THE EFFECT OF ORANGE JUICE ON THE CALCIUM,PHOSPHORUS, MAGNESIUM, AND NITROGEN

RETENTION AND URINARY ORGANICACIDS OF GROWING CHILDREN.*

By MARGARET S. CHANEY AND KATHARINE BLUNT.

(From the Nutrition Laboratory of the Department of Home Economics ofthe University of Chicago, Chicago.)

(Received for publication, September 18, 1925.)

The purpose of this investigation was to observe the effect onmineral and nitrogen metabolism of the addition of orange juiceto the diet of growing children. Observations were also made onthe urinary organic acids, ammonia, and hydrogen ion concentra-tion. As oranges have been shown to produce an improved con-dition in the underweight child and as the retention of mineralsand nitrogen by the growing organism is considered an indicationof normality and health, it has seemed possible that oranges mightfunction by favoring a better assimilation.

REVIEW OF LITERATURE.

Orange Juice and Growth in Children.-That oranges are of value incausing a gain in weight of malnourished children has been demonstratedby Newell and Miller (1) who worked with fourteen underweight childrenchosen because they had shown little or no improvement as a result ofgroup instruction in health habits. When 45 cc. of orange juice wereadded daily to the diet of these children and all other conditions were keptunchanged, the percentage of expected gain of the group was increasedfrom 49 for the 4 months preceding the experiment to 132 during the 3months in which orange juice was fed. Another study (2) made in Cali-fornia on 256 malnourished children demonstrated a marked stimulationto growth when oranges were fed as a mid-morning lunch. During 2months in the winter those children who received an orange a day, in

* The data are taken from the thesis of Margaret S. Chaney submittedin partial fulfillment of the requirements for the degree of Doctor ofPhilosophy, in the Ogden Graduate School of Science, University ofChicago, 1925.

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addition to their normal diet, gained an average of 141 per cent above thepredicted, while the children who received no mid-morning lunch gained28 per cent above the expected amount. In the spring the comparativegain above that predicted was 118 for the orange group as against 18 forthe controls.

Components of Orange Juice Which Have Been Shown to Affect Metabolism.-The orange has several components which are of value in regulatingmetabolism. All of the commonly accepted vitamins have been shown tobe present in oranges. Givens and McClugage (3) state that orangeshead the list of foods protecting against scurvy, and Sherman (4) goesso far as to say that "better growth, higher stamina, and better generalhealth and disposition are induced by the feeding of vitamin C in theform of orange or tomato juice." Investigations also show the potencyof oranges in the fat-soluble vitamin (5, 6, 7).

The importance of the antineuritic factor in oranges was demonstratedby Byfield and Daniels (8) who observed that babies not gaining normallyon the prescribed diet of modified milk and 15 cc. of orange juice daily,received a marked stimulation to growth when the juice was increased to45 cc., and they proved that the acceleration in growth was due to a rela-tively large amount of water-soluble B in the fruit, as a similar growthwas not observed when this vitamin was removed. Hess (9) likewise gota gain in weight in infants when oranges were added to the diet; the omis-sion of orange juice was accompanied by a period of stationary weight.

The organic constituents of oranges may aid by supplementing a defi-ciency of minerals in other foods; or by causing a shift in acid-base balancefrom the acid condition of a diet high in meat and cereal foods. Orangesyield a residue basic in character, 5.61 cc. normal base in 100 gm. of orangejuice as calculated by Sherman and Gettler (10). Blatherwick and Long(11) found that the ingestion of from 600 to 2400 cc. of orange juice dailyincreased the pH and the organic acids of the urine and diminished theammonia.

Some Factors Influencing the Retention of Minerals by the Body.

Vitamins.-Investigations on the relationship between the intake ofvitamins A, B, and C and calcium retention have lead to no marked orconsistent conclusions, although a slight favorable influence on calciummetabolism was observed by Miyadera (12) in growing dogs when butterand germinating grains were used instead of the lard and wheat of thebasal diet, and by Bogert and Trail (13) in adult women when butter fator yeast was added to a practically vitamin-free diet. Recently the specialantirachitic factor or vitamin which decidedly influences calcium deposi-tion has been demonstrated (14), and many detailed studies made. Orangejuice has not been proved to contain it.

