Effect of lactose on nitrogen metabolism and phenol excretion in infants

EFFECT

OF L A C T O S E ON N I T R O G E N M E T A B O L I S M A N D PHENOL EXCRETION IN INFANTS

DO~aLD A. COW,ELY, M.D., LEwis A. BARNESS, M.D., A~D

PAUL GYORGY, M.D. PHILADELPHIA, PA. E T E N T I O N of p r o t e i n b y the inis related to the biological value of the protein in the diet, the a d e q u a e y of the intestinal tract, and other factors. D u n c a n 1 has summarized, in an excellent review, present k n o w l e d g e of the physiologicaI effects of Iaetose. These effects include increased calcium absorption, acidurie intestinal flora with p r e d o m i n a n c e of Lactobacitlus bifidus, and alterations in absorption of fats, vitamins, and other earbohydrates. D u n c a n states t h a t lactose in the diet " p r o d u c e s healthy, firm-fleshed babies with few gastrointestinal u p s e t s . " The effect of d i e t a r y lactose on protein metabolism is unknown. In the elassie s t u d y b y Gordon and associates 2 on n i t r o g e n retention b y premature infants, h u m a n milk, whieh contains lactose as the only c a r b o h y d r a t e , was c o m p a r e d with s k i m m e d c o w ' s m i l k fortified w i t h D e x t r i - ~ a l t o s e and olive oil or eMeium paraeaseinate. W h e n these two milk f o r m u l a s were fed at equiprotein, equiealorie levels, n i t r o g e n r e t e n t i o n was a p p r o x i m a t e l y the same. Metabolic p r o d u c t s of protein metabolism include urea, ammonia,

R fant

From the Philadelphia General Hospital. and the Department of Pediatrics, School of lV[ecl i e i n e, University of Pennsylvania. Studies were carried out in the Pediatric Department of the Philadelphia Genezal Hospital.

amino a r i d s , and polypeptides. B r e a k d o w n p r o d u c t s of the a r o m a t i c amino acids include phenols and phenolic compounds. I t m i g h t be expected t h a t changes in food composition would alter u r i n a r y phenol excretion, either t h r o u g h change in intestinal flora or differences in interm e d i a r y protein metabolism. Phenols are n o r m a l l y excreted in small a m o u n t s in h u m a n urine or m a y be f o u n d in increased quantities in patients w i t h p h e n y l k e t o n u r i a 3 and following ingestion of certain phenolic toxic agents. Phenols are m a i n l y detectable in the urine a s metabolic p r o d u c t s of t r y p t o p h a n e and tyrosine metabolism, ~ or as a b s o r p t i o n products of intestinal p u t r e f a c t i o n ? Levine and co-workers 6 have noted the excretion o f p-hydroxyphenyllactie and p - h y d r o x y p h e n y l p y r u v i e acids in the urine of p r e m a t u r e i n f a n t s fed tyrosine and phenylalanine. These phenolic acids decreased in the urine of the i n f a n t s a f t e r a d m i n i s t r a t i o n of aseorbie acid or a f t e r decreasing the protein intake. I n l a r g e quantities, free phenols are toxic. Less is k n o w n of the significance of the phenolic acids. Phenols are usually detoxified in the liver. Detoxifieation p r o d u c t s include glueuronides, sulfates, and combinations with glyeine. 40

CORNELY

ET

AL. :

EFFECT

OF

LACTOSE

The present s t u d y was designed to determine protein retention after feeding" commonly used i n f a n t formulas. The chief difference between the formulas lay in the presence of lactose or Dextri-Maltose as the source of carbohydrate. METHOD

Five healthy male infants were seIected f r o m the n u r s e r y of a large general hospitaV ~ and placed on balance studies. T h e y had not previously been breast fed n o r had they received antibiotics. Two concentrated, sterilized liquid ~ormulas were p r e p a r e d as follows: 1. Lactose FormulM Cow's milk protein Blend of vegetable and animal Lactose

fats

2. D.IVL Formula~ Cow's milk protein Butterfat Dextri-Ma~tose No. 1

1.5% 3.5% 7.0% 1.5% 3.6%

&8% (to make total carbohydrate 6.8%)

I n the foreperiod of seven days, the lactose formula was fed. The lactose f o r m u l a was t h e n fed to each of the babies f o r a period of twelve to t w e n t y days, followed b y the Dextri-MaRose f o r m u l a f o r a similar period, a f t e r which the lactose formula was reinstituted for the t h i r d period of the study. All babies were given daily supplements of oral multiplc vitamin drops. The ages of the babies and length of feeding periods are shown in Table I. *Philadelphia General l-Iospital. "~Lactose formula (S-M-A, food formula for infants) kindly furnished by Wyeth Laboratories. $D.M. formula prepared from available ingredients in a manner similar to S-M-A by Wyeth Laboratories.

