Cardiovascular Lesions, Blood Lipids, Coagulation and Fibrinolysis in Butter-induced Obesity in the R at* 1

SHAPUR NAIMI, GEORGE F. WILGRAM, MARTIN M. NOTHMAN AND SAMUEL PROGER Pratt Clinic-New England Center Hospital and the Department of Medicine, Tufts University School of Medicine, Boston, Massachusetts

ABSTRACT A study was made of obesity in the rat, induced by feeding a high butter fat diet over a long period and possible effects of obesity on the development of cardiovascular lesions. A group of 17 male W istar albino rats was fed a diet that contained 40% butter by weight (providing about 65% of the calories). To evaluate the independent contribution of obesity to the development of cardiovascular disease, repeated measurements were made of other parameters that might be affected by fats, namely, blood lipids, coagulation and fibrinolysis. The animals became grossly obese but did not develop any significant changes in blood lipids, coagulation and fibrinolysis throughout the period of the experiment (average of 428 days). Nor were there any significant cardiovascular lesions at the end of this period, beyond those normally observed in the aging rat. It was concluded that under the conditions of this experi­ ment where no significant changes occurred in blood lipids, coagulation and fibrino­ lysis, butter-induced obesity in the rat does not materially predispose to the develop­ m ent of cardiovascular lesions.

Obesity has been implicated frequently tion and fibrinolysis over a period of several in the development of cardiovascular dis­ months, it was necessary to compare the ease (1, 2), and yet the clinical evidence measurements in the experimental group for this is by no means incontrovertible with that of a normal group to safeguard (3-6). Moreover, in view of the emphasis against any variation in the activity of placed on this aspect of obesity in man, reagents used at different periods of the long-term studies of the atherogenic effect experiment. This was particularly im­ of experimental obesity have not received portant for coagulation studies where dif­ the attention they might. It was there­ ferent batches of substrates and reagents fore considered worthwhile to study ex­ may show considerable variation in activ­ perimental obesity in the rat as induced ity. The normal group selected for this by a high fat diet; and in addition, to study purpose was a chow-fed group, since in the independent effect of obesity on the de­ our previous studies (7), we have had velopment of atherosclerosis by the meas­ extensive experience with the level of blood urement of some other parameters that lipids, coagulation, fibrinolysis and the might be affected by fats, such as blood normal variation thereof in this group, and lipids, coagulation and fibrinolysis. Also, since this is the accepted normal diet of a high butter fat diet was selected for the laboratory rats about which extensive data study because of the presumed atherogenic have accumulated over the years. effect of saturated fats. The statistical study both in the initial and the terminal phases of the experiment MATERIALS AND METHODS was carried out between the experimental Two groups of rats were used. Each group and the normal group. However, group consisted of 17 male Wistar albinos. this was used only as an indirect way to The experimental group received a diet detect the possible changes in the level of the composition of which appears in table blood lipids, coagulation and fibrinolysis in 1. The butter content of this diet was Received for publication March 8, 1965. 40% by weight which provided about 65% 1 Supported by Public Health Service Research of the calories. Since the studies involved Grants no. HE-03753 and GM-9726 from the National Institutes of Health, and by a Training Grant from the measurement of blood lipids, coagula­ the National Heart Institute (5 T1 HE 5391).

J. N utrition, 86 : ’65 325 326 S. NAIMI, G. F. WILGRAM, M. M. NOTHMAN AND S. PROGER

TABLE 1 used for 2 frozen sections. Thus, a mini­ Composition of the diet of the experimental group mum of 10 frozen sections, not more than 5 a thick, was prepared from each rat heart zvt % so that as many cardiac lesions as possi­ Powdered cellulose 1 6 .0 ble would be detected. The aortas were C a s e in 2 2 0 .0 Choline chloride 0 .2 first stained in the gross and examined for Salt m ixture 3 4 .0 lesions with a magnifying lens. They were S u c ro s e 2 7 .8 then cut longitudinally on the freezing mi­ Vitamin mixture 4 2 .0 crotome, mounted, and searched for le­ 4 0 .0 B u tte r sions under the microscope. The frozen 1 Alphacel, Nutritional Biochemicals Corporation, sections were stained with oil red O in C leveland. 2 Casein (purified), Nutritional Biochemicals Corpo­ triethylphosphate. Livers and kidneys were ra tio n . 3 The salt mixture (Nutritional Biochemicals Corpo­ embedded in paraffin and stained rou­ ration) was the Wesson modification of the Osborne tinely with hematoxylin and eosin. and Mendel salt mixture. The composition in per cent by weight is as follows: calcium carbonate, 21.000; Blood coagulation and lipid studies and copper sulfate ( 5 H 2O), 0.039; ferric phosphate, 1.470; manganous sulfate (anhyd.), 0.020; magnesium sul­ methods. The studies of blood lipids, fate (anhyd.), 9.000; potassium aluminum sulfate, coagulation, and fibrinolysis were carried 0.009; potassium chloride, 12.000; potassium dihydro­ gen phosphate, 31.000; potassium iodide, 0.005; sodium out in both groups simultaneously at chloride, 10.500; sodium fluoride, 0.057; tricalcium phosphate, 14.900. monthly intervals, in both the early phase 4 Each kilogram of the vitamin mixture contained the following triturated in dextrose: vitamin A con­ of the experiment, that is, the first 4 centrate (200,000 U /g), 4.5 g; vitamin D concentrate months, and the late phase, that is, the (400,000 U/g), 0.25 g; a-tocopherol, 5.0 g; ascorbic acid, 40.5 g; inositol, 5.0 g; niacin, 4.5 g; riboflavin, last 4 months of the experiment. Rats 1-0 g; pyridoxine-HCl, 1.0 g; thiamine-HCl, 1.0 g; Ca were fasted for 16 hours before blood was pantothenate, 3.0 g; biotin, 20 mg; folic acid, 90 mg; and vitamin B 12, 1.35 m g. collected from the femoral vein. Since a number of blood coagulation and lipid de­ the experimental group over the many terminations require relatively substantial months of the experiment. As such the volumes of blood, it was not feasible to normal group should not be considered as obtain enough blood from a single animal a control group. The studies on the ex­ (unless killed) for all the studies. How­ perimental group in the initial phase of ever, to minimize the possible errors from the experiment provide the true control for this source, the same number and type of the studies in this same group toward the blood coagulation and fibrinolysis tests end of the experiment. were made in both groups at each veni­ The average starting weight of the ani­ puncture. Each rat was anesthetized with mals in the normal group was 410 g and ether, and blood was drawn with a 21- in the experimental group 390 g (the gauge siliconized needle and syringe by a small differences were not statistically sig­ 2-syringe technique. The volume of blood nificant). All animals were housed in in­ drawn at each venipuncture varied from dividual cages and were fed ad libitum. 3 to 6 ml. Three per cent sodium citrate Pathological studies and methods. Six was used as the anticoagulant throughout, rats in the experimental group were killed and 9 volumes of blood were added to 1 for pathological studies after they had re­ volume of citrate. Blood was centrifuged ceived the diet for 6 months. The rest of at 2500 rev/min for 10 minutes. Blood the animals in both groups were main­ and plasma were kept in melting ice tained in the experiment for an average of throughout. Observations were made in 428 days (range 200 to 580 days). Complete duplicate. Since the trends were similar autopsies were done on all animals, but only both in the early and the late phase of the heart, aorta, liver and kidney were sub­ experiment, in each phase of the study re­ mitted to histological preparations. These sults were pooled for statistical purposes. organs were removed and were fixed in 10% calcium formalin after death or kill­ Coagulation methods ing of the animals. Hearts and aortas were Prothrombin time. The one-stage method then embedded in gelatin for the prepara­ of Quick (8) was used except that a saline tion of frozen sections. Each heart was cut extract of acetone-dried human brain was into at least 5 slices, each of which was used as a source of thromboplastin. STUDIES IN BUTTER-INDUCED OBESITY 327

Stypven time. Russell viper venom2 lary tube. The end point was taken at the was diluted 1:10,000 with distilled water, instant that the liquid level in the capil­ then diluted 1:100,000 with 0.9% saline. lary tube reached the upper surface of the Platelet-poor plasma, 0.1 ml, was incubated clot. with 0.1 ml of freshly prepared venom Platelet count. The method of Brecher for 30 seconds at 37°; then 0.1 ml of and Cronkite (15) was used. 0 . 0 2 m calcium chloride was added and the Plasma fibrinogen. The method of clotting time determined. Ratnoff and Menzie as modified by Holburn Prothrombin levels. Plasma prothrom­ (16) was used. bin was quantitatively determined by a Statistical studies. Comparison of the modification of the Owren technique as means was carried out by the t test method described by Alexander (9). (17) . The differences were taken to be Factor V (labile factor). Activity was statistically significant when P was less measured by determining the corrective than 0.05. effect on the prothrombin time of a sub­ strate devoid of factor V but containing Chemical methods the other factors (prothrombin, factors The serum cholesterol and cholesterol VII and X, and fibrinogen) that affect the esters were determined by the method of prothrombin time (10). Schoenheimer and Sperry (18). The lipid Factors VII and X activity. The pro­ phosphorus was obtained by extracting the cedure used was a modification of the serum with the Bloor reagent and petro­ Owren technique for determining procon­ leum ether. The lipid phosphorus was con­ vertin activity (11). This technique is now verted to inorganic phosphorus by diges­ known to measure the combined activity tion and the inorganic phosphorus so of factors VII and X. obtained was measured by the procedure Modified thromboplastin generation. of Fiske and Subbarow (19). A factor of The test, as modified by Hicks and Pitney 25 was used to convert the lipid phospho­ (12) for determining the thromboplastin rus to phospholipids. The total serum generation from whole plasma was used. fatty acids were determined by the method Soybean phospholipid3 was added as a of Stoddard and Drury (20). For the de­ platelet substitute. This procedure meas­ terminations of ethanolamine phosphatide ures the development of plasma or blood and serine phosphatide, the method of thromboplastin. Axelrod et al. (21) was used. Fibrinolysis. The method described by Loomis (13) was used as modified by RESULTS Scott and Thomas (14). Streptokinase- Course of the experiment. Throughout streptodornase4 was used to activate fi­ the experiment the animals of the experi­ brinolysis. Rat plasma, 0.025 ml, was mental group gained more weight than added to 0.05 ml of a 1:5 dilution in sa­ those of the normal group. The caloric line of citrated human platelet-poor plasma. intake of the experimental group was esti­ The mixture was incubated at 37° for 30 mated to be about one-and-a-half times seconds and then 0.1 ml of streptokinase- that of the normal group (26kcal/100g streptodornase containing 2000 U of strep- rat/day in the experimental group com­ tokinase/ml was added to the mixture. pared with 16 in the normal group). The Thirty seconds after the reagents had been average weight of the rats in the experi­ thoroughly mixed, 0.1 ml of bovine throm­ mental group that were maintained with bin 5 containing 50 U/ml was added to the the diet for the full course of the experi­ mixture and the stop watch was started. ment was 1072 g (range 915 to 1570), Thirty seconds later, at which time a clot and that of the normal group after a had already formed, the open end of a 2 Stypven, Burroughs Wellcome and Company, capillary tube was inserted perpendicularly Tuckahoe, New York; prepared at Wellcome Research Laboratories, Beckenham, England. into the bottom of the clot. The tube was 3 Inosithin, Associated Concentrates, Inc., Woodside, New York. . left undisturbed in a glass water bath at 4 Varidase, Lederle Laboratories, American Cyana- 37°. Once fibrinolysis had begun, the mid Company, Pearl River, New York. s Obtained from the Upjohn Company, Kalamazoo, liquefied plasma ascended into the capil­ M ichigan. 3 2 8 S. NAIMI, G. F. WILGRAM, M. M. NOIHMAN AND S. PROGER comparable period of time was 553 g rine phosphatides were higher. The differ­ (range 410 to 664). ences, although statistically significant in Results of blood lipid studies. The respect to some determinations, are again mean results of the blood lipid determina­ noted to be small. tions in the early phase of the experiment Comparing the late phase of the study appear on table 2. Each result is the to the early phase, there was some de­ average of all the determinations in a crease in certain lipid parameters, namely, particular group. The animals of the ex­ total fatty acids, cholesterol, and not quite perimental group tended to have a some­ to the same extent as for total phospho­ what higher level of blood lipids as indi­ lipids, in the experimental group. This cated by total cholesterol, total fatty acids, has resulted in a decrease in total lipids- and total phospholipids including ethanol- to-phospholipid ratio which may be sig­ amine and serine phosphatides. In a num­ nificant. However, a similar decrease ber of determinations the differences are occurred in the total lipids-to-phospholipid statistically significant, yet considered in ratio of the chow-fed group. Conversely the context of other experiments in diet- there was an increase in the phosphatides induced lipemia in the rat (7), the in the experimental group during the increase of blood lipids in the experi­ period of study. These various changes are mental group compared with the normal pointed out since many investigators group is minor in absolute terms. might consider them significant. However, The mean results of the blood lipid the observed changes were small in ab­ determinations in the late phase of the solute terms compared with the changes experiment appear in table 3. Total cho­ we have observed in previous studies of lesterol and fatty acids were somewhat experimental atherosclerosis in the rat lower in the experimental group in this (7). For this reason and because some phase of the experiment, but total phos­ of the changes in the lipids in the chow- pholipids including ethanolamine and se­ fed group showed a similar trend, we did

TABLE 2 Mean of blood lipid determinations in the normal and experimental groups in the early phase of the experiment

N o rm a l Experimental group group SED 1

Total cholesterol, m g/100 ml 6 7 .4 ( 1 9 ) 2 7 4 .7 ( 1 9 ) 5 .6 Cholesterol esters, % 7 9 .4 ( 1 9 ) 7 0 .6 ( 1 9 ) 3 .2 * Total fatty acids, m g/100 ml 2 0 5 .0 ( 1 8 ) 3 0 1 .0 ( 1 8 ) 4 0 .3 * Total phospholipids, m g/100 ml 1 3 3 .0 ( 1 5 ) 2 2 6 .0 ( 1 5 ) 4 4 .3 * Ethanolamine phosphatide, mg/100 ml 3 .7 ( 1 5 ) 5 .0 ( 1 5 ) 0 .6 * Serine phosphatide, m g/100 ml 4 .9 ( 1 5 ) 5 .8 ( 1 5 ) 0 .5

1 sed indicates standard error of the difference. 2 The number of the determinations on which each mean is based appears in parentheses. * Statistically significant.

TA BLE 3 Mean of blood lipid determinations in the normal and experimental groups in the late phase of the experiment

N o rm a l Experimental g roup gro u p SED 1

Total cholesterol, m g/100 ml 8 6 .3 ( 1 1 ) 2 6 5 .3 ( 1 1 ) 8 .5 * Cholesterol esters, % 8 6 .0 ( 2 ) 7 8 .5 ( 4 ) 4 .9 Total fatty acids, m g/100 ml 1 5 4 .0 ( 8 ) 1 2 2 .0 ( 8 ) 2 7 .9 Total phospholipids, m g/100 ml 1 7 9 .2 ( 8 ) 2 0 2 .3 ( 8 ) 3 4 .8 Ethanolamine phosphatide, mg/100 ml 3 .5 ( 8 ) 6 . 8 ( 8 ) 1.8 Serine phosphatide, m g/100 ml 4 .7 ( 8 ) 8 . 4 ( 8 ) 1 .7 *

1 s e d indicates standard error of the difference. 2 The number of the determinations on which each mean is based appears in parentheses. * Statistically significant. STUDIES IN BUTTER-INDUCED OBESITY 329 not consider these changes to be biologi­ clotting time was shorter in the experi­ cally significant and therefore withhold mental group, the difference being statis­ further comment, particularly since dif­ tically significant. The biological signifi­ ferent interpretations would be placed on cance of this test, however, is not entirely various changes in lipid parameters and clear (22). The average results of throm­ ratios thereof by different investigators. boplastin generation test in the early Results of blood coagulation and fibrin­ phase of the experiment appear in table 5 olysis studies. The average results of and show no statistically significant differ­ blood coagulation and fibrinolysis studies ences between the 2 groups. in the early phase of the experiment ap­ The results of the studies of blood co­ pear on table 4. There are no significant agulation and fibrinolysis in the late phase differences between the 2 groups as indi­ of the experiment appear in table 6. The cated by the Quick prothrombin time, difference between the 2 groups generally Owren determinations of prothrombin, appears to be slight in most of the deter­ factor V and factors VII and X, platelet minations. If anything, there may be a count, hematocrit, and streptokinase- shift in the balance of blood coagulation induced fibrinolysis. Russell viper venom and fibrinolysis away from clot formation

TA BLE 4 Mean of blood coagulation and fibrinolysis studies in the normal and experimental group in the early phase of the experiment

N o rm a l Experimental SED 1 g ro u p gro u p

Quick (8) prothrom bin time, sec 1 8 .4 ( 3 0 ) 2 1 8 .1 ( 3 0 ) 0 .8

Russell viper venom tim e,3 sec 2 3 .1 ( 2 9 ) 2 0 .1 ( 2 9 ) 1.4 *

Owren determ inations ( 9 ) Prothrombin, % 1 0 1 .8 ( 2 9 ) 1 0 6 .9 ( 2 9 ) 4 .6 F a c to r V , % 1 0 0 .2 ( 2 9 ) 9 3 .3 ( 2 9 ) 5 .3 Factors VII and X, % 9 6 .1 ( 3 0 ) 1 0 4 .1 ( 3 0 ) 4 .8

Platelet count, in 1000/cm 3 1 0 8 9 .5 ( 2 6 ) 1 0 0 3 .0 ( 2 6 ) 8 2 .7

Hematocrit, % 3 9 .7 ( 3 1 ) 3 9 .5 ( 3 1 ) 0 .7

Streptokinase 4 fibrinolysis, sec 1 5 8 .2 ( 2 8 ) 1 6 6 .6 ( 2 8 ) 9 .2

1 sed indicates standard error of the difference. 2 The number of the determinations on which each mean is based appears in parentheses. 3 Stypven, Burroughs Wellcome and Company, Tuckahoe, New York. 4 Varidase, Lederle Laboratories, American Cyanamid Company, Pearl River, New York. * Statistically significant.

TA BLE 5 Mean of the modified thromboplastin generation test in the normal and experimental groups in the early phase of the experiment

Clotting time In c u b a tio n tim e N o rm a l Experimental SED 1 g roup g roup m in sec sec sec 2 9 1 .0 ( 2 5 ) 2 9 2 .1 ( 2 5 ) 10.1 3 6 3 .9 ( 2 1 ) 5 7 .3 ( 2 1 ) 7.5 4 5 4 .2 ( 2 4 ) 5 0 .6 ( 2 4 ) 8.1 5 4 4 .3 ( 2 3 ) 3 7 .0 ( 2 3 ) 6 .9 6 3 8 .1 ( 2 5 ) 3 0 .9 ( 2 5 ) 5 .2 7 3 1 .8 ( 2 4 ) 2 7 .5 ( 2 4 ) 3 .0 8 2 7 .8 ( 2 4 ) 2 6 .0 ( 2 4 ) 3 .7 9 2 6 .2 ( 2 4 ) 2 3 .7 ( 2 4 ) 2 .8 10 2 6 .2 ( 2 1 ) 2 4 .3 ( 2 1 ) 2 .7

1 se d indicates standard error of the difference. 2 The number of the determinations on which each mean is based appears in parentheses. 330 S. NAIMI, G. F. WILGRAM, M. M. NOTHMAN AND S. PROGER in the experimental group. The differences be made indirectly, that is, in each phase between the 2 groups, however, are not of the experiment the simultaneous stud­ great in absolute terms and are not ies on the normal group should be taken thought to be significant. The results of into consideration to make allowance for average thromboplastin generation in the the differences in measurements due to late phase of the study appear in table 7 variation in the activity of different and similarly indicate no significant dif­ batches of substrates and reagents.) ferences between the 2 groups. Results of pathological studies. Patho­ To summarize: The comparison be­ logical studies in 6 rats in the experimen­ tween the blood coagulation and fibrinol­ tal group that were maintained with the ysis studies of the experimental group in diet for 6 months showed them to be free the early phase of the experiment and of cardiovascular lesions. Apart from gross those in the late phase indicate that there obesity and a fatty liver, there were no was no significant change in the period of remarkable findings at the autopsy of the experiment. (This comparison has to these rats.

TA BLE 6 Mean of blood coagulation and fibrinolysis studies in the normal and experimental groups in the late phase of the experiment

N o rm a l Experimental SED 1 g ro u p g ro u p

Quick (8) prothrom bin time, sec 1 7 .4 ( 1 1 ) 2 1 8 .1 ( 1 1 ) l . i

Russell viper venom tim e,3 sec 3 2 .5 ( 1 1 ) 3 2 .7 ( 1 1 ) 5 .8

Owren determinations ( 9 ) Prothrombin, % 1 0 9 .2 ( 1 1 ) 1 2 2 .1 ( 1 1 ) 9 .5 F a c to r V , % 1 1 0 .9 ( 1 1 ) 8 5 .8 ( 1 1 ) 9 .0 * Factors VII and X, % 1 2 8 .0 ( 1 1 ) 1 2 5 .0 ( 1 1 ) 9 .0

Platelet count, in 1000/cm 3 1 0 4 9 .7 ( 1 0 ) 8 8 1 .3 ( 1 0 ) 1 4 1 .0

Hematocrit, % 3 7 .3 ( 1 1 ) 3 6 .0 ( 1 1 ) 2 .0

Streptokinase 4 fibrinolysis, sec 1 5 7 .2 ( 1 1 ) 1 2 8 .6 ( 1 1 ) 2 4 .9

Fibrinogen, m g/100 ml 3 9 9 .0 ( 5 ) 3 8 2 .8 ( 5 ) 3 5 .8

1 se d indicates standard error of the difference. 2 The number of the determinations on which each mean is based appears in parentheses. 3 Stypven. 4 Varidase. * Statistically significant.

TABLE 7 Mean of the modified thromboplastin generation test in the normal and experimental groups in the late phase of the experiment

Clotting time In c u b a tio n tim e N o rm a l Experimental g ro u p g roup SED 1 m in sec sec sec 2 144.7(10) 2 1 3 4 .8 ( 1 0 ) 2 6 .5 4 9 7 .6 ( 1 0 ) 7 7 .1 ( 1 0 ) 2 2 .4 6 4 8 .8 ( 1 0 ) 4 7 .2 ( 1 0 ) 9 .5 8 3 5 .3 ( 1 0 ) 3 1 .5 ( 1 0 ) 6 .7 9 3 3 .3 ( 1 0 ) 2 7 .8 ( 1 0 ) 6.1 10 2 9 .9 ( 1 0 ) 2 4 .2 ( 1 0 ) 4 .6 11 2 8 .9 ( 1 0 ) 2 4 .0 ( 1 0 ) 3 .8 12 2 7 .4 ( 1 0 ) 2 3 .3 ( 1 0 ) 2 .7 13 2 5 .9 ( 1 0 ) 2 3 .8 ( 1 0 ) 1.9 14 2 5 .7 ( 1 0 ) 2 4 .2 ( 1 0 ) 1.5

1 s e d indicates standard error of the difference. 2 The number of the determinations on which each mean is based appears in parentheses. STUDIES IN BUTTER-INDUCED OBESITY 331

Pathological studies on 11 animals in influenced by different factors and hence each group that continued in the experi­ caution must be used in the extrapolation ment for a longer period of time, that is, of the experimental data obtained in the for an average of 428 days, showed occa­ rat to the occurrence of obesity in man. sional vascular lesions in both groups, Yet, it is perhaps worth noting that in the identical in appearance and severity to clinical field, too, in recent years, the asso­ those observed in aging rats. There were ciation between obesity per se and in­ essentially 2 types of lesions. One type of creased levels of blood lipids has been lesion was in 2 animals in each group. questioned. Thus, Ross and Proger (24) It consisted of lipid infiltration of the wall reported no significant increase in the of the coronary artery. The other type of incidence of hypercholesterolemia (cho­ lesion was noted in one rat in the experi­ lesterol over 250 mg/100 ml) in obese sub­ mental group and 2 rats in the normal jects compared with matched controls. group. The most striking feature of this More recently Spain and associates (6) type of lesion was the formation of a have reported similar results in a study plaque containing subintimal fibrin-like of 5000 male subjects in New York. These material and lipids. These lesions may latter authors conclude that it is the over­ have been inflammatory in nature as in­ weight due to increased muscle mass dicated by some round cell infiltration in rather than that due to excess fat that is the adventitia. Occasionally foci of round related to both serum cholesterol level and cell infiltration were observed in the myo­ the development of coronary heart dis­ cardium of both groups representing inter­ ease; as such they make a clear distinction stitial myocarditis. This type of lesion has between obesity and overweight. been described previously in aging rats With respect to the relationship between (23). obesity and cardiovascular disease, our Histological examination of the liver in results support the hypothesis that obesity the experimental group gave a picture per se is not responsible for the develop­ identical to that commonly observed in ment of cardiovascular disease (5), and obese rats (predominantly a periportal that the increased risk of coronary heart type of lipid accumulation). disease in obesity may well be accounted for by the common association of hyper­ DISCUSSION tension and decreased glucose tolerance A diet containing a very large amount with this disorder (24). This is also in of animal fat did not, under the circum­ accord with the more recent observations stances of this experiment, produce in the of the Framingham Study. This group rat changes in blood lipids or vascular originally (25) attributed to obesity an lesions that might have been anticipated. increased risk in the development of cor­ The absence of such adverse changes, de­ onary heart disease. More recently, how­ spite the development of gross obesity in ever, they have noted (26) that once the these animals, may be significant, since effect is taken into consideration of other both obesity and animal fats have been factors that may accompany obesity such considered to be associated with lipemia as hypercholesterolemia and hypertension, and vascular lesions. It may be suggested the independent contribution of obesity to that other dietary factors might have pro­ the risk of the development of coronary tected the experimental group against heart disease becomes relatively small. such changes. Yet, even if this happens As to the relationship between lipemia to be the case, it should not detract from and changes in blood coagulation and the significance of the fact that large fibrinolysis, and the possible relevance of amounts of saturated fat and obesity are these to the development of cardiovascular not necessarily associated with lipemia lesions, the current studies appear to sup­ and vascular lesions. plement some of our previous results (7). With respect to the possible clinical sig­ Previously in the rat fed a diet high in nificance of these results, it must be em­ cholesterol, sodium cholate, thiouracil and phasized that the pathogenesis of athero­ butter, we observed a concurrence among sclerosis in various species may well be gross increase of blood lipids, marked 3 3 2 S. NAIMI, G. F. WILGRAM, M. M. NOTHMAN AND S. PROGER changes in the balance of blood coagula­ 10. Goldstein, R., A. Le Belloc’h, B. Alexander tion and fibrinolysis in the direction of and E. Zonderman 1959 Preparation and properties of prothrombin. J. Biol. Chem., clot formation, and the development of 2 3 4 : 2 8 5 7 . severe cardiovascular lesions. In the pres­ 11. Alexander, B. 1955 Determination of Spca ent investigations, however, we find that (convertin, factor VII). In The Coagulation a diet that does not lead to lipemia and of Blood, ed., L. M. Tocantins. Grune and changes in blood coagulation and fibrinol­ Stratton, New York, p. 141. 12. Hicks, N. D., and W. R. Pitney 1957 A ysis does not result in the development of rapid screening test for disorders of throm bo­ cardiovascular lesions, despite the develop­ plastin generation. Brit. J. Haemat., 3: 227. ment of gross obesity. Thus, in the con­ 13. Loomis, E. C. 1955 Estimation of fibrino- text of previous experiments the present lysin activity of human plasma or serum. observations may further support the con­ In The Coagulation of Blood, ed., L. M. Tocantins. Grune and Stratton, New York, cept that in the pathogenesis of cardio­ p . 163. vascular disease the role of obesity is of 14. Scott, R. F., and W. A. Thomas 1957 little importance beyond that of certain Methods for comparing effects of various variables that may be associated with it. fats on fibrinolysis. Proc. Soc. Exp. Biol., 9 6 : 2 4 . ACKNOWLEDGMENT 15. Brecher, G., and E. P. Cronkite 1950 Mor­ phology and enumeration of human blood The authors gratefully acknowledge the platelets. J. Appl. Physiol., 3: 365. technical assistance of Betty Busiek and 16. Holburn, R. R. 1955 Estimation of fibrino­ Eve Magic. gen in small samples of plasma (method of Ratnoff and Menzie). In The Coagulation LITERATURE CITED of Blood, ed., L. M. Tocantins. Grune and Stratton, New York, p. 155. 1. Dublin, L. I., and H. H. Marks 1951 Mor­ tality among insurance overweights in re­ 17. Brownlee, K. A. 1957 Industrial Experi­ cent years. 60th Annual Meeting, Associa­ mentation. Her Majesty’s Stationery Office, tion of Life Insurance Medical Directors. London, p. 34. 2. Stamler, J. 1958 Epidemiology as an in­ 18. Schoenheimer, R., and W. M. Sperry 1934 vestigative method for the study of human A micromethod for the determination of atherosclerosis. J. Nat. Med. A., 50: 161. free and combined cholesterol. J. Biol. 3. Keys, A. 1954 Obesity and degenerative C h e m ., 1 0 6 : 7 4 5 . heart disease. Am. J. Public Health, 44: 864. 19. Fiske, C. H., and Y. Subbarow 1925 The 4. Yater, W. M., A. H. Traum, W. G. Brown, colorimetric determination of phosphorus. J. R. P. Fitzgerald, M. A. Geisler and B. B. Biol. Chem., 66: 375. Wilcox 1948 Coronary artery disease in 20. Stoddard, J. L., and P. E. Drury 1929 A men eighteen to thirty-nine years of age. titration method for blood fat. J. Biol. Am. Heart J., 36: 334. C h e m ., 8 4 : 7 4 1 . 5. Proger, S. 1958 Current status of diet and 21. Axelrod, J., J. Reichenthal and B. B. Brodie lipid metabolism in the pathogenesis of 1953 The determination of phosphatidyl atherosclerosis. Prog. Cardiovasc. Dis., 1: ethanolamine and phosphatidyl serine in 134. plasma and red blood cells. J. Biol. Chem., 6. Spain, D. M., D. J. Nathan and M. Gellis 2 0 4 : 9 0 3 . 1963 Weight, body type and the prevalence 22. O’Brien, J. R. 1957 Dietary fat and cor­ of coronary atherosclerotic heart disease in onary disease. Brit. Med. J., 2: 297. males. Am. J. Med. Sci., 245; 63. 23. Wilgram, G. F. 1957 A survey of experi­ 7. Naimi, S., R. Goldstein, M. M. Nothman, m ental arteriopathies in the rat. Arch. Path., G. F. Wilgram and S. Proger 1962 Cardio­ 6 4 : 6 2 9 . vascular lesions and changes in blood co­ 24. Ross, C. F., and S. Proger 1958-1959 Obes­ agulation and fibrinolysis associated with ity, associated findings, and their possible diet-induced lipemia in the rat. J. Clin. relationship to heart disease. Proc. New I n v e s t., 4 1 : 17 0 8 . England Cardiovasc. Soc., 17: 15. 8. Biggs, R., and R. G. Macfarlane 1962 One- 25. Dawber, T. R., F. E. Moore and G. V. Mann stage prothrombin time. In Human Blood 1957 Coronary heart disease in the Fra­ Coagulation and Its Disorders, ed. 3. F. A. mingham Study. Am. J. Pub. Health, 47 Davis Company, Philadelphia, p. 385. (suppl. no. 4, part 2): 4. 9. Alexander, B. 1955 Estimation of plasma 26. Dawber, T. R., W. B. Kannel, N. Revotskie prothrombin by the one-stage method. In and A. Kagan 1962 The epidemiology of The Coagulation of Blood, ed., L. M. To­ coronary heart disease — the Framingham cantins. Gruñe and Stratton, New York, p. 89. Enquiry. Proc. Roy. Soc. of Med., 55: 265. Amino Acid Requirements of Children: QUANTITATIVE AMINO ACID REQUIREMENTS OF GIRLS BASED ON NITROGEN BALANCE METHOD

ITSIRO NAKAGAWA, TETSUZO TAKAHASHI, TAKESHI SUZUKI a n d KATSUMI KOBAYASHI Department of Nutrition and Biochemistry, Institute of Public Health, Tokyo, Japan

ABSTRACT The problem of whether the minimal requirements of essential amino acids and of total nitrogen established previously for boys were also adequate for girls was examined. For a period of 12 to 19 days, 6 healthy 8- to 13-year-old girls were given minimal quantities of the 8 essential amino acids studied previously with boys of the same age group, but with total nitrogen varied. The youngest of the group maintained positive balance with an intake of 8 g nitrogen. Of the three 12-year-old girls, two remained in positive balance with 8 g, and the other with 12 g. Two 13-year- old girls also maintained positive balance with a nitrogen intake of 12 g. The large variation in total nitrogen requirement is attributed to individual differences in the time of onset and degree of the pubertal growth spurt. Body weight and excretion of creatinine and 17-ketosteroids remained almost constant throughout the experimental p e rio d .

In the previous papers of this series method. The average daily excretion of (1—6), the requirements for each of the 8 fecal nitrogen was obtained by dividing the essential amino acids and for total nitro­ total output by the number of days in the gen for 10- to 12-year-old boys were re­ period. In addition, urinary creatine and ported. The present paper extends this creatinine were determined by the methods work to the amino acid requirements for reported previously (1, 2), and 17-keto­ girls of the same age group. steroids by our modification of the Zimmer­ mann reaction method (7). EXPERIMENTAL PROCEDURE The results obtained are shown in table AND RESULTS 3. All children rem ained healthy and m ain­ Six healthy girls 8 to 13 years of age tained constant body weight throughout served as experimental subjects (table 1). the experimental period. Subject Y.S., the The general procedures of the experiment youngest girl, maintained positive balance have been described previously (1-4). For with a daily intake of 8 g of total nitrogen the first 3 days, the subjects consumed a (mixture 1) for 16 days. Two other sub­ normal diet having about the same nitro­ jects (K.T. and Y.N.) remained in posi­ gen and caloric levels as the experimental tive balance not only with the intake of basal diet. This period served to accustom 10 g nitrogen (mixture 2), but also with the subjects to living in a laboratory and 8 g. The study on subject K.T. could not to minimize prior dietary effects. The basal be continued because of the occurrence of diet contained cornstarch, corn oil, butter her menstrual period. Subject T.K.. al­ fat, mineral and vitamin mixtures, and the though of the same age as K.T. and Y.N., amino acid mixture. The same minimal failed to maintain positive balance with quantity of the 8 essential amino acids the intake of 10 g, but remained in posi­ which was established for boys, was given tive balance with 12 g of nitrogen (m ix­ to the girls, but the total nitrogen was ture 3). Two middle-school girls (K.Y. and varied as shown in table 2. The nitrogen S.K. ) maintained positive nitrogen balance balance was measured throughout the ex­ with 12 g, but not with 10 g. When they perimental period of 19 days. The amount were fed 12 g, the balance became positive of nitrogen in the diet, urine and feces was analyzed by the semimicro-Kjeldahl Received for publication March 9, 1965.

J. N utrition, 86: ’65 333 334 I. NAKAGAWA, T. TAKAHASHI, T. SUZUKI AND K. KOBAYASHI

TABLE 1 Age, height, weight, basal metabolism and energy intake of subjects

Avg Body Body B asal daily Subject Age h eigh t w t m etabolism energy intake 1

years, h e a l/ k g / day k e a l/k g m o n th s cm kg Y.S. 8 6 1 2 8 .6 2 1 .1 41 6 0 K.T. 11 9 1 4 9 .8 3 8 .9 3 0 3 9 Y.N. 11 10 1 5 0 .7 3 6 .0 31 4 2 T.K. 11 11 1 3 8 .7 3 6 .4 30 4 9 K.Y. 13 3 1 5 9 .3 4 4 .5 25 3 4 S.K. 13 3 1 5 5 .9 3 6 .6 2 9 3 7

1 Calories derived from the am ino acid mixture are not included.

TABLE 2 Composition and daily intake of amino acid mixtures

M ixture 1 M ixture 2 M ixture 3 Com ponent D aily N itrogen D aily N itrogen D aily N itrogen intake content intake content intake content

9 9 9 9 9 9 Essential amino acids L-Isoleucine 1 .0 0 0 .1 0 7 L -L eu cin e 1 .5 0 0 .1 6 0 n -L y sin e 1 .6 0 0 .3 0 7 t-M ethionine 0 .8 0 0 .0 7 5 L-Phenylalanine 0 .8 0 0 .0 6 8 L-Threonine 1 .0 0 0 .1 1 8 L-Tryptophan 0 .1 2 0 .0 1 7 L -V aline 0 .9 0 0 .1 0 8

Nonessential amino acids L -A la n in e 5 .6 6 0 .8 9 0 7 .3 4 1 .1 5 4 9 .0 1 1 .4 1 6 L -A rgin in e 6 .6 4 2 .1 3 5 8 .6 0 2 .7 6 6 1 0 .5 6 3 .3 9 6 L-Aspartie acid 6 .6 1 0 .6 9 5 8 .5 6 0 .9 0 1 1 0 .5 1 1 .1 0 6 L-Glutamic acid 6 .6 1 0 .6 2 9 8 .5 6 0 .8 1 5 10.51 1 .0 0 1 L-Sodium glutamate 7 .6 7 0 .6 3 5 9 .9 4 0 .8 2 3 1 2 .2 1 1.011 G ly c in e 4 .7 1 0 .8 7 4 6 .0 8 1 .1 2 9 7 .5 0 1 .3 9 3 L-Histidine 1 .0 0 0 .2 7 1 1 .0 0 0 .2 7 1 1 .0 0 0 .2 7 1 L -P rolin e 2 .8 3 0 .3 4 4 3 .6 7 0 .4 4 7 4 .5 0 0 .5 4 8 L -Serine 4 .2 5 0 .5 6 7 5 .5 0 0 .7 3 3 6 .7 6 0 .8 9 8

Total (essential + nonessential) 8 .0 0 0 1 0 .0 0 0 1 2 .0 0 0 again. The excretion of creatinine and 17- was examined in the present study. The ketosteroids remained almost constant results showed that the minimal amounts throughout the experiment. of essential amino acids established for boys were also adeqate for girls, although DISCUSSION the minimal requirement of individual Amino acid requirements of female essential amino acids for girls has not been adults have been reported by Leverton investigated. It is difficult to define the (8-12), Jones (13), Clark (14) and zone for allowance of retention that is Swendseid (15, 16), but the requirements necessary not only for maintenance of of growing girls had not been studied pre­ positive nitrogen balance, but also for viously. Therefore the problem of whether growth and development. We estimate that the mixture of minimal requirements of 0.38 g of nitrogen is a safe daily intake for 8 essential amino acids established previ­ retention consistent with the demands for ously for boys is also adequate for girls growth in 10- to 12-year-old boys. AMINO ACID REQUIREMENTS OF GIRLS 335

TA RLE 3

Nitrogen balance and urinary excretion of creatinine, creatine and 17-ketosteroids

D aily Avg daily N output Avg Avg daily urinary excretion S u b ject P erio d d aily w t N in ta k e U rin e Feces N C rea­ C rea­ 17-Keto- b a la n ce tin in e tin e stero id s

days kg 9 9 9 9 mg mg mg Y.S. 2 1 .1 1 4 6 3 2 2 6 0 2 4 2 1 .4 8 .1 6 7 .5 0 ) + 0 .4 7 4 7 4 2 4 5 0 .1 9 4 2 1 .2 8 .1 6 6 .4 3 / + 1 .5 4 4 6 9 2 2 0 4 2 1 .1 8 .1 5 6 .5 9 / + 1.31 5 5 0 2 7 2 0 .2 5 4 2 0 .5 8 .1 5 7 .1 5 / + 0 .7 5 4 2 1 3 3 3

K.T. 3 8 .9 1 7 3 7 2 9 9 2 4 3 8 .4 1 0 .1 8 7 .6 1 / + 2 .4 3 79 8 151 1.09 0 .1 4 4 3 8 .8 1 0 .1 8 8 .4 7 / + 1 .5 7 7 4 7 113 0 .8 8 4 3 8 .8 8 .1 8 6 .5 4 0 .1 4 + 1 .5 0 7 5 2 60 0 .9 5

Y.N. 3 6 .0 1 5 6 3 2 1 3 6 2 4 3 6 .3 1 0 .1 7 6 .6 7 / + 3 .3 5 5 7 6 2 2 7 0 .7 2 0 .1 5 4 3 7 .0 1 0 .1 7 6 .8 3 / + 3 .1 9 4 4 3 141 0 .5 5 4 3 6 .2 8 .1 7 5 .1 9 / + 2 .7 6 5 4 4 188 0 .7 0 0 .2 2 4 3 5 .9 8 .1 7 5 .4 1 / + 2 .5 4 4 5 9 2 0 0 0 .6 3

T.K. 3 6 .4 1 77 1 2 2 1 8 2 4 3 6 .0 10 .1 8 1 0 .8 1 / - 0 . 9 2 9 1 7 3 5 6 1 .1 6 0 .2 9 4 3 6 .5 1 0 .1 8 1 0 .1 2 / - 0 . 2 3 81 1 2 9 0 1 .1 2 4 3 7 .3 1 2 .1 8 1 1 .2 8 / + 0 .3 7 78 3 4 0 5 1 .0 5 0 .5 3 4 3 6 .1 1 2 .1 8 1 1 .3 6 / + 0 .2 9 77 5 4 7 2 1 .2 7

K.Y. 4 4 .5 1 1 0 7 4 2 31 2 4 4 3 .8 1 2 .1 8 1 0 .2 6 / + 1 .6 3 1 0 4 0 6 7 1 .6 4 0 .2 9 4 4 4 .2 1 2 .1 8 1 1 .4 0 / + 0 .4 9 1 1 0 0 1 66 1.28 4 4 4 .2 1 0 .1 6 1 0 .2 1 / - 0 . 3 1 1 1 3 0 2 5 6 1 .1 3 0 .2 6 3 4 4 .6 1 0 .1 6 9 .8 2 / + 0 .0 8 10 8 8 2 8 5 1 .1 4 4 4 4 .6 1 2 .1 6 1 0 .9 8 0 .2 4 + 0 .9 4 1 0 0 0 1 96 1 .1 0

S.K. 3 6 .6 1 8 1 0 2 13 2 4 3 6 .5 1 2 .1 6 8 .0 5 / + 3 .8 5 7 3 2 11 1.01 0 .2 6 4 3 7 .3 1 2 .1 6 9 .7 6 / + 2 .2 0 8 2 0 3 5 1 .1 4 4 3 6 .9 1 0 .1 6 9 .8 3 / - 0 . 1 1 9 3 5 128 1 .1 7 0 .4 4 3 3 6 .5 1 0 .1 6 9 .6 1 / + 0 .1 1 8 6 8 6 7 1 .2 3 4 3 6 .4 1 2 .1 6 1 1 .5 2 0 .5 1 + 0 .1 3 8 9 7 4 7 1 .0 6

1 Initial body weight. 2 Average of urinary excretion for 3 days, when consuming a normal diet.

In a previous experiment, boys main­ maintained. In the present study, how­ tained positive nitrogen balance at levels ever, the excretion of 17-ketosteroids re­ of 11 and 12 g of nitrogen. Girls of the mained almost constant throughout the same age group showed greater individual experiment. variation with respect to nitrogen in main­ taining positive nitrogen balance. Possibly ACKNOWLEDGMENTS this is because the growth spurt in girls We wish to express our appreciation to occurs earlier than in boys of this age. Taeko Ishihara, Youko Masana, Terumi Moreover, individual variation in the onset Sagara and Fusa Ohki for their technical and degree of the pubertal growth spurt assistance. may be related to the difference in require­ ments among the girls of the same age. LITERATURE CITED Further studies are required to clarify this 1. Nakagawa, I., T. Takahashi and T. Suzuki point. Yoshimura (17) reported that after 1960 Amino acid requirements of children. J. Nutrition, 71: 176. feeding a low protein diet the urinary ex­ 2. Nakagawa, I., T. Takahashi and T. Suzuki cretion of 17-ketosteroids decreased, al­ 1961 Amino acid requirements of children: though the nitrogen equilibrium was Isoleucine and leucine. J. Nutrition, 73; 186. 3 3 6 I. NAKAGAWA, T. TAKAHASHI, T. SUZUKI AND K. KOBAYASHI

3. Nakagawa, I., T. Takahashi and T. Suzuki acid requirements of young women. III. 1961 Amino acid requirements of children. Tryptophan. J. Nutrition, 58: 219. Minimal needs of lysine and methionine 11. Leverton, R. M., N. Johnson, J. Ellison, D. based on nitrogen balance method. J. Nutri­ Geschwender and F. Schmidt 1956 The tion, 74: 401. quantitative amino acid requirements of 4. Nakagawa, I., T. Takahashi, T. Suzuki and young women. IV. Phenylalanine with and K. Kobayashi 1962 Amino acid require­ without tyrosine. J. Nutrition, 58: 341. ments of children: Minimal needs of threo­ 12. Leverton, R. M., J. Ellison, N. Johnson, J. nine, valine and phenylalanine based on Pazur, F. Schmidt and D. Geschwender 1956 nitrogen balance method. J. Nutrition, 77: The quantitative amino acid requirements of 6 1 . young women. V. Leucine. J. Nutrition, 58: 5. Nakagawa, I., T. Takahashi, T. Suzuki and 3 5 5 . K. Kobayashi 1963 Amino acids require­ 13. Jones, E. M., C. A. Baumann and M. S. Rey­ ments of children: Minimal needs of trypto­ nolds 1956 Nitrogen balances of women phan, arginine and histidine based on nitro­ maintained on various levels of lysine. J. gen balance method. J. Nutrition, 80: 305. Nutrition, 60; 549. 6. Nakagawa, I., T. Takahashi, T. Suzuki and 14. Clark, H. E., S. P. Yang, W. Walton and K. Kobayashi 1964 Amino acid require­ E. T. Mertz 1960 Amino acid requirements ments of children: Nitrogen balance at the of men and women. II. Relation of lysine minimal level of essential amino acids. J. requirement to sex, body size, basal caloric Nutrition, 83: 115. expenditure and creatinine excretion. J. 7. Zimmermann, W. 1937 Colorimetrische Nutrition, 71: 2 2 9 . Bestimmung der Keimdriisenhormone. 2. 15. Swendseid, M. E., I. Williams and M. S. Mitteilung. Ztschr. Physiol. Chem., 245: 47. Dunn 1956 Amino acid requirements of 8. Leverton, R. M., M. R. Gram, M. Chaloupka, young women based on nitrogen balance E. Brodovsky and A. Mitchell 1956 The data. I. The sulfur-containing amino acids. quantitative amino acid requirements of J. Nutrition, 58: 495. young women. I. Threonine. J. Nutrition, 16. Swendseid, M. E., and M. S. Dunn 1956 5 8 : 59. Amino acid requirements of young women 9. Leverton, R. M., M. R. Gram, E. Brodovsky, based on nitrogen balance data. II. Studies on M. Chaloupka, A. Mitchell and N. Johnson isoleucine and on minimum amounts of the 1956 The quantitative amino acid require­ eight essential amino acids fed simultane­ ments of young women. II. Valine. J. Nutri­ ously. J. Nutrition, 58; 507. tion, 58: 83. 17. Yoshimura, H. 1961 Adult protein require­ 10. Leverton, R. M., N. Johnson, J. Pazur and ments. Federation Proc., 20 (no. 1, part 3, J. Ellison 1956 The quantitative amino suppl. no. 7): 103. Effect of Age and Dietary Fat on Serum Protein Components of the Rat

FLORENCE L. LAKSHMANAN a n d MILDRED ADAMS Human Nutrition Research Division, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland

ABSTRACT The effects of age and dietary fat on the concentration of the protein components in the sera of rats were investigated. Two groups of diets were studied: 1 ) a semipurified diet with the level and kind of fat varied; and 2) the same semi- purified diet but with 25% replaced by dried, cooked egg and the kind but not level of fat varied. Of the serum protein components — pre-albumin, albumin + ai-globulin, a2-, [3- and 7-globulins — pre-albumin concentration was most dependent on diet. Amount and incidence of pre-albumin varied with age and with diet, particularly with the kind and level of fat. Differences observed with the kind of fat did not appear to relate to any specific characteristic of the fat. The age at which high levels of pre­ albumin occurred varied with dietary fat. The albumin and ai-globulin, the /3- and the 7-globulin all tended to be influenced by diet with the differences generally related to level of fat. The 112-globulin was least influenced by diet. At high levels of pre­ albumin, concentration of all except aa-globulin decreased. With age, albumin + ai-globulin decreased and the other globulins increased.

Several factors — heredity, environment, Mendel salt mixture (8), 2 g cellulose age, sex, water intake, nutritional history flour and 52 g sucrose/100 g of diet. — may affect the electrophoretic distribu­ Diet 2: SP 8 HVO containing 0.6 g cry­ tion of the major, well-recognized serum stalline cholesterol,/100 g of diet. protein components (1-3). Proteins mi­ Diet 3: SP 16 HVO, as no. 1 except grating more rapidly than albumin (pre­ 16 g HVO and 44 g sucrose/100 g of diet. albumin) have also been observed in serum Diet 4: SP 16 Blend, as diet 3 except (4). Halliday and Kekwick (5) reported HVO was replaced by a blend of fats simi­ that the concentration of serum pre­ lar in fatty acid composition to diets in the albumin was higher in young rats than United States based on the Household Food in older animals. In the sera of normal Consumption Survey of 1955 (9). The individuals in a state of alimentary hyper­ approximate composition of the blend was lipemia, Herbst and Hurley (6) have ob­ 39% saturated fatty acids, 46% oleic acid served significant amounts of this com­ and 9% linoleic acid. ponent following intravenous injection of Diet 5: SPE-HVO, 75 g SP 8 HVO + 25 g heparin. Investigations in this laboratory dried cooked egg. have shown the presence of high levels of Diets 6 to 10: The 6 g HVO in diet 5 was this component in the sera of rats fed a replaced by 6 g corn oil, safflower oil, lard, diet containing 25% whole egg (7). This butter or the blend of fats. Each rat re­ report deals with further studies to deter­ ceived weekly percomorph oil supplying mine the combined effect of diet and age approximately 3000 units of vitamin A and and the extent to which dietary fat may 400 units of vitamin D and in addition influence the presence or absence of pre­ 36 mg of a-tocopheryl acetate in 0.01 ml albumin as well as the proportion of the cottonseed oil; 10 g fresh kale were fed other serum proteins. twice a week. The fat content of diets 1 and 2 was 9% , of diets 3 and 4, 17% , and EXPERIMENTAL of diets 5 to 10, 19% . The cholesterol con­ The diets fed were as follows: tent of the egg-containing diets was 0.5% , Diet 1: SP 8 HVO, a semipurified diet approximately that of diet 2. containing 16 g casein, 8 g lactalbum in, 8 g hydrogenated vegetable oil (HVO),* 1 10 g Received for publication February 8, 1965. 1 Crisco, Procter and Gamble, Cincinnati; contain­ brewer’s yeast, 4 g modified Osborne and ing approximately 10% linoleic acid.

J. N utritio n , 86 : ’65 337 3 3 8 FLORENCE L. LAKSHMANAN AND MILDRED ADAMS

Male BHE (10) rats were weaned at 21 The effect of diet and age on the occur­ days of age and caged individually in a rence and concentration of serum pre­ room in which the temperature and humid­ albumin is shown in table 1. In this table, ity were maintained at approximately as well as in succeeding tables, data for 26.7° and 40%, respectively. From wean­ the serum components are expressed as ing, each animal was fed ad libitum one of percentage of total protein. Data are not the 10 experimental diets, with water avail­ included for animals that were losing able at all times. At scheduled intervals weight consistently or that were moribund rats were killed following an overnight when killed. The results have been com­ fast. At the time of killing, the animal bined when there were no consistent dif­ was anesthetized with sodium amytal (10 ferences between age groups. m g/100 g of body weight), the blood re­ Pre-albumin was observed in sera from moved from the left ventricle of the heart some of the rats, regardless of the diet fed. and tissues excised for histological exami­ The percentage of sera containing pre­ nation. albumin, as well as the relative concen­ Prior to electrophoretic analysis, the tration of this component varied with diet serum was diluted 1:5 with veronal buffer and with age. The addition of cholesterol (pH 8.6, 0.1 a) and dialyzed against the to the semipurified diet (8 HVO) caused buffer overnight at 10°. The protein com­ no significant change in level of pre­ ponents of the serum were analyzed by albumin nor incidence of pre-albumin in moving boundary electrophoresis (Aminco sera of these animals. With these two low portable apparatus) and the percentages fat-containing diets the percentage of pre­ and mobilities of the individual compo­ albumin was significantly higher (P < nents were calculated from the ascending 0.01) in the sera of 550-day-old rats than Schlieren patterns. The total protein and in the sera of younger rats. nonprotein nitrogen content of some of the With the 2 diets containing the higher sera were determined by a micro-Kjeldahl level of fat without egg, the percentage of method using a 2-step mixed indicator pre-albumin tended to be high at all ages (11). Significant differences within a and was significantly higher in sera of rats group were determined by means of the 350 and 450 days old than in rats of com­ t test and comparisons were made at the parable age fed at the lower level of fat 5 and 1% levels of probability. without egg. RESULTS AND DISCUSSION Although the diets containing egg were Total serum protein concentrations (5.7 identical in all respects except for the kind to 6.9) fell within the range generally of fat accompanying the egg fat, some sig­ reported for the rat (12). Although some nificant differences were noted. When the differences related to age and diet were fat was HVO, the results for rats 250 days significant, they were not sufficiently large or younger were similar to those obtained to influence interpretation of the results when the diet contained the low level of included in this report. HVO without egg. By 350 days, the per­ The mobilities of the major protein com­ centage of rats with pre-albumin in their sera2 as well as the concentration was ponents — albumin, a,-, a2-, 3- and y-globu- lin — were also comparable with values significantly higher (P < 0.01) than was reported in the literature for the rat (13). observed in the younger rats and was simi­ Neither diet nor age influenced the mobili­ lar to that observed when the diets con­ ties of these components. Although pro­ tained a high level of fat without egg. teins migrating more rapidly than albumin With the blend of fats or with safflower oil were not always present, they were ob­ the results were similar in general to those served in the sera of some of the rats with observed with HVO. When the SPE diet each of the experimental diets. Pre-albumin contained butter, lard or com oil, levels of mobility was not influenced by age and was pre-albumin were generally low, with this consistently lower when rats were fed component absent in 250-day-old rats fed SP 8 HVO (6.7 cm2 v-1 sec-1) than when lard or corn oil. In contrast, pre-albumin fed any of the other diets (7.2 to 7.6 cm2 2 W hen dealing with percentage of rats with pre­ album in in their sera, significance was based on Chi- V 1 sec-1). square analysis. AGE AND DIETARY FAT ON RAT SERUM PROTEINS 3 3 9

TABLE 1 Effect of diet and age on the occurrence and concentration of pre-albumin (PA) in rat serum

T o t a l R a t s Diet and age (days) n o . o f w i t h p r e - P A , % o f protein avg 1 r a t s a l b u m i n

% SP Diet w ithout egg (SP) 8 H V O 1 5 0 5 2 0 3 . 3 2 5 0 6 3 3 4 . 9 ± 1 . 6 2 3 5 0 , 4 5 0 1 0 4 0 3 . 0 ± 0 . 3 5 5 0 6 5 0 1 0 . 5 ± 3 . 6

8 HV O-(-cholesterol 2 5 0 4 5 0 2 . 8 ± 0 . 2 3 5 0 , 4 5 0 1 0 4 0 4 . 2 ± 0 . 4 5 5 0 4 7 5 9 . 3 ± 2 . 6

1 6 H V O 3 5 0 9 6 7 9 . 3 ± 2 . 0 4 5 0 9 5 6 8 . 2 ± 2 . 2 5 5 0 5 4 0 1 0 . 1 ± 2 . 6

1 6 B l e n d 3 5 0 7 7 1 1 3 . 8 ± 2 . 3 4 5 0 1 1 4 5 7 . 4 ± 1 .6

SP Diet w ith egg (SPE) HVO 1 5 0 5 0 2 5 0 1 8 3 9 5 . 0 ± 1 .0 3 5 0 1 3 1 0 0 1 0 . 8 ± 1 . 6 4 5 0 , 5 5 0 1 0 4 0 1 1 . 6 ± 6 . 6

B l e n d 2 5 0 8 7 5 8 . 0 ± 2 . 2 3 5 0 , 4 5 0 1 2 8 3 1 2 . 8 ± 2 . 7

Safflow er oil 2 5 0 8 7 5 9 . 8 ± 4 . 1 3 5 0 , 4 5 0 1 0 8 0 1 1 . 3 ± 3 . 3

B u t t e r 2 5 0 5 1 0 0 5 . 7 ± 1 .1 350, 450, 550 1 1 8 2 6 . 1 ± 0 . 8

L a r d 2 5 0 5 0 350,450, 550 1 2 5 8 5 . 9 ± 1 .2

C o r n o i l 2 5 0 4 0 350, 450, 550 1 1 7 3 5 . 0 ± 0 . 9

1 Average based on values for rats with PA in their sera. 2 M ean ± s e . was present in 100% of the sera from 250- to relate to any specific characteristic of day-old rats fed the butter-containing diet. the fat ingested. Lard with a high concen­ The level of pre-albumin in the sera of the tration of saturated fatty acids yielded re­ older rats fed butter or corn oil was sig­ sults similar to those with corn oil, which nificantly lower (P < 0.05) than that in has a high concentration of polyunsatu­ the sera of rats of comparable age fed rated fatty acids. The level of pre-albumin SPE-HVO. Although the results reported in the sera of rats fed the diets containing indicate that the presence and concentra­ corn or safflower oil differed significantly, tion of a rapidly moving component(s) is although both of these oils contained high influenced by the kind and level of dietary concentrations of polyunsaturated fatty fat, the differences observed do not appear acids. 3 4 0 FLORENCE L. LAKSHMANAN AND MILDRED ADAMS

Preliminary investigations in this labo­ of 1.0 or less), both the incidence and con­ ratory on the chemical nature of pre­ centration of pre-albumin showed a con­ albumin have shown this fraction to con­ sistent increase with increasing kidney tain a lipid moiety.3 By the use of isotopes damage. Significant amounts of a rapidly and thin-layer chromatography Glover and moving component in sera of some neph­ Joo4 have determined the kinds of lipid rotic patients have also been reported material in a pre-albumin fraction obtained (16, 17). on paper electropherograms. They have Data to show the influence of diet and concluded that this fraction may play a age on the five major protein components significant part in the transport and uptake are presented in table 3. A combined value of lipids by tissues since it is a molecular for albumin and cu-globulin is given be­ aggregate smaller in size than serum lipo­ cause of the poor separation of these com­ proteins and the lipid components are more ponents in some sera. The results are re­ labile than those in the lipoproteins. In ported for 2 levels of pre-albumin, zero contrast, Losticky (14) has reported that to 6%, and 6% and over, because the the lipid pre-albumin of immunoelectro- presence of an additional component in pherograms is formed by the binding of appreciable amounts necessarily influences unesterified fatty acids to cu-lipoprotein the proportions of the other proteins. The after the unesterified fatty acids are re­ influence of the presence of pre-albumin leased by lipase from triglycerides of low- on the relative concentration of the other density lipoproteins. More information is serum proteins was too small to be signifi­ needed to establish the chemical nature of cant when the level of pre-albumin was these rapidly moving components and to below 6%. Data for rats more than 450 determine their role in the transport of fat. days old were limited due to poor survival Nephrosis has been found to be the with some of the diets, particularly those major cause of death of BHE rats and to be containing egg. accelerated by certain dietary combina­ The influence of diet on the relative per­ tions (15). This condition occurred even centage of the various serum proteins was in animals that were maintaining weight apparent in the results for 250- to 450-day- at the time of killing. Summarized in table old rats having less than 6% pre-albumin 2 are data relating pre-albumin concentra­ in their sera. The concentrations of the tion of serum to extent of kidney damage proteins in the sera of rats fed the 3 diets based on microscopic examination. The containing HVO as the chief source of fat procedure used for rating damage in the were similar except for y-globulin concen- kidney is described in detail elsewhere 3 Lakshmanan, F. L. 1963 Factors influencing the (15). Although small amounts of pre­ presence of rapidly migrating serum protein com­ ponentes), PA. Federation Proc., 22: 608 (abstract). albumin were present in the sera of 36% 4 Glover, J., and C. N. Joo 1963 Nature and origin of the animals with kidneys showing no or of the lipids in the pre-albumin fraction of electro­ pherograms of blood serum. Biochem. J., 88: 65P little evidence of structural change (rating (abstract).

TABLE 2 Relation of the degree of nephrosis to the occurrence and concentration of pre-albumin (PA) in rat serum

Kidney histology 1 Total Rats no. of with pre­ PA, % of Hyalin Cystic Glomerular rats albumin protein avg 2 % 0.0 0.0 0.0 50 20 3 .9 ± 0.4 3 1.0 0.0 0.0 79 47 5.5 ± 0.5 2.0 0.0 0.0 14 71 6 .3 ± 1.1 1.0 1.2 0.3 11 82 7.7 ± 1 .3 2.0 1.0 0.0 25 84 10 .3 ± 1.6 2.0 2.4 1.3 22 91 12.1 + 1.5 3.0 2.7 1.5 20 100 12.3 + 1.9 1 Rating based on a maximal rating of 4.0 for each type of damage. 2 Average based on values for rats with PA in their sera. 3 M ean + s e . AGE AND DIETARY FAT ON RAT SERUM PROTEINS 3 4 1

TABLE 3 Effect of diet and age on the protein components of sera classified according to level of pre-albumin

Concentration of protein components, % o f p r o t e i n T o t a l n o . o f D i e t Albumin and r a t s

Zero to 6% pre-album in, 250 to 450 days of age S P 8 H V O 1 0 65.2 ± 1.6 1 8 . 3 + 0 . 4 1 6 . 7 + 0 . 7 8 . 6 + 1 . 1 SP 8 HVO + cholesterol 1 0 6 8 . 0 ± 1 .1 7 . 9 + 0 . 5 1 6 .1 + 0 . 5 6 . 3 + 0 . 8 S P 1 6 H V O 1 2 6 9 . 6 ± 1 .8 8 . 2 + 0 . 4 1 5 . 6 + 1 . 0 4 . 8 + 0 . 7 SP 16 Blend 9 7 3 . 0 ± 1 .3 7 . 9 + 0 . 7 1 4 . 5 + 0 . 9 4 . 2 + 0 . 6 SPE-HVO 8 7 2 . 9 ± 2 . 1 6 . 9 ± 0 . 5 1 4 . 2 ± 1 . 3 4 . 4 + 0 . 9 S P E - B l e n d 4 7 3 . 3 ± 2 . 7 7 .1 + 0 . 6 1 2 . 8 + 3 . 3 5 . 2 + 1 . 4 SPE-Safflower oil 5 7 2 . 3 ± 1 .2 7 . 9 + 0 . 9 1 3 . 8 + 0 . 9 3 . 5 + 0 . 7 S P E - B u t t e r 7 7 3 . 4 ± 1 .7 8 . 4 + 0 . 7 1 1 . 6 + 1 . 0 3 . 3 ± 0 . 5 S P E - L a r d 6 7 1 . 4 + 1 . 8 8 . 4 + 0 . 6 1 4 . 4 + 1 . 2 5 . 2 ± 0 . 9 SPE-Corn oil 8 6 5 . 8 ± 1 .0 9 . 1 + 0 . 8 1 5 . 6 + 0 . 9 6 . 8 + 0 . 3

M ore than 6% pre-album in, 250 to 450 days of age S P 1 6 H V O 6 6 2 . 2 ± 0 . 8 9 . 0 + 1 .3 1 2 . 6 ± 1 .5 3 . 9 + 0 . 7 SP 16 Blend 9 6 4 . 3 + 1 .4 8 . 7 + 0 . 6 1 2 . 1 + 1 .0 3 . 5 + 1 . 0 SPE-HVO 1 3 6 6 . 9 + 1 .6 7 . 8 + 0 . 5 9 . 4 + 0 . 7 2 . 6 + 0 . 6 S P E - B l e n d 8 6 7 . 5 + 2 . 5 6 . 8 + 0 . 5 8 . 7 + 0 . 6 1 . 9 + 0 . 6 SPE-Safflower oil 5 6 9 . 9 + 2 . 3 7 .1 + 1 .3 6 . 5 + 1 . 2 0 . 9 ± 0 . 5 S P E - L a r d 2 7 3 . 5 + 2 . 2 7 . 0 + 2 . 3 8 . 2 + 2 . 4 2 . 0 + 0 . 7

Z e r o t o 6 % pre-album in, over 450 days of age S P 8 H V O 3 5 4 . 4 ± 2 . 0 1 1 . 2 + 1 . 5 2 1 . 6 + 1 . 3 1 2 . 7 + 1 . 8 SP 8 HVO + cholesterol 2 5 6 . 3 ± 1 .5 1 1 .1 ± 1 .6 1 9 . 6 ± 0 . 4 1 0 . 8 + 0 . 4 S P 1 6 H V O 3 6 2 . 8 + 1 .9 9 . 0 + 1 . 4 2 1 . 3 + 1 .9 7 . 0 + 1 . 6 SPE-HVO 2 6 4 . 5 ± 0 . 6 1 0 . 3 ± 0 . 8 1 8 . 2 + 0 . 4 6 . 9 + 0 . 3 S P E - L a r d 2 4 6 . 0 + 6 . 0 1 2 . 0 + 2 . 2 2 5 . 6 + 0 . 7 1 4 . 4 + 2 . 2 SPE-Com oil 2 5 9 . 1 + 0 . 3 1 2 . 6 + 0 . 2 1 5 . 9 ± 0 . 8 9 . 0 + 1 . 4

1 M ean ± se. tration which was significantly higher with some diets. With this group of sera (P < 0.01) in sera of rats fed SP 8 HVO having a high concentration of pre­ than in sera of rats fed SP 16 HVO. In albumin, (3-globulin was the component general, the distribution of proteins in the most influenced by diet and tended to be sera of rats fed SP 16 Blend and the egg- low when the diet contained egg. The con­ containing diets was similar except for centration of Y-globulin was extremely low SPE-corn oil. W hen compared with the with the diet containing safflower oil. results for SP 8 HVO, the m ajor differences When the concentration of pre-albumin were in the higher levels of albumin and exceeded 6% there was a consistent tend­ ai-globulin and in the lower levels of ency for the levels of albumin and ai-globu- y-globulin, which were significant for all lin, (3-globulin and Y-globulin to decrease except y-globulin when the diet was SPE- when compared with the levels in sera of Blend. In contrast, the results with com rats fed the same diet but with a concen- oil resembled those obtained with SP 8 tration of pre-albumin of less than 6%. HVO rather than those obtained with the The decrease in albumin and co-globulin other egg-containing diets. The concentra­ concentration was significant (P < 0.01) tion of (3-globulin was significantly lower when SP 16 HVO or SP 16 Blend was fed; (P<0.01) in the sera of rats fed SPE- the decrease in (3-globulin concentration butter than in the sera of rats fed SP 8 was significant (P<0.01) when SPE- HVO with or without cholesterol. The safflower oil was fed. The sera of rats serum protein component least affected by fed SPE-HVO had significantly reduced diet was co-globulin. amounts of albumin and co-globulin (P < When the level of pre-albumin exceeded 0.05) as well as (3-globulin ( P < 0 .0 1 ) . 6% , diet comparisons were limited because Y-Globulin concentrations tended to be of the lack of high levels of this component lower when pre-albumin levels were high. 3 4 2 FLORENCE L. LAKSHMANAN AND MILDRED ADAMS

These differences in concentration may 5. Halliday, R., and R. A. Kekwick 1957 actually represent decreases in the a- or Electrophoretic analysis of the sera of young 146: [3-lipoproteins instead of the globulins, rats. Proc. Roy. Soc. of London, 4 3 1 . 6. Herbst, F. S. M., and N. A. Hurley 1954 since each boundary of the electrophoretic Effects of heparin on alimentary hyper­ pattern is the total amount of protein, lipo­ lipem ia, an electrophoretic study. J. Clin. protein, and glycoprotein present. Herbst I n v e s t . , 33: 9 0 7 . and Hurley (6) have demonstrated that 7. Adams, M. 1964 Diet as a factor in length sera of normal hyperlipemic (alimentary- of life and in structure and composition of tissues of the rat with aging. USDA Home induced) individuals injected with heparin Economics Research Report no. 24. U. S. had decreased amounts of [3-globulin in D epartm ent of Agriculture, W ashington, D. C. their moving boundary electrophoretic pat­ 8. Osborne, T. B, and L. B. Mendel 1919 terns as well as the presence of a compo­ The nutritive value of the w heat kernel and nent migrating ahead of albumin. In their its m illing products. J. Biol. Chem ., 37.- 557. study simultaneous examination of the 9. Irwin, M. I., and H. F. W iese 1961 Varia­ tions in linoleic acid content of dietary fat lipoprotein and protein patterns obtained in relation to m etabolism of fat, nitrogen and by paper electrophoresis revealed that the m inerals, and to changes in blood lipids. J. lipid band, normally present in the [3-globu­ N utrition, 74: 2 1 7 . lin area, was now in the area of a2- or 10. M arshall, M. W ., and H. E. Hildebrand 1963 globulin. Instead of the lipid band, nor­ Differences in rat strain response to three mally present in the ai-globulin area, lipid diets of different composition. J. N utrition, 79: 2 2 7 . material migrated ahead of albumin. 11. Sher, I. H. 1955 Two-step mixed indicator Despite the limited numbers in the older for Kjeldahl nitrogen titration. Analyt. age group in this present study, it was Chem ., 27: 831. obvious that the relative concentration of 12. Dittmer, D. S. 1961 Blood and Other Body the various serum proteins was influenced Fluids. Federation Am. Soc. for Exp. Biol., by age. Albumin and co-globulin decreased W ashington, D. C., p. 186. with age; a2-, [3- and y-globulin increased 13. Dittmer, D. S. 1961 Blood and Other Body Fluids. Federation Am. Soc. for Exp. Biol., with age. These differences are similar to W ashington, D. C., p. 61. those reported in the literature for humans 14. Losticky, C. 1963 The formation of lipid and animals (18-20). pre-album in. Clin. Chim. Acta, 8: 8 5 9 . 15. Durand, A. M. A., M. Fisher and M. Adams ACKNOWLEDGMENT 1964 Histology in rats as influenced by age The authors are grateful to A. M. Allen and diet. I. Renal and cardiovascular sys­ 77: Durand, M.D., and M. Fisher (biologist) tems. Arch. Path., 2 6 8 . 16. Aly, F. W ., and K. H. Niederhellm ann 1958 for their evaluation of the renal changes Investigations for pre-albumin in hum an in the animals. serum . Klin. W chnschr., 36: 954. 17. Peetoom, F., and E. Kramer 1962 Immuno- LITERATURE CITED electrophoretic investigation of the p-proteins (pre-album ins) of some pathological sera. 1. Bier, M. 1959 Electrophoresis: Theory, Vox Sanguinis, 7: 298. M ethods and Applications. Academic Press, New York, p. 391. 18. Das, B. C., and S. K. Bhattacharya 1961 Changes in hum an serum protein fractions 2. Dimopoullos, G. T. 1961 Plasm a Proteins in Health and Disease. Annals of the New with age and weight. Canad. J. Biochem. 39: York Academy of Sciences, vol. 94, part 4. P h y s i o l . , 5 6 9 . N e w Y o r k . 19. Costanzo, F., and P. Cognasso 1961 Serum 3. Putnam, F. W. 1960 The Plasma Proteins, protein levels in relation to aging. Acta vol. 2. Academic Press, New York, chapters G erontol., 11: 1 2 7 . 1 1 , 1 9 . 20. Rafsky, H. A., A. A. Brill, K. G. Stern and 4. Deutsch, H. F., and M. B. Goodloe 1945 H. Corey 1952 Electrophoretic studies on An electrophoretic survey of various anim al the serum of “norm al” aged individuals. Am. plasm a. J. Biol. Chem., 161: 1. J. M ed. Sci., 224: 522. Effect of High Intakes of Thiamine, Riboflavin and Pyridoxine on Reproduction in Rats and Vitamin Requirements of the Offspring 1 *

SISTER M. F. SCHUMACHER, M. A. WILLIAMS a n d R. L. LYMAN Department of Nutritional Sciences, University of California, Berkeley, California

ABSTRACT Female rats were fed during pregnancy and lactation a control diet containing 0.25 mg pyridoxine, 0.4 mg thiam ine, and 0.4 mg riboflavin/100 g, or a high pyridoxine, high thiam ine, or high riboflavin diet containing the specified vita­ m in at 25 tim es the level of the control diet. The levels of these vitam ins in the car­ casses of the young at birth and in the liver at w eaning were determ ined. The effect of m aternal vitam in intake on the vitam in requirem ents of the young was tested in 2 ways: 1) by comparing the rate of depletion of the young of females fed the high vitam in diets with the rate of depletion of the control group, and 2) by the growth response of the depleted young to graded levels of the vitam in fed in excess in the m aternal diet. It was concluded that high intakes of thiam ine, riboflavin, or pyridox­ ine during the reproductive period had no effect on the young, as shown by litter size at birth, growth until w eaning, or their vitam in requirem ents after weaning.

Relatively little attention has been given diet did not appear to increase the vitamin to the effects of high intakes of B vitamins Be requirement of young rats as indicated on reproduction. Massive doses of thia­ by rate of vitamin B6 depletion. If this had mine have been reported to interfere with been the case, then vitamin Be deficiency lactation, produce cannibalism, and de­ should have developed more rapidly in the crease fertility in rats (1, 2). Richards young from the dams fed the high pyri­ ('3) also reported that excess thiamine fed doxine diets. No test was made, however, to female rats increased mortality and de­ of the pyridoxine requirement of the young creased the weights of the young at wean­ rats after they had been depleted of pyri­ ing. However, Morrison and Sarett (4) doxine. This point is important, since an reported normal reproduction in rats fed increased need for pyridoxine in the young high levels of thiamine. An abnormally during the depletion period may have been high need for pyridoxine was reported in masked by the higher liver stores of the a newborn human infant whose mother young from the dams fed the high pyri­ had received large doses of pyridoxine in­ doxine diets. Greater liver stores were tramuscularly during the first trimester of pregnancy (5). It was suggested that ex­ reported by Morrison and Sarett (4). cess pyridoxine intake during gestation The following experiment tested the ef­ may have increased the pyridoxine require­ fect of maternal intake of pyridoxine, thi­ ment of the infant. Later, Hunt (6) re­ amine, or riboflavin on the rate at which ported that the pyridoxine intake of the the young rats were depleted of each vita­ dam during gestation did not affect the min, as well as on the subsequent growth rate of vitamin B6 depletion or increase the response of the depleted young to graded occurrence of convulsions in young rats levels of the vitamins. after birth. Morrison and Sarett (4) also reported that a high intake of pyridoxine METHODS during pregnancy did not affect reproduc­ Animals. Thirty female rats (about tion or increase the rate of vitamin B6 de­ 300 g) of the Long-Evans strain, were used pletion in young rats in the period from 3 for each of 3 experiments. In each experi- to 5 weeks of age. The preceding studies have shown that Received for publication March 31, 1965. high levels of pyridoxine in the maternal 1 Supported in part by USDA Project W-57.

J . N u t r i t i o n , 8 6 : ’6 5 343 3 4 4 SISTER M. F. SCHUMACHER, M. A. WILLIAMS AND R. L. LYMAN ment, 15 females were fed the basal diet.2 Once depletion was established, the ani­ The other rats were divided into 3 groups mals were fed the appropriate vitamin at of 5 rats each. One group was fed the a below-minimal or minimal level. The high pyridoxine diet, the second group the minimal level for pyridoxine was 15 ug/ high thiamine diet, and the third group rat/day (4);3 for thiamine, 10 ug/rat/day the high riboflavin diet. The high vitamin (8); for riboflavin, 24 ng/rat/day (7). diets contained 25 times the level of pyri­ The below-minimal levels were one-half of doxine, thiamine, or riboflavin used in the the minimal level. Negative controls (with­ basal diet. The basal diet contained: (in out pyridoxine, thiamine, or riboflavin) mg/100 g) pyridoxine, 0.25; thiamine, also continued to be fed the deficient diets. 0.4; and riboflavin, 0.4 (7). The high vita­ Each level of the vitamin was made up min diets contained: (in mg/100 g) pyri­ in a 20% ethyl alcohol solution. Two milli­ doxine, 6.25; thiamine, 10; and riboflavin, liters of this solution were fed 3 times a 10. Food and water were provided ad week. The total supplement over a period libitum. Weight gain and food intake were of one week was equivalent to 7 times the recorded 3 times weekly. daily amount indicated. The females were mated after they had Vitamin analyses. The carcasses of the been fed the experimental diets for 2 newborn and the livers of the weanlings weeks, and these diets were continued were homogenized and analyzed for pyri­ during gestation and lactation. The num­ doxine, thiamine, and riboflavin. Pyridox­ ber and average weight of the newborn ine was determined microbiologically with were recorded as soon as possible after the Saccharomyces carlsbergenesis in a modi­ birth of each litter. Some of the newborn fication of the method of Rabinowitz and from each diet group were decapitated, the Snell (9). Thiamine was determined by stomachs and intestinal tracts removed, the thiochrome method and riboflavin was and the carcasses weighed and frozen. determined fluorometrically (10). The remaining young were nursed for 21 days, when they were weaned. The young RESULTS had access to the maternal diet during the Reproduction. Reproduction data are nursing period. At weaning, representa­ shown in table 1. The reproduction of the tive young from each group were decapi­ control group and the high thiamine group tated, the livers removed, weighed and was considerably better (68% for both frozen. A number of the male weanlings, groups) than that of the high riboflavin or weighing from 40 to 50 g, were saved for high pyridoxine groups (38 and 47%, re­ the growth study. spectively). The value of 68% was slightly The growth study was divided into a de­ less than that usually found for the stock pletion period and a supplementation colony females of this laboratory (70 to period. The rats were housed individually 75% ). in galvanized, screen-bottom cages. Food The average birth weight, number of and tap water were given ad libitum. young per litter, and average weight of Weight gains and food intakes were re­ the young at weaning were not signifi- corded 3 times weekly. Four to five ani­ 2 Composition of basal diet: (g/100 g) vitamin-free mals from the high vitamin maternal casein, 18; sucrose, 61.8; USP Salts 14, 4; Cellu Flour (Chicago Dietetic Supply House, Chicago), 5; cotton­ groups were fed the basal control diet, with seed oil, 9; vitamin-fortified cottonseed oil, 1; vitamin the omission of the vitamin which had mix in sucrose, 1; choline chloride, 0.15. The fortified oil provided per 100 g diet: vitamin A, 1700 ITJ; been fed in excess in the maternal diet. v ita m in D 2, 100 IU; a-tocopheryl acetate, 6.7 mg. The vitamin mixture in sucrose provided: (m g/100 g diet) Weanlings from the dams fed the basal diet thiamine, 0.4; riboflavin, 0.4; pyridoxine, 0.25; Ca were also fed the vitamin-deficient diets. pantothenate, 2.0; inositol, 10.0; biotin, 0.01; folic acid, 0.1; niacinamide, 1.0; vitamin B 12 (0.1% B 12 The remaining rats from each maternal triturate in mannitol), 0.02; menadione, 0.5. group served as controls and were fed the 3 The minimal requirement for pyridoxine for maxi­ mal growth in the rat beyond 6 weeks of age has complete basal ration. All of the diets recently been shown to be greater than 30 ¿ig/day (Beaton, G. H., and M. C. Cheney, Federation Proc., were fed until the deficient groups ceased 24: 624, 1965; Williams, M. A., Federation Proc., to gain (weight plateau) or until their rate 24: 624, 1965). However 15 ¿¿g/day will produce nearly maximal growth in the rat for the first 2 weeks of gain was significantly less than that of after weaning or the first 2 weeks of repletion of pre­ viously depleted rats (Williams, M. A., unpublished the appropriate control group. resu lts). Reproduction data on animals receiving a diet containing levels of thiamine, riboflavin or pyridoxine 25 times that of the control level 3 CN<3 t “ ft to S H«H 3 2 «HU3 «HDO jh 0

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cantly influenced by increasing the levels difference caused by maternal diet in the of pyridoxine, thiamine, or riboflavin in tissue concentration of pyridoxine at birth the maternal diet. The number of off­ did not appear at weaning (21 days after spring per litter from the dams fed the birth). high thiamine ration was somewhat Growth studies — depletion and supple­ smaller than that from the other maternal mentation. Figure 1 shows the growth groups. The average birth weight, how­ curves of the different groups when fed ever, was somewhat greater in the high diets deficient in pyridoxine, thiamine, or thiamine group. The higher mortality riboflavin, as well as the control groups. (birth to weaning) in the high riboflavin The only difference observed during de­ group reflected the complete loss of one pletion was that the onset of thiamine de­ litter (10 young). Two control litters were ficiency was delayed slightly in the rats also lost because the mothers failed to from the dams fed the high thiamine diet. nurse. More data are needed to determine Maternal diet did not affect the develop­ whether the high riboflavin diet increased ment of riboflavin deficiency, in which both mortality. groups reached a plateau in body weight by Tissue vitamin storage. High levels of day 21. The depletion period of vitamin thiamine or riboflavin in the maternal diet Be was arbitrarily ended on day 28 when did not increase the levels of vitamins in the weight gains of the deficient groups the newborn (table 2). The level of pyri­ were significantly less than the gains of doxine in the newborn from the dams fed the groups fed the control diet. Maternal the high pyridoxine diet was significantly pyridoxine intake did not affect pyridoxine higher than the level for the control group depletion in the young (fig. 1). Neither (P < 0.01). At weaning, the offspring of did the maternal diet affect the growth the females fed the high thiamine diet had response of any of the groups to the con­ nearly twice as high a level of liver thia­ trol diet. mine as did the controls. In contrast with Figure 2 shows the growth response of the effect of thiamine, high levels of ribo­ the vitamin-depleted rats to graded levels flavin or pyridoxine in the maternal diet did of the corresponding vitamin. All groups not influence the storage of these 2 vita­ received the supplements for 14 days. mins in the livers of the weanlings. The Growth reflected the level of supplementa-

Fig. 1 Influence of excess thiamine, riboflavin, or pyridoxine in maternal diet on weight gain of male weanling rats fed a diet deficient in the respective vitamin, a ------A, control young fed adequate diet; O----O, experimental young fed adequate diet; A ------A , control young fed depletion diet; • ------•, experimental young fed depletion diet. Numbers in parentheses indicate number of rats in the group. VITAMINS Bn, Bj, AND B6 IN REPRODUCTION 3 4 7

Fig. 2 Influence of excess thiamine, riboflavin, or pyridoxine in maternal diet on weight gain of previously depleted male rats fed graded levels of that vitamin. A------A, control young; O------O, experimental young. Numbers in parentheses indicate number of rats in the group. tion for each vitamin, and maternal diet weights, all of the high vitamin groups and had no significant effect. The greater vari­ the control group compared favorably in ability in the growth of both pyridoxine- performance with the laboratory breeding depleted groups to supplements of 7.5 or colony. The results from the high thia­ 15 Mg pyridoxine is typical of the response mine diet confirm the conclusions of Mor­ of pyridoxine-depleted rats fed at these rison and Sarett (4) that high intakes of levels of the vitamin.4 Nevertheless, the thiamine did not affect the overall repro­ rats from the dams fed the high pyridoxine ductive performance, in contrast with the diet made weight gains similar to those report of Richards (3). from the control dams. If the need for The level of thiamine (0.93 ug/g) in the pyridoxine had been greater in the young tissues of the newborn from either the from the high pyridoxine dams, then their control or the high thiamine group was weight gains at both levels of pyridoxine less than that reported by Barrett and Ever­ would have been less than those of the son (11) and by Brown and Snodgrass control young. This lack of a difference (12). Barrett and Everson reported a in pyridoxine need between the young value of 3.0 ug/g with a maternal diet from the high pyridoxine dams and the containing 1.32 mg/100 g. Brown and control group is supported by the similar Snodgrass reported a level of approxi­ weight changes in the pyridoxine-depleted mately 2 ug/g with a maternal diet con­ groups that continued to be fed the pyri- taining 0.25 or 0.50 mg/100 g. There is doxine-deficient diet. no explanation for these differences ex­ cept that the analyses were not made on DISCUSSION comparable tissues. In the present study, The reduced number of litters for the the vitamin analyses were made on the high riboflavin and high pyridoxine groups exsanguinated carcass, with the head, may be related to the high maternal in­ stomach, and intestinal tract removed. The take of these vitamins. More data are other reports imply that the analyses were needed to establish this point. With re­ made on the entire animal. Fetal storage spect to the number of young per litter of riboflavin (3.34 ug/g) was similar to and the average birth and weaning 4 Williams, M. A., unpublished results. 348 SISTER M. F. SCHUMACHER, M. A. WILLIAMS AND R. L, LYMAN the values of Barrett and Everson (2.5 ug/ rats from the high thiamine maternal diet g). No values for fetal storage of pyridox- probably reflects the increased tissue thia­ ine were found in the literature. mine storage at weaning. At weaning, liver storage of thiamine re­ In conclusion, high levels of thiamine, flected the thiamine level of the maternal riboflavin, or pyridoxine ingested during diet although the maternal diet did not af­ the reproductive period had no effect on fect the carcass thiamine of the newborn. the young, as shown by litter size at birth, The higher level at weaning may reflect growth until weaning, or vitamin require­ the amount of maternal diet eaten by the ments after weaning. young before weaning. The level in the control weanlings (5.8 ug/g) is higher LITERATURE CITED than the value (1.9 u/g) observed by Mor­ rison and Sarett whose control diet con­ 1. Perla, D. 1937 Toxic effects of an excess of vitamin Bi in rats. Proc. Soc. Exp. Biol. tained only 0.15 ug th iam in e /100 g, in Med., 37: 169. contrast with 0.4 mg/100 g in the present 2. Perla, D., and M. Sandberg 1939 Meta­ study. Ochoa and Peters (13) and Byer- bolic interdependence of vitamin Bi and rum and Flokstra (14) had reported pre­ manganese. Reciprocal neutralization of viously that increasing the dietary thia­ their toxic effects. Proc. Soc. Exp. Biol. mine above that needed for maximal Med., 41: 522. 3. Richards, M. B. 1945 Imbalance of vita­ growth increased the tissue concentrations min B factors. Pyridoxine deficiency caused of thiamine. by addition of aneurin and chalk. Brit. Med. High levels of riboflavin or pyridoxine J„ 1: 433. in the maternal diet did not influence the 4. Morrison, A. B., and H. P. Sarett 1959 Ef­ liver storage of these 2 vitamins at wean­ fects of excess thiamine and pyridoxine on ing. The value for riboflavin (21.5 ug/g) growth and reproduction in rats. J. Nutri­ tion, 69: 111. was similar to the value observed by 5. Hunt, A. D„ J. Stokes, W. W. McCrory and Decker and Byerrum (15) in rats 30 days H. H. Stroud 1954 Pyridoxine dependency; after birth (24.5 ug/g). The similarity of report of a case of intractable convulsions in values for liver pyridoxine in both the high the infant controlled by pyridoxine. Pedi­ atrics, 13: 140. pyridoxine and the control groups differs 6. Hunt, A. D. 1957 Abnormally high pyri­ from the results of Morrison and Sarett, doxine requirement. Am. J. Clin. Nutrition, who observed increased levels of pyridox­ 5: 561. ine in the livers of weanlings from dams 7. Brown, R. A., and M. Sturtevant 1949 The fed high pyridoxine diets (7.5 mg/100 g) vitamin requirements of the growing rat. Vitam ins and Hormones, 7: 171. in contrast with a control diet containing 8. Elvehjem, C. A. 1944 Symposium on hu­ 0.15 mg/100 g. Their values were 8.8 man vitamin requirements. Federation Proc., ug/g for the high pyridoxine group and 3: 158. 4.8 ug/g for the control group. In the 9. Rabinowitz, J. C., and E. E. Snell 1947 present paper the values were 7.1 ug/g for Extraction procedures in the microbiological determination of vitamin Bi. Ind. Eng. Chem. the high pyridoxine group and 7.4 ug/g (Anal, ed.), 19: 277. for the control. Perhaps the level of pyri­ 10. Johnson, B. C. 1944 Methods of Vitamin doxine in the diet of Morrison and Sarett Determination. Burgess Publishing Company, (4) was too low for maximal tissue stor­ Minneapolis, Minnesota. 11. Barrett, M., and G. Everson 1951 Deposi­ age, although this is above the stated re­ tion of B vitamins in normally developing quirement (16). fetuses as evidence for increased vitamin The growth studies gave no evidence for needs of the rat for reproduction. I. Thia­ any beneficial effect in the young result­ mine and riboflavin. J. Nutrition, 45: 493. ing from high maternal intakes of these 12. Brown, M., and C. E. Snodgrass 1965 Ef­ fect of dietary level of thiamine on repro­ vitamins during pregnancy and lactation. duction in the rat. J. Nutrition, 85: 102. In addition, there was no evidence that the 13. Ochoa, S., and R. A. Peters 1938 Vitamin vitamin requirements of the offspring were Bj and cocarboxylase in animal tissues. Bio- increased, that is, the rats were made more chem. J., 32: 1501. vitamin-dependent, as a result of high 14. Byerrum, R. U., and J. H. Flokstra 1951 Cocarboxylase and thiamine in tissues of maternal intakes. The slight delay in the rats receiving different concentrations of development of thiamine deficiency in the vitamin Bi in the diet. J. Nutrition, 43: 17. VITAMINS B,. B2, AND B6 IN REPRODUCTION 349

15. Decker, L. E., and R. U. Byerrum 1954 16. National Research Council, Committee on The relationship between dietary riboflavin Animal Nutrition 1962 Nutrient require­ concentration and tissue concentration of ments for laboratory animals, pub. 990. Na­ riboflavin-containing coenzymes and enzymes. tional Academy of Sciences — National Re­ J. Nutrition, 53: 303. search Council, Washington, D. C. Determination of First Limiting Nitrogenous Factor in Corn Protein for Nitrogen Retention in Human Adults 1 *

CONSTANCE KIES, ELEANOR WILLIAMS a n d HAZEL METZ FOX Department of Food and Nutrition, School of Home Economics, University of Nebraska, Lincoln, Nebraska

ABSTRACT Nitrogen retention of adult men fed isocaloric diets was significantly greater when white corn meal provided 8.0 g nitrogen/subject/day than when either 4.0 or 6.0 g nitrogen were supplied from corn protein. When a suboptimal intake of corn meal was fed (6.0 g nitrogen/subject/day), the optimal nitrogen retention level was re-established by the addition of 2.0 g nitrogen from any of several purified essential amino acids or other purified sources of nitrogen, or from both. Therefore, “non­ specific” nitrogen, that is, nitrogen from any metabolically usable, non-toxic source, is the first limiting nitrogenous factor in corn protein for nitrogen retention in adult men.

Cereal grains are the primary source of The 100-day project was divided into a dietary protein for many peoples through­ 3-day introductory period and 3 studies of out the world ( 1 ). Most of the knowledge 31, 50, and 16 days, respectively, con­ dealing with human utilization and metab­ ducted consecutively with no lapse in time olism of cereal proteins has been derived between studies. from laboratory studies based on the as­ Previous studies (9-10) have shown sumption that the essential amino acid that subjects attain nitrogen equilibrium pattern of cereal proteins is the factor that more rapidly in response to controlled diets limits their nutritional value. Accordingly, when they are fed a low nitrogen diet for these studies (2-8) have dealt with at­ a few days preceding the experimental tempts to achieve optimal nitrogen reten­ period. Hence, the purposes of the intro­ tion by supplementing cereal proteins with ductory period were to hasten the attain­ purified essential amino acids or intact ment of nitrogen equilibrium by the sub­ proteins to provide a theoretically balanced jects eating experimental diets more readily pattern of amino acids. through an initial use of a very low nitro­ The purposes of the present project were gen diet, to introduce the subjects to their to establish the minimal corn protein re­ duties and responsibilities, and to deter­ quirement for adult men to attain positive mine the caloric requirements of the in­ nitrogen balance and to determine the first dividual subjects for weight maintenance. limiting nitrogenous factor in corn protein During this time, nitrogen intake was for the maintenance of this degree of nitro­ 2.00 g/'subject/day, 1.30 from white, de- gen retention. germinated corn m eal3 and the remainder from the basal diet composed of a few low- PROCEDURE protein fruits and vegetables. Cornstarch Pertinent data regarding the 10 men, wafers in varying amounts were used to inmates of the Nebraska Penal and Cor­ meet individual caloric requirements for rectional Complex,2 who were subjects for maintenance of weight. the project are shown in table 1. Institu­ Study 1 was divided into 3 experimental tion medical records, verified by physical periods of 10 days each arranged at ran- examinations conducted by a physician, certified that all were in good health at the Received for publication March 25, 1965. 1 Published with the approval of the Director as beginning and throughout the project. paper no. 1705, Journal Series, Nebraska Agricultural Experiment Station. Subjects were housed and received meals 2 The feeding phase of this project was pursued at as a group in an isolated section of the in­ the Reformatory Unit of the Nebraska Penal and Correctional Complex. stitution; however, all maintained their us­ 3 White, degerminated corn meal used in this project was a gift of the Gooch Milling and Elevator Company, ual work assignments. Lincoln, Nebraska.

3 5 0 J . N u t r i t i o n , 8 6 : ’6 5 FIRST LIMITING NITROGENOUS FACTOR IN CORN 3 5 1

TABLE 1 Age, height, weight, and caloric intakes of the subjects Daily caloric intake Calories furnished by fat Subject no. 1 Age Height Weight 2 Studies Study Studies Study 1, 2 3 1, 2 3

years cm kcal/kg kcal/hg kg body tut body tut % % 50 28 185 90.7 41.0 51.0 25 40 51 29 180 73.9 44.5 54.5 15 31 52 34 171 61.2 50.0 60.0 15 29 53 37 175 72.6 41.5 51.5 7 25 54 32 185 81.6 40.2 50.2 15 — 55 26 190.5 88.4 34.1 44.1 7 28 56 31 185 65.8 50.0 60.0 15 29 57 22 183 84.4 38.9 48.9 15 32 58 35 175 65.8 49.9 59.9 15 29 59 30 179 65.9 40.0 50.0 15 — 1 Subjects for studies 1, 2, and 3. Subjects 54 and 59 did not participate in study 3. Subject 59 did not participate in periods 7, 8, 9 and 10 of study 2. 2 Weight at the beginning of the project. All subjects maintained their initial weight or gained weight slightly during studies 1 and 2. During study 3 all subjects showed some gain in weight due to the in­ creased caloric intake. TABLE 2 Comparison of diets used at three levels of intact corn protein intake Intake/subject/day for diet 1 2 3

9 9 9 Corn meal (white, degerminated) 1 354 531 708 Cornstarch 345 175 0 Corn oil ( as required by each subject for weight maintenance) 2 4 2 0 Tomato paste 100 100 100 Peaches 100 100 100 Applesauce 100 100 100 Coffee (dry, instant) 11 11 11 Jelly 30 30 30 Vitamin supplement, daily 3 Mineral supplement, daily 4 Low calorie soft drink, ad libitum N/subject/day provided by corn meal 4.0 6.0 8.0 N/subject/day provided by other dietary items 0.7 0.7 0.7

1 Amino acid composition as follows: (mg amino acid/g of corn nitrogen) tryptophan, 38.5; threonine, 216; isoleucine, 286; leucine, 958; lysine, 135; methionine + cystine, 237; phenylalanine -{-tyrosine, 637; valine, 307. 2 The corn meal contained 0.01 g fat/g of meal. Additional corn oil was added for weight maintenance of individual subjects as shown in table 1. 3 Composed of the following: (m g/subject/day) thiamine• HC1, 1.2; ascorbic acid, 50; riboflavin, 1.5; pyridoxine-HCl, 0.3; niacin, 6.0; vitamin B12, 12.0; and folic acid, 50.0 mEq; vitamin A, 5000 IU; and vitamin D, 400 IU. 4 Composed of the following: (g/subject/day) Ca, 1.00; P, 1.001; Mg, 0.199; Fe, 0.015; Cu, 0.002; I, 0.00015; Mn, 0.002; Zn, 0.0009. dom. White, degerminated corn meal varying among the subjects as shown in was fed in amounts to provide 4.00, 6.00, table 1. Other items in the diet provided or 8.00 g of nitrogen/subject/day as shown 0.70 g of nitrogen/subject/day. Vitamin in table 2. As the amount of corn meal in and mineral supplements and other dietary the diet was increased or decreased, the components were as shown in table 2. Individual allotments of corn meal amount of cornstarch 4 5 was adjusted so as cornstarch, corn oil, mineral supplements to maintain a constant caloric intake for each individual. Individual caloric require­ 4 See footnote 3. 5 Cornstarch and corn oil used in this project was ments were met by varying the corn oil6 a gift of the Corn Products Company, New York, New York. intake, constant for each individual but 6 See footnote 5. 3 5 2 CONSTANCE KIES, ELEANOR WILLIAMS AND HAZEL METZ FOX

and baking powder for each day were mixtures. The purified nitrogen mixtures mixed together and then divided into 3 were identical to 3 of those used in study 2 equal portions to be combined with water, and included non-specific nitrogen (as rep­ cooked and consumed at each of the 3 resented by a mixture of glycine, diam­ daily meals. monium citrate, and glutamic acid), the Study 2 included a preliminary adjust­ essential amino acids and non-essential ment day and 10 experimental periods of amino acids as found in 2.0 g corn protein 5 days each arranged in a Latin square nitrogen, and tryptophan as found in 2.0 g design. During this study, nitrogen intake corn protein nitrogen. In all cases, the from corn was kept constant at 6.00 g/sub- mixture of glycine, diammonium citrate, ject/day, an amount established as sub- and glutamic acid was used to maintain optimal for nitrogen retention for all sub­ diets isonitrogenous. Other details regard­ jects in study 1. Various individual amino ing composition and administration of acids in purified form, thought to be pos­ diets were as previously described. sible first limiting nitrogenous factors, The amino acid content of the corn were added in amounts found in 2 g corn was determined microbiologically by the protein nitrogen during the 10 experi­ method of Steele et al. (14). Nitrogen in mental periods. These were selected on corn, low protein foods, purified nitrogen the basis of results of several other investi­ mixtures, and excreta was determined ac­ gators (2, 3, 6, 7) and by comparison of cording to the boric acid modification of the essential amino acid pattern of corn the Kjeldahl method (15). Urine samples with the FAO pattern (11), with the mini­ were preserved under toluene and were mal requirements of the essential amino analyzed daily. Daily fecal nitrogen values acids as reported by Rose (12), and with were based on 5-day fecal composites dur­ the essential amino acid pattern of egg ing studies 1 and 2 and on 4-day compo­ protein (11). A mixture composed of 4 sites during study 3. Creatinine determin­ parts nitrogen each from glycine and ations were made on the 24-hour urine diammonium citrate and 2 parts glutamic collections by the method of Folin (16). acid was used to maintain diets isonitrog- enous at 8.70 g/subject/day; that is, RESULTS 6.00 g nitrogen from corn protein, 2.0 g Study 1. Individual mean daily nitro­ nitrogen from various purified sources, and gen balances of the 10 subjects for the last 0.70 g nitrogen from the ordinary foods in 5 days of each 10-day experimental period the diet. The purified nitrogen compo­ as well as the collective means of all sub­ nents of the diets were presented to the jects are shown in figure 1. As the amount subjects in water solutions or suspensions. of nitrogen provided by corn protein was Other details regarding caloric intake, vita­ increased from 4.00 to 6.00 to 8.00 g/sub­ min and mineral supplementation, and ject/day, nitrogen retention of each indi­ diet preparation were essentially as de­ vidual increased steadily. scribed in study 1. Analysis of variance gave an F value Study 3 was divided into 4 experimental which was significant at the 0.5% level of periods of 4 days each arranged in a probability. double Latin square design (only 8 of the No subject was in positive nitrogen bal­ original 10 subjects participated in this ance while receiving 4.00 g, two were in part of the project). Appropriate addi­ positive balance while receiving 6.00 g, and tions of corn oil to the diet increased ca­ 8 were in positive nitrogen balance while loric intake by 10 kcal/kg of body weight/ receiving 8.00 g nitrogen from corn pro­ day over that established during studies 1 tein. All subjects showed an improvement and 2 as adequate for weight maintenance in nitrogen retention between the 6.00- for each subject. Total nitrogen intake was and 8.00-g corn nitrogen levels, individual maintained at 8.70 g/subject/day; 0.70 increments being 0.51, 0.37, 1.77, 0.60, from ordinary foods in the diet plus 8.00 g 1.18, 1.46, 0.33, 0.15, 1.77, and 1.66 g nitrogen from white corn meal or 6.00 g nitrogen. Similarly, increments in nitro­ nitrogen from white corn meal and 2.00 g gen retention between the 4.00 and 6.00 g nitrogen from one of 3 purified nitrogen corn nitrogen levels were 0.22, 0.33, 0.34, FIRST LIMITING NITROGENOUS FACTOR IN CORN 3 5 3

Fig. 1 Average nitrogen balances of subjects fed at 3 levels of nitrogen from white, degerminated corn meal. Points represent the average nitrogen balances of individual sub­ jects for each 5-day period. The line indicates the average nitrogen balances for all subjects at each intake level.

1.51, 0.24, 0.68, 0.49, 0.44, 0.30, and 0.92 tion of nitrogen retention. A comparison g nitrogen. of the results of studies 1 and 2 revealed Fecal nitrogen excretion tended to paral­ no differences between nitrogen balances lel the level of corn nitrogen in the diet. achieved by subjects receiving 8.00 g ni­ Average daily fecal nitrogen values for all trogen from corn protein and those shown subjects while receiving 4.00, 6.00, and by the subjects while consuming 6.00 g 8.00 g nitrogen from corn protein were nitrogen from corn protein plus 2.00 g ni­ 1.73, 1.99, and 2.28 g nitrogen, respec­ trogen from any of the 10 purified nitrogen tively. Statistical analysis indicated that mixtures. However, nitrogen retention was these values did not differ significantly. significantly greater when diets provided Study 2. Mean daily nitrogen balances 6 g nitrogen from corn protein and 2 g for each 5-day experimental period are nitrogen from any of several supplemen­ shown in table 3. The results indicated no tary sources than when 6 g nitrogen from significant differences among the various corn alone were provided. Analysis of purified nitrogen mixtures in the promo­ variance of these differences all gave F 3 5 4 CONSTANCE KIES, ELEANOR WILLIAMS AND HAZEL METZ FOX

TABLE 3 have demonstrated an apparent discrep­ Determination of first limiting nitrogenous factor ancy between calculated minimal require­ in corn for nitrogen retention in adult men ments of specific natural proteins based on Possible first Avg N their amino acid composition and experi­ limiting factor 1 balance 2 mentally determined minimal require­ Essential + non-essential amino acids + 0.65 ments of the same proteins for mainten­ Essential amino acids + 0.72 ance of nitrogen equilibrium in humans. Tryptophan + 0.57 Evidence has been obtained indicating Lysine + 0.52 that “unessential” nitrogen (nitrogen from Sulfur-containing amino acids + 0.51 Isoleucine + 0.58 sources other than the essential amino Tryptophan + sulfur-containing acids) is the first limiting nitrogenous fac­ amino acids + 0.68 tor in proteins of high biological value Tryptophan+ lysine + 0.74 such as those contained in milk and eggs Isoleucine + threonine + 0.52 “Non-essential” nitrogen (glycine, by Synderman et al. (17), Clark et al. (18) diammonium citrate, and Kies.7 glutamic acid) + 0.69 All of the purified nitrogen mixtures 1 Limiting factor in purified form fed in amounts used in the present project were equally found in 2.0 g corn protein nitrogen. Intact corn effective in the re-establishment of positive provided 6.0 g N/day. Diets maintained isonitrogenous at 8.0 g N/day by additions of glycine, diammonium nitrogen balance when added to a diet pro­ citrate and glutamic acid. 2 Average nitrogen balance of 10 subjects for 5 viding insufficient corn protein for sup­ days with each diet. porting nitrogen retention. Apparently “non-specific” nitrogen, that is, nitrogen values which were significant at greater from any metabolically usable, non-toxic than the 0.5% level of probability. source, is the first limiting nitrogenous Study 3. Mean daily nitrogen balances factor in corn protein, a protein of low bio­ for each 4-day experimental period are shown in table 4. All subjects retained logical value. slightly more nitrogen with the high ca­ At the 6.00-g corn nitrogen level all es­ loric diets than with similar diets contain­ sential amino acids, except tryptophan, ing sufficient calories for weight main­ were provided in amounts which met or tenance. Comparison of the nitrogen exceeded the requirements as established balances of subjects receiving the 4 dietary by Rose (12). Although this amount pro­ variations at the increased caloric intake vided only 91% of the tryptophan require­ ment, the addition of purified nitrogen mix­ were similar to results obtained at caloric intakes sufficient to maintain body weight. tures containing tryptophan were no more effective in the re-establishment of positive DISCUSSION nitrogen balance than were the purified The concept that the nutritional quality mixtures not containing tryptophan. All of a protein is dependent upon its amino diets were supplemented with niacin along acid composition has been prevalent for with other vitamins and minerals (table the last half century. However, several 7 Kies, C. V. 1963 Effect of essential to non- essential amino acid relationships in man and in the papers as reviewed by Synderman (17) rat. Ph.D. thesis, University of Wisconsin.

TABLE 4 Effect of caloric intake on determination of first limiting nitrogenous factor in com

Avg N balance Possible first lim iting factor High Moderate caloric caloric diet diet g N/day g N/day Essential + non-essential amino acids + 0.88 + 0.65 Tryptophan + 0.96 + 0.57 Non-essential nitrogen (glycine, diammonium citrate, glutamic acid) + 0.92 + 0.69 8 g nitrogen from intact com diet + 0.66 + 0.49 FIRST LIMITING NITROGENOUS FACTOR IN CORN 3 5 5

2). Thus, it may be that the tryptophan mentation to some theoretically balanced requirement was spared by the elimina­ pattern in order for nitrogen retention to tion of tryptophan conversion to niacin. be achieved. Furthermore, in the establishment of the Since cereal proteins contain a consider­ minimal requirements of the essential ably lower percentage of essential amino amino acids, Rose (12) defined adequacy acids as compared with non-essential as the amount of each amino acid required amino acids, Jansen and Howe (1) con­ by the subject with the greatest apparent clude that it is unlikely that total nitrogen need for maintenance of positive nitrogen can be the limiting factor with a cereal balance. Individual variation in require­ diet, thus suggesting that supplementation ment being great, it is possible that the of protein-deficient cereal diets with non- actual tryptophan requirements for the essential nitrogen is definitely contra-indi­ subjects in the present study were less than cated. The results of the present project the requirement of the one individual offer experimental evidence indicating whose need, in essence, determined the that, in fact, non-essential or non-specific tryptophan requirement as expressed by nitrogen is the first limiting factor in corn Rose. protein. Therefore, additional research on Supplementation studies in which pos­ the possible beneficial effects of non-essen­ sible “non-specific” nitrogen requirements tial nitrogen supplementation of cereal of the subjects were not considered cannot proteins is suggested. be compared directly with the present study. Results reported by Truswell and ACKNOWLEDGMENT Brock (7) on the effect of supplementing The authors wish to thank Warden corn protein with purified amino acids in­ Maurice Sigler, Deputy Warden John B. dicated that lysine is the first limiting Greenholtz, Associate Warden E. Scar­ amino acid in corn. Either arbitrary borough, Medical Officer G. Lewis, M.D., amounts or the Rose “safe” requirement the officers of the Nebraska Penal and Cor­ pattern were used in determining levels of rectional Complex — Reformatory Unit, the essential amino acid supplementation, and the participating inmates for their thus markedly altering the corn amino acid cooperation. pattern. In addition, no attempt was made to keep total nitrogen intake at an optimal LITERATURE CITED level or to determine the possible effect of 1. Jansen, G. R., and E. E. Howe 1964 World non-essential nitrogen intake on the re­ problems in protein nutrition. Am. J. Clin. sults. Nutrition, 15: 262. The results of a series of experiments 2. Scrimshaw, N. S., R. Bressani, M. Behar and F. Viteri 1958 Supplementation of carried out by researchers connected with cereal proteins with amino acids. I. Effect the Institute of Central America and of amino acid supplementation of corn-masa Panama (2-6) and designed to determine at high levels of protein intake on nitrogen the effectiveness of amino acid supple­ retention of young children. J. Nutrition, 66; 485. mentation of cereal proteins for young 3. Bressani, R., N. S. Scrimshaw, M. Behar children indicate that total protein intake and F. Viteri 1958 Supplementation of influence the effectiveness of the various cereal proteins with amino acids. II. Effect supplements and that the effect of amino of amino acid supplementation of corn-masa at intermediate levels of protein intake on acid supplementation of particular cereals the nitrogen retention of young children. cannot always be predicted from compari­ J. Nutrition, 66; 501. son with a reference amino acid pattern or 4. Bressani, R., D. L. Wilson, M. Behar and from the results of animal studies. Using N. S. Scrimshaw 1960 Supplementation of cereal proteins with amino acids. III. Effect weight gain and nitrogen retention of of amino acid supplementation of wheat young children as the criteria of evalua­ flour as measured by nitrogen retention of tion, tryptophan and lysine were impli­ young children. J. Nutrition, 70; 176. cated as being the first limiting amino 5. Bressani, R., D. W ilson, M. Behar, M. Chung and N. S. Scrimshaw 1963 Supplementa­ acids in corn-masa. However, these stud­ tion of cereal proteins with amino acids. ies also assumed that the corn protein pat­ IV. Lysine supplementation of wheat flour tern is so imbalanced as to require supple­ fed to young children at different levels of 3 5 6 CONSTANCE KIES, ELEANOR WILLIAMS AND HAZEL METZ FOX

protein intake in the presence and absence Committee. Nutritional Studies Series 16. of other amino acids. J. Nutrition, 79: 333. Food and Agriculture Organization of the 6. Bressani, R., D. Wilson, M. Chung, M. United Nations, Rome. Behar, and N. S. Scrimshaw 1963 Supple­ 12. Rose, W. C. 1957 The amino acid require­ mentation of cereal proteins with amino ments of adult man. Nutrition Abstr. Rev., acids. V. Effect of supplementing lime- 27: 631. treated corn with different levels of lysine, 13. Orr, M. L., and B. K. W att 1957 Amino tryptophan and isoleucine on the nitrogen acid content of foods. Home Economics Re­ retention of young children. J. Nutrition, search Report no. 4. U. S. Department of 80: 80. Agriculture, Washington, D. C. 7. Truswell, A. S., and J. F. Brock 1961 14. Steele, B. F., H. E. Sauberlich, M. S. Rey­ Effects of amino acid supplements on the nolds and C. A. Baum ann 1949 Media for nutritive value of maize protein for human Leuconostoc mesenteroid.es P-60 and Leu- adults. Am. J. Clin. Nutrition, 9: 715. conostoc citrovorum 8081. J. Biol. Chem., 8. National Academy of Sciences — National 177: 533. Research Council 1961 Progress in meet­ 15. Scales, F. M., and A. P. Harrison 1920 ing the protein needs of infants and pre­ Boric acid modification of the Kjeldahl school children. Proceedings of an Inter­ method for crop and soil analysis. J. Ind. national Conference under the auspices of Eng. Chem., 12: 350. The Committee on Protein Malnutrition, 16. Folin, O. 1914 On the determination of Food and Nutrition Board, and The Nutri­ creatinine and creatine in urine. J. Biol. tion Study Section, National Institutes of Chem., 17: 469. Health. Publication 843. Washington, D. C. 17. Snyderman, S. E., L. E. Holt, Jr., J. Dancis, 9. Linkswiler, H., H. M. Fox and P. C. Fry E. Roitman, A. Boyer and M. E. Balis 1962 1960 Availability to man of amino acids “Unessential” nitrogen: A limiting factor from foods. IV. Isoleucine from corn. J. for human growth. J. Nutrition, 78: 57. Nutrition, 72: 397. 18. Clark, H. E., M. A. Kenney, A. F. Goodwin, 10. Hegsted, D. M., A. G. Tsongas, D. B. Abbott K. Goyal and E. T. Mertz 1963 Effect of and F. J. Stare 1946 Protein requirements certain factors on nitrogen retention and of adults. J. Lab. Clin. Med., 31: 261. lysine requirements of adult human sub­ 11. Food and Agriculture Organization 1957 jects. IV. Total nitrogen intake. J. Nutri­ Protein requirements. Report of the FAO tion, 81: 223. Effect of "Non-specific" Nitrogen Intake on Adequacy of Cereal Proteins for Nitrogen Retention in Human Adults 1 *

CONSTANCE KIES, ELEANOR WILLIAMS a n d HAZEL METZ FOX Department of Food and Nutrition, School of Home Economics, University of Nebraska, Lincoln, Nebraska

ABSTRACT The optimal intake of “non-specific” nitrogen for apparent nitrogen retention of human adults when the essential amino acids are provided by intact corn protein was studied. During 5 periods of 10 days each arranged in a double Latin square design, nitrogen balances of 10 adult men fed 6.0 g nitrogen/day from white degerminated corn meal plus 0.0, 2.0, 4.0, 6.0 or 8.0 g nitrogen/day from “non-specific” nitrogen (an isonitrogenous mixture of glycine and diammonium citrate) were deter­ mined. Other foods provided 0.68 g nitrogen/day. Average nitrogen balances for all subjects during the last 5 days of each period when total nitrogen intake was 6.68, 8.68, 10.68, 12.68 or 14.68 g day, were —0.33, +0.44, +0.85, +1.32, and +1.22 g nitro­ gen/day, respectively. The step-by-step increases in nitrogen retention among the first 4 values were statistically significant at the 0.5% level of probability.

It has been generally accepted that puri­ constant amounts of the essential amino fied diets must provide sufficient nitrogen acids as contained in intact corn protein is for synthesis of the non-essential amino reported in the present paper. acids as well as adequate amounts of the essential amino acids in order to meet PROCEDURE human protein requirements (1, 2). Data The study was composed of a 3-day pre­ reported by Snyderman et al. (3) indicate liminary period and 5 experimental periods that “unessential” nitrogen, i.e., nitrogen of 10 days each, arranged in a double Latin from sources other than the essential amino square design. acids, may be the first limiting nitrogenous During the preliminary period nitrogen factor in milk for growth and nitrogen re­ intake per subject per day totaled 1.68 g; tention of human infants. Recent evidence that is, 1.00 g nitrogen from white, de­ indicates that a broader term, “non­ germinated corn m eal2 and 0.68 g from the specific” nitrogen, i.e., nitrogen from a basal diet composed of cornstarch, fat, in­ metabolically useable, non-toxic source in­ stant coffee, and a few low protein fruits cluding excess essential amino acid nitro­ and vegetables. Purposes of this period gen not utilized in meeting essential amino included the introduction of subjects to acid needs, might be more meaningful in their duties and responsibilities, determina­ defining this suggested requirement (4). tion of individual caloric requirements for Apparent nitrogen retention of adult men weight maintenance, and utilization of a fed 6.0 g nitrogen/day from intact corn very low nitrogen diet to speed the adjust­ protein was improved equally by the addi­ ment of subjects to the later experimental tion of 2.0 g nitrogen/day from any of diets. several purified essential amino acids or During all experimental periods, intake “unessential” nitrogen sources, or both, or of the essential amino acids was main­ from intact corn protein. Thus, “non­ tained constant in the amounts shown in specific” nitrogen was implicated as the table 1 as provided by 6.00 g corn protein first factor in corn protein limiting nitro­ nitrogen (472 g white, degerminated com gen retention in human adults. Received for publication March 25, 1965. The possible extent of quantitative im­ 1 Published with the approval of the Director as Paper no. 1704, Journal Series, Nebraska Agricultural provement of nitrogen retention in human Experiment Station. adults with graded increases in “non-spe­ 2 The white, degerminated, unenriched com meal used in this study was furnished gratis by the Gooch cific” nitrogen added to diets providing Milling and Elevator Company, Lincoln, Nebraska.

J. N u t r it io n , 86: ’65 357 3 5 8 CONSTANCE KIES, ELEANOR WILLIAMS AND HAZEL METZ FOX

TABLE 1 TABLE 2 Composition of diet Vital statistics and the caloric intakes of subjects A m oun t/ su b ject/ Subject Age Weight Height Caloric day intake years kg cm kcal/day 9 Corn meal (white, degerminated, 60 25 81.8 180 3780 unenriched ) 4 7 2 b2 61 30 67.3 173 3119 Cornstarch, varied z-3 62 26 67.3 173 3119 Corn oil or butter oil, varied 2-3*4 63 28 91.4 185 3815 J e lly 3 0 64 25 74.5 178 3447 A p p le sa u c e 1 00 65 36 77.3 180 3580 Tomato paste 10 0 66 28 72.3 183 3354 Instant coffee (powder) 10 67 33 70.4 180 3264 Low calorie, carbonated beverages 68 28 71.4 179 3313 (protein and fat-free), varied 69 38 90.4 180 3877 Vitam in supplem ent, daily 5 * M ineral supplem ent, daily z-e Glycine and diammonium citrate, varied 7 cases fat provided 20% of the calories. As shown in table 1, one-half the subjects re­ 1 The amino acid analysis of the corn meal gave the following values: (in mg of amino acid/g of corn ceived butter oil and one-half received corn nitrogen) tryptophan, 38.5; threonine, 216; isoleucine, 286; leucine, 958; lysine, 135; methionine -f cystine, oil as the source of dietary fat. Vitamin 237; phenylalanine + tyrosine, 637; and valine, 307. The corn meal contained 0.0127 g nitrogen/g of corn. and mineral supplements were also in­ 2 Com meal, cornstarch, fat source, mineral sup­ cluded (table 1). plement, and baking powder for each subject for each day were combined, divided by weight into 3 equal Methods used in the preparation and portions, mixed with appropriate amounts of water, cooked in several standard forms (mush, muffins, administration of the purified nitrogen pizza), and served. mixture, corn meal, cornstarch, and other 3 Cornstarch and fat were added in amounts needed to meet individual caloric requirements for mainte­ food items were basically the same as out­ nance of body weight. 4 Subjects 60-64 received butter oil as the dietary lined in an earlier paper (4). fat source; subjects 65—69 received corn oil. Fat was Details regarding the 10 men, inmates fed in amounts to provide 20% of the total caloric intake for each individual. of the Nebraska Penal and Correctional 5 The vitamin supplement in pill form provided the following/subject/day: (in mg) thiamine HC1, Complex, who were subjects for the study 1.2; riboflavin, 1.5; pyridoxine-HCl, 0.3; ascorbic acid, 50.0; niacin, 6.0; vitamin B 12, 12.0; and folic acid, are shown in table 2. Institutional health 50.0 mEq. Also, vitamin A, 4000 IU; and vitamin D, records indicated that all were in good 400 IU. 6 The mineral supplement provided the following/ health; medical consultation of a physician su b ject/d a y : (in g ram s) CaC(>3, 1.36; KH3PO4, 1.31; MgC03 Mg(0H)2-3H20, 0.80; FeC6H50 7 -6H 20 , 0.095; was available to the subjects throughout CuS04-5H20, 0.008; KI, 0.0002; MnCl2-4H20, 0.007; the study. Subjects were housed and ate ZnCl2, 0.002. In addition, baking powder provided approximately NaHCOs, 2.0 g and Ca(H2P04)2H 20, meals together in a separate part of the 2.9 g. 7 An isonitrogenous mixture of glycine and diam­ institution; however, usual work assign­ monium citrate was given in solution to provide 0.0, ments were maintained. 2.0, 4.0, 6.0, or 8.0 g of nitrogen/subject/day de­ pending upon the experimental period. Each daily Nitrogen content of corn meal, other allotment was divided into 3 portions of equal size foods, glycine and diammonium citrate for consumption at each of the 3 daily meals. mixtures, and excreta was determined ac­ m eal3) per subject per day. A mixture of cording to the boric acid modification of glycine and diammonium citrate in iso­ the Kjeldahl method (5). Creatinine in nitrogenous amounts provided 0.00, 2.00, urine was analyzed by the method of Folin 4.00, 6.00, or 8.00 g nitrogen/subject/day (6) in order to check the accuracy of col­ during the 5 experimental periods. Thus, lections. Urinary nitrogen and creatinine with the inclusion of nitrogen provided by excretions were determined daily on each the basal diet (0.68 g, daily, as shown in 24-hour collection and fecal nitrogen data table 1) total nitrogen intake per subject were obtained from 5-day composite col­ per day was systematically varied accord­ lections for each individual. Amino acid ing to the Latin square design from 6.67 content of the corn meal was determined to 14.67 g. Caloric intake for each indi­ microbiologically by the method of Steele vidual was kept constant at the level re­ et al. (7). quired for weight maintenance (table 2) 3 See footnote 2. by varying the intake of cornstarch 4 and 4 The cornstarch and corn oil used in this study were supplied gratis by Com Products Company, Argo, fat among the subjects; however, in all Illinois. EFFECT OF “NON-SPECIFIC” NITROGEN INTAKE 3 5 9

Fig. 1 Effect of several levels of dietary “non-specific” nitrogen on nitrogen balances of adult men fed a constant amount of corn protein (to provide 6.0 g nitrogen/subject/day). Dots represent average nitrogen balances of each individual for the last 5 days of each 10-day experimental period. Crosses represent mean balances of all subjects at each level of “non-specific” nitrogen intake.

