ACIDIFICATION OF THE URINE BY INFANTS FED HUMAN MILK AND WHOLE COW'S MILK

(1)

ACIDIFICATION

OF

THE

URINE

BY INFANTS

FED

HUMAN

MILK

AND

WHOLE

COW’S

MILK

By Samuel J. Fomon, M.D., Dorris M. Harris, M.D.,

and Robert L. Jensen, B.S.

Department of Pediatrics, College of Medicine, State University of Iowa

113

T

HE STUDIES to be reported were under-taken to define the extent of the differ-ence in acidification of the urine by two groups of infants receiving quite different feedings. One group received human milk.

The intake of protein and of salts of this

group was therefore relatively low. The

other group received a high-protein feeding

of cow’s milk providing relatively large amounts of salts.

SUBJECTS AND PROCEDURES

Eight normal infants were studied before and after administration of ammonium chloride. Group I consisted of four infants (M.G., V.5., S.P. and EQ.), fed fresh or pasteurized human milk, and Group II, of four infants (A.H., T.W., L.T. and MO.) fed either evaporated milk diluted with an equal volume of water or homogenized whole cow’s milk. Additional vitamins were given to infants of each group. Carbohydrate was not added to the feedings and none of the infants received solid foods. Four or five feedings were given daily between 6 A.M. and 10 P.M.

Two infants of each group (MG. and V.5. in Group I; A.H. and T.W. in Group II) lived

in a previously described1 Metabolism Ward

from the time of discharge from the newborn

nursery (5 to 8 days of age) until completion

of the studies reported here. The other infants lived at home with their parents and were admitted to the Metabolism Ward only for col-lection of the specimens of urine. E.Q. and S.P. were breast-fed from the time of birth until completion of the studies reported here;

M.G. and V.5. had received pasteurized human

milk for 55 and 31 days, respectively; L.T. and T.W. had received evaporated milk and water for 91 and 85 days, respectively; A.H. and

MO. had received homogenized whole cow’s

(Accepted July 14, 1958; submitted June 14.)

Supported by grants from Mead Johnson and Co.,

ADDRESS: (S.J.F.) Iowa City, Iowa.

milk for 63 and 36 days, respectively.

All collections of urine were made during the 12-hour period from 7 i.isi. to 7 AM. Urine

was collected from boys with the apparatus previously described,1 the collecting jar being surrounded by ice throughout the collection. No preservative was employed. By close

ob-servation of the girls it was usuall\ possible to

remove urine from the container into which

it was voided before it became contaminated

with stool. Aliquots of urine were stored in a refrigerator. On several occasions a specimen of urine was contaminated with stool. Such a specimen was strained, measured, and dis-carded, the discarded specimen being assumed to have the same composition as the remainder of the 12-hour specimen.*

Specimens of venous blood were drawn from an external jugular vein 1 or 2 hours after termination of the collection of urine. The content of carbon dioxide and the pH of the blood were determined immediately. The con-centration of urea iii blood and urine and the concentration of ammonia in urine, as well as the titratable acidity, were determined within a few hours after completion of the 12-hour collection of urine.

After initial study of each infant, ammonium chloride in a cinnamon-flavored syrup was ad-ministered in dosage of 4 gm (i.e., 77 meq of chlonide)/m2/24 hr. Divided doses were given between 7 AM. and 10 P.M. for 2 days. A second 12-hour specimen of urine was then obtained. The last dose of ammonium chloride

was given after the beginning (7 P.M.) of the 12-hour period of collection of urine for

analysis.

0 The instances in which urine was contaminated

with stool and the volume so contaminated were as follows: MG. age 118 days, 34 ml; VS. age 108

days, 76 ml; age 111 days, 90 ml; L.T. age 96

days, 94 ml; age 99 days, 99 ml.

Ross Laboratories and National Dairy Council.

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114

METHODS

Blood was drawn in a heparinized syringe and the pH determined with a Beckman Model

GS, pH meter, using a Beckman 290-31 elec-trode immersed in a water bath at 37#{176}C.Con-tent of carbon dioxide in whole blood was

determined by a slight modification of the

method of Conway,2 with 0.2 normal solution of barium hydroxide in the central well of the Conway “unit.”