Acidity of the Gastrointestinal Contents.-The hydrogen ion concentra-tion of the contents of the stomach and upper small intestine controls toa large extent the absorption of calcium and phosphorus. Zacker, Johnson,

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and Barnett (15) have found that a diet favorable to the absorption ofthese elements could be changed to one producing rickets by the additionof alkali. According to Telfer (16) absorption of calcium is hinderedby achlorhydria, by hypoacidity, or by a pathological state in the uppersmall intestine. Jones (17) has successfully used hydrochloric acid ther-apy in rickets.

Acid-Base Ratio.-A diet giving an alkaline residue in metabolism, ac-cording to Shohl (18) is essential in infancy where the body is rapidlydeveloping. Forbes and Keith (19), in a complete review of the work doneon phosphorus metabolism up to 1914, conclude that acid formation in thebody increases calcium and phosphorus excretion especially in the urine,and that the ingestion of acids and acid salts results in a diminished basebalance. Experiments by Bogert and Kirkpatrick (20) on adult womenshowed a somewhat better calcium retention on a base-forming diet thanon a balanced or acid-forming one. Steenbock, Nelson, and Hart (21)have found decalcification of the bones in calves and swine on a highingestion of acid-forming foods but noted no serious effect on growth andreproduction.

Several complete acid-base metabolism studies have been made todetermine the effect of acid and alkali on mineral retention by the body.Baumann and Howard (22) on adding orange juice to the diet of a man sickwith scurvy found a greater retention of calcium, magnesium, sodium,potassium, chlorine, and nitrogen, but the phosphorus stayed in negativebalance throughout. In order to determine the effect of acidosis on theretention of minerals, Sawyer, Baumann, and Stevens (23) substitutedfat for an equal number of calories of carbohydrate in the diet of twonormal children, boys 5 and 8 years old, with the result that the alkali andnitrogen retention was much reduced.

In a complete acid-base metabolism experiment on normal infantsShohl and Sato (24) observed that the normal positive base balance is 10.0cc. L 2 cc. of 0.1 N per kilo per day; acid added to the diet of these infantscaused a smaller mineral retention than normal, and alkali a salt retentionmore alkaline in character.

Intake as Related to Output.-A metabolism experiment conducted bySherman and Hawley (25) on children 3 to 13 years of age shows that onan adequate intake of calcium an average of 0.01 gm. of calcium per kiloper day is stored regardless of the age of the child. The amount of cal-cium retained was shown to vary with the milk (and calcium) intake,the optimum retention amounting to 0.017 gm. per kilo per day when 1000cc. milk were taken. The phosphorus retention was slightly less than thecalcium, 0.008 gm. per kilo per day.

Little has been written concerning the magnesium requirement ofman but the consensus of opinion is that if the calcium quota is suffi-cient the magnesium demand of the body will also be met. Bogert andMcKittrick (26) concluded from metabolism experiments on four adultsthat the magnesium requirement of the body is probably somewhat lowerthan the calcium.

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Calcium-Phosphorus Ratio.-The proportion of calcium to phosphorusin the diet is important in mineral retention. It has been demonstratedon cattle (27) and on rats (28) that an excess of either element in the dietwill cause an increase in the concentration of that element in blood plasma,and a decrease in the other. In experiments on infants by Orr, Holt,Wilkins, and Boone (29) and on children by Schabad (30) similar resultswere obtained-an excess of sodium phosphate improving phosphorusretention and impairing calcium assimilation, while calcium chloridecaused a diminution in phosphorus retention.

EXPERIMENTAL WORK.

In order to ascertain the effect of orange juice on growing chil-dren a metabolism study was conducted without and with orangesin the diet, the retention by the body of calcium, phosphorus,magnesium, and nitrogen determined during both periods, andthe urinary organic acids, ammonia, and hydrogen ion concentra-tion observed daily.

