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Bilirubin Metabolism
Bilirubin Metabolism DAVID YI-YUNG HSIA, M.D.
THE CAUSES OF NEONATAL JAUNDICE
The conjugation of bilirubin is carried out by means of the following reaction:
Bilirubin {indirectreacting)
+
GLUCURONYL UDPGA
-------~) Bilirubin glucuronide
(Uridine diphosphate glucuronic acid)
TRANSFERASE
(directreacting)
+
UDP (Uridine diphosphate)
There is general agreement that hyperbilirubinemia in the newborn infant results either from excessive breakdown of red cells resulting in the accumulation of unusual amounts of indirect-reacting bilirubin or from a functional immaturity of glucuronyl transferase resulting in the failure of conversion of indirect-reacting bilirubin to direct-reacting bilirubin glucuronide, or both. The extensive knowledge in this area accumulated over the past decade has been reviewed. 2 , 46 The present discussion will be confined to the three factors which have recently been shown to influence the degree of jaundice in the newborn infant. Glucose-6-Phosphate Dehydrogenase Deficiency For some years we have known that hyperbilirubinemia in the neonatal period can be caused by hemolysis induced by blood group incompatibility, sepsis, and certain erythrocyte enzyme defects such as congenital nonspherocytic hemolytic anemia with glucose-6-phosphate From the Genetic Clinic of the Children's Memorial Hospital and the Department of Pediatrics, Northwestern University Medical School, Chicago. Aided by a grant from the Association for the Aid to Crippled Children. Prepared during the tenure of a Special Fellowship from the National Institute of Child Health and Human Development (IF 3 HD 2984) at the Kinderspital, Zurich, Switzerland.
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dehydrogenase (G-6-P D) or pyruvate kinase deficiency. It is only recently, however, that a deficiency of G-6-P D without obvious hemolytic anemia has been implicated as a cause for jaundice in the newborn infant. In 1960 Smith and Vella 38 described 14 cases of kernicterus without blood group incompatibility among Chinese infants in Singapore. All except one were found to have a deficiency of G-6-P D. Since G-6-P D deficiency is seen also in other races in Singapore, they attributed the high incidence of jaundice and kernicterus to the use of drugs related to traditional Chinese methods of caring for the mother and baby during the puerperium. One year later Doxiadis, Fessas and Valaes 9 noted a high incidence of G-6-P D deficiency among infants with severe neonatal jaundice of unknown cause in Greece. In this survey involving 22 such infants, 13 of the 17 males and one of the five females were found to be G-6-P D-deficient. Of the remaining five females, three of the fathers were reactors, making their daughters at least heterozygotes for the abnormal gene. Increased jaundice has not always been noted among G-6-P Ddeficient male infants. O'Flynn and Hsia,32 working with American Negro males, and Szeinberg and his co-workers,40 working with infants in Israel, were unable to detect any significant difference in the bilirubin levels between those with normal and those with deficient G-6-P D levels. Other workers 24 , 45 expressed the view that although Negro infants with G-6-P D deficiency might be more jaundiced in some instances, the deficiency of this enzyme is not a main cause of jaundice. Finally, the administration of menadione (Synkayvite) or vitamin Kl (Konakion) did not result in excessive rises of bilirubin among the infants with G-6-P D deficiency.7,45 These conflicting observations led Fessas, Doxiadis and Valaes l l to re-examine the Greek population. In a random survey of 786 newborn infants they found 23 with G-6-P D deficiency. Only one out of 21 such infants had severe hyperbilirubinemia. In contrast, 11 out of the 43 siblings of the original families had hyperbilirubinemia. In a more recent survey carried out in Lesbos, where the incidence of severe hyperbilirubinemia without incompatibility is unusually high, Doxiadis and his co-workers 1o found serum bilirubin levels of 16 mg. per 100 ml. in 34 per cent of G-6-P D-deficient newborn males and 9.1 per cent of normal newborn males, both unusually high. These data have led the authors to postulate that there may be at least two and possibly three types of G-6-P D-deficient populations in Greece. The common type behaves much in the same manner as those with G-6-P D deficiency in other Mediterranean populations. There appears, however, to be another group or groups of individuals in whom G-6-P D deficiency results in a greater tendency to jaundice in the neonatal period. Kirkman and his co-workers 20 have described the physicochemical properties of eight Greek children with G-6-P D deficiency known to