The effective osmolanity of the urine was de-termined from the depression of the freezing

point as measured with a Fiske osmometer.

Concentrations of urea in plasma and urine and ammonia in urine were determined by the microdiffusion method of Conway and O’Malley.3 The concentration of chloride in plasma and urine was determined

polarographi-cally by the method of Zimmerman and

Lay-ton,’ and concentrations of sodium and p0-tassium by flame photometry. The

concentra-tion of inorganic phosphorus in urine was

de-termined by the method of Fiske and

Sub-barow.5 Titratable acidity was measured by titration of an aliquot of urine to pH 7.4, using a Beckman Model H-2, pH meter.

RESU LTS

Results of study of blood and urine of infants fed human milk (Group I) are pre-sented in Table I and those of infants fed whole cow’s milk or evaporated milk (Group II) are presented in Table II. A summary of the findings is presented in Table III.

Findings in Blood

It may be seen that the dosage of am-monium chloride was sufficient to produce

metabolic

acidosis, usually mild, in at least

some of the infants. Acidosis in the infants

of Group II tended to be somewhat better

compensated. The concentration of chloride did not change significantly in the blood of infants of either group. Concentrations of

urea were high, as was to be expected, in

blood

of infants receiving diets with greater content of protein (Group II). Increase in concentration of urea after administration

of

ammonium chloride was not observed in either group.

Findings in the Urine

BEFORE ADMINIsm&TIoN OF AMMONIUM

Cin.omnE: The mean pH of the urine of in-fants of Group I was 6.4 compared with 5.3 in the urine of infants of Group II

(

Table III). The mean rate of excretion of

total

solutes and of each of the determined

individual solutes was greaten by infants

of Group II than by infants of Group I. The mean rate of excretion of ammonia by

in-fants of Group I (7 meq/m2/12 hr) was

somewhat greater than the titratable acidity

(

3 meq/m2/12 hr), while the reverse was true with infants of Group II: the mean

rate of excretion of ammonia (26 meq/m2/12

hr)

was somewhat less than the titratable

acidity (32 meq/m2/12 hr). The mean rate

of excretion of phosphorus was 3 mmol/

m2/12 hr in Group I compared with 35 mmol/m2/12 hr in Group II.

AFTER ADMINIsTitTIoN OF AMMONIUM

CHLORIDE: The mean pH of the urine of

in-fants

of Group

I decreased

from 6.4 to 4.7

while that of infants of Group II decreased

from

5.3 to 5.0. The rate of excretion of total

solutes

by all of the infants except T.W.

increased after administration of ammonium

chloride.

In Group I the mean rate of excre-tion increased from 104 to 170 mosmol/m2/

12 hr and in Group II from 485 to 532

mosmol/m2/12 hr. The increase in both

groups

was attributed primarily to increases

in rates of excretion of chloride and

am-monium ions. The mean increase in titrat-able acidity after administration of

am-monium chloride (from 3 to 6 meq/m2/12 hr for Group I and from 32 to 34 meq/m2/

12 hr for Group II) was small in comparison

with the mean increase in rate of excretion

of

ammonia (from 7 to 21 meq/m2/12 hr for

Group I and from 26 to 49 meq/m2/12 hr

for Group II).

The mean augmentation in excretion of

ammonia after administration of ammonium chloride was significantly greater for in-fants of Group II than for infants of Group

I (p<O.01).

Because only the last dose of ammonium

(3)

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SUMMARY OF FINDINGS IN BLOOD AND URINE BEFORE AND ArI’ER ADMINISTRATION OF AMMONIUM CHLORIDE TO INFANTS FED lItMAN MILK AND WHOLE Cow’s MILK

Specimen

human Mill Whole Con’s Milk

Before ?S114(l After N!14(’l

mean mean

(range) (range)

Before ?‘i’114C1 After N114(’l

mean mean

(range) (range)

Whole blood

p11

(202 content (rninol/l)

Plasma

Cl (meq/l)

1_Trea nitrogen

(mg/100 nil)

103 (93-111)

104 9-1 13)

241 219

(‘206-’275) (135-325)

5.3 5.0

(4.9-5.6) (4.6-5.4)

485 532

(437-556) (5()5-63)

7.41 7.31

(7 .40-7 .42) (7.24-7 .40)