General Procedure.-Two girls, 10 and 11 years old, from theChicago Home for Jewish Orphans were selected as being normalsubjects with no known organic trouble shown by a medicalexamination, with some indications of malnutrition, but "free togain." Two experiments were conducted, the second a duplicateof the first, lasting respectively from April 25 to May 5, 1924,and from July 18 to August 10, 1924. During the two periods thechildren lived away from the Home under the constant care of oneof the writers (M. S. C.) and led a regular, happy life with plentyof sunshine, outdoor air, and sleep.

Each experiment was divided into a non-orange juice and anorange juice period. The procedure was to give the children theexperimental diet for a preliminary 3 days in the first experimentand 5 in the second, in order to attain equilibrium. Next, for 3days the basal diet was continued and corresponding urine andfeces collected. Then after the necessary delay of a day or twofor the lag in the feces, 600 cc. to 700 cc. of the unstrained juice ofCalifornia Valencia oranges were added to the diet and the experi-ment repeated. The diet was continued until the last collection'of feces.

Diet.-The basal diet during the two experiments consisted ofoatmeal, rice, Irish potatoes, white bread, flour, sugar, filteredoleomargarine fat, ground lean beef, and Klim whole milk powder.

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c.p. sodium chloride was kept constant (1.6 gm. per child per day)because of the possibility of its affecting the calcium output (31).Distilled water was used ad libitum for drinking, and also for cook-ing purposes. Table I gives the detail of the food eaten by thechildren during the preliminary and orange juice periods of oneof the experiments. The basal diet was less varied but similar tothat to which the children were accustomed in the Home, whichwas largely carbohydrate, with an adequate supply of calories andprotein, a low fat, mineral, and vitamin quota, and a decidedlyacid residue as computed by use of Sherman and Gettler's figures(10). In the experimental diet the fat was increased over theHome diet so as to cause a' good absorption of calcium, and waskept uniform in amount (32). The food of the non-orange juiceperiod was acid-forming but the addition of oranges changed it toone predominantly basic.

Collection of Urine and Feces.-The 24 hour urines were collectedseparately, beginning with the first meal of the test, and preservedwith toluene in Pyrex flasks on ice. After measuring and makingup to volume, a part was removed for the nitrogen and otheranalyses to be done immediately, and an aliquot set aside to becombined with the other two days collections, for mineral analyses.For the marking of stools, capsules, each containing 0.2 gm. ofpurified wood charcoal which had been thoroughly washed withacid to remove mineral matter, were taken at the beginning andend of each collection period. The feces immediately upon obtain-ing were dried to constant weight on the water bath, alcohol beingadded during the drying, then ground, thoroughly mixed, andpreserved in glass-stoppered bottles in desiccators.

Chemical Analyses.-Oatmeal, rice, flour, and milk powderwere used for analysis as purchased without further drying. Inorder to obtain a representative sample of the ground beef andriced potatoes, a quantity of each sufficient for the 3 day diet wasprepared, and a sample of it dried to constant weight in an elec-tric oven at a temperature below 100°C. A slice of bread fromeach loaf was likewise dried. The orange juice was first evapo-rated to a thick syrup on a water bath and was then dried in theoven. The dried food samples were pulverized finely and pre-served in air-tight containers. The oleomargarine fat and sugarwere not analyzed as they were considered 100 per cent pure; and

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TABLE 1.

Excess in. ~ residue.'*

Food. Measure. A r

Experimental diet, Period 1, without oranges.

gm. ga. gm. CC.N CC.N

Breakfast:Oatmeal ................... 4 cup. 20 80 3.2 1.4 2.6Milk ...................... " 50 33 1.7 2.0 1.2Sugar .................... 2 teaspoons. 10 40Bread ..................... 3 slices. 82 216 7.5 1.1 5.8Oleomargarine fat ......... 1 tablespoon. 12 100 12.0Milk ..................... 1 cup. 150 105 5.0 6.0 3.6

Dinner:Beef ....................... cake. 100 150 23.2 2.5 13.9Potato .................... 1 cup. 200 166 4.4 0.2 14.4Milk* .................... 4 " 40 26 1.3 1.6 0.9Bread ..................... 3 slices. 76 200 7.0 1.0 5.4