BILIRUBIN METABOLISM
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have had severe jaundice during infancy and four other Greek children with G-6-P D deficiency who were not jaundiced. Although there were no distinctive differences between the two groups and they all followed the customary pattern seen in Sephardic-Sardinian patients with G-6-P D deficiency, the more jaundiced children showed a wider range of variability in terms of (1) the degree of bimodality of the pH optimum curves, and (2) the utilization of 2 desoxy glucose-6-phosphate as a substrate varying from 3 to 65 per cent instead of the usual 23 to 37 per cent. Although this does not provide evidence of a difference in the molecular structure of G-6-P D in the two groups, it does emphasize the need of studying this unusual group of infants further to determine the: mechanism leading to hyperbilirubinemia. It is possible that a deficiency of G-6-P D may result in a whole spectrum of clinical manifestations varying all the way from those who will not hemolyze even in the presence of drugs to those who hemolyze constantly even in the absence of drugs, as in congenital nonspherocytic hemolytic anemia. In these Greek and perhaps also in the Chinese babies the birth process, possibly with its associated hypoxia and hypoglycemia, in some way adversely affects the G-6-P D and results in a temporary increase in the breakdown of red cells. It would be interesting to find out if, later in life, such persons show an abnormal response to the fava bean or other drugs. The Role of Inhibitors Physiologic jaundice in the full-term and particularly in the premature infant is believed to be caused primarily by a functional immaturity of liver glucuronyl transferase. 6 Although it would be attractive to bring about an adaptive increase of this enzyme during the critical first few days of life, repeated efforts to do this have so far been unsuccessful. During recent years increased attention has been turned toward whether the presence of inhibitors might be responsible for the decrease of glucuronyl transferase in the newborn. In 1958 Lathe and Walker22 first reported that serum from pregnant women inhibited the conjugation of bilirubin by liver slices and demonstrated a similar effect with progestational steroids in vitro. Two years later Hsia and his co-workers18 found a similar inhibition upon o-aminophenol conjugation by rat liver homogenate. It was found that serum from pregnant women showed 64.0 -+- 21.8 per cent inhibition, from immediately postpartum women 42.8 -+- 22.5 per cent inhibition, and from newborn infants 48.2 -+- 20.0 per cent inhibition. In contrast, adult males' serum showed only 1.3 -+- 1.0 per cent inhibition, and adult nonpregnant females' serum showed only 26.8 -+- 9.8 per cent inhibition. Pooled serum from pregnant women was purified, and the predominant fraction of the inhibitor substance was found to be pregnanediol. In the meantime two separate clinical syndromes characterized by hyperbilirubinemia in the newborn infant have been described. In 1960
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Lucey, Arias and McKay26 reported on transient familial neonatal hyperbilirubinemia. This condition is manifested by transient nonhemolytic unconjugated hyperbilirubinemia within hours after birth, occurs in siblings and is unrelated to breast feeding. The serum bilirubin level ranged from 25 to 65 mg. per 100 ml. during the first four days of life, and kernicterus was frequently observed. From the second trimester of pregnancy, mothers of infants with this syndrome have in their plasma three to five times higher titers of an inhibitor of glucuronyl transferase than that seen in other pregnant women. Although it would be attractive to postulate that these women have a constitutional or genetic inability to handle pregnanediol or other progestational agents during late pregnancy, no evidence for this has so far emerged. In our own laboratory we have not been able to find any difference in the excretion of pregnanediol in the urine of such women as compared with other women. About the same time a number of reports appeared describing prolonged neonatal jaundice associated with breast feeding. a. 29 In all such instances the removal of human milk and the substitution of cow's Inilk resulted in a rapid disappearance of the hyperbilirubinemia. Milk obtained from the mothers of seven such infants consistently inhibited glucuronyl transferase activity in vitro, while Inilk obtained from other mothers did not demonstrate this