ON NITRO(~EN

AND

PHENOLS

41

A 1-month-old baby was fed frozen and reheated human milk containing 1.1 per cent protein, and a diluted cow's milk formula containing 1.1 per cent protein, 7 p e r eent lactose, and 3.2 p e r cent fat. Phenol excretions were d e t e r m i n e d during these feedings. The total excretions of urine and of stools were collected. The techniques of p r e p a r a t i o n of urine and stools and methods of analysis for tile nitrogen components were similar to those described in a previous study. 7 E x c r e t i o n of phenolic compounds in the urine was d e t e r m i n e d b y a method essentially similar to that described by B r a y and Thorpe s and b y l~ogers and associates2 Ten cubic centimeters of urine was b r o u g h t to p H 8 with 2 per eent N a O t t and saturated with NaC1. The urine was placed in s e p a r a t o r y funnels in an automatic shaker and ext r a c t e d t h r e e times with 25 c.c. ether for ten minutes. Ten milliliters of w a t e r was added to the combined extracts, and the ether carefully evaporated. One milliliter of the aqueous residue was diluted to 10 ml. with water, and I ml. Folin-Cioealteau reagent and 2 ml. of 2 per cent sodium carbonate were a d d e d ; the blue color developed b y the ether-extraetable phenols at p i t 8 was read in a photoelectric colorimeter with a red filter in comparison with phenol as a standard. The urine in the s e p a r a t o r y funnel was then brought to p i t 1 with 10 N I-I~SOd, and immediately e x t r a c t e d in the same way. The color was developed and read in a similar manner. The s t a n d a r d used was p-hydroxyphenylacetic acid.

TABLE I.

BALANCE

DATA

28

3.18

3.54 3.85 4.30 4.55

15 24 18

AVG. WT. GAIN (G~./ DAY) 21 19 7

2.

31

3.20

3.50 3.68 4.15 4.40

15 18 18

12 26 14

I II l

19.40 23.96 19.87

3.

17

3.56

3.63 3.81 4.08 4.28

15 15 15

12 18 13

I II I

19.25 19.04 19.73

5,76 6.95 9.37'

3.05 2.84 4.23

10,44 9.25 6.]3

56 49 32

4.

17

2.45

2.53 2.82 2.96 3.07

15 12 18

19 12 6

I II I

17.85 22.81 19.91

4.77 4.72 9.62

4.36 2.00 2.63

8.72 6.09 7.66

49 47 39

5.

24

3.20

3.40 3.67 3.87 3.98

15 15 15

18 13 14

I %t I

18.67 18.50 18.78

8.]3 8.98 8.50

3,30 3.15 2.58

7.24 6.37 7.60

39 35 41

]6 18 11

I II I

1.27 1.27 1.22

0.53 0.64 0.68

0.26 0.24 0.21

0.48 0.39 0.33

38 31 28

BABY NO. 1.

AGE

BIRTH WT. ]VT. *

(DA.) [ (KG.)

DAYSDE ITON

Daily averago

%

~t

~t

Nf

FORMULA I II I

INTAKE GM. 19.94 32.03 23.88

URINE GI~. 11.65 20.26 16.25

STOOLS G1Vs 3.70 7.69 4.35

TOTAL BAL. 4.59 4.08 3.28

RETEN TION 23 13 24

10.52 14.26 13.46

5.03 5.72 3.47

3.85 3.98 2.94

20 16 15

-

* W e i g h t in k i l o g r a m s . F i r s t t h r e e w e i g h t s for each b a b y w e re d e t e r m i n e d a t t h e s t a r t of each new f e e d i n g period. The f o u r t h w e i g h t r e p r e s e n t s t he w e i g h t a t t h e conclusion of the l a s t f e e d i n g period. t T o t a l n i t r o g e n for the t o t a l n u m b e r of d a y s of the s t u d y on each formula. r|~ABLE l I .