Supper:Rice ................... ... cup. 37 130 3.0 0.1 3.0Bread ..................... 5 slices. 212 558 19.5 2.8 15.1Flour ..................... 1 teaspoon. 8 18 0.9 1.1 0.8Oleomargarine fat ......... 1 tablespoon. 14 100 14.0Milk ...................... 13 cups. 200 140 6.6 8.0 4.7

Total ........................................ 2062 83.3 52.7146.6124.8

Experimental diet, Period 2, with oranges.

Foods except bread andorange juice as inPeriod 1 ................. 1088 49.247.9 20.324.8

Bread ................... 11 slices. 353 92832.5 4.6 25.1Orange juice . .............. 3 cups. 700 350 5.6 1.4 39.3

Total ........................................ 236687.353.9 45.464. 1

* Calculations from Bulletin 28 (37).f Analysis by M.S.C.$ Calculations from Sherman and Gettler (10).

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the phosphorus in flour and rice, and the calcium and magnesiumin flour, rice, oatmeal, and beef were not determined becauseof the extremely low percentage of the element in the food and thesmall amounts eaten. It was computed that in the foods notanalyzed a possible variation from the values given by Sherman(33) would have no appreciable effect on the results.

Calcium and magnesium were determined by McCrudden'smethod (34), but using methyl red as an indicator in order to ascer-tain the correct pH for the precipitation of the calcium oxalate.It was found that using the amounts of sodium acetate called forby McCrudden, the solution was on the acid margin, and thatlarger quantities were preferable. The calcium was ignited andweighed in platinum crucibles as calcium oxide; the magnesiumwas precipitated as magnesium ammonium phosphate and weighedas pyrophosphate. The total phosphorus was determined inurine after asking by Neumann's method (35) as directed byMcCandless and Burton (36), precipitating as ammonium phos-phomolybdate and magnesium ammonium phosphate and weigh-ing as pyrophosphate. For all mineral determinations on foodsand feces the ashed samples were used and the same methodsemployed as in urine. Ashing was done according to the OfficialMethod (37), using an electric muffle furnace kept at approxi-mately 450°C.

The micro Kjeldahl method of Koch and McMeekin (38) wasemployed for the nitrogen of the urine, and, with several modifica-tions, for the food and feces. In order to get the two latter intosolution and to work with weighable quantities, amounts con-taining from 30 to 100 mg. of nitrogen were digested for severalhours in a small Kjeldahl flask with 50 cc. of concentrated sulfuricacid. To complete the oxidation several cc. of the 30 per centhydrogen peroxide were then added in small portions, heatingafter each addition, the volume was made up to 100 cc. with dis-tilled water, and 1 cc. used for Nesslerization.

The organic acids of the urine were determined daily by theVan Slyke and Palmer method (39); the creatinine, creatine, andammonia by Folin's (40); and the hydrogen ion concentration bya modification of Henderson and Palmer's method described byHaskins (41). Qualitative tests for sugar, acetone, acetoaceticacids, and albumin were made daily and gave negative results inall cases.

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Ca, P, Mg, and N Retention

All precautions of analytical work were used. Volumetric wareand weights were calibrated, blanks run on all materials, and eachmethod was checked using chemicals of known composition. Allanalyses were done in duplicate or triplicate, usually in triplicate.For all nitrogen, organic acids, ammonia, and pH determinationsfresh, doubly distilled water was used.

RESULTS AND DISCUSSION.

The figures for the analyses of the food are given in Table II.For comparison, food composition figures from Sherman are alsoincluded, and the marked differences in the inorganic constituents,even more than in the nitrogen, illustrate well the necessity ofanalyzing the actual foods used in even approximate metabolismexperiments rather than trusting to published figures. Theresults of the analyses of the urine and feces and the comparisonwith the intake are given in Tables III, IV, V, and VI.