inhibition. In contrast, the serum from these women did not inhibit glucuronyl transferase any more than that of other women at the same postpartum stage. From the inhibitory Inilk, pregnane-3a 20 ,a-diol has been consistently isolated, while none has been found in noninhibitory Inilk. It is estimated that 1 mg. of this steroid has been secreted through breast milk by the mother daily. Since pregnanediol in low concentrations acts as a competitive inhibitor of glucuronyl transferase,17 it has been postulated that this material from the milk inhibits an already immature enzyme system and causes increased jaundice due to the inability to conjugate bilirubin. This has been tested in normal full-term infants clinically;12 0.33 mg. per kilogram of pregnane-3a 20 ,a-diol was given to four infants six, eight, 34 and 66 days old and to one adult male. In the two younger infants the serum bilirubin level rose significantly, while no such increase was noted in the two older infants and the adult. It has been suggested that in such situations breast-feeding should be temporarily curtailed for a few days and then resumed. In a survey carried on at several maternity hospitals in our area, we have estimated that this syndrome occurs in about one in 200 to one in 500 births. The Influence of '1atrogenic" Substances A few years ago it was suggested that the majority of instances of unexplained jaundice during the neonatal period were caused by "iatrogenic" substances. As a result, efforts were made to prevent pregnant
BILIRUBIN METABOLISM
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mothers and their newborn babies from receiving drugs and other substances. Although this is a good idea in principle, up until the present only three groups of drugs has been convincingly shown either to alter the bilirubin level or to increase the susceptibility to development of kernicterus. The role of vitamin K analogues in hyperbilirubinemia is well known. Several groups5. 28 have presented clear evidence that the use of large doses of vitamin K will result in increased levels. of indirect bilirubin in the serum, particularly in the premature infant. Surprisingly, the mechanism for this increased hemolysis in non-G-6-P D-deficient erythrocytes is not properly understood. Zinkham 44 suggested that Synkayvite might lower the glucose level of newborn erythrocytes more rapidly than adult erythrocytes and that this would cause increased hemolysis. The role of Gantrisin in the development of kernicterus among premature infants is also well known. In 1956 Silverman and his coworkers 37 carried out a controlled study on the relative effectiveness of two prophylactic antibacterial regimens. Quite unexpectedly, they found 29 examples of kernicterus among 141 necropsies. Of these, 27 instances occurred among infants receiving penicillin and sulfisoxazole and only two instances among infants receiving oxytetracycline. The subsequent studies of Ode1l30 have shown that this occurs because the sulfonamide prevents bilirubin from attaching to serum proteins. The free bilirubin can thus diffuse through protein-impermeable membranes and cause kernicterus. This may occur even if the circulating serum bilirubin levels are not excessively high. In 1961 Sutherland and Keller39 showed that novobiocin can cause hyperbilirubinemia in the newborn infant. They found that jaundice developed in 60 out of 688 infants (9 per cent) who received 50 mg. of novobiocin three times daily, while the incidence was only 50 out of 2044 (2 per cent) in the preceding six months and 67 out of 1809 (3 per cent) in the subsequent six months when novobiocin was not administered. When novobiocin was injected into newborn rats, they became jaundiced, and there was also a decreased excretion of bilirubin in rabbits after the administration of novobiocin. The excessive jaundice caused by novobiocin occurs because this antibiotic is an inhibitor of glucuronyl transferase. l4 • 25 Although a large number of other compounds have been implicated as "iatrogenic" substances in hyperbilirubinemia of the newborn, they have not stood up to rigid control trials. For example, many clinical observations have been made showing that babies born of mothers receiving Demerol or Phenergan are more jaundiced than those not receiving these drugs. At least three controlled trials have been carried out showing that there is no difference in the serum bilirubin levels between
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DAVID YI-YUNG HSIA
those recelvmg and those not recelvmg the drugs. 4. 21.34 These data emphasize the need of using rigid trial conditions for documenting clinical observations.
THE TREATMENT OF NEONATAL JAUNDICE
The primary objective of therapy is to prevent the formation of indirect-reacting bilirubin and to promote its removal from the circulation. For the former, we have discussed the prevention of red cell breakdown, the removal of inhibitors, and the exclusion of "iatrogenic" substances. Despite these measures, some infants will still accumulate excessive amounts of indirect-reacting bilirubin. This section will discuss the current thinking on methods of removal. Hydration In 1962 Hubbell and his co-workers 19 observed that infants of diabetic mothers who were not fed for 48 hours become more significantly jaundiced than those who were fed shortly after birth. Haworth and Medovy15 did not find this in a group of healthy premature infants. They found that a group of infants fed 15 ml. of 5 per cent glucose water per kilogram per day, starting at four hours of age and fed every three hours, showed the same bilirubin level as a similar group of infants receiving no feedings for the first 36 hours. Although obviously more data are needed on this important point, it would appear logical that the bilirubin levels in the serum should be lower in the better hydrated infants. Whether this decrease is meaningful or not in terms of the development of kernicterus remains unknown. Chelating Agents In 1962 Lester, Hammaker and Schmid23 found that when Gunn rats with a genetic deficiency of glucuronyl transferase were given cholestyramine resin, there was a decrease of serum bilirubin levels and an increase of bilirubin-l-C14 clearance. It was felt that this compound, which exhibits a high affinity for bilirubins, irreversibly binds intestinal bilirubin and prevents its reabsorption. In this way the efficiency of pigmentation disposition would be increased and the plasma level would be reduced to a new, lower plateau. According to this same reasoning, cholestyramine was given to a group of premature infants 24 hours after birth. There was little difference between the mean 48-hour bilirubin levels of the resin and the control groups. More recently Ulstrom and Eisenklam41 have performed similar studies using activated charcoal (N. F. Mallinckrodt) in full-term infants. They found that if the charcoal was administered at 12 hours of age, it had no effect on the serum bilirubin levels. But if the charcoal was given at four hours of age, the treated group showed a peak serum
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719
bilirubin level of 6.2 -+- 3.1 mg. per 100 ml., while the control group showed a peak level of 8.9 -+- 3.6 mg. per 100 ml., a difference which was significant (p = 0.036). Although we are not at the stage where these agents can readily be used therapeutically in the treatment of hyperbilirubinemia, they nevertheless offer a fresh approach to the problem. Exchange Transfusion This remains the only effective means of reducing serum bilirubin levels and preventing kernicterus. During the past few years there has been continued controversy as to what should be regarded as the critical serum bilirubin level for exchange transfusions in full-term and premature infants. Although there is no magic in the level of 20 mg. per 100 ml. of indirect bilirubin, charts to aid in the interpretation of bilirubin levels at various ages are available! and provide a rough guide as to when an initial exchange transfusion should be performed. More important is the need for establishing uniformity in the measurement of serum bilirubin levels in different hospitals. For example, Lucey and his co-workers27 found that in the same community over the same period, one hospital had performed exchange transfusions on 44 out of 160 newborn infants, while the other had performed only four in 156 newborn infants, even though the same criteria had been used as guidelines for both hospitals. A careful investigation showed that the bilirubin measurements in the two hospitals were very different. Similarly, Westphal, Viergiver and Roth43 reported that 13 different teaching hospitals had an average discrepancy of 3.6 mg. per 100 ml. Because of this wide variability, the Committee on the Fetus and Newborn of the American Academy of Pediatrics 33 has recommended that an acceptable primary bilirubin standard must give a i-cm. molar absorptivity at 453 millimicrons in chloroform at 25°C. between 59,100 and 62,300. Only in this way can the results from one hospital be effectively compared with those of another hospital. Odell and Cohen3 ! have suggested that the removal of bilirubin from infants by exchange transfusion might be augmented if albumin was given before the exchange transfusion. An average of 41 per cent more bilirubin was removed in the first exchange transfusion from jaundiced infants when they were primed with 1 gm. per kilogram of birth weight of albumin one to two hours before exchange transfusion. Although this approach was proposed nearly five years ago, no definitive data are available on whether it is of any significant value in reducing the incidence of kernicterus. Sequelae of Hyperbilirubinemia Although considerable information is available on the neurologic sequelae of infants with kernicterus, much less is known about the effects of hyperbilirubinemia upon those who fail to show gross abnormalities
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DAVID YI-YUNG HSIA
during the neonatal period. In a study undertaken before the era of widespread use of exchange transfusions, Gerver and D ay l3 reported that for a group of 68 children who recovered from erythroblastosis fetalis without suffering obvious motor nerve damage, the mean intelligence quotient was 11.8 points lower than that of their unaffected older brothers and sisters. When these same studies were carried out in an equal group of similar children after the introduction of exchange transfusions, Day and Haines 8 showed that the difference was only 5.7 points. But when the data were analyzed further, they found that the most anemic infants with hemoglobin values of less than 15 gm. per 100 m!. were 8.9 points lower, the infants who appeared most jaundiced were 14.5 points lower, and those infants who were both most anemic and most jaundiced were 23.1 points lower. Recently, data have also been accumulated on infants of low birth weight without erythroblastosis fetalis. Heimer and her co-workers l6 compared the peak bilirubin levels and the Gesell Gross Motor Schedule at one year in 68 males and 94 females of low birth weight. They found a negative coefficient of correlation of -0.34 (significant at 0.10 level) for males and -0.11 (not significant) for females. Schiller and Silverman 35 undertook a three-year follow-up of a group of 110 infants of low birth weight from whom serum bilirubin levels were obtained during the neonatal period. They were unable to detect any significant difference in either the neurologic or mental status between these infants with peak serum bilirubin levels which were over and under 18 mg. per 100 m!. Vuckovich and his co-workers 42 in a four-year follow-up on a group of 61 infants of low birth weight could not find any striking correlation between the peak serum bilirubin levels and the psychologic and neurologic tests carried out at six months, one year, three years and four years. These findings would lead us to believe that kernicterus appears in general to be an all-or-none phenomenon. If the infant escapes gross neurologic damage in the neonatal period, he is likely to show both motor and mental development very similar to that of other infants of the same gestation length and birth weight.
REFERENCES 1. Allen, F. H., Jr., and Diamond, L. K.: Erythroblastosis FetaUs. Boston, Little, Brown & Company, 1958. 2. Arias, I. M.: The Chemical Basis of Kernicterus. Adv. Clin. Chern., 3:35, 1960. 3. 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 20j3-diol in Maternal Milk That Inhibits Glucuronide Formation in Vitro. J. CUn. Invest., 43:2037, 1964. 4. Boggs, T. R., Lucine, A. A., and Needleman, H. L.: Promethazine Hydrochloride and Neonatal Bilirubin Concentrations. ]. Pediat., 62: 160, 1963. 5. Bound, J., and Telfer, T.: Effect of Vitamin K Dosage on Plasma Bilirubin Levels in Prematures. Lancet, 1:720, 1956.
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6. Brown, A. K., and Zuelzer, W. W.: Studies on the Neonatal Development of the Glucuronide Conjugating System. J. Clin. Invest., 37:332,1958. 7. Capps, F. P. A., Gilles, H. M., Jolly, H., and Worlledge, S. M.: Glucose-6-Phosphate Dehydrogenase Deficiency and Neonatal Jaundice in Nigeria. Lancet, 2:379, 1963. 8. Day, R., and Haines, M. D.: Intelligence Quotients of Children Recovered from Erythroblastosis Fetalis since the Introduction of Exchange Transfusion. Pediatrics, 13:333, 1954. 9. Doxiadis, S. A., Fessas, P., and Valaes, T.: Glucose-6-Phosphate Dehydrogenase Deficiency. A New Aetiological Factor of Severe Neonatal Jaundice. Lancet, 1 :297, 1961. 10. Doxiadis, S. A., Valaes, T., Karaklis, A., and Stavrakakis, D.: Risk of Severe Jaundice in Glucose-6-Phosphate Deficient Newborns. Differences in Population Groups. Lancet, 2:1210,1964. 11. Fessas, P., Doxiadis, S. A., and Valaes, T.: Neonatal Jaundice in Glucose-6-Phosphat Dehydrogenase Deficient Infants. Brit. M.J., 2: 1359, 1962. 12. Gartner, L. M., and Arias, 1. M.: Production of Unconjugated Hyperbilirubinemia in Full-Term Newborn Infants Following Administration of Pregnane-3a 20/3diol. J. Pediat., 65:1045,1964. 13. Gerver, J. M., and Day, R.: Intelligence Quotient of Children Who Have Recovered from Erythroblastosis Fetalis. ]. Pediat., 36:342, 1950. 14. Hargreaves, T., and Holton, J. B.: Jaundice of the Newborn Due to Novobiocin. Lancet, 1:839, 1962. 15. Haworth, J. C., and Medovy, H.: The Effect of Early and Late Feeding and Glucagon upon Blood Sugar and Serum Bilirubin Levels of Premature Infants. J. Pediat., 63:498,1963. . 16. Heimer, C. B., and others: The Sequelae of Neonatal Hyperbilirubinemia of Prematurity at the Age of One Year. Am. J. Dis. Child., 100:495, 1960. 17. Hsia, D. Y.-Y., Riabov, S., and Dowben, R. M.: Inhibition of Glucuronosyl Transferase by Steroid Hormones. Arch Biochem. & Biophys., 103:181, 1963. 18. Hsia, D. Y.-Y., Dowben, R., Shaw, R., and Grossman, A.: Inhibition of Glucuronyl Transferase by Progestational Agents from Serum of Pregnant Women. Nature, 187:693,1960. 19. Hubbell, J. P., and others: "Early" versus "Late" Feeding of Infants of Diabetic Mothers. New England]. Med., 265:835, 1961. 20. Kirkman, H. N., Doxiadis, S. A., Valaes, T., Tassopoulos, N., and Brinson, A. G.: Diverse Characteristics of Glucose-6-Phosphate Dehydrogenase from Greek Children. ]. Lab. & Clin. Med., 65:212, 1965. 21. de Lamerens, S., Tuttle, A. H., and Aballi, A. J.: Neonatal Bilirubin Levels after Use of Phenothiazine Derivatives for Obstetrical Analgesia. J. Pediat., 65:925, 1964. 22. Lathe, G. H., and Walker, M.: Inhibition of Bilirubin Conjugation in Rat Liver Slices by Human Pregnancy and Neonatal Serum and Steroids. Quart. J.. Exp. Physiol., 43:257, 1958. . 23. Lester, R., Hammaker, L., and Schmid, R.: A New Therapeutic Approach to Unconjugated Hyperbilirubinaemia. Lancet, 2:1257, 1962. 24. Levin, S. E., Charlton, R. W., and Freiman, I.: Glucose-6-Phosphate Dehydrogenase Deficiency and Neonatal Jaundice in South African Bantu Infants. J. Pediat., 65:757, 1964. 25. Lokietz, H., Dowben, R. M., and Hsia, D. Y.-Y.: Studies on the Effect of Novobiocin on Glucuronyl Transferase. Pediatrics, 32:47, 1963. 26. Lucey, J., Arias, I., and McKay, R.: Transient Familial Neonatal Hyperbilirubinemia. Am. J. Dis. Child., 100:787, 1960. 27. Lucey, J. F., Phillips, C. L., Utterback, J. G., and McKay, R. J.: A Difference in the Incidence of Hyperbilirubinemia among Premature Infants in Two Hospitals. Pediatrics, 30:3, 1962. 28. Meyer, T. C., and Angus, J.: The Effect of Large Doses of "Synkavite" in the Newborn. Arch Dis. Childhood, 31:212, 1956. 29. Newman, A. J., and Gross, S.: Hyperbilirubinemia in Breast-Fed Infants. Pediatrics, 32:995, 1963.
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30. Odell, G. B.: Studies in Kernicterus. I. The Protein Binding of Bilirubin. J. Clin. Invest., 38:823, 1959. 31. Odell, G. B., and Cohen, S.: Albumin Priming in the Management of Hyperbilirubinemia by Exchange Transfusion. Am. J. Dis. Child., 102:699, 1961. 32. O'Flynn, M. E. D., and Hsia, D. Y.-Y.: Serum Bilirubin Levels and Glucose-6Phosphate Dehydrogenase Deficiency in Newborn American Negroes. J. Pediat., 63:160,1963. 33. Recommendations on a Uniform Bilirubin Standard. Pediatrics, 31:878, 1963. 34. Rowley, W. F., Tannrikulu, 0., Grossman, A., and Hsia, D. Y.-Y.: A Controlled Study on Effect of Promethazine Hydrochloride and Meperidine Hydrochloride upon Serum Bilirubin Levels in the Newborn Infant. J. Pediat., 62:934, 1963. 35. Schiller, J. G., and Silverman, W. A.: Uncomplicated Hyperbilirubinemia of Prematurity. Am.]. Dis. Child., 101:587, 1961. 36. Schmid, R., Forbes, A., Rosenthal, I. M., and Lester, R.: Lack of Effect of Cholestyramine Resin on Hyperbilirubinaemia of Premature Infants. Lancet, 2:938, 1963. 37. Silverman, W. A., Andersen, D. H., Blanc, W. A., and Crozier, D. N.: A Difference in Mortality Rate and Incidence of Kernicterus among Premature Infants Allotted to Two Prophylactic Antibacterial Regimens. Pediatrics, 18:614, 1956. 38. Smith, G. D., and Vella, F.: Erythrocyte Enzyme Deficiency in Unexplained Kernicterus. Lancet, 1:1133, 1960. 39. Sutherland, J. M., and Keller, W. H.: Novobiocin and Neonatal Hyperbilirubinemia. Am. J. Dis. Child., 101:447, 1961. 40. Szeinberg, A., Oliver, M., Schmidt, R., Adam, A., and Sheba, C.: Glucose-6-Phosphate Dehydrogenase Deficiency and Haemolytic Disease of the Newborn in Israel. Arch. Dis. Childhood, 38:23, 1963. 41. Ulstrom, R. A., and Eisenklam, E.: The Enterohepatic Shunting of Bilirubin in the Newborn Infant. ]. Pediat., 65:27, 1964. 42. Vuckovich, D. M., Haimowitz, N., Bowers, N. D., Cosbey, J., and Hsia, D. Y.-Y.: The Influence of Serum Bilirubin Levels upon the Ultimate Development of Low Birthweight Infants. ]. Ment. Def. Res., to be published. 43. Westphal, M., Viergiver, E., and Roth, R.: Analysis of a Bilirubin Survey. Pediatrics, 30: 12, 1962. 44. Zinkham, W. H.: An in-Vitro Abnormality of Glutathione Metabolism in Erythrocytes from Normal Newborns: Mechanisms and Clinical Significance. Pediatrics, 23: 18, 1959. 45. Idem: Peripheral Blood and Bilirubin Values in Normal Full-Term PrimaquineSensitive Negro Infants: Effect of Vitamin K. Pediatrics, 31:983, 1963. 46. Zuelzer, W. W., and Brown, A. K.: Neonatal Jaundice. Am. J. Dis. Child., 101:87, 1961. Children's Memorial Hospital 707 Fullerton Avenue Chicago, Ill. 60614
THE CAUSES OF NEONATAL JAUNDICE
The conjugation of bilirubin is carried out by means of the following reaction:
Bilirubin {indirectreacting)
+
GLUCURONYL UDPGA
-------~) Bilirubin glucuronide
(Uridine diphosphate glucuronic acid)
TRANSFERASE
(directreacting)
+
UDP (Uridine diphosphate)
There is general agreement that hyperbilirubinemia in the newborn infant results either from excessive breakdown of red cells resulting in the accumulation of unusual amounts of indirect-reacting bilirubin or from a functional immaturity of glucuronyl transferase resulting in the failure of conversion of indirect-reacting bilirubin to direct-reacting bilirubin glucuronide, or both. The extensive knowledge in this area accumulated over the past decade has been reviewed. 2 , 46 The present discussion will be confined to the three factors which have recently been shown to influence the degree of jaundice in the newborn infant. Glucose-6-Phosphate Dehydrogenase Deficiency For some years we have known that hyperbilirubinemia in the neonatal period can be caused by hemolysis induced by blood group incompatibility, sepsis, and certain erythrocyte enzyme defects such as congenital nonspherocytic hemolytic anemia with glucose-6-phosphate From the Genetic Clinic of the Children's Memorial Hospital and the Department of Pediatrics, Northwestern University Medical School, Chicago. Aided by a grant from the Association for the Aid to Crippled Children. Prepared during the tenure of a Special Fellowship from the National Institute of Child Health and Human Development (IF 3 HD 2984) at the Kinderspital, Zurich, Switzerland.
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dehydrogenase (G-6-P D) or pyruvate kinase deficiency. It is only recently, however, that a deficiency of G-6-P D without obvious hemolytic anemia has been implicated as a cause for jaundice in the newborn infant. In 1960 Smith and Vella 38 described 14 cases of kernicterus without blood group incompatibility among Chinese infants in Singapore. All except one were found to have a deficiency of G-6-P D. Since G-6-P D deficiency is seen also in other races in Singapore, they attributed the high incidence of jaundice and kernicterus to the use of drugs related to traditional Chinese methods of caring for the mother and baby during the puerperium. One year later Doxiadis, Fessas and Valaes 9 noted a high incidence of G-6-P D deficiency among infants with severe neonatal jaundice of unknown cause in Greece. In this survey involving 22 such infants, 13 of the 17 males and one of the five females were found to be G-6-P D-deficient. Of the remaining five females, three of the fathers were reactors, making their daughters at least heterozygotes for the abnormal gene. Increased jaundice has not always been noted among G-6-P Ddeficient male infants. O'Flynn and Hsia,32 working with American Negro males, and Szeinberg and his co-workers,40 working with infants in Israel, were unable to detect any significant difference in the bilirubin levels between those with normal and those with deficient G-6-P D levels. Other workers 24 , 45 expressed the view that although Negro infants with G-6-P D deficiency might be more jaundiced in some instances, the deficiency of this enzyme is not a main cause of jaundice. Finally, the administration of menadione (Synkayvite) or vitamin Kl (Konakion) did not result in excessive rises of bilirubin among the infants with G-6-P D deficiency.7,45 These conflicting observations led Fessas, Doxiadis and Valaes l l to re-examine the Greek population. In a random survey of 786 newborn infants they found 23 with G-6-P D deficiency. Only one out of 21 such infants had severe hyperbilirubinemia. In contrast, 11 out of the 43 siblings of the original families had hyperbilirubinemia. In a more recent survey carried out in Lesbos, where the incidence of severe hyperbilirubinemia without incompatibility is unusually high, Doxiadis and his co-workers 1o found serum bilirubin levels of 16 mg. per 100 ml. in 34 per cent of G-6-P D-deficient newborn males and 9.1 per cent of normal newborn males, both unusually high. These data have led the authors to postulate that there may be at least two and possibly three types of G-6-P D-deficient populations in Greece. The common type behaves much in the same manner as those with G-6-P D deficiency in other Mediterranean populations. There appears, however, to be another group or groups of individuals in whom G-6-P D deficiency results in a greater tendency to jaundice in the neonatal period. Kirkman and his co-workers 20 have described the physicochemical properties of eight Greek children with G-6-P D deficiency known to
BILIRUBIN METABOLISM
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have had severe jaundice during infancy and four other Greek children with G-6-P D deficiency who were not jaundiced. Although there were no distinctive differences between the two groups and they all followed the customary pattern seen in Sephardic-Sardinian patients with G-6-P D deficiency, the more jaundiced children showed a wider range of variability in terms of (1) the degree of bimodality of the pH optimum curves, and (2) the utilization of 2 desoxy glucose-6-phosphate as a substrate varying from 3 to 65 per cent instead of the usual 23 to 37 per cent. Although this does not provide evidence of a difference in the molecular structure of G-6-P D in the two groups, it does emphasize the need of studying this unusual group of infants further to determine the: mechanism leading to hyperbilirubinemia. It is possible that a deficiency of G-6-P D may result in a whole spectrum of clinical manifestations varying all the way from those who will not hemolyze even in the presence of drugs to those who hemolyze constantly even in the absence of drugs, as in congenital nonspherocytic hemolