Studies of prolonged neonatal jaundice in the breast-fed infant

Studies of prolonged neonatal jaundice in the breast-fed infant

54 T h e Journal o[ P E D I A T R I C S Studies of prolonged neonatal jaundice in the breast-fed infant The clinical course o[ 20 breast-led in[ants...

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54

T h e Journal o[ P E D I A T R I C S

Studies of prolonged neonatal jaundice in the breast-fed infant The clinical course o[ 20 breast-led in[ants with prolonged unconjugated hyperbilirubinemia is described. Severe jaundice began during the latter part o[ the first or beginning o[ the second week o[ li[e and maximum concentrations o[ unconjugated bilirubin in the serum ranged [rom 9.0 to 26.0 rag. per 100 ml. Milk [rom the mothers o[ the jaundiced in[ants markedly inhibited hepatic glucuronyl trans[erase activity in vitro. Pregnane-3(a),20(fl)-diol was isolated [rom milk obtained [rom 4 mothers o[ infants with prolonged jaundice, but has not been isolated #ore normal human milk. The relationship between intensity and duration o[ inhibition by milk and intensity and duration o[ hyperbilirubinemia in the in[ants is illustrated. Two thirds o[ the breast-led siblings o[ the in[ants studied had a history o[ prolonged jaundice, whereas none o[ the artificially led siblings had a history o[ prolonged jaundice.

Lawrence M. Gartner, M.D.,* and Irwin M. Arias, M.D. NEW

YORK~

N.

Y.

A S Y N D R O M ~ of prolonged, unconjugated hyperbilirubinemia occurring in fullterm, breast-fed newborn infants 1' 10, x~ and not in their bottle-fed siblings has previously been described? Extensive study of these infants failed to reveal any cause of jaundice. Milk from mothers of the jaundiced infants significantly inhibited hepatic glucuronyl transferase activity in vitro when compared with milk from mothers of normal infants? Glucuronyl transferase catalyzes the transfer From the Departments of Pediatrics and Medicine, Albert Einstein College o[ Medicine and the Bronx Municipal Hospital Center. This investigation was supported in part by Grant AM 02019 [rom the National Institute o[ Arthritis and. Metabolic Disease, United States Public H~alth Service. *Special Fellow, National Institute o[ Child Health and Human Development I-F3-HD-23073, United States Public Health Service. Address, Dep.artment o[ Pediatrics, Albert Einstein College o[ Medicine, New York, N. Y. 10461.

of glucuronic acid from uridine diphosphate glucuronic acid to bilirubin, forming bilirubin glucuronide, the major bilirubin conjugate. Pregnane-3 (~),20 (/3) -diol was isolated from inhibitory milk of 3 mothers of jaundiced, breast-fed infants and was demonstrated to be a competitive inhibitor of glucuronyl transferase activity in vitro? Reversible, nonhemolytic, unconjugated hyperbilirubinemia was produced in 2 healthy, newborn infants by administration of synthetic pregnane-3 (a),20(/3)-diol for 4 to 5 days beginning on the sixth and eighth days of life, respectively. 2 Administration of the steroid to 2Jolder infants and one adult failed to produce hyperbilirubinemia. The present report describes the clinical and laboratory findings in 20 infants with this syndrome and in their mothers, the relationship between the intensity and duration

Volume 68 Number 1

of glucuronyl transferase inhibition by breast milk and the intensity and duration of hyperbilirubinemia in the infants, and studies of the familial incidence of the syndrome. CLINICAL MATERIAL AND

METHODS

Twenty full-term, breast-fed infants with prolonged, unconjugated hyperbilirubinemia not due to known causes were studied. Seven of the infants were seen at the Bronx Municipal Hospital Center. Clinical and laboratory data and appropriate specimens in the other 13 infants were supplied by the referring pediatricians. Family histories were obtained from hospital records, physicians' records, and parents. Conventional procedures were used for all clinical laboratory studies. Serologic tests for syphilis and hemoglobin determinations were performed in each infant. Direct Coombs tests were performed in 19 infants. Blood smears from 17 infants were examined for erythrocyte morphology. Glucose-6-phosphate dehydrogenase activity was estimated in erythrocytes from 3 infants. Urinary sediments from 4 infants were examined for cytomegalic inclusion bodies. Roentgenograms of the skull were obtained in 3 infants and serum protein bound iodine concentration was estimated in one infant. Total and directreacting bilirubin concentrations were estimated in the serum according to the method of Malloy and Evelyn? Mother's milk was collected manually or with the aid of a breast pump and frozen until examined. One hundred and thirtyseven specimens of milk were obtained from the 20 mothers of jaundiced infants from the fifth to the eighty-eighth postpartum day. From one to 29 milk specimens, each from a different day, were obtained from each mother. Ninety-nine specimens of milk were obtained from the second to the one-hundred and fiftieth postpartum day from 71 mothers whose infants did not demonstrate prolonged jaundice. A single serum specimen was obtained from 14 of the 20 mothers of infants with pro-