BABY

No. 1.

FORMuLA I Ii i

PI{ENOL EXCRETION AND STOOL p~-~

I~G./DAY* PIIEI'70LS ~EEEt EAcID~/ ~ 0 ~ { 3.3 7.2 3.8 14.3 6.0 14.9 6.3 27.2 3.7 11.9 4.8 21.4

CREATININE I ]~ILLIMOLES

/pay

PHENOL

X 10/0r

]FBEEtI~7OO~7~.~]TOTAL

STOOL

p~

4.5 6.9 7.1

7.3 8.7 5.2

16.0 21.6 16.8

8.5 9.1 6.8

31.8 39.4 28.8

5.1 6.8 7.0

2.

I II I

9.2 5.7 3.6

7.2 11.4 8.1

3.2 3.6 2.3

19.6 20.7 14.0

4.9 5.1 5.7

] 9.2 ]1.2 6.3

15.0 22.4 14.2

6.7 7.] 4.0

40.9 40.7 24.5

4.2 6.5 6.4

3.

I II ]

3,1 3.7 6.2

7.3 9.0 11.8

2.8 3.4 2.1

13.2 16.1 20.1

6.6 5.0 4.2

4,7 7.4 14.8

11.1 18.0 28.0

4.2 6.8 5.0

20,0 32,2 47.8

5.9 6.4 6.0

4.

I II I

4.6 5.7 6.1

10.0 20.1 17.6

].7 3.5 5.1

16.3 29.3 28.8

3.9 4.6 5.3

11.8 12.4 11.5

25.6 43.8 33.2

4.4 7.6 9.6

41.8 63.8 54.3

5.4 6.2 6.0

5.

I II I

6.9 8.7 10.1

9.9 ].7.4 15.5

2.5 2.9 3.8

19.3 29.0 29.4

4.4 5.4 5,8

15.7 16.1 17.4

22.5 32.3 26.7

5.7 5.4 6.4

43.9 53.8 50.5

5.4 6.5

Daily average

I II I

5.4 6.0 5.9

8,3 14.5 13.0

2.8 3.9 3.6

16.5 24.4 22.5

4.8 5.4 5.6

11,3 11.3 10.5

17.3 26.9 23.2

5.8 7.2 6.4

34.4 45.2 40.2

5.2 6.5 6.3

*Expressed as rag. p-hydroxyphenylacetic acid. tExtraetable at pH 8. S E x t r a e t a b l e a t p H 1. w phenols a f t e r h y d r o l y s i s w i t h H2SO4, e x t r a c t e d a t pH 8.

COI%NELY E T A L . :

E F F E C T OF L A C T O S E O N N I T R O G E N A N D P H E N O L S

Conjugated phenols were determined after hydrolysis. The urine in the separatory funnel was transferred to a flask, an equal volume of 10 N H~SO, was added, and the mixture boiled under reflux for one hour. The urine was adjusted to pH 8 and reextracted as for free phenol. The coIor was developed and read as be/ore. RESULTS

Total nitrogen intake, excretion, and balance are shown in Table I. Daily nitrogen intake was approximately constant and averaged 1.27 Gin. for the first feeding of the lactose formula, 1.27 Gin. for the D.M. formula, and 1.22 Gin. for the last feeding of the lactose formula. Nitrogen retention averaged 38 per cent for the first feeding of the lactose formula, 31 per cent for the ]).lV[. formula, and 28 per cent for the last feeding of the lactose formula. All of the babies retained more nitrogen during the initial feeding of lactose formula. In three of the five babies, retention was greater in the second period (I).IVL formula) than in the third period (lactose formula). Excretion of phenols and phenolic products is shown in Table II. The average total phenolic excretion was highest after feeding D.1VL i,ormula, averaging 24.4 rag., as compared to 16.5 and 22.5 mg. after feeding lactose formula in the first and third periods, respectively. The most marked differences were noted in t h e phenolic compounds extractable at pH 1. Excretion of this fraction of phenolic compounds was higher in all except one of the feedings of the I).M. formula.