RESULTS increased when the total nitrogen intake Mean nitrogen balances of each indi­ was increased from 8.67 to 10.67 g and vidual subject for the last 5 days of each from 10.67 to 12.67 g/day, average incre­ experimental period as well as collective ments being 0.46 and 0.41 g nitrogen, re­ m eans for all subjects axe shown in spectively. Analysis of variance of these figure 1. data also gave F values significant at All subjects showed increases in ap­ greater than the 0.5% level of probability. parent nitrogen retention when the total Nitrogen retention was not significantly nitrogen intake was increased from 6.67 greater when total nitrogen intake was to 8.67 g nitrogen/day, the average incre­ 14.67 g/day as compared with values ob­ ment being 0.72 g nitrogen. Analysis of tained on the 12.67 g total nitrogen intake variance gave an F value significant at level. greater than the 0.5% level of probability. Fecal nitrogen excretion remained rela­ Similarly, average nitrogen retention was tively constant for all subjects regardless 3 6 0 CONSTANCE KIES, ELEANOR WILLIAMS AND HAZEL METZ FOX

of total nitrogen intake although differ­ of total essential amino acids and the ences existed among subjects. Average greatly unbalanced proportions of several fecal nitrogen excretion values per day for amino acids make it extremely unlikely subjects 60 through 69 were 1.23, 1.42, that total nitrogen is the limiting factor in 1.01, 2.25, 1.73, 2.00, 2.16, 1.56, 1.59, and a cereal diet. The results of the present 1.45 g, respectively. study indicate that nitrogen retention of adults eating a corn diet can be increased DISCUSSION greatly by additions of “non-specific” nitro­ Well-known discrepancies exist among gen, thus by increasing the total nitrogen the apparent minimal requirements of the intake. various essential amino acids for human Although the results of this study offer adults reported by several investigators (9). interesting possibilities for supplementa­ Although some variations existed in experi­ tion of cereal diets on a practical basis, mental procedure and criterion of evalua­ much additional information is needed on tion, the lack of consistency in level of the effects of “non-specific” nitrogen sup­ nitrogen intake from sources other than plementation of cereal diets fed children, the essential amino acids may have had a long-term effects of “non-essential” nitro­ greater effect on the results than formerly gen intake, and whether a high degree of supposed. As discussed in an earlier paper apparent nitrogen retention always repre­ (4), several authors have also noticed a sents the optimal in protein nutrition. discrepancy between calculated minimal requirements of certain intact proteins ACKNOWLEDGMENT based on their essential amino acid compo­ The authors wish to thank and to ac­ sition and actual minimal requirements knowledge the cooperation of Warden based on the results of nitrogen balance Maurice Sigler, Deputy Warden John B. studies. This also may be the result of Greenholtz, Associate Warden Ed Scar­ failure to take the “non-essential” or “non­ borough, Medical Officer Robert Moore, specific” nitrogen requirements into con­ M.D., the staff of the Nebraska Penal and sideration. Correctional Complex—Reformatory Unit, The 472 g of white corn meal fed daily and, particularly, the 10 inmates who were to each subject provided sufficient amounts subjects for this project. of all the essential amino acids except tryptophan as judged by the Rose (1) cri­ LITERATURE CITED teria of the minimal requirements of the 1. Rose, W. C. 1957 The amino acid require­ essential amino acids. Experimental data ments of adult man. Nutrition Abstr. Rev., from the previous study (4) indicated that 27: 631. sufficient amounts of tryptophan (91% of 2. Leverton, R. M. 1959 Amino acid require­ the Rose minimum requirement) were pro­ ments of young adults. In: Protein and vided at this level of corn protein intake Amino Acid Nutrition, ed., A. A. Albanese. Academic Press, New York, p. 477. and that higher intakes of tryptophan 3. Snyderman, S. E., L. E. Holt, Jr., J. Dancis, would result in no improvement in nitro­ E. Roitman, A. Boyer and M. E. Balis 1962 gen retention. “Unessential” nitrogen: A limiting factor for Several authors have reported that human growth. J. Nutrition, 78; 57. adults fed constant amounts of the essen­ 4. Kies, C., E. Williams and H. M. Fox 1965 Determination of first limiting nitrogenous tial amino acids retain greater amounts factor in corn protein for nitrogen retention of nitrogen with diets containing medium in human adults. J. Nutrition, 85: 350. or high levels of “non-specific” nitrogen as 5. Scales, F. M., and A. P. Harrison 1920 compared with those containing relatively Boric acid modification of the Kjeldahl method for crop and soil analysis. J. Ind. low amounts (8-11). In these studies the Eng. Chem., 12: 350. essential amino acids were provided in 6. Folin, O. 1914 On the determination of accordance with the egg protein propor­ creatinine and creatine in urine. J. Biol. tionality pattern in the form of whole egg, Chem., 17: 469. purified amino acids, or cereals plus puri­ 7. Steele, B. F., H. E. Sauberlich, M. S. Reynolds and C. A. Baum ann 1949 Media for Leuco- fied amino acids. In a recent paper, Jansen nostoc mesenteroides P-60 and Leuconostoc and Howe (12) stated that the lower level citrovorum 8081. J. Biol. Chem., 177: 533. EFFECT OF ‘NON-SPECIFIC NITROGEN INTAKE 3 6 1

8. Clark, H. E., M. A. Kenney, A. F. Goodwin, K. essential nitrogen on nitrogen balance in Goyal and E. T. Mertz 1963 Effect of cer­ young adults. J. Nutrition, 71: 105. tain factors on nitrogen retention and lysine 11. Kies, C., L. Shortridge and M. S. Reynolds requirements of adult human subjects. IV. 1965 Effect on nitrogen retention of men Total nitrogen intake. J. Nutrition, 81: 223. of varying the total dietary nitrogen with 9. Linkswiler, H. M., H. M. Fox and P. C. Fry essential amino acid intake kept constant. 1960 Availability to man of amino acids J. Nutrition, 85: 260. from foods. III. Threonine from com. J. 12. Jansen, G. R., and E. E. Howe 1964 World Nutrition, 72: 389. problems in protein nutrition. Am. J. Clin. 10. Swendseid, M. E., C. L. Harris and S. G. Nutrition, 15: 262. Tuttle 1960 The effect of sources of non­ B-Complex Vitamin Content of Cheddar Cheese ’'*2'3'4

KAY M. NILSON, JAYANTKUMAR R. VAKIL a n d KHEM M. SHAHANI Department of Dairy Science, College of Agriculture and Home Economics, University of Nebraska, Lincoln, Nebraska

ABSTRACT The retention of B-vitamins of milk in Cheddar cheese curd, loss of the vitamins in whey, and the effect of temperature and length of ripening upon the vitamin content of cheese were investigated. The vitamin content of the milk used varied widely. The vitamin content of the cheese curd varied with that of the milk. The significant loss of vitamins in the whey produced during the cheese-making process is indicated by the fact that from 10 to 40% of the vitamin content of the milk was retained in the cheese curd. The temperature and length of ripening mark­ edly affected the vitamin content of cheese. The changes in the vitamin content proceeded most rapidly in the cheese ripened at 15.6°, more slowly at 10°, and slowest at 4.4°. The content of four of the six vitamins increased during the initial period of ripening. The pantothenic acid and vitamin Bi2 content, however, decreased during the initial period and then increased. The increase in at least the niacin and vitamin B6 content during the early stages of ripening may be related to the lactose metabo­ lism in Cheddar cheese. The data revealed that mild and sharp Cheddar cheese and a mixture of sharp cheese with fresh Cheddar cheese curd as used in the manufacture of process cheese are richer in B-vitamin content than medium Cheddar cheese by itself.

The content of B-vitamins varies widely 70% of the vitamin B6 content and 43 to not only with varieties of cheese, but also 60% of the vitamin B12 content of milk with different samples of the same variety. were lost in the whey produced during the Shahani et al. (1) observed that the nia­ manufacture of cheese. cin content in 278 samples of 23 varieties In general, the proteolytic and mold- of cheese varied between 21 and 3,416, ripened cheese varieties such as Lim- with an average of 691 ug/100 g. Similar burger, Camembert, and Roquefort con­ wide variations were observed in the vita­ tain much higher concentrations of min Bc, pantothenic acid, biotin, and folic vitamins than the hard and semi-hard acid content. In 12 varieties of cheese, varieties, such as Cheddar, Swiss, Moz­ Sullivan et al. (2) also reported that the zarella, and others. The vitamin content niacin, pantothenic acid, and biotin of cheese is greatly affected by cultures ranged from 30 to 1,600, 130 to 960, and used in its manufacture and the subse­ 1.1 to 7.6 ug/100 g, respectively. Karlin quent ripening conditions (1, 2, 7). Eman- (3, 4) observed that the vitamin Be con­ uilov and Nachev5 observed that in 3 tent in 19 varieties of cheese ranged be­ types of Kachkaval Bulgarian cheese the tween 40 to 198, and the vitamin Bi2 vitam in Bi2 content increased during rip­ content of 4 varieties ranged between 1.2 ening. However, in Camembert, Bleu, and 2.55 ug/100 g of cheese. Port-Salut, and Gruyere the vitamin B12 Variations in the vitamin content of content decreased during the initial few cheese have been attributed to the differ­ days of ripening and then increased to ences in the milk and in the cheese-mak­ beyond the original level (4). ing and curing processes used. The vita­ Received for publication February 18, 1965. min content of milk, which varies with ^Supported in part by a grant from the American Dairy Association. season, stage of lactation, and feed (5), 2 Published with the approval of the Director as paper no. 1651, Journal Series, Nebraska Agricultural may affect markedly the resultant vitamin Experiment Station, Lincoln. content of fresh cheese curd. Collins and 3 Part of this paper was presented at the 58th Annual Meeting of American Dairy Science Associa­ Yaguchi (6) reported that a considerably tion, 1963. higher concentration of vitamin Bi2 was 4 Paper no. 3 in a series: B-Complex Vitamin Con­ tent of Cheese. lost in rennet whey than in an acid whey. 5 Emanuilov, L, and L. Nachev 1959 Sudurjhanie na vitamin B 12 v tvurdite sireneta. Dairy Sci. Abstr., Karlin (3, 4) also observed that 54 to 21: 470 (abstract).

362 J. N utritio n , 8 6 : ’65 B-VITAMIN CONTENT OF CHEDDAR CHEESE 3 6 3

Fagen et al. (8) and Sjostrom (9) re­ Vitamin assay methods. The micro­ ported that the small quantity of lactose biological methods of Gregory (11) and present in fresh cheese curd is readily those of Shahani et al. (1) were used for metabolized within the initial 3 to 14 days the determination of niacin, vitamin B0, of ripening. Since the nature of fermenta­ folic acid, biotin, and pantothenic acid in tion in cheese is greatly influenced by its milk, whey, and cheese. The method re­ lactose content (10), the vitamin content ported by Lichtenstein et al. (12) was of the cheese, particularly during the early used for vitamin Bi2. Vitamin extracts stages of ripening, may vary with the from milk, whey, or cheese were prepared amount of lactose present. separately for each vitamin and assayed, The paucity of information concerning using Lactobacillus plantarum 8014 as the the factors affecting the B-vitamin content assay organisms for niacin and biotin; of Cheddar cheese and the need for stand­ Saccharomyces carlsbergenesis 9080 for ardizing methods to produce cheese rich vitamin B6; Streptococcus faecalis 8043 in vitamins prompted this investigation. for folic acid; Lactobacillus casei 7469 for pantothenic acid; and Lactobacillus leich- METHODS m annii 7830 for vitamin B,2. The work was directed toward the de­ Effect of lactose metabolism upon the termination of niacin, vitamin B„, panto­ biosynthesis of niacin and vitamin Be. thenic acid, biotin, folic acid, and vitamin Since during ripening of cheese most of B12 content of the milk, the Cheddar the lactose is metabolized in the first 2 cheese curd, and the whey produced. The weeks (8, 9) and since in the preceding effect of ripening conditions upon the vita­ phase of work the content of four of the min content of Cheddar cheese was also vitamins in cheese increased rapidly dur­ determined. Also, studies were made to ing the first few weeks of ripening, it was determine the effect of lactose metabolism thought that the increase in vitamin levels upon the vitamin content of the cheese during the initial stages of ripening might during early stages of ripening. be associated with the metabolism of lac­ tose in the cheese. Studies were therefore Manufacture of cheese and handling of made to investigate the relationship be­ samples. The milk used in this study tween the lactose metabolism and the was grade A milk produced by the Uni­ vitamin synthesis. In these trials a lactose versity dairy herd and was pasteurized at “feed back” technique was used. the University dairy plant. Using five Five additional batches of cheese were different lots of milk five separate batches manufactured as described above and of Cheddar cheese were manufactured by allowed to ripen at 10°. Samples were the commercial method. The starter con­ taken at 2-day intervals for 14 days and sisted of a commercial Cheddar cheese assayed for the lactose, niacin, and vitamin starter. The cheese curd was hooped in B6 content. The lactose content was deter­ 10-kg Wilson hoops and pressed overnight, mined by the method of Folin-Wu, as after which the cheese was removed from described by Hawk et al. (13). It was the hoops, cut into approximately 1-kg found that most of the lactose present in loaves, wrapped in “Parakote” and pressed cheese had disappeared at the end of about for an additional 24 hours. Vitamin as­ 14 days of ripening. The cheese samples says of fresh samples of milk, whey, and were then divided into 2 lots. To one lot the Cheddar cheese curd were run imme­ 2% lactose was added aseptically and the diately. Also, each lot of cheese was di­ cheese was macerated, put into sterile con­ vided into 3 sublots, ripened at 4.4, 10, tainers and covered with paraffin. The and 15.6° and assayed for vitamins at the other lot, serving as the control, was end of 1 week, 2 weeks, and 1, 2, 4, 6, 9, treated the same way except that no lac­ and 12 months of ripening. Simulta­ tose was added. The samples were ripened neously with the vitamin assays the cheese at 10° and assayed for lactose, niacin, and samples were examined organoleptically vitamin B6 every two or three days for the for flavor, and for body and texture. next 6 weeks. 364 KAY M. NILSON, JAYANTKUMAR R. VAKIL AND KHEM M. SHAHANI

RESULTS ripened at 4.4, 10, and 15.6°. At prede­ B-vitamin content of milk, whey, and termined intervals representative samples Cheddar cheese curd. In table 1 is pre­ were withdrawn and assayed for the vari­ sented a summary of the data recorded on ous vitamins. Simultaneously with the the B-vitamin content of milk, whey, and vitamin assays, the cheese samples were fresh Cheddar cheese curd and the per­ judged organoleptically for flavor and for centage vitamin content of milk retained body and texture. in the cheese curd. All 5 lots of cheese were of good quality. The 5 lots of milk used for the manu­ As the ripening time progressed, the facture of different batches of cheese cheese developed typical ripened cheese varied widely in vitamin content. The flavor and texture. The rate of ripening average content of niacin, vitamin Br„ was slightly faster at 15.6° than at 10 or pantothenic acid, biotin, folic acid, and 4.4°. At the end of 2 to 3 months of vitamin B,2 of the milk were 76, 17, 340, ripening all the cheese samples possessed 29, 3.8, and 0.48 ug/100 g, respectively. a typical ripened cheese flavor. The average values in whey were 89, 14, The data showing the effects of temper­ 387, 26, 4.3, and 0.41 ug/100 g, respec­ ature and length of ripening time upon tively, indicating that considerable quan­ the niacin, vitamin Bn, folic acid, and tities of the vitamins of milk are lost biotin content of cheese are presented in through the whey produced in the process. figure 1. The temperature and time of In general, for each vitamin the higher ripening affected markedly the vitamin the content in the milk the higher the content of the cheese. In general, the vitamin content of the cheese curd ob­ changes (increase or decrease) in the vita­ tained from it. Average values for the min content proceeded most rapidly in 5 lots showed that 100 g of the cheese the cheese samples ripened at 15.6°, more curd contained 169 ug of niacin, 33 ug of slowly at 10°, and slowest at 4.4°. vitamin B6, 912 ug of pantothenic acid, Niacin. The changes in the niacin 29 ug of biotin, 15.2 ug of folic acid and content during ripening are shown in 0.52 ug of vitamin B]2. The percentage figure 1, block a. In all the 5 trials, there vitamin retention in the cheese curd was a progressive increase in the niacin amounted to 22% for niacin, 19% for content during ripening. It increased rap­ vitamin B6, 28% for pantothenic acid, idly during the first 3 to 4 weeks at all 3 10% for biotin, 40% for folic acid, and temperatures; thereafter, it leveled off at 11% for vitamin Bi2, based on averages the 4.4° temperature, but continued to for the 5 lots. increase at 10 and 15.6°. There was a Effect of temperature and ripening time marked difference between the maximal upon the vitamin content of Cheddar level in vitamin content of the cheese rip­ cheese. To determine the effect of tem­ ened at the lowest temperature (210 ug/ perature and ripening time upon the vita­ 100 g) compared with that in cheese min content of the cheese, each lot of the ripened at the higher temperature (318 cheese was divided into 3 sublots and ug/100 g).

TABLE 1 B-Vitamin content of milk, whey and Cheddar cheese curd percentage and retention of vitamins of milk in cheese curd Milk Whey Cheese curd Vitamin Retention Range Avg i Range Avg i Range Avg 1

/i g / 1 0 0 g lig / 1 0 0 g fig / 1 0 0 g fig / 1 0 0 g fig / 1 0 0 g fig / 1 0 0 g % N iacin 61-100 76 42-138 89 140-295 169 22 Vitamin Bo 4 -2 7 17 8 -2 6 14 12-57 33 19 Pantothenic acid 300-380 340 3 6 0 ^ 1 2 387 864-960 912 28 Biotin 11^18 29 6 -38 26 11-67 29 10 Folic acid 1.0-6.0 3.8 0.3-13.0 4.3 1.0-35.0 15.2 40 Vitamin BJ2 0.39-0.56 0.48 0.38-0.42 0.41 0.50-0.56 0.52 11 1 Average of 5 trials. B-VITAMIN CONTENT OF CHEDDAR CHEESE 3 6 5

Fig. 1 Effect of temperature and length of ripening upon the niacin, vitamin B6, folic acid and biotin content of Cheddar cheese (average of 5 lots).

Vitamin Be. As shown in block b of temperature and length of ripening are figure 1, the vitamin Be content increased presented in figure 2. The changes during rapidly during the first 1 to 2 weeks from ripening were different from those of the 33 to about 70 ug/100 g and then de­ 4 vitamins discussed above in that irre­ creased to a level of 35 to 38 ug/100 g at spective of the temperature of ripening, a the end of 2 months’ ripening. Thereafter, decline in their content was observed dur­ there was a gradual increase to a range of ing the initial ripening period. The panto­ 87 to 116 ug/100 g at the end of 12 thenic acid content decreased sharply dur­ months. The greatest increase was noted ing the first 4 to 8 weeks, followed by a at 15.6°, next at 10°, and the least at 4.4°. slow increase during the remaining period Folic acid. Like the vitamin Be content, of ripening. The vitamin Bi2 content de­ the folic acid content also increased very creased sharply during the first week (from rapidly at all the three ripening tempera­ 0.52 to 0.27 ug/100 g) and thereafter in­ tures during the first week of ripening, creased slowly up to the end of the 9- increasing from 15 to 44 to 60 ug/100 g month ripening period and then very rap­ (fig. 1, block c). Thereafter, it decreased idly during the last 3 months of ripening. rapidly for 6 weeks and at a slower rate Effect of lactose metabolism upon the for the remainder of the ripening period. niacin and vitamin Be synthesis. The data Biotin. The biotin content (fig. 1, showing the effect of lactose metabolism block d), increased for the first 2 months, on vitamin content of the cheese are pre­ then decreased sharply to below the orig­ sented in figure 3. As observed by Fagen inal level at the end of the next 2 months, et al. (8) and Sjostrom (9), the initial with little change thereafter. lactose present in cheese was utilized al­ Pantothenic acid and vitamin B,° con­ most completely during the first 14 days tent. The data on the pantothenic acid (block a). The niacin content (block b) and vitamin Bi= content as affected by increased during the same period; the 3 6 6 KAY M. NILSON, JAYANTKUMAR R. VAKIL AND KHEM M. SHAHANI

LENGTH OF RIPENING (MONTHS)

Fig. 2 Effect of temperature and length of ripening upon the pantothenic acid and vitamin B,a content of Cheddar cheese (average of 5 lots).

LENGTH OF RIPENING (DAYS) Fig. 3 Relationship between lactose metabolism and the biosynthesis of niacin and vitamin Bi in Cheddar cheese (average of 5 lots). vitamin B6 (block c) also increased but in the control cheese. At the end of 56 only for 7 days and then decreased. The days of ripening (42 days after the addi­ extra 2% lactose that was “fed back” was tion of extra lactose) the niacin content also utilized very rapidly. Simultaneously, in the lactose-added cheese had increased the niacin content of the lactose-added from 170 to 472 pg/100 g of cheese as cheese increased much more rapidly than compared with 170 to 282 ag in the con­ B-VITAMIN CONTENT OF CHEDDAR CHEESE 3 6 7 trol cheese. Similarly, the vitamin B6 con­ the temperature and length of ripening tent of the cheese increased rapidly for had a very pronounced effect upon the 10 days after the addition of lactose, vitamin content of the cheese. The reaching 70 ng/100 g and then decreasing. changes in the vitamin content were great­ The vitamin Bs content of the control est at 15.6°, next at 10°, and least at 4.4°. cheese continued to decrease, as was ob­ The content of four out of the six vita­ served in the previous trials (fig. 1, mins, niacin, vitamin B6, biotin, and folic block b). acid, increased during the initial period of The increase in niacin and vitamin B6 ripening, and the increase at least in the levels after the addition of lactose to the case of niacin and vitamin Bs content 14-day-old Cheddar cheese may be attrib­ appears to be related to the available lac­ uted to the added lactose. As the lactose tose in the cheese. was utilized the vitamin content increased, Cheddar cheese constitutes by far the but after the lactose was completely uti­ most widely consumed cheese in the lized the niacin content tended to level American diet, and is used in considerably off, and the vitamin B6 content decreased, higher quantities than any other variety as in the case of the regular cheese sam­ of cheese. It is a good source of the B- ples (fig. 1). The data presented herein, vitamins which play an important role in therefore, suggest that the increase in the human nutrition. The B-vitamin content vitamin content of cheese during the ini­ of Cheddar cheese, as available to the con­ tial period of ripening may be related sumer, will vary depending upon the ini­ directly to the lactose content and lactose tial vitamin content of the milk from utilization in the cheese. These results are which it was made and upon the time and in harmony with the observations of Teply temperature of ripening. et al. (14) that the addition of lactose to No definitive information is available the ration enhanced the synthesis of nia­ with respect to the amount of Cheddar cin and folic acid by intestinal micro­ cheese produced currently in this country organisms of rats. that is marketed or consumed as mild, medium, or sharp Cheddar; however, it is DISCUSSION estimated that about 31 to 50% is con­ sumed as mild 6 and 33% as sharp (17). The niacin, vitamin B«, pantothenic The data obtained in this study revealed acid, biotin, folic acid, and vitamin Bia that, by and large, mild cheese ripened content of milk used in the manufacture for only 2 months or so and sharp Cheddar of cheese varied widely among different ripened for 9 to 12 months contain higher lots of milk. On an average, the milk levels of vitamins than medium cheese contained 76 ug of niacin, 17 ag of vita­ ripened for 4 to 6 months. min Be, 340 ag of pantothenic acid, 29 ag Also, considerable quantities of fresh of biotin, 3.8 ag of folic acid, and 0.48 ag Cheddar cheese curd, and mild and sharp of vitamin B,2/100 g of milk. These values are fairly typical of the values reported in Cheddar cheese go into the manufacture of “pasteurized process” cheese. Since the literature (2, 11, 15, 16). Consider­ able portions of the vitamin content of most of these vitamins in this are heat stable, no great loss of the vitamins would milk are lost through the whey produced occur during processing. Consequently, during the manufacture of cheese. The vitamin content of the milk retained in the vitamin content of “pasteurized proc­ ess” cheese would be comparable to that the cheese curd ranged between 10 and of mild or sharp Cheddar which are richer 40% for the various vitamins. in B-vitamins than medium Cheddar. The vitamin content of fresh cheese curd varied with the vitamin content of the original milk, in that the higher the ACKNOWLEDGMENT vitamin content of the milk, the higher The technical assistance of Clara Zoz is the resultant vitamin content in cheese gratefully acknowledged. curd. In addition, as suggested by Burk­ holder et al. (7) and Sullivan et al. (2), 6 Krett, O. J. 1964 Personal communication. 3 6 8 KAY M. NIL SON, JAYANTKUMAR R. VAKIL AND KHEM M. SHAHANI

LITERATURE CITED 10. Babcock, S. M., H. L. Russell, A. Vivian and 1. Shahani, K. M., I. L. H athaway and P. L. E. G. Hastings 1901 Influence of sugar on Kelly 1962 B-complex vitamin content of the nature of the fermentations occurring cheese. II. Niacin, pantothenic acid, pyri- in milk and cheese. 18th Annual Report of doxine, biotin and folic acid. J. Dairy Sci., the Wisconsin Agricultural Experiment Sta­ 45: 833. tion, Madison, p. 162. 2. Sullivan, R. A., E. Bloom and J. Jarmal 11. Gregory, M. E., J. E. Ford and S. K. Kon 1943 The value of dairy products in nutri­ 1958 The B-vitamin content of milk in tion. III. The riboflavin, pantothenic acid, relation to breed of cow and stage of lacta­ nicotinic acid and biotin content of several varieties of cheese. J. Nutrition, 25: 463. tion. J. Dairy Res., 25: 447. 3. Karlin, R. 1961 Sur le taux de vitamine B6 12. Lichtenstein, H., A. Beloian and E. W. dans les fromages variations au cours de la Murphy 1961 Vitamin B12 microbiological maturation. Internat. Ztschr. Vitaminforsch., assay methods and distribution in selected 31: 176. foods. Home Economics Research Report 4. Karlin, R. 1961 Evolution de la teneur en no. 13. U. S. Department of Agriculture, vitam ine B 12 des fromages pendant la fabri­ Washington, D. C. cation, la maturation et la conservation. 13. Hawk, P. B., B. L. Oser and W. H. Summer- Internat. Ztschr. Vitaminforsch., 31: 326. son 1947 Practical Physiological Chemis­ 5. Karlin, R. and D. Portafaix 1960 Nouvelle try. Maple Press Company, York, Pennsyl­ etude sur la teneur en vitamine B12 du lait vania, p. 215. de vache. C. R. Soc. Biol. Paris, 154: 1810. 14. Teply, L. J., W. A. Krehl and C. A. Elvehjem 6. Collins, E. B., and M. Yaguchi 1959 Vita­ 1947 The intestinal synthesis of niacin and m in B12 wheys prepared by coagulating milk folic acid in the rat. Am. J. Physiol., 148: 91. with acid and rennet. J. Dairy Sci., 42: 1927. 7. Burkholder, P. P., J. Collier and D. Moyer 15. Hardinge, M. G., and H. Crooks 1961 1943 Synthesis of vitamin content of fancy Lesser known vitamins in foods. J. Am. cheeses. Food Res., 8: 314. Dietet. A., 38: 240. 8. Fagen, H. J., J. B. Stine and R. V. Hussong 16. Teply, L. J., P. H. Derse and W. V. Price 1952 The identification of reducing sugars 1958 Composition and nutritive value of in Cheddar cheese during the early stages of cheese produced from milk treated with ripening. J. Dairy Sci., 35: 779. hydrogen peroxide and catalase. J. Dairy 9. Sjostrom, G. 1956 The velocity of sugar Sci., 41: 593. breakdown in cheese. Proc. XIV Interna­ 17. The American Dairy Association 1960 The tional Dairy Congress. Ill (2). p . 562. Household Cheese Market, Chicago, Illinois. Effects of Exercise and Diet on Nitrogenous Constituents in Several Tissues of Adult Rats 1,2

D. A. CHRISTENSEN3 a n d E. W. CRAMPTON Department of Animal Science, Macdonald College, Province of Quebec, Canada

ABSTRACT The effects of exercise and dietary protein level on amounts of DNA, RNA and total nitrogen in livers, gastrocnemius muscles and skin samples, and on serum albumin-to-globulin ratios were examined. The purpose was to test the stability of DNA and hence the validity of nitrogen-to-DNA and RNA-to-DNA ratios where exercise of various durations was imposed; also to evaluate the hypothesis that exercise might reduce protein reserves by converting labile to stable nitrogen compounds, and that protein in excess of the maintenance requirements might compensate for this conversion. One group of rats was preconditioned by intermittent periods of exercise prior to this test while two other groups underwent no previous exercise. The one- month period of exercise imposed during this test led to reduced serum albumin-to- globulin ratios. Feeding a sub-maintenance protein diet had the same effect, but in combination these treatments did not result in a greater decrease. Preconditioning brought about a marked elevation of plasma globulin level. Preconditioned rats when exercised during this trial had greatly increased amounts of DNA, RNA, and total nitrogen in their gastrocnemius muscles. This increase did not occur in rats exercised daily for 28 days. Liver DNA content was increased in the preconditioned group. Neither exercise nor diet brought about statistically significant changes in skin com­ position. It was concluded that albumin-to-globulin ratios and DNA ratios could give misleading results in comparisons of exercised and idle rats.

During the past century numerous ex­ Watkin also observed that exercise led to an periments have been conducted to deter­ increased loss of nitrogen in the sweat and mine the effect of exercise on dietary concluded that this contributed to increased protein requirement. The earlier evidence nitrogen requirement. This claim un­ that exercise increased urinary nitrogen doubtedly has merit; however, the amount output (1, 2) must be discounted because of dermal nitrogen loss depends to a large of the observation that this increase is not extent upon environment rather than proportional to severity or duration of ex­ amount of work done (6, 7). Even if this ercise, but is a consequence of tissue cata­ sweat loss is considered as a separate re­ bolism resulting from insufficient caloric quirement, and it is assumed that exercise intake in relation to the increased energy does not increase protein requirement by expenditure (3). In fact, where energy reducing nitrogen retention, it does not intake is adequate, reduced urinary nitro­ necessarily follow that exercise does not in­ gen loss during exercise has been reported crease protein requirement. In fact, Cramp- (4). Although there is sufficient evidence ton (8) has concluded that most nutrient to conclude that exercise does not increase requirements, including that of protein, are protein requirement through increased uri­ related to energy intake. The experiment nary nitrogen loss, various claims have reported here was carried out to test our been made for beneficial effects of supple­ mentary nitrogen during exercise. Kraut Received for publication December 10, 1964. 1 This work is part of a doctoral dissertation by and Lehmann (5) reported that additional D. A. C hristensen. protein was necessary to m aintain effi­ 2 Financial support for this work was received from the Defence Research Board, Canada Department of ciency of work and prevent psychic depres­ National Defence. 3 Present address: Department of Animal Science, sion. More recently Watkin4 concluded University of Saskatchewan, Saskatoon, Saskatchewan. that increased protein was required to 4 Watkin, D. M., J. B. Das and M. C. McCarthy 1964 Protein for strenuous physical work. Federa­ maintain performance of strenuous work. tion Proc., 23: 399 (abstract).

J . N u t r i t i o n , 8 6 : ’6 5 3 6 9 3 7 0 D. A. CHRISTENSEN AND E. W. CRAMPTON hypothesis that exercise might reduce the age and preconditioning. The rats in group body’s reserve of labile nitrogen com­ 1 were 12.5 months old at the end of this pounds through their conversion to more 6-week experiment and had been precon­ stable compounds, possibly involving trans­ ditioned by exercising daily as described location to another tissue. If such a reduc­ below during their fifth, seventh and ninth tion in reserve protein occurred it might months. Groups 2 and 3 were not precon­ lead to reduced resistance to intoxication ditioned and were 10.5 and 11.5 months and infection (9, 10). We further hypoth­ old, respectively, at the end of the experi­ esized that an increased protein level ment. They received a 22% mixed protein in the diet of working individuals might diet prior to this experiment. be effective in producing an increase in In each group 3 subgroups comprised of labile protein which would tend to offset two exercised and two control rats received any reduction in reserves induced by work. a diet containing 3.75, 7.5 or 15.0% of One of the problems in testing this hypo­ casein, respectively. This diet was made thesis is selection of a sensitive means of up of the following: (in per cent) partially expressing the relative amounts of the hydrogenated vegetable oil, 14; cellulose,5 various nitrogenous components. David­ 5; salt mix (USP no. 2), 4; vitamin mix,6 1; son (11) observed that under various regi­ and starch plus casein, 76. The 7.5% mens DNA is the least variable tissue com­ casein diet was designed to meet the essen­ ponent and concluded that the ratio of tial and non-essential amino acid require­ tissue constituent to DNA is the most sat­ ments of adult rats, assuming a gross isfactory means of expression. Mendes energy requirement of 121 kcal X W °75 kg and Waterlow (12) arrived at the same (15). The amino acid composition of the conclusion after observing that neither casein was estimated (16). starvation nor protein-deficient diets re­ The exercise equipment consisted of 2 duced total liver DNA or DNA per nucleus. aluminum drums, each 90 cm in diameter Allison (13) has established that the and 80 cm long, mounted by means of a amount of serum albumin is a sensitive single axle supported by bearings on a index of protein reserves, and because metal frame. The drums were rotated at changes in globulin level tend to correct 5 rev/min by an electric motor acting changes in blood volume, albumin-to- through a fixed speed transmission. Twelve globulin ratios are the most sensitive index individual acrylic plastic (Lucite) cages, of the amount of albumin. These criteria fitted with individual watering devices have been established for measuring pro­ were supported by the frame so that the tein deficiency under varied dietary regi­ concave lower edges of the sides were just mens and although effect of exercise on above the upper surfaces of the drums, the serum protein level has been reported (14) arrangement being such that the drums the effects of exercise on DNA and RNA formed the floor of the cages. Three brass have not been determined in animals fed electrodes were mounted in one end of graded protein levels. The experiment re­ each cage just above the surface of the ported here was carried out to measure the drums. As the drum rotated, these elec­ effect of exercise on the total amount of trodes were charged intermittently as an these compounds in relation to changes in aid in training the rats to walk. total nitrogen in representative tissue of The control rats, and, during rest peri­ adult rats to determine the reliability of ods, the exercised rats, were housed in DNA as a base for comparing tissue com­ cages 20 X 15 X 15 cm in length, width position, and to reach a preliminary evalu­ and height, respectively. ation of our hypothesized effect of exercise on reserve nitrogen. 5 Alphacel, Nutritional Biochemicals Corporation, C leveland. EXPERIMENTAL 6 The vitamin mix had the following composition : (in per cent) vitamin A acetate (500,000 IU/g), 0.488; vitamin D 3 (1654 IU /g), 17.15; dZ,ct-tocopheryl Three groups of 12 female rats were acetate, 2.12; thiamine-HC1, 0.285; riboflavin, 0.285; selected. Six rats in each group were ex­ niacin, 1.27; Ca pantothenate, 0.855; pyridoxine-HCl, 0.285; choline chloride, 21.27; inositol, 1.42; folic acid, ercised as described below, and six were 0.258; biotin, 0.006; menadione, 0.634; vitamin B 12 (0.1% ), 0.48; and cellulose (Alphacel, Nutritional kept as controls. The groups differed in Biochemicals Corp., Cleveland), 53.194. EFFECTS OF EXERCISE AND DIET ON ADULT RATS 3 7 1

The duration of the experiment was 42 RESULTS AND DISCUSSION days. During the first 14 days the rats The results of this 2X3X3 factorial were fed their respective diets ad libitum experiment were subjected to analyses of but were not exercised. Training of the variance (23). Exercise brought about sig­ rats in the exercised groups was begun on nificant reductions in total serum protein, day 15, and the duration of exercise was albumin, globulin, and albumin-to-globulin gradually increased until day 25. The rate ratio (table 1). In contrast with the ob­ of walking was constant at 14.3 m/minute servations of Yoshimura (14) on young throughout the experiment. The daily ex­ men in a shorter trial, this reduction oc- ercise consisted of cycles of 25 minutes of cured regardless of the dietary protein exercise followed by 5 minutes rest. The level. Albumin-to-globulin ratios decreased daily number of these cycles was gradually to the same level in exercised groups fed increased so that on the twenty-fifth day, 15 and 3.75% casein (0.61 and 0.59, re­ and thereafter, the rats walked for 180 spectively). Groups fed the 3.75% casein minutes. diet had significantly lower (P<0.01) On day 42 of the experiment blood sam­ total protein, albumin and albumin-to- ples were collected following decapitation globulin ratios than those fed the diet con­ while the rats were under light ether an­ taining 7.5% casein. In keeping with the esthesia. Exercised rats were decapitated observations of Allison (13), feeding pro­ 2 to 3 hours after exercise to avoid short­ tein in excess of the requirement did not term effects on blood volume and composi­ increase blood protein levels. tion (17, 18). The difference in age between the two Just before decapitation the lumbar re­ youngest groups did not result in changes gion of each rat was shaved; immediately in amounts of blood proteins, and hence it following blood collection a uniform area may be concluded that the increased total of skin was removed, the liver and the serum protein and reduced albumin-to- gastrocnemius muscles of the hind legs globulin ratios which was due to increased were quantitatively removed and weighed. globulin level in the 12.5-month-old group These tissues were analyzed for DNA and was associated with preconditioning. Blood RNA according to the methods described volume was not measured, hence it is by Ceriotti (19, 20) and for nitrogen by not known to what extent blood volume the micro-Kjeldahl method (21). changes affected globulin level. From this The blood plasma was analyzed for non­ observation it appears that where long-term protein nitrogen (22), and total nitrogen exercise is imposed, albumin-to-globulin (21), and albumin and globulin separation ratios could give misleading results. was carried out with the Spinco model R In agreement with the observations of electrophoresis system. Summers and Fisher (24) and of Mendes

TABLE 1 Effect of exercise, dietary protein level, and conditioning on total serum proteins, albumin, globulins, and albumin-to-globulin ratios of adult female rats 1 Total Albumin-to- Treatment group No./group serum Serum Serum globulin protein albumin globulin ratio g/100 ml g/100 ml g/100 ml No exercise 18 6.57a 2.65a 3.92a 0.68» Excercise 18 6.00» 2.30» 3.70» 0.63» 15% Casein diet 12 6.44» 2.50» 3.94» 0.65*» 7.5% Casein diet 12 6.49» 2.67» 3.82» 0.70» 3.75% Casein diet 12 5.92» 2.25» 3.67» 0.62» Not conditioned (10.5 months old) 12 6.08» 2.45» 3.63» 0.68» Not conditioned (11.5 months old) 12 6.10» 2.48» 3.62» 0.68» Preconditioned (12.5 months old) 12 6.68» 2.51» 4.17» 0.61»

sd of the general mean 0.42 0.24 0.30 0.07 1 Means within a column and treatment group are not significantly different (P < 0.05) if followed by the same superscript. 3 7 2 D. A. CHRISTENSEN AND E. W. CRAMPTON and Waterlow (12) dietary protein level did not influence the amount of DNA in 05 d d d d d d « CM O H 0 )0 5 o t> d the liver (table 2), nor did it influence CD t> I > 00 c d co n5 co co co CO CO CO CO CO co amount of RNA; but as observed by numer­ ous other workers (12, 25, 26) there was Cb 2* d id d d « ci n d 5= q CM q ^ o r-j cq q a reduction in liver nitrogen with reduced G cq CO UO d «0 CM d d d d CO dietary protein level. \ co co co co co CO CO co Although this experiment supported the g use of DNA as a basis for measuring -i d - - « « r d uo changes in liver composition when protein oo cq iq d cm cq q «h o o o d d o ’ O d level is varied, preconditioning resulted in r-f r - ( *-« a significant increase (P < 0.05) in liver DNA (table 2). Hence, where intermittent exercise is involved DNA ratios could not be used satisfactorily. i1 ^(M In contrast with the effect of precondi­ tioning, a single period of exercise brought P about a decrease (P<0.07) in total liver q DNA (table 2). As shown in table 3, this t> o d d d d o £ cm co CM CM (M CM CM CO reduction occurred mainly in those groups 03 which had not been preconditioned. These £ observations indicate that whereas a sin­ Q o q cq ^ o OON gle exercise period inhibits liver DNA for­ d d d d d d d d mation, intermittent periods of exercise stimulate its formation. It is possible that o g V in the former case formation of polyploid g s Oh nuclei is inhibited, whereas during the rest CM CO I> rd ^ O N w CO U0 b- CD CO ^ CO 05 periods following exercise the rate of for­ CM regardless of previous exercise history, it q ^ eo (M q CO

TA BLE 3 Effect of conditioning and exercise on liver DNA 1

G roup No exercise E x ercise m g /liv e r m g /live r Not conditioned (10.5 months old) 21.9bc 18.7C Not conditioned (11.5 months old) 22.3abc 18.I e Preconditioned (12.5 months old) 24.6“ 24.5ab

1 Six rats per mean. Means are not significantly different (P < 0.05) if followed by the same superscript.

TA BLE 4 Interaction of exercise and conditioning on muscle DNA, RNA and nitrogen in adult female rats

No exercise E x e rc ise G roup DNA RNA N itro g e n DNA RNA N itro g e n mg/both gastrocnemius muscles mg/both gastrocnemius muscles N o t conditioned (10.5 months old) 4.0 1 26.8 109 4.0 28.8 118 2 Not conditioned (11.5 months old) 4.1 27.7 114 3.9 27.7 115 Preconditioned (12.5 months old) 3.9 27.1 110 5 .5 3A 34.0 2-4 139 2,4

1 Six rats per mean. 2 Different from corresponding non-exercised group (P < 0.05). 3 Different from corresponding non-exercised group (P < 0.01). 4 Different from both corresponding groups of non-conditioned rats (P < 0.05).

TA BLE 5 Interaction of diet and conditioning on muscle RNA and nitrogen in adult female rats

RNA Nitrogen G roup Casein, % Casein, % 15.0 7.5 3.75 15.0 7.5 3.75 mg/both gastrocnemius muscles mg/both gastrocnemius nmscles Not conditioned (10.5 months old) 27.1 1 29.3 25.1 2 117 117 1072 Not conditioned (11.5 months old) 30.5 25.22 27.4 128 1042 110 Preconditioned (12.5 months old) 27.9 32.7 31.1 118 131 126

1 F o u r r a ts p e r m e a n . 2 Different from corresponding preconditioned group (P < 0.05). 3 7 4 D. A. CHRISTENSEN AND E. W. CRAMPTON action resulted from a tendency for re­ body weight increased to an extent equal duced muscle RNA and nitrogen content in to those fed the 15.0% casein diet. non-conditioned rats when the two lower In agreement with earlier observations protein diets were fed, whereas in the pre­ (11, 12, 24) this experiment showed that conditioned groups the levels of muscle DNA content of tissues is not affected by RNA and nitrogen were maintained re­ variation in dietary protein level. Exercise, gardless of protein level in the diet. however, particularly intermittent periods Analyses of variance of DNA, RNA and of exercise, resulted in significant changes nitrogen in skin samples showed that its in total DNA in liver and muscle which composition was remarkably constant in would rule out its use as a base for meas­ that none of the treatments resulted in uring other components. Moreover, inter­ statistically significant effects on amounts of these components (table 2). mittent periods of exercise increased blood Total feed consumed during the 42-day globulin levels and hence the use of period, expressed as therm intake/100 g albumin-to-globulin ratios to compare ex­ of initial weight, was reduced by exercise ercised and unexercised rats is question­ (table 6). A similar observation was re­ able. ported by Mayer (29) in rats subjected The skin clearly does not contain reserve to strenuous exercise. Moreover, weight protein in a form which can be withdrawn change was significantly reduced (P < during exercise or when low protein diets 0.01) by exercise as a result of exercised are fed. The loss of serum albumin as a rats not maintaining their initial weight, consequence of exercise cannot be modified whereas the cage-idle rats gained mark­ by changes in dietary protein level. The edly. main benefit of feeding higher levels of Preconditioning did not significantly in­ protein to the exercised rats was that a fluence energy intake or weight change greater increase in muscle RNA and nitro­ (table 6). Rats fed the diet containing gen was permitted in those rats that had 15% casein had a significantly lower en­ not been preconditioned. ergy intake than rats fed either of the The hypothesis that exercise might lead other 2 diets. Despite this lower intake to a reduction in reserve protein therefore they gained weight, whereas those rats fed cannot be verified from the results of this the 3.75% casein diet could not maintain experiment. Nevertheless, additional stud­ their weight even though they consumed ies on the effects of exercise on the individ­ more feed. The group fed the 7.5% casein ual components of the labile nitrogen pool diet did not differ in energy intake from should be carried out to illuminate the those fed the 3.75% casein diet but their effects of exercise when the protein intake

T A B L E 6 Effect of exercise, dietary protein level and conditioning on energy intake and body weight change 1

Energy Final wt Treatment group intake/ No./group 100 g minus initial wt initial wt therms 9 No exercise 18 0.887» + 18» Exercise 18 0.786» - 5» 15% Casein diet 12 0.784» + 11» 7.5% Casein diet 12 0.855» + 12» 3.75% Casein diet 12 0.870» — 4» Not conditioned (10.5 months old) 12 0.861a + 9» Not conditioned (11.5 months old ) 12 0.852» + 8» Preconditioned (12.5 months old) 12 0.797» + 2» sd of the general mean 0.083 13.8 1 Means within a column and treatment group are not significantly different (P < 0.05) if followed by the same superscript. EFFECTS OF EXERCISE AND DIET ON ADULT RATS 3 7 5 is at or below the estimated maintenance 15. National Research Council, Committee on requirement. Animal Nutrition 1962 Nutrient require­ ments of laboratory animals, publ. 990. Na­ tional Academy of Sciences — National Re­ LITERATURE CITED search Council, Washington, D. C. 1. Nakayama, T. 1938 Relationship between 16. Joint United States Canadian Tables of Feed muscular exercise and nitrogen equilibrium. Composition 1959 Pub. 659, National Acad­ Japanese J. Exp. Med., 16: 347. emy of Sciences — National Research Coun­ 2. Cathcart, E. P., and W. A. Burnett 1926 cil, Washington, D. C. 17. Darling, R. C., and E. Shea 1951 The effect The influence of muscle work on metabolism of a short period of strenuous exercise on in varying conditions of diet. Proc. Roy. Soc., hemoconcentration. Arch. Phys. Med., 32: B99: 405. 292. 3. Keys, A. 1943 Physical performance in re­ 18. DeLanne, R., J. R. Barnes and L. Broula lation to diet. Federation Proc., 2: 164. 1958 Changes in concentration of plasma 4. Young, D. R., and R. Price 1961 Utiliza­ protein fractions during muscular work and tion of body energy reserves during work in recovery. J. Appl. Physiol., 13: 97. dogs. J. Appl. Physiol., 16: 351. 19. Ceriotti, G. 1952 A microchemical deter­ 5. Kraut, H., and G. Lehmann 1948 Der mination of deoxyribose nucleic acid. J. Biol. Eweisshedarf des Schwerarbeiters. 1. Physi- Chem., 198: 297. ologisches und funktionelles Eiweissmini- 20. Ceriotti, G. 1955 Determination of nucleic mum. Biochem. Ztschr., 3 19: 228. acids in animal tissues. J. Biol. Chem., 214: 6. Best, C. H., and N. B. Taylor 1961 The 59. Physiological Basis of Medical Practice. The 21. Association of Official Agricultural Chemists Williams and Wilkins Company, Baltimore. 1960 Official Methods of Analysis, ed. 9. 7. Lyburn, E. E. St. J. 1956 A comparison of Washington, D. C. the composition of sweat induced by dry heat 22. Hawk, P. B., B. L. Oser and W. H. Summer- and by wet heat. J. Physiol., 134: 207. son 1954 Practical Physiological Chemis­ 8. Crampton, E. W. 1964 Nutrient-to-calorie try. The Blakiston Company, New York. ratios in applied nutrition. J. Nutrition, 82: 23. Snedecor, G. W. 1956 Statistical Methods. The Iowa State College Press, Ames. 353. 24. Summers, J. D., and H. Fisher 1962 Nutri­ 9. Whipple, G. H. 1942 Hemoglobin and tional requirements of the protein depleted plasma proteins: their production utilization chicken. 2. Effect of different protein deple­ and interrelation. Am. J. Med. Sci., 203: 477. tion regimens on nucleic acid and enzyme 10. Cannon, P. R. The importance of proteins activity in the liver. J. Nutrition, 76: 187. in resistance to infection. J.A.M.A., 128: 25. Williams, J. N. 1961 Response of the liver 360. to prolonged protein depletion. 1. Liver 11. Davidson, J. N. 1960 The Biochemistry of weight, nitrogen, and deoxyribonucleic acid. the Amino Acids. John Wiley and Sons, J. Nutrition, 73: 199. N ew York. 26. Addis, T., L. J. Poo and W. Lew 1936 The 12. Mendes, C. B., and J. C. W aterlow 1958 quantities of protein lost by various organs The effect of a low protein diet, and of re­ and tissues of the body during a fast. J. Biol. feeding on the composition of liver and mus­ Chem., 115: 111. cle in the weanling rat. Brit. J. Nutrition, 12: 27. Joubert, D. M. 1956 An analysis of factors 74. influencing post-natal growth and develop­ 13. Allison, J. B., and W. H. Fitzpatrick 1960 ment of muscle fiber. J. Agr. Sci., 45: 59. Dietary Proteins in Health and Disease. 28. Cowdry, E. V. 1934 A Textbook of His­ Charles C Thomas Comapny, Springfield, tology. Lea and Febiger, Philadelphia. Illinois. 29. Mayer, J., N. B. Marshall, J. J. Vitale, J. H. 14. Yoshimura, H. 1960 Proteins and Amino Christensen, M. B. Mashsigekhi and F. J. Acids in Nutrition. Adult Protein Require­ Stare 1954 Exercise, food intake and body ments. Fifth International Congress on Nutri­ weight in normal rats and genetically obese tion. Washington, D. C. adult m ice. Am. J. Physiol., 177: 544. Utilization of Algae as a Protein Source for Humans 1,2

RICHARD DAM, SUNGHEE LEE, PEGGY C. FRY a n d HAZEL FOX Departments of Biochemistry and Nutrition, and Food and Nutrition, University of Nebraska, Lincoln, Nebraska

ABSTRACT Two experiments were conducted using algae as the principle source of nitrogen for human subjects. This represented approximately 90 to 95% of the total nitrogen intake. In the first experiment Scenedesmus obliquus, 7.1 g N/day, was fed as the whole green lyophilized algae. At the end of 5 days the mean nitrogen balance for 5 subjects was +0.20 g N/day. In the second experiment, using 5 differ­ ent subjects, 2 levels of ethanol-extracted Chlorella pyrenoidosa were fed (6.0 and 10.0 g N/day), each for 10 days. The mean nitrogen balance for 6.0-g algae nitrogen was negative, 0.84 g N/day, whereas the mean nitrogen balance for 10.0-g algae nitrogen level was positive, 0.61 g N/day. High fecal excretion of nitrogen was char­ acteristic of all levels of algae. As a result, the algae used in these studies had low apparent nitrogen digestibility, 68% for dried green Scenedesmus obliquus and 58% for ethanol extracted Chlorella pyrenoidosa. This apparent digestibility was the major contributing factor to positive or negative nitrogen balance. The second experiment demonstrated that human subjects were capable of consuming algae as the principle source of protein in the diet for a 20-day period without ill effect.

During the past several years, many un­ EXPERIMENTAL conventional foodstuffs have been investi­ In the first experiment, 5 inmates of the gated as potential food sources for man. Nebraska State Penitentiary were used as One of these foodstuffs was algae which subjects. These men were 27 to 33 years has received especial attention because of of age and weighed from 68 to 82 kg. All its possible use in closed ecological sys­ of the men had been subjects in previous tems. Even though interest in algae is diet studies. They were selected for this widespread, few studies have been con­ experiment because information was avail­ ducted on the nutritional adequacy of this able on their caloric and nutrient require­ material for man (1-5). Earlier workers ments and on the basis of their previous (3) have reported the “true digestibility” cooperation. Mixed amounts of 2 strains of of decolorized Chlorella to be 83.0%. They Scenedesmus obliquus ATCC 11457 and reported that the residual smell and taste 11477 were used in the first experi­ of the decolorized Chlorella were intoler­ ment. Each strain was cultured separately able and impaired appetite. In later stud­ and grown autotrophically in the double ies (4) “true digestibilities” of 75% for strength medium of Chorney et al. (6). blanched algae and 86% for methanol- Cultures were grown under constant il­ extracted algae were reported. In these lumination and were continually aerated 2 studies approximately 30 g of algae were with a mixture of 95% air 5% CCb. Cells fed per day. This supplied about one-third were harvested by continuous centrifuga­ of the total protein. Only one study (5) tion and then lyophilized. The dried algae reports feeding amounts of algae in excess was autoclaved at 121° for 30 minutes. of 35 g (dry weight) per day. Subjects tol­ Ethanol extraction was carried out in a erated amounts up to 100 g/day quite well, large Soxhlet extractor for 48 hours. The but acute toxicity symptoms developed at extracted material was air dried. The proxi­ 200- and 500-g levels. The present paper presents data from mate analyses of the 2 forms of algae are shown in table 1. 2 separate nitrogen balance studies with human subjects, using an experimental diet in which algae was the principle Received for publication March 1, 1965. 1 Published with the approval of the Director as source of protein nitrogen. Algae con­ paper no. 1663, Journal Series, Nebraska Agricultural tributed 90 to 95% of the total dietary Experiment Station. 2 Supported in part by University of Nebraska Re­ nitrogen intake. search Council Fund W4315-10A.

376 J. N utritio n , 86: ’65 ALGAE AS A PROTEIN SOURCE FOR HUMANS 3 7 7

TA BLE 1 Chemical composition of algae samples 1

Scenedesmus obliquus, Chlorella. pyrenoidosa, ATCC 11457, 11477 mixed thermophillic strain 71105 Green 48-hour- Green 144-hour- lyophilized extracted lyophilized extracted % dry w t % dry w t % dry w t % dry w t Moisture 3.7 2.6 5.8 6.2 Crude protein (N X 6.25) 48.2 58.7 56.5 69.2 Ether extract 6.8 0.8 2.2 1.5 Crude fiber 6.8 9.7 2.5 5.1 Carbohydrate (by difference) 27.3 19.4 25.8 9.7 Ash 7.2 8.8 7.2 8.3 Total 100 100 100 100 Calcium 0.36 0.44 0.07 0.09 Phosphorus 1.74 2.05 1.79 1.97 Isoleucine 2 2.20 2.76 Leucine 4.41 6.40 Lysine 3.50 4.31 M ethionine 0.81 1.18 Phenylalanine 2.65 3.24 Threonine 2.02 2.80 Valine 3.01 3.78 1 Standard AOAC procedures (Association of Official Agricultural Chemists 1960 Official Methods of Analysis, ed. 9. Washington, D. C.). 2 All amino acid values were obtained microbiologie ally using Streptococcus spp. ATCC 8042, after acid hydrolysis.