The orange juice used in the second experiment was found byMiss Honora English to contain 0.98 per cent citric acid, 1.5 percent total organic acid (by a modification of the Van Slyke andPalmer method for urine), and 1.03 per cent titratable aciditytoward phenolphthalein.

The addition of orange juice to the diet of the children resultedin an increased retention by the body of calcium, phosphorus,magnesium, and nitrogen.

Calcium Metabolism.-The children both showed a slight posi-tive calcium balance on the basal diet, but this was much increasedin the orange juice period, chiefly through diminished excretion inthe feces (Table III). The extra calcium retention in the orangejuice period was considerably more than the little excess calciumin the diet of that period. For example, in Experiment II withD. B., when the intake of calcium was increased 0.20 gm. for the3 days by the orange juice, an excess of 0.29 gin. was retained; andwith L. F., 0.22 gm. additional calcium ingested resulted in 0.32gm. extra retained. The percentage retained was also increasedfrom 20.6 per cent without oranges, to 31.8 per cent with orangesin the case of D. B., and from 9.9 per cent to 23.2 per cent in thecase of L. F.

The basal intake of calcium was kept at approximately that towhich the children had been accustomed at the Home, and neither

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TABLE II.Mineral and Nitrogen Content of Foods, in Percentage.*

Calcium:Experiment I..................

" II .................

Sherman's figures ..............

Phosphorus:Experiment I..................

" II .................

Sherman's figures ..............

Magnesium:Experiment I..................

" II.................

Sherman's figures ..............

Nitrogen:Experiment I..................

" II.................

Sherman's figures ..............

Beef. Potato

0.01

0.1670. 208

0.1690.174

0.243

0.027

3.663.44

3.223.23

3.71

0.00140.001(

0.00150.002C

0.014

0.0200.031

0.0410.037

0.058

0.0180.025

0.0110.012

0.028

0.330.25

D.393.42

3.35

Klim.

0.934

0.930

0.960'

0.704

.727

0.7441

0.096

0.093

o.096

4.25

4.25

4.22t

Bread.

0.0240.025

0.0610.06&

0.027

0.1020.102

0.0970.091

0.093

0.0300.029

0.0420.049

0.023

1.681.64

1.611.67

1.47

Oat-meal.

0.069

0.31

0.31

0.392

0. 110

2.44

2.65

2.58

Orangejuice.

0.0085

D.0110

3.029

0.019

0.017

0.016

).0137

).0130

).011

0.123

).148

0.128

* Each percentage represents the average of two or three analyses ofthe food; when two percentages are given each represents the food used ina single period.

t This is eight times the value given for whole milk, since Klim dilutedto eight times its weight is the equivalent of cow's whole milk.

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it nor the slightly increased amount of the orange juice period wasup to the optimum of 1 gm. per day recommended by Sherman andHawley (25). It might, therefore, be thought that the increasedretention with the orange juice was due to increased intake withits nearer approach to the optimum. However, the intake of thebasal period in the second experiment was higher than that of thefirst, due to increased bread consumption, and yet the percentageretention was decidedly less and the variation in the actual reten-

TABLE III.

Calcium Balance, Expressed in 3 Day Periods.

D.B. L.F.

Period 1, Period 2, Period 1, Period 2,without oranges. with oranges. without oranges. with oranges.

Experiment I.

gm. per cent gm. per cent gm. per ment gm. per cent

Intake .............. 1.19 100.0 1.33 100.0 1.17 100.0 1.33 100.0Feces .............. 0.72 60.6 0.70 53.0 0.72 61.0 0.76 57.5Urine .............. 0.16 13.1 0.21 14.8 0.13 11.4 0.16 11.7Total output ........ 0.88 73.7 0.91 67.8 0.85 72.4 0.92 69.2Retention .......... 0.31 +26.3 +0.42 +32.2 +0.32 +27.6 +0.41 +30.8

Experiment II.