‘26 16

(4.4-6.8) (7.1 -19.0)

107 107

(104-110) (91-126)

6 7

(5.3-7.4) (4.3-9.9)

234 ?13

(13’2-386) (9’2-340)

6.4 4.7

(5.8-6.9) (4.3-5.0)

104 170

(74-135) (143-19’2)

7

(5.9-7.4) (15.6-24.2)

3 6

(0.6-5.1) (3.9-7.6)

32 44

(24-49) (30-60)

8 16

(-3) (9-3)

14 43

(5-’26) (38-45)

16 13

(12-24) (6-20)

3 4

(0.1-5.3) (1.7-8.1)

hour period of collection of urine, the amount of urea formed by the liver from the ammonia during this period would not

be expected to be great. With infants of

p11

Total solutes

(mosmol/iii2/1 hr)

Anuiioiiia

(meq/m2/12 lir)

L’itratal)le aei(lity

(Ineq/IIi2/12 lir)

Ijrea

(IflIllOl/IIl2/12 lir)

Sodium

(meq/In2/1 lir)

Chloride

(meq/in2/12 hr)

Potassium

(meq/rn2/12 hr)

Phosphorus

(iiiinol/m2/1’2 1w)

3 34

(27.0-39.4) (24.1-39.6)

209 196

(17O-25) (178-35)

35 (31-38)

41 (31-51)

55

(3-77)

f29

(H-57)

71

(53-102)

44

(‘21-57)

TABLE III

Urine

Volume (ml)

7.43 7.39

(7.41-7.44) (7.36-7.42)

23 18

(0.3-28.1) (16.3-18.5)

2O

(14.4-25.8) (16.7-26.3)

26 49

(17.5-30.8)

(44.4-58.5)

35 35

(8.1-43.5)

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in-fants after administration of ammonium

chloride. With infants of Group II, a

de-crease in rate of excretion was observed

with three infants and an increase with only

one.

The augmentation in rate of excretion of

chloride after administration of ammonium

chloride

was similar with each group (Table

III). The rate of excretion of carbon dioxide in the urine (not shown in the tables) was never greater than 1.2 mmol/m2/12 hr.

DISCUSSION

Acidification of the urine, as the phrase

is used here, applies to the secretion of hydrogen ions, whether measured as

titrat-able acidity or combined with free ammonia

(

NH) to form ammonium ion (H4#{247}).

Hunt#{176}has emphasized the importance of

the dietary intake of sulfur on acidification of the urine of adult subjects. The greater

concentration of sulfur in cow’s milk (30

mg/100 ml) than in human milk (14 mg/100

ml)7

may be responsible, at least in part,

for the greater acidification of the urine of

infants of Group II than of those of Group

I.

Titratable Acidity

The similarity in rates of excretion of

phosphorus and titratable acidity (Tables I

and II) is not fortuitous but a reflection of the pre-eminent importance of phosphate in

formation of titratable acidity. Other

urinary buffers are much less important.

The presence of dibasic phosphate in the

urine permits conservation of 1 mmol of

cation for each millimol of hydrogen ion

secreted by the renal tubular cells. The

greater amount of phosphate in the urine

of infants of Group II permits the produc-tion of greater titratable acidity.

At pH 6.4, the mean pH of the urine of infants of Group I before administration of

ammonium chloride, approximately 24% of

the phosphate is in the dibasic form.

How-ever, because the amount of phosphate in

the urine is small, there is little opportunity for saving of fixed cations through exchange with hydrogen ions and resultant conversion

of dibasic to monobasic phosphate. The

situ-ation is different with infants fed whole

cow’s milk. Although the amount of

phos-phate in the urine is 10 times as great, the pH of the urine with this feeding is so low (mean pH 5.3) that only about 3% of the

phosphate is in the dibasic form. Therefore, the possibility of conserving base by further

decrease in pH of the urine is limited with

this feeding also.

Excretion of Ammonia

The

rate

of excretion

of ammonia may be

considered to be dependent on the rate of

formation of ammonia within the tubular

cell and the rate of transport of ammonia from tubular cell into the fluid within the

tubular

lumen. According to current

con-cepts,8” this rate of transport is dependent

on the availability of hydrogen ions in the fluid within the tubular lumen. Ammonia is

believed

to diffuse

passively in the

union-ized state (NH3) from tubular cell into the

fluid within the tubular lumen where

com-bination with hydrogen ion results in the

formation of ammonium ion (NH4).

After

administration of ammonium

chlo-ride, the pH of the urine was less in Group I

than in Group II.* The greater rate of

excretion of ammonia by infants of Group II is evidence that the rate of excretion of

ammonia is not primarily dependent on pH. The greater excretion of ammonia by

in-fants of Group II must, therefore, be

as-cribed

to a greater rate of formation of

am-monia within the tubular cell. The factors known to influence the rate of formation of ammonia are amount and duration of acid

intake, presumably resulting in variation in intracellular activity of the deammnating mechanism,1214 and availability of

precur-sons of ammonia.1517

After administration of ammonium

chlo-ride,

the greater rate of formation of

am-monia by infants of Group II than by those of Group I may be attributed jointly to

0 The greater decrease in pH of the urine of

infants of Group I may be attributed to the smaller

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ARTICLES

1) the prolonged ingestion of the high

pro-tein (i.e., acid-ash) diet, and to the greater

total

amount of acid intake (resulting from

the cumulative effect of ingesting am-monium chloride and high protein diet), and, possibly, 2) the greater availability of glutamine, its precursors, and other amino

acids for deamination in renal tubular cells.

In whole cow’s milk the concentrations of glutamine, glutamic acid, ornithine, and

other precursors of glutamine are

considera-bly greater than in human milk.7

It is probable that after administration of

ammonium chloride for 2 days the rate of excretion of ammonia was not maximal with

either group of infants.15 Whether

adminis-tration of ammonium chloride for a longer

period

of time would have resulted in more

nearly equal rates of excretion by infants

of

the two groups cannot be stated.

SUMMARY

Acidification of the urine was studied with two groups of infants, each group

con-sisting of four infants between 2 and 6

months

of age: Group I received fresh or

pasteurized

human milk and Group II

re-ceived

whole cow’s milk on evaporated milk

and water without additional carbohydrate. The mean titratable acidity of the urine

of

infants of Group I was 3 meq/m2/12 hr compared with 35 meq/m2/12 hr for infants of Group II. The rates of excretion of

am-monia were 7 and 28 meq/m2/12 hr by

infants of Group I and II, respectively. After administration of ammonium chloride (4 gm/m2/day) for 2 days, the titratable

acidity increased to 6 meq/m2/12 hr in

Group I and to 34 meq/m2/12 hr in Group II. The mean rates of excretion of ammonia

increased

to 21 and 36 meq/m2/12 hr,

re-spectively.

The greater titratable acidity of the urine

of infants of Group II is attributed

pri-manly

to the greater amounts of phosphate in the urine (mean, 35 mmol/m2/12 hr

ex-creted by infants of Group II compared with 3 mmol/m2/12 hr by infants of Group I). The greater rate of excretion of ammonia

by infants of Group II is attributed jointly

to the prolonged administration of a diet

with relatively great residue of anions and,

perhaps, to the greater availability of

gluta-mine and other precursors of ammonia.

REFERENCES

1. Fomon, S.

J.,

Thomas, L. N., Jensen, R. L., and May, C. D. : Determination of nitro-gen balance of infants less than 6 months

of age. PEDIATRICS, 22:94, 1958.

2. Conway, E.

J.

: Microdiffusion Analysis and

Volumetric Error, 3rd Ed. New York, Van Nostrand, 1950, p. 208 if.

3. Conway, E.

J.,

and O’Malley, E. :

Micro-diffusion methods. Ammonia and urea

using buffered absorbents (revised

methods for ranges greater than 10 p.g.N). Biochem.

J.,

36:655, 1942.

4. Zimmerman, W.

J.,

and Layton, W. M.,

J

r. : A polarographic micromethod for the determination of blood chloride.

J.

Biol. Chem., 181:141, 1949.

5. Fiske, C. H., and Subbarow, Y. : The colonimetnic determination of phos-phorus.

J.

Biol. Chem., 66:375, 1925. 6. Hunt,

J.

N. : The influence of dietary

sul-fur on the urinary output of acid in man.

Clin. Sc., 15:119, 1956.

