk 150 Fourth Day Breakfast Grapefruit juice 100 Egg 100 Bread 20 Butter (total for day) 20 Milk 150 Lunch Swiss steak 125 Cauliflower 100 ‘Milk -150 Dinner Roast pork 125 Beets 100 Apricots 100 Milk 150 TABLE 8 (continued) 60 Fifth Day Meal Food Weight SIDS 0 Breakfast Tomato juice 100 E88 100 Bread 20 Butter (total for day) 15 Milk 150 Lunch Baked whitefish 125 Butter sauce 15 Baked sweet potato 100 Milk 150 Dinner Salmon salad l25 Celery 25 Lettuce 10 Mayonnaise 25 Green beans 100 ‘Milk 150 Sixth Day Breakfast Grapefruit and orange juice 100 Egg 100 Bread 20 Butter (total for day) 1 Milk 15 Lunch Hamburg 125 Tossed salad 100 French dressing 5 Milk 150 Dinner Pork roast 125 Peas 75 Lettuce 50 Canned peaches 100 Milk 150 61 TABLE 8 (continued) __._-I- w -’ Seventh Day Meal Food weight gins. Breakfast Orange (1) Sweet roll 25 Butter (total for day) 20 Milk 150 Dinner Roast chicken 150 Fresh tomato 100 Lettuce 50 French dressing 5 Milk 150 Supper Cold meat loaf 125 Bread 50 Apple 150 B. FOODS ADDED TO WEIGHT REDUCTION DIET DURING WEIGHT MAINTENANCE PERIOD W First Day Food Weight gins e Plums 100 Raw carrot 15 Green beans - 100 Cookies 20 Butter 50 Second Day A8paragus lOO Butter 20 Cake 55 TABLE 8 (continued) 62 Third Day Food Weight gms. Pineapple 100 Cottage cheese 50 Butter 20 Lettuce 50 Cookies 20 Fourth Day Fruit cocktail 100 Potato 100 Butter 20 Fifth Day Orange sections 75 Canne d t omato 90 Royal.Ann cherries 100 Cupcake with frosting 0 Butter 0 Sixth Day Fresh grapefruit 100 Cheddar cheese 38 Potato 1 Butter 20 Seventh Day Baked potato 80 Ice cream 100 Cookies 20 Butter 15 TABLE 9 INDIVIDUAL DATA ON AVERAGE DAILY CALCIUM METABOLISM 02' ALL SUBJECTS DURING SELF-SELECTION AND DURING WEIGHT REDUCTION’PERIODS m J j Excretion . Subject Period Intake Retention Urinary Fecal gin/d sm/d sm/d sm/d EH 1 0.24 0.72 1.06 0.10 2 0.25 0.79 0. 1 -—0.1 3 0.1 0.24 0. 0 0.0 4 002 0. g 0. 9 -0014. g 0.18 0.4 0. 1 0.15 0.23 0.77 0.91 —0.09 Meang 0.22 0.65 0.84 -—0.03 AW 1 0.23 0.71 0.98 0.0 2 0. 0.5; 0.33 0. 3 00 g 002 00 '0006 4 0.2 0. 2 0.39 —0.11 2b 0.30 0072 O. l ‘0021 0.38 0.93 1.17 -0.14 Mean 0.30 0.61 0.84 -—0.08 MI 1 0.21 0.66 0.9; 0.06 2 0.31 0.60 0.9 0.00 3 0. 6 0.47 0.78 0.05 4 0.23 0.32 0.77 0.19 2 0.2g 0. 0.59 -—o.22 0.2 0.93 0. 9 ——0.32 Mean 0.27 0.62 0.82 —0.06 a Mean of weight reduction periods(2,3,4,5,6). b Maintenance diet values, not included in mean. 54 TABLE 9 (continued) Excretion Retention Intake Period Subject Fecal Urinary am/d sm/d em/d gm/d 867261 2.419.223 OOOOOAUo 12342 ‘*0.02 0.56 0.83 0.28 Mean 55340 011110 000000 62012.10 108779 000000 100000 7105912114 nvnzfizvalfiu 000000 483040 110111 000000 12 3450 '0.00(4) 0.67 0.81 0.15 Mean 94.4) 04 011 11 o o co 00 000000 12 340 VA 0.81 0.12 mean REFERENCES CITED Adolph, wm. 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'.‘ 3’-" fi.‘ ' . ‘5 w h‘ .- ‘ _ AZ. . :. Lg”. Q; *2 r fir - I ' ‘ '~ ‘ A I‘. . ‘ h 5 Z): .1 ‘h 'V:. -A 3 ii...‘ 2': ‘ O H“) J I ”.32, .4 I ~ - " XIA- " . l': -' ' . ‘ a ‘ , . - - ~, d' . '2'!“ f. Q “ I' z , 4 “‘ .~ g. '3 3' 41 (‘h- - 1'“ i”. D In!“ 1.3.4 ‘ ._. “ 1 " . f ' -: E.“- .s' t. a U‘ ‘ 4 - . . A . I~ ' .. , \o.r~"Ml . " ' . ‘ . " ,_ 1 . ~ I ~.. . . . . ‘ ,- v 131:? k. . . - A, ewe-'4‘”, Z .' s v ‘I l , . l .. .‘;"‘~'{.-J‘I . . . . _ ‘ 6 . ‘ 0 9‘4?” '.-‘-3 02"” ' a; I . o"' _ ‘1 ,. .x . .1 . 1.... . , ...... .. ..... \ “..fi. .1. -.u.. ..w¥-4MI . . .,. :y‘m‘§ } an... .\. IL.. in . 1.3.5.... M...“ Mr: . . . .3591.“ w*. s 3ND. #14 fib‘h. I...» fig: .. - 4w. . . _ . ..‘3’L/4 fixfifi . 7‘1. 1%.... “(6“.13 -. , A. n .1 A. . . _ u _ —.m.':_. A... _ .mcitwwu uanuvfl .Ia. Hm“..y.r.fi.:v:xww§£.o.. ..vJ4+ . "Hlal- 1....-. a... . . . .. . .u'b.‘0( ..3.....n‘u.1._?.a .. p—Vfi A» ....0. WOW“. 3 1 93 03178 6407 III I II I II I] l II II III II l !