43

For comparison, stool pH is listed in the last column of Table II. In genera% the lower the stool pH, the lower the phenol excretion. The average daily excretion of phenols, expressed as p-hydroxyphenylacetic acid, during the lactose formula, was: free phenoIs--(free) 5.6 rag.; phenolic compounds extracted at pH 1 (pH l ), 10.6 rag.; conjugated phenols (conjugated), 3.2 mg.; total phenols (total), 19.4 rag.; and during the I).M. formula: free, 6.0 rag.; pH 1, 14.5 rag., conjugated, 3.9 rag., total 24.4 rag. The average daily excretion of phenolic compounds by the 1-monthold baby fed human milk with 1.1 per cent protein was: free, 5.0 rag.; pH 1, 11.1; conjugated 2.7, total 18.8; and for this baby during the feeding of the cow's milk formula containing 1.1 per cent protein and 7 per cent lactose: free 4.0 rag., p i t 1, 8.2 rag., conjugated 1.6 rag., total 13.8 mg. DISCUSSION

The ingredients of most widely used infant formulas include cow's milk, carbohydrate, and water. Two of the carbohydrates in common use are lactose, the natural sugar in all mammalian milks, and a mixture of maltose and dextrins obtained by incomplete starch hydrolysis. It has been known for a long' time that lactose is absorbed from the intestine in a manner different from the absorption of other sugars. Intestinal flora also have been changed when another carbohydrate is substituted for lactose in infant feeding, Lactobacillus bifidus being better maintained when lactose is fed. Calcium

44

T H E J O U R N A L OF PEDIATRICS

and magnesium absorption are increased when feedings contain lactose. I t is said t h a t the body tissue of babies fed lactose contains less fat than does the tissue of those fed other sugars. It also has been postulated t h a t galaetose has a role in the formation of cerebrosides. These and other effects of lactose in the human organism have been documented by Duncan.

a

Since excreted phenols p a r t l y represent " b a c t e r i a l p u t r e f a c t i o n of aromatic amino acids in the intestine, ''~ it was expected t h a t a change of intestinal flora would alter phenol excretion. Aterman ~~ and Rogers and their associates ~ have shown such a change with antibiotics. The results of this s t u d y indicate that phenolic excretion, especially of the phenolic acids, is higher when D e x t r i - ~ a l t o s e is substituted for lactose in i n f a n t feeding. Since this s t u d y was designed to measure phenolic excretion in infants fed two commonly used formulas, the lactose was included iu the formula which contained a mixture of animal and vegetable fats, and the Dextri-lVfaltose was used in the formula which contained b u t t e r f a t and some lactose. It would be expected t h a t the fat would contribute little to the phenol excretion, but it cannot be stated t h a t Dextri-Maltose alone was responsible for the increase in phenol excretion. The data indicating increase in pheno]ie excretion b y infants fed the formula containing Dextri-Maltose are too scant to permit statistical analysis. The increase occurred in all babies when the lactose formula was followed b y the Dextri-iV[altose formula. Since, a f t e r reinstitution of

the lactose formula, the pI-I of the stools did not us.ually r e t u r n immediately to the average observed during the initial period, it might be expected t h a t longer collection periods might have p e r m i t t e d a more significant change in phenolic excretion. Nitrogen retention was 5 to 10 per cent greater in all babies fed the lactose formula, whereas the nitrogen intake was constant on both formulas. Following feeding of the D e x t r i - ~ a l tose-eontaining formula, three of the five babies showed decreased nitrogen retention when the lactose formula was reinstituted. Since phenol excretion is related to protein metabolism, it would be expected that a n y t h i n g that increases excretion of phenol would lower nitrogen retention. In spite of V o l t e r r a ' s 11 comment that nitrogen intake in adults does not affect phenolic excretion, our unpublished data in infants and the data of Levine and co-workers 6 indicate that "phenolic excretion m a y increase with increase in nitrogen intake. However, both formulas used in the present s t u d y contained protein in equal amounts, similar to the protein content of human milk (1.5 per cent, or slightly less t h a n one-half t h a t of cow's milk). The i d e n t i t y of the phenols and phenolic acids is unknown. W h e t h e r the levels of phenols or phenolic acids in urine have any physiological significance is also unknown. The data in this p a p e r indicate a free phenol excretion in infants of 3 to 10 rag. a day, compared with 11 to 70 rag. a day in adults. 12 H y d r o x y acids excreted b y the infants amounted to 7 to 20 rag., as compared to an excretion of 49 to 80 rag. b y adults. 12 Excretion of phenols b y the one i n f a n t