Breast [eeding and jaundice

55

longed jaundice from the fifth to the twentyninth postpartum day and from 11 of the 20 jaundiced infants from the fifth to the twenty-eighth day of life. The effect of milk and serum on glucuronyl transferase activity was estimated in vitro using o-aminophenol as glucuronide receptor, an excess of uridine diphosphate glucuronic acid ( U D P G A ) and guinea pig liver homogenate as the source of glucuronyl transferase as previously described? T h e results are expressed as per cent inhibition of o-aminophenol glucuronide formation resulting from the addition of milk or serum as compared with the addition of water to the incubation medium. Isolation of pregnane-3 (~) ,20 (fi) -diol from milk and its identification were performed as previously described? RESULTS

The average age at delivery of the 20 mothers whose infants had prolonged jaundice was 27.5 years and ranged from 16 to 38 years. Sixteen of the mothers were Caucasian, 3 were Negro, and one was Chinese. Each mother had had an uncomplicated full-term pregnancy and vaginal delivery. During labor, 2 mothers received Demerol and scopolamine; one mother received Demerol, scopolamine, and chlorpromazine; and one mother was delivered under nitrous oxide and ether anesthesia. Chlorothiazide was given to 2 mothers during the last trimester of pregnancy and prochlorperazine was given to one mother one week prior to delivery. Prenatal vitamin preparations were taken by all mothers. Eight mothers received vitamin K (10 mg.) by intramuscular injection prior to delivery. At no time during pregnancy, labor, or lactation did any mother receive steroids, hormonal medication, or known hepatotoxic drugs. The mothers were well and without clinical evidence of liver disease or jaundice. The 10 male and 10 female infants were healthy and vigorous at delivery. T w o infants received vitamin K (2.5 rag.) by intramuscular injection immediately after delivery. Four infants were sufficiently icteric

56

January 1966

Gartner and Arias

during the first week of life that serum bilirubin concentrations were determined. T h e total serum bilirubin concentrations in these 4 infants were 10.3, 11.6, 12.3, and 15.5 mg. per 100 ml. on the third to fifth days of life. Slight jaundice was noted in 11 infants during the first week of life and considered to be "physiologic" jaundice. Five infants had no clinical icterus during the first week of life. I n the 11 infants with mild "physiologic" jaundice, icterus either receded partially or disappeared before the onset of severe jaundice. T h e day of onset of severe jaundice in 16 of the 20 patients is indicated in Table I. I n 4 infants the data were not sufficient to

Table I. D a y of onset of severe jaundice in 16 infants with prolonged hyperbilirubinemia Day of onset of severe jaundice

No. of infants

Days 3 to 5 Day 7 Day 8 Day 9 Day 12

4 3 5 1 3

specify the exact onset of severe jaundice which was present by the twelfth day of life. T h e m a x i m u m serum indirect-reacting bilirubin concentrations ranged from 6.7 to 26.0 mg. per cent and were observed from the fifth to the twenty-seventh day of life (Table I I ) . T h e m a x i m u m serum directreacting bilirubin concentrations of the same specimens were 0.3 to 2.4 mg. per cent. The m a x i m u m serum bilirubin concentrations may have been higher in some infants and may have occurred earlier since several infants were not seen by a physician until jaundice had been present for from 8 to 20 days. Six mothers abruptly discontinued breast feeding their infants on the sixteenth to eighteenth postpartum day, and the serum biIirubin concentrations returned to normal in 4 to 6 days. O n e mother began alternating nursing and bottle feeding her infant on day 19 and the infant's serum bilirubin concentration did not return to normal until 12 days later. Breast feeding of 7 infants was continued and the serum bilirubin concentrations became normal by the twenty-eighth to the seventy-ninth postpartum day in 6

Table II. Sex, birth weight, and m a x i m u m serum bilirubin concentrations observed in 20 infants with prolonged hyperbilirubinemia

Infant

Sex

Birth weight (grams)

A B C D E F G H I J K L M N O P Q R S T

F M M F F F M M M M M M F F M M F F F F

2,740 3,540 3,440 3,520 3,405 3,060 3,400 3,220 3,300 3,270 3,600 3,450 3,480 3,360 3,240 3,500 2,580 3,940 3,520 3,025

Maximum serum bilirubin concentration Total Direct (mg./lO0 ml.) (mg./lO0 ml.)

15.0 17.0 23.1 16.0 11.0 27.0 14.0 7.0 13.4 10.5 11.8 17.2 13.5 18.4 20.0 12.0 15.5 17.0 18.2 10.0

2.0 1.2 2.4 1.0 0.8 1.0 1.0 0.3 1.2 0.6 0.4 1.0 0.6 1.1 2.0 2.0 2.0 2.0 2.1 1.0

Day on which maximum was observed

19 11 15 16 16 9 15 27 18 15 17 18 20 9 8 7 8 11 5 10

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Breast feeding and jaundice

infants. I n one i n f a n t who c o n t i n u e d breast feeding, the serum bilirubin concentration was 7.3 mg. p e r cent on d a y 26 a n d she was not seen by h e r physician subsequently. I n 6 infants breast feeding was temporarily discontinued for f r o m 4 to 7 days beginning between the eighth a n d the twenty-seventh p o s t p a r t u m d a y ( T a b l e I I I ) . T h e total serum bilirubin concentrations decreased 4.0 to 15.7 mg. p e r 100 ml. Breast feeding was then resumed a n d in 5 cases the total serum bilirubin concentrations rose 0.4 to 2.4 rag. p e r 100 ml. after 4 to 7 days of nursing. I n the sixth infant, the serum bilirubin concentration r e m a i n e d constant after resumption of breast feeding. Physical e x a m i n a t i o n of each infant failed to reveal any a b n o r m a l findings except for

57

jaundice. T h e r e was no evidence of kernicterus or other neurological a b n o r m a l i t y in any of the infants d u r i n g the p e r i o d of study. F i v e infants were examined at one year of age at the Bronx M u n i c i p a l H o s p i t a l Center a n d f o u n d to be neurologically a n d developmentally normal. T h e m a j o r blood groups and R h types were c o m p a t i b l e in 11 m o t h e r - i n f a n t pairs. T h e r e was potential A B O i n c o m p a t i b i l i t y in 2 cases a n d potential R h i n c o m p a t i b i l i t y in 3 cases. T h e blood types of 3 infants a n d one m o t h e r are unknown. Coombs tests were negative in 19 infants. H e m o g l o b i n concentrations were n o r m a l in all infants a n d erythrocyte m o r p h o l o g y in 17 infants was normal. Reticulocyte counts which were perf o r m e d in 14 infants did n o t exceed 2.8 p e r

T a b l e I I I . T o t a l serum bilirubin concentration in infants w h e n breast feeding was temporarily discontinued a n d then resumed

Infant

Day o/ l~e nu~ing stopped

Totalbilirubin (mg./lO0 ml.) on day nu~ing stopped

No. of days on bottle feed

G H K M N Q

17 27 20 21 11 9

12.8 7.0 9.4 13.5 17.5 15.4

5 6 4 6 5 7

Total bilirubin I (mg./lO0 ml.) I Days on breast at end of after bottle feed resumption 5.6 3.0 1.3 8.1 1.8 5.3

Net change in total bilirubin (mg./lO0 ml.) after nursing

3 7 4 5 4 4

+2.4 +0.9 +0.4 +1.3 +1.9 -0.4

T a b l e I V . History of prolonged j a u n d i c e a m o n g siblings of infants studied as related to breast or bottle feedings ~

Infant

Total no. siblings

A B C D E F G I K L R S T Total

1 3 1 2 2 3 2 2 1 3 3 4 2 29

Breast-fed No. proNo. [edbreast- [ longed jaundice 1 2 0 1 1 0 1 2 0 2 2 4 2 '18

eInfants not included i n this table had no siblings.

Bottle-led NO. prO-

No. not jaundiced

0 2

1 0

I 1

0 0

1 2

0 0

2 2 0 1 12

0 0 4 ~1 6

No. bottle[ed 0 1 1 1 1 3 1 0 1 1 1 0 0 11

longed jaundice

No. not jaundiced

0 0 0 0 0 0

1 1 1 1 3 1

0 0 0

1 1 1

0

11

58

January 1966

Gartner and Arias

glucuronide formation by 137 specimens of milk obtained on the third to the eightyeighth postpartum day from mothers of the 20 infants with prolonged, unconjugated hyperbilirubinemia was 69.9 +_ 22.1 per cent (S.D.) (Fig. 1). Milk specimens obtained on the second to one-hundred and fiftieth postpartum day from mothers of infants without prolonged jaundice inhibited oaminophenol glucuronide formation by 11 • 4.1 per cent (S.D.). From one to 29 specimens of milk were examined from each of the 20 mothers whose infants had prolonged jaundice. In each case at least one specimen inhibited glucuronyl transferase activity by 78 per cent or more. Eight specimens of milk from 3 mothers of jaundiced infants produced an inhibitory effect within 3 standard deviations of the mean for control milks (Fig. 1). These lower degrees of inhibition were produced by milk obtained either prior to the fourth postpartum day or after the twenty-third postpartum day. Each of 71 specimens of milk obtained from the sixth to the twenty-first postpartum day from mothers of jaundiced infants inhibited o-aminophenol glucuronide formation by 40 per cent or more. The inhibitory effect of each mother's milk varied from day to day. This variability is illustrated in Fig. 2, which presents the maximum and mini-