Intake .............. 2.03 100.0 2.23100. 100. 100.0Feces .............. 1.30 64.3 1.30 58.2 1.41 69.8 1.37 61.2Urine ............... 0.31 15.1 0.22 10.0 0.41 20.3 0.35 15.6Total output ........ 1.61 79.4 1.52 68.2 1.82 90.1 1.72 76.8Retention .......... +0.42 +20.6 +0.71 +31.8 +0.20 +9.9 +0.52 +23.2

tion irregular (with one child greater and the other less). Sher-man and Hawley, also, when they increased the milk intake of theirchildren, had an increase in actual amount of calcium retained,but, in many cases, according to calculations from their figures,a lowered percentage retained. Thus it seems to be demonstratedthat some factor in the orange juice, beside the additional calcium,must have caused the retention of calcium.

The results with phosphorus were similar to those with calcium(Table IV). In the first experiment the children had, during theingestion of the basal diet, a slightly negative balance of 0.06 gm.

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and 0.14 gm. phosphorus, but when the orange juice was added thebalance became positive, 0.52 gm. and 0.57 gm. respectively.The results of the second experiment showed a slightly positivebalance at the beginning and a decided increase in retention on theaddition of oranges. As in all cases but one (Experiment II,L. F.) the additional amount of phosphorus retained on theingestion of oranges was greater than the amount of phosphorusadded by the oranges, it seems probable that here, as in the case

TABLE IV.

Phosphorus Balance, Expressed in S Day Periods.

D.B. L.F.

Period 1, Period 2, Period 1, Period 2,without oranges. with oranges. without oranges. with oranges.

Experiment I.

g. Per cent gm. per cent gm. per am cent gm. per cent

Intake ............. 2.61 100.0 2.91 100.0 2.56 100.0 2.91 100.0Feces ............... 0. 30.7 0.72 24.8 0.72 28.2 0.55 19.6Urine ............... 1.87 71.5 1.66 57.1 1.98 77.3 1.79 61.6Total output.. 2.67 102.2 2.38 81.9 2.70 105.5 2.34 81.2Retention ...... -0.06 -2.2 +0.53 +18.1-0.14 -5.5 +0.57 +18.8

Experiment II.

Intake ............. 3.13 100.0 3.36 100.0 3.14 100.0 3.37 100.0Feces ............... 0.89 28.6 0.96 28.7 0.86 27.3 0.67 20.0Urine ............... 2.11 67.2 1.94 57.6 2.20 70.2 2.45 72.5Total output.. 3.00 95.8 2.90 86.3 3.06 97.5 3.12 92.5Retention ..... +0.13 +4.2 +0.46 +13.7 +0.08 +2.5 +0.25 +7.5

of calcium, there was some factor present which assisted the bodyin making an economical use of the mineral.

Magnesium Metabolism.-In spite of the slightly increased uri-nary and fecal output of magnesium during Period 2, the retentionof magnesium by the body was increased (Table V), but this isless marked than is that of calcium and phosphorus, in which thebody output was actually diminished when oranges were eaten.

Nitrogen Metabolism.-The intake of protein was made fairlyhigh to relieve the monotony of the diet, approximately 13 percent of the total calories in Experiment I, and 15 per cent inExperiment II, and in the latter experiment it was kept the same

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840 Ca, P, Mg, and N Retention

during both periods, the additional nitrogen consumed in theorange juice being counterbalanced by a smaller bread consump-tion.

A marked retention of nitrogen resulted from the ingestion ofthe orange juice (Table VI). This is especially well demonstratedin the second experiment where with a constant nitrogen intakethe nitrogen retained is increased by 2.05 gm. and 1.32 gm. in the3 days when oranges are taken.

TABLE V.

Magnesium Balance, Expressed in 3 Day Periods.

D. B.

Period 1, Period 2,withoutoranges, with oranges.

L. F.

Period 1, Period 2,without oranges. with oranges.

Experiment I.

. per cent gm. per cent m. per cet gm per cent

Intake.............. 68 100. 0.87 100. 0.66 100.0 0.86 100.0Feces .............. 0.25 37.5 0.26 30.2 0.16 23.7 0.22 25.0Urine ............... 0.28 40.5 0.33 38.0 0.21 32.0 0.31 36.4Total output........ 0.53 78.0 0.59 68.2 0.37 55.7 0.53 61.4Retention .......... .15 +22. 0. 28+31 +0.29 +44.3 +0.33 +38.6

Experiment II.