7. Macs’, I. G., Kelly, H.

J.,

and Sloan, R. E.:

The Composition of Milks. A

Compila-tion of the Comparative Composition and Properties of Human, Cow and Goat Milk, Colostrum, and Transitional Milk. Washington, D.C., National Academy of Sciences-National Research Council, Pub-lication 254, 1953.

8. Pitts, R. F. : Renal excretion of acid. Fed.

Proc., 7:418, 1948.

9. Pitts, R. F. : Acid-base regulation by the

kidneys. Am.

J.

Med., 9:356, 1950.

10. Gilman, A., and Brazeau, P. : The role of the kidney in the regulation of acid-base metabolism. Am.

J.

Med., 15:765, 1953. 11. Orloif,

J.,

and Berlinger, R. W. : The mechanism of the excretion of ammonia in the dog.

J.

Clin. Invest., 35:223, 1956.

12. Davies, B. M. A., and Yudkin,

J.

: Studies

on biochemical adaptation. The origin of urinary ammonia as indicated by the effect of chronic acidosis and alkalosis on some renal enzymes in the rat.

Bio-chem.

J.,

52:407, 1952.

13. Rector, F. C., Jr., Seldin, D. W., and

Copenhaver,

J.

H. : The mechanism of

ammonia excretion during ammonium chloride acidosis.

J.

Clin. Invest., 34:20, 1955.

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120

ammonia excretion in the rat. Am.

J.

Physiol., 182:131, 1955.

15. Van Slyke, D. D., Phillips, R. A.,

Hamil-ton, P. B., Archibald, R. M., Futchen, P. H., and Hiller, A. : Glutamine as

source material of urinary ammonia.

J.

Biol. Chem., 150:481, 1943.

16. Lotspeich, W. D., and Pitts, R. F. : The role of amino acids in the renal tubular

secretion of ammonia.

J.

Biol. Chem.,

168:611, 1947.

17. Kamin, H., and Handler, P. : The metabo-lism of parenterally administered amino acids. III. Ammonia formation.

J.

Biol.

Chem., 193:873, 1951.

18. Ryberg, C. : On the formation of ammonia

in the kidneys during acidosis. Acta

physiol. scandinav., 15:114, 1948.

PEDIAi-mc INDEX, Edwin F. Patton, M.D.

St. Louis, C. V. Mosby Co., 1958, 640 pp.,

$13.50.

While one must admire the diligence and

dedication which enabled the author to spend

“six years of laborious reading, study, and writing” in the preparation of this book, it is difficult to evaluate its usefulness. It can only be suggested that if a practitioner feels the need of another “index,” similar to The Corn-pleat Pediatrician, and believes that this sort

of compilation actually facilitates the clinical management of patients, this book deserves to be examined. If he is faced with an infant with diarrhea, termed entenitis in the Index, he will be advised to rule out mononucleosis,

brucellosis, influenza, virus disease, food

pois-oning, various types of arthritis, meningitis, food intolerance, etc.! Treatments listed range from mild sedation and restriction of diet through a list of antibiotics which have been recommended for the treatment of diarrhea extending from the suffonamides to Synnema-tin B#{174}.For correction of dehydration and acid-base disturbances, one will learn that

sev-eral suitable solutions are available commer-cially. Other general considerations pertain-irig to parenteral-fluid therapy are essentially abbreviations of material readily available in

textbooks of pediatrics.

It is difficult to escape the conclusion that if physicians feel the need for this sort of book,

the education received in medical school has not been very effective. It cannot be denied that occasionally a particular fact may be found in this single volume more conveniently than by turning to several standard textbooks or the indices of the pediatric journals.

The original suggestion may be

repeated-each person will have to judge the usefulness

of this book for himself. The misgivings of a single reviewer should not be considered a representative reaction. It would certainly lighten the burden of clinical teaching if one could acquire faith in the usefulness of the approach represented by this Pediatric Index.

Perhaps the author will forgive an expression of skepticism, and the sales and longevity of the book will be “the proof of the pudding.”

(9)

1959;23;113

Pediatrics

Samuel J. Fomon, Dorris M. Harris and Robert L. Jensen

WHOLE COW'S MILK

ACIDIFICATION OF THE URINE BY INFANTS FED HUMAN MILK AND

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