CORNELY E~J: AL. :

EFFECT OF LACTOSE ON NITROGEN AND PHENOLS

fed human milk was somewhat lower than the average for babies fed either cow's milk formula. Even if phenolic excretion, per se, is not an indication of a circulating toxic metabolite, it seems likely that excessive phenolic excretion, like excessive nitrogen or water excretion, may represent a potential overloading of the excretory mechanism of the y o u n g infant. SU~{[MARY

Phenolic excretion was measured in five infants fed ~ariations of common infant formulas. The main difference in the formulas was in substitution of Dextri-Maltose for part of the lactose in one feeding mixture. The nitrogen content was the same in type and quantity. Nitrogen retention was greater in the infants fed the lactose-containing formuIa. The infants tended to excrete more phenols during feeding of the formula containing Dextri-Maltose than during feeding periods on the formula containing lactose as the sole carbohydrate. Excretion of phenolic acids was much greater in the infants fed the formula containing Dextri-N[alrose. The nature and significance of these phenolic acids are unknown. The range of normal phenolic acid excretion for infants is presented.

45

REFERENCES 1. Duncan~ D. L.:

The Physiological Ef & Rev. 25: 309, 1.955. 2. Gordon, I-L :[I., Levine, S. :V., Wheatley, hs A., and Marples, E.R.: Respiratory IVfetabolism in Infancy and Childhood: The :Nitrogen iVIetabolism in Premature Infants; a Comparison of }Iuman and Cow's Milk, Am. J. Dis. Child. 54: 1030, 1937. 3. Boscott, 1%. J., and Bickel, H.: Detection of Some New A b n o r m a l ~Ietabolites in the U r i n e of P h e n y l k e t o n u r i a , Sca~d i n a r . J. Clin. Lab. Invest. 5: 380, 195;~. 4. Armstrong, 3s D., and Robinson, K. S.: On the Excretion of tndole Derivatives in P h e n y l k e t o n u r i a , Arch. Biochem. 52: 287, 1954. 5. Williams, R. T.: Detoxication NIeeha nisms, New York, John Wiley & Son~, Inc., 1949, pp. 68-80. 6. Levine, S. F., Marples, E., and Gordon. J:I. H.: A Defect in the Metabolism of Aromatic Amino Acids in :Premature Infants, t. Identification and Assay of I n t e r m e d i a r y Products, J. Clin. Invest. 20: 199, 1941. 7. Barness, L. A., Baker, D., Gnilbert, P., Torres, F. E., and Gybrgy, P.: Nitrogen lv[etabolism of I n f a n t s Fed H u m a n and Cow's Milk, J. PEDIAT. 51: 29~ 1957. 8. Bray, tI. G., and Thorpe, W . V . : Analysis of Phenolic Compounds of I n t e r e s t in Metabolism. Methods of ]~iochemi~ cal Analysis, vol. I, D a v i d G]ick, ed., N e w York, 1954, Interseience Publishers, Inc., p. 27. 9. Rogers, W. F., Burdick, M. P., and Burnett, G . R . : The Effect of Antibiotics on the E x c r e t i o n of Phenolic Corn pounds, J. Lab. & Clin. lV[ed. 45: 87~ 1955. 10. Aterman, ]~., Boscott, R. J., and Cooke, W. T.: The Significance of U r i n a r y p-Hydroxyphenylaeetlc Acid, Biochem. J. 55: 17, 1953. 11. Volterra, i~.: U r i n a r y Phenols. II. T h e i r Signlfieanee in Normal and Pathologieal Conditions, Am. ~. Clin. Path. 12: 580, 1942. 12. Volterra, M,: Urinary Phenols. I. Methods of Determination, Am. J. Clin. Path. 12: 525, 1942. fects of Lactose, Nutrition Abstr.