Intake .............. 0.85 100.0 1.18 100.0 0.85 100.0 1.19 100.0Feces............... 0.37 43.0 0.47 39.3 0.34 40.0 0.40 33.5Urine .............. 0.30 34.7 0.34 20.9 0.25 29.0 0.35 29.6Total output ........ 0.67 77.7 0.81 60.2 0.59 69.0 0.75 63.1Retention .......... +0.18 +22.3 +0.37 +39.8 +0.26 +31.0 +0.44 +36.9

It is interesting to compare these results with the work ofYushiue (42) who found that avitaminosis was unfavorable to apositive nitrogen balance, even if a large amount of protein wasfed, and also with the work of Borak (43) who secured less nitro-gen retention on a predominantly acid-forming diet.

Organic Acids, Ammonia, and Acidity.-The addition of theorange juice to the diet increased the pH from 5.6 or 5.9, to 6.45,diminished the ammonia, and increased the organic acid. (TableVII gives the results for the second experiment. The first experi-ment showed no important differences from the second.) The

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results of Blatherwick and Long (11) are thus confirmed and alsothose of McLaughlin and Blunt (44) who found increased urinaryorganic acids after eating other fruits. The increase of organicacid for the two children in the first experiment is equivalent to6.1 per cent and 6.5 per cent of the 20.5 gm. of citric acid ingestedin the orange juice, and in the second to 7.5 per cent and 7.4 percent of the 21.6 gm. ingested.

Creatinine and Creatine.-These two substances were determinedas a step in the correct determination of the organic acids (Table

TABLE VI.

Nitrogen Balance, Expressed in S Day Periods.

D.B. L.F.

Period 1, Period 2, Period , Period 2,without oranges. with oranges. without oranges,. with oranges.

Experiment I.

gm. per cent g. per cent gm. per cent am. per cent

Intake .............. 31.58 100.0 34.48 100.0 30.98 100.0 34.53 100.0Feces ............... 2.46 7.8 2.54 7.4 3.83 12.4 3.47 10.0Urine............... 28.22 89.0 27.86 80.8 25.31 81.7 24.96 72.3Total output........ 30.68 96.8 30.40 88.2 29.14 94.1 28.43 82.3Retention.......... +0.90 +3.2 +4.08 +11.8 +1.84 +5.9 +6.10 +17.7

Experiment II.

Intake.............. 41.20 100.0 41.22 100.0 41.29 100.0 41.42 100.0Feces............... 2.44 5.9 3.89 9.4 4.06 9.8 3.65 8.8Urine ............... 32.00 77.7 28.52 69.2 30.81 74.6 30.03 72.5Total output ....... 34.44 83.6 32.41 78.6 34.87 84.4 33.68 81.3Retention ..... +6.76 +16.4 +8.81 +21.4+6.42 +15.6 +7.74 +18.7

VII). The creatine excretion of the girls was remarkably high,several times half of the total creatinine. Apparently it wasuninfluenced by the orange juice.

Gain in Weight during the Experiments.-When orange juicewas added to the diet both children showed marked gain in weight(Table VIII). During the ingestion of the basal diet there was amuch better gain than that obtained while at the Home and greaterthan that expected for children of their ages, an improvementwhich may be attributed to the quieter life led by the girls and

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842 Ca, P, Mg, and N Retention

especially to the larger food intake; but on the addition of oranges,the gain in weight was three to four times as much daily as in theperiod when oranges were not included in the menu.

This is hardly explainable merely on the basis of the additionalcalories ingested-about 300 more when oranges were added to

TABLE VII.

Total Acidity, Ammonia, Organic Acids, Creatinine, and Creatine inthe 24 Hour Urine.

Experiment II.

D. B., Period 1:Day 1 ........................

2........................It 3........................

Average ......................Period 2:

Day 1 ........................" 2........................

3........................

Average ......................

L. F., Period 1:Day ........................

2........................

3........................Average ......................

Period 2:Day ........................

2........................3........................

Vol-ume.

cc.

94012501040

136012681390

12401235

935

121012151426

pH

5.95.95.9

6.456.456.45

5.95.95.9

6.456.456.45

Average ................... .........

Ammo-nia

nitro-gen.

mg.

523549474515

446446386426

500500500500

403369414

395

Or-ganicacid0.1 N.

cC.

290161229227

287336308310

182218238213

288282319

296

Pre-formedcreati-nine.

mg.

793704739745

721867758782

730632666676

646769678

698

Crea-tine.

mg.

331407332357

446446386426

402534503480

492323411

409

the diet-for an even larger increase in calories from the Home dietto that of the basal diet of the experiment did not cause the markedstimulation in growth. In Experiment II in the 9 days of theorange period D. B. gained 2.9 pounds as against 1.2 pounds in the14 days of the basal diet, or figured per day, 3.7 times as much in

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M. S. Chaney and K. Blunt 843

the orange period. L. F.'s gain increased from 1.2 pounds inthe basal diet period to 3.3 pounds when oranges were eaten,being 4.3 times as rapid when oranges were fed.

How the orange brings about the favorable nitrogen and mineralretention is unknown. The effect may be due to one or several ofthe following factors-the vitamins which promote the economicaluse of elements already present but not efficiently used; the addi-tional calcium and phosphorus which induces the retention ofthese and possibly other elements in excess of the amount added;some factor which stimulates a greater flow of hydrochloric acid in

TABLE VIII.

Gain in Weight as Influenced by Orange Feeding.

Experiment. Subject. Length of First Last Gain in weightperiod. weight. weight. per day.

Period 1, without oranges.

days lbs. lbs. lb.

I D. B. 5 65.6 66.2 0.12I L. F. 5 61.2 61.7 0.10

II D. B. 14 66.6 67.8 0.085II L. F. 14 61.1 62.3 0.085

Period 2, with oranges.

I D. B. 5 66.2 67.1 0.18I L. F. 5 61.7 63.4 0.34

II D. B. 9 67.8 70.7 0.32II L. F. 9 62.3 65.6 0.37

the stomach, causing a greater acidity in the upper part of thesmall intestine and a greater absorption of minerals; or the basicresidue which may favor normal activities in the growing body.Work on the solution of these problems is planned.

SUMMARY.

A study was conducted on two growing girls, 10 and 11 yearsold, to determine the effect of orange juice on calcium, phosphorus,magnesium, and nitrogen metabolism.

The basal diet consisting of oatmeal, rice, flour, bread, potato,

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844 Ca, P, Mg, and N Retention

sugar, oleomargarine fat, lean beef, and whole milk powder wasingested for a preliminary adjustment period and a 3 day collec-tion period. Then orange juice, 600 to 700 cc. daily, was added tothe diet; another adjustment period and collection period followed.After an interval of 2½ months the experiment was again performedon the same girls.

Calcium assimilation was decidedly benefited when orangesformed a part of the diet, the increased retention being consider-ably greater than the calcium added in the oranges and greaterthan might be expected from a stimulus to retention caused by alarger calcium intake.

The increase in phosphorus retention was even more markedthan that of calcium, more than three times as much phosphorus,both in gm. and percentage of intake, being assimilated whenorange juice was added.

The magnesium retention was also increased, although to a lessmarked extent than that of calcium and phosphorus.

Nitrogen assimilation was greater when orange juice wasingested, even though the nitrogen intake was not altered.

Urinary ammonia was decreased, and urinary pH and organicacids increased, thus confirming the work of Blatherwick and Long.The increase of organic acids amounted to approximately 7 percent of the citric acid of the orange juice ingested.

A marked increase in the children's weight was observed.

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Margaret S. Chaney and Katharine BluntORGANIC ACIDS OF GROWING CHILDREN

NITROGEN RETENTION AND URINARY ANDCALCIUM, PHOSPHORUS, MAGNESIUM,

THE EFFECT OF ORANGE JUICE ON THE

1925, 66:829-845.J. Biol. Chem. 

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