A preliminary adjustment period of 5 grams) calcium, 2.1; phosphorus, 1.3; days preceded a 5-day algae trial period sodium, 2.7; potassium, 3.6; for the algae using dried whole green Scenedesmus ob- period the intakes were calcium, 1.3; liquus as the primary source of protein. phosphorus, 1.9; sodium, 3.2; and potas­ The first trial period was followed by a sium, 1.9. In experiment 2 the calculated second brief adjustment period, then a average daily intakes were: (in grams) second 5-day algae trial period during calcium, 0.9 to 1.7; phosphorus, 1.3 to 2.2; which time ethanol-extracted Scenedesmus sodium, 4.4 to 7.7; and potassium, 0.4 to obliquus served as the primary source of 1.1. In experiment 2 the range of the protein. During the perliminary period values reflects differences in intake of subjects were fed 2500 to 3000 keal/day. algae and caloric supplements. The values All subjects received 6.5 g of nitrogen as for sodium and potassium in experiment 2 protein nitrogen in the form of a dietary do not include amounts present in the beverage,3 fortified with non-fat dry milk. extracted algae, since analytical data on The subjects also received canned fruits this material are not available. The sodium and instant coffee or tea. The total nitro­ content of this material is estimated to be gen intake varied then from 7.2 to 7.3 g almost nil and the potassium content about depending on these supplements. A daily 0.5 g. During the first algae experimental vitamin supplement4 provided the follow­ period, 92.5 g whole green lyophilized algae ing: (in milligrams) thiamine, 2; ribo­ provided 7.4 g nitrogen/day. Supplemental flavin, 2.5; ascorbic acid, 50.0; pyridoxine, nitrogen from fruits and coffee increased 1; niacinamide, 20; pantothenic acid, 1; the levels to a total of 7.8 to 8.2 g/day. and vitamin Bi2, 1 ag; vitamin A, 5000 IU; Caloric intake was maintained at the same and vitamin D, 500 IU. A daily mineral level as in the preliminary period. For the supplement was not given. Daily intakes first 4 days, algae was given in biscuit of calcium, phosphorus, sodium and potas­ 3 Metrecal, Edward Dalton Company, Evansville 12, sium were estimated from analytical data Indiana. 4 Miles Products, Division of Miles Laboratories, Inc., available for algae and tabulated values Elkhart, Indiana. 5 Watt, B. C., and A. L. Merrill 1963 Composition for other dietary ingredients.5 In experi­ of Foods — Raw, Processed, Prepared. Agriculture ment 1 the calculated average daily intakes Handbook no. 8, Consumer and Food Economics Re­ search Division, ARS, U.S. Department of Agriculture, during the preliminary period were: (in Washington, D. C. 3 7 8 RICHARD DAM, SUNGHEE LEE, PEGGY C. FRY AND HAZEL FOX form only. On the fifth day part of the occasion during the first 10-day period and algae was incorporated into other foods on 3 occasions during the second 10-day in the diet. One-half of the algae (46 g) period one biscuit was replaced by an algae was added to the following foodstuffs in pizza. This pizza was essentially the same the amounts indicated: tomato juice, 10 g/ as the biscuit, but contained some added 473 ml; flavored gelatin,6 7 g/118 ml; starch and seasoning, and was topped with canned plums, 5 g/100 g; pineapple drink, tomato paste. Variable ingredients in diets 18 g/708 ml; and beef bouillon, 6 g/236 consisted of extra calorie wafers, butter, ml. The algae biscuit formula was: (in jelly, coffee or tea, sugar, carbonated soft grams) wheat starch,7 160; sugar, 20; bak­ drink, roll candies and pickles. These were ing powder, 14.4; salt, 2; butter oil, 100; used to adjust the fat and caloric intake algae, 92.5 and water, 80. This formula of the subjects. These ingredients allowed provided one day’s supply of 12 biscuits some selection by the subject, but once per subject. selection was established they were held A second experiment was conducted constant. A sample of each ingredient using a commercial preparation of Chlorel- used in the experiment was analyzed for la pyrenoidosa, thermophillic strain 71105. nitrogen content by the Kjeldahl method. This was grown in the medium described In both experiments, total urine and by Lubitz (7).8 The entire sample was fecal collections were made daily. Creati­ autoclaved for 15 minutes at 121°. This nine determinations were carried out daily material was extracted for 144 hours with on urine samples as a check on the com­ ethanol. The chemical analysis of this pleteness of individual collections. Car­ material is shown in table 1. Subjects were mine was used as a marker to indicate the selected from staff and graduate students beginning or ending of fecal collection at the University of Nebraska. A group periods. of 12 prospective subjects was fed 3 meals RESULTS typical of those that they would have dur­ The first experiment was designed to ing the experimental period. Five sub­ determine nitrogen utilization and accepta­ jects, 4 male and one female, were selected bility of green and ethanol-extracted Scene- from this group. These subjects ranged desmus obliquus. In determining the ac­ in age from 24 to 35 years, and in weight ceptability of this material it also seemed from 55 to 97.5 kg. Algae nitrogen was important to determine whether individ­ fed at 2 levels, 6.0 and 10.0 g, each for a uals would prefer to receive this material period of 10 days. These levels were in a single concentrated form such as a equivalent to 54.2 and 90.3 g of extracted biscuit or to have it distributed throughout algae, respectively. Two of the subjects the diet mixed with foods that might coun­ started with the diet at the 10.0-g level teract the algae flavor. During the first 4 and three at the 6.0-g level. At the end of days, the subjects were able to ingest the 10 days these levels were reversed. The diet containing green algae although they first algae period was preceded by a 3-day complained about the green color of the depletion period during which time the biscuits, the bitter taste of the whole algae, subjects received 2.8 to 3.0 g of nitrogen of upset stomach, and of a bloated feeling. per day. Caloric intake was maintained at Also of concern was algae halitosis, which approximately 45 kcal/kg body weight for made the subjects somewhat less accepta­ the men and 40 kcal/kg for the female ble to their compatriots. On the fifth day subject. Fat intake was adjusted to 40% when algae was mixed with other foods, all of the total caloric intake. of these complaints were greatly accen­ The algae was fed primarily in the form tuated, resulting in nausea. Nitrogen bal­ of a more concentrated biscuit. This bis­ ance results for the preliminary and first cuit formula was: (in grams) wheat algae periods are presented in table 2. starch, 50; sugar, 55; salt, 0.5; butter, 40; Mean nitrogen balance approached equi- algae, 90.3 and water, 60. This provided 6 Jello, General Foods, Inc., New York. one day’s supply of 4 biscuits per subject. 7 Generously supplied by Hercules Powder Company, To diminish further the bitter taste, the Harbor Beach, Michigan. 8 Purchased from General Dynamics Corporation, algae was first fried in butter. On one Electric Boat Division, Groton, Connecticut. ALGAE AS A PROTEIN SOURCE FOR HUMANS 3 7 9

TABLE 2 Nitrogen balance and apparent digestibility of algae diets

T reatm ent F ecalN N balance Apparent N digestibility g/day g/day % Experiment 1 Preliminary 0.75 ± 0 .1 0 1 — 0.19 ± 0.66 1 90 Green lyophilized S. obliquus 2.60 ± 0.31 + 0.20 ±0.46 68 Experiment 2 Extracted C. pyrenoidosa, low level 2.86 ± 0 .2 3 — 0.84 ± 0.30 57 Extracted C. pyrenoidosa, high level 4.35 ± 0 .2 2 + 0.61 ± 0.25 59

1 s e o f m e a n . librium in both periods with the response is presented in table 2. One subject com­ being slightly improved in the period of pleted only the first 5 days at this level. algae feeding. However, one individual Two days later he withdrew from the had an increased nitrogen excretion with study. However, balance data indicated the green algae diet. he was apparently in more positive nitro­ The response to the ethanol-extracted gen balance than any of the other subjects material was rather disappointing. This during this period. The remaining 4 sub­ material was tolerated less well than the jects were in negative balance for the over­ fresh material, which is contrary to re­ all period. In order to have at least 5 sub­ ports from other laboratories. This extrac­ jects studied at the 10-g level of algae tion process had removed almost all of the nitrogen, another subject was added to the green color and the bitter taste. A faint- experiment. He was given a 2-day deple­ forage-like flavor remained. Four of the tion diet and then started to eat the diet subjects became nauseated after complet­ providing 10 g nitrogen from algae. The ing one meal and were forced to discon­ summary of results of nitrogen balance tinue the study. However, one subject com­ data for the 10-g level are also presented pleted the study period without any ill in table 2. The mean balance for the 10- effect. Whether this unfavorable response day period was positive, +0.62 g nitrogen was due to the extracted algae itself, or compared with a negative balance, —0.84 g due to psychological or physiological fac­ nitrogen, when subjects were fed 6.0 g tors from recent association with the fresh nitrogen from algae. Four of the five sub­ algae, is not known. It was concluded that jects were in positive balance for this 10- algae was most acceptable when it was day period. The acceptability of the algae incorporated into one food in the diet. In­ diet in experiment 2 was markedly im­ corporation of this green material in sev­ proved over that in experiment 1. In the eral foodstuffs aggravated any objections second experiment no complaints of subjects might have to the algae. nausea, bloated feeling or bitter taste were A second experiment was conducted to attributed to the algae. Those complaints obtain further information on the level of that were registered concerned the vari­ dietary algae nitrogen necessary to main­ able ingredients in the diet, that is, the tain a positive nitrogen balance. Chlorella extra calorie wafers, roll candy, jelly, etc., pyrenoidosa thermophillic strain 71105 was and the monotomy of the diet. used in this experiment. Sufficient material A high fecal excretion of nitrogen was was available to feed algae nitrogen at 2 characteristic of. all levels of algae in both levels, 6.0 and 10.0 g. These levels were experiments. In experiment 1 the contrast slightly lower and higher, respectively, in fecal nitrogen excretion between the than the level of algae nitrogen required preliminary period and the algae period is in the first experiment to maintain nitro­ very marked, 0.75 g nitrogen/day, average, gen equilibrium. versus 2.60 g nitrogen/day, average. The A summary of the nitrogen balances of variation in fecal excretion between in­ these subjects on 6.0 g of algae nitrogen dividuals eating the algae diet was also 3 8 0 RICHARD DAM, SUNGHEE LEE, PEGGY C. FRY AND HAZEL FOX quite marked. Response to the algae diet Two reasons for the poor digestibility of indicated a mean apparent digestibility of algae may be considered: 1) resistance of 68% (range 59 to 77% ) in contrast with the algae cell wall to disruption by the nor­ a value of 90% (range 87 to 94% ) for the mal digestive process, and 2) release of diet fed during the preliminary period. nitrogen from the cell in a form which can­ High fecal nitrogen excretions were also not be digested or absorbed from the diges­ characteristic of both levels of algae nitro­ tive tract. gen used in the second experiment. The Assuming complete disruption of the apparent nitrogen digestibility of the 6.0-g cell, the chemical form and anatomical nitrogen diet was 58% (range 51 to 65% ), location in the algal cell of the nitrogen- and that of the 10.0-g nitrogen diet, 59% containing compounds may exert some in­ (range 54 to 66% ). Again there was vari­ fluence on digestibility. Data on Chlorella ation between individuals in fecal nitrogen pyrenoidosa (8) suggest that approxi­ excretion, particularly with the 10.0-g mately 3.7% of the total protein of dry nitrogen level. The difference in apparent algae is in the cell wall. This percentage digestibility of the algae was the major of the total protein would be increased contributing factor to a positive or nega­ after ethanol extraction. This protein is tive nitrogen balance. part of the continuous matrix and is prob­ ably resistant to attack by proteolytic en­ DISCUSSION zymes in the digestive tract. If allowance In these studies algae was fed as the was made in the nitrogen balance data for primary source of protein to human sub­ this material, the digestibility data would jects. The authors are unaware of previ­ be somewhat improved. However in no ous studies in which algae has served as way could this account for the 30 to 40% the primary source of protein for a period of non-absorbed nitrogen. as long as 20 days. A major observation Another possible source of non-digestible in the present experiments is that nitrogen nitrogen is the non-protein nitrogen frac­ balance can be maintained in human sub­ tion. This fraction represents 15 to 20% jects using algae as the protein source. The of the total nitrogen. Nitrogen-containing data presented in table 2 suggest that 8.0 compounds in algae, other than amino to 10.0 g of algae nitrogen would be suffi­ acids, are chlorophyll, amides, purines and cient. A second observation in the present pyrimidines, and amino sugars. The chloro­ studies is the low apparent nitrogen diges- phyll content of Chlorella pyrenoidosa may tability of the algal species used. Over vary from 3.3 to 6.6% of the total weight 30% of the nitrogen present in the diet in depending upon the light intensity under experiment 1, and over 40% in experiment which it is grown (9, 10). It is conceiv­ 2 was apparently not absorbed from the able that chlorophyll may account for 3 digestive tract. This utilization would un­ to 5% of the total nitrogen in algae. doubtedly be improved through use of a Ammonia or amide nitrogen accounts for processing technique to rupture the cell 8.1 to 8.9% of the total nitrogen in this walls before the incorporation of algae in species (11). Nitrogen provided by gluco­ the diet. samine amounts to a much smaller frac­ The digestibility values presented in this tion of the total, about 0.4% (8). Of these paper are much lower than those reported non-protein forms of nitrogen it appears by others (3, 4) for human subjects. In most likely that only chlorophyll would the present study a direct method was used affect digestibility, since the phytyl side to measure nitrogen digestibility, whereas chain would make this compound suffi­ in the previous studies an indirect method ciently insoluble to be poorly absorbed. If was used. Thus differences between the the cell wall protein and the chlorophyll present study and prior studies may have are considered essentially inert, unabsorb- reflected differences in methodology used. able forms of nitrogen, the digestibility If algae is to be considered as a major data for the non-extracted algae (exp. 1) source of dietary protein the direct method are somewhat improved. In experiment 2, is preferred since this method more ade­ ethanol extraction would have removed a quately reflects digestibility. large portion of the chlorophyll. In this ALGAE AS A PROTEIN SOURCE FOR HUMANS 3 8 1 case, the effect on digestibility would be ments (15) were not continued long less pronounced. enough to permit an evaluation of toxicity Even if these factors are considered, the or differences in acceptability with sterile value for fecal nitrogen excretion would versus “contaminated” algae. Indeed, com­ still be very high, 25 to 30% of the total positional changes resulting in difference nitrogen. The explanations remaining are in amounts of cell wall material as well as that the bulk proteins of algae are not in carbohydrates and lipids may have ex­ digestible per se, or that an intact cell wall plained the difference in digestibility be­ prevents or retards access of the digestive tween the 2 sterile forms. Furthermore, enzymes to the bulk proteins. The latter is the sterile and contaminated autotrophic supported by the presence of intact algae cultures were not grown under the same cell membranes in the feces of human sub­ laboratory conditions so that compositional jects (12) and that increased destruction changes resulting from such factors as dif­ of the algal cell wall results in increased ferences in illumination, age of cell at har­ nitrogen utilization in rats (13). That vest and components of media may have nitrogen equilibrium was maintained in been more prominent than the effect of the present studies indicates that the nitro­ bacterial contamination in explaining the gen made available to the individual was difference in response to the 2 autotrophic well utilized. cultures. Another possible cause of the A problem area in algae feeding has gastrointestinal disturbances might be the been that of palatability. The whole green presence of antibacterial substances re­ algae has a bitter taste and a forage-like ported present in these algal species (16- flavor. The latter appears to result in a 19).9 Previous workers (5) reported that definite after-taste when this product is symptoms were most marked at the be­ consumed in foods. Ethanol extraction ginning of each new algae period, and that greatly alleviated these conditions. This is the subjects readapted to each level. A in agreement with the previous reports of similar adaptation phenomenon was noted other workers (3). In the present studies, in experiment 1 of our studies. Part of treatment of the extracted algae by frying this adaptation was undoubtedly due to in butter before incorporation into food­ taste. Part of it might also represent an stuffs appeared to further reduce these ob­ adaptation or alteration of the microflora jectionable properties. Others (14) report of the gastrointestinal tract. Whatever the elimination of this problem by a photo- the explanation, gastrointestinal disturb­ bleaching process. Such material remains ances were not a problem in experiment 2 to be tested in larger scale nutritional stud­ of the present studies. Either the proces­ ies. It appears, however, from the present sing treatment was sufficient to overcome studies, and those of other workers, that these factors, or else the factors were not the problem of palatability can be over­ present in this species of algae. come. Feeding algae to humans as a major A second problem of some concern has dietary item has been a virtually unex­ been that of possible toxicity of algal prep­ plored field of investigation. Many ques­ arations. Previous workers (5) reported tions remain to be answered. What effects that gastrointestinal symptoms occurred do species and culture conditions have on when 100 g or more of algae were fed. digestibility? What processing conditions Similar observations were made in experi­ will yield the most acceptable and nutri­ ment 1 using whole green Scenedesvms tious product? What is the availability of obliquus as the source of algae. That algae the vitamin and caloric content of algae? toxicity evidenced by gastrointestinal dis­ Can some intermediate food producer, tress may be caused by bacterial contami­ plant or animal, be profitably inserted in nation has been suggested (15). These the food chain between algae and man? workers, in a digestion trial with rats, com­ These remain a challenge. As the present pared sterile heterotrophic and autotrophic 9 Levina, R. I. 1961 Antagonism between plank­ cultures of Chlorella pyrenoidosa with a tonic algae and microflora in biological ponds. In contaminated culture of this algae. It has The Purification of Waste Waters in Biological Ponds. Minsk. Akad. Nauk USSR p. 136 (cited in Biol. Abstr., been pointed out (14) that these experi­ 42: 18828, 1963). 3 8 2 RICHARD DAM, S UNGHEE LEE, PEGGY C. FRY AND HAZEL FOX studies indicate that it is possible to con­ structure of the cell wall of Chlorella pyre- noidosa. Biochem. J., 70: 391. sume algae as a major dietary component 9. Sargent, M. C. 1940 Effect of light inten­ without ill effect for longer periods than sity on the development of the photosynthetic previous studies have indicated, it should mechanism. Plant Physiol., 15: 275. be possible to investigate these questions. 10. Myers, J. 1946 Culture conditions and the development of the photosynthetic mecha­ nism. III. Influence of light intensity on ACKNOWLEDGMENTS cellular characteristics of Chlorella. J. Gen. We would like to acknowledge the tech­ Physiol., 29: 419. nical assistance of Mrs. Nelle S. Bennett, 11. Leveille, G. A., H. E. Sauberlich and M. E. McDowell 1962 Nutrient content of vari­ Mrs. Sue Jenkins, Mrs. Donna Gesch- ous algae and amino acid adequacy for wender and Mrs. Agnes Bray. We also growth of rats and chicks. In Biologistics for thank Warden Maurice Sigler, Deputy Space Systems Symposium. 6570th Aerospace Warden John Greenholtz, the staff and Med. Res. Labs. Tech. Documentary Report no. AMRL-TDR-62-116, 1962. Wright-Patter­ inmates of the Nebraska State Penitentiary son Air Force Base, Ohio, p. 405. for their cooperation. 12. Tamura, E., H. Baba, A. Tamura and Y. Kobatake 1958 Nutritional studies on LITERATURE CITED Chlorella, Report 12. Electron microphoto of digested Scenedesmus. Annual Report of The 1. Jorgensen, J., and J. Convit 1953 Culti­ National Institute of Nutrition, Toyamacho, vation of complexes of algae with other fresh­ Tokyo, Japan, p. 20. water microorganisms in the tropics. In 13. Meffert, M. E., and W. Pabst 1963 Uber Algal Culture from Laboratory to Pilot Plant, die Verwertbarkeit der Substanz von Scene­ ed., J. S. Burlew, pub. 600. Carnegie Institu­ desmus obliquus als Eiweissquelle in Ratten- tion of Washington, Washington, D. C., p. Bilanz-Versuchen. Nutritio Dieta, 5: 235. 190. 14. McDowell, M. E., and G. A. Leveille 1963 2. Morimura, Y., and N. Tamiya 1954 Pre­ Feeding experiments with algae. Federation liminary experiments in the use of Chlorella Proc., 22: 1431. as human food. Food TechnoL, 8: 179. 15. Vanderveen, J. E., E. G. Sander, C. M. Cox 3. Hayami, H., and K. Shino 1958 Nutri­ and K. M. Offner 1962 Nutrition value of tional studies on decolorized Chlorella. II. algae grown under sterile conditions. In Studies on the rate of absorption of the de­ Biologistics for Space Systems Symposium. colorized Chlorella on adult men. Annual 6570th Aerospace Med. Res. Labs. Tech. Report of the National Institute of Nutrition. Documentary Report no. AMRL-TOR-62-116, Toyamacho, Tokyo, Japan, p. 59. 1962. Wright-Patterson Air Force Base, Ohio, 4. Hayami, H., K. Shino and Y. Morimura p. 357. 1961 Chemical composition and applicabil­ 16. Pratt, R., T. C. Daniels, J. J. Eiler, J. B. ity as food and feed of mass-cultured uni­ Gunnison, W. D. Kumler, J. F. Oneto, L. A. cellular algae. IV. Test of digestibility with Strait, H. A. Spoehr, G. J. Hardin, H. W. Mil­ human beings. Final Report, no. 1, Contract ner, J. H. C. Smith and H. H. Strain 1944 NODA 92-557-FEC 33129, U. S. Army Re­ Chlorellin, and antibacterial substance from search Development Group 9852. Far East. Chlorella. Science, 99: 351. 5. Powell, R. C., E. M. Nevels and M. E. 17. Spoehr, H. A., J. H. C. Smith, H. H. Strain, McDowell 1961 Algae feeding in humans. H. W. Milner and G. J. Hardin 1949 Fatty J. Nutrition, 75: 7. acid antibacterials from plants, pub. 586. 6. Chorney, W., N. J. Scully, H. L. Crespi and Carnegie Institution of Washington, Wash­ J. J. Katz 1960 The growth of algae in ington, D.C. deuterium oxide. Biochim. Biophys. Acta, 18. Jorgensen, E. G. 1962 Antibiotic sub­ 37: 280. stances from cells and culture solutions of 7. Lubitz, J. A. 1961 The protein quality, unicellular algae with special reference to digestibility and composition of Chlorella some chlorophyll derivatives. Physiologia 71105, ASD Technical Report 61-535, Con­ Plantarum, 15: 530. tract no. AF 33(616)-7373, Project no. 61 19. Smith, L. W. 1944 Chlorophyll: An experi­ 7-6373, Task no. 63124. Wright Air Develop­ mental study of its water-soluble deriva­ ment Center, Wright-Patterson Air Force tives. I. Remarks upon the history, chemis­ Base, Ohio. try, toxicity and antibacterial properties of 8. Northcote, D. H., K. J. Goulding and R. W. water-soluble chlorophyll derivatives as ther­ Horne 1958 The chemical composition and apeutic agents. Am. J. Med. Sci., 207: 647. Iron Deficiency in Rats:

CHANGES IN BODY AND ORGAN WEIGHTS, PLASM A PROTEINS, HEMOGLOBINS, MYOGLOBINS, AND CATALASE 1,2

RUTH P. CUSACK a n d W. DUANE BROWN Institute of Marine Resources, Department of Nutritional Sciences, University of California, Berkeley, California

ABSTRACT Changes in body and organ weights; plasma protein components: he­ moglobins; skeletal and heart muscle myoglobins; and liver catalase were investigated in rats consuming a diet lacking in iron. Iron-deficient rats displayed gray rather than black fur, an early marked cardiomegaly, and a delayed massive splenomegaly. Total plasma protein concentration in deficient animals, 6.49 g/100 ml, was significantly greater than that of control animals, 5.58 g/100 ml. This difference was due to a higher level of /l-globulins in the deficient groups, 2.75 g/100 ml as compared with 1.80 g/100 ml in the control group. Albumin levels in deficient animals, 2.65 g/100 ml, did not differ significantly from control animals, 2.45 g/'100 ml. Myoglobin concentra­ tion in hind leg muscle did not change significantly during 12 weeks of dietary iron deprivation. In weanling rats, this myoglobin concentration was 0.71 ± 0.28 mg/100 mg N, and in deficient animals at 15 weeks of age it was 0.61 ± 0.10 mg/100 mg N. During this time, however, myoglobin concentration in skeletal muscle of pair-fed control animals increased to 1.90 ± 0.31 mg/100 mg N. Heart myoglobin concentra­ tion increased in both deficient and control animals. Because of the massive increase in size, total myoglobin content of deficient hearts was much greater than that of con­ trols. Weanling rat hearts contained an average of 0.29 mg myoglobin; after 6 weeks on the diets hearts from deficient animals contained an average of 1.43 mg myoglobin, and hearts from pair-fed controls, an average of 0.53 mg. Catalase activity did not differ in control and deficient animals. Unit activity did not change significantly dur­ ing the study, but as liver size increased with age, total catalase activity of liver in­ creased also. Heterogeneity of hemoglobin and myoglobin was demonstrated by elec­ trophoretic separation of 4 hemoglobin fractions and 2 myoglobin fractions. However, bands from deficient animals appeared similar to those of control animals.

Lowered blood hemoglobin as a result of The present study was designed to in­ lack of dietary iron is well documented, vestigate the progressive effects of dietary but the fate of the smaller iron compart­ iron deficiency in the rat on myoglobin, ments, such as myoglobin, the cytochrome catalase and plasma protein concentra­ enzymes and catalase, is less certain. tions as related to changes in hemoglobin. Hahn and Whipple (1) observed both myo­ In addition, the presence and possible globin and iron-containing enzymes to be change of multiple forms of hemoglobin normal in iron-deficient dogs and suggested and myoglobin under the influence of that tissue iron was “inviolable.” On the severe deprivation of iron were investi­ other hand, Gubler and co-workers (2) gated, and changes in general appearance noted cytochrome c and myoglobin levels and sizes of livers, spleens, kidneys, and to be severely reduced in iron-deficient hearts were noted. pigs. The experiments of Beutler and Blaisdell (3) with iron-deficient rats indi­ EXPERIMENTAL cated decreased levels of cytochrome c in Male weanling rats of the Long-Evans liver and kidney and of cytochrome oxi­ strain from the colony of the Nutritional dase in kidney, with no change in catalase Sciences department were used in the ex­ in liver or red cells. These later workers periment. They were housed in individual reported cases of iron deficiency in hu­ mans not accompanied by anemia and Received for publication February 27, 1965. 1 Supported in part by Public Health Service Re­ noted subjective improvement in other search Grant no. (GM-09899) from the National Insti­ patients before an improvement in the tutes of Health. 2 Presented in part at the 6th International Congress hemoglobin level. of Nutrition, Edinburgh, Scotland, 1963.

J. N u t r it io n , 86; ’65 383 384 RUTH P. CUSACK AND W. DUANE BROWN

galvanized iron cages equipped with bottles control and 0.2 ml from deficient animals. of distilled water. The iron-poor diet, fed Absorption was measured at 540 mu in a ad libitum, was whole dry milk with 10 mg Bausch and Lomb model 20 spectropho­ CuS04-5H20, 250 mg MnSCh, 1 mg K I03 tometer. The method was standardized and 1.8 g choline bitartrate added/kg of against the total iron determination of milk. The control diet contained in addi­ Wong (5 ). tion 300 mg FeS04-7H20/kg and was fed Total plasma protein concentration was to 2 groups. Animals in one group were determined by the Biuret method (6) us­ pair-fed to the experimental group, receiv­ ing the Bausch and Lomb spectrophotom­ ing food 3 times weekly. The second con­ eter. The method was standardized us­ trol group was fed ad libitum. The defi­ ing crystalline bovine plasma albumin.4 cient diet contained 6 ppm iron and the For determination of individual protein supplemented diet 66 ppm. components, plasma was separated from A complete vitamin mixture was given erythrocytes by centrifuging at 3000 rev/ to all animals 3 times weekly in glass min for 20 minutes in a refrigerated cen­ castor cups. The water-soluble vitamin trifuge. The proteins were separated in a mix contained: (in milligrams) thiamine- Spinco Model R paper electrophoresis HC1, 84; riboflavin, 126; niacinamide, 945; chamber using barbiturate buffer, pH 8.6, Ca pantothenate, 630; pyridoxine-HCl, 147; ionic strength 0.075, and a current of 12 folic acid, 21; biotin, 21; vitamin Bi2, 0.63; ma for 16 hours. Bands were stained with and menadione, 21; dissolved in 1500 ml Bromphenol Blue and their density meas­ 5% ethanol. Two milliliters of this solu­ ured by a Spinco Model RB Analytrol; the tion were given each time along with 3 percentage of each protein was calculated drops of an oil solution containing 2 g from the densitometer tracings. vitamin A distillate (500,000 IU/g), 250 Erythrocytes were washed 3 times with mg irradiated ergosterol (400,000 IU/g) 0.9% NaCl, then frozen in small plastic and 5 g dba-tocopheryl acetate and 993 g test tubes. To remove hemoglobin, the cottonseed oil. cells were thawed, then lysed with an Animals were taken for analysis at equal volume of water. A half volume of weekly intervals for 6 weeks, then every chloroform was added, the mixture shaken, other week for six additional weeks. A then centrifuged, and the clear red super­ total of 113 animals was analyzed; five natant decanted. Multiple forms of hemo­ weanlings, five in each deficient and pair- globin were separated by electrophoresis in fed control group and two in each ad columns of acrylamide gel using the libitum control group. They were anes­ Canalco Model 12 disc electrophoresis ap­ thetized with intraperitoneal injections of paratus.5 pentobarbital sodium 3 at levels of 9 mg/ Myoglobin concentration in heart and 100 g of body weight. The abdominal wall leg muscle was determined by the method was opened, blood was withdrawn from the of Brown (7). The frozen muscle was heart with a syringe coated with a 1% sliced with a scalpel and weighed. It was solution of heparin and transferred to test ground with sand in a mortar and pestle, tubes containing 0.15 mg heparin/ml of then transferred to a large centrifuge tube blood as anticoagulant. Liver, kidneys, with a minimum of cold water and re­ heart, and spleen were removed, the heart frigerated overnight. Following centrifug­ was washed in saline solution and all or­ ing for 30 minutes at 10,000 rev/min, the gans were blotted and weighed. The liver supernatant was decanted and measured. and heart were wrapped in individual A one-milliliter sample of this extract was water-impermeable packages and frozen applied to a 1X 6 cm column of DEAE immediately in dry ice. In addition the cellulose equilibrated with 0.05 m tris hind leg muscle was removed from the buffer, pH 8.6. On elution with buffer, bone and frozen. Hemoglobin concentration was deter­ 3 Nembutal, Abbott Laboratories, Inc., North Chi­ mined as the metcyano derivative by the cago, Illinois. 4 Armour Laboratories, Chicago. method of Collier (4) modified to use 5 The procedure as outlined in newsletter no. 3 of the Canal Industrial Corporation, Bethesda, Maryland, larger volumes of blood, i.e., 0.1 ml from on hemoglobin analysis was followed. IRON DEFICIENCY IN RATS 3 8 5 hemoglobin remains at or near the top of Sandell (9) was used except that 0.5% the column and myoglobin passes through. 2,2-bipyridyl was used for the color forma­ The concentration of the colored eluate tion instead of ortho-phenanthroline. was measured in a Cary model 11 spectro­ photometer by recording the absorption of RESULTS the Soret peak between 410 and 420 mu. General appearance. Deficient animals A standard absorbancy of A,% = 80 (at the appeared different from the controls in sev­ peak) was used for calculating concentra­ eral ways. The coat was more sparse and tion. unkempt. The hair of the young deficient Multiple forms of myoglobin were sepa­ rats was softer than that of the controls, rated from a concentrated solution ob­ but was matted and harsh to the touch in tained by passing a water extract of mus­ older animals. A red scaly deposit was ob­ cle through a column using 0.1 m tris served on the skin of some of the deficient buffer. At this ionic strength the myoglo­ animals, especially on the back. bin band is less diffuse than at the lower The animals were all black or black and molarity and can be recovered in a more gray hooded. By the end of the first month concentrated eluate. The ionic strength of a definite graying of the hair of the iron- the muscle extract results in a movement deficient group was noticed. This obser­ of hemoglobin also, but the band is dis­ vation has been reported elsewhere (10). tinct and slower moving than that of myo­ The two long inciser teeth of the defi­ globin so the two can be separated. cient animals were pearly-white as com­ The eluate was applied to cellulose ace­ pared with the brown-tinged ones of the tate strips (Sepraphore III) in a Gelman controls. The paws of the deficient group electrophoresis chamber containing 0.1 m were white in contrast with the pink paws tris-EDTA-borate buffer, pH 8.6. A cur­ of the controls. These changes were ap­ rent of 250 volts was applied for 90 min­ parent by the end of the first week. utes and the strips stained with Amido Body and organ weights. Weights given Schwartz. in tables 1-5 are those at autopsy and are Catalase activity was determined in one- the average of 5 animals for deficient and to 2-g samples of liver. Samples were pair-fed control groups and 2 animals for the ad libitum control groups. By the homogenized with 5 ml 0.067 m phosphate twelfth week, iron-deficient animals had buffer pH 6.8 in a glass tissue grinder held in a beaker of ice. The homogenate was larger hearts, spleens and kidneys, but the rate of increase varied for each organ. transferred to a 25-ml graduated cylinder, Cardiomegaly was pronounced by the first refrigerated for one hour, then filtered week. By the sixth week of the study, the through glass wool. The filtrate was diluted heart from any deficient animal was about 1:50, then one milliliter was used to de­ twice the size of that from its pair-fed termine catalase activity by the peroxide control, and this ratio was maintained for method of Sumner and Dounce (8). Dur­ ing the iodine reaction the solution was TA BLE 1 placed under a stream of nitrogen. Titra­ Body weights of iron-deficient, pair-fed control, tion was with 0.05 n sodium thiosulfate and ad libitum control rats using paragon starch indicator for the end P a ir-fe d 1 Ad libitum 2 point. The formula for calculating units W eek Deficient 1 9 9 9 of activity was: K = \ In in which 0 5 7 ± 1 5 7 ± 1 5 7 ± 1 Co = hydrogen peroxide content at zero 1 8 3 ± 3 8 3 ± 11 9 9 ± 7 time and Ct = peroxide content at time t. 2 1 0 4 ± 7 1 0 4 ± 5 1 1 6 ± 13 3 1 0 2 ± 2 3 12 4 ± 6 1 3 8 ± 11 At the time of sampling for catalase ac­ 4 1 0 6 ± 41 12 1 ± 2 9 1 73 ± 0 tivity or myoglobin, 300- to 500-mg sam­ 5 1 2 6 ± 2 8 14 9 ± 2 0 2 0 7 ± 5 ples of liver or muscle were removed for 6 12 8 ± 7 13 3 ± 3 0 2 6 2 ± 4 nitrogen analysis by the Kjeldahl method. 8 1 9 5 ± 3 0 1 9 9 ± 3 6 2 8 3 ± 4 10 2 3 8 ± 2 8 2 4 8 ± 38 2 9 4 ± 4 5 Iron content of livers was determined from 12 2 5 4 ± 4 5 2 8 6 ± 2 3 3 7 3 ± 1 8 an aliquot of this same sample after di­ 1 Mean from 5 animals and standard deviation. gestion with sulfuric acid. The method of 2 Mean from 2 animals (except five at zero week) 3 8 6 RUTH P. CUSACK AND W. DUANE BROWN

TABLE 2 was not influenced by diet. The appear­ W eight of hearts from iron-deficient, pair-fed ance of the liver, however, differed greatly control and ad libitum control rats in deficient and control animals. Control Week D eficient1 Pair-fed 1 Ad libitum 2 fivers were dark red and tender in texture;

9 9 9 fivers from deficient animals were yellow- 0 0.29 ± 0 .0 5 0.29 ± 0 .0 5 0.29 ± 0 .0 5 brown and tough when cut. 1 0.47 ± 0.03 0.35 ± 0 .0 4 0.37 ± 0.01 Plasma proteins. Total plasma protein 2 0 .6 2 ± 0 .1 1 0 .3 8 ± 0.01 0.40 ± 0 .0 5 concentration was higher in deficient ani­ 3 0.74 ± 0 .2 0 0.41 ± 0 .0 2 0.51 ± 0 .0 6 4 0.75 ± 0.10 0 .3 9 ± 0.09 0.57 ± 0 .0 3 mals than in pair-fed controls (table 6). 5 0 .8 9 ± 0 .3 2 0.47 ± 0.05 0.62 ± 0 .0 3 The difference was apparently due to an 6 0 .9 0 ± 0 .1 7 0.43 ± 0 .0 9 0.77 ± 0.01 increase in the (3-globulin fraction. In table 8 1.09 ± 0.16 0.54 ± 0 .0 7 0.79 ± 0 .0 3 10 1 .3 3 ± 0 .0 8 0.66 ± 0 .0 6 0 .8 2 ± 0 .1 4 7 these differences are expressed on a 12 1.41 ± 0.17 0.74 ± 0 .1 1 0.96 ± 0 .0 4 statistical basis. The mean plasma protein 1 Mean from 5 animals and standard deviation. concentration in the deficient animals was 2 Mean from 2 animals (except five at zero week). 6.49 g/100 ml and in the control animals TABLE 3 5.58 g/100 ml. 3-Globulin concentration in Weight of spleens from iron-deficient, pair-fed the deficient group was 2.74 g/'lOO ml control and ad libitum control rats and in the control group 1.80 g/100 ml. Week Deficient 1 Pair-fed 1 Ad libitum 2 Both of these differences were highly sig­ nificant (P < 0.001) but the small change 9 9 9 in albumin concentration was not signifi­ 0 0 .3 2 ± 0.05 0.32 ± 0 .0 5 0 .3 2 ± 0.05 1 0.31 ± 0 .0 6 0.37 ± 0.08 0.50 ± 0.04 cant. 2 0.40 ± 0.13 0.38 ± 0 .0 7 0.42 ± 0 .0 1 3 0.48 ± 0 .0 6 0.37 ± 0.03 0.48 ± 0 .0 0 TABLE 4 4 0.81 ± 0.19 0.32 ± 0 .0 9 0.54 ± 0 .0 6 Weight of kidneys from iron-deficient, pair-fed 5 0.92 ± 0 .2 9 0.41 ± 0 .1 2 0.64 ± 0 .1 2 control and ad libitum control rats 6 1.31 ± 0 .2 2 0 .3 6 ± 0 .1 0 0.59 ± 0 .1 6 8 1.60 ± 0 .2 0 0.42 ± 0 .0 8 0.58 ± 0 .1 0 Week Deficient 1 Pair-fed 1 Ad libitum 2 10 2 .1 0 ± 0 .6 6 0.47 ± 0.12 0.61 ± 0 .1 1 9 9 12 2.34 ± 0 .5 1 0.61 ± 0.12 0.62 ± 0 .0 1 9 0 0.70 ± 0 .0 6 0.70 ± 0 .0 6 0.70 ± 0.06 1 Mean from 5 animals and standard deviation, 1 0.84 ± 0 .1 8 0 .9 5 ± 0 .1 1 1.04 ± 0 .0 3 2 Two animals (except five at zero week). 2 1.17 ± 0.11 1 .1 7 ± 0.08 1 .1 2 ± 0 .1 1 3 1.22 ± 0.21 1.03 ± 0 .1 1 1.18 ± 0.08 the remaining 6 weeks. This increase in 4 1.31 ± 0 .2 6 1.1 3 ± 0 .2 8 1.38 ± 0 .2 3 weight was accompanied by a decrease in 5 1.44 ± 0 .3 7 1 .2 8 ± 0 .1 6 1 .5 0 ± 0.01 color, the deficient hearts being much 6 1.54 ± 0.13 1 .1 0 ± 0 .1 6 1 .7 4 ± 0 .1 0 paler. 8 1.87 ± 0.26 1.35 ± 0.21 1 .8 2 ± 0.15 10 1.97 ± 0.31 1.49 ± 0.18 1.95 ± 0.18 The increase in size of spleens did not 12 2.36 ± 0 .2 6 1 .6 7 ± 0 .1 6 2.07 ± 0 .1 6 start until the third week but was more 1 Mean from 5 animals and standard deviation. pronounced than that of the hearts during 2 Two animals (except five at zero week). the latter half of the experimental period. During the first 10 weeks, the size of TABLE 5 W eight of livers from iron-deficient, p a ir -fe d spleens in the deficient animals increased control and ad libitum control rats some eightfold, from 0.32 g at weaning to 2.34 g at 12 weeks. W eek Deficient 1 Pair-fed 1 Ad libitum 2 After the third week, kidneys were also 9 9 9 larger in the deficient animals. Even at 0 2.33 ± 0.09 2.33 ± 0 .0 9 2.33 ± 0 .0 9 12 weeks, however, the difference in size 1 2.85 ± 0.27 3.13 ± 0.75 4.22 ± 0 .8 5 was not as marked as that of hearts or 2 3.66 ± 0.37 4.76 ± 0 .3 9 4.30 ± 0 .3 0 3 3.63 ± 0.57 4.16 ± 0 .4 5 5.69 ± 0 .1 7 spleens, being only about one-half again 4 4.30 ± 1.60 4.59 ± 1 .2 3 7.25 ± 0 .1 0 as large.- 2.36 g compared with 1.67 g for 5 4.47 ± 1.03 4 .9 0 ± 0 .7 2 8.07 ± 0.99 the pair-fed controls. The color of the kid­ 6 4.80 ± 0.53 4.05 ± 0.75 9.81 ± 0 .0 1 neys in deficient animals was pale yellow, 8 6.49 ± 0.99 5.70 ± 0 .9 5 9.31 ± 0 .4 2 10 7.87 ± 0.88 6.34 ± 1 .2 2 8.83 ± 1 .2 0 as contrasted with deep red in controls. 12 8.09 ± 1.54 7.68 ± 0 .6 8 11.59 ± 1.16 The size of the liver increased with in­ 1 Mean from 5 animals and standard deviation. creasing body weight in all animals and 2 Two animals (except five at zero week). IRON DEFICIENCY IN RATS 3 8 7

TABLE 6 Plasma protein concentration in iron-deficient and pair-fed control rats

Week Diet Total /3-Globulin a-Globulin Albumin g/100m l I g/100m l g/100 ml g/100m l 0 5.1 ± 0 .5 1.3 ± 0.2 0 .6 ± 0 .1 3.2 ± 0 .2 4 — Iron 6.3 ± 0 .9 2 .6 ± 0.4 1.4 ± 0.4 2.3 ± 0 .9 -flron 6.1 ± 0.9 2 .0 ± 0 .5 1.5 ± 0.4 2.6 ± 0 .5 5 — Iron 2 5.8 ± 1 .4 2.4 ± 0 .4 1 .6 ± 0 .2 1 .8 ± 1.0 + Iron 5.4 ± 0 .5 1.7 ± 0.2 1 .2 ± 0.2 2 .6 ± 0.7 6 — Iron 6.4 ± 1.1 2 .5 ± 0 .7 1.3 ± 0.1 2.6 ± 0 .8 -flron 5.1 ± 0.7 1.5 ± 0.2 0.9 ± 0.2 2.7 ± 0.5 8 — Iron 6.4 ± 0 .7 2 .6 ± 0.3 1.2 ± 0.1 2.5 ± 0.3 -f Iron 3 5.3 ± 0 .2 1.5 ± 0 .3 1.0 ± 0.3 2.8 ± 0 .4 10 — Iron 7.4 ± 2 .6 3.4 ± 0 .8 1 .0 ± 0 .2 3.0 ± 0.7 -flron 5.7 ± 0.3 1 .9 ± 0 .2 1.0 ± 0.3 2.8 ± 0 .2 12 — Iron 6.4 ± 0 .6 2.9 ± 0 .4 1.2 ± 0.2 2.4 ± 0 .2 -flron 5.7 ± 0.5 2.1 ± 0.5 1 .2 ± 0.2 2.5 ± 0 .3 1 Mean of 5 animals and standard deviation with exceptions noted in footnotes 2 Three animals. 3 Four animals.

TABLE 7 Statistical analysis of plasma protein concentration in iron -deficient and pair-fed control rats

Plasma Concentration Difference protein (A) (B) A — B 1 t P 2 fraction — Iron + Iron g/100 ml g/100 ml Total 6.49 5.58 0.91 ± 0.24 3.81 < 0.001 /3-Globulin 2.74 1.80 0.94 ± 0 .1 5 6.36 < 0.001 Albumin 2.45 2.65 — 0 .2 0 ± 0.15 1.36 ns 1 Average difference between paired means and standard error. Data from 27 pairs of animals, from 4 to 12 weeks postweaning. 2 Students t test (27).

Hemoglobin. Hemoglobin levels are pre­ In the deficient animals, the myoglobin sented in table 8. In the deficient animals concentration of the leg muscle was the there was a rapid decrease to 4.5 g/100 same at the end of the experimental period ml after one week from the already low as at the beginning. Although the mean value at weaning, 7.2 g/100 ml. During concentration of deficient muscle, 0.61 this first week the level increased to 10.8 mg/100 mg nitrogen, was somewhat less g/100 ml in pair-fed control and to 11.2 g/100 ml in ad libitum control animals. TABLE 8 Changes after the first week were more Hemoglobin concentration in blood of iron- deficient, pair-fed control and ad libitum gradual; levels dropped in the deficient control rats animals to about 3 g/100 ml at 4 weeks and increased in the pair-fed controls to Week Deficient1 Pair-fed 1 Ad libitum 2 about 15 g/100 ml at 6 weeks. Changes g/100 ml g/100m l g/100 ml thereafter were not significant. In general, 0 7 .2 ± 0.5 7 .2 ± 0 .5 7.2 ± 0.5 hemoglobin concentration in the ad libitum 1 4.5 ± 0.7 10.8 ± 1 .6 11.2 ± 0.7 controls was lower than that of the pair- 2 4 .6 ± 0.7 12.2 ± 0 .6 11.7 ± 0.9 3 3.8 ± 0 .8 1 3 .6 ± 0.8 13.2 ± 0.5 fed group of the same age, especially after 4 3.2 ± 0 .4 13.7 ± 0.8 13.7 ± 1 .0 the fifth week. 5 3.4 ± 0 .4 14.4 ± 0 .7 13.1 ± 0.5 Myoglobin. Myoglobin concentration in 6 3 .2 ± 0 .5 14.9 ± 0.9 14.1 ± 0.4 8 3.3 ± 0 .3 15.4 ± 0 .9 14.0 ± 0 .0 hind leg muscle of 3-week-old weanlings 10 3.4 ± 0 .4 15.5 ± 0 .2 15.3 ± 0 .6 and of animals fed the deficient diet or 12 3.2 ± 0 .6 1 5 .0 ± 0 .6 14.3 ± 0 .2 pair-fed the control diet for 12 weeks is 1 Mean from 5 animals and standard deviation. presented in table 9. 2 Two animals (except five at zero week). 388 RUTH P. CUSACK AND W. DUANE BROWN

TABLE 9 Myoglobin concentration in hearts from Myoglobin concentration in hind leg muscles of deficient animals was higher than that weanling, iron-deficient and pair-fed from pair-fed controls some weeks and control rats lower in others, but differences were small. W eek D iet Myoglobin 1 Data are not from individual animals but m g/g wet m g/100 mg from 4 or 5 pooled hearts and cannot be tissue nitrogen analyzed statistically. However, the myo­ 0 0 .2 1 ± 0 . 0 8 0 .7 1 ± 0 . 2 8 12 — I r o n 0 .2 1 ± 0 . 0 3 0 . 6 1 ± 0 .1 0 globin concentration of the heart of the 12 + I r o n 0 .6 4 ± 0 .1 1 1 .9 0 ± 0 .3 1 deficient animal was not decreased as much as in the leg muscle. The myo­ 1 M ean from 5 animals and standard d e v iatio n . globin concentration of the heart, in fact, than the 0.71 mg/100 mg nitrogen for the increased while the animal ate an iron-poor weanlings, this difference is not statisti­ diet up to twice the original value of 3.5 cally significant because of the wide varia­ mg/100 mg nitrogen. tion in the weanling values. Since the heart of the deficient animal On the other hand, leg myoglobin con­ was larger than that of the control, the centration in the control group increased total heart myoglobin was greater in the to 1.90 mg/100 mg nitrogen at 15 weeks deficient animal than the control for all of age, which is 3 times that of the defi­ weeks except the first, when the difference cient group of the same age. The nitrogen was small (table 11). For some weeks the content of deficient and control muscle of total myoglobin content of the deficient the same age was similar; hence this dif­ hearts was more than twice that of the con­ ference in myoglobin concentration is ap­ trols, e.g., at 6 weeks 0.53 mg in the con­ parent also when expressed on the basis trol and 1.43 mg in the deficient animals. of wet weight, namely 0.64 mg vs. 0.21 Multiple hemoglobin and myoglobin. m g/g. Figure 1 shows the result of electrophoretic Table 10 illustrates the change of myo­ separation of hemoglobin on acrylamide globin concentration of the heart with age. gel. The bands are not stained and the Beginning at a level of 3.5 mg/100 mg picture was taken immediately after com­ nitrogen at 3 weeks, the myoglobin level pletion of the electrophoresis run. Four at 15 weeks was increased to 8.2 mg in bands are clearly visible. The hemoglobin the unrestricted control and to 7.9 mg in is from the red cells of animals receiving the pair-fed control animals. The concen­ the experimental diets for 12 weeks. The tration increased more rapidly in the pair- gel on the right shows separation of hemo­ fed group. This doubling of the myoglobin globin from a control animal compared concentration in the heart during the 12 with that of a deficient animal on the left. weeks of the experiment is similar to the No differences between the two can be 2.5-fold increase in concentration in the seen, either in the number or location of leg during the same period. the bands or in their apparent concentra-

TABLE 10 Myoglobin concentration in hearts of iron-deficient, pair-fed control and ad libitum control rats

Ad libitum W eek Deficient :i Pair-fed Ad libitum Deficient 1 P a ir-fed co n tro l 1 c o n tro l 2 c o n tro l 1 c o n tro l 2 m g/g wet tissiie m g/100 mg nitrogen 0 0 .9 6 0 .9 6 0 .9 6 3 .5 3 .5 3 .5 1 1 .2 2 1 .7 0 4 .4 6.1 1 .0 9 3 .7 2 1 .5 8 1 .6 5 5 .9 5 .7 3 1 .2 9 1 .7 5 4 .6 6.1 1 .2 7 4 .3 4 2 .0 7 1 .7 0 7 .2 5 .6 5 1.31 1 .7 5 5 .5 1 .8 8 6 .3 6 1.63 1 .3 7 5 .9 4 .5 8 2 .3 6 1.73 7.7 5.8 10 2 .0 6 1 .9 7 7.8 6 .5 12 2 .3 3 2 .4 2 7.9 8 .2 1 Five hearts pooled for the determination. 2 Four hearts, weeks 1 to 6; 2 hearts, weeks 8 to 12. IRON DEFICIENCY IN RATS 389

TABLE 11 Total myoglobin content of hearts from iron-deficient, pair-fed control and ad libitum control rats

Heart weight Myoglobin content W eek Deficient 1 P air-fed 1 Ad libitum 2 D e fic ie n t1 P a ir-fe d 1 Ad libitum 2

9 9 9 mg mg mg 0 0 .3 0 0 .3 0 0 .3 0 0 .2 9 0 .2 9 0 .2 9 1 0 .4 4 0 .3 4 0 .5 4 0 .5 8 0 .3 9 0 .4 2 2 0 .6 0 0 .3 7 0 .9 5 0.61 3 0 .8 2 0 .3 9 1.06 0 .6 8 0 .5 1 0 .6 5 4 0 .7 3 0 .3 7 1.51 0 .6 3 5 0 .8 6 0 .4 3 1.13 0 .7 5 0 .6 7 1 .2 6 6 0 .8 8 0 .3 9 1 .4 3 0 .5 3 8 0 .5 3 0 .7 5 1 .2 5 1 .3 0 10 0 .6 4 0 .7 5 1 .3 2 1 .5 4 12 0-72 0 .9 3 1.68 2 .2 5

1 Five hearts pooled for the determination. 2 Four hearts, weeks 1 to 6; 2 hearts, weeks 8 to 12.

Electrophoresis on cellulose acetate strips of myoglobin from either deficient or control animals produced 2 components which appeared identical in location and similar in quantity. On acrylamide gel, myoglobin also separated into 2 bands, one of much greater quantity than the other. Catalase. Catalase activity in deficient * and control livers did not differ (table 12) i and did not change as the animals grew older, remaining essentially the same whether related to the wet weight or ni­ trogen content of the liver or to the total body weight of the animal. The variation 1 k * n k i within any group was considerable, how­ ever. Total catalase activity of the liver in­ creased as liver size increased, but the Fig. 1 Electropheretic separation on acryla­ size of the liver was related to body weight, mide gel of hemoglobin from blood of iron-defi­ and not to the presence or absence of iron cient and pair-fed control rats. Column on the in the diet. left from iron-deficient and on the right from pair-fed control animal. Bottom band is brom- Liver iron. Iron content of livers from phenol blue tracking dye. Hemoglobin bands pair-fed control animals from weeks 6 unstained. through 12 is shown in table 13. The con­ centration remained about the same dur­ tion. The hemoglobin concentration in the ing this time, averaging from 0.19 mg to blood of the control animal was 15.6 g/ 0.24 mg/g, but with increasing liver size, 100 ml and that of the deficient animal total liver iron content increased steadily was 3.8 g/100 ml. Although the quantity with age, from 0.9 mg at 6 weeks to 1.6 of hemoglobin being produced was de­ mg at 12 weeks. Iron levels in livers from creased fourfold, the types of hemoglobin weanling or deficient animals were below apparently remained the same. the limit of detection of the method used. 3 9 0 RUTH P. CUSACK AND W. DUANE BROWN

TA B LE 12 Catalase activity in livers from iron-deficient and pair-fed control rats

Week Diet Catalase activity 1

u n i t s / g 7 i n i t s / g u n i t s / u n i t s / g t i s s u e n i t r o g e n l i v e r b o d y w t 0 81 ± 15 2.49 ± 0 .3 8 188 ± 4 1 3.26 ± 0.71 2 — Iron 95 ± 2 3 2.87 ± 0.62 344 ± 9 1 3.29 ± 0.76 + Iron 72 ± 1 2 2.34 ± 0 .3 3 314 ± 39 2.77 ± 0.43 3 — Iron 80 ± 3 0 2.47 ± 0.88 291 ± 1 4 1 2 .7 6 ± 0 .9 5 + Iron 86 ± 2 3 2.59 ± 0 .6 1 348 ± 7 9 2.99 ± 0 .6 9 4 — Iron 78 ± 2 3 2.42 ± 0 .6 6 3 1 3 ± 153 3 .0 0 ± 0.79 + Iron 88 ± 1 0 2.53 ± 0 .2 5 4 0 4 ± 156 3.24 ± 0 .5 4 5 — Iron 83 ± 12 2.56 ± 0 .3 9 370 ± 8 4 3.01 ± 0.59 + Iron 93 ± 2 7 2.68 ± 0 .6 7 445 ± 1 3 6 2.99 ± 0 .7 8 6 — Iron 83 ± 1 4 2.57 ± 0.34 392 ± 6 0 3.08 ± 0.47 + Iron 90 ± 8 2.42 ± 0 .2 3 357 ± 65 2.73 ± 0 .4 2 8 — Iron 99 ± 1 6 3 .0 5 ± 0 .4 8 642 ± 66 3.28 ± 0 .4 0 -(-Iron 104 ± 2 0 2.98 ± 0.64 687 ± 3 5 4 3.33 ± 1 .2 3 10 — Iron 97 ± 14 3.00 ± 0 .4 3 750 ± 9 9 3.18 ± 1.02 -(-Iron 111 ± 22 2.97 ± 0.66 715 ± 2 4 5 2.83 ± 0 .6 1 12 — Iron 109 ± 3 5 3 .4 9 ± 1.13 949 ± 4 1 4 3.64 ± 1 .1 5 + Iron 131 ± 28 3 .7 3 ± 0 .7 1 1013 ± 2 7 2 3.42 ± 0 .7 0

1 Mean of 5 animals and standard deviation.

TA B LE 13 in anemic women. Morgan and co-workers Iron content of livers from rats receiving a diet (16) reported graying in milk-fed rats that adequate in iron but limited in quantity (pair- fed to animals eating an iron-deficient diet) was partly remedied by iron and copper feeding. Week of experiment1 Liver iron 2 Splenomegaly is not common in human nutritional anemia. The cause of the mas­ m g /g w et m g /live r 6 0.24 ± 0 .0 8 0 .9 ± 0 .4 sive splenic enlargement observed in this 8 0.19 ± 0 .0 5 1.1 ± 0.2 experiment is not known. It may have 10 0.23 ± 0 .0 3 1.4 ± 0.2 been secondary to changes in the liver. 12 0.21 ± 0 .0 3 1 .6 ± 0 .3 The livers became hard and yellow early 1 Dietary treatment begun with weanling animals, 3 in the experiment, whereas massive splenic w eeks old. 2 Mean of 5 animals and standard deviation. enlargement occurred only after the fifth week. Blaustein and Diggs (17) report DISCUSSION that chronic congestive splenomegaly can The cause of the graying of the fur in result from portal hypertension caused by iron-deficient animals is not known. Re­ cirrhosis of the liver. The irregular size views of iron deficiency (11) do not men­ and shape of erythrocytes in severe iron tion this phenomenon although the bleach­ deficiency makes them more susceptible to ing of the incisors is indicated as a culling by the spleen, and this greater classic symptom of iron deficiency in the work may result in hyperplasia. The re­ albino rat. The metal commonly associ­ tention of a normal, dark red color by the ated with achromotricia is copper rather spleens of iron-deficient animals noted in than iron (12), but changes in fur in mink this study is not understood. Whether this and of feathers in chickens have been at­ color is due to the presence of ferritin, tributed to a dietary lack of iron. A cotton- hemosiderin or hemoglobin, it is not ex­ fur effect noted in mink fed a fish diet was pected that the spleen would sequester subsequently attributed to lack of iron in iron rather than release it in the face of the diet (13). Davis and associates (14) drastically lowered blood hemoglobin reported a depigmentation from red to levels. white in feathers of iron deficient chickens Significant changes in the 3-globulin eating treated soybean protein diets. Win- fraction were observed in the present trobe (15) mentions, without references, study. Since the diets used were relatively that early graying of hair is often observed high in fat, it might be assumed that IRON DEFICIENCY IN RATS 3 9 1 there is an increase in blood cholesterol hypertrophy was only moderate, the total levels with an accompanying increase in myoglobin content of the heart was still those 0-globulins used for transport of less than that of the control, namely 194 cholesterol. However, the control animals vs. 322 mg. However, these workers did were pair-fed, and we therefore assume find blood hemoglobin levels reduced from that the increase in p-globulins in defi­ 13.2 to 3.2 g/100 ml and leg muscle myo­ cient animals represents an increase in globin reduced from 0.44 mg to 0.12 circulating transferrin. Although trans­ mg g. These values are similar to levels ferrin is considered to be a plasma pro­ observed in this experiment in which tein, experiments with isotopic tracers hemoglobin was reduced from 15.0 to 3.2 have shown that more than 50% of the g/100 ml and leg muscle myoglobin from exchangeable pool of transferrin is extra- 0.64 to 0.21 mg/g of wet tissue. vascular (18), that is, on the surface of Poel (20) noted an increase in myo­ cells, in the intestinal mucosa and in the bone marrow. The role of transferrin globin concentration in the heart and a in iron absorption is under dispute at the decrease in the leg in simulated altitude present time, as are other facets of iron tests with rats after 152 days. Hearts were absorption. Wheby and Jones (19) indi­ also grossly hypertrophied and the hemo­ cate that transferrin saturation does not globin level higher than normal. Poel influence absorption. They do not indi­ suggests, then, that the myoglobin content cate, however, whether the amount of of muscle is determined by muscular ac­ iron in the intestine influences the distri­ tivity rather than anoxia. The skeletal myo­ bution of transferrin. Why the transferrin globin change noted by Poel is opposite to would leave the site of absorption in an that observed by Reynafarje (21) in na­ iron-deficient animal and enter the vas­ tive Peruvians who had lived at sea level cular compartment, where it is not needed or at high altitude all their lives. Reyna­ for iron transport, is not clear. Some farje reported the myoglobin concentra­ protein needs to enter the bloodstream to tion in the altitude-adapted men, 7 mg g. maintain blood volume, or in the instance to be significantly higher than that in of maintained volume, to maintain os­ men who lived at sea level, 6 mg /g. motic pressure when the packed cell vol­ In the vitamin E-deficient guinea pig, ume decreases. The response of utilizing myoglobin concentration was higher after a protein already formed rather than syn­ the animals had been fed a deficient diet thesizing more albumin, for instance, for 15 days, but lower after 30 days (22). would represent remarkable physiological At 30 days the levels in the gastrocnemius economy on the part of the animal. muscle were 1.14 mg/g in the deficient The expected lowering of hemoglobin and 1.51 mg/g in the supplemented ani­ levels in deficient animals occurred, al­ mals. In the red masseter muscle, the though the fact that the low levels of decrease was from 3.93 mg in the control about 3 g /100 ml were reached after the to 3.14 mg/g in the deficient group. diet had been consumed for 4 weeks was On the other hand, severe folic acid somewhat unexpected. The ability of the deficiency in the guinea pig resulted in animal to increase the myoglobin concen­ an increase in myoglobin concentration tration and content of the heart and to (23). Typical figures for the combined maintain the myoglobin concentration in gastrocnemius and soleus muscles were growing leg muscle while consuming an 1.64 mg/g in the deficient and 1.12 mg/g iron-poor diet is in contrast with this im­ in the control animals. mediate decrease in hemoglobin level. Although the myoglobin content changes Our data on myoglobin concentration under the influence of these various stres­ in hearts is at variance with that reported ses, it is not the only physiological re­ previously. Gubler and associates (2), us­ sponse occurring and the situations can­ ing iron-deficient pigs, observed that myo­ not be exactly equated with one another. globin concentration in the deficient heart The increase in hemoglobin in anoxic was less than half that of the control anoxia has been mentioned. Changes in heart, 1.4 mg vs. 3.0 mg g. Since cardiac blood dissimilar to those occurring in 3 9 2 RUTH P. CUSACK AND W DUANE BROWN iron deficiency in the other stresses men­ LITERATURE CITED tioned are an increase in red cell fragility 1. Hahn, P. F., and G. H. Whipple 1936 in vitamin E deficiency and leukopenia II. Iron metabolism. Its absorption, storage and utilization in experimental anemia. Am. and macrocytic anemia in folic acid de­ J. Med. Sci., 191: 24. ficiency. 2. Gubler, C. J., G. E. Cartwright and M. M. Of the three iron compartments studied, Wintrobe 1957 Studies on copper metab­ hemoglobin showed the greatest percent­ olism. XX. Enzyme activities and iron me­ tabolism in copper and iron deficiencies. J. age difference between deficient animals Biol. Chem., 224; 533. and controls at the end of the experi­ 3. Beutler, E., and R. K. Blaisdell 1960 Iron mental period. Myoglobin was substan­ enzymes in iron deficiency. V. Succinic de­ tially reduced in skeletal muscle, and hydrogenase in rat liver, kidney, and heart. catalase was not changed. (The total Blood, 15: 30. 4. Collier, H. B. 1944 The standardization of amount of iron present in catalase is blood hemoglobin determinations. Canad. insignificant compared with that in hemo­ Med. Assoc. J., 50: 550. globin and myoglobin.) The limited ex­ 5. Wong, S. Y. 1928 Colorimetric determ ina­ periments carried out on multiple hemo­ tion of iron and hemoglobin in blood. J. globins and myoglobins give no evidence Biol. Chem., 77; 409. 6. Layne, E. 1957 Spectrophotometric and for a preferential alteration of any species. turbidimetric methods for measuring pro­ A gradually increasing liver iron con­ teins. III. Biuret method. In Methods in centration, from 0.06 to 0.12 mg/g has Enzymology, vol. 3. eds., S. P. Colowick and been reported in 4- to 36-week-old rats N. O. Kaplan. Academic Press, New York. 7. Brown, W. D. 1961 Chromatography of eating a laboratory chow diet (24). Total myoglobin on diethylaminoethyl cellulose col­ liver iron also increased from 0.2 to 2.1 umns. J. Biol. Chem., 236; 2238. mg. Although iron concentration noted 8. Sumner, J. B., and A. L. Dounce 1955 in the study reported here is higher, the Liver catalase. In Methods in Enzymology, total liver content is about the same for vol. 2. Academic Press, New York. 9. Sandell, E. B. 1944 Colorimetric Deter­ animals of the same age. mination of Traces of Metals, vol. 3. Inter­ McCall et al. (25, 26) measured hemo­ science Publishers, New York. globin, tissue cytochrome c, plasma iron, 10. Cusack, R., and W. D. Brown 1964 Achro- and carcass iron in rats fed iron-poor diets motricia in iron deficient rats. Nature, similar to those used in our study, and 204; 582. 11. Follis, R. H. 1958 Deficiency Diseases. suggested that when insufficient iron is Charles C Thomas Company, Springfield, available, the relative competitive power Illinois. of some body component for iron may 12. Frost, D. V. 1948 The relation of nutri­ increase at the expense of others. These tional deficiencies to graying. Physiol. Rev., workers believe that the tissue concentra­ 28; 368. 13. Stout, F. M., J. E. Oldfield and J. Adair tion does not fall below a given minimal 1960 Aberrant iron metabolism and the level, and that the animal grows within “cotton-fur” abnormality in mink. J. Nutri­ the limiting amount of iron available. tion, 72; 46. Overall results indicate that several 14. Davis, P. N., L. C. Norris and F. H. Kratzer 1962 Iron deficiency studies in chicks using adaptive changes in the metabolism of treated isolated soybean protein diets. J. the rats enable them to survive in the Nutrition, 78; 445. face of extremely low hemoglobin levels 15. Wintrobe, M. M. 1947 Physiological impli­ resulting from lack of dietary iron during cations of the anemic state. In Nutritional a period of rapid growth. These include Anemia, ed., A. Lejwa. The Robert Gould maintenance of original skeletal myo­ Research Foundation, Cincinnati, Ohio. 16. Morgan, A. F., L. Perlman and M. Groody globin concentration; increase in heart 1944 Hemoglobin-regenerating properties of muscle myoglobin concentration accom­ prunes. Food Res., 9: 154. panied by massive increase in heart size; 17. Blaustein, A. U., and L. W. Diggs 1963 increase in 6-globulins and hence total Pathology of the spleen. In The Spleen, ed., plasma protein concentration; and main­ A. Blaustein. McGraw-Hill Book Company, N ew York. tenance of heterogeneous forms of both 18. Katz, J. H. 1961 Iron and protein kenetics hemoglobin and myoglobin. studied by means of doubly labeled hyman IRON DEFICIENCY IN RATS 3 9 3

crystalline transferrin. J. Clin. Invest., on myoglobin concentration in skeletal mus­ 40: 2143. cle of the guinea pig. J. Nutrition, 66: 361. 19. W heby, M. S„ and L. G. Jones 1963 Role 24. Kaufman, N., J. V. Klavins and T. D. Kinney of transferrin in iron absorption. J. Clin. 1962 Iron content of liver in relation to Invest., 42: 1007. age. J. Nutrition, 78: 338. 20. Poel, W. E. 1949 Effect of anoxic anoxia 25. McCall, M. G., G. E. Newm an, J. R. P. on myoglobin concentration in striated mus­ O’Brien, L. S. Valberg and L. J. W itts 1962 cle. Am. J. Physiol., 156: 44. Studies in iron metabolism. I. The experi­ 21. Reynafarje, B. 1962 Myoglobin content mental production of iron deficiency in the and enzymatic activity of muscle and alti­ growing rat. Brit. J. Nutrition, 16: 297. tude adaptation. J. Appl. Physiol., 17: 301. 26. McCall, M. G., G. E. Newm an, J. R. P. 22. Schottelius, B. A., D. D. Schottelius and O’Brien and L. J. Witts 1962 Studies in A. D. Bender 1959 Effect of vitamin E on iron metabolism. 2. The effects of experi­ myoglobin content of guinea pig skeletal mental iron deficiency in the growing rat. muscle. Proc. Soc. Exp. Biol. Med., 302: 581. Brit. J. Nutrition, 3 6: 305. 23. Faulkner, W. R., F. R. Blood and W. J. 27. Snedecor, G. W. 1956 Statistical Methods, Darby 1958 Effect of folic acid deficiency ed. 5. Iowa State College Press, Ames. Vitamin B12 Distribution in Cow's Milk 1,2

YOUNG PARK KIM,3 EVANGELOS GIZIS, J. R. BRUNNER and B. S. SCHWEIGERT Department of Food Science, Michigan State University, East Lansing, Michigan

ABSTRACT The amounts of free and bound vitamin B i2 in cow’s milk were de­ termined microbiologically with the use of the test organism, Lactobacillus leichmannii. Bound vitamin B i2 was released by autoclaving the samples in the presence of acid and cyanide. Approximately 95% of the total vitamin BJ2 in milk was in the bound form. Milk proteins were separated into gross fractions by isoelectric precipitation, coagulation with rennin, centrifugation, and salting-out with ammonium sulfate. Bound forms of the vitamin occurred in all protein fractions, although the whey pro­ teins contained more than the casein proteins, based on the amount of bound vitamin B 12 per mg of protein. The amount of bound vitamin BJ2 in the original milk was accounted for by analysis of the casein and whey protein fractions.

Attention has been given to the deter­ scribed below, and the results are presented mination of the vitamin B)2 content of milk in table 1. from various species, to methods for re­ a) Twenty milliliters of milk were di­ leasing bound forms in milk, and to the luted with an equal volume of water and binding of the vitamin by a highly purified 0.1 n HC1 was added until the pH was protein prepared from sow’s milk (1-5). reduced to 4.6. Twenty drops of a 1% The binding of vitamin Bi2 by intrinsic fac­ (w/v) NaCN solution were added and the tor and other protein preparations has been mixture was autoclaved for 10 minutes at reviewed by Glass (6). These studies 115°. After cooling, the extract was diluted showed that greater quantities of vitamin to 100 ml with water and filtered. Five B12 were bound by sow’s milk proteins than milliliters of the filtrate were adjusted to by the proteins from the cow or human (7). pH 6.8 with 0.1 n NaOH and diluted with The present study was undertaken to deter­ water to a predetermined volume for assay. mine the amount of bound vitamin Bi2 b) Twenty milliliters of 0.1 m sodium present in protein fractions prepared from acetate buffer, pH 4.6, and 20 drops of a cow’s milk. 1% (w/v) NaCN solution were added to a 20-ml sample of milk. The mixture was EXPERIMENTAL AND RESULTS heated in steam for 30 minutes, cooled to Methods for measuring the “free” and room temperature, diluted to 100 ml, ad­ “bound” forms of vitamin B,s in milk and justed to pH 4.6 and filtered. Five milli­ milk protein fractions. Vitamin B[2 was liters of solution were adjusted to pH 6.8 measured by microbiological assay with and diluted to volume with water in prepa­ Lactobacillus leichmannii ATCC 7830 as ration for the vitamin B12 assay. the test organism and the procedures de­ c) For enzymatic digestion with papain, scribed by Gregory (1), Scheid and 20 ml of milk were mixed with 20 ml of Schweigert (8 ), USP XVI (9 ) and the 0.1 m sodium acetate buffer, pH 4.6, AOAC (10). The amount of free vitamin warmed to 60°, and one gram of powdered B12 was determined in Seitz or membrane papain preparation in 10 ml water was ultrafiltrates of the samples (table 1). added. The enzyme was activated by the *123 Three methods of obtaining maximal re­ Received for publication March 4, 1965. lease of the bound form of vitamin Bi2 1 Published with the approval of the Director of were compared: a) autoclaving in the the Michigan Agricultural Experiment Station as Journal Article no. 3581. presence of HC1 and cyanide; b) autoclav­ 2 Supported in part by grant no. EF-284 from the Division of Environmental Health and Food Protec­ ing in sodium acetate buffer with added tion, U.S. Public Health Service. cyanide, and c) digestion with papain. 3 Research taken in part from a thesis submitted in partial fulfillment of the requirements of the M.S. These procedures were carried out as de­ degree in Food Science, 1964.

394 J. N u t r it io n , 86; ’65 V IT A M IN B 12 IN M IL K 3 9 5

TABLE 1 Vitamin B 12 content of pasteurized milk

Total vitamin B12 Treated Free vitamin B12 Treated with with cyanide Treated Membrane Seitz cyanide and with ultra- ultra­ and HC1 sodium papain filtrate filtrate acetate m/ig/mlmilh mfig/mlmilh m/ig/ml milk vifig/mlmilk m/ig/mlmilk Average 1 269 255 267 16 14 Range 210-357 180-294 255-303 8-28 4-17 1 Average of 3 samples. addition of 10 drops of a 1% (w /v ) NaCN Determination of the free and bound solution. The mixture was incubated for vitamin Bv, in milk protein fractions and one hour at 60°, steamed for 10 minutes the methods used in the preparation of to inactivate the enzyme, made to pH 4.6, these fractions. Whole casein was ob­ diluted to 100 ml with water and filtered. tained from skim milk by 3 procedures: Five milliliters of solution were adjusted a) ultracentrifugation, b) coagulation with to pH 6.8 and diluted for assay. rennin, and c) isoelectric precipitation at In view of the results obtained and the pH 4.6. The details of the procedures used simplified procedures involved, Seitz filtra­ are as follows: tion prior to analysis for free vitamin B,. a) Ten milliliters of 2 M CaCT were and autoclaving of the samples in the pres­ added to 200 ml of skim milk and the solu­ ence of HC1 and cyanide prior to measure­ tion centrifuged at 21,000 rev min for 90 ments of total vitamin Bu were the proce­ minutes in a type 21 rotor with a Spinco dures adopted for subsequent experiments. model L centrifuge. Whey was separated In this report, bound vitamin Bia is defined from the casein by decantation. The pel­ as the difference between the total and letized casein was resuspended in 250 ml free vitamin B,2 when determined by micro­ of 0.085 m NaCl solution. One hundred biological assay. milliliters of 1.5 m potassium oxalate along Subsequent experiments showed that the with sufficient oxalic acid to maintain a free and total vitamin Bi2 content of raw pH of 6.7 were added. The resulting cal­ milk, pasteurized milk, and pasteurized cium oxalate precipitate was removed by skim milk were similar. centrifuging at 2,000 rev, min.

TABLE 2 Vitamin Bv> content of shim milk and its constituent casein and whey fractions

Method of fractionation Vitamin B12 content of Coagulation Isoelectric Exp. no. unfractionated Centrifugation with rennin precipitation skim milk Casein Whey Casein Whey Casein Whey m/ig/100 ml uifig/100 ml skim, milk m/j.g/100 ml skim milk mfig/100 ml skim, milk

Total vitamin B12 1 332 128 120 156 139 147 103 2 350 156 — 156 102 217 131 3 332 125 — 147 171 176 127 4 350 140 139 146 140 187 147 Avg 341 137 130 151 138 182 127 (40% ) 1 (38% ) (44% ) (41% ) (53% ) (37% )

Free vitamin B12 1 21 2 10 12 11 50 14 2 26 6 13 5 14 — 18 1 Percentage of vitamin B12 accounted for in fractions. 3 9 6 Y. P. KIM, E. GIZIS, J. R. BRUNNER AND B. S. SCHWEIGERT

RAW MILK Separate 3 X at 40°C

Cream - SKIM MILK Adjust to pH 4.6 with 0.1 N HC1, filter

Casein -

WHEY

Adjust to pH 6.5 with 0.1 N NaOH, add (NH4)2S04 to 0.5 satr. Centrifuge at 2000 X G for 15'.

Precipitate Redissolve at 3% cone, adjust Supernatant to pH 4.6, add (NH4)2S04 to Dialyze, adjust to 0.25 satr. Centrifuge at 25,000 pH 4.6, filter. X G for 15'.

Ppt -

Ppt -

Supernatant Supernatant Filter through glass wool, adjust Adjust to pH 6.5, to pH 6.0, add (NH4)2S04 to 0.4 add (NH4)2S04 to satr. Centrifuge at 25,000 XG 0.5 satr. Centrifuge for 15'. at 2000 X G for 15'.

Supernatant - Ppt-

Precipitate Supernatant Redissolve at 3% cone., adjust to Dialyze pH 4.5, filter. Lyophilize

Ppt -

LACT ALB U MIN FRACTION * Supernatant Dialyze Lyophilize

I LACTOGLOBULIN FRACTION * * Stored at — 10°C until assayed.

Fig. 1 Procedure for isolating lactoglobulin and lactalbumin fractions from cow’s milk. V IT A M IN B 12 IN M IL K 3 9 7

TABLE 3 Electrophoretic evaluation of lactalbumin and lactoglobulin fractions (Veronal; pH 8.6, T/ 2 = 0.1) Electrophoretic mobility (A = x 10-5, cm2, V - 1 , sec-1 ) Fraction Proteins Ascending Descending present1 Peak pattern pattern Experiment no. 1 Lactalbumin 1 - 4 .1 2 - 3 .9 0 o-lactalbumin (75% ) 2 - 5 .5 8 - 5 .4 9 /3-lactoglobulin (25% )

Lactoglobulin 1 - 2 .2 1 - 2 . 0 immune globulin fraction (60% ) 2 - 3 .8 5 - 3 .6 5 a-lactalbumin (40% ) Experiment no. 2 Lactalbumin 1 - 3 .2 5 - 4 . 0 a-lactalbumin (74% ) 2 - 4 .1 7 - 5 . 4 /3-lactoglobulin (19% ) 3 - 5 .4 6 - 7 . 2 blood serum albumin ( 7 % )

Lactoglobulin 1 - 2 .3 0 - 2 .1 3 immune globulin fraction (73% ) 2 - 4 .5 4 - 4 .2 3 a-lactalbumin (27% ) 1 Identifications were based on data presented in the 1960 report of the Committee on Milk Protein Nomenclature and Methodology, American Dairy Science Association (13).

b) Approximately 2 ml of rennin (0.45 Total vitamin Bi2 was determined by mg/ml) were added to 200 ml of skim grinding the casein samples with a mortar milk in a water bath at 36 to 37° until co­ and pestle until the casein was in the form agulation was complete (30 minutes). The of fine particles. The ground casein was coagulum was dispersed and centrifuged at transferred to a 250-ml volumetric flask 2.000 rev/min for 30 minutes. Whey was and made to 250 ml with water. Five milli­ separated from the casein pellet by de­ liters of the diluted casein sample, as well cantation. The casein was washed with as suitable portions of the whey samples, water and centrifuged at 2,000 rev/min were assayed for vitamin BJ2 as described for 30 minutes. previously. The results obtained from c) Fifty milliliters of water were added these experiments are presented in table 2. to 200 ml of skim milk and the pH of the When these data are considered on the mixture was adjusted to 4.6 with 1 n HC1. basis of concentration of vitamin Bi2 per mg The mixture was stirred with a magnetic of protein, vitamin B12 is more concentrated stirrer and centrifuged at 2,000 rev/min in the whey proteins. Consequently, the for 30 minutes. Whey was removed from whey proteins were separated into lacto­ the casein by decantation and the casein globulin and lactalbumin fractions accord­ was washed with water and centrifuged at ing to the scheme outlined in figure 1. 2.000 rev/min for 30 minutes. The protein distribution in these fractions By these methods, about 20 to 30 g of was assayed by free-boundary electrophor­ moist casein and 170 to 180 ml of whey esis (table 3). The amount of vitamin Bi» were obtained from 200 ml skim milk. The in the major milk proteins, expressed in whey and washings from the casein were micromicrograms per milligram of pro­ combined and treated as the whey fraction teins, is listed in table 4. for these experiments. DISCUSSION TA BLE 4 The values for total vitamin B12 content Vitamin B,3 content of milk protein fractions of milk and skim milk reported herein are Vitamin B12 content in good agreement with those obtained in Fraction No. of samples Range Avg other laboratories (11, 12). Extraction of vitam in B by autoclaving with cyanide fißg/mg protein 12 Skim milk 6 150-209 186 and HC1, or with cyanide and NaAc, and Casein 5 109-183 126 digestion with activated papain proved to Whey 9 274-495 380 be satisfactory methods for the release of Lactalbumin 16 231-482 363 the protein-bound form of vitamin Bi>. Re­ Lactoglobulin 13 138-326 203 covery studies with added cyanocobalamin 3 9 8 Y. P. KIM, E. GIZIS, J. R. BRUNNER AND B. S. SCHWEIGERT resulted in the detection of from 95 to vitam in Bi2 content of the major milk pro­ 101% of the original amount added. tein fractions, no unique or predominant Free vitamin IF» accounted for approxi­ binding by one fraction occurred. Experi­ mately 5% of the total vitamin IF» content ments are now in progress to determine the of milk. Both methods, Seitz filtration and magnitude of binding of added vitamin BJ2 membrane ultrafiltration, gave 60 to 80% by specific milk protein fractions and to recoveries of cyanocobalamin added to ascertain whether specific peptides ob­ milk. Gregory (1) showed that when cow’s tained from milk proteins have different milk was extracted by cyanide or papain binding capacities. before ultrafiltration, 50 to 75% of the total vitamin B12 present in the milk could LITERATURE CITED be recovered in the ultrafiltrate. Her ex­ 1. Gregory, M. E. 1954 The microbiological planation for this low recovery was that assay of “vitamin Bi2” in the milk of different animal species. Brit. J. Nutrition, 8: 340. some substances present in regenerated 2. Anthony, W. B., J. R. Couch, I. W. Rupel, cellulose tubing may combine with free M. B. Henderson and C. Brown 1951 Vita­ vitamin Bia. The same phenomenon might min Bi» in blood of newborn and colostrum- account for the low recoveries observed in fed calves and in colostrum and normal milk of Holstein and Jersey cows. J. Dairy Sci., these studies. 34: 749. The data in table 2 show that the whey 3. Collins, R. A., R. E. Boldt, C. A. Elvehjem , samples from three different methods of E. B. Hart and R. A. Bomstein 1953 Fur­ casein preparation gave slight but probably ther studies on the folic acid and vitamin Bi2 content of cow’s milk. J. Dairy Sci., 36: 24. nonsignificant differences in vitamin BJ2 4. Gregory, M. E., and E. S. Holdsworth 1953 content ranging from 103 to 171 mug of The combination of some vitamin Bi»-like total vitamin Bi2 in the whey prepared from compounds with sow’s milk whey and “in­ 100 ml of skim milk. Also, these data trinsic factor” concentrates. Biochem. J., 55- 830. show that vitamin Bi2 was approximately 5. Gregory, M. E., J. E. Ford and S. K. Kon equally distributed between the casein and 1952 A vitamin Bi2-binding factor in sow’s whey fractions. However, the free vitamin milk. Biochem. J., 51: xxix. B i2 in isoelectrically prepared casein 6. Glass, G. B. J. 1963 Gastric intrinsic fac­ showed an unusually high value. No ra­ tor and its function in the metabolism of vitamin Bi2. Physiol. Rev., 43: 529. tionale is apparent to explain this observa­ 7. Gregory, M. E., and E. S. Holdsworth 1960 tion. The binding of cyanocobalamin and its nat­ Seventy-eight to 95% of the bound vita­ urally occurring analogues by certain body min Bi2 present in milk was accounted for fluids and tissue extracts. Biochim. Biophys. Acta, 42: 462. in the casein and whey protein fractions. 8. Scheid, H. E., and B. S. Schweigert 1951 Of this, 90% of the vitamin present in the Liberation and microbiological assay of vita­ whey fraction was in the lactalbumin and m in Bi2 in animal tissues. J. Biol. Chem., lactoglobulin fractions. These results indi­ 193: 299. cate that significant losses of the bound 9. United States Pharmacopoeia 1960 United States Pharmacopoeial Convention, Inc., vitamin did not occur as a result of the Washington, D. C. fractionation procedures. As shown in 10. Association of Official Agricultural Chemists table 4, the concentration of vitamin Bi» 1960 Official Methods of Analysis. Wash­ per unit of protein was two to three times ington, D. C. 11. Kawashima, R., S. Uesaka and M. Mitsuhashi higher in the whey proteins than in the 1962 Studies on the vitamin Bt2 content of caseins. Also, it may be concluded that cows’ milk, produced by the dairy farmers in vitam in Bi2 is bound by all the milk protein Kyoto Prefecture of Japan. Proc. Silver Fubi- fractions or that the specific protein-Bi» lee Lab., Anim. Husb., Coll. Agric., Kyoto Univ. (In Japanese) (Cited in Dairy Sci. complex was a contaminant in all protein Abstr., 24: 156. 1962.) fractions. The vitamin Bj2 content of the 12. Gregory, M. E. 1955 The microbiological lactalbumin and lactoglobulin fractions assay of the B-vitamins in milk. Dairy Sci. varied with individual samples but was Abstr., 17: 187. approximately equal. 13. Brunner, J. R., C. A. Ernstrom, R. A. Hollis, B. L. Larson, R. McL. W hitney and C. A. It appears, therefore, that although some Zittle 1960 Nomenclature of the proteins differences were observed in the bound of bovine milk. J. Dairy Sci., 43: 901. Effects of Dietary Lipid and Diethylstilbestrol upon Liver Fatty Acids of Choline-deficient Rats * 1

GLENN J. MILLER a n d WILLIAM W. ELLIS Division of Biochemistry, University of Wyoming, Laramie, Wyoming

ABSTRACT The effects of various dietary lipids upon the composition of liver fatty acids in choline-supplemented and choline-deficient rats with and without diethyl­ stilbestrol (DES) treatment, are presented and discussed. Changes in liver fatty acids of choline-deficient rats receiving different dietary lipids appear to be due to specific accumulation of triglycerides with their characteristic fatty acid compositions. In most cases DES treatment resulted in increases in liver monoenoic acids and decreases in stearic and arachidonic acids, particularly in the cholesterol ester fraction. How­ ever, these fatty acid effects appeared to be unrelated to the lipotropic action of DES.

Lipid accumulation in livers of choline- lipids were estimated by a method de­ deficient rats is influenced by the type of scribed by Sunderman et al. (7). Com­ dietary lipid (1), yet there is not a great posite samples of some of the liver lipids deal of information concerning the effects were separated into sterol esters, trigly­ of dietary lipids upon liver fatty acid com­ cerides and phospholipids by the micro­ position in choline-deficiency. Even though method of Lis et al. (8). it is known that estrogenic substances pro­ For gas-liquid chromatographic analysis duce lipotropic effects in choline-deficient all lipids were saponified and the free acids rats little is known of the influence, if any, methylated with a sulfuric acid-methanol of these substances upon liver fatty acid (1%) reagent. The methyl esters were composition. This present work was an then determined by an instrument employ­ attempt to study these conditions and ing a thermal conductivity detector. The effects. stainless steel column (213 cm X 0.6 cm ) contained 15% ethyleneglycol-succinate EXPERIMENTAL on 60/80 mesh Chromosorb W-HMDS, and Weanling, male albino rats (about 3 was operated at 187° with a helium flow weeks old) from a stock colony were pro­ rate of about 80 ml/min. Injector tem­ vided with tap water and experimental perature was 250° and detector tempera­ diets ad libitum. The basic diet was the ture was 220°. Each methyl ester prepara­ same as reported previously (2) except tion was run at the same sensitivity to that several different dietary lipids were Received for publication March 1, 1965. used.2 These lipids were selected for their 1 Approved as Journal Paper no. 177 by Director, Wyoming Agricultural Experiment Station. varying content of saturated, monoenoic 2 The diet consisted of the following: (% ) soy pro­ and dienoic fatty acids. The types and tein (Drackett Assay Protein C-l, Archer-Daniels- Midland Co., Minneapolis), 20; sucrose, 68; salt mix- sources of the lipids are as follows: ture-U.S.P. XIV, 4; cellulose, 3; and lipid, 5. Vitamins: (mg/kg diet) thiamine-HCl, 5; riboflavin, 5; Ca A, lard;3 B, palm oil;4 C, corn oil;5 D, olive pantothenate, 10; niacin, 50; pyridoxine, 10; p-amino- oil;6 E, soybean oil;7 F, safflower oil.8 Vita­ benzoic acid, 50; inositol, 250; biotin. 0.2; folic acid, 0.2; menadione, 1; choline (when present), 1500. mins and diethylstilbestrol9 (10 ppm) Vitamin A alcohol, calciferol and tocopherol were dissolved in 50% ethanol and administered p er os. were administered as reported (3). Five Diethylstilbestrol (10 ppm) was added to the diets in animals received each experimental diet 95% ethanol solution. 3 Rex Brand, Cudahy Packing Company, Omaha, for 28 days. N ebraska. 4 The Matheson Company, Inc., East Rutherford, Liver lipids were extracted by the pro­ New Jersey. cedure of Hanson and Olley (4) and the 5 Mazola, Corn Products Company, New York. 6 Pompeian Olive Oil Corporation, Baltimore, Mary­ percentages of total lipids determined land. 7 Crisco Oil, Procter and Gamble Company, Cin­ gravimetrically. Total sterol was deter­ cinnati, Ohio. mined by the procedure of Pearson et al. 8 Gratuitously supplied by Vegetable Oil Products Company, Inc., Wilmington, California. (5) as further modified (6). Phospho­ 9 See footnote 2.

J. N utritio n , 86: ’65 399 4 0 0 GLENN J. MILLER AND W ILLIAM W. ELLIS increase reproducibility. Relative peak receiving the various dietary lipids is areas were measured as heighth times shown in table 2. width at half heighth. A magnifying lens Choline-supplemented rats. Varying lev­ with inscribed scale was used to measure els of dietary 16:0 had little influence upon the widths. After multiplying each peak its level in liver lipids. The percentage of area by the square root of the molecular liver 16:0 remained fairly constant over a weights of the component, weight percent­ dietary range of 8 to 46%. With the ex­ age compositions were calculated. Quan­ ception of rats receiving lard, liver 16:1 titative results with National Heart Insti­ was higher than might be expected from tute Fatty Acid Standards B and D agree its level in the dietary lipids. This would with the stated composition data with a indicate that a proportion of liver 16:1 relative error less than 0.5% for both was of endogenous origin. Like 16:0, liver major and minor components. 18:0 was relatively unaffected by its level in the diet. Liver 18:1 was high in all Where applicable, data were tested for cases but was highest when dietary 18:1 significance by means of analysis of vari­ was the predominant dietary fatty acid. ance (9). In all cases where significant The percentage of liver 18:2 was strongly results are discussed, P < 0.05. reflected by the proportion of this acid in the dietary lipid. It appeared that dietary RESULTS AND DISCUSSION lipids high in 18:1 but low in 18:2 (lard, The content of liver lipids in rats re­ palm oil and olive oil) caused decreased ceiving the various treatments was not levels of liver 20:4. influenced by the dietary lipids. There­ Choline-deficient rats. The percentage fore, the values were combined and the of liver lipid 16:0 in choline-deficient rats average percentages of total liver lipids was not significantly affected by the level with standard deviations are as follows: of this acid in the dietary lipid. Changes choline-supplemented rats, 4.9 ± 0.4; cho­ in dietary lipids did not influence the rela­ line-deficient rats, 9.7 ± 2.0; choline-sup­ tive percentage of 16:1 in liver lipids. plemented and diethylstilbestrol (DES)- Regardless of its percentage in dietary treated rats, 4.8 ± 0.3; choline-deficient lipids, the percentage of 18:0 in liver and DES-treated rats, 6.1 ± 0.8. The val­ lipids was always decreased during cho­ ues are expressed on a fresh-weight basis line deficiency. On the other hand, liver and show the expected results. Choline- lipid 18:1 was always increased during deficiency increased liver lipids, and DES choline deficiency regardless of its level produced a lipotropic action in choline- in the dietary lipids. The percentage of deficient rats. 18:2 in liver lipid was only increased when this acid was the predominant dietary A. Fatty acid composition of fatty acid (soybean and safflower oils). total liver lipids Liver lipid 20:4 was always decreased dur­ The fatty acid composition of the dietary ing choline deficiency. It is clear that lipids appears in table 1 and the fatty acid liver lipid accumulation is not due to sim­ composition of total liver lipids in rats ple accumulation of dietary lipids.

TA BLE 1 Dietary lipid fatty acid composition

F a tty (A) (B) (C) (D) (E) (F) acids L a rd P a lm oil C o rn oil O live oil S oybean Safflow er oil oil % % % % % % 1 6 :0 31 4 6 i l 10 13 8 1 6 :1 7 1 < 1 < 1 < 1 2 1 8 :0 22 0 0 2 3 < 1 1 8 :1 3 5 4 4 5 0 83 2 5 8 1 8 :2 4 9 3 9 5 59 8 2 2 0 :4 1 0 0 0 0 0 T A B LE 2 Total liver lipid fatty acid composition ö o s oQ 'S -S.Sw -SoQ s£ .s * ¿* ;§ £ 5 J o.SW ¡3 gw ^ oO Î Ê-8+ 0 ^ 2 ¿3 o 5 j P ® « « m ‘ u 8

3W rt 0 4 h + I . +1 +1 +1 +1 +1 +| ^ ^ pq +1^ +1 +1 +1 +1 +1 & u +1 +1 +1 +1 +1 a +1 +1 +1a +1$ +1 +1 g >3 g CO ^ CO LO 05 05 in 05 W ^COHHCOtNH !S MHHCOH O C H H (M ^ " C H TP H < /"N CO ( H H CO H H H (N C 0 0 M H H (M g H H O C d H M C £ N M M 10 CM ^ (M O IN f ' î O O O O N i 5 5 5 N CD IN 05 05 05 M CO H CM M O H fO H CO (M CD ^ CO O 05 M H H (M H H d I—I H (M »"H(M H CM CO H H H Cl CD ^ CD ^ CM CD H (N H H H H (?J O M ( 1 + CO CO CD CO Ö O (M CO H CO LO CD IN LO IN O V ™ 1 + Mri O C iH r CO ^P(M 05 (M O IN H CO H d H H +i +1II II II II + 1+ 1+ 1+ 1+ 1 O - H CO H CO r-i O d H H H CO H LO (M CO CD O ^P +1 +1 +1 II II II O M CO rM CO N N rM IN IN O II II II II II Il O H o H d tp CD CDd CO CO H CO O o H O 1 1 l I I I I II II II II II Il I — r- r( ( 0>1 -( r r—( -i r —I r—Ir

!i 10 1 + c h h 1 +

D O H LO CD H h h o c h +1 1 + h h +1

(M +1

Cl H H H Cl 1 1 1 + +1 O

h h +1 co h 1 +

(M

v +1 +1 ITR LPDDSLVR AT ACIDS FATTY LIPID-DES-LIVER DIETARY IN 1 +

Zs o d co r ^ w £ a M M 5 5 ^P 05 > a>H H d CCS CD 05 CO 05 Ö CO O II II II II II CMO II rt t O

h M K C O +1 +1 +1 +1 +1 h

H Sp d rH O —1 h h 1 + +1 +1 +1 h N

dd (M (M CO (M h +1

ci d ci +1 +1 +1 +1

h h h h +1 +1 +1 +1 +1

d d

+[ 1-1 1-1 h +1 +1 +1 +1 +1 +1

ences throughout the groups of rats are are rats of groups the therefore, throughout and ences values, these upon formed were lipids these of esters, percentages acid cholesterol fatty safflower into and oil separated olive were lard, oil receiving rats esters. Lard contained more 16:0 and and 16:0 more contained Lard esters. in liver lipid 18:0 and 20:4. Liver lipid lipid Liver 20:4. and fected. 18:0 decreases lipid liver consistent were in acids monoenoic monoenoic lipid the in liver the increase of consistent percentages almost the was ihs lvl f 82 s n choline-defi­ in as 18:2 the of ciency. containing level lipids dietary highest receiving rats of effects these the of both reflect to composition acid appeared rats fatty liver The lipid contained more 16 :0 and 18 :0 than than 18 :0 and 16 :0 more contained lipid ny ag dfeecs r osset differ­ discussed. consistent or differences large table only in appear the values These and determined. phospholipids and triglycerides did not significantly increase this fatty fatty this increase DES and significantly high not already was did was 18:1 lipid exception liver An 18:1. and 16:1 acids, deficient and DES-treated rats. Liver lipid lipid Liver rats. DES-treated decreased ent. and treatm were deficient 20:4 DES and 18:0 and lipid Liver choline-deficiency esters of rats fed this lipid contained the the contained lipid this fed rats con­ oil of Olive esters groups. 2 this other fed the of rats esters in esters cholesterol liver the . nlss f aine ol nt e per­ be not could variance of Analysis 3. acid. Accompanying the increases in these these in rats increases the these In Accompanying acid. oil). (olive over 18:1 containing lipid of dietary a 80% fed rats the rats choline-supplemented of ent treatm rats. and liver lipid 18:2 was increased in those those in increased was 18:2 lipid liver and choline- both in as increased was 18:1 and highest level of 18:1. However, 18:1 was was 18:1 However, 18:1. of cholesterol level the and highest 18:1 80% over tained lesterol ester 18:2 was also influenced by by influenced also all was in 18:2 ester ester lesterol cholesterol inant predom the 60 n 1: wr nt infcnl af­ significantly not were 18:2 and 16:0 16:1 was increased as in DES treatm ent ent treatm DES in as increased was 16:1 i poue te ihs lvl f hs cho- this of level highest the produced oil Cho­ level. dietary its of regardless groups and oil safflower and oil olive than 18:0 the quantity of dietary 18:2 since safflower safflower since 18:2 dietary of quantity the hln-eiin ad E-rae rats. DES-treated and Choline-deficient Composite samples of the liver lipids of of lipids liver the of samples Composite hln-upeetd n DES-treated and Choline-supplemented hln-upeetd rats. Choline-supplemented The most striking effect of DES DES of effect striking most The . at ai cmoiin of compositions acid Fatty B. ie lpd fractions lipid liver Cholesterol Cholesterol

1 0 4

2 0 4

Fatty acid composition of liver lipid fractions to* «*§ S'SQ i f sa 's § 2-3W 6.Sw cS.Sg o.Sw ߣcO fl-s i> ^ rßfi > H u 0.3 i” f ! oP ■rß + o ~

o + « d 3W P

MI N WI AM W. IS L L E . W M IA L IL W AND R E L IL M . J N N E L G D 03 C CO CO 3 CO 0 (N ID O rt rH - o S' a v s 0 o v rl CO W ri H (O vO ö H H C H CO H H ) O N Vö * rt* O) O) " vQ v“ V 5 Ç C C ^ H CD W H CO CO ^ .Ç, - *- v“3 V D O > CÛ O l>p O t O rH E"- CO T-H M f(3 H (M - v*V V V h r-l O V V h > in i> ^ CO CO CM ^ h V V O H CO CM ^ CO rH O O O T—I CO CO ^ O O O

ca CM V V V V V V V V V V

1 Diethylstilbestrol. DIETARY LIPID-DES-LIVER FATTY ACIDS 4 0 3

lesterol ester. The presence of very high in this lipid fraction after choline-defi­ levels of dietary 18:1 (olive oil) caused a ciency. decrease in cholesterol ester 20:4. Choline-supplemented and DES-treated Triglycerides. Liver triglyceride 16:0 rats. Cholesterol esters. DES treatment was relatively unaffected by dietary levels almost doubled the percentage of liver of this acid. The triglycerides were char­ cholesterol ester 16:1 regardless of the acterized by low levels of 18:0 and only dietary lipid fed. Cholesterol ester 18:1 traces of 20:4. The level of 18:0 remained also increased in those rats fed lipids con­ low even when the dietary lipid (lard) taining low or moderate levels of 18:1 contained 22% of this acid. In the tri­ (lard and safflower oil). In those rats fed glycerides of rats fed lard and olive oil, lipid with a high content of 18:1 (olive 18:1 made up over 50% of the total acids. oil), liver cholesterol ester 18:1 was not Large quantities of dietary 18:2 (safflower increased. However, this acid already con­ oil) caused quite high levels of triglyceride tained about 60% of the cholesterol ester 18:2 with a corresponding decrease in fatty acids in choline-supplemented rats. 18:1. Accompanying the increases in 16:1 and Phospholipids. As in triglycerides, the 18:1, were consistent decreases in choles­ level of phospholipid 16:0 was unaffected terol esters 18:0 and 20:4. by levels of dietary 16:0. In all groups the Triglycerides. In all cases DES treat­ predominant acid in the phospholipids was ment caused a slight increase in liver tri­ 18:0 and its percentage was not influenced glyceride 16:1 and a slight decrease in by its dietary level. The feeding of lipid 18:2. DES treatment did not increase tri­ containing high levels of 18:1 (olive oil) glyceride 18:1 in livers of those rats fed did not cause an increase in phospholipid lard and safflower oil, but this acid was 18:1. High levels of dietary 18:2 (safflower high in the triglycerides of the choline- oil) did increase phospholipid 18:2, and supplemented rats. There was a definite this increase was accompanied by a de­ increase in triglyceride 18:1 in livers of crease of 18:1 as in the triglycerides. The those rats fed safflower oil after DES treat­ phospholipids contained appreciable quan­ ment. The percentage of triglyceride 18:0 tities of 20:4 and the percentage of this was low in the choline-supplemented rats acid was not influenced by the changes in and DES did not decrease it further. dietary lipids. Phospholipids. There were consistent, Choline-deficient rats. Cholesterol esters. although small, increases in liver phospho­ Lard, containing mainly 16:0, 18:0 and lipid 16:1 and 18:1 after DES treatment, 18:1, produced no changes in 16:0, a de­ and these were accompanied by consistent crease of 18:0 and an increase of 18:1 in and small decreases in phospholipid 18:0 the liver cholesterol ester of choline-defi­ and 20:4. cient rats. Olive oil which contained high Choline-deficient and DES-treated rats. levels of 18:1 did not cause an increase There were indications that both choline in cholesterol ester 18:1 during choline- deficiency and DES treatment were influ­ deficiency. However, the percentage of this encing the fatty acid composition of liver acid in the cholesterol ester of choline- cholesterol esters and phospholipids in all supplemented rats was high. High levels 3 groups. Only the influence of DES was of dietary 18:2 (safflower oil) did not noted in the liver triglyceride fatty acids of cause appreciable increase in cholesterol choline-deficient rats since the composition ester 18:2. of these acids was unaffected by choline Triglycerides. Choline-deficiency did deficiency. not appear to have any effect upon the It is well known that the increase of fatty acid compositions of liver triglycer­ liver lipids in choline-deficient rats is ides in any of the 3 groups. mainly due to an accumulation of trigly­ Phospholipids. There was a consistent, cerides. Data presented here indicate that slight increase in liver phospholipid 18:0 choline-deficiency itself has no effect on during choline-deficiency in all 3 groups. the fatty acid composition of liver trigly­ There were no consistent or large changes cerides. However, due to an accumulation in the percentages of the other fatty acids of this fraction with its specific fatty acid 4 0 4 GLENN J. MILLER AND WILLIAM W. ELLIS

TABLE 4 Liver sterols 1

Sterols Dietary With Without lipid With Without choline choline choline choline + DES 2 + DES % % % % (A ) Lard 0.22±0.01 3 0.27 ± 0.05 0.52 ± 0 .0 3 0.43 ± 0 .0 1 (D ) Olive oil 0.35 ± 0 .0 3 0.43 ± 0 .0 8 0.47 ± 0.05 0.58 ± 0 .0 8 (F) Safflower oil 0.34 ± 0 .0 6 0.39 ± 0 .0 2 0.43 ± 0 .0 1 0.43 ± 0 .0 1 1 Expressed as percentage of fresh weight. 2 Diethylstilbestrol. 3 SD.

composition, choline-deficiency should pro­ 20:4 in these cholesterol esters (table 3). duce effects on the fatty acid composition These effects, coupled with small but con­ of total liver lipids. Values in table 3 show sistent increases of 16:1 in both trigly­ that the liver triglycerides of choline-sup­ cerides and phospholipids and decreases plemented rats fed lard and olive oil con­ of 18:0 and 20:4 in phospholipids noted tained a high level of 18:1 and low levels after DES treatment (table 3), could there­ of 18:0 and 20:4. Furthermore, these fore mean increases of 16:1 and decreases proportions were not changed by choline- of 18:0 and 20:4 in the total liver lipids deficiency. Therefore, an accumulation of of these 3 groups of choline-supplemented these triglycerides during choline-defi­ rats after DES treatment. Data in table 2 ciency could cause an increase in the total show this to be true. Even though DES treatment produced liver lipid percentage of 18:1 and de­ specific effects upon the levels of certain creases in 18:0 and 20:4. Table 2 shows fatty acids in rat livers, there did not this to be true. The liver triglycerides of appear to be any correlation between these rats receiving safflower oil contained ap­ effects and the lipotropic effect. Conse­ preciable quantities of 18:1 and 18:2 and quently DES does not cause decreased low quantities of 18:0 and 20:4. Accu­ accumulation of liver lipid during choline- mulation of these triglycerides might deficiency by decreasing the levels of spe­ cause increases in percentage of total liver cific fatty acids. lipid 18:1 and 18:2 and decreases in 18:0 Liver lipid phospholipids were calcu­ and 20:4. Table 2 also indicates that this lated in the livers of the rats receiving is true. However, the liver triglycerides in lard, olive oil and safflower oil but these all 3 groups of choline-supplemented rats values were not significantly influenced by contained high levels of 16:0 and yet this the dietary lipids. Therefore, the values acid was not significantly increased in the were combined and the average percent­ total liver lipids after choline-deficiency. ages of liver phospholipids with standard This could be explained by the fact that deviations are as follows: choline-supple­ phospholipids and triglycerides contain mented rats, 2.6 ± 0.25; choline-deficient similar percentages of 16:0 (table 3). rats, 2.1 ± 0.50; choline-supplemented Therefore, an increase in one of these and DES treated rats, 2.3 ± 0.14; choline- fractions would not necessarily increase deficient and DES treated rats 2.4 ± 0.20. the proportion of 16:0 in the total. It is apparent that the lipotropic action of The DES treatment significantly in­ DES is involved in a specific decrease, or creased liver cholesterol in choline-supple­ inhibition in accumulation, of liver tri­ mented rats fed lard, olive oil and saf­ glycerides. flower oil (table 4). It is known that this increase in liver cholesterol is due to a LITERATURE CITED specific increase in cholesterol esters (10). 1. Renton, D. A., H. E. Spivey, F. Quiros-Perez, Furthermore, this study has shown that A. E. Harper and C. A. Elvehjem 1957 Effect of certain dietary fats on choline DES caused considerable increases of 16:1 requirements of rats. Proc. Soc. Exp. Biol. and considerable decreases of 18:0 and Med., 94: 100. DIETARY LIPID-DE S-LIVER FATTY ACIDS 4 0 5

2. Miller, G. J., W. W. Ellis and I. Rosenfeld 7. Sunderman, W. F, W. F. Sunderman, Jr. and 1961 Hypo- and hyperthyroidism and the E. C. Heinmiller 1960 Method for lipid lipotropism of diethylstilbestrol in choline-de­ phosphorus in serum. In Lipids and the ficient rats. J. Nutrition, 74: 408. Steroid Hormones in Clinical Medicine, eds., 3. Miller, G. J., W. Ellis, P. G. Rand and R. H. F. W. Sunderman and F. W. Sunderman, King 1958 Comparative effects of cotton­ Jr. J. B. Lippincott Company, Philadelphia, seed oil, fatty acids and diethylstilbestrol p. 28. upon choline-deficient weanling albino rats. 8. Lis, E. W., J. Tinoco and R. Okey 1961 J. Nutrition, 66: 425. A micromethod for fractionation of lipids by 4. Hanson, S. W. F., and J. Olley 1963 Ap­ silicic acid chromatography. Analyt. Bio- plication of the Bligh and Dyer Method of chem., 2: 100. lipid extraction to tissue homogenates. Bio- 9. Li, J. C. R. 1957 Introduction to Statistical chem. J., 89: 101 P. Inference. Edward Brothers, Ann Arbor, 5. Pearson, S., S. Stern and T. H. McCavack M ichigan. 1954 A rapid accurate method for the de­ 10. Müler, G. J., E. David, W. W. Ellis and I. termination of total cholesterol in serum. Rosenfeld 1963 Plasma and liver choles­ Analyt. Chem., 25: 813. terol and fatty acid composition of esterified 6. Miller, G. J. 1960 Some characteristics of cholesterol in the rat as influenced by di­ kidney and liver lipids from the American ethylstilbestrol. J. Colorado-Wyoming Acad Antelope. J. Am. Oil Chem. Soc., 37: 247. Sei., 5(4): 25. Dietary Phosphorus and Magnesium Deficiency in the R at* 1

G. E. BUNCE,2 3 H. E. SAUBERLICH, P. G. REEVES 3 a n d T. S. OBA U. S. Army Medical Research and Nutrition Laboratory, Fitzsimons General Hospital, Denver, Colorado

ABSTRACT Calcium, phosphorus and magnesium balance was studied in weanling rats, using 3 dietary levels of magnesium (130, 260 and 1,000 ppm) at each of 3 levels of phosphorus (0.3, 0.5 and 1.0% ). The effect of changes in dietary phosphorus on magnesium balance varied and was dependent on the dietary magnesium concen­ tration; high intakes of phosphorus lowered the apparent absorption of magnesium when magnesium was ample, but improved absorption when magnesium was limit­ ing. Similarly, high intakes of magnesium reduced the apparent absorption of phosphorus only when ample quantities of phosphate were consumed. Variations in magnesium intake caused a pattern of calcium absorption which closely resembled the pattern obtained for fecal phosphorus, implying a possible effect of magnesium on calcium phosphate solubility in the gut. Accumulation of calcium in the kidneys was aggravated by either an increase in phosphorus or a decrease in magnesium. Microscopic examination of the kidneys after staining with alizarin red S revealed urolithiasis alone when the accumulation of excess calcium was minimal, but both urolithiasis and nephrocalcinosis when considerable calcium was present. This evi­ dence supports the observations of others that intratubular cast deposition is the primary lesion in magnesium deficiency and thus suggests that magnesium might be of value in the prevention or control of spontaneous urolithiasis.

An elevation of dietary phosphorus has information on the interrelationship of been reported both to antagonize and to these nutrients. accelerate the signs of magnesium defi­ ciency in the guinea pig (1-5), rat (5), EXPERIMENTAL chick (6) and dog (7). The mechanism of Nine diets were prepared yielding 3 this effect, however, is not yet clear. levels of phosphorus (0.3, 0.5 and 1.0% ) O’Dell et al. (4) studied the effect of at each of 3 levels of magnesium (130, high phosphorus intakes on magnesium 260 and 1,000 ppm). The calcium level balance in the quinea pig and concluded was kept constant and averaged 0.65% of that excess (1.8% ) phosphorus interfered the diet. The basal diet (0.3% P, 130 ppm with magnesium absorption. Tibbets and Mg) was composed of casein, 20% ; su­ Aub (8) and Meintzer and Steenbock (9), crose, 62% ; cottonseed oil,4 8.0% ; cellu­ however, observed phosphorus to be with­ lose,5 5.0% ; mineral mixture,6 4.5% ; vita­ out effect on magnesium absorption in the min mixture,7 0.4% ; and choline chloride, human and rat, respectively, although the 0.1%. Elevation of the phosphorus level levels used were considerably lower than from 0.3 to 0.5% was achieved by replace­ those of O’Dell’s group. More recently, ment of potassium carbonate in the basal Forbes (10) has reported that an increase mineral mix with the necessary amount of in dietary phosphorus from 0.19 to 0.50% Received for publication March 18, 1965. actually improved magnesium absorption 1 The principles of laboratory animal care as and balance although precipitating a mag­ promulgated by the National Society for Medical Re­ search were observed. nesium deficiency. He attributed the del­ 2 Present address: U.S. Army Tripler General Hos­ pital, Box 234, APO 438, San Francisco. eterious effect of phosphorus in this in­ 3 Present address: Department of Biochemistry, stance to the faster rate of gain with the School of Agriculture, Virginia Polytechnic Institute, Blacksburg, Virginia. higher phosphorus intake. 4 Wesson Oil, Wesson Oil Sales Company, Fullerton, California. The present study was designed to meas­ 3 Cellu Flour, The Chicago Dietetic Supply House, ure calcium, phosphorus and magnesium Chicago. 6 Jones-Foster salt mix (11) with MgS04 deleted and balance at several levels of phosphorus phosphorus content adjusted to the desired level by replacement of a portion of the KH2PO4 with K2CO3. and magnesium intake to gain additional 7 Identical to that used by Leveille et al. (12).

406 J. N utrition, 86: ’65 PHOSPHORUS AND MAGNESIUM 4 0 7

KH2PO4. An additional 2.05 g of NafTPCh nesium. As expected, all groups fed a 130- (anhydrous)/100 g diet replaced an equal ppm magnesium ration developed the char­ weight of sucrose in the preparation of the acteristic visible magnesium deficiency 1.0% P diet. Magnesium was added when syndrome within 2 weeks. No difference, necessary as anhydrous magnesium sulfate however, could be detected in the severity with a corresponding reduction in sucrose. or rate of appearance of the symptoms as Thus, a relatively constant potassium level a direct function of the dietary phosphorus. was maintained and the small variation in Weight gains (table 1) also followed the dietary sodium was assumed to be without expected pattern with one exception. The significance on the questions under study. gain of the rats fed 0.3% phosphorus was A total of 108 weanling male rats of the significantly less than that of the 0.5% Holtzman strain, weighing between 40 and group when magnesium was limiting (130 50 g each, were divided into 9 groups and ppm), although no difference was noted allowed to consume the various rations for when magnesium intakes were adequate 2 weeks. They were then placed in metab­ (260 or 1,000 ppm). Presumably, this olism cages for a 14-day period. Aliquots was a result of the diminished magnesium of the feces and urine and triplicate sam­ balance discussed in the next paragraph. ples of each diet were then wet-ashed (13) The mean cumulative balance of cal­ and analyzed for calcium and magnesium cium, phosphorus and magnesium for all using a slight modification of an EDTA- groups during the 14-day balance period is eriochrome black T method (14) and for presented in table 2. The effect of varia­ phosphorus, with the method of Fiske and tions in dietary phosphorus on magnesium Subbarow (15). At the conclusion of the balance is shown in figure 1. When dietary balance trial, the animals were killed and magnesium was present at a concentra­ samples of kidney were fixed in 10% buff­ tion of 130 ppm, each increase of phos­ ered formalin and stained with hematox­ phorus resulted in an increased apparent ylin eosin and alizarin red S for histopath- absorption of magnesium. With higher in­ ological examination. The remaining takes of magnesium, however, apparent kidney tissue was dried to a constant absorption of magnesium was significantly weight, wet-ashed and also analyzed with reduced with the 1.0% phosphorus diet as the methods noted above. Aliquots of the compared with levels of 0.3 or 0.5% phos­ serum collected at the time of killing were phorus. Urinary magnesium excretion did analyzed directly without prior ashing. not simply reflect the apparent absorption. Urinary magnesium loss appeared to be RESULTS generally encouraged by the lowest phos­ With a 20% protein diet, the lowest level phorus regimen. The absence of this trend of magnesium (130 ppm) was expected to at the 260-ppm plane of magnesium intake cause visible signs of deficiency, that is, may well have been caused by small but hyperemia, ulcerations of the skin, and physiologically significant variations in the reduced weight gain. The highest level of actual phosphorus concentration of the phosphorus was also expected to suppress various diets. Urinary magnesium content weight gain at all levels of dietary mag­ was not further reduced by the increase in

t a b l e 1 Effect of the level of d ie ta ry p h o s p h o ru s a n d m a g n e s iu m o n iv e ig h t g a in o f w e a n lin g ra ts

Group no. 1 2 3 4 5 6 7 8 9 Dietary supplement: Phosphorus, % 0.3 0.5 1.0 0.3 0.5 1.0 0.3 0.15 l.U Magnesium, ppm 130 130 130 260 260 260 1000 1000 1000

9 9 9 9 9 9 9 9 9 Days on experiment 7 21.8 28.6 25.2 32.2 31.7 31.0 2 8 .9 36.1 33.8 14 49.2 68.2 48.5 72.5 72.7 63.3 71.8 76.9 69.9 21 79.5 104.3 74.8 113.6 110.3 94.8 117.0 118.2 105.3 28 98.9 124.8 86.0 149.9 145.0 125.8 145.3 143.5 128.1 408 G. E. BUNCE, H. E. SÄUBERLICH, P. G. REEVES AND T. S. OBA

TABLE 2 Effect of the level of dietary phosphorus and magnesium on mineral balance in rats

Group no. 1 2 3 4 5 6 7 8 9 Dietary supplement: Phosphorus, % 0.3 0.5 1.0 0.3 0.5 1.0 0.3 0.5 1.0 Magnesium, ppm 130 130 130 260 260 260 1000 1000 1000 Mean cumulative balance for 14 days Calcium Intake, mg 1064 1219 999 1214 1181 1018 910 942 957 Feces, mg 338 370 318 542 494 369 170 309 361 Feces, % 32 29 31 45 42 36 19 33 38 Urine, mg 40 3 3 21 4 4 74 8 11 Urine, % 4 0.2 0.3 2 0.3 0.4 8 1 1 Retention, mg 675 888 686 651 683 646 666 633 585 Retention, % 64 71 69 53 58 64 73 66 61 (mg/g gain) 13.6 15.7 18.3 8.4 9.5 10.3 9.1 9.5 10.1 Phosphorus Intake, mg 458 958 1590 609 1171 1857 469 797 1651 Feces, mg 61 132 150 157 181 234 44 128 254 Feces, % 14 14 9 26 15 13 9 16 15 Urine, mg 3 258 740 22 342 897 3 154 809 Urine, % 1 27 47 4 29 48 1 19 49 Retention, mg 387 568 700 430 648 726 422 515 588 Retention, % 85 59 44 71 55 39 90 65 36 (mg/g gain) 7.8 10.1 18.7 5.6 9.0 11.6 5.7 7.7 10.1 M agnesium Intake, mg 22.5 27.9 17.1 52.6 51.2 44.9 185.3 203.3 191.2 Feces, mg 24.5 20.4 11.3 28.2 23.8 30.6 46.9 55.4 81.7 Feces, % 109 73 66 54 46 68 25 27 43 Urine, mg 6.5 3.4 3.8 6.4 7.3 7.2 89.6 34.9 36.8 Urine, % 29 12 22 12 14 16 48 17 19 Retention, mg - 8 . 5 4.1 2.0 18.0 20.1 7.1 48.8 113.0 72.7 Retention, % - 3 8 15 12 34 39 16 26 55 38 (p g /g gain) - 1 7 1 72.4 53.3 232.5 278.0 113.6 663.9 1696.7 1249.4 Weight gain, 14 days, g 49.7 56.6 37.5 77.4 72.3 62.5 73.5 66.6 58.2 Ca/P ratio, retained/g gain 1.74 1.56 0.97 1.51 1.05 0.89 1.58 1.23 1.00 dietary phosphorus from 0.5 to 1.0% de­ Moreover, the variations at the 0.3% plane spite the diminished apparent absorption followed a different pattern and were which accompanied this change at the highly significant. These results suggest higher magnesium intakes. that interactions with other dietary con­ If increases in dietary inorganic phos­ stituents might be of considerable impor­ phorus caused a reduced apparent absorp­ tance. The influence of magnesium on tion of magnesium because of the forma­ urinary phosphorus excretion also varied, dependent on the level of dietary phos­ tion of relatively insoluble complexes, then phorus. No effect was observed with a it would also be expected to find greater 1.0% phosphorus ration. A trend towards quantities of fecal phosphorus following greater phosphorus retention accompanied increases in dietary inorganic magnesium. the highest magnesium intake at the other This expected trend was observed (fig. 2) 2 planes of phosphorus, but a reduction of when phosphorus was in excess (1.0%) equal magnitude was also noted with the and to a lesser degree at the 0.5% plane. lowest magnesium level and 0.3% phos­ It is unexpected however, that an eleva­ phorus. Fractionation of the fecal and tion of dietary magnesium from 260 to urinary phosphorus into their organic and 1,000 ppm had no greater effect quantita­ inorganic constituents might have been tively than an elevation from 130 to 260. helpful in explaining these results. PHOSPHORUS AND MAGNESIUM 4 0 9

MEAN URINARY MAGNESIUM

ppm Mg 130 260 1000 % P .3 .5 1 .3 .5 1 .3 .5 GROUP No. 1 2 3 4 5 6 7 8

1 VS 2 4 vs 5 7 vs 8 N. ; ¡»P« < 0.001 Ip-<0.025 FECAL i vs 3 J 4 vs 6 J 7 vs 9 1 ^P»- 2 vs 3 P* < 0.05 5 vs 6 P - <0.001 8 vs 9 J

1 vs 2 P»< 0.001 4 vs 5 1 7 vs S'! r p> URINARY i vs 3 P«< 0.005 4 vs 6 fN. S. 7 vs 9 J 2 vs 3 N. S. 5 vs 6 J 8 vs 9 N.S

Fig. 1 Effect of variations in dietary phosphorus on magnesium balance.

The effect of dietary variations of both unaffected at the 130-ppm magnesium phosphorus and magnesium on calcium level. Changes in dietary magnesium pro­ balance are also of interest. Calcium might duced a pattern of fecal calcium (fig. 4) have been expected to respond to modifica­ similar to that obtained for fecal phos­ tions in the dietary phosphorus (fig. 3) in phorus (fig. 2), an observation which im­ a manner similar to that observed for mag­ plies that the concentration of magnesium nesium (fig. 1). The urinary excretory in the gut may have an important effect patterns were similar in that significantly on the solubility of calcium phosphate dur­ larger quantities of calcium were excreted ing absorption. The urinary excretory pat­ in the urine with the lowest phosphorus tern showed a general tendency towards ration as compared with the higher levels. increased excretion of calcium with the Fecal calcium also increased in response highest magnesium intake at all levels of to elevations of dietary phosphorus when dietary phosphorus. the dietary magnesium was high. Calcium Analysis of the kidneys for total calcium apparent absorption, however, was im­ (table 3) showed that at every level of proved by increases in dietary phosphorus magnesium, an increase in dietary phos­ at the 260-ppm plane of magnesium and phorus from 0.5 to 1.0% resulted in an 4 1 0 G. E. BUNCE, H. E. SAUBERLICH, P. G. REEVES AND T. S. OBA

MEAN FECAL PHOSPHORUS

MEAN URINARY PHOSPHORUS

ppm Mg 130 260 1000 130 260 1000 130 260 1000 % P 0.3 0.5 1.0 GROUP No. 1 4 7 2 5 8 3 6 9

1 vs 4 P- <0.001 2 vs 5 P < 0.0 05 3 vs 6 ■ < 0 001 FECAL 1 vs 7 P- < 0.005 2 vs 8 P < 0.010 3 vs 9 Jl p' 4 vs 7 P- < 0.001 5 vs 8 N. S. 6 vs 9 P • < 0.025

1 vs 4 p . < 0.001 2 vs 5 N. S. 3 vs 6 1 URINARY 1 vs 7 N.S. 2 vs 8 P ■< 0.010 3 vs 9 s. r 4 vs 7 P - < 0.001 5 vs 8 P -< 0.010 6 vs 9 j Fig. 2 Effect of variations in dietary magnesium on phosphorus balance. increase in renal calcium. With the lowest thiasis (intratubular calcified casts) and magnesium intake, an increase in phos­ nephrocalcinosis (intracellular calcium de­ phorus from 0.3 to 0.5% also caused a position) were widespread in the kidneys marked increase in the quantity of kidney of those animals from the 2 groups with a calcium. In particular, group 1 (130 ppm mean kidney calcium in excess of 1,000 Mg, 0.3% P) had normal renal calcium mg/100 g dry weight. Urolithiasis alone values despite definite hyperemia and der­ was observed in the 2 groups with mean mal lesions, whereas groups 6 (260 ppm kidney calcium values between 10 and Mg, 1.0% P) and 9 (1,000 ppm Mg, 1.0% 1,000 mg/100 g dry weight. P) were free of visible signs of magnesium deficiency, yet had large accumulations DISCUSSION of renal calcium. Microscopic examina­ Increases in dietary phosphorus have tion showed that those groups with a mean been reported to precipitate or aggravate kidney calcium of less than 10 mg/100 g magnesium deficiency in several species. dry weight appeared normal. Both uroli­ The mechanism of this effect, however, is PHOSPHORUS AND MAGNESIUM 4 1 1

MEAN FECAL CALCIUM

MEAN URINARY CALCIUM

ppm Mg 130 260 1 0 0 0 % P .3 .5 1 .3 ,5 1 .3 .5 1 GROUP No. 1 2 3 4 5 6 7 8 9

1 vs 2 1 4 VS 5 N. S. 7 vs 8 Ï f*P = <0.001 PECAL i vs 3 S N.S. 4 VS 6 P - < 0.01 7 vs 9 2 vs 3 J 5 VS 6 P - < 0.05 8 vs 9 N. S.

1 vs 2 1 4 vs 5 1 7 vs 8 1 ^ p . < 0.001 ^P = < 0.001 > P=< 0.001 URINARY 1 vs 3 4 vs 6 7 vs 9 2 vs 3 N. S. 5 vs 6 N. S. 8 vs 9 N. S. Fig. 3 Effect of variations in dietary phosphorus on calcium balance. still in question. O’Dell et al. (4) com­ high. At lower levels of dietary magne­ pared the effect of increases in dietary sium. however, magnesium apparent ab­ phosphorus from 0.4 to 0.9 and 1.8% with sorption was actually enhanced by the 2 levels of magnesium (1,000 and 3,000 higher phosphate intakes. Furthermore, no ppm) and concluded that a high content consistent elevation of serum inorganic of dietary phosphorus contributes to the phosphate was observed as a consequence magnesium deficiency syndrome primarily of elevations of the dietary phosphate up to by interfering with magnesium absorption 1.0% . The importance of this mechanism and secondarily by maintaining a high at physiological levels, therefore, is ques­ blood phosphate level. This study confirms tionable. that dietary phosphate appears to interfere Forbes (10) has reported that increases with magnesium absorption so long as the in dietary phosphorus from 0.19 to 0.50% dietary magnesium content is relatively improved magnesium balance with diets 4 1 2 G. E. BUNCE, H. E. SÄUBERLICH, P. G. REEVES AND T. S. OBA

MEAN FECAL CALCIUM

5 0 , U. ° UJ

UQ J *< £ 5 g -j 2H £ ? “= ? si

z MEAN URINARY CALCIUM O

H ppm Mg 130 260 1000 130 260 1000 130 260 1000 % P 0.3 0.5 1.0 GROUP No. I 4 7 2 5 8 3 6 9

I vs 4 - 2 vs 5 P - < 0 .0 0 l 3 vs 6 N.S. FECAL I VS 7 ‘ p . <0.001 2 vs 8 N.S. 3 vs 9 P» < 0.0 5 4 vs7 - 5 vs 8 P «<0.001 6 vs 9 N .S.

I vs 4 2 vs 5 ~| 3 vs 6 N.S URINARY I vs 7 ■P < 0.001 2 vs 8 L n .S. 3 vs 9 j>P-< 0.001 4 vs 7 5 vs 6 J 6 vs 9 Fig. 4 Effect of variations in dietary magnesium on calcium balance.

TABLE 3 Effect of variation in level of dietary phosphorus and magnesium on tissue mineral composition Group no. 1 2 3 4 5 6 7 8 9 Dietary supplement: Phosphorus, % 0.3 0.5 1.0 0.3 0.5 1.0 0.3 0.5 1.0 Magnesium, ppm 130 130 130 260 260 260 1000 1000 1000 Mean content Kidney Calcium, mg/100 g dry tissue i 120 2028 3 7 1679 0 4 72 Phosphorus, mg/100 g dry tissue 1174 1398 2149 1101 1386 2433 1373 1397 1450 Serum Ca, mg/100 ml 12.6 11.5 12.4 14.2 12.9 13.3 14.2 9.2 10.4 P. mg/100 ml 11.0 8.9 11.6 11.4 10.8 9.9 8.9 8.6 8.2 Mg, mg/100 ml 0.89 1.45 1.26 2.57 1.97 1.96 2.12 2.45 2.42 PHOSPHORUS AND MAGNESIUM 413

containing either 0.4 or 0.8% calcium and LITERATURE CITED 142 or 420 ppm magnesium, while simul­ 1. Hogan, A. G., W. O. Regan and W. R. House taneously aggrevating the external signs of 1950 Calcium phosphate deposits in guinea magnesium deficiency. He attributed this pigs and the phosphorus content of the diet. apparent contradiction to the greater mag­ J. Nutrition, 41: 203. nesium requirement associated with the 2. House, W. B., and A. G. Hogan 1955 In­ jury to guinea pigs that follows a high in­ faster rate of gain at the higher phosphorus take of phosphates. The modifying effect of intake. Our results are in agreement since magnesium and potassium. J. Nutrition, 55; an increase in dietary phosphorus from 507. 0.3 to 0.5% was accompanied by an im­ 3. Maynard, L. A., D. Boggs, G. Fiske and D. provement in magnesium balance at the Seguin 1958 Dietary mineral interrelations 130 and 260 ppm levels of magnesium. as a cause of soft tissue calcification in guinea pigs. J. Nutrition, 64; 85. Furthermore, in this instance, the lowest 4. O’Dell, B. L., E. R. Morris, E. E. Pickett and phosphorus level was not limiting of itself A. G. Hogan 1957 Diet composition and and, as a consequence, no aggrevation of mineral balance in guinea pigs J. Nutri­ the external signs of magnesium deficiency tion, 63: 65. were detected. 5. O’Dell, B. L., E. R. Morris and W. O. Regan Kidney calcification is a well-known fea­ 1960 Magnesium requirement of guinea pigs and rats. Effects of calcium and phos­ ture of magnesium deficiency in the rat. phorus and symptoms of magnesium defi­ Orginally, it was thought to occur as a ciency. J. Nutrition, 70; 103. sequel of cellular death and tubular de­ 6. Nugara, D., and H. M. Edwards, Jr. 1963 generation. Ko et al. (16) and Whang et Influence of dietary calcium and phosphorus al. (17), however, conducted careful histo- levels on the magnesium requirement of the chick. J. Nutrition, 80: 181. pathological studies and concluded that 7. Bunce, G. E., Y. Chiemchaisri and P. H. intratubular cast deposition precedes rather Phillips 1962 The mineral requirements of than follows cellular death. Ko’s group the dog. IV. Effect of certain dietary and proposed that magnesium might be neces­ physiological factors upon the magnesium sary to prevent calcium phosphate precipi­ deficiency syndrome. J. Nutrition, 76: 23. 8. Tibbets, D. M., and J. C. Aub 1937 Mag­ tation within the tubule. These results sup­ nesium metabolism in health and disease. port this concept in that only urolithiasis I. The magnesium and calcium excretion of occurred when renal calcium accumula­ normal individuals, also the effects of mag­ tion and magnesium deficiency were mini­ nesium, chloride and phosphate ions. J. Clin. Invest., 16; 491. mal. Furthermore, urolithiasis is not an 9. Meintzer, R. B., and H. Steenbock 1955 imperative consequence of magnesium de­ Vitamin D and magnesium absorption. J. ficiency; that is, it may not be observed Nutrition, 56; 285. when the phosphate intake is low, even 10. Forbes, R. M. 1963 Mineral utilization in when the other characteristic symptoms the rat. I. Effects of varying dietary ratios of calcium, magnesium and phosphorus. J. are evident. Thus, it appears that a critical Nutrition, 80; 321. dietary intake of magnesium is necessary to 11. Jones, J. H., and C. Foster 1942 A salt prevent urolithiasis and that the absolute mixture for use with basal diets either low amount necessary is directly dependent or high in phosphorus. J. Nutrition, 24; 245. upon the phosphate load and possibly other 12. Leveille, G. A., H. E. Sauberlich and J. W. Shocldey 1962 Protein value and the factors as well. It further suggests that a amino acid deficiencies of various algae for magnesium imbalance may well be a key growth of rats and chicks. J. Nutrition, 76; nutritional factor in spontaneous uroli­ 423. thiasis (18, 19). The mechanism whereby 13. Reitz, L. L., W. H. Smith and M. P. Plumlee magnesium might promote solubilization 1960 A simple wet oxidation procedure for biological materials. Analyt. Chem., 32; of calcium phosphate in the urine is not 1728. known. Morris et al. (20), however, have 14. Robinson, H. M. C., and J. C. Rathbun 1959 recently presented strong evidence of a role The determination of calcium and magne­ for magnesium in the maintenance of the sium in blood using one indicator. Canad. integrity of body mucopolysaccharides and, J. Biochem. Physiol., 37; 225. 15. Hawk, P. B., B. L. Oser and W H. Summer- since a mucoid matrix is an essential com­ son 1947 Practical Physiological Chemis­ ponent of all calculi, this is a logical area try, ed. 12. McGraw-Hill Book Company, for further investigation. Blakiston Division, Philadelphia, p. 579. 4 1 4 G. E. BUNCE, H. E. SÄUBERLICH, P. G. REEVES AND T. S. OBA

16. Ko, K. W., F. X. Fellers and J. M. Craig 19. Moore, C. A., and G. E. Bunce 1964 Re­ 1962 Observations on magnesium deficiency duction in frequency of renal calculus for­ in the rat. Laboratory Invest., 11: 294. mation by oral magnesium administration. 17. Whang, R., J. Oliver, M. McDowell and L. Invest. Urol., 2: 7. G. Welt 1962 The renal lesion of magne­ 20. Morris, M. L., Jr., W. R. Featherston, P. H. sium depletion. Clinical Res., 10: 257. Phillips and S. H McNutt 1963 Influence 18. Säuberlich, H. E., G. E. Bunce, C. A. Moore of lactose and dried skim milk upon the and O. G. Stonington 1964 Oral magne­ magnesium deficiency syndrome in the dog. sium administration in the treatment of renal II. Pathological changes. J. Nutrition, 79.- calculus formation. Am. J. Clin. Nutrition, 437. 14: 240. Nutritional Value of Haitian Cereal-Legume Blends * 1

KOSOL SIRINIT,2 ABDEL-GAWAD M. SOLIMAN, ALI T. VAN LOO a n d KENDALL W. KING Department of Biochemistry and Nutrition, Virginia Polytechnic Institute, Blacksburg, Virginia

ABSTRACT The nutritional value of various beans indigenous to Haiti was evaluated in the rat, both as a sole source of protein and when blended with rice, sorghum, or corn. For the beans alone maximal PER and feed efficiency were ob­ served at 15 to 18% dietary protein. Amino acid supplementation of bean protein confirmed, in general, the deficiencies predicted from chemical analysis, the con­ spicuous exception being failure of added isoleucine to stimulate growth. Blends of beans with cereals showed maximal PER and feed efficiency when beans constituted 20 to 30% of the mixture, the higher level being particularly advantageous with sorghum. Amino acid supplementation of the blends resulted in growth responses generally predicted from chemical analysis. The quality of the protein in most of the blends was significantly inferior to casein, but comparable to Incaparina. The ability of the blends to contribute to human nutrient requirements other than for protein and calories was evaluated by comparing composition with National Research Council recommendations.

Formulation of low-cost blends of tions. An extensive nutritional survey con­ leguminous and cereal foods in which centrating on the dominantly rural popu­ the various components compensate each lation was conducted by Sebrell et al. (2). other’s amino acid deficiencies is one of Jelliffe and Jelliffe (3) simultaneously several useful approaches to combating studied the nutritional status of pre-school the malnutrition which is characteristic of children. These results are in general the emerging nations. The Institute of agreement with those of Grant and Groom Nutrition for Central America and Pan­ (4) and other nutrition-related studies ama (INCAP) pioneered practical use of (5, 6). this principle through its development of Several characteristics of the Haitian Incaparina (1). Both the cultural and situation led us to conclude that cereal- economic practicality of the INCAP ap­ legume blends even simpler than Incapa­ proach is now established by INCAP’s rina were essential to making progress in success in introducing the blend through alleviating the nutritional stress on normal channels of trade in Central Amer­ Haitian children. The fact that the Haitian ica and Northern South America. The economy is the most depressed in the public health significance of the INCAP Western Hemisphere places products even blend is perhaps most clearly apparent as inexpensive as Incaparina beyond the from the fact that under present commer­ reach of the population groups most in cial conditions the cost of 454 g of protein need of improved nutrition. The fact that as Incaparina approximately equivalent in very little food in Haiti passes through quality to milk is about one-tenth the cost anything resembling a wholesale channel of 454 g of protein purchased as milk limits the effectiveness of any manufac­ itself. In many areas of the world prod­ tured food item. Because the bulk of the ucts of this type have a major role to play rural population is illiterate and extremely in alleviating the multiple nutritional naive in its nutritional understanding, any stresses to which large segments of the blend to be encouraged through rural population are subjected, particularly chil­ Received for publication February 5, 1965. dren of pre-school age. 1 Supported by the Williams-Waterman Fund, Re­ search Corporation, New York, New York. In most ways, the Republic of Haiti 2 Present address: U.S. Army Medical Research and presents a nutritional picture which is Nutrition Laboratory, Irradiated Foods, Chemical Division, Fitzsimons General Hospital, Denver, Colo­ typical of technically underdeveloped na­ rado.

J . N u t r it io n , 8 6 : ’6 5 4 1 5 416 KOSOL SIRINIT, ABDEL-GAWAD M, SOLIMAN, ALI T. VAN LOO AND KENDALL W. KING health centers, home economists, agricul­ beans from Central America. The foods tural field workers, and governmental or were ground to pass a 40-mesh screen missionary school programs must be ex­ before formulation into diets. tremely simple. For economic and cultural The results of proximate analyses (9, reasons the exclusive use of indigenous 10) are shown in table 1. foods seems dictated. Three cereals pro­ In all experiments except that carried vide the bulk of the calories in the Haitian out according to the method of Campbell diet, (rice, sorghum and corn) but in (11) weanling male rats derived from the specific areas only one of these is often Sprague-Dawley strain were fed ad libitum used. As a result, separate blends for each for one week a stock diet containing: (in of the cereals are needed. grams) cottonseed oil, 8; salt mixture USP We have, therefore, set as objectives the XVI, 5; powdered cellulose,3 2.4; vitamin development of a series of binary cereal- mixture, 1; vitamin-free casein, 15; su­ legume blends of indigenous foods having crose, 63.6; and water, 5. The vitamin a level and quality of protein adequate to mixture contained: (in grams) Ca panto­ prevent clinical protein-calorie deficiency thenate, 2; niacin, 1; inositcl, 1; thiamine disease, but not necessarily adequate to •HC1, 0.5; menadione, 0.4: riboflavin, 0.3; support maximal growth and development pyridoxineHCl, 0.3; folic acid, 0.02; bi­ of children or to correct the endemic vita­ otin, 0.01; vitamin Bi2, 0.001; and sucrose min and mineral deficiencies that are to make 1000. At the end of this period known to exist. the animals were divided by weight into In developing those binary blends, reli­ groups of 6 rats each and fasted for 24 able information was needed on the amino hours before being weighed. Water was acid composition, total protein content, available at all times. Vitamins A, D, and annual production, and cost of the staple E were administered orally each week in foods potentially useful in blending. These cottonseed oil. Experimental periods are data describing 86 samples including all indicated in footnotes to the individual varieties of all seed-crop foods indigenous tables. to Haiti have been published elsewhere In the balance study feces were collected (7). The present data describe rat per­ daily and frozen. Urine was collected in formance when fed blends were selected 6 n HC1 under toluene and frozen daily. on the basis of chemical composition, cost, All diets were equalized for fat, mois­ and annual production to be most likely ture, ash, and fiber at 8, 6.2, 5, and 6% , to be useful in combating malnutrition respectively, by appropriate addition of in Haiti. cottonseed oil, water, USP XVI mineral EXPERIMENTAL mixture, and powdered cellulose.4 Protein levels were determined assuming protein All of the natural foods were samples = NX 6.25. Varying protein levels were collected in local Haitian markets (7) introduced at the expense of sucrose. Diets brought into the United States without were refrigerated, and fresh food was pro­ sterilization. On arrival, seeds damaged vided daily. Any uneaten diet was dis­ by insect infestation or fungal infection carded. were removed, and the samples were The experiment carried out according to stored frozen. The refuse and surplus sup­ the method of Campbell (11) was con­ plies were sterilized at 121° for one hour ducted precisely as recommended despite before being discarded to prevent introduc­ reservations concerning the value of cer­ tion of new insect pests or plant patho­ tain of the procedural details. gens. All beans were heated at 121° for 15 minutes in 2 volumes of water and then RESULTS dried at 80° for 18 hours to destroy trypsin Table 1 shows the proximate analysis inhibitor and other toxins. These condi­ of the foods used here. In each instance tions were selected on the basis of the the samples were pooled from the several detailed study by Bressani et al. (8) of the effect of heat on the toxicity and nu­ 3 Solka Floe, Brown Company, Berlin, New Hamp­ shire. tritional value of taxonomically identical 4 See footnote 3. VALUE OF HAITIAN FOOD BLENDS 4 1 7 varieties grown in different areas of the The extent to which a blend would country because no significant differences contribute to solution of nutritional prob­ within varieties were noted in the earlier lems other than protein-calorie malnutri­ chemical studies (7). tion is of importance also. In Haiti the Table 2 summarizes the PER’s and feed most conspicuous clinical problems of in­ efficiencies at several levels of dietary pro­ adequate vitamin intake involve riboflavin tein of the 3 beans that were indicated by and vitamin A. Therefore it was deter­ amino acid composition and current pro­ mined whether the beans cooked long duction rates to be most promising for enough to accomplish the necessary détox­ blending with cereals. ifications would retain enough riboflavin The results of nitrogen balance studies of the same 3 varieties of bean and of to represent a significant contribution to Cajanus indicus are shown in table 3. control of ariboflavinosis. Diets were pre­ These evaluations were carried out at the pared from the red, black, and lima bean levels of dietary protein indicated earlier samples of Phaseolus vulgaris to provide to result in optimal PER. 18% dietary protein. In addition to the Comparison of the amino acid composi­ usual vitamin mixture, batches were pre­ tion of these beans with the estimates of pared containing no riboflavin and one- Rama Rao et al. (12) of the requirements half the usual amount of riboflavin. Diets for the growing rat indicated methionine like those described earlier were then pre­ to be first limiting and isoleucine second pared using each type of bean but having limiting. In C. indicus the order of defi­ no added riboflavin, one-half the usual ciency from most to least was trypto­ amount, and the usual amount. Over a phan, isoleucine, valine, and methionine. 4-week period of ad libitum feeding no Whether this was biologically true was significant differences between growth assessed in the experiment summarized in rate or PER of weanling male rats were table 4. observed with the 9 diets.

TABLE 1 Proximate analysis of Haitian foods

Ether- Crude N-free Food Protein Ash Moisture extractable fraction fiber extract

% % % % % % Phaseolus vulgaris, rouge 1 23.2 3.4 6.7 1.6 6.8 58.3 Phaseolus vulgaris, noir 25.5 3.5 6.0 2.7 5.6 56.7 Phaseolus vulgaris, blanc 24.3 3.5 5.1 1.0 7.7 58.4 Phaseolus lunatus, beurre 24.8 3.5 6.2 2.5 6.3 56.7 Cajanus indicus, Congo 21.9 2.8 5.9 2.5 8.1 58.8 Zea m ays (c o m ) 11.1 1.2 11.9 5.3 1.8 68.7 Sorghum vulgare (so rg h u m ) 10.6 0.6 12.0 1.3 0.2 75.3 Oryza sativa (ric e ) 8.1 0.3 11.2 0.7 0.3 79.4 1 Rouge, noir, blanc, and beurre are common names referring to the red, black, white, and lima beans in Haiti. Pois congo refers to C. indicus. TABLE 2 Effect of dietary protein level on PER and feed efficiency of Haitian beans

Dietary protein level, % 1 Protein 18 20 source 10 12 15 PE R 2 Eff 3 PER Eff PER Eff PER Eff PER Eff

Phaseolus vulgaris, rouge 0.69 0.08 0.83 0.12 1.14 0.21 1.16 0.25 0.92 0.23 Phaseolus vulgaris, noir 1.00 0.13 0.83 0.12 1.06 0.19 1.17 0.26 1.09 0.27 Phaseolus lunatus, beurre 0.90 0.11 0.96 0.14 1.34 0.25 0.98 0.21 1.02 0.25 Casein+ 0.2% L-cystine 3.45 0.42 2.99 0.44 2.44 0.45 2.09 0.46 1.81 0.44 1 Experimental period: 10 days. 2 PER indicates protein efficiency ratio; all PER values adjusted to casein (unfortified) = 2.50. 3 Feed efficiency in g of gain/g of diet consumed. 418 KOSOL SIRINIT, ABDEL-GAWAD M. SOLIMAN, ALI T. VAN LOO AND KENDALL W. KING

TABLE 3 Multiple assessments of nutritional value of Haitian bean proteins 1

Dietary protein sources 15% 15% Pois Parameter 2 10% 18% 18% rouge + None Casein 3 Pois Pois Pois pois rouge 4 noir beurre congo mix 5 N-intake, g 0 1.33 2.14 2.34 1.94 1.97 Fecal N, g 0.06 0.14 0.74 0.80 0.63 0.69 Urinary N, g 0.10 0.25 0.77 0.79 0.68 0.61 Weight gain, g - 8 33.0 17.0 21.0 18.0 18.0 PER (casein = 2.5) — 3.25 1.06 1.17 1.15 1.16 Feed efficiency — 0.40 0.23 0.27 0.21 0.21 True digestibility — 94.0 68.2 68.4 70.6 68.0 Net protein ratio — 4.88 1.87 1.99 2.15 2.11 Protein retention efficiency — 78.1 29.9 31.8 34.4 33.8 Biological value — 88.0 54.0 57.0 58.0 62.0 Net protein utilization — 0.83 0.37 0.39 0.41 0.42 1 Experimental period: 7 days. 2 Calculated values as described (11). 3 Casein fortified with 0.2% L-cystine...... 4 Pois rouge, noir and beurre are common names referring to the red, black and lima beans in Haiti. Pois congo refers to C. indicus. 5 One-half of the total protein in this mixture came from each of the 2 beans.

TABLE 4 Effect of amino acid supplementation on the nutritive value of Haitian beans 1

Dietary PER 2 Feed Protein source Supplement protein (casein efficiency 3 level + 2.5) % Phaseolus vulgaris, rouge none 18.0 0.98 0.21 m eth 4 18.2 1.63 0.36 isoleu 18.2 0.82 0.18 meth + isoleu 18.4 1.51 0.33 Phaseolus vulgaris, noir none 18.0 0.94 0.12 meth 18.2 1.67 0.36 isoleu 18.2 1.09 0.23 meth + isoleu 18.4 1.49 0.33 Phaseolus lunatus, beurre none 15.0 0.91 0.17 m eth 15.2 1.54 0.28 isoleu 15.2 1.11 0.20 meth + isoleu 15.4 1.83 0.34 Cajanus indicus, congo none 15.0 1.47 0.27 tryp 15.2 1.53 0.28 tryp + isoleu 15.4 1.34 0.24 tryp + val 15.4 1.67 0.30 tryp + isoleu + val + meth 15.7 2.00 0.36 isoleu + val + meth 15.5 1.45 0,27 Casein cys 10.0 3.10 0.39 1 Experimental period: 10 days. 2 PER indicates protein efficiency ratio. 3 Feed efficiency in g of gain/g of feed consumed. 4 Supplementation with chromatographically pure L-isomers at 0.2% of the diet in all instances except 0.1% methionine in the C. indicus diets.

The effectiveness of the 4 beans as sup­ in the blends from comparison of chemi­ plements at the 10, 20, and 30% levels cal data with the requirement of the rat to rice, corn, and sorghum was evaluated were actually limiting in growth perform­ in terms of weight gains and PER with ance. Composition of the amino acid the results shown in table 5. blends selected as representative of the An indication was sought as to whether more promising combinations was calcu­ the amino acids predicted to be limiting lated. These values were compared with VALUE OF HAITIAN FOOD BLENDS 4 1 9

TABLE 5 Weight gain and protein efficiency ratio (PER) of rats fed cereal-legume blends varying in composition 1

Com-bean diets 2 Sorghum-•bean diets Rice-bean diets Legume blended Gain PER 3 Gain PER Gain PER % 9 9 9 None 17 1.40 6 0.53 27 2.38 Pois rouge 4 10 5 26 1.78 17 1.22 34 2.60 Pois rouge 20 30 2.05 30 1.83 41 3.03 Pois rouge 30 30 1.98 30 2.13 36 2.90 Pois noir 4 10 27 1.78 23 1.65 38 2.83 Pois noir 20 36 2.30 36 2.13 39 2.95 Pois noir 30 32 2.10 43 2.45 36 2.75 Pois blanc 4 10 23 1.68 21 1.50 36 2.65 Pois blanc 20 28 1.83 35 2.25 39 2.80 Pois blanc 30 31 2.00 36 2.30 39 2.98 Pois beurre 4 10 32 1.53 20 1.43 41 2.78 Pois beurre 20 36 2.28 33 1.98 42 2.90 Poie beurre 30 35 2.13 38 2.45 30 2.35 1 Experimental period: 2 weeks. 2 Corn and sorghum diets contained 8.3% crude protein. Rice diets contained 6.4% crude protein. 3 All PER values adjusted to casein = 2.5. 4 Rouge, noir, blanc, and beurre are common names referring to the red, black, white and lima beans in Haiti. 5 Per cent represents the % by weight of beans in the bean-cereal mixture.

the estimates of Rama Rao et al. (12) of teria for the blends have been met. A the requirements of growing rats. Diets number of the blends (table 7) have PER were then prepared which were supple­ values of 2.0 to 2.2 at the 10% protein mented with chromatographically pure, level, and at the 15% protein levels under allo-free, L-amino acids in amounts to which actual field use is anticipated, the raise them to 100% of Rama Rao’s esti­ PER values are 2.0 to 2.3 with corn, 2.1 mates of requirement. Growth and PER to 2.5 with sorghum, and 2.4 to 3.0 with were then measured over a 2-week period, rice (table 5). Total crude protein levels the results being summarized in table 6. in blends containing 20% beans are ap­ For purposes of comparing the nutritive proximately 13.7, 13.3, and 11.3% for value of these blends with that of similar corn, sorghum, and rice, respectively; at preparations a final determination of PER 30% beans the corresponding crude pro­ was made following the procedure de­ tein levels are 15.0, 14.7, and 12.9% . The scribed by Campbell (11). These results cost of 20% bean blends and 30% bean are summarized in table 7. With all its blends compared with that of the 3 basic limitations Campbell’s procedure is suffi­ cereals alone has been estimated from the ciently well-described that reproducibility market data of Sebrell et al. (2). The of PER values from different laboratories cost of rice and beans being equivalent, should be good except for variability intro­ blending does not add to the price of 11 duced by differences in the genetic line of cents/454g. For sorghum a blend with the rats and in the environmental condi­ 30% beans would cost about 8.9 cents/ tions in the animal room. 454 g compared with 8 cents/454 g for sorghum alone. Corn values fall in the DISCUSSION range of 5 cents/454 g, and a corn-bean The usefulness of blends of this type blend with 30% beans would cost about remains to be determined in clinically 6.8 cents/454 g. Market costs5 as of controlled field tests in Haiti. With the January, 1965 were similar to those re- data presented here, however, it appears 5 Current Haitian food prices supplied by Mile. warranted to proceed with testing the Gladys Dominique, Dietitian, Bureau of Nutrition, Department of Public Health, Republic of Haiti, blends in humans because the major cri­ Port-au-Prince. 420 KOSOL SIRINIT, ABDEL-GAWAD M. SOLIMAN, ALI T. VAN LOO AND KENDALL W. KING

TABLE 6 Effect of amino acid supplementation of cereal-legume blends 1

Cereal-legume Gain PER 2 mixture Supplementation 9 Com-pois rouge none 47 2.41 (70-.30) 3 0.28 m eth 4 58 2.60 0.28 meth, 0.36 lys 42 2.35 0.28 meth, 0.36 lys, 0.19 thre 66 2.69 0.28 meth, 0.36 lys, 0.19 thre, 0.21 isoleu 43 2.38 0.28 meth, 0.36 lys, 0.19 thre, 0.21 isoleu, 0.14 val 43 2.45 Sorghum-pois noir none 56 2.54 (7 0 :3 0 ) 0.30 m eth 58 2.61 0.30 meth, 0.40 lys 64 3.05 0.30 meth, 0.40 lys, 0.21 thre 77 3.29 0.30 meth, 0.40 lys, 0.21 thre, 0.21 isoleu 67 3.16 0.30 meth, 0.40 lys, 0.21 thre, 0.21 isoleu, 0.13 val 69 3.33 0.30 meth, 0.40 lys, 0.21 thre, 0.21 isoleu, 0.13 val, 0.07 tyro, 0.01 hist 72 3.41 Rice-pois rouge none 57 2.85 (8 0 :2 0 ) 0.25 meth 62 2.98 0.25 meth, 0.24 thre 57 2.93 0.25 meth, 0.24 thre, 0.41 lys 67 3.20 0.25 meth, 0.24 thre, 0.41 lys, 0.22 isoleu 63 3.69 0.25 meth, 0.24 thre, 0.41 lys, 0.22 isoleu, 0.09 tyro 70 3.68 0.25 meth, 0.24 thre, 0.41 lys, 0.22 isoleu, 0.09 tyro 0.12 val, 0.08 leu, 0.03 hist 70 3.74 Corn-pois beurre none 52 2.50 (8 0 :2 0 ) 0.26 meth 51 2.56 0.26 meth, 0.41 lys 46 2.49 0.26 meth, 0.41 lys, 0.20 thre, 0.22 isoleu 49 2.94 0.26 meth, 0.41 lys, 0.20 thre, 0.22 isoleu, 0.15 val, 0.05 tyro, 0.02 tryp 65 3.09 Sorghum-pois beurre none 50 2.44 (7 0 :3 0 ) 0.29 meth 50 2.47 0.29 meth, 0.41 lys 77 3.27 0.29 meth, 0.41 lys, 0.20 thre 65 3.18 0.29 meth, 0.41 lys, 0.20 thre, 0.18 isoleu, 0.11 val, 0.06 tyro 66 3.28 Rice-pois beurre none 62 3.08 (8 0 :2 0 ) 0.25 meth 63 3.13 0.25 meth, 0.24 thre 66 3.17 0.25 meth, 0.24 thre, 0.39 lys 73 3.66 0.25 meth, 0.24 thre, 0.39 lys, 0.21 isoleu, 0.07 tvro, 0.08 leu, 0.06 val, 0.02 hist 72 3.82 1 Experimental period, 2 weeks. 2 PER indicates protein efficiency ratio. 3 Ratios are weight of cereal ¡weight of bean. 4 Number preceding each amino acid supplement is the grams of amino acid added per 100 g of diet.

ported by Sebrell et al. (2) being as fol­ the problem of low total protein is less lows: pois blanc, 10; pois rouge, 14.5; pois acute than with rice. beurre, 12; pois noir, 10; sorghum, 6; corn, Reasonable estimates of cereal consump­ 4; and rice, 10 cents (American) per 454 tion in Haiti were developed by Sebrell et g. Prices fluctuate considerably with sea­ al. (2) as 120 pounds [55 kg] of corn, 95 son and with crop yields, but in general pounds [43 kg] of sorghum and millet, 23 this pattern is pertinent. In terms of pro­ pounds [10 kg] of rice, and 20 pounds [9 tein quality, total protein, and cost, the kg] of wheat per person per year. Consider­ 30% blends meet the first criteria sought. ing the relatively large consumption of There may even be use for the 20% blends, sorghum and its extremely low nutritional particularly with corn and sorghum where value (table 5), special attention to en- VALUE OF HAITIAN FOOD BLENDS 4 2 1

TA BLE 7 Groivth performance and protein efficiency ratio (PER) of cereal-legume blends, Incaparina and casein determined as described by Campbell (11)

Multiple Cereal Bean 1 Beau in Protein 2 Avg Avg range 4 mixture in diet PER 3 gain test groupings

% % 9 Sorghum rouge 20 10.00 1.45 48.5 Sorghum blanc 5 20 9.88 1.72 59.0 Sorghum blanc 30 10.31 1.77 63.6 Corn rouge 20 10.06 1.88 71.0 Incaparina 6 — — 10.25 1.78 72.4 Sorghum rouge 30 10.13 2.02 72.8 Sorghum noir 20 9.81 1.85 74.2 Rice noir 30 9.63 2.04 76.1 Rice rouge 20 9.31 2.04 77.6 Sorghum noir 30 10.00 1.90 84.0 Corn noir 20 10.25 1.99 86.3 Rice rouge 30 9.94 2.12 86.8 Rice noir 20 9.38 2.19 88.7 Com blanc 30 10.31 1.97 90.0 Corn rouge 30 10.13 2.12 94.0 Rice blanc 30 10.00 2.10 94.1 Corn blanc 20 9.81 2.04 95.8 Corn noir 30 10.19 2.09 95.9 Rice blanc 20 9.31 2.56 116.0 Casein — — 10.00 2.50 131.9 1 Rouge, noir, and blanc are common names referring to the red, black and white beans in Haiti. 2 Determined by Kjeldahl analysis of diets as fed. 3 Adjusted to casein = 2.50. 4 Bars connect values of mean gains that are not distinguishably different at P = 0.05 as described by Duncan (13). 5 In this experiment only the pois blanc samples were lyophilized rather than dried at 80°. 6 Incaparina generously supplied by Dr. Ricardo Bressani. couraging use of sorghum-bean blends is needs is to be anticipated, particularly warranted. among the vulnerable pre-school age chil­ As in many other parts of the world, the dren. nutritional problem in Haiti involves more Only in the experiment summarized in than protein-calorie malnutrition. Ribo­ table 7 was the exact procedure of Camp­ flavin, vitamin A, and iodine deficiencies bell (11) used in determining PER’s. Be­ are prevalent, and it is probable that the cause the procedure carries the sanction of intakes of iron and calcium are marginal the Committee on Protein Malnutrition of (2). The extent to which increased uses the Food and Nutrition Board of the Na­ of such blends would contribute to alleviat­ tional Research Council, it can be expected ing these nutritional stresses in addition to to serve at least informally as a “standard” the protein stress is, therefore, of impor­ procedure. The principal procedural dif­ tance. In table 8 a general summary of ferences in the earlier experiments here the nutritional value of blends containing were (a) use of protein levels other than 30% beans and 70% of each of the three 10% ; (b) use of 6 rather than 10 animals basic cereals is presented. Assuming that per group; and (c) use of experimental 75% of the daily caloric requirement of periods of less than 28 days. infants, children, and adults is supplied by Two practical and two theoretical rea­ the blends, the percentages of the NRC sons led to our use of these modifications. recommended dietary allowances (14) of From our own experience and the pub­ other major nutrients have been calculated. lished reports of Bender and Doell (16), it Significant amounts of iron, thiamine, ribo­ is apparent that in general, 28-day PER flavin, and niacin are present particularly values are lower than those at 10 days and in the corn and sorghum blends; but little 2 weeks, but the relative values remain the contribution to the vitamin A and calcium same. Using shorter periods and some- 4 2 2 KOSOL SIRINIT, ABDEL-GAWAD M. SOLIMAN, ALI T. VAN LOO AND KENDALL W. KING

what smaller groups of animals here per­ 10 W3 I>OcO(N05(NWHO CO H ^ IS CO (M IS CO H mitted answers of satisfactory reliability COI « 51 t-H ^ CO rH rH from experiments approximately one- fourth as large. The resulting economy in OG0L0rHl/3lL0G303O e ^10 0)0 ^ CO 03 time, equipment, labor, and materials is o rH CM CO rH & obvious. Use of protein levels other than 10% is CO fH I « .wootNOHtDcoo justified on 2 bases. Experimental vari­ co £ ^ 1 0 CM rH CO CO rH rH Csl CO rH £ ability is inherently minimal in the region -£ ^COTtCOrHCOOOt-tSO of optimal response for any experimental O S« h o 05 in o rH OJ rH rH system. For blends of the type studied $ o here 15 to 18% protein is optimum in COOCOrHlOtSOitSO terms of PER. In addition an indication 10 ^ 03 rH rH Cq of possible behavior at the protein levels anticipated for field use was sought. Cer­ tainly Campbell’s procedure has value as 10 w .OOOOH^-tOO a detailed experimental protocol facilitat­ gc CD cq ^ CM CO CO rH CO CO ing inter-laboratory comparisons of pro­ tein sources. Rigid adherence to its details lO 71 CO H ^oqintscocooirfoo in all PER measurements, however, is both I ^ ^ rH in co io cq LO CO V IN rH ^ rH rH CO rH unsound and beyond the stated intention o of the Committe on Protein Malnutrition. £ + CD M ^HOO)(N^(NOO I « cq io is f l co ^ rH r H CO ACKNOWLEDGMENTS cn CO M I « . q Ojcoqcoohnoo Dr. H. A. Wood, Chief Public Health rH 0) ^ CO LO o> io CO Officer of the United States Overseas Mis­ co £h O sion (AID) played vital roles in discussing W J PQ 0 CDCOlSNIOCqcOOO the most useful type of cereal-legume ec co 03 cq Tp co i * rH cq blends for use in Haiti and in arranging for his colleagues in agriculture to collect,

•XJ identify botanically, and ship the samples £ lO a> CO u to the United States. Dr. William Fougere, I Ctf .0O3cqOdCOHOO co g> ^ cq io ^ co is rH >> rH Cq rH Head of the Bureau of Nutrition of the Haitian Public Health Service, and Dr. CO >H .COOCOOCOO’iO O Ivan Beghin, WHO-PAHO also contributed COI w ctf ^ cq cq rH T f ^ o rH to establishing the criteria sought in the in flS rH £ present blends. o COI os H iniocoocq^rHoo CO £ cq rH cq rt co co h 05 LITERATURE CITED rO Ph « 1. Bressani, R., L. G. Elias, A. Aguirre and N. r£ vCsO r H C O O Q 0 0 3 I S O O <3 Haiti. Am. J. Clin. Nutrition, 7: 538. > 'S 3. Jelliffe, D. B„ and E. F. P. Jelliffe 1961 The s g nutritional status of Haitian children. Acta CjO QJft' Tropica, 18: 1. < 0) 03 Q 4. Grant, F. W., and D. Groom 1958 A die­ b .H > .a £ tary study in Haiti. J. Am. Dietet. A., 34: 0 to g s - S f * s 708. CS a os o O o hQ . to o 5. King, K. W., J. Foucauld, W. Fougere and &hO h > h p 3 2 ; < > E. L. Severinghaus 1963 Height and weight VALUE OF HAITIAN FOOD BLENDS 4 2 3

of Haitian children. Am. J. Clin. Nutrition, emy of Sciences — National Research Coun­ 13; 106. cil, Washington, D. C., p. 31. 6. King, K. W., W. H. Sebrell, E. L. Severing- 12. Rama Rao, P. B., H. W. Norton and B. C. haus and W. O Storvick 1963 Lysine forti­ Johnson 1964 Amino acid compositions fication of wheat bread fed to Haitian school and nutritive value of proteins. V. Amino children. Am. J. Clin. Nutrition., 12; 36. acid requirements as a pattern for protein 7. King, K. W. 1964 Development of all- evaluation. J. Nutrition, 82; 88. plant food mixture using crops indigenous 13. Duncan, D. B. 1955 Multiple range and to Haiti: Amino acid composition and pro­ multiple F tests. Biometrics, 11: 1. tein quality. Econ. Botany, IS; 311. 8. Bressani, R., L. G. Elias and A. T. Valiente 14. National Research Council, Food and Nutri­ 1963 Effect of cooking and of amino acid tion Board 1964 Recommended dietary al­ supplementation on the nutritive value of lowances, ed. 6. National Academy of Sci­ black beans (Phaseolus vulgaris). Brit. J. ences — National Research Council Wash­ Nutrition, 17; 69. ington, D. C. 9. Association of Official Agricultural Chemists 15. Leung, W. W., and M. Flores 1961 INCAP - 1960 Official Methods of Analysis, ed. 9, ICNND food composition table for use in Washington, D. C. Latin America. Interdepartmental Com­ 10. Whitehouse, K., A. Zarow and H. Shay 1945 mittee on Nutrition for National Defense, Rapid method of determining “crude fiber” National Institutes of Health, Bethesda, in distillers’ dried grain. J. A. Off. Agr. Maryland. Chem., 28; 147. 16. Bender, A. E., and B. H. Doell 1958 Bio­ 11. Campbell, J. A. 1963 In Evaluation of logical evaluation of proteins, a new aspect. Protein Quality, pub. 1100. National Acad­ Brit. J. Nutrition, 11: 140. Decrease in Appetite and Biochemical Changes in Amino Acid Imbalance in the R at* 1

JUAN C. SANAHUJA, MARIA E. RIO a n d MARIA N. LEDE Department of Biochemistry, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina

ABSTRACT Rats fed imbalanced diets containing 10 and 15% of wheat gluten supplemented with lysine, to which a 3.1% mixture of essential amino acids lacking threonine was added, had lower “appetite quotient” values than control animals fed balanced diets. This confirms our assumption that decreased food intake is a primary effect of the imbalance. The plasma amino acid pattern produced by the imbalance was similar to that described in earlier experiments: the concentration of threonine, limiting amino acid of the diet decreased markedly, and the ratios between the con­ centration of other essential amino acids to threonine increased markedly. Animals fed the imbalanced diets showed an increased liver weight correlated with a high glycogen content, when expressed per 100 g of body weight. The liver lipid of im­ balanced animals was decreased possibly as a consequence of the lipotropic effect of lysine and threonine that were ingested in amounts higher than needed for growth. These changes could be related to the mechanism responsible for food intake de­ pression. The similarity between some of these observations and some of the changes occurring with kwashiorkor supports the theory that imbalance of amino acids in the diet plays an important role in the incidence of that disease.

Food intake of rats fed a diet in which If appetite is in some way affected di­ there is an imbalance of amino acids de­ rectly by the diet, the food consumption creases rapidly. Some earlier observations of experimental animals measured as me­ suggested that the decrease in food intake tabolizable energy would not be propor­ could be secondary to some change in tional to their maintenance requirements amino acid metabolism producing a de­ (5). On this basis Carpenter (6) estab­ crease in the efficiency of utilization of lished a method for calculating the food the limiting amino acid of the diet and, intake of ad libitum-fed rats, in terms of hence, a retarded growth rate (1,2). their body size, as a measure for the detec­ In previous studies concerning the ef­ tion of differences in their appetite accord­ fect of amino acid imbalance on food ing to the diets they receive. He called intake and preference (3) we observed this the “appetite quotient,” representing that protein-depleted rats given a choice between an imbalanced and a protein-free the ratio of intake of metabolizable energy diet, showed a preference for the protein- to body weight0 88. free diet after the third day, and rejected In the present investigation, we used the imbalanced diet. Further experiments Carpenter’s method to determine the ap­ demonstrate that rats that have been pre­ petite quotient of rats fed imbalanced viously starved or fed ad libitum showed diets. The results showed that these val­ a similar preference after various periods ues are significantly lower than those ob­ of time (4). The preference for the pro­ tained in rats fed balanced diets; this tein-free diet over the imbalanced diet confirms our assumption that food intake cannot be attributed to any superiority in is directly affected by the dietary amino the nutritive value of the protein-free diet acid pattern. because it did not support growth, whereas Data are also presented to show the the imbalanced diet did. These observa­ influence of imbalanced diets on body tions indicate that an amino acid imbal­ Received for publication January 4, 1965. ance may affect directly some basic mech­ 1 Supported in part by grants from the Research Corporation and from the Consejo Nacional de In­ anism regulating food intake. vestigaciones Científicas y Técnicas (Argentina).

424 J. N u t r it io n , 86: ’65 APPETITE AND AMINO ACID IMBALANCE 4 2 5

composition in protein-depleted and non- subsequent lipid determination. A third depleted rats. In previous experiments it piece weighing approximately one gram was observed that changes in plasma was placed in 30% KOH for glycogen amino acid pattern precede changes in determination. appetite or occur at the same time (7, 8). Gastrocnemius muscle. The right gas­ As a working hypothesis it was assumed trocnemius muscle, was weighed, placed that this altered blood amino acid pattern in 10% perchloric acid and blended in a triggers an appetite-depressing mechanism Virtis homogenizer. Aliquots of this sus­ that causes the decrease in food intake pension were used for protein determina­ (8 ,9 ). tion. The results obtained in this study, indi­ cate that concurrently with the variations Chemical analysis in the plasma amino acid pattern, some Amino acids. Plasma amino acids were changes occur in weight and composition determined by the method of Levy as of liver in rats fed imbalanced diets; these modified by Peraino and Harper (10). changes may be also related to the mech­ Protein. Tissue protein was determined anism responsible for curtailing appetite. by the method of Marenzi et al. (11). Lipid. Lipid content of liver was deter­ EXPERIMENTAL mined by the procedure described by Sid- Groups of 6 male rats of the Wistar ransky and Baba (12). strain were used in these experiments. Glycogen. Liver glycogen was deter­ Nondepleted rats. Rats weighing an mined according to the method indicated average of 50 to 60 g were fed the experi­ by Hawk et al. (13). mental diets ad libitum for 2 weeks. Diets. Two different basal diets con­ Protein-depleted rats. Animals weigh­ taining 10 and 15% of wheat gluten, re­ ing on the average 80 to 85 g were de­ spectively, were used in the experiments, pleted of protein by feeding them a pro­ as shown in table 1. In both diets the tein-free diet for 6 days. Rats losing 15 to content of lysine was increased to 0.9% ; 17 g were selected; they were fed the therefore threonine, the next limiting experimental diets ad libitum for 2 weeks. amino acid of the wheat gluten remained The rats were housed in individual sus­ as the first limiting amino acid of the pended cages with screen bottoms and diets. Supplemented diets were made by water was offered ad libitum. In all the adding 0.1% of DL-threonine to the basal experiments, food intake was measured diets. Imbalanced diets were obtained by and recorded daily, and the animals were further addition to the supplemented diets weighed twice a week. The appetite quo­ of 3.1% of a mixture of essential amino tient was calculated according to Car­ acids lacking threonine.2 The protein-free penter (6). diet had the same composition as the basal diets, except that wheat gluten and Collection of material lysine were replaced by dextrin. Plasma. At the end of the experimental periods, the rats were anesthesized with RESULTS ether, and blood was withdrawn by heart The effect of the addition of DL-threo­ puncture. The blood of each group was nine, and DL-threonine plus the imbal­ pooled and plasma separated from the anced amino acid mixture lacking threo­ heparinized blood by centrifugation. The nine, to the basal diets is shown in table 2. samples of plasma were deproteinized As expected, growth rate of nondepleted with 10% of perchloric acid for the sub­ rats increased by the supplementation of sequent amino acid analysis. both basal diets with threonine; groups 2 Liver. After weighing the whole liver and 5 fed the supplemented diets had a of exsanguinated rats a piece was placed higher intake and gained more weight, in 10% perchloric acid and blended in a respectively, than groups 1 and 4 fed the Virtis homogenizer; suitable aliquots of 2 Sanahuja, J. C., M. E. Rio and M. N. Lede 1964 this suspension were used for protein de­ Effect of amino acid imbalance on nutritive value of supplemented proteins. Federation Proc., 23: 878 termination. A large piece was frozen for (abstract). 4 2 6 JUAN C. SANAHUJA, MARIA E. RIO AND MARIA N. LEDE basal diets. But the improvement ob­ their performance being similar to that tained by the addition of DL-threonine was observed in groups receiving the basal completely offset by the further addition diets. of the amino acid mixture; rats of groups As the effect of the imbalance in non- 3 and 6, fed the imbalanced diets, showed depleted rats was markedly higher in ani­ a considerable depression in food con­ mals fed diets containing 10% wheat glu­ sumption and in growth rate compared ten than in groups receiving diets with with those fed the supplemented diets, 15% wheat gluten, only the former diets

TA BLE 1 Composition of diets

A B c D E F B asa l Supplemented Im b a la n c e d B asa l Supplemented Imbalanced % % % % % % Wheat gluten 1 10 10 10 15 15 15 L-Lysine 2 0.80 0.80 0.80 0.75 0.75 0.75 DL-Threonine — 0.10 0.10 — 0.10 0.10 Amino acid mixture 3 — — 3.10 — — 3.10 Minerals 4 5 5 5 5 5 5 Vitamin mixture 4 0.25 0.25 0.25 0.25 0.25 0.25 Choline chloride 0.15 0.15 0.15 0.15 0.15 0.15 Corn o il5 5 5 5 5 5 5 Dextrin 6 78.80 78.70 75.60 73.85 73.75 70.65 Total protein content 9.18 9.28 12.38 12.57 12.67 15.77 Total threonine content 0.20 0.30 0.30 0.30 0.40 0.40 1 Containing 13.4% N (83.8% of protein), 2% of threonine and 1% of lysine. 2 As E-lysine *HC1. 3 The amino acid mixture provided in % in the diet: E-arginine-HC1, 0.2; E-lysine • HC1, 0.4; DL-isoleucine, 0.4; L-leucine, 0.3; DL-methionine, 0.4; DL-phenylalanine, 0.2; L-histidine-HCl, 0.4; DL-tryptophan, 0.4; DL-valine, 0.4. 4 Harper, A. E. (14). 5 Fat-soluble vitamins were included in the com oil (14). 6 Moist cornstarch heated at 121° in an autoclave for 3 hours.

TA BLE 2 Food intake, change in weight and “appetite quotient” of rats fed the experimental diets ad libitum for 2 weeks

Group Diet Food Threonine Appetite intake intake Wt gain quotient 1 g /day ■mg 1 day 9 Nondepleted i A Basal (10% wheat gluten) 2 8.3 16.6 15.0 ± 1.3 3 0.992 ±0.020 2 B Supplemented (A + 0.1% DL-threonine) 13.6 40.8 36.8 ± 2 .0 5 1.071 ±0.020 3 C Imbalanced (B + 3.1% A A m ix)4 7.0 21.0 19.5 + 2.1 0.864 ±0.027 5 4 D Basal (15% wheat gluten) 2 11.2 33.6 42.3 ± 2 .9 1.008 ± 0 .0 2 0 5 E Supplemented (D + 0.1% DL-threonine) 14.0 56.0 59.0 ± 2 .4 5 1.017 + 0.027 6 F Imbalanced (E + 3.1% AAmix) 4 11.0 44.0 4 8 .6 ± 2 .4 0.908 ±0.014 5

Protein-depleted 7 A Basal (10% wheat gluten) 2 10.8 21.6 1 7 .5 ± 3 .1 0.900 ± 0 .0 2 0 8 B Supplemented (A + 0.1% DL-threonine) 15.5 46.5 54.8 ± 5 .4 5 1.053 ± 0 .0 2 3 9 C Imbalanced (B + 3.1% AAmix) 4 10.5 31.5 23.8 ± 2 .8 0.784 ±0.025 4 Food intake x metabolizable calories per gram J W eight0-88 d.t. 2 Plus L-lysine, as L-lysine-HCl, up to 0.9% in the diet. 3 Mean value + s e of m ean . 4 For composition, see footnote 3, table 1. 5 Highly significantly (P < 0.01) different from basal group mean. APPETITE AND AMINO ACID IMBALANCE 4 2 7 were used in subsequent experiments with the balanced diets (basal or supplemented), protein-depleted rats. but were significantly lower (P < 0.01) in During 2 weeks, the protein-depleted rats receiving the imbalanced diets (groups rats of group 7, fed the basal diet, regained 3, 6 and 9). only the weight lost during the depletion The results of analysis of amino acids period. Protein-depleted rats of group 8 in plasma of nondepleted rats after the receiving the supplemented diet had a experimental period of 2 weeks are shown much higher food intake and regained in table 3. The plasma threonine concen­ more than 3 times the weight lost during tration of rats fed the imbalanced diets the protein depletion period. But here (groups 3 and 6), decreased markedly, again this improvement was offset by the being lower at the end of the experiment imbalance: the weight increase of rats fed than that for the groups fed the balanced the imbalanced diet (group 9) was poorer diets (basal or supplemented), despite the than that for group 8 and similar to that fact that the threonine intake was higher observed in rats fed the basal diet (group in the imbalanced groups than in groups 1 7), even though the concentration of threo­ and 4 receiving the basal diets (table 2). nine was 50% higher in the imbalanced During the experiment the concentrations than in the basal diet. Table 2 also shows of other essential amino acids increased the appetite quotient values for all experi­ in the blood of animals fed imbalanced mental groups. These values were similar diets and after 2 weeks were higher than in depleted or nondepleted rats consuming in rats fed the supplemented or basal diets.

TA B LE 3 Plasma amino acid concentration of nondepleted rats fed the experimental diets ad libitum for 2 weeks (analysis from pooled blood of 6 rats)

G roup 1 2 3 4 5 6 (d ie t A ) 1 (diet B) 2 (dietC) 3 ( diet D ) 4 ( diet E ) 5 (d ie t F ) 6 limóles/100 ml plasma limoles / 100 ml plasma limoles / 100 ml plasma Threonine 6.0 10.0 3.0 6.0 24.0 3.0 Valine 25.0 23.5 40.0 28.5 43.0 51.5 Leucine + isoleucine 13.0 13.0 37.0 15.0 41.5 52.5 Lysine 19.0 24.0 47.0 59.0 43.0 90.0 Glutamic acid 18.0 27.0 28.0 116.0 57.0 102.5 Proline + OH proline 65.0 70.0 59.0 71.0 46.0 Glycine 21.5 17.0 20.5 56.0 39.5 43.5 Alanine 66.0 45.0 64.0 74.5 78.5 67.0 Serine 46.5 30.0 40.5 110.0 73.0 51.0

1 Basal: 10% wheat gluten plus L-lysine, as L-lysine-HCl, up to 0.9% in the diet. 2 Supplemented : diet A + 0.1% DL-threonine. 3 Imbalanced: diet B + 3.1% amino acid mixture (for composition, see footnote 3, table 1). 4 Basal: 15% wheat gluten, plus L-lysine, as L-lysine-HCl, up to 0.9% in the diet, s Supplemented: diet D + 0.1% DL-threonine. 6 Imbalanced: diet E + 3.1% amino acid mixture (for composition, see footnote 3, table 1).

TA B LE 4 Ratio of selected indispensable amino acids to threonine in plasma of nondepleted rats fed the experimental diets ad libitum for 2 weeks

Group 1 2 3 4 5 6 (diet A) 1 (diet B ) 2 (diet C) « ( diet D ) 4 (dietE ) s (diet F )

V aline/ threonine 4.1 2.3 13.3 4.7 1.8 17.1 Leucine + isoleucine/threonine 2.1 1.3 10.6 2.5 1.7 17.5 Lysine/threonine 3.1 2.4 15.6 9.8 1.8 30.0

1 Basal: 10% wheat gluten plus L-lysine, as L-lysine-HCl, up to 0.9% in the diet. 2 Supplemented: diet A + 0.1% DL-threonine. 3 Imbalanced: diet B + 3.1% amino acid mixture (for composition, see footnote 3, table 1). 4 Basal: 15% wheat gluten, plus L-lysine, as L-lysine-HCl, up to 0.9% in the diet. 3 Supplemented: diet D + 0.1% DL-threonine. 6 Imbalanced: diet E + 3.1% amino acid mixture (for composition, see footnote 3, table 1). 8 2 4

Weight and composition of liver and gastrocnemius muscle of rats fed the experimental diets ad libitum for 2 weeks O UAN C S A, Í E RO N MARA LEDE . N RIA A M AND RIO E. RÍA A M , JA U H A N SA C. N A JU O > O O bo CD "0 «“H 2T) 03 03 -M C33 A s h 03 03 S 0+4 £?H 03 S'® O) o S’® 1 ) O rQ rQ o p

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Highly significantly (P < 0.01) different from supplemented group mean. Probably significantly (P between 0.01 and 0.05) different from supplemented group mean. Highly significantly (P < 0.01) different from basal group mean. APPETITE AND AMINO ACID IMBALANCE 4 2 9

Of the dispensable amino acids the con­ of the animals fed the supplemented diets centrations of glutamic acid and serine (groups 2 and 5), respectively. Protein- reached a high value in the animals fed depleted rats fed the basal diet (group 7) the basal diet D containing 15% wheat showed only a slight difference in liver gluten. In rats fed the imbalanced diets lipid content in respect to the zero time these changes resulted in substantial in­ value, but the fat levels were decreased in creases in the plasma ratios of various groups 8 and 9 receiving the supplemented indispensable amino acids to threonine and the imbalanced diet, respectively. (table 4); these ratios had lower values The most important differences were and showed only little differences in rats observed in the liver glycogen content: fed the balanced diets. nondepleted rats fed the imbalanced diets Table 5 shows the changes in weight of (groups 3 and 6) had a markedly higher liver and gastrocnemius muscle of protein- glycogen content than the groups fed the depleted and nondepleted rats expressed balanced diets (supplemented or basal as grams per 100 grams of body weight diets). The same picture was observed in after the experimental periods. depleted animals: the amount of glycogen The mean wet weight of the liver in in the liver of imbalanced rats (group 9) nondepleted rats was significantly elevated was significantly higher than in group 7, in groups 3 and 6, fed the imbalanced fed the basal diet and group 8, the supple­ diets, compared with groups 2 and 5, re­ mented one. spectively, receiving the supplemented diets. In contrast, no significant differ­ DISCUSSION ences were observed between the liver Appetite and amino acid imbalance. The weights of the animals fed the supple­ concept of an appetite quotient developed mented and the basal diets. Protein-de­ by Carpenter (6), based on the studies of pleted rats fed the basal and the imbal­ Hegsted and Heffenreffer (5), provides a anced diets (groups 7 and 9) maintained practical method for determining whether their liver weight during the experiment the decreased appetite is a primary effect at the higher level that was obtained at of the imbalance or whether it is only a zero time after depletion; on the other consequence of a diminished nutritive hand, the liver weight of animals of group value of the diet. 8, fed the supplemented diet, reached a The appetite quotient values obtained lower and more normal value after the in these experiments were similar for the experimental period than the weight at groups fed the balanced diets (basal or zero time. supplemented) despite the differences in The right gastrocnemius muscle weighed nutritive value, but in the groups fed the more or less the same in all the groups imbalanced diets the appetite quotient val­ when expressed as grams per 100 grams ues were significantly lower (P<0.01), of body weight. indicating that the appetite of these rats The liver and gastrocnemius muscle was subnormal during the experimental composition, expressed in milligrams of period. constituent per total tissue per 100 g of This suggests that the depression in body weight, is also shown in table 5. food consumption showed by imbalanced The liver protein content was slightly animals would not be a consequence of higher in nondepleted rats fed the im­ the lower nutritive value of the imbalanced balanced diets than in animals fed the diet but of some specific physiological ef­ supplemented diets, but no differences fects that affect directly the food intake. were observed in the liver of protein- These results may also explain the ob­ depleted rats fed the experimental diets. servations described in a previous study The gastrocnemius muscle protein was in which rats failed to discriminate be­ essentially similar in all the experimental tween 2 balanced diets, one of higher groups of animals. nutritive value than the other, but always The liver lipid diminished in nonde­ discriminating and refusing an imbal­ pleted rats fed the imbalanced diets anced diet when it was offered simulta­ (groups 3 and 6) and was lower than that neously with a balanced diet. 4 3 0 JUAN C. SANAHUJA, MARÍA E. RIO AND MARÍA N. LEDE

The changes in the plasma amino acid Similar fiver weight increase and its pattern of nondepleted rats resemble those higher glycogen content was also observed previously observed in animals fed imbal­ in protein-depleted rats after 24 hours of anced diets containing fibrin in which the being fed the imbalanced diet C.3 altered amino acid pattern was associated Sidransky and Faber (17) reported that with the depression in food intake (8). the fiver of rats force-fed a threonine- Here too, rats fed the imbalanced diets devoid diet contained high amounts of in which the appetite quotient was subnor­ glycogen; these authors failed to observe mal, showed a severely altered plasma an increased synthesis of fiver lipid start­ amino acid pattern that resulted in very ing from the carbon chains of amino acids high values in the ratios of concentrations not used for protein synthesis. Since the of several indispensable amino acids to fiver lipid content was not increased in threonine, the most limiting amino acid of rats fed the imbalanced diets, the possible the diet. In contrast, in animals fed bal­ utilization of the excess of amino acids anced diets, in which the appetite quotient provided by the imbalanced mixture for values were higher, the plasma pattern of the synthesis of glycogen cannot be dis­ amino acids was not altered. regarded. Similar results were obtained in short­ These high levels of liver glycogen ob­ term experiments; plasma threonine con­ served in rats fed the imbalanced diets centration decreased markedly in rats fed could be associated with the depression in the imbalanced diet C within the first appetite; Anliker and Mayer (18) pointed 24 hours, when the decreased food intake out that in mice a relationship exists be­ became apparent.3 tween feeding behavior and fiver glycogen. This relationship between appetite quo­ This possibility, that would provide an tient and altered plasma amino acid pat­ alternative to the hypothesis that the de­ tern supports the assumption that the pression in food intake may be the conse­ desire for food could be curtailed when quence of the altered plasma amino acid, the ratio of the plasma concentration of appears worthy of further investigation. each indispensable amino acid to that of The fiver protein content showed only the one lacking in the amino acid mixture, little difference between nondepleted or reaches some critical level. This could be protein-depleted rats fed the imbalanced a part of a homeostatic mechanism tend­ or balanced diets. Similar results were ing to prevent the animal from ingesting obtained in short-term studies:4 the fiver of an excessive amount of an imbalanced protein-depleted rats pair-fed the imbal­ diet (8, 15). Krauss and Meyer (16) anced diet C and the supplemented diet B suggested that this regulating mechanism showed an identical increase in their total may be independent of the satiety centers protein content within 24 hours. These of the hypothalamus. observations agree with those of Kumta Amino acid imbalance and liver compo­ and Harper (19) and Spolter and Harper sition. The most interesting of the changes (20) indicating that the direct inhibition observed in liver composition were those of protein synthesis could not be the pri­ related to the content of glycogen, that mary cause for the reduced food intake. reached nearly 10% of the liver weight of After the 2-week experiment, total body nondepleted rats fed the imbalanced diets. protein synthesis was diminished, since This high level of glycogen would account growth rate was depressed; but it is con­ for the abnormally high liver weight in ceivable that this effect is secondary to these animals since it is known that gly­ the decrease in food intake. cogen is stored with nearly twice its water The differences in fiver lipid content weight. could be related to the amounts of threo­ In protein-depleted rats fed the imbal­ nine and lysine ingested during the ex­ anced diets, even though the liver glycogen perimental period since it is known that content was not as high as in nondepleted rats, the values obtained were also signifi­ 3 Sanahuja, J. C., M. N. Lede and M. E. Sambucetti 1964 Appetite quotient and biochemical changes in cantly higher than those for control ani­ rats fed imbalanced diets. Federation Proc., 23: 185 (abstract). mals fed the balanced diets. 4 See footnote 3. APPETITE AND AMINO ACID IMBALANCE 4 3 1

these amino acids could exert a marked 2. Desphande, P. D., A. E. Harper and C. A. lipotropic action (21, 22); the higher re­ Elvehjem 1958 Amino acid imbalance and nitrogen retention. J. Biol. Chem., 230; 335. quirements for the indispensable amino 3. Sanahuja, J. C., and A. E. Harper 1962 acids in nondepleted animals with a rapid Effect of amino acid imbalance on food in­ growth rate (groups 2, 4 and 5) could take and preference. Am. J. Physiol., 202: produce deficiencies of threonine, the most 165. 4. Sanahuja, J. C., and A. E. Harper 1963 limiting amino acid of the diet, or of lysine Effect of amino acid imbalance on self-selec­ that would result in a higher liver fat tion of diet by the rat. J. Nutrition, 81: 363. content. 5. Hegsted, D. M., and V. K. Haffenreffer 1949 Caloric intakes in relation to the quantity In nondepleted rats fed the imbalanced and quality of protein in the diet. Am. J. diets, the amounts of lysine and threonine Physiol., 157: 141. ingested may be excessive for the require­ 6. Carpenter, K. J. 1953 The concept of an ments as a consequence of the slow growth “appetite quotient” for the interpretation of ad libitum feeding experiments. J. Nutri­ rate due to the imbalance; this could ex­ tion, 51: 435. plain the very low liver lipid content ob­ 7. Kumta, U. S., and A. E. Harper 1962 served in groups 3 and 6. Amino acid balance and imbalance. IX. The lipotropic effect of threonine may Effect of amino acid imbalance on blood explain also the differences observed in amino acid pattern. Proc. Soc. Exp. Biol. Med., 110: 512. the liver fat content of protein-depleted 8. Sanahuja, J. C., and A. E. Harper 1963 rats; the high lipid content at zero time, Effect of dietary amino acid pattern on that resulted from the protein-free diet plasma amino acid pattern and food intake. consumed during the protein-depletion Am. J. Physiol., 204: 686. 9. Harper, A. E., P. Leung, A. Yoshida and period was greatly reduced in animals fed Q. R. Rogers 1964 Some new thoughts on the diets supplemented with threonine amino acid imbalance. Federation Proc., (groups 8 and 9). In contrast, group 7, 23: 1089. fed a diet not supplemented with threonine 10. Peraino, C., and A. E. Harper 1961 Quan­ titative paper chromatography of plasma showed a high liver fat content, similar to amino acids. Modification of the dinitro- that of protein-depleted animals (zero phenylation procedure of Levy. Analyt. tim e). Chem., 33; 1863. Some of the results of this study are 11. Marenzi, A., J. Moglia and F. Vilallonga 1945 Estudio comparativo de algunos mé­ similar to those observed in kwashiorkor in todos de valoración de las proteínas del which the decrease of appetite is a char­ suero. I. Proteínas totales. An. Cent. Invest. acteristic sign. Whitehead (23) reported Tis., 9: 134. an altered blood amino acid pattern in 12. Sidransky, H., and T. Baba 1960 Chemical Pathology of acute amino acid deficiencies. children suffering from severe kwashiorkor III. Morphologic and biochemical changes in that is not observed in successfully treated young rats fed valine- or lysine-devoid diets. cases. Stuart et al. (24) and Waterlow J. Nutrition, 70: 436. (25) have also observed that in infants 13. Hawk, P., B. Oser and W. Summerson 1954 Practical Physiological Chemistry. McGraw- with kwashiorkor, liver glycogen can reach Hill Book Company, New York, p. 1071. values of 10 to 12% accounting for about 14. Harper, A. E. 1959 Amino acid balance Dne-half of the fat-free dry weight of the and imbalance. I. Dietary level of protein liver. and amino acid imbalance. J. Nutrition, 68: 405. The similarity of these observations sup­ 15. Munro, H. N., and J. B. Allison 1964 Mam­ ports the assumption that the depression in malian Protein Metabolism. Academic Press, food intake resulting from the consump­ Inc., New York, p. 131. tion of poorly balanced proteins, as in the 16. Krauss, R., and J. Mayer 1963 Effect of excess dietary amino acids and protein on case of kwashiorkor, could be related to food intake in hyperphagic rats. Nature, the imbalanced amino acid pattern of the 200; 1213. diet (26). 17. Sidransky, H., and E. Farber 1958 Chem­ ical pathology of acute amino acid deficien­ LITERATURE CITED cies. II. Biochemical changes in rats fed threonine- or methionine-devoid diets. Arch. 1. Salmon, W. D. 1954 The tryptophan re­ Path., 66: 135. quirement of the rat as affected by niacin 18. Anliker, J., and J. Mayer 1957 The regu­ and level of dietary nitrogen. Arch. Biochem. lation of food intake. Am. J. Clin. Nutrition, Biophys., 51: 30. 5: 148. 4 3 2 JUAN C. SANAHUJA, MARÍA E. RIO AND MARÍA N. LEDE

19. Kumta, U. S., and A. E. Harper 1961 23. Whitehead, R. G. 1963 The imbalance of Effect of dietary additions of amino acids on serum amino acids in children with kwashi­ food intake and blood urea concentration of orkor. Biochem. J., 89: 84P. rats fed low-protein diets containing fibrin. 24. Stuart, K. L., G. Bras, S. J. Patrick and J. C. J. Nutrition, 74: 139. Waterlow 1958 Further clinical and in­ 20. Spolter, P. D., and A. E. Harper 1961 Leu­ vestigative uses of liver biopsy. An analysis cine-isoleucine antagonism in the rat. Am. of five hundred twenty-seven biopsies. Arch. J. Physiol., 200: 513. Intern. Med., 101: 67. 21. Harper, A. E., D. A. Benton, M. E. Winje, 25. Waterlow, J. C. 1964 Diet and Bodily W. J. Monson and C. A. Elvehjem 1954 Composition. Ciba Foundation Study Group. Effect of threonine on fat deposition in the J. Churchill Ltd., London, p. 65. livers of mature rats. J. Biol. Chem., 26. Sanahuja, J. C. 1964 Effect of amino acid 209: 165. imbalance on food intake and food selection. 22. Desphande, P. D., A. E. Harper and C. A. Vitalstoffe Zivilisationskrankheiten J., 42: Elvehjem 1958 Amino acid imbalance on 150. low fibrin diets. J. Biol. Chem., 230: 327. Absorption of Calcium and Phosphorus Along the Gastrointestinal Tract of the Laying Fowl as Influenced by Dietary Calcium and Egg Shell Formation * 1

S. HURWITZ A N D A. BAR The Volcani Institute of Agricultural Research, The National and University Institute of Agriculture, Rehovot, Israel

ABSTRACT The apparent absorption of calcium, phosphorus and total dry matter along the gastrointestinal tract of the laying hen, was followed using yttrium-91 as a non-absorbed tracer. Percentage calcium and phosphorus absorption appeared to be greater in the proximal parts of the intestine than in the distal parts. This differ­ ence was smaller for total dry matter. Percentage calcium absorption was not signifi­ cantly influenced by its dietary level, nor did the latter influence the absorption of dry matter. Percentage absorption of phosphorus was, however, depressed by the higher dietary calcium level. Egg shell deposition was associated with increased calcium and to a smaller degree, phosphorus absorption. It did not influence dry matter ab­ sorption. A heavy endogenous phosphorus excretion was observed in the duodenum.

The identification of the sites of mineral available carbohydrate absorption along absorption is important for understanding the tract of chickens. Marcus and Lenge- the mechanism involved in the mineral mann (3) reported that 91Y was not ab­ absorption. This problem was studied sorbed in the rat. mainly by in vitro and in situ experimenta­ Preliminary trials in this laboratory tion. The in vitro studies such as those of showed that this isotope was not absorbed Schachter and associates (1, 2) provide by colostomized chickens. Following an information on the capacity of various oral dose of 91Y mixed in some feed, 93% intestinal segments to absorb calcium. of the dose was detected in the feces voided However, the rate and percentage of cal­ during the first 24-hour period, and 3% in cium absorption may also depend on the the following 24-hour period. No activity rate of passage of food, which is quite could be detected in the urine of these variable in the various intestinal segments animals. (3, 4). Therefore, in vitro methods can Therefore, 91Y was used in the present not measure the relative contribution of trial to follow the absorption of calcium each segment to total calcium absorption and phosphorus along the intestinal tract in vivo. A different method for studying of laying hens. The possibility that dietary calcium absorption is the use of oral doses calcium and the presence of a calcifying of 45Ca or its injection into different parts shell in the uterus may modify the pattern of the intestine (5, 6). This method, how­ of calcium and phosphorus absorption was ever, measures mainly the outflux of cal­ also investigated. cium from the intestine and not the net absorption which is the difference between EXPERIMENTAL the outflux and the influx. The experimental diets are shown in Chandler and Cragle (7) used ‘"“Ce as table 1. These diets were all-vegetable, of a non-absorbed tracer to study calcium and the practical type, and were prepared as phosphorus absorption in calves. The ratio follows: the calcium carbonate and the of calcium and phosphorus to 144Ce at any dicalcium phosphate portions of each ra-

point in the intestine measures the cumu­ Received for publication March 3, 1965. lative percentage absorption up to this i Contribution from the National and University Institute of Agriculture, Rehovot, Israel, 1965, Series point. Similarly, Bolton (8) used cellulose no. 840-E. Supported by a grant-in-aid from the U.S. as a tracer for estimation of protein and Department of Agriculture, project no. A10-AH-20.

J. N u t r it io n , 86: ’65 433 434 S. HURWITZ AND A. BAR

TA B LE 1 oviposition. The time of killing ranged Composition of the experimental diets between 7 and 11:30 a .m . Since the egg

D iet 1 D iet 2 is laid shortly after the end of shell calci­ fication, results obtained with this group % % Yellow corn 3 0 .0 0 3 0 .0 0 represent absorption during the period of Soybean oil meal (45% protein) 2 4 .0 0 2 4 .0 0 late calcification, (c) Five birds of each Soybean oil, refined 4 .0 0 4 .0 0 lot were killed between 9 and 11 a .m . Vitamin m ixture 1 0 .2 5 0 .2 5 Those birds had not laid any eggs during M ineral mixture 2 0 .3 0 0 .3 0 DL-Methionine 0 .1 0 0 .1 0 the morning, but uncalcified egg was found Alfalfa meal, dehydrated 2 .0 0 2 .0 0 either in the magnum or in the isthmus Calcium carbonate 3 .5 0 7 .5 0 at the time of killing. Results from this Dicalcium phosphate 1 .7 0 1 .7 0 M ilo 3 4 .1 5 3 0 .1 5 group indicate the absorption of calcium and phosphorus when no shell is formed. Calcium content, assayed, % 1 .9 0 3 .5 6 The gastrointestinal tract of each hen Phosphorus content, assayed, % 0 .6 3 0 .6 3 (excluding the esophagus and crop) was 1 Commercial premix, supplied per kg of diet : vita­ removed and separated into gizzard, duo­ min A, 10,000 IU; vitamin D 3 , 1400 ICU; riboflavin, 3 mg; Ca pantothenate, 3 mg; niacin, 10 mg; choline denum, upper jejunum, lower jejunum, chloride, 400 mg; vitamin B 12, 8 ¿ig; b u ty la te d h ydroxy- toluene, 125 mg. upper ileum, lower ileum and colon. The 2 Feed grade salt supplied in mg per kg of diet: Mn, 60; zinc, 50; iodine, 1.2; cobalt, 0.2; iron, 25; cecums were not taken for analysis. copper, 2. Meckel’s diverticulum was arbitrarily taken as the point of demarcation between the tion were mixed well. A solution contain­ jejunum and ileum, which were each di­ ing 91Y was added to the mixture. After vided into 2 parts of equal length. The drying in an oven, the latter was passed contents of each segment were emptied 3 times through a thin-mesh wire screen. into a crucible, oven-dried and ashed at Count rates of samples were taken to as­ 700°. The ash was then dissolved in 2 n sure proper mixing. The Ca-91Y mixture hydrochloric acid. For 91Y assay, an aliquot was then added to the other pre-mixed die­ was dried on a glass planchet and counted tary ingredients, all ground in a hammer in a gas-flow detector. Within the range of mill. This was followed by thorough mix­ the weights of samples measured, there ing. Concentration of 91Y was approxi­ was no variation due to self absorption. mately 25 uc/kg and 55 uc/kg in diets 1 Calcium was determined by EDTA titra­ and 2, which contained 1.90% (low Ca) tion as described previously (9) except and 3.56% (high Ca) calcium, respec­ that murexide was replaced by hydroxy- tively. naphthol blue.2 Phosphorus was deter­ Forty White Leghorn laying hens, 16 mined by the method of Gomori (10). months old, were selected for the experi­ ment on the basis of previous egg produc­ Results are expressed as ratios of cal­ tion records. For a 1-week preliminary cium, phosphorus or dry matter to counts period they were fed a control diet identi­ per minute (cpm) X lO-4 of 91Y observed cal to diet 2, but containing no radioactive in the respective segment. This ratio de­ supplement. At the end of this period they creases with progressive absorption of the were divided into 2 lots receiving the re­ test nutrient. The cumulative percentage spective labeled low calcium and high cal­ absorption in any segment is given by cium diets. After 3 days of feeding the ex­ 1 0 0 (\ nutrient/91Y in intestine \ perimental diets, the birds were killed by ^ \ nutrient/91Y in feed / dislocation of the neck in the following Because of the differences in the 91Y con­ order, (a) Four birds of each lot were killed tent of the 2 diets, the appropriate correc­ at 8 p .m . after the presence of a shelled tions were made to bring the results to the egg in their uteri had been established. At same scale. this time calcification had already pro­ Analyses of variance of calcium, phos­ ceeded for 3 to 5 hours. Results from this phorus and dry matter to 91Y ratios in each group represent the absorption during the intestinal segment were run according to period of early calcification, (b) Four birds Dixon and Massey (11). Pooled standard of each lot were killed immediately after 2 Mallinkrodt. CALCIUM AND PHOSPHORUS ABSORPTION 435 errors (n = 4) were calculated from the error term of the analysis of variance.

RESULTS Since the trial was designed as a fac­ torial of 2 calcium levels and 3 physiologi­ cal states with respect to egg shell forma­ tion, the results are grouped according to the factorial variables. With only one exception, there was no significant effect of any of the experimen­ tal variables on the dry-matter contents of the various intestinal segments. These were in milligrams as follows (average — s e ) : gizzard, 3018 ± 254; duodenum, 671 ± 151; upper jejunum, 785 ± 85; lower jejunum, 1266 ± 333; upper ileum, 1075 ± 279; lower ileum, 845 ± 169; and colon, 268 ± 74. The calcium, phosphorus and dry matter to 91Y ratios in the various intestinal seg­ ments are shown in figures 1,2, and 3 re­ spectively. The probability levels for the effect of each factorial variable in any intestinal segment are shown in table 2. Following an increase from the gizzard to Fig. 2 P /9IY ratio in gastrointestinal segments the duodenum, there was a marked decline of laying hens fed 91Y-labeled diets. Upper dia­ gram, comparison between 1.90% calcium (# ) and 3.56% calcium diets (O ). Lower diagram, comparison among periods of early egg shell calcification (A ), late shell calcification (A ) and of no shell formation (□ ). Standard errors are as follows: gizzard, 0.40; duodenum, 6.14; upper jejunum , 2.67; lower jejunum , 1.03; upper ileum, 1.27; lower ileum, 0.41; colon, 0.32.

in the Ca/91Y ratio down to the lower jejunum with a smaller change in the following segments. The average cumulative percentage ab­ sorption of calcium was 50.3% at the lower jejunum and 53.2% at the colon. The Ca/91Y ratio in the duodenum was lower than that of feed, suggesting considerable absorption of calcium in this segment. In the high calcium hens with no shell pres­ ent, the upper ileum showed an increase in the ratio, indicating net endogenous ex­ cretion of calcium. There was no signifi­ Fig. 1 Ca/91Y ratio in gastrointestinal seg­ cant effect of dietary calcium level on the m ents of laying hens fed 91Y-labeled diets. Upper percentage absorption of this mineral. diagram, comparison between 1.90% calcium (• ) and 3.56% calcium (O ) diets. Lower diagram, Hence, the total amount of calcium ab­ comparison among periods of early egg shell cal­ sorbed was higher in the high calcium cification (A ), late shell calcification (A ) and birds. Percentage calcium absorption was with no shell formation (□ ). Standard errors significantly greater during the 2 periods were as follows: gizzard, 0.90; duodenum, 1.24; upper jejunum , 0.85; lower jejunum , 1.09; upper of shell calcification than when no forming ileum, 1.16; lower ileum, 0.81; colon, 1.09. shell was present. However, there was no 436 S. HURWITZ AND A. BAR

significant difference in calcium absorp­ tion between periods of early and late calcification. The former difference was observed along the entire intestine but did not reach significance in the duodenum. The calculated cumulative percentage of calcium absorption in the colon was 67 and 39%, during periods of calcification and no calcification, respectively. Changes in the P /91Y ratios (fig. 2) were similar to those observed for calcium, indi­ cating that the main sites of phosphorus absorption are at the anterior intestine, i.e., jejunum. The P /91Y ratio in the duo­ denum is much higher than that of feed, suggesting a large endogenous phosphorus excretion in this segment. Unlike calcium, the relative absorption of phosphorus was influenced by dietary calcium level; the high calcium level increased the P/81Y ratio in the 3 lower portions of the intes­ tine. During the 2 periods of egg shell calcification, there was a significant in­ crease in phosphorus absorption in the lower portions of the intestine, although the same tendency was observed in the upper segments as well. There was also a significant interaction between dietary cal­ cium and shell calcification on phosphorus Fig. 3 Total dry m atter/91Y ratio in gastro­ cumulative absorption in the colon. intestinal segments of laying hens fed 9IY-labeled Dry m atter/91Y ratios in the various in­ diets. Upper diagram, comparison between 1.90% (•) and 3.56% (O) calcium diets. Lower dia­ testinal segments are shown in figure 3. gram, comparison among periods of early egg The ratio was higher in the gizzard and shell calcification (A ), late shell calcification duodenum than in feed. It then decreased (A ), and of no shell formation (□ ). Standard at a progressively lower rate along the errors were as follows: gizzard, 479; duodenum, 370; upper jejunum, 278; lower jejunum, 84; rest of the intestine. The calculated cumu­ upper ileum, 62; lower ileum, 26; colon, 18. lative dry-matter absorption in the colon

TABLE 2 Levels of probability for the effects of the several factorial components for the various feed ingredients and intestinal segments 1

F a c to ria l Dietary calcium Diurnal variation v a ria b le ( shell form ation ) Interaction

P a ra m e te r C a/Y P/Y D m /Y 2 C a/Y P/Y D m /Y C a/Y P/Y D m /Y

G iz z a rd n s n s n s n s n s 0 .0 1 0 .0 1 n s n s D u o d e n u m n s n s n s n s n s n s n s n s n s Upper jejunum n s n s n s 0 .0 1 n s n s n s n s n s Lower jejunum n s n s n s 0 .0 1 n s n s n s n s n s Upper ileum n s 0 .0 1 0 .0 5 0 .0 1 0 .0 1 0 .0 5 n s 0 .0 5 n s Lower ileum n s n s n s 0 .0 1 0 .0 5 n s n s n s n s C o lo n n s 0 .0 1 n s 0 .0 1 0 .0 1 0 .0 1 n s 0 .0 1 n s

1 Probability levels calculated on the basis of 21 analyses of variance, each for one feed ingredient and intestinal segment. 2 Dm = dry matter. CALCIUM AND PHOSPHORUS ABSORPTION 437 was about 73%, on the average. Although phosphorus and yttrium are assumed to there is some tendency for dry matter to be move at an equal rate along the small better absorbed by the low calcium birds, intestine. Preliminary results in this labo­ this effect is only significant in one seg­ ratory appear to support this assumption.4 ment. There was also some, and at times Therefore, nutrient/91Y ratios appear to be significant, diurnal variation in dry mat- suitable for calculation of cumulative ab­ ter/91Y ratio. However, this variation ap­ sorption along the intestine. However, more pears small and inconsistent. evidence is necessary to clarify this point. Nevertheless, the comparison of the pat­ DISCUSSION terns of calcium and phosphorus absorp­ On the basis of the known functions of tion as influenced by dietary and diurnal the gizzard it appears unlikely that large variations, is apparently valid since it ap­ amounts of calcium and phosphorus could pears unlikely that the movement of 91Y be absorbed in this organ, as appears to be could be influenced either by dietary cal­ suggested by the lower Ca and P /91Y ratios cium or egg shell deposition. In this re­ compared with those of the feed. It also spect it should be recalled that the late- appears unlikely that very large quantities calcification hens and the no-calcification of dry matter are secreted in this organ hens were killed at the same time of the as suggested by the very high dry mat- day. ter/91Y ratio. The examination of the giz­ Those factors with respect to the rate zard contents revealed the presence of of passage of the test nutrient relative to relatively large feed particles, mostly the tracer, demonstrate clearly the difficul­ grains, with few smaller ones. Apparently, ties in the use of such tracer. therefore, the gizzard retains the larger The pattern of dry m atter/9IY indicates feed particles longer than the smaller absorption of dry matter along the entire ones and the solutes. Calcium and phos­ intestine with a somewhat reduced rate at phorus which are mainly in solution in the posterior segments. The high dry- the gizzard (12) might be emptied more matter/91Y ratio in the duodenum is prob­ rapidly than the larger grain particles. On ably due to secretion of digestive sub­ the other hand, 91Y tends to be partially stances in this organ. The pattern in adsorbed on solid particles (3) even at a general is similar to that observed by low pH such as that in the gizzard. This Bolton (8) in chicks, for the absorption of isotope will therefore be slower than cal­ protein and available carbohydrate. cium and phosphorus to leave the gizzard, Assuming the same passage time for but more rapid than the solids. This inter­ calcium, phosphorus and yttrium along the pretation is also supported by a prelimi­ intestine, the present results appear to in­ nary study in this laboratory with a single dicate that the major portion of calcium dose of 45Ca and 91Y and may explain the and phosphorus absorption is located at low Ca and P /91Y ratios and the high dry the anterior parts of the intestine. It can matter/91Y ratio in the gizzard.3 In the be easily shown that calcium concentration steady state of feed passage from the giz­ in the total dry matter is actually increas­ zard it may be assumed that the latter ing along the intestine and therefore could passes the various feed components at not be a factor in the reduced absorption constant rate. This assumption is sup­ in the posterior parts. The latter may be ported by the dry-matter/91Y ratios which due either to the greater capacity of the show only slight variations that appear anterior segments to absorb calcium (1,2, to be more of diurnal nature rather than 5, 14, 15) or to the reduced solubility of related to shell deposition. There was calcium due to increase in pH with the dis­ also no significant difference in the nutri­ tance from the gizzard (12, 16). ent,/9^ ratios in the duodenum, suggesting Dietary calcium, at the levels used, did uniform emptying of yttrium, calcium, not influence the relative absorption of cal­ phosphorus and total solids from the cium, in agreement with previous balance gizzard. studies (9). The absence of a calcifying In accordance with the observations of 3 Unpublished data, S. Hurwitz and A. Bar, 1965. Marcus and Lengemann (13), calcium, 4 See footnote 3. 438 S. HURWITZ AND A. BAR shell was associated with a reduced rate 3. Marcus, C. S., and F. W. Lengemann 1962 of calcium absorption. This effect was Use of radioyttrium to study food movement in the small intestine of the rat. J. Nutrition, identical for the high calcium and low 7 6 : 179. calcium lots. The mechanism responsible 4. Cramer, C. F., and D. H. Copp 1959 Prog­ for this variation is not clear, although it ress and rate of absorption of radiostrontium may be explained in terms of the greater through intestinal tracts of rats. Proc. Soc. Exp. Biol. Med., 102: 514. need for calcium during periods of egg 5. Lengemann, F. W., R. H. Wasserman and shell formation. It is accepted that absorp­ C. L. Comar 1959 Studies on the enhance­ tion alone cannot supply all the calcium ment of radiocalcium and radiostrontium needed during egg shell formation and that absorption by lactose in the rat. J. Nutrition, 6 8 : 4 4 3 . bone reserves have to be utilized (17), 6. Lengemann, F. W., and C. L. Comar 1961 even when birds are fed sufficient calcium. Distribution of absorbed strontium-85 and This conclusion is based on daily averages calcium-45 as influenced by lactose. Am. J. of calcium retention not considering any Physiol., 200: 1051. 7. Chandler, P. T., and R. G. Cragle 1962 diurnal variations in calcium absorption. Gastrointestinal sites of absorption and en­ The greater calcium absorption during dogenous secretion of calcium and phos­ shell deposition, observed in this study, phorus in dairy calves. Proc. Soc. Exp. Biol. could mean that the importance of bone M e d ., Ill: 4 3 1 . 8. Bolton, W. 1961 The absorption of food as a calcium reserve in birds fed sufficient from the gut of the fowl. Proc. Nutrition calcium, is not as great as previously be­ Soc., 20: xxvi. lieved. 9. Hurwitz, S., and P. Griminger 1960 Ob­ The absorption of phosphorus appears servations on the calcium balance of laying to be closely related to that of calcium, and hens. J. Agr. Sci., 54: 3 7 3 . 10. Gomori, G. 1942 A modification of the is probably influenced by the latter. The colorimetric phosphorus determination for depressing effect of high levels of dietary use with photometric colorimeter. J. Lab. calcium on phosphorus absorption is well Clin. Med., 27: 955. known (18). The significant increase in 11. Dixon, W. J., and F. J. Massey, Jr. 1957 Introduction of Statistical Analysis. McGraw absorption of phosphorus during shell for­ Hill Book Company, New York, p. 139. mation is not as striking as with calcium 12. Tyler, C. L. 1946 Studies on the absorp­ absorption, and appears to be secondary tion and excretion of certain minerals by to the latter. poultry. II. J. Agr. Sci., 35: 275. 13. Marcus, C. S., and F. W. Lengemann 1962 From the P /B1Y ratios in the duodenum, Absorption of Ca45 and Sr85 from solid and it is apparent that large quantities of en­ liquid food at various levels of the alimen­ dogenous phosphorus are emptied into this tary tract of the rat. J. Nutrition, 77: 155. segment. It may be calculated that the 14. W illiams, G. A., E. N. Bowser, W. J. Hender­ son and V. Uzgiries 1961 Effects of vita­ ratio of endogenous to dietary phosphorus min D and cortisone on intestinal absorption in this segment is at least 4:1. This ob­ of calcium in the rat. Proc. Soc. Exp. Biol. servation is consistent with the heavy 32P M e d ., 1 0 6 : 6 6 4 . secretion into the duodenum, observed in 15. Harrison, H. E., and H. C. Harrison 1960 the laying hen by Shirley et al. (19). Transfer of Ca45 across intestinal wall in vitro in relation to action of vitamin D and cortisol. Am. J. Physiol., 1 9 9 : 2 6 5 . ACKNOWLEDGMENT 16 Coates, M. E., and E. S. Holdsworth 1961 The authors wish to acknowledge the Vitamin D3 and absorption of calcium in the technical assistance of Mrs. M. Cotter. chick. Brit. J. Nutrition, 15: 131. 17. Simkiss, K. 1961 Calcium metabolism and avian reproduction. Biol. Rev., 36: 321. LITERATURE CITED 18. deLange, D. J. 1961 Some factors which 1. Schachter, D., and S. M. Rosen 1959 influence the absorption and retention of Active transport of Ca45 by the sm all intestine calcium and phosphorus in rats on a high and its dependence on vitamin D. Am. J. cereal diet. Proc. Nutrition Soc. S. Africa, 2: P h y s io l., 1 9 6 : 3 5 7 . 76. 2. Kimberg, D. V., D. Schachter and H. Schenker 19. Shirley, R. L., J. C. Driggers, J. T. McCall and 1961 Active transport of calcium by intes­ G. K. Davis 1952 Excretion of P32 and Ca45 tine: effects of dietary calcium. Am. J. into the various alim entary segments of hens. Physiol., 200: 1256. Poultry Sci., 3 1 : 3 1 6 . Proceedings of the Twenty-ninth Annual Meeting of the American Institute of Nutrition

SHELBURNE HOTEL, ATLANTIC CITY, NEW JERSEY APRIL 9-14, 1965

COUNCIL MEETINGS The following were elected: The Council of the American Institute of P r e s id e n t: O. L. Kline Nutrition met Thursday evening, April 8, President-elect: A. E. Schaefer Secretary-elect: W. N. Pearson and Friday morning and evening, April 9. T r e a s u r e r : W. A. Krehl The actions of the Council were presented C o u n c ilo r : E. L. R. Stokstad at the Institute business meetings and are Nominating Committee: included in the report of those meetings. G. H. Beaton, Chairman R. S. Harris SCIENTIFIC SESSIONS H. Linkswiler G. M a n n A total of 306 abstracts of papers was R. J. Young submitted to the Institute; 9 of them were transferred to other societies; 50 were ac­ III. Constitutional Amendments cepted from other societies, making a total By over two-thirds of all votes cast, the of 347 papers programed by the Institute. following amendments to and changes in These were arranged into 29 regular and the constitution and bylaws were adopted: 3 intersociety (atherosclerosis) sessions. A. By vote of 588 for to 8 against, the In addition, two informal conferences were following addition was made to Article I: held, Poultry Nutrition and Ruminant Section 7. Emeritus Members. A m e m ­ Nutrition, and the following half-day sym­ ber in good standing for 25 years or posia were presented: upon reaching the age of 65 may apply 1. Biological Availability of Different Forms to the Secretary for Emeritus Member of Vitamin A and E status. Members who wish Emeritus 2. Adolescent Obesity status because of prolonged disability from work may also apply. Emeritus 3. Amino Acid Release, Transport and Members are exempt from dues but re­ M e ta b o lis m tain all other privileges of membership. 4. Nutritional Significance of the Non- They may subscribe to Federation and/ Nutrient Components of Food or Institute publications at m em ber rates. 5. Nutrition in Relation to Certain Aspects of Lipid Metabolism B. By vote of 555 for to 36 against, the following addition w'as made to Article I: BUSINESS MEETINGS Section 8. Supporting Members. M e m ­ Business meetings were held on Satur­ bers who voluntarily contribute $25 or day, April 10 and Tuesday, April 13. Dr. more annually over and above regular assessment in support of the general R. W. Engel presided at both meetings. activities of the Institute shall become I. Minutes of 1964 meeting Supporting Members. The minutes as published in the Journal C. By vote of 542 for to 47 against, the following addition was made to Article I: of Nutrition, 83: 385, 1964, were approved. Section 9. Sustaining Associates. A n y II. Elections association, corporation, institution or individual desiring to support the Society The 609 ballots were counted by Drs. may be invited by the President to be­ Dorothy Arata and Lewis R. Arrington. come a Sustaining Associate.

J. N u t r it io n , 8 6 : ’65 439 440 AMERICAN INSTITUTE OF NUTRITION

D. By vote of 580 for to 15 against, President, President-Elect, Past-President, Article III, Section 2, has been changed to Secretary, Treasurer and three additional m e m b e rs . read as follows: S e c tio n 2. C o u n c il. The President, Past- IV. Membership Status President, President-Elect, Secretary, Treasurer, Secretary-Treasurer of the As of April 1, 1965, there were 999 Clinical Division, and three additional members of the Institute: 891 active, 92 councilors, one of whom shall be elected retired and 16 honorary. There was an annually to serve a term of three years, increase of 61 members over last year. shall constitute a nine-member Board of Trustees and shall be known as the The Clinical Division had a total of 162 Council. The President-Elect shall be members. considered as a Vice-President and serve Notice of the deaths of these members in the absence of the President. was received this year: E. By vote of 566 for to 19 against, W illiam E. Abbott, December 27, 1963 Article IV, Section 1, has been changed to James B. Allison, September 25, 1964 Hazel M. Hauck, April 23, 1964 read as follows: Martha Koehne (Charter Member), Section 1. Powers. The general man­ August 4, 1961 agement of the Society during the inter­ Henry W. Loy, December 1, 1964 vals between meetings shall be vested in Roe E. Remington, May 15, 1964 the Council, which shall regularly per­ V. P. Sydenstricker, December 12, 1964 form the ordinary duties of an executive Martha F. Trulson, January 3, 1965 committee and possess all the powers Max W achstein, January 15, 1965 conferred upon the Board of Trustees of an educational institution chartered by The following resolution in honor of the Education Department of the Uni­ versity of the State of New York. A Dr. Allison was read: permanent charter was issued to the RESOLVED: That the American Insti­ American Institute of Nutrition under tute of Nutrition, assembled at Atlantic date of November 16, 1934. The Council City, New Jersey, at its annual meeting, may appoint and compensate an Execu­ April 10, 1965, wishing to express its tive Secretary who shall assist in carry­ deep regret and sorrow at the passing of ing on the functions of the Society. one of its most distinguished members, James Boyd Allison, place this statem ent F. By vote of 586 for to 6 against, in its minutes for permanent record. Article VI, Section 1, has been changed Dr. Allison served as President of the to read as follows: New York Academy of Sciences, he was a member of the editorial board of the Section 1. Dues. The dues shall be an Journal of Nutrition, Treasurer of the annual assessment which shall be deter­ mined by majority vote at the annual American Institute of Nutrition, and President-Elect of our society for this meetings, upon recommendation of the year. He served on important commit­ Council, plus the annual cost of a mem­ tees of the World Health Organization, ber subscription to one journal, as fol­ the Food and Agriculture Organization, lows: A. Division members, when re­ the American Medical Association, and quired to subscribe to the official journal other national and international organi­ of their division, shall not be obligated to subscribe to the official journal z a tio n s . He carried out research in m any fields, of the American Institute of Nutrition. B. All other members shall subscribe to including marine biology, kidney func­ tion, gravity shock, and cancer. How­ the official journal of the Institute. Dues shall be paid by September 1. All mem­ ever, his most important contributions bers have the privilege of subscribing to were in the field of protein nutrition and any official journal of the Institute or metabolism, in which he was recognized its division at members’ rates. as an international authority. He was also teacher, consultant, and adm inistra­ G. By vote of 572 for to 7 against, tor and at all he excelled. Paragraph 3 of the Constitution (below) To everyone, everywhere, he was is now deleted since it duplicates Article known as Jim Allison, for he was every­ one’s friend. He was patient and con­ III, Section 2 (see D above): siderate in his dealings with others and Paragraph 3. The management of the modest about his own accomplishments. American Institute of Nutrition shall be His death is a great loss to our Society vested in a council consisting of the and to his countless friends. PROCEEDINGS 441

The following resolution in honor of HONORARY MEMBERS Dr. Koehne was read: The following scientists were elected to RESOLVED: That the American Insti­ Honorary membership: tute of Nutrition, assembled at Atlantic City, New Jersey, at its annual meeting, Joachim Kiihnau April 10, 1965, place in its minutes for Professor of Medicine perm anent record this statem ent of deep University of Hamburg, Germany regret and sorrow at the passing of one Conrado R. Pascual of its Charter Members, M artha Koehne. Research Director Dr. M artha Koehne was one of the dis­ Food and Nutrition Research Center tinguished women who received their The Philippines doctorates under Professor Lafayette B. Mendel at Yale. Early in her career she VI. Office of the Executive was a member of the staff of Professor Secretariat E. V. McCollum at the School of Hygiene and Public Health at Johns Hopkins Uni­ In accord with the wishes of the society, versity. She contributed much to the re­ as voted at the Annual Meetings in April, search of nutrition in relation to dental 1963 and 1964, the Institute has estab­ health when at the University of Michi­ gan. She was teacher and administrator lished a full-time office of the Secretariat at at the University of Washington, the Beaumont House, 9650 Wisconsin Avenue, University of Tennessee and for more Bethesda, Maryland. Dr. James Waddell, than 15 years, chief nutritionist with the formerly with E. I. duPont de Nemours and Ohio Department of Health. While at Ohio, Dr. Koehne played a major role in Company, was appointed Executive Secre­ promoting the passage of legislation for tary on a part-time basis. the enrichm ent of flour. The activities of the office will be in conformity with the broad objectives of V. New Members such an office as adopted by the society The Membership Committee considered and as published in the Journal of Nutri­ the qualifications of 77 nominees. The tion, 83: 385, 1964. following 50 nominees recommended by The office, with the advice and assist­ the Committee were approved for mem­ ance of the Committee on Nutrition Re­ bership at the business meeting: search and Training, Food and Nutrition Board, National Academy of Sciences, Na­ NEW MEMBERS — 1965 * tional Research Council (NAS-NRC), and Lilia Aftergood Montague Lane (C) Thomas A. Anderson Hugh B. Lofland, Jr. with the concurrence of the Council, has Lloyd W. Beck Mary E. Lojkin submitted a proposal to the National In­ W alter D. Block Paul Baker Me Cay David F. Brown (C) D. S. M cL aren (C ) stitutes of Health (NIH) for the support C. E. Butterworth, Jr. (C) W alter Mertz of a project entitled “National Program to John Edward Canham (C) James K. Miller W illiam W alter Carlton Leonard V. Packett Promote Nutrition in Health.” The objec­ Yet-Oy Chang Albert M. Pearson Herbert L. Chapman, Jr. Karoly G. Pinter (C) tives are to define better the scope of nutri­ Albert J. Clawson Clifford Julius Pollard tion research, to identify those areas that W illiam J. Culley Raymond Reiser Indrajit D. Desai George H. Reussner are being neglected, to emphasize the need W illiam N. Garrett Daniel Rudman (C) J. A. Goyco-Daubon Irving I. Rusoff for increasing numbers of adequately Barbara E. Gunning Herta Spencer trained personnel, to evaluate the current James G. Hamilton P. R. Sundaresan Robert W. Harkins H o w ard S. T eag u e status of the science of nutrition and its James Raymond Howes André G. van Veen Jin Soon Ju Fernando E. Viteri (C) accomplishments under its present support Kung-Ying Tang Kao Marjorie Grant W hiting structures, and to review and identify cur­ Fred Kern, Jr. C. S. W ilson D. Wei Cheng King Herbert G. W indmueller rent training programs and centers. F. A. Klip stein (C ) P e te r D. S. W ood Lennart Krook Paul Lee W right The application has been scheduled for NIH review in June 1965. (C) Clinical Division. # * For institutional affiliations and addresses of new members, see the Federation Directory of Members VII. Treasurer’s Report to be published in the Fall of 1965. Dr. Douglas V. Frost presented the Drs. Leonard A. Luhby, Irvin C. Plough, Treasurer’s report which was approved. and Joseph J. Vitale, already members of The Auditing Committee, Drs. Joseph H. the American Institute of Nutrition, were Roe and Callie Mae Coons, reported that elected to membership in the American they had examined the books and records Society for Clinical Nutrition. of the Treasurer and found them in order. 442 AMERICAN INSTITUTE OF NUTRITION

TREASURER’S REPORT

April 3, 1964 to April 1, 1965

B a la n c e i n c h e c k in g a c c o u n t, A p ril 3 , 1 9 6 4 ...... $ 9,321.99 C itiz e n s N a t i o n a l B a n k o f W a u k e g a n ...... 6 ,1 9 4 .0 2 F i r s t W e s te r n S a v in g s a n d L o a n ...... 1 0 ,0 4 0 .5 1 U . S. S e rie s K B o n d ...... 5 0 0 .0 0

$ 2 6 ,0 5 6 .5 2

9 ,3 2 1 .9 9

BEGINNING BALANCE IN CHECKING ACCOUNT RECEIPTS: A IN d u e s , 8 9 2 m e m b e r s ...... $ 4 ,4 6 0 .0 0 Federation rebate from 1964 annual meeting ...... 4 ,3 3 5 .2 6 W istar Institute for Editorial Office, J. Nutrition, 4 quarters at $2,425 . .. 9,700.00 W is t a r I n s t i t u t e , a d d itio n a l n e t s u b s c r ip ti o n s , 171 a t 1 1 .2 5 ...... 1 ,9 2 3 .7 5 Page Charge collections, J. Nutrition ...... 7 ,6 7 0 .2 6 U . S. S e rie s K B o n d I n t e r e s t ...... 1 3 .8 0 C itiz e n s N a tio n a l B a n k , I n t e r e s t ...... 2 4 7 .7 6 [Nutrition Foundation Support for Award Winner Reception -j Borden 419.10 [Mead Johnson D u e s b a c k p a y m e n ts ...... 3 9 .5 0 Canadian-U. S. Nutrition Conference (repayment for advance) 300.00

$ 2 9 ,1 0 9 .4 3 O v e r p a y m e n t f r o m F A S E B f o r A IN d u e s ...... 3 6 5 .0 0

TOTAL RECEIPTS 2 9 ,4 7 4 .4 3

S u b -to ta l 3 8 .7 9 6 .4 2

DISBURSEMENTS: W istar Institute, subscriptions to J. Nutrition for 1963-64 ...... 2 ,1 9 0 .0 0 Barnes, R. H., for J. Nutrition Editorial Office 12,600.00 S e c r e ta r y ’s O ffice e x p e n s e ...... 1 ,1 5 7 .8 8 O ffice o f E x e c u tiv e S e c r e ta r y ...... 4 ,0 0 0 .0 0 F A S E B — A d v a n c e f o r m a n a g e m e n t s e r v i c e s ...... 2 0 0 .0 0 M a n a g e m e n t s e rv ic e s , c o lle c tio n 1 . 7 5 / m e m b e r ...... 1 ,5 6 1 .0 0 R e t u r n o n o v e r p a y m e n t ...... 3 6 5 .0 0 Coffee Lounge, Annual Meeting ...... 1 8 6 .6 8 N a tio n a l D a iry C o u n c i l ...... 7 0 .4 6 A w a r d W in n e r R e c e p tio n ...... 4 1 9 .1 0 Canadian-U. S. Nutrition Conference (advance) ...... 3 0 0 .0 0 P r i n t i n g C o sts ...... 2 4 8 .7 8 M is c e lla n e o u s ...... 4 8 .3 7 Deposited (Allstate Savings and L o an ) ...... 1 0 ,0 0 0 .0 0

TOTAL DISBURSED 3 3 ,3 4 7 .2 7

Balance on hand in checking account, April 1, 1965 $ 5 ,4 4 9 .1 5

TOTAL ASSETS: Bank Balance (Checking account) — April 1, 1965 5 ,4 4 9 .1 5 F ir s t W e s te r n S a v in g s a n d L o a n ...... 1 0 ,5 1 7 .4 0 Allstate Savings and L o an ...... 1 0 ,2 8 5 .6 1 Citizens National Bank of W aukegan ...... 6 ,1 9 4 .0 2 U . S. S e rie s K B o n d ...... 5 0 0 .0 0

New April 1, 1965 TOTAL ASSETS BALANCE $32,946.18 PROCEEDINGS 443

VIII. Dues IX. Editor’s Report — President Engel reported on the recom­ Journal of Nutrition mendation of the Council that the AIN The editor of the Journal of Nutrition, annual dues be increased $2.00. He dis­ Dr. Richard H. Barnes, submitted his report cussed the needs for an increase in dues for the calendar year 1964. Special atten­ because of the desire of the Council that tion was given to the reasons listed by the the income from dues defray at least one- Editorial Board for rejecting manuscripts half of the cost of the recently established submitted in 1962 and 1963. The rate of AIN Secretariat. acceptance and rejection of manuscripts The motion was made, seconded and for that period was also reviewed. The unanimously passed by the membership to report and proposed budget were approved increase the dues by $2.00. and are summarized below :

Editing and Publication Operations

Calendar year 1 9 6 2 1 9 6 3 1 9 6 4 Volumes published 7 6 , 7 7 ,7 8 7 9 , 8 0 ,8 1 8 2 ,8 3 , 84 Pages published (including papers, biographies, announcements, and proceedings) 1 4 4 7 1 4 3 6 1 3 3 9 (Scientific papers only) ( 1 4 0 3 ) ( 1 3 8 3 ) ( 1 2 6 7 ) Papers published (including 3 biographies) 2 0 9 2 0 4 191 Papers submitted 3 2 1 3 1 2 2 9 3 Papers rejected 85 113 1 12 Rejection rate (based on no. papers submitted) 2 6 % 3 6 % 3 8 % Supplements published 1 Letters (to the editor) l

Operating Schedule Avg time from date of receipt of manuscript to mailing to Wistar: Avg no. days with reviewer 2 0 2 1 .2 2 0 .7 Avg no. days out for revision 1 7 .9 2 4 .8 2 2 .6 Avg no. days in office, in mail or in unavoidable delay 3 2 .6 3 4 .9 2 9 .2

Avg total days 7 0 .5 8 0 .9 7 2 .5

Avg time from date of receipt of manuscript to mailing by Wistar: Avg no. m onths with Editorial Office 2 .4 2 .6 2 .4 Avg no. months with W istar Press 3 .4 3 .2 3 .2

A v g total months from date of receipt to mailing of Journal 5.8 5 .8 5 .6

Summary of Finances in the Operation of the X. Secretary’s Report E d i to r ’s O ffice, Journal of Nutrition July 1, 1964 — June 30, 1965 Secretary Mickelsen informed the mem­ Balance brought forward ...... $ ( — 1 1 5 .2 9 ) bership of the following procedures ap­ Receipts, AIN ...... 1 2 ,0 0 0 .0 0 proved by the Council during their April 1965 meetings: Total receipts and Beginning next year, ballots for the elec­ balance available ...... 1 1 ,8 8 4 .7 1 tion of officers and Nomination Committee Expenditures, partially estimated p e r s c h e d u le ...... 1 1 ,2 8 4 .7 1 members, and voting proposed constitu­ tional amendments shall be received and Estimated balance counted by the Office of the Execeutive Sec­ July 1, 1965 $ 600.00 retary. The ballots are to be turned over The Council approved the budget proposed by Dr. Barnes for the Editor’s Office for the year to the Tellers’ Committee for verification starting July 1, 1965. and final count at the Spring Meeting. 444 AMERICAN INSTITUTE OF NUTRITION

The Executive Secretary may select a C. President Engel announced the estab­ committee to assist in arranging the scien­ lishment of a new award, the Conrad A. tific program for the annual meeting. The Elvehjem Award for Public Service in travel expenses, if any, of this committee Nutrition, sponsored by the Wisconsin shall be borne by the Institute. Alumni Research Foundation. Invitation There has been a favorable reaction to for nominations for this award appears at the newly established AIN Nutrition Notes the end of these Proceedings. which fills a long-felt need. The news­ letter is available to non-members at the XII. Business Management subscription rate of $2.00 per year; it was On the recommendation of Mr. John also decided that the newsletter should be Rice, Federation Business Manager, it was copyrighted. A vote of appreciation and decided that the Institute’s fiscal year be thanks go to Dr. M. R. Spivey Fox, Editor, made to coincide with the calendar year and J. William Boehne, Associate Editor, beginning January 1, 1966. for their efforts in getting the newsletter A new agreement will be signed whereby started. all financial matters of AIN will be handled by the Federation Business Office. XI. President’s Report The Council expressed appreciation to A. International Union of Nutrition the Federation Business Office for its ex­ Sciences: cellent handling of AIN business last year. 1. Council approved the expenditures anticipated for the travel of Dr. Glenn XIII. Reports of Committees and King, U. S. representative to the IUNS Representatives Executive Council, to Helsinki. Dr. King, A. Standing Committee on Experimental as a member of the Executive Council, Animal Nutrition: G. F. Combs will be involved in the final arrange­ The Committee was organized to emphasize the ments for the Vllth International Con­ role that research workers in basic and applied gress of Nutrition to be held in Hamburg. experimental animal nutrition have played and will continue to play in the development of the This meeting will also consider the science of nutrition and to arouse greater interest means whereby the International Union on the part of these scientists in the functions of of Nutrition Sciences can be elected to AIN. the International Congress of Scientific Standing Operating Procedure for the Commit­ tee has been approved by the AIN Council. Unions. Dr. King has been proposed by The activities of the Committee included the the U. S. National Committee as the organization of one symposium and two confer­ President of IUNS to succeed Dr. David ence programs at the 1965 annual meeting of Cuthbertson whose term expires shortly. AIN as follows: a) “The Biological Availability of Different 2. The Institute has made applica­ Forms of Vitamins A and E to Animals” tion to the National Institutes of Health chaired by L. D. Matterson. (Symposium) and the National Science Foundation for b) “Sixth Annual Ruminant Nutrition Con­ ference” chaired by R. S. Emory and R. L. funds ($25,000 each) to be used for R e id . defraying travel costs of U. S. scientists c) “Informal Poultry Nutrition Conference” to the 1966 International Nutrition Con­ chaired by H. M. Edwards, Jr., and F. H. gress. In addition, $15,000 remaining K ra tz e r. A sub-committee on Nutrition and Education, from the 1960 Congress will be added chaired by J. K. Loosli, has been formed. This to these funds making approximately committee proposed that, at the 1966 Federation $65,000 to support travel grants. This Meetings, a symposium be held on formal nutri­ $15,000 and the $500 (Dr. King’s ex­ tion education. In considering this proposal, the penses) are to be taken out of the Pine sub-committee established liaison with the Com­ mittee on Educational Policy in Agriculture, Na­ Street Fund only when actually needed, tional Research Council. When this suggestion to avoid any interest loss. for a symposium was presented to the Council of the Institute, it was endorsed with the sugges­ B. Dr. D. Mark Hegsted is the AIN tion that the scope be expanded to include ani­ Council representative to the annual meet­ mal nutrition, clinical nutrition, dental nutrition and dietetics. The Symposium Committee is con­ ing of the Nutrition Society of Canada, sidering this as one of the regularly scheduled June 11 and 12. symposia for 1966. PROCEEDINGS 445

The Committee is also considering the possibil­ can Institute of Nutrition, one of the Feder­ ity of assembling a slide collection depicting nu­ ation of American Societies for Experimental tritional deficiency diseases in experimental ani­ Biology and mals for use in nutrition instruction. Investigators WHEREAS the science of nutrition by its having m aterial which illustrates either gross or multidisciplinary nature is an important microscopic nutritional lesions suitable for inclu­ component in the study of diseases which sion in such a collection are requested to contact are the concern of the National Institutes for Dr. Gerald F. Combs, Department of Poultry Sci­ Cancer, Heart, Dental Research, Child Health ence, University of Maryland, College Park, and Development, Arthritis and Metabolic M a r y la n d . Diseases, Mental Health, Neurological Dis­ eases and Blindness, as well as the Institute B. Finance Committee Report: G. M. of General M edical Sciences, and Briggs WHEREAS the advancement of the science of nutrition deserves recognition of its sig­ This Committee was to assist the Council in nificance in the training of investigators for matters of finance. From the time of its appoint­ the conduct of research to increase our under­ ment in July until December, the Committee’s standing of normal and diseased states, major concern expressed to the Council was the urgent need for the establishm ent of an Executive BE IT RESOLVED that the Institute of Secretariat. We felt that by this means the In­ General Medical Sciences be urged to estab­ stitute’s financial structure would be benefitted lish a training committee for Nutrition Train­ more than by any other means. ing Grants to provide a continuing, consistent and effective procedure for the review and To underwrite the costs of a Secretariat and appraisal of applications for support of nutri­ to strengthen all activities of the Institute we have tion training. urged that some of the present surplus in the treasury be used, and that additional funds be obtained from such sources as a) a small increase D. Ad hoc Committee on Nutrition in membership dues; b) requests for funds to the Careers Brochure: F. J. Stare NIH and to private foundations; c) new classes The revised career brochure was submitted by of membership known as “supporting members” the Committee. The Council expressed appreci­ and “sustaining associate members”; d) a portion ation to the Committee on the completion of the of the page charges from the Journal of Nutrition task and moved that the Committee be discharged. and of the income to the Institute from the Fed­ The membership was urged to purchase the eration Meeting registration fees; e) increased brochure in lots of 100 ($4.00 per 100) and to advertising revenue from Institute journals; f) disseminate them through regular office mailings sale of a proposed collection of biographies from as a means of reaching interested groups or in­ past issues of the Journal of Nutrition; g ) th e d iv id u a ls . possible establishment of other journal(s); and h) other suggested means requiring approval of E. International Nutrition Committee: the Council and the membership. The Finance Committee commends the Council W. N. Pearson under Dr. Engel’s leadership for taking prompt The Committee recently carried out a survey action in the establishment of the Executive of the membership to determine their possible Secretariat, for the development of formal pro­ association with international nutrition programs. posals to outside agencies for funds, and in taking Of the approximately 1,000 questionnaires sent other action necessary to finance the ever-increas­ out, 407 were returned. Of those answering the ing activities of the Institute. In the development questionnaire, 141 had been affiliated with foreign of these m atters by the Council the advice of the nutrition programs during the past 5 years, and Finance Committee was sought at all times and 83 AIN members had actually worked in a foreign we were kept informed of all progress. We be­ country. A large number of members (111) an­ lieve that this has been a most im portant year for ticipated that they would participate in foreign the future of the American Institute of Nutrition. nutrition programs during the next few years, and 258 members indicated that they would be C. Committee on Nutrition Training and interested in working in a foreign country. The Fellowships: L. C. Norris Committee files now contain the names and cur­ rent addresses of 625 foreign students trained in The following resolution was adapted the U. S. by members of the AIN. Copies of this by the membership at its meeting on April report are available from the Executive Secretary. 13, 1965 and was sent to the Director of Dr. Pearson urged AIN membership participa­ the National Institutes of Health on April tion in the VITA program (Volunteers for Inter­ national Technical Assistance, Inc). The AIN 26, 1965: Council appointed Dr. James Waddell as AIN’s RESOLUTION: WHEREAS the science of liaison with VITA. The program calls upon sci­ nutrition includes elements of physiology, entists to provide technical assistance for under­ biochemistry, pathology and related fields, developed countries in one or more of the follow­ it is also recognized as a distinct discipline ing ways: 1) act as area representative; 2) give in its own right as evidenced by the adher­ general assistance (entertain students, collect ence of over one thousand scientists to its technical books, etc.); 3) prepare state-of-the art leading national scientific society, the Ameri­ reports on technical subjects; 4) participate in 446 AMERICAN INSTITUTE OF NUTRITION research and development projects which do not Biological Program under The Division of Biology conflict with one’s full-time job; and 5) work up and Agriculture. Dr. Roger Revelle will serve as replies to specific technical questions. chairman. Dr. George K. Davis, who is a member of this committee, also is international chairman F. Report on International Biological of the section on Use and M anagement of Biologi­ Program: G. K. Davis cal Resources, in which nutrition is a specific area. A working group in nutrition has been As the result of the July 22-25, 1964 meetings, established to develop basic guide lines for projects called under the auspices of the International in nutrition. Council of Scientific Unions in Paris, to consider The Food and Nutrition Board (FNB), in its the activation and implementation of an Inter­ 25th year, sponsored an International Conference national Biological Program, a Special Committee on Prevention of M alnutrition in the Pre-school for the International Biological Program (SCIBP) Child which was held in the Academy, December was established and charged with developing the 7—11 and a symposium on Significance of Plasm a various program sections. Free Amino Acid Levels for Evaluation of Protein A special working group has been set up con­ Nutrition at Rutgers University, February 1—2 as sisting of representatives from the International a memorial to Dr. James B. Allison. Union of Nutrition Sciences, the section on Hu­ Recent publications of this board include: man Adaptability and the sections on Use and NRC Pub. 1195, "An Evaluation of Public M anagement. This group is to prepare the defini­ Health Hazards from Microbiological Contamina­ tive programs for nutrition. tion of Foods” and NRC Pub. 1225, Food Habits The National Academy of Sciences has approved Research; Problems of the 1960’s.” NRC Pub. 398. the establishm ent of a National Committee for the “The Use of Chemical Additives in Food Proc­ International Biological Program under its section e s s in g ” has been revised and is in press. Two on biology and agriculture. other publications in preparation are “T h e R o le of Cereals in World Nutrition” a n d “M o n o g r a p h G. Publication Management Committee: on the Geographic Occurrence and Characteristics W. J. Darby of Nutrition Diseases.” No meeting of this Committee was held during The Food Protection Committee, FNB, has de­ the year. The newly appointed Executive Secre­ veloped provisional specifications for approxi­ tary is to be the continuing Secretary to this Com­ mately 400 food-grade chemicals and has issued mittee and should explore all means by which 415 pages in loose-leaf segments to date. The the journals of AIN can best serve the member­ final bound book defining 500 chemicals is ex­ s h ip . pected to be published next year. The Advisory Committee on Marine Protein H. Representative to the Food and Agri­ Concentrates, FNB, has been requested by the culture Organization: B. S. Schweigert Department of Interior to plan for the evaluation of the feasibility of introducing new marine pro­ FAO continues to be active in many areas of tein products into commercial outlets. significance to nutritionists and has sponsored At the April 2 meeting of the Food and Nutri­ several im portant conferences and the publication tion Board, Mr. Alan Berg, Assistant to the Direc­ of monographs on timely and important topics. tor, Food for Peace, emphasized the intensified Examples of recent publications include: specifi­ focus on nutrition in future programs for Food cations for the identity and purity of food addi­ for Peace through the Agency for International tives and their toxicological evaluation; emulsi­ Development (AID). fiers, stabilizers, bleaching and m aturing agents; The Agricultural Board, Division of Biology and legumes in hum an nutrition; news bulletin of the Agriculture, has produced the following revised Protein Advisory Group on the protein rich foods re p o rts : program and on aflotoxins (joint with WHO and 1964 NAS Pub. 1192, “Nutrient Requirements UNICEF); contemporary views on world dairy o f S w i n e ”; 1964 NAS Pub. 1193, “N u t r i e n t R e ­ education; and a biomonthly publication high­ quirements of Sheep”; and 1965 NAS Pub. 1232, lighting activities of the Freedom from Hunger C a m p a ig n . “Joint U. S.-Canadian Tables of Feed Composi­ ti o n .” In addition its Committee on Educational We can look forward to expanded attention by Policy in Agriculture has produced an illustrated FAO, other international agencies and national brochure, “Threads of Life” for use by high school groups to coordinate agricultural production, nu­ guidance counselors. Also the Institute of Labora­ trition and health, food processing and preserva­ tory Animal Resources has published standards tion, and population problems in their programs for the breeding, care and m anagement of labora­ in the future. tory dogs, cats and rabbits, augmenting the cur­ I. Representative to National Research rently accepted standards on mice, rats, ham sters, and guinea pigs. Council Divisions and Boards: G. F. Combs Proceedings of the annual meeting of the Dr. A. Geoffrey Norman, Vice President for Agricultural Research Institute, October 12-13, research at the University of Michigan, succeeds 1964, are available. The central theme of this Dr. T. C. Byerly as chairman of the Division of meeting was “Advancement of W orld Agriculture.” Biology and Agriculture. Proceedings of the Conference on Nutrition in The National Academy of Sciences has estab­ Space and Related W aste Problems, April 27-30, lished a National Committee for the International 1964, are available (NASA, SP-70). PROCEEDINGS 447

The National Committee, International Union ASCN is initiating the McCollum Award, of Nutrition Sciences, has obtained the support of the first of which will be presented during the NAS for admission of IUNS into the Inter­ national Council of Scientific Unions. This will the dinner meeting on May 1. greatly improve the opportunity for nutrition sci­ The McCollum Award is sponsored by entists to participate in international programs the National Dairy Council and the ASCN such as IBP. and is presented for outstanding research J. Report of the Federation Board Rep­ in the field of clinical nutrition. The recog­ resentative: Grace A. Goldsmith nition consists of $1000 and a commem­ The proposed revisions of the Federation Con­ orative scroll. stitution and Bylaws are still in the draft stage The Norman Jolliffe Fellowship Award and have gone back to the Advisory Committee will be publicized and sponsored by the for review. AIN approved the draft as circulated ASCN; the custodian of contributions will by the Federation. The Federation Directory of Members will here­ be the Nutrition Clinics Fund. The pur­ after include zip codes of the members. pose of this Award is to stimulate an inter­ Upon the recommendation of Dr. Maurice est in clinical nutrition among medical Visscher, the FASEB Representative on the Na­ students in the United States and to give tional Advisory Committee to the Accreditation Board of the Animal Care Panel, the Federation financial support to those active in the will appoint a representative (and one alternate) teaching of clinical nutrition in American to become a member of the newly formed Ameri­ medical schools. The Fellowship carries a can Council on Accreditation of Laboratory Ani­ minimum of $4000 annually for the next mal Care. FASEB has approved a contribution five years. of $1000 in support of this national accreditation p r o g r a m . XV. Metric System Resolutions Exploration is continuing relative to the estab­ lishm ent of a Biology Joint Council. This Council The following resolutions were adopted is envisioned as a supra-organization which could by the membership at its meeting on April be a spokesman for all of biology and provide 10. 1965: an environment for the discussion of problems of m utual interest to its constituent organizations WHEREAS the United States led the world and a means for facilitating communication with decimal currency and among all biologists. Further plans will be brought WHEREAS Congress legalized metric weights to the attention of members as they develop. and measures, July 28, 1866, The following proposals for standardization of BE IT THEREFORE RESOLVED that the rules for the submission of abstracts (annual American Institute of Nutrition, at its An­ meeting) were considered but not accepted: a) nual Meeting, April 10, 1965, recommends A member may be responsible (responsible author issuance of a metric centennial commemora­ or member) for only one paper on the program tive postage stamp on July 28, 1966. of each Society to which he belongs, b) A non­ WHEREAS more than 90% of the world’s member’s name may appear only once in the population now operates under the metric program of the Federation. system, and whereas the Journal of Nutrition, Present Federation regulations limit any in­ Poultry Science, Journal of Animal Science, dividual to present orally only one 10-minute Journal of Dairy Science, Food Chemicals paper. The following rule was adopted: Any C o d e x , and publications of the National Society may, at its discretion, elect to program Academy of Sciences-National Research for delivery only a fraction of the total number Council now use or will use metric weights of papers submitted to it or transferred to it. and measures exclusively, Those abstracts not scheduled, under these cir­ BE IT THEREFORE RESOLVED that the American Institute of Nutrition in its Annual cumstances, may then be published in the ab­ Meeting, April 10, 1965, recommends passage stracts volume. Dr. Milton Lee will retire as Executive Director of the bills now before Congress to study feasibility and practicability of conversion to of the Federation on July 1, 1965. He will con­ tinue to serve in an advisory capacity to the the metric system of weights and measures for general use in the United States. Chairman of the Federation Board until the time BE IT FURTHER RESOLVED that copies of of his retirement at age 65. The Advisory Com­ this resolution be sent to committees con­ mittee of the Federation has been asked to search cerned with metric conversion study bills for a replacement for Dr. Lee. Suggestions from S. 774, H.R. 2626, H R. 38, and H.R. 1154 to the AIN Council were invited. achieve the above objective. XIV. Report of the Clinical Division: The first resolution was sent to the U. S. J. F. Mueller Postmaster General on May 7, 1965 with The fifth annual meeting of the Clinical copies to the President of the Metric Asso­ Division of AIN will be held May 1, at the ciation and Director of the Project for Seaside Hotel, Atlantic City, New Jersey. Metric Research. 448 AMERICAN INSTITUTE OF NUTRITION

The second resolution was sent to Sen­ Biochemical Lesions Due to Dietary Im­ ators Warren Magnuson, Norris Cotton and balance, will be published in the Canadian Claii'borne Pell; Representatives G. P. Journal of Biochemistry and the Journal Miller, Robert McClory and James Roose­ of the Canadian Medical Association. velt; Drs. F. Seitz and A. G. Norman, NAS; Although the cost for printing the ab­ Editor of Science; Secretary of Commerce; stracts and programs was over $1000, the and Dr. D. F. Hornig, President’s Science Federation met approximately one-half this Advisory Committee. cost. XVI. The Joint Canadian — Sincere thanks were rendered Dr. Wil­ U. S. Nutrition Conference: liams for his unstinting services on behalf Dr. H. H. Williams of the Conference. The first Conference of Nutrition spon­ XVII. Future Federation meetings: sored jointly by the Nutrition Society of 1966, April 11-16, Atlantic City Canada and the AIN was held at the Royal 1967, April 16-21, Chicago York Hotel In Toronto, Canada, on Septem­ 1968, April 7-12, Atlantic City ber 14 and 15, 1964. The total registration XVIII. The next Council meeting will was 310 (248 active, 62 associate and be held on October 30, 1965 in Washing­ graduate). Fifty-two scientific papers were ton, D. C. presented (14 Canadian, 33 United States and 5 from other countries). Two hundred ANNUAL DINNER AND and seventy-six attended the dinner given PRESENTATION OF FELLOWS by the Province of Ontario. A N D A W A R D S Total receipts were $4405 against $3730 The annual banquet was held Monday, in expenses. The small balance will be April 12, 1965 at the Shelburne Hotel, used to purchase reprints of the symposia with 372 individuals attending. Dr. Engel presented at the meeting. The two sym­ presided. posia 1) Nutrition, Aging and Longevity, Dr. James Waddell introduced the newly and 2) Development and Significance of appointed Fellows, whose citations follow:

R a y m o n d W . S w i f t — for his out­ standing contributions to our knowledge of energy metabolism in animals and in man, and the effect of such factors as environ­ P a u l G y ô r g y — for a lifetime de­ ment and nutrient balance on the G e n e v i e v e St e a r n s — for a dis­ voted to both experimental and efficiency of energy utilization. tinguished career as investigator applied nutrition; for basic con­ During his thirty-eight years of and lecturer; a recognized author­ tributions to our knowledge of association with, and his fourteen ity on the mineral and protein pyridoxine, riboflavin, biotin, cho­ years as director of, the group metabolism of infants, children, line, and vitamin E; for studies working with the Armsby respira­ adolescents, and adult women; on the prevention of hepatic in­ tion calorimeter he provided on the vitamin requirements of jury by dietary means and on sound leadership as exemplified infants and children; on the protein malnutrition. A physician by his efforts in refining and im­ changes in body metabolism in for fifty years, he has applied his proving feeding standards and in diseases of bone. She served on knowledge to the improvement of the nutritive evaluation of for­ the U. S. Committee to the World the nutrition of infants and chil­ ages. A meticulous and resource­ Health Organization Seminars on dren. He has received previously ful investigator, a lucid writer Infant Nutrition held in Leiden from this Society both the Borden and an inspiring teacher, he has and Stockholm (1950). She re­ Award and the Osborne and Men­ greatly enhanced the status of ceived the AIN Borden Award d el A w ard. the science of nutrition. (with P. C. Jeans) in 1946.

PAUL GYÒRGY RAYMOND W. SWIFT GENEVIEVE STEARNS PROCEEDINGS 449

AMERICAN INSTITUTE OF NUTRITION

Founded April 11, 1933; Incorporated November 16, 1934; Member of Federation 1940

OFFICERS, 1965-1966 Joint Committee on Biochemical Nomenclature (joint with American Society of Biological P r e s id e n t: O. L. Kline, Food and Drug Adminis­ Chemists): S. R. Ames (1966), chairman; tration, W ashington, D. C. P. L. Harris (1966), H. H. Williams (1966). President-Elect: A. E. Schaefer, Interdepartmen­ Committee on Publication Management: W . J. tal Committee on Nutrition for National De­ Darby (1966), chairm an; P. L. Harris (1966), velopment, National Institutes of Health, J. K. Loosli (1966), D. M. Hegsted (1967), Bethesda, Maryland. F. S. Doft (1967), James Waddell (contin­ S e c r e ta r y : Olaf Mickelsen, Department of Foods uing), secretary. Ex officio, Olaf Mickelsen, and Nutrition, College of Home Economics, J. F. Mueller. Michigan State University, East Lansing, Nominating Committee for Mead Johnson Award: M ichigan (1966). B. C. Johnson (1966), chairman; P. L. Day T r e a s u r e r : W. A. Krehl, Clinical Research Cen­ (1967), A. R. Kemmerer (1968). ter, University of Iowa, Iowa City, Iowa Nominating Committee for Borden Award: L . D. ( 1 9 6 8 ) . W right (1966), chairm an; E. L. Hove (1967). Councilors: D. M. Hegsted (1966), N. S. Scrim­ P. H. Weswig (1968). shaw (1967), E. L. R. Stokstad (1968). Nominating Committee for Osborne-Mendel A w a r d : N. S. Scrimshaw (1966), chairman; COMMITTEES M. O. Schultze (1967), Alex Black (1968). Nominating Committee for Conrad A. Elvehjem Nominating Committee: G. H. Beaton, chair­ Award for Public Service in Nutrition: B . S. man; R. S. Harris, Hellen M. Linkswiler, Schweigert (1966), chairman; E. E. Howe G. V. Mann, R. J. Young. (1967), F. W. Quackenbush (1968).

Borden Award in Nutrition The 1965 Borden Award of $1000 and a gold medal was pre­ sented to Dr. Alfred E. Harper, Osborne and Mendel Award Professor of Nutrition, Massa­ chusetts Institute of Technology. The 1965 Osborne and Mendel The award was presented for a Award of $1000 and a scroll was series of investigations on the presented to Dr. David Mark interrelationship of amino acids Hegsted, Professor of Nutrition, Mead Johnson Award for in nutrition. His contributions Harvard School of Public Health. This was given for his research Research in Nutrition have advanced our knowledge of the concepts of amino acid im­ in the areas of mammalian cal­ The 1965 Mead Johnson and balances, toxicides, and antago­ cium and protein requirements Company Award of $1000 and a nisms — factors of importance in and for his studies of cholesterol scroll was awarded to Dr. John the prediction of the nutritive metabolism. He has systemati­ G. Bieri, Chief, Section on Nutri­ value of proteins. His recent ob­ cally investigated various methods tional Biochemistry, National In­ servations that amino acid im­ of evaluating the role diet may stitute of Arthritis and Metabolic balance exerts its effect in large play in the development of cardio­ Diseases. It was presented for re­ part by depressing the appetite vascular disease. He has _ com­ search on the utilization, metab­ and that this can be correlated bined a continuing interest in ex­ olism, function and nutritional with blood levels of amino acids perimental nutrition while at­ requirements of vitamin E in the are of particular significance. tempting also to improve the chick, rat and man; the interrela­ These investigations have con­ nutritional condition of man both tionships of selenium, antioxi­ tributed to a clearer understand­ in this country and abroad. Dur­ dants, lipids, sulfur amino acids, ing of the role played by amino ing a distinguished academic and ubiquinone in the metabolism acids in nutrition, particularly in career, he has trained many stu­ of vitamin E; and analytical populations where low levels of dents who have become outstand­ methods for the measurement of protein of poor quality are con­ ing investigators in many parts tocopherols in animal tissues. sum ed. of the world.

JOHN G. BIERI ALFRED E. HARPER DAVID MARK HEGSTED 450 AMERICAN INSTITUTE OF NUTRITION

Fellows Committee: Charlotte M. Young (1966), (1968). Ex officio (non-voting) members: chairman; W. D. Salmon (1966), S. L. A. G. Norman, R. K. Cannan, Harrison Hansard (1967), P. E. Johnson (1967), Brown, E. C. Rowan. W. H. Griffith (1968). Public Information Committee: P. L. White REPRESENTATIVES (1966), chairman; M. S. Read (1966), A. D. National Research Council Boards and Divisions: Tillman (1966), L. J. Teply (1966), A. L. G. F. Combs (1968). Forbes (1967), C. J. Ackerman (1967), Ruth American Association for the Advancement of L. Pike (1967), E. D. Wilson (1967). Ex S c ie n c e : R. L. Lyman (delegate), Ruth M. officio, Olaf Mickelsen. Leverton (alternate). Committee on Honorary Memberships: Paul National Society for Medical Research: H. C. Gyorgy, chairman; Grace A. Goldsmith, P. L. Spruth. Day. Food and Agriculture Organization: B. S. Schweig- Auditing Committee: R. E. Hodges (1966), C. P. ert (1966). Berg (1966). Federation Public Information Committee: P. L. Symposium Committee: N. S. Scrimshaw, chair­ White (1966). man; G. F. Combs, R. L. Jackson. Federation Proceedings Editorial Board: P. L. Ad hoc Committee on International Nutrition: Harris (1967). W. N. Pearson, chairman; Guillermo Arro- Federation Proceedings Translation Supplement: yave, C. F. Asenjo, K. W. King, W. C. F. P. Stekol (1968). Unglaub, C. W. Woodruff. Ad hoc Committee on Nutrition Training and Editorial Board Fellowships: L. C. Norris, chairman; James The Journal of Nutrition McGinnis, co-chairman; C. O. Chichester, R. H. Barnes, editor; H. H. Williams, associate J. S. Dinning, W. H. Griffith, R. E. Hodges, editor; E. Neige Todhunter, biographical edi­ O. N. Miller, Clara A. Storvick. tor; John G. Bieri, George M. Briggs, Harry Ad hoc Committee on Recommended Constitu­ P. Broquist, George K. Davis, Samuel J. tional Changes: A. E. Axelrod, chairman; Fomon, R. M. Forbes, R. Gaurth Hansen, G. M. Briggs, R. E. Hodges, R. E. Olson. L. M. Henderson, F. W. Hill, Jules Hirsch, Marion E. Swendseid. Ralph T. Holman, E. E. Howe, Gennard Committee on Experimental Animal Nutrition: Matrone, Paul M. Newberne, Boyd L. O’Dell. G. F. Combs, chairman; E. W. Crampton, H. E. Sauberlich, E. L. R. Stokstad and F. W. Hill, F. H. Kratzer, J. K. Loosli, R. W. Clara A. Storvick. Luecke, L. D. Matterson, W. F. Pfander, R. L. Reed, R. W. Engel (ex officio). Officers, American Society for Clinical Nutrition Ad hoc Committee on Travel Funds to the 1966 (A division of the American Institute of Nutrition) Nutrition Congress: James Waddell, chair­ man; G. F. Combs, Dorothy Arata, A. E. President, C. S. Davidson; President-Elect, R. E. Schaefer, E. L. R. Stokstad, O. L. Kline (ex Hodges; Past President, W. A. Krehl; Sec­ officio). retary-Treasurer, J. F. Mueller, Brooklyn- Cumberland Medical Center, 121 DeKalb Ad hoc Committee on Sustaining Associates: Avenue, Brooklyn, New York; Councilors: C. H. Krieger, chairman; W. R. Graham. H. S. Olcott, F. E. Shideman. T. B. Van Itallie, R. M. Kark, W. S. Hartroft. Membership Committee: G. P. Barron (1966), chairman; M. L. Scott (1967), K. W. King Editorial Board (1968), Dena C. Cederquist (1969), G. V. American Journal of Clinical Nutrition Mann (1970). W. A. Krehl, editor-in-chief; Robert E. Hodges, Finance Committee: H. H. Williams, chairman; associate editor; Margaret J. Albrink, Ernest D. V. Frost, P. E. Johnson, ex officio; W. A. Beutler, George J. Gabuzda, D. Mark Hegsted. Krehl, treasurer (1968). Robert Jackson, Francisco Grande, Sami A. U. S. National Committee, IUNS: G. F. Combs Hashim, Robert E. Olson, Donald M. Watkin, (1966) , Charlotte M. Young (1966), O. L. Maurice E. Shils, Victor Herbert and Carroll Kline (1966), C. G. King (1967), W. J. Darby M. Leevy. (1967) , W. A. Krehl (1967), W. M. Beeson J a m e s W a d d ell, Executive Secretary (1968) , A. L. Forbes (1968), R. W. Engel American Institute of Nutrition Invitation for Nominations for 1966 American Institute of Nutrition Awards

Nominations are requested for the 1966 in the United States and Canada which annual awards administered by the Ameri­ has emphasized the nutritive significance can Institute of Nutrition to be presented of milk or its components. The award will at the next annual meeting. Nominations be made primarily for the publication of may be made by anyone, including mem­ specific papers during the previous cal­ bers of the Nominating Committees and endar year, but the Jury of Award may non-members of the Institute. recommend that it be given for important The following information must be sub­ contributions made over a more extended mitted : (1) Name of the award for which period of time not necessarily including the candidate is proposed. (2) A brief con­ the previous calendar year. Employees of vincing statement setting forth the basis the Borden Company are not eligible for for the nomination. A bibliography and this award nor are individuals who have supporting letters are not to be submitted. received a Borden Award from another (3) Five copies of the nominating letter administering association unless the new must be sent to the chairman of the appro­ award be for outstanding research on a priate nominating committee before Octo­ different subject or for specific accom­ ber 1, 1965, to be considered for the 1966 plishment subsequent to the first award. awards. Former recipients of this award are: General regulations for A.I.N. aivards. 1944 - E. V. McCollum 1955-A. G. Hogan Membership in the American Institute of 1945 - H. H. Mitchell 1956 - F. M. Strong 1946 - P. C. Jeans and 1957 - no award Nutrition is not a requirement for eligibility Genevieve Stearns 1958 - L. D. W right 1947 — L. A. M ay n a rd 1959 - H. Steenbock for an award and there is no limitation as 1948 - C . A. C ary 1960 - R. G. Hansen to age except as specified for the Mead 1949-H . J. Deuel, Jr. 1961 - K. Schwarz 1950 - H. C. S h e rm a n 1962-H . A. Barker Johnson Award. An individual who has 1951 - P. György 1963 — A rthur L. Black 1952 - M. Kleiber 1964 - G. K. Davis received one Institute award is ineligible to 1953 - H. H. W illiams 1965 — A. E. H arper 1954 - A. F. Morgan and receive another Institute award unless it is A. H. S m ith for outstanding research subsequent to or not covered by the first award. A Jury of N o m in a t in g C o m m it t e e : Award composed of A.I.N. members, which L . D. W r ig h t , C h a ir m a n makes final selection and remains anony­ E . L. H ove mous, may recommend that an award be P. H . W e s w ig omitted in any given year if in its opinion Send nominations to: the work of the candidates nominated does not warrant the award. An award is usu­ D r . L . D. W r ig h t Graduate School of Nutrition ally given to one person, but, if circum­ Savage Hall, Cornell University stances and justice so dictate, a Jury of Ithaca, New York Award may recommend that any particu­ lar award be divided between two or more 1966 Osborne and Mendel Award collaborators in a given research. Presentation of awards will be made at The Osborne and Mendel Award of the annual dinner at the annual meeting. $1000 and an inscribed scroll has been established by the Nutrition Foundation, 1966 Borden Aivard in Nutrition Inc., for the recognition of outstanding re­ The Borden Award in Nutrition, con­ cent basic research accomplishments in sisting of $1000 and a gold medal, is made the general field of exploratory research available by the Borden Company Founda­ in the science of nutrition. It shall be tion, Inc. The award is given in recogni­ given to the investigator who, in the tion of distinctive research by investigators opinion of a Jury of Award, has made the

451 AMERICAN INSTITUTE OF NUTRITION most significant published contribution in Former recipients of this award are: approximately the calendar year preceding 1939 - C. A. Elvehjem 1946 - E. E. Snell 1940-W. H. Sebrell, Jr. 1947 - W. J. Darby the annual meeting of the Institute, or J. C. K eresztesy P. L. D ay who has published recently a series of J. R. Stevens E. L. R. Stokstad S. A. H a rris 1948 — F. Lipm ann papers of outstanding significance. Nor­ E. T. Stiller 1949 - Mary S. Shorb K. Fo lk ers K. Folkers mally preference will be given to research 1941 - R. J. W illiams 1 9 5 0 - W . B. C astle 1942 - G. R. Cowgill 1951 — no a w ard workers in the United States and Canada, 1943 - V. du Vigneaud 1952- H. E. Sauberlich but investigators in other countries, espe­ 1 9 4 4 - A. G. H ogan 1964 - J. S. D in n in g cially those sojourning in the United States 1945 — D. W. Woolley 1965-J. G. Bieri or Canada for a period of time, are not ex­ N o m in a t in g C o m m it t e e : cluded from consideration. B. C o n n o r J o h n s o n , C h a ir m a n Former recipients of this award are: P. L. D ay 1949 - W . C. Rose 1958 - P. Gy orgy A. R. Ke m m e r e r 1950 — C. A. E lv eh jem 1959 — Grace A, Goldsmith 1951 -E . E. Snell 1960 — N. S. S crim sh aw Send nominations to: 1952 - Icie Macy Hoobler 1961 - Max K. Horwitt 1953 — V. d u V ig n eau d 1962 - W illiam J. Darby D r . B. C o n n o r J o h n s o n 1954 — L. A. M ay n a rd 1963 - James B. Allison University of Oklahoma 1955 - E. V. McCollum 1964 - L. E m m e tt H olt, Jr. 1956 — A. G. H o g an 1965 - D. M. H egsted School of Medicine 1957 - G. R. Cowgill Oklahoma City, Oklahoma 73104

N o m in a t in g C o m m it t e e : N e v in Sc r im s h a w , C h a ir m a n 1966 Conrad A. Elvehjem Award for M. O. Sc h u l t z e Public Service in Nutrition Al e x B la ck The American Institute of Nutrition is Send nominations to: pleased to announce the establishment of D r . N e v in Sc r im s h a w the Conrad A. Elvehjem Award for Public Department of Nutrition and Service in Nutrition. It is to consist of Food Science $1000 and an inscribed scroll and will be Massachusetts Institute of made available annually by the Wisconsin T e c h n o lo g y Cambridge, M assachusetts 02139 Alumni Research Foundation. The award is to be bestowed in recognition of distin­ guished service to the public through the 1966 Mead Johnson Award for science of nutrition. Such service, pri­ Research in Nutrition marily, would be through distinctive activi­ The Mead Johnson Award of $1000 and ties in the public interest in governmental, an inscribed scroll is made available by industrial, private, or international institu­ Mead Johnson and Company to an investi­ tions but would not exclude, necessarily, gator who has not reached his 46th birth­ contributions of an investigative character. day during the calendar year in which the N o m in a t in g C o m m it t e e : Award is given. Selection by the Jury of B. S. Sc h w e ig e r t , C h a ir m a n Award will be based primarily on a single E. E. H o w e outstanding piece of research in nutrition F. W . Q u a c k e n b u s h published in the year preceding the annual meeting or on a series of papers on the Send nominations to: same subject published within not more D r . B. S. Sc h w e ig e r t , C h a ir m a n Food Science Department than the three years preceding the annual Michigan State University meeting. East Lansing, Michigan 48823

452 Invitation for Nominations for 1966 American Institute of Nutrition Fellows

The Fellows Committee of the American Institute of Nutrition in­ vites nominations for Fellows in the Society. Eligible candidates are active or retired members of the Society who have passed their sixty- fifth birthday (by the time of the annual meeting) and who have had distinguished careers in nutrition. Up to three Fellows will be chosen each year. Nominations may be made to the Chairman of the Fellows Com­ mittee by any member of the Society, including members of the Com­ mittee. Nominations (in 5 copies) are due by October 1. A supporting statement giving the reason for the nomination is desirable. Final selection will be made by the Fellows Committee and a suit­ able citation will be presented at the Annual Dinner in April.

Fellows Committee:

Ch a r l o t t e M. Yo u n g , C h a irm a n W . D. Sa l m o n S. L. H a n sa rd P a u l E. J o h n so n W . H . Gr if f it h

Send nominations to:

D r . Ch a r l o t t e M. Youn g Cornell University Savage Hall Ithaca, New York 14850

The following persons have been elected previously as Fellows of the Society: J. B. Brown (1964) Elmer V. McCollum (1958) Thorne M. Carpenter (1958) Harold H. Mitchell (1958) George R. Cowgill (1958) Agnes Fay Morgan (1959) Henrik Dam (1964) John R. Murlin (1958) Eugene F. DuBois (1958) Leo C. Norris (1963) R. Adams Dutcher (1961) Helen T. Parsons (1961) Ernest B. Forbes (1958) Lydia J. Roberts (1962) Casimir Funk (1958) William C. Rose (1959) Wendell H. Griffith (1963) W. D. Salmon (1962) Paul György (1965) Arthur H. Smith (1961) Albert G. Hogan (1959 ) Genevieve Stearns (1965) Icie Macy Hoobler (1960) Harry Steenbock (1958) Paul E. Howe (1960) Hazel K. Stiebeling (1964 ) J. S. Hughes (1962) Raymond W. Swift (1965) C. Glen King (1963) Robert R. Williams (1958 ) Leonard A. Maynard (1960)

453 Invitation for Nominations for Honorary Membership in the American Institute of Nutrition

The Committee on Honorary Memberships of the American Institute of Nutrition invites nominations for Honorary Members. Distinguished individuals of any country who are not members of the American Institute of Nutrition and who have contributed to the advance of the science of nutrition shall be eligible for proposal as Honorary Members of the Society. Nominations may be made to the Chairman of the Committee on Honorary Memberships by two members of the Society. Nominations (in three copies) are due by October 1. A supporting statement giving the reason for the nomination is desirable but not necessary. Final selection of nominees will be made by the Council of the American Institute of Nutrition and such nominations submitted to the Society at the spring meeting. Election requires a two-thirds majority of the ballots cast. Honorary members pay no membership fees but are eligible to subscribe to the official joumal(s) at member’s rates.

Committee on Honorary Memberships: P a u l György, Chairman P a u l D ay Grace Go l d s m it h

Send nominations to: D r . P a u l György University of Pennsylvania Pennsylvania General Hospital Philadelphia, Pennsylvania 19104

The following persons have been elected previously as Honorary Members of the Society: Kunitaro Arimoto Herbert M. Evans W. R. Aykroyd Joachim Kiihnau Frank B. Berry Tosino Oiso Edward Jean Bigwood Lord John Boyd Orr Frank G. Boudreau Conrado R. Pascual Robert C. Burgess V. N. Patwardhan Harriette Chick Emile F. Terroine F. W. A. Clements Eric John Underwood David P. Cuthbertson Artturi I. Virtanen

454 Index

A B e n n e t t , M il d re d J. See Wiese, Hilda F., 271. B e n t o n , D. A. See Romberg, B., 289. A b e r n a t h y , R. P. See Miller, Josephine, 309. B l o c k , W a l t e r D., M ara E. M arkovs a n d B e t t y A b e r n a t h y , R. P., a n d J o s e p h in e M il l e r . Effects F. St e e l e . Effect of protein and of free L- of imbalances or antagonisms among non- methionine intake on amino acid excretion by essential amino acids on growth and nitrogen human subjects, 256. utilization by rats, 231. Body composition, effect of intermittent food re­ Absorption, calcium and phosphorus, along GI striction in rats, 265. tract of laying fowl, influence of dietary cal­ ------and energy metabolism of rats fed low pro­ cium and egg shell formation, 433. tein diets, effect of type of carbohydrate, 289. — of radioactive Cu, Zn, Mo, and Fe from ligated — weight, organ weights, effect of iron deficiency segments of rat GI tract, 120. in rats, 383. Ad a m s , M il d r e d . See Lakshmanan, Florence L., B o r c h e r s , R a y m o n d , Sarah M o e n t e r An d e r s e n 337. a n d J u d y Sp e l t s . Rate of respiratory C14 di­ Age and dietary fat, effect on serum protein oxide excretion after injection of C14-amino components of the rat, 337. acids in rats fed raw soybean meal, 253. Algae utilization as protein source for humans, B o y d e n , R u t h E., a n d S. E . E r ik s o n . Metabolic 376. patterns in preadolescent children. XVI. Ribo­ Alfalfa in purified diet for reproduction of ewes, flavin utilization in relation to nitrogen intake, 154. 82. —, value of selenium in for prevention of Se B b o w n , W. D u a n e . See Cusack, Ruth P ., 383. deficiencies in chicks and rats, 213. B r u n n e r , J. E. See Kim, Young Park, 394. A l l a w a y , W. H. See Mathias, M. M., 213. B r u s h , M. K. See Fisher, H., 113. American Institute of Nutrition invitation for B u n c e , G. E ., H. E. Sä u b e r l ic h , P. G. R e e v e s nominations for 1965 awards, 221, 451. a n d T. S. Oba. Dietary phosphorus and mag­ Amino acid(s) deficiencies, chemical pathology. nesium deficiency in the rat, 406. VIII. Influence of amino acid intake on morpho­ B u n c e , Geo r g e E. See Kinnamon, Kenneth E ., logic and biochemical changes in young rats 225. fed threonine-devoid diet, 73. ------, free, patterns of blood plasma of chicks B u n n , Clara R. See Matrone, Gennard, 154. fed fishmeal proteins, 37. Butter-induced obesity in rats, cardiovascular ------imbalance and tryptophan-niacin metabo­ lesions, blood lipids, coagulation and fibrinol­ lism. I. Effect of excess leucine on urinary ysis, 325. excretion of tryptophan-niacin metabolites in rats, 100. c ------imbalance, appetite decrease in, in rats, 424. ------, nonessential, effects of imbalances or an­ Cadmium, effects on life span, tumors and tissue tagonisms on growth and nitrogen utilization levels in rats, 51. by rats, 231. Cajanus indicus, of Haiti, proximate analysis, ------patterns of blood plasma of chicks fed 415. sesame and raw, heated and overheated soy­ Calcinosis, magnesium, in dog and rat, effect of bean meals, 45. fluoride, 23. ------, radioactive, injection of rats fed raw soy­ Calcium absorption along GI tract of laying fowl bean meal, rate of respiratory C14 dioxide ex­ as influenced by dietary calcium and egg shell cretion, 253. formation, 433. ------requirements of girls, nitrogen balance — and phosphorus metabolism in man, effect of method, 333. high phosphorus intake, 125. An d e r s e n , Sarah M o e n t h e r . See Borchers, Ray­ —, high and low intake, radioactive zinc metab­ mond, 253. olism in man, 169. A n d e r s o n , J o s e p h T. See Grande, Francisco, 313. —, phosphorus and magnesium balance in baby Appetite decrease in amino acid imbalance in pigs, effects of dietary magnesium level, 209. rats, 424. C a l v e r t , C. C. See Menge, H., 115. Carbohydrate(s), dietary, and fat, effect of kind and amount on lipid metabolism of puppies, B 271. B a la ssa , J o s e p h J. See Schroeder, Henry A., 51. —, effect of type on energy metabolism and body Ba r , A. See Hurwitz, S., 433. composition of rats fed low protein diets, 289. B a r n e s , R ic h a rd H., a n d E va K w o n g . Effect of — of leguminous seeds, wheat and potatoes on soybean trypsin inhibitor and penicillin on serum cholesterol concentration in man, 313. cystine biosynthesis in the pancreas and its Carbon, radioactive, dioxide, respiratory, rate of transport as exocrine protein secretion in the excretion after injection of C14-amino acids in intestinal tract of the rat, 245. rats fed raw soybean meal, 253.

4 5 5 456 INDEX

Cartilage formation, abnormal, in young chicks, — , radioactive, absorption from ligated segments nutritional, genetic and morphological studies, of rat GI tract, 120. 236. Corn-bean diets, Haitian, weight gain and PER Casein and soy protein diets, effect on growth of rats fed, 415. of ducklings, 17. — protein, determination of first limiting nitrog­ Catalase, effect of iron deficiency in rats, 383. enous factor, for hum an adults, 350. Cereal-legume blends, Haitian, nutritional value C o r n a t z e r , W. E. See Glende, Eric A., Jr., 178. (rats), 415. C r a m p t o n , E. W. See Christensen, D. A., 369. — proteins, adequacy for nitrogen retention in Creatine metabolism in muscle, liver and brain human adults, 350. of rabbits fed choline-deficient diet, 298. Cheese, Cheddar, B-vitamin content, 362. C u s a c k , R u t h P ., a n d W . D u a n e B r o w n . I r o n Chicken(s), absorption of calcium and phos­ deficiency in rats: changes in body and organ phorus along GI tract of laying fowl as in­ weights, plasma proteins, hemoglobins, myo­ fluenced by dietary calcium and egg shell globins and catalase, 383. formation, 433. Cystine biosynthesis in the pancreas and its trans­ — and rats, value of selenium in alfalfa for pre­ port as exocrine protein secretion in the in­ vention of Se deficiencies, 213. testinal tract of the rat, effect of soybean tryp­ —, effect of dietary lipids on fatty acid composi­ sin inhibitor and penicillin, 245. tion of neutral lipids and phosphatides on liver Cytochrome oxidase in mice and rats fed meat and bile, 143. d ie t, 3 0 3 . — fed deficient-to-excess protein and lysine, and infected with tuberculosis, protein metabolism in livers, 133. D —, free amino acid patterns of blood plasma of D a ja n i, R a s h id M ., L a t if e h Gh a n d u r -Mn a y m - chicks fed sesame and soybean meals, 45. n e h , M e r l e H a r r iso n a n d T a m e r N a ssa r . T h e —, free amino acid patterns of blood plasma utilization of ethanol. III. Liver changes in­ when fed fishmeal proteins, 37. duced by alcohol, 29. —, linoleic acid and reproduction in hens, 115. D a m , H e n d r ik . See Glenn, Joseph L., 143. —, metabolism of ethylenediaminetetraacetic D a m , R ic h a r d , Su n g h e e Le e , P eggy C . F r y a n d acid, 187. H a z e l F o x . Utilization of algae as a protein —, young, nutritional, genetic and morphological source for hum ans, 376. studies of abnormal cartilage formation, 236. D a r w is h , N a z e k M ., a n d F . H . Kr a t z e r . C hiemchaisri, Y., a n d P a u l H. P h i l l i p s . Cer­ Metabolism of ethylenediaminetetraacetic acid tain factors including fluoride which affect (EDTA) by chickens, 187. magnesium calcinosis in the dog and rat, 23. D e n t o n , C. A. See Menge, H., 115. Children, amino acid requirements of girls, nitro­ D e v i, A. See Srivastava, Uma, 298. gen balance method, 333. Diethylstilbestrol and dietary lipid, effect on liver —, preadolescent, excretion of niacin or trypto­ fatty acids of choline-deficient rats, 399. phan metabolites when fed controlled diets sup­ Diet(s) and exercise, effect on nitrogenous con­ plemented with nicotinamide, 309. stituents in rat tissue, 369. —, preadolescent, riboflavin utilization in rela­ —, casein and soy protein, effect on growth of tion to nitrogen intake, 82. ducklings, 17. Chlorella pyrenoidosa utilization as protein source —, purified, effect on abnormal cartilage forma­ for humans, 376. tion in young chicks, 236. Cholesterol metabolism, influence of medium- —, — , for growth and reproduction of rum inant, chain triglyceride in rats, 67. 1 5 4 . —■, serum, concentration in man, effect of carbo­ Dog(s) and rat, effect of fluoride on magnesium hydrate of wheat, leguminous seeds and po­ calcinosis, 23. tatoes, 313. —, puppies, effect of kind and amount of fat Choline deficiency, fatty acid composition of rat and dietary carbohydrate on lipid metabolism liver lipids during, 178. o f, 2 7 1 . — -deficient diet, nucleic acid, phosphorus and D o is y , E . A ., J r . See M atschiner, John T., 93. creatine metabolism in muscle, liver and brain D uck(s), effect of casein and soy protein diets on of rabbits fed, 298. growth, 17. ----deficient rats, effect of dietary lipid and di- Dystrophy, muscular, nucleic acid, phosphorus ethylstilbestrol on liver fatty acids of, 399. and creatine metabolism in muscle, liver and C h r is t e n s e n , D. A., a n d E. W. C r a m p t o n . Effects brain of rabbits fed choline-deficient diet, 298. of exercise and diet on nitrogenous constituents in several tissues of adult rats, 369. Chromium, effects on life span, tumors and E tissue levels in rats, 51. Coagulation, blood, in butter-induced obesity in Egg(s) shell formation and dietary calcium, in­ rats, 325. fluence on calcium and phosphorus absorption C o o n , E d m u n d . See Wiese, Hilda F., 271. along GI tract of laying fowl, 433. Copper, effect on radioactive zinc tissue distribu­ — production, effect of linoleic acid, 115. tion and excretion in rats, 225. E ic h n e r , J o e l l e n T. See Hurley, Lucille S., 201. — metabolism in germfree and conventional rats, E l l is , W il l ia m W. See Miller, Glenn J., 399. 159. E r ik s o n , S. E . See Boyden, Ruth E., 82. INDEX 457

Ethanol, liver changes induced by, in rats, 29. G u g g e n h e im , K. See Havivi, E ., 3 0 3 . Ethylenediaminetetraacetic acid, metabolism by Guinea pig(s), pantothenic acid deficiency in chicks, 187. pregnant and non-pregnant, effect on fetus, 201. Ew e(s), reproduction with purified diets, 154. Exercise and diet, effect on nitrogenous constitu­ ents in rat tissue, 369. H Haiti, beans indigenous to, nutritional value F (rats), 415. H a r r is o n , B e r t h a N e a l . See Spivey Fox, M. R., Fat(s), dietary, and age, effect on serum protein 8 9 . components of the rat, 337. H a r r is o n , M e r l e . See Dajani, Rashid M., 29. —- — , effect of kind and amount on lipid metab­ H a v iv i, E ., a n d K. G u g g e n h e im . Cytochrome oxi­ olism of puppies, 271. dase in mice and rats maintained with a meat Fatty acid(s) composition of neutral lipids and d ie t, 3 0 3 . phosphatides in chick liver and bile, effect of Heart, cardiovascular lesions in butter-induced dietary lipids, 143. obesity in rats, 325. — — composition of rat liver lipids during cho­ H e g g e n e s s , Fr a n k l in W. Effect of interm ittent line deficiency, 178. food restriction on growth, food utilization and F is h e r , H ., P . Gr im in g e r , E . R . So s t m a n a n d body composition of the rat, 265. M . K. Br u s h . Dietary pectin and blood cho­ Hemoglobin, effect of iron deficiency in rats, 383. lesterol (Letter to the Editor), 113. H o e f e r , J. A. See M iller, E. R., 209. Fishmeal proteins, free amino acid patterns of H o g u e , D. E. See M athias, M. M., 213. blood plasm a of chicks fed, 37. H um an(s) adults, determination of first limiting Fluoride, effect on magnesium calcinosis in dog nitrogenous factor in corn protein, 350. and rat, 23. — adults, effect of “non-specific” nitrogen intake Food restriction, interm ittent, effect on growth, on nitrogen retention, in hum ans, 357. food utilization and body composition of the — , effect of protein and free L-methionine intake r a t , 2 6 5 . on amino acid excretion, 256. —utilization, effect of interm ittent food restric­ -—, use of algae as protein source for, 376. tion in rats, 265. H u r l e y , L u c il l e S., N e l d a E. V o l k e r t a n d F o r r e s , R. M. M ineral utilization in the rat. V. Jo e l l e n T . Eic h n e r . Pantothenic acid defi­ Effects of dietary thyroxine on m ineral balance ciency in pregnant and non-pregnant guinea and tissue mineral composition with special pigs, with special reference to effects on the reference to magnesium nutriture, 193. fetus, 201. F o x , H a z e l . See Dam, Richard, 376. H u r w i t z , S., a n d A . B a r . Absorption of calcium F o x , H a z e l M e t z . See Kies, Constance, 350, 357. and phosphorus along the gastrointestinal tract F r y , P e g g y C. See Dam, Richard, 376. of the laying fowl as influenced by dietary calcium and egg shell formation, 433. G I G a r d n e r , R. W. See M athias, M. M., 213. Genetic selection of chicks, effect on occurrence Iron deficiency in rats; changes in body and of abnormal metaphyseal cartilage, 236. organ weights, plasma proteins, hemoglobins, G h a n d u r -M n a y m n e h , L a t if e h . See Dajani, myoglobins and catalase, 383. Rashid M., 29. — metabolism in germfree and conventional rats, G i z i s , E v a n g e l o s . See Kim, Young Park, 394. 159. G l e n d e , E r ic A ., Jr ., a n d W . E . C o r n a t z e r . — , radioactive, absorption from ligated segments Fatty acid composition of rat liver lipids during of rat GI tract, 120. choline deficiency, 178. G l e n n , Jo s e p h L ., a n d H e n d r ik D a m . I n f lu e n c e K of dietary lipids on the fatty acid composition of neutral lipids and phosphatides in chick K e y s , A n c e l . See Grande, Francisco, 313. liver and bile, 143. K i e s , C o n s t a n c e , E l e a n o r W i l l i a m s a n d H a z e l Glucose metabolism of chromium-deficient rats M e t z F o x . Determination of the first limiting raised in controlled environment, 107. nitrogenous factor in corn protein for nitrogen G o p a l a n , C. See Raghuramulu, N., 100. retention in human adults, 350. G r a n d e , F r a n c is c o , Jo s e p h T. A n d e r s o n a n d K i e s , C o n s t a n c e , E l e a n o r W i l l i a m s a n d H a z e l A n c e l Ke y s . Effect of carbohydrates of legum­ M e t z F o x . Effect of “non-specific” nitrogen in­ inous seeds, wheat and potatoes on serum cho­ take on adequacy of cereal proteins for nitrogen lesterol concentration in m an, 313. retention in hum an adults, 357. G r im in g e r , P. See Fisher, H., 113. Ki m , Y o u n g P a r k , E v a n g e l o s G i z i s , J. E. Br u n ­ Growth and nitrogen utilization by rats, effects of n e r a n d B . S. Sc h w e ig e r t . V ita m in B i2 d is ­ imbalances or antagonisms among nonessential tribution in cow’s milk, 394. amino acids, 231. K in g , K e n d a l l W. See Sirinit, Kosol, 415 — and reproduction in ruminant, with purified K in n a m o n , K e n n e t h E ., a n d G eorge E . B u n c e . diets, 154. Effects of copper, molybdenum, and zinc on — , effect of interm ittent food restriction in rats, zinc-65 tissue distribution and excretion in the 2 6 5 . r a t , 2 2 5 . 458 INDEX

K o b a y a s h i, K a t s u m i . See Nakagawa, Itsiro, 333. Magnesium calcinosis in dog and rat, effect of K r a t z e r , F. H. See Darwish, Nazek M., 187. fluoride, 23. Kr it c h e v s k y , D a v id , a n d Sh ir l e y A. T e p p e r . — , calcium and phosphorus balance in baby pigs, Influence of medium-chain triglyceride (MCT) effects of dietary magnesium level, 209. on cholesterol metabolism in rats, 67. — deficiency in the rat and dietary phosphorus, Kwashiorkor, appetite decrease in amino acid im­ 4 0 6 . balance, 424. — , effect of thyroxine on response to varying in­ Kw o n g , E v a . See Barnes, Richard H., 245. takes, in rats, 193. M a r io n , M. V. See M athias, M. M., 213. M a r k o v s , M a r a E. See Block, W alter D., 256. L M a t h ia s , M. M., W. H. A l l a w a y , D . E . H o g u e , M . V. M a r io n a n d R . W . G a r d n e r . V a lu e o f L a k s h m a n a n , F l o r e n c e L . , a n d M i l d r e d A d a m s . Effect of dietary fat and age on the serum selenium in alfalfa for the prevention of protein components of the rat, 337. selenium deficiencies in chicks and rats, 213. Lam b(s), growing-finishing, effect of protein in­ M a t r o n e , G e n n a r d , C l a r a R . B u n n a n d J. J. take on blood urea nitrogen, 281. M c N e il l . Study of purified diets for growth —, growth with purified diets, 154. and reproduction of the rum inant, 154. Langworthy, Charles Ford — a biographical M a t s c h in e r , Jo h n T ., a n d E . A. D o i s y , J r . E ffe c t s k e tc h , 1. of dietary protein on the development of vita­ m in K deficiency in the rat, 93. L e a c h , R . M ., J r ., a n d M . C . N e s h e i m . N u t r i ­ tional, genetic and morphological studies of an M e n c z e l , Ja c o b . See Spencer, Herta, 125. abnormal cartilage formation in young chicks, M cN e il l , J. J. See Matrone, Gennard, 154 2 3 6 . M e n g e , H ., C . C. C a l v e r t a n d C. A . D e n t o n . Lead, effects on life span, tumors and tissue levels Further studies of the effect of linoleic acid on in rats, 51. reproduction in the hen, 115. L e d e , M a r i a N. See Sanahuja, Juan C., 424. M e r t z , W a l t e r , E d w a r d E. R o g in s k i a n d H e n r y L e e , S u n g h e e . See Dam, Richard, 376. A. Sc h r o e d e r . Some aspects of glucose metab­ Legume(s)-cereal blends, Haitian, nutritional olism of chromium-deficient rats raised in a value (rats), 415. strictly controlled environment, 107. Leucine, excess, effect on urinary excretion of Metabolism, calcium and phosphorus in man, tryptophan-niacin metabolites in rats, 100. effect of high phosphorus intake, 125. L e w i n , I s a a c . See Spencer, Herta, 125, 169. —, cholesterol, influence of medium-chain tri­ Life span, effect of chromium, cadmium and glyceride in rats, 67. lead, in rats, 51. — , energy, and body composition of rats fed low Linoleic acid, effect on reproduction in hens, 115. protein diets, effect of type of carbohydrate, 289. Lipid(s), blood, in butter-induced obesity in rats, — , glucose, of chromium-deficient rats raised in 3 2 5 . controlled environment, 107. — , dietary and diethylstilbestrol, effect on fatty — of ethylenediam inetetraacetic acid by chickens, acids of choline-deficient rats, 399. 187. — , — , effect on fatty acid composition of neutral — of iron, copper and manganese in germfree lipids and phosphatides in chick liver and bile, and conventional rats, 159. 143. — , radioactive zinc, in m an during low and high — metabolism of puppies as affected by kind and calcium intake, 169. am ount of fat and of dietary carbohydrate, 271. — ■,r tryptophan-niacin and amino acid imbalance. — , rat liver, fatty acid composition during cho­ I. Effect of excess leucine on urinary excre­ line deficiency, 178. tion of tryptophan-niacin metabolites in rats, Liver changes induced by alcohol, in rats, 29. 100. — fat of rats fed amino acid imbalanced diets, M ethionine ( l - ) and protein intake, effect on 4 2 4 . amino acid excretion by hum ans, 256. — weight of rats, increase with amino acid im­ Mice and rats fed meat diet, cytochrome oxidase, balanced diets, 424. 3 0 3 . L u e c k e , R. W. See Miller, E. R., 209. Milk, cow’s, vitamin B 12 distribution in, 394. Lysine and protein, deficient-to-excess, effect on M il l e r , E. R., D. E. U l l r e y , C. L. Z u t a u t , J . A. livers of chicks infected with tuberculosis, 133. H o eeer a n d R . W . L u e c k e . Mineral balance L y m a n , R. L. See Schumacher, Sister M. F,, 343. studies with the baby pig: effects of dietary magnesium level upon calcium, phosphorus M and magnesium balance, 209. M il l e r , G l e n n J ., a n d W il l i a m W . E l l i s . Man, effect of carbohydrates of leguminous seeds, Effects of dietary lipid and diethylstilbestrol wheat and potatoes on serum cholesterol con­ upon liver fatty acids of choline-deficient rats, centration of, 313. 3 9 9 . —, effect of high phosphorus intake on calcium M il l e r , Jo s e p h in e , a n d R . P . A b e r n a t h y . M e ta ­ and phosphorus metabolism in, 125. bolic patterns in preadolescent children. XIV. —, radioactive zinc metabolism with low and Excretion of niacin or tryptophan metabolites high calcium intake, 169. by girls fed controlled diets supplemented with Manganese metabolism in germfree and conven­ nicotinam ide, 309. tional rats, 159. M il l e r , Jo s e p h in e . See Abernathy, R. P., 231. INDEX 459

M ineral(s) balance studies with baby pig; effects P f ä n d e r , W. H. See Preston, R. L., 281. of dietary magnesium level on calcium, phos­ Phaseolus lunatus, lim a beans of Haiti, proximate phorus and magnesium balance, 209. analysis, 415. — balance, tissue mineral composition, and mag­ — v u lg a r is , red, black and white beans of Haiti, nesium nutriture, in rats, 193. proximate analysis, 415. M i t c h e l l , E l i z a b e t h A. See Van Campen, Dar­ P h i l l i p s , P a u l H. See Chiemchaisri, Y., 23. rell R., 120. Phosphorus absorption along GI tract of laying M olybdenum, effect on radioactive zinc tissue dis­ fowl as influenced by dietary calcium and egg tribution and excretion in rats, 225. shell form ation, 433. — , radioactive, absorption from ligated segments — , calcium and magnesium balance in baby pigs, of rat GI tract, 120. effects of dietary magnesium level, 209. Myoglobins, effect of iron deficiency in rats, 383. — , dietary, and magnesium deficiency in the rat, 4 0 6 . — intake, high, effect on calcium and phos­ N phorus metabolism in man, 125. • - metabolism in muscle, liver and brain of rab­ N a i m i , Sh a p u r , G eorge F . W il g r a m , M a r t in M. bits fed choline-deficient diet, 298. N o t h m a n a n d Sa m u e l P r o g e r . Cardiovascular lesions, blood lipids, coagulation and fibrinol­ P l e a s a n t s , J. R. See Reddy, B. S., 159. ysis in butter-induced obesity in the rat, 325. Potatoes, effect of carbohydrate on serum choles­ terol concentration in man, 313. N a k a g a w a , I t s ir o , T e t s u z o T a k a h a s h i, T a k e s h i Pregnancy and pantothenic acid deficiency in Su z u k i a n d K a t s u m i K o b a y a s h i. Amino acid requirements of children: quantitative amino guinea pigs, 201. acid requirements of girls based on nitrogen P r e s t o n , R. L., D. D. S chankenberg a n d W . H . balance method, 333. P f ä n d e r . Protein utilization in ruminants. N a r a s in g a R a o , B. S. See Raghuramulu, N., 100. I. Blood urea nitrogen as affected by protein N a s s a r , T a m e r . See Dajani, Rashid M., 29. intake, 281. N e s h e im , M. C. See Leach, R. M ., J r ., 236. Proceedings of the 28th annual meeting of the Niacin or tryptophan metabolites, excretion by American Institute of Nutrition, 439. girls fed controlled diets supplemented with P r o g e r , S a m u e l . See Naimi, Shapur, 325. nicotinamide, 309. Protein(s), casein and soy, diets, effect on growth Nicotinamide-supplemented diets, excretion of of ducklings, 17. niacin or tryptophan metabolites by girls fed, — and free L-methionine intake, effect on amino 309. acid excretion by humans, 256. N i l s o n , K a y M ., Ja y a n t k u m a r R . V a k il a n d — , cereal, effect of “non-specific” nitrogen intake K h e m M . Sh a h a n i . B-complex vitam in content on nitrogen retention of hum an adults, 357. of Cheddar cheese, 362. —, cereal, first limiting nitrogenous factor in Nitrogen balance method, amino acid require­ corn, in hum an adults, 350. ments of girls vs. boys, 333. —, dietary, effect on development of vitamin K —, DNA and RNA in rat tissues, effects of exer­ deficiency in rat, 93. cise and diet, 369. — , exocrine, secretion in intestinal tract of rats, — intake in relation to riboflavin utilization in effect of soybean trypsin inhibitor and penicillin preadolescent children, 82. on cystine biosynthesis in the pancreas and its , “non-specific,” intake, effect on adequacy of transport as, 245. cereal proteins for nitrogen retention in hum an — , fishmeal, free amino acid patterns of blood adults, 357. plasma of chicks fed, 37. N o t h m a n , M a r t in M. See Naimi, Shapur, 325. —, free amino acid patterns of blood plasma of Nucleic acid metabolism in muscle, liver and chicks fed sesame and soybean meals, 45. brain of rabbits fed choline-deficient diet, 298. — intake, effect on blood urea nitrogen in rum i­ nants, 281. __, low, diets, effect of type of carbohydrate on o energy metabolism and body composition of rats f e d , 2 8 9 . O b a , T. S. See Bunce, G. E., 406. — metabolism in livers of chicks fed deficient-to- Oryza sativa (Haiti) proximate analysis, 415. excess protein and lysine and infected with tuberculosis, 133. — , plasm a, effect of iron deficiency in rats, 383. P . plasma, effect of zinc deficiency in Japanese Pantothenic acid deficiency in pregnant and non­ q u a il, 8 9 . pregnant guinea pigs, effects on fetus, 201. — source for humans, algae as, 376. Pathology, chemical of acute amino acid defi­ Pyridoxine intake, high, effect on reproduction ciencies. VIII. Influence of amino acid intake and vitamin requirements of offspring of rats, on morphologic and biochemical changes in 3 4 3 . young rats fed threonine-devoid diet, 73. Penicillin and soybean trypsin inhibitor, effect on Q cystine biosynthesis in pancreas, and its trans­ port as exocrine protein secretion in the in­ Quail, Japanese, effect of zinc deficiency on testinal tract of rats, 245. plasm a proteins, 89. 460 INDEX

R R e e v e s , P. G. See Bunce, G. E., 406. Reproduction and growth of rum inant with puri­ Rabbit(s) fed choline-deficient diet, nucleic acid, fied diets, 154. phosphorus and creatine metabolism in muscle, — in hens, effect of linoleic acid, 115. liver and brain, 298. — of rats and vitam in requirements of offspring, R a g h u r a m u l u , N ., B . S. N a r a s i n g a R a o a n d C. effect of high intakes of thiamine, riboflavin G o p a l a n . Amino acid imbalance and trypto­ and pyridoxine, 343. phan-niacin metabolism. I. Effect of excess Riboflavin intake, high, effect on reproduction and leucine on the urinary excretion of tryptophan- vitam in requirements of offspring of rats, 343. niacin metabolites in rats, 100. —utilization in relation to nitrogen intake, pre­ R at(s), absorption of radioactive Cu, Zn, Mo, and adolescent children, 82. Fe from ligated segments of GI tract, 120. Rice-bean diets, Haitian, weight gain and PER — and chicks, value of selenium in alfalfa for prevention of Se deficiencies, 213. of rats fed, 415. — and dog, effect of fluoride on magnesium cal­ R ic h e r t , D a n A ., a n d W . W . W e s t e r f e l d . E f fe c t cinosis, 23. of casein and soy protein diets on the growth — and mice fed meat diet, cytochrome oxidase, of ducklings, 17. 3 0 3 . R io , M a r ia E. See Sanahuja, Juan C., 424. — , choline-deficient, effect of dietary lipid and R o g in s k i, E d w a r d E. See Mertz, W alter, 107. diethylstilbestrol on liver fatty acids, 399. R o m b e r g , B ., a n d D . A. B e n t o n . Effect of type of —, chromium-deficient, raised in controlled en­ carbohydrate on energy metabolism and body vironment, glucose metabolism, 107. composition of rats fed low protein diets, 289. —, dietary phosphorus and magnesium defici­ e n c y , 4 0 6 . —, effects of chromium, cadmium and lead on s life span, tumors and tissue levels, 51. Sa m a c h s o n , Jo s e p h . See Spencer, Herta, 125, —, effects of copper, molybdenum and zinc on 169. radioactive zinc tissue distribution and ex­ Sa n a h u j a , Ju a n C ., M a r ia E . R io a n d M a r ia N . cretion, 225. L e d e . Decrease in appetite and biochemical — , effect of dietary fat and age on serum protein changes in amino acid imbalance, 424. components, 337. Sa r k a r , N. K. See Srivastava, Uma, 298. —, effect of dietary protein on development of Sa u b e r l ic h , H. E. See Bunce, G. E., 406. vitamin K deficiency, 93. —, effect of dietary thyroxine on mineral bal­ Scenedesmus obliquus utilization as protein source ance and tissue m ineral composition — magne­ for hum ans, 376. sium nutriture, 193. Schankenberg , D. D. See Preston, R. L., 281. — , effect of excess leucine on urinary excretion SCHROEDER, HENRY A., JOSEPH J. BALASSA AND of tryptophan-niacin metabolites, 100. W i l l i a m H . V i n t o n , Jr . Chromium, cadmium — , effects of imbalances or antagonisms among and lead in rats: effects on life span, tumors nonessential amino acids on growth and nitro­ and tissue levels, 51. gen utilization, 231. Sc h r o e d e r , H e n r y A. See Mertz, W alter, 107. —, effect of intermittent food restriction on Sc h u m a c h e r , Sis t e r M. F., M. A. W i l l ia m s and growth, food utilization and body composition, R . L . L y m a n . Effect of high intakes of thia­ 2 6 5 . mine, riboflavin and pyridoxine on reproduction — , effect of soybean trypsin inhibitor and peni­ and vitam in requirements of the offspring, 343. cillin on cystine biosynthesis in the pancreas Sc h w e ig e r t , B. S. See Kim, Young Park, 394. and its transport as exocrine protein secretion Sc o t t , H. M. See Smith, R. E., 37, 45. in intestinal tract of rats, 245. Seeds, leguminous, effect of carbohydrate on — fed low protein diets, effect of type of carbo­ serum cholesterol concentration in man, 313. hydrate on energy metabolism and body com­ Selenium, value in alfalfa for prevention of Se position, 289. deficiencies in chicks and rats, 213. — , germfree and conventional, iron, copper and Sesame and soybean meals, free amino acid pat­ manganese metabolism, 159. tern of blood plasma of chicks fed, 45. —, influence of medium-chain triglyceride on Sh a h a n i , K h e m M . See Nilson, Kay M., 362. cholesterol metabolism, 67. Si d r a n s k y , H e r s c h e l , a n d E t h e l V e r n y . C h e m ­ — liver lipids, fatty acid composition during ical pathology of acute amino acid deficiencies. choline deficiency, 178. VIII. Influence of amino acid intake on the — , rate of respiratory C14 dioxide excretion after morphologic and biochemical changes in young injection of C14-amino acids, raw soybean meal rats force-fed a threonine-devoid diet, 73. d ie t, 2 5 3 . Sie g e l , H e n r y . See Squibb, Robert L., 133. — with butter-induced obesity, cardiovascular Si r i n i t , K o s o l , A b d e l -Ga w a d M . So l i m a n , A l i lesions, blood lipids, coagulation and fibrinol­ T . v a n L oo a n d Ke n d a l l W . Ki n g . Nutritional y s is , 3 2 5 . value of Haitian cereal-legume blends, 415. — , young, fed threonine-devoid diet, influence of Sm i t h , R . E ., a n d H . M . Sc o t t . Use of free amino amino acid intake on morphologic and bio­ acid concentrations in blood plasma in evaluat­ chemical changes, 73. ing the amino acid adequacy of intact proteins R e d d y , B. S., B . S. W o s t m a n n a n d J . R . P l e a s ­ for chick growth. I. Free amino acid patterns a n t s . Iron, copper, and manganese i n g e rm - of blood plasma of chicks fed unheated and free and conventional rats, 159. heated fishmeal proteins, 37. INDEX 461

Sm i t h , R . E ., a n d H . M . Sc o t t . Use of free amino Tryptophan or niacin metabolites, excretion by acid concentrations in blood plasma in evalu­ girls fed controlled diets supplemented with ating the amino acid adequacy of intact pro­ nicotinamide, 309. teins for chick growth. II. Free amino acid Tuberculosis, protein metabolism in livers of patterns of blood plasma of chicks fed sesame chicks fed deficient-to-excess protein and lysine and raw, heated and overheated soybean meals, and infected with, 133. 4 5 . Tumors, effect of chromium, cadmium and lead So l im a n , A b d e l -G a w a d M. See Sirinit, Kosol, 415. in rats, 51. Solotorovsky , M o r r is . See Squibb, Robert L., 133. u Sorghum-bean diets, Haitian, weight gain and U l l r e y , D. E. See Miller, E. R., 209. PER of rats fed, 415. Urea, blood, nitrogen, effect of protein intake in Sorghum vulgare (H aiti) proximate analysis, 415. rum inants, 281. So s t m a n , E. R. See Fisher, H., 113. Soy and casein protein diets, effect on growth of V ducklings, 17. Soybean(s) meal, raw, rate of respiratory C14 di­ V a k i l , Ja y a n t k u m a r R. See Nilson, Kay M., 362. oxide excretion after injection of C14-amino V a n C a m p e n , D a r r e l l R ., a n d E l iz a b e t h A. acids in rats fed, 253. M it c h e l l . Absorption of C u64, Z n 63, M o " a n d — and sesame meals, free amino acid pattern of Fe39 from ligated segments of the rat gastro­ blood plasm a of chicks fed, 45. intestinal tract, 120. — trypsin inhibitor and penicillin, effect on cys­ V a n k in s c o t t , V e r n ic e . See Spencer, Herta, 169. tine biosynthesis in pancreas, and its transport v a n L o o , A l i T. See Sirinit, Kosol, 415. as exocrine protein secretion in the intestinal V e r n e y , E t h e l . See Sidransky, Herschel, 73. tract of rats, 245. V i n t o n , W i l l i a m H . Jr . See Schroeder, Henry Sp e l t s , Ju d y . See Borchers, Raymond, 253. A ., 5 1 . Sp e n c e r , H e r t a , Ja c o b M e n c z e l , I saac L e w in V itam in(s) B complex content of Cheddar cheese, a n d Jo s e p h Sa m a c h s o n . Effect of high phos­ 3 6 2 . phorus intake on calcium and phosphorus me­ — B 12 distribution in cow’s milk, 394. tabolism in man, 125. — K deficiency, effect of dietary protein in rats, Sp e n c e r , H e r t a , V e r n ic e V a n k in s c o t t , I saac 9 3 . L e w in a n d J o s e p h Sa m a c h s o n . Z in c -6 5 m e ­ — requirements of offspring of rats, effect of tabolism during low and high calcium intake high intakes of thiamine, riboflavin and pyri- in man, 169. doxine on, 343. Sp iv e y F o x , M . R ., a n d B e r t h a N e a l H a r r is o n . V o l k e r t , N e ld a E. See Hurley, Lucille S., 201. Effects of zinc deficiency on plasm a proteins of young Japanese quail, 89. w Sq u ib b , R o b e r t L ., H e n r y Sie g e l a n d M o rris Solotorovsky . Protein metabolism in livers of W e s t e r f e l d , W. W. See Richert, Dan A., 17. chicks fed deficient-to-excess quantities of pro­ W heat, effect of carbohydrate on serum choles­ tein and lysine and infected with tuberculosis, terol concentration in man, 313. 133. W ie s e , H il d a F ., M ild r e d J . Be n n e t t , E d m u n d Sr iv a s t a v a , U m a , A. D e v i a n d N . K. Sa r k a r . C o o n a n d W i l l i a m Y a m a n a k a . Lipid metabo­ Biochemical changes in progressive muscular lism of puppies as affected by kind and amount dystrophy. III. Nucleic acid, phosphorus and of fat and of dietary carbohydrate, 271. creatine metabolism in the muscle, liver and W il g r a m , G eorge F. See Naimi, Shapur, 325. brain of rabbits maintained with a choline- W i l l i a m s , E l e a n o r . See Kies, Constance, 350, deficient diet, 298. 3 5 7 . St e e l e , B e t t y F. See Block, W alter D., 256. W i l l i a m s , M. A. See Schumacher, Sister M. F., Su z u k i , T a k e s h i. See Nakagawa, Itsiro, 333. 3 4 3 . Sw a n s o n , P e a r l . Charles Ford Langworthy — a W o s t m a n n , B. S. See Reddy, B. S., 159. biographical sketch, 1. Y

T Y a m a n a k a , W i l l i a m . See Wiese, Hilda F., 271. T a k a h a s h i, T e t s u z o . See Nakagawa, Itsiro, 333. T e p p e r , Sh ir l e y A. See Kritchevsky, David, 67. z Thiam ine intake, high, effect on reproduction and vitam in requirements of offspring of rats, 343. Z e a m a y s , (H aiti), proximate analysis, 415. Threonine-devoid diet and influence of amino acid Zinc deficiency, effect on plasma proteins of intake on morphologic and biochemical Japanese quail, 89. changes in young rats, 73. — , effect on radioactive zinc tissue distribution Thyroxine, dietary, effect on m ineral balance and and excretion in rats, 225. tissue m ineral composition, in rats, 193. — , radioactive, absorption from ligated segments Triglyceride, medium-chain, influence on choles­ of rat GI tract, 120. terol metabolism in rats, 67. — , radioactive, metabolism during low and high Tryptophan-niacin metabolites, effect of excess calcium intake in man, 169. leucine on urinary excretion of, in rats, 100. Z u t a u t , C. L. See M iller, E. R., 209.