cent. Studies of erythrocyte glucose-6-phosphate dehydrogenase activity in 3 infants were normal. Serologic tests for syphilis were negative in all infants or mothers. Examination of the urine from 8 infants was negative for cells and reducing substances. Urinary sediment from 4 infants did not contain cytomegalic inclusion bodies. Estimation of serum concentration of albumin and globulin, thymol turbidity and cephalin cholesterol flocculation, and glutamicoxaloacetic and glutamic-pyruvate transaminase activities were normal in 15 infants. Radiologic examination of the skull w a s normal in 3 infants. Thirteen mothers had 29 previous infants, 18 of whom were breast fed (Table IV). Of these breast-fed infants, 12 had a history of severe and prolonged jaundice. Eleven artif i c i a l l y fed infants did not have prolonged jaundice during the early months of life. Nine of the previous breast-fed jaundiced infants were seen by pediatricians at one to 5 y e a r s of age and found to be normal. Intensive search among the relatives of the mothers failed to reveal any infants with a history of severe or prolonged jaundice. No parents, siblings, grandparents, uncles, or aunts of the parents of the jaundiced infants ever had unexplained jaundice. The mean inhibition of o-aminophenol

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30 40 50 60 DAYS POST PARTUM

70

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Fag. 1. Inh'ibition of o-aminophenol glucuronide formation by milk from 20 mothers of infants with prolonged hyperbilirubinemia (dots) as compared with mothers of normal infants (control) in vitro. T h e inhibition by milk from control mothers is indicated as the mean + 3 standard deviations (shaded area).

Volume 68

Number 1

Breast [eeding and j a u n d i c e

59

Q)

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30 40 50 60 70 80 90 DAYS POST PARTUM Fig. 2. Maximum and minimum inhibition of o-aminophenol glucuronide formation produced by milk specimens from 20 mothers of infants with prolonged jaundice. The inhibition by milk from control mothers is indicated as the mean + 3 standard deviations (shaded area).

Table V. Maternal and infant serum inhibition of OAP-glucuronide formation Infant and mother

B C D E H I j L M N Q Average Standard deviation

Maternal serum .. Day obtained I Per cent inhibition

9 7 13 12 28 13 21 14 20 11 5

11 13 14 18 14 12 14 6 7 12 17

13.9

12.5 •

m u m inhibition by milk from each of the 20 mothers. Pregnane-3 (a),20 (/3) -diol was isolated from milk specimens obtained from four mothers (Cases A, K, L, and N ) . Isolation of a steroid was not attempted from milk obtained f r o m the remaining 16 mothers. Serum obtained on the fifth to the twentyeighth postpartum day from 11 mothers of infants with prolonged jaundice inhibited o-aminophenol glucuronide formation by 12.5 -+ 3.5 per cent (S.D.) (Table V ) . Serum obtained from 15 control mothers during the same postpartum interval inhibited o-aminophenol glueuronide formation by 9.0 + 4.3

In[ant serum Day obtained [Per cent inhibition

9 8 13 12 28 13 20

10 6 3 4 8 4 6

--

--

31 11 5

10 18 o

15.0

6.9 •

per cent (S.D.). Serum obtained from 10 jaundiced infants on the fifth to the thirtyfirst day of life inhibited o-aminophenol glucuronide formation by 6.9 + 4.7 per cent

(S.D.). CASE R E P O R T S T h e following 5 cases illustrate the relation between the intensity and duration of inhibitory activity in milk and the intensity and duration of hyperbilirubinemia in the infants. Case L. (Fig. 3.) This 3,450 gram Caucasian male infant was born to a 26,year-old mother after an uneventful pregnancy. Two sib-

60

Gartner and Arias

]anuary 1966

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lings were breast fed and had prolonged jaundice. Another sibling was artificially fed and remained anicteric. Mild transient jaundice was noted during the first 4 days of life. On day 7 intense jaundice was first noted. The total serum bilirubin concentration on day 9 was 19.0 rag. per cent (1.0 mg. per cent direct reacting). Breast feeding was continued for 6 days and the serum bilirubin concentration remained between 17.5 and 18.0 rag. per cent. On day 17, breast feeding was discontinued. Three days later the serum bilirubin concentration was 5.3 rag. per cent. On day 22, the serum bilirubin concentration was 1.4 mg. per cent. Milk from days 9 to 15 inhibited o-aminophenol glucuronide formation by 89 to 97 per cent. Pregnane-3 (a) ,20 (fl) -diol was isolated from this mother's milk. This case illustrates an abrupt decline in the infant's serum bilirubin concentration when breast feeding was discontinued. From the ninth through the fifteenth day, milk remained markedly inhibitory. Case Q. (Fig. 4). This 2,580 gram female Chinese infant was born at term to a 26-yearold primigravid mother after an uneventfuI pregnancy and delivery. Nursing began at 17 hours of age and jaundice was first noted on the third day. The total serum bilirubin concentration was 15.5 mg. per cent (0.7 mg. per cent direct-reacting) on the fourth day. By the sixth day, the serum bilirubin con-

centration declined to 11.2 mg. per cent and rose again to 15.7 rag. per cent by the ninth day at which time breast feeding was discontinued. The serum bilirubin concentration declined to 5.1 rag. per cent after 6 days of artificial feeding. Breast feeding was resumed at this time and during the next 5 days the serum bilirubin concentration remained essentially unchanged. On day 19, the serum bilirubin concentration was 4.8 nag. per cent and subsequently declined slowly despite continued breast feeding. Milk obtained from the fourth to the seventeenth postpartum days inhibited o-aminophenol glucuronide formation by 40 to 66 per cent. Milk from day 21 inhibited o-aminophenol glucuronide formation by 86 per cent. In this case, interruption of breast feeding resulted in rapid decline in the infant's serum bilirubin concentration. With resumption of breast feeding, the serum bilirubin concentration remained stable for 5 days. Milk was moderately inhibitory during the first 2 weeks post partum and became markedly inhibitory during the third week. Case N. (Fig. 5.) This 3,360 gram Caucasian female was born at term to a 22-year-old primigravid mother. Pregnancy was uneventful except for mild edema requiring administration of chlorothiazide. Breast feeding was begun 24 hours after birth and jaundice was observed on the eighth day of life. On the ninth day, the total serum bilirubin

Volume 68

Number 1

Breast feeding a n d ~aundice

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DAYS PosT PARTUM Fig. 5. Case N. Duration of breast feeding, inhibition of o-aminophenol glucuronide formation by milk ( ......... ), and infant's total serum bilirubin concentration rag. per cent ( ).

concentration was 18.4 rag. per cent (1.1 per cent direct-reacting). The following day serum bilirubin concentration was 17.5 rag. cent and breast feeding was discontinued. abrupt fall in serum bilirubin concentration noted during the subsequent 6 days. On

rag. the per An was the

fifteenth day, the serum bilirubin concentration was 1.8 mg. per cent. Breast feeding was resumed the following day. Four days later, the serum bilirubin concentration was 3.7 rag. per cent and breast feeding was permanently discontinued. Milk obtained on the eleventh and the twelfth

62

Gartner and Arias

January 1966

day inhibited o-aminophenol glucuronide formation by 42 and 46 per cent, respectively. Milk obtained on the nineteenth day inhibited o-aminophenol glucuronide formation by 93 per cent. During the following 4 days the inhibitory effect of the milk progressively declined. In this infant, hyperbilirubinemia decreased markedly when breast feeding was discontinued. Four days after resumption of nursing, the serum bilirubin concentration had increased 1.9 mg. per cent. The inhibitory activity of breast milk rapidly disappeared during the third postpartum week. Case R. (Fig. 6.) This 3,490 gram Negro female infant was born to a 33-year-old mother after an uneventful pregnancy and labor. Two previous infants were breast fed and had severe jaundice throughout the first 3 weeks of life. One sibling was artificially fed and, although premature, never demonstrated icterus. Jaundice was noted on the fifth day of life and the total serum bilirubin concentration was 12.3 mg. per cent (1.0 mg. per cent direct-reacting). Breast feeding began on the first day and was continued for 90 days. Hyperbilirubinemia increased from the sixth to the eleventh day of life. Despite continued breast feeding, the serum bilirubin concentration decreased rapidly during the subsequent 4 days, and became normal on the twenty-sixth day of life. Milk obtained on the fifth day inhibited o-aminophenol glucuronide formation by 33 per

cent. Milk obtained during the following 9 days inhibited o-aminophenol glucuronide formation by 44 to 78 per cent. Milk obtained on the twenty-sixth and the eighty-eighth postpartum day inhibited o-aminophenol ghicuronide formation by 43 and 20 per cent, respectively. In this case breast feeding was continued. The infant's serum bilirubin concentration and the inhibitory effect of maternal milk simultaneously decreased. Case O. (Fig. 7.) This 3,240 gram Caucasian male was born to a 29-year-old primigravid mother after an uneventful pregnancy and labor. Breast feeding was begun on the first day and continued through the seventy-second day. The serum bilirubin concentration was 12.0 rag. per cent on the fourth day and reached a maximum of 20 mg: per cent on the eighth day. The serum bilirubin concentration slowly returned to normal by the sixty-seventh day. Clinical icterus was noted until the fortieth day of life. Milk obtained from the ninth to the fiftieth postpartum day consistently inhibited o-aminophenol glucuronide formation by 70 per cent or more. The infant's serum bilirubin concentration declined although breast milk remained markedly inhibitory. The serum bilirubin concentration remained elevated for a longer period of time than in the other cases and did not become normal until after 40 days of age.

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Volume 68 Number 1

Breast feeding and jaundice

DISCUSSION

6. Administration of pregnane-3 (a) ,20 (fl)-diol by mouth to 2 infants during the first week of life resulted in the development of unconjugated hyperbilirubinemia. In 75 per cent of the infants, severe hyperbilirubinemia was first observed at the end of the first or beginning of the second week of life. Colostrum obtained from 2 mothers during the first 4 postpartum days had negligible inhibition; however, subsequent milk specimens from these same mothers were markedly inhibitory? These observations suggest that the inhibitory steroid is probably not present in colostrum before approximately the fourth postpartum day. Milk from mothers of the jaundiced infants varied from mother to mother in intensity and duration of inhibition. These variations may reflect dilution of the inhibitor in differing volumes of milk secreted or variations in the secretion of inhibitor into the milk. Milk from each of the 20 mothers of jaundiced infants significantly inhibited o-aminophenol glucuronide formation for at least 21 days postpartum and in 3 mothers remained strongly inhibitory for at least 49 days. Regardless of the duration of inhibitory activity in milk, hyperbilirubinemia in all infants began to remit at about 20 days and disappeared between 30 and 60 days of age. In

The syndrome of prolonged unconjugated hyperbilirubinemia in breast-fed infants is believed to result from ingestion of maternal milk containing pregnane-3 (a),20 (/3)-diol. This steroid competitively inhibits glucuronyl transferase activity in vitro. The following observations support this postulate: 1. Clinical and laboratory studies of the 20 infants failed to demonstrate any known causes of hyperbilirubinemia. 2. Prolonged unexplained jaundice occurred in 12 of 18 breast-fed siblings and in none of 11 bottle-fed siblings. 3. Milk from each of the mothers of the 20 infants inhibited glucuronyl transferase activity significantly more than did milk from mothers of normal infants. 4. Abrupt discontinuation of breast feeding in 5 infants resulted in rapid decrease in hyperbilirubinemia. Continuation of breast feeding in 8 infants resulted in persistent hyperbilirubinemia for 4 to 11 weeks. In 5 infants, temporary interruption of breast feeding resulted in decreased hyperbilirubinemia and resumption of breast feeding was accompanied by subsequent increased hyperbilirubinemia. 5. Pregnane-3 (~) ,20 (/3) -diol was isolated from nfilk obtained from 4 mothers.

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Gartner and Arias

those cases in which the inhibitory effect of milk declined progressively during this period, the decrease in hyperbilirubinemia may result from reduced ingestion of the inhibitory steroid (Case R, Fig. 6). Decline in the infant's serum bilirubin concentration without simultaneous decline in the inhibitory effect of maternal milk suggests additional mechanisms (Case O, Fig. 7). Hepatic glucuronyl transferase activity in human neonates and in the newborns of most laboratory animals is reduced in comparison to the activity found in adults. 4, ~, ~ The transient unconjugated hyperbilirubinemia common to most infants during the first week of life has been attributed to a deficiency in glucuronyl transferase. 4, ~ Therefore, during the newborn period, an infant may be more likely than an older child to develop jaundice following ingestion of an inhibitor of glucuronyl transferase because the inhibition is superimposed on an already deficient enzyme. Supporting evidence for this concept was obtained following the administration of pregnane-3(a),20(fl)-diol to normal infants of different ages. 2 Oral administration of pregnane-3 (a),20(fl)-diol to 2 infants during the second week of life resulted in unconjugated hyperbilirubinernia. Administration of the steroid in an amount adjusted for body weight to infants at one and 2 months of age and to an adult did not produce hyperbilirubinemia. Presumably, glucuronyl transferase is fully developed in the older infants and in the adult, and, therefore, they are less likely to develop hy-perbilirubinemia after ingestion of the inhibitory steroid. The disappearance of hyperbilirubinemia in infants who continue to ingest the steroid in milk may result from maturation of hepatic glucuronyl transferase (Case O , Fig. 7). Six previous breast-fed infants of the mothers studied did not develop prolonged jaundice. It is possible that the mothers secreted very little or no inhibitor of glucuronyl transferase following earlier pregnancies or that the development of glucuronyl transferase was particularly rapid in these infants. The occurrence, intensity, and duration of

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hyperbilirubinemia in the infant probably depend on the available glucuronyl transferase as well as on the amount of inhibitory steroid ingested. Since intestinal absorption of pregnane-3(a),20(fi)-diol has not been studied in infants, it is not possible to relate this factor to the problem: Five per cent of mothers of normal infants secrete milk which inhibits glucuronyl transferase by more than 20 per cent? Only rarely, however, does the per cent inhibition found in milk from these mothers approach the high levels found in milk obtained from mothers of intensely jaundiced infants. The failure of some infants to develop jaundice when ingesting inhibitory milk has been discussed above. There is nothing unique about the infants of mothers whose milk contains pregnane3(a),20(fi)-diol, since it was possible to reproduce the syndrome in normal, unrelated infants by oral administration of the steroid2 Pregnane-3(a),20(fl)-diol has not previously been identified in human tissues or secretions, although it is an important metabolite in guinea pigs and cattle, z The major progesterone metabolite in the human is pregnane-3 (a),20(a)-diol. s Production of the unusual isomer could occur in breast tissue or in nonmammary tissue. The usual precursor of pregnanediol is progesterone, but secretion of progesterone is believed to cease after expulsion of the placenta2 Therefore, any explanation for the presence of pregnane-3(a),20(fi)-diol in human milk must consider not only the production of an u n u sual isomer but also the source and precursors of the steroid. There is no evidence from the family histories that this syndrome has occurred in infants of relatives. This suggests that the syndrome may not be inherited. If the syndrome is acquired, there is no evidence to suggest that it results from ingestion of drugs or contact with known toxic substances. Furthermore, it is unlikely that the inhibitor would recur repeatedly in the same mother if an exogenous substance were the etiologic agent. The most likely explanation is that

Volume 68 Number 1

these mothers have an abnormality in the metabolism of progestational steroids. The nature of the defect is unknown, as is its mode of acquisition. Eleven cases of prolonged, unconjugated hyperbilirubinemia persisting into the second month of life in breast-fed infants have been reported by Newman and Gross. 1~ Although inhibition studies were not performed with milk from their cases, the clinical descriptions are similar to the cases in this report. Silverberg has also described cases of this syndrome. 11 Breast-fed infants with prolonged, unconjugated hyperbilirubinemia without inhibitor in breast milk have been observed. In some of these infants, jaundice decreased when breast feeding was interrupted. The etiology of jaundice in these cases must be sought in mechanisms other than inhibition of glucuronyl transferase by an inhibitor in breast milk. Inhibition of glucuronyl transferase activity occurs in other syndromes of neonatal jaundice. Transient familial neonatal hyperbilirubinemia is a syndrome of severe, transient, unconjugated hyperbilirubinemia beginning during the first 48 hours of life in all infants of certain mothers? 2 It is associated with an inhibitor of glucuronyl transferase in the sera of both mother and infant? 3 Although normal sera obtained late in pregnancy and sera from normal mothers and infants during the first week after delivery inhibit glucuronyt transferase activity to a moderate degree, ~4 the inhibition caused by sera from mothers of infants with transient familial neonatal hyperbilirubinemia is several fold greater. This serum inhibitor factor has not been identified. Sera from mothers of the jaundiced breast-fed infants never demonstrated inhibition greater than that found in mothers of normal infants. Administration of novobiocin has been associated with unconjugated hyperbilirubinemia in infants? s Novobiocin is a noncompetitive inhibitor of glucuronyl transferase activity in vitro? s None of the mothers or infants whom we studied received this antibiotic.

Breast feeding and jaundice

SUMMARY

AND

65

CONCLUSIONS

The clinical course and laboratory studies in 20 full-term, breast-fed infants with prolonged, unconjugated hyperbilirubinemia have been described. Milk from mothers of each of these infants markedly inhibited hepatic glucuronyl transferase activity in vitro. The relationship between the intensity and duration of glucuronyl transferase inhibition by breast milk and the intensity and duration of hyperbilirubinemia in the infants has been discussed. All infants had intense hyperbilirubinemia during the first 2 weeks of life, beginning after the fourth day of life. Severe hyperbilirubinemia persisted throughout the period of nursing or for at least 2 weeks if breast feeding was not interrupted. Interruption of breast feeding was accompanied by a prompt and rapid decline in serum bilirubin concentration. Resumption of breast feeding resulted in a small rise in serum bilirubin concentration or in maintenance of a constant bilirubin concentration. Hyperbilirubinemia diminished progressively in all infants, even when nursing continued without interruption. The rate of decline of serum bilirubin concentrations varied considerably among the infants. The duration of inhibitory effect in milk varied from 3 weeks to more than 2 months postpartum. Early decrease in inhibitory effect of milk is generally accompanied by early disappearance of hyperbilirubinemia in the infant, whereas persistence of marked inhibitory effect of milk is accompanied by prolonged duration of hyperbilirubinemia. Prolonged jaundice occurred in two thirds of the previously breast-fed infants of the 20 mothers studied and none of 11 artificially fed infants. Whether this syndrome in the mothers occurs as an inherited or acquired defect is uncertain at this time. The unconjugated hyperbilirubinemia in the infants with this syndrome is not associated with neurological abnormalities dur-

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ing early infancy or at one year of age. I n view of this observation a n d the rarity of this syndrome, we feel that the advantages of breast feeding far outweigh any possible disadvantage. I t is our policy to p e r m i t infants with this syndrome to continue nursing unless the total serum bilirubin concentration approaches 20 mg. per 100 ml. d u r i n g the first week of life. I n these cases interr u p t i o n of nursing for two or three days permits sufficient lowering of serum bilirubin concentration so that breast feeding may be resumed. We are grateful to the many physicians who have sent specimens and case histories to us.

REFERENCES

1. Arias, I. M., Gartner, L. M., Seifter, S., and Furman, M.: Prolonged neonatal unconjugated hyperbilirubinemia associated with breast feeding and a steroid pregnane-3(a), 20(/3)-diol, in maternal milk which inhibits glucuronide formation in vitro, J. Clin. Invest. 43: 2037, 1964. 2. Arias, I. M., and Gartner, L. M.: Production of unconjugated hyperbilirubinemia in fullterm new-born infants following administration of pregnane-3(a),20(fl)-diol, Nature 203: 1292, 1964. 3. Malloy, H., and Evelyn, K.: Determination of bilirubin with the photo-electric colorimeter, J. Biol. Chem. 119: 481, 1937. 4. Gartner, L. M., and Arias, I. M.: Developmental pattern of glucuronide formation in rat and guinea pig liver, Am. J. Physiol. 205: 663, 1963.

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5. Brown, A., and Zuelzer, W.: Studies on the neonatal development of the glucuronide conjugating system, J. Clin. Invest. 37: 332, 1958. 6. Lathe, G. H., and Walker, M.: An enzyme defect in human neonatal jaundice and in Gunn's strain of jaundiced rats, Biochem. J. 67: 9P, 1958. 7. Pearlman, W, H.: Recent experiences in the detection, estimation, and isolation of progesterone and related C 21 steroids, Recent Prog. Hormone Res. 9: 27, 1954. 8. Hsia, D., Dowben, R., Shaw, R., and Grossman, A.: Inhibition of glucuronyl transferase by progestationaI agents from serum of pregnant women, Nature 187: 69, 1960, 9. Deshpande, G. N., Turner, A. K., and Sommerville, I. F.: Plasma progesterone and pregnanediol in human pregnancy, during labor and post-partum, J. Obst. & Gynaec. Brit. Emp. 67: 954, 1960. 10. Newman, A. J., and Gross, S.: Hyperbitirubinemia in breast-fed infants, Pediatrics 32: 995, 1963. 11. Silverberg, M.: Personal communication. 12. Lucey, J., Arias, I., and McKay, R.: Transient familial neonatal hyperbilirubinemia, Am. J. Dis. Child. 100: 175, 1960. 13. Arias, I. M., Wolfson, S., Lucey, J. F., and McKay, J., Jr.: Transient familial neonatal hyperbilirubinemia. In press. 14. Lathe, G. H., and Walker, M.: Inhibition of bilirubin conjugation in rat liver slices by human pregnancy and neonatal serum and steroids, Quart. J. Exper. Physiol. 43: 257, 1958. 15. Sutherland, J. M., and Keller, W. H.: Novobiocin and neonatal hyperbilirubinemia, Am. J. Dis. Child. 101: 447, 1961. 16. Lokietz, H., Dowben, R. M., and Hsla, D. Y.: Studies on the effect of novoblocin on glucuronyl transferase, Pediatrics 32: 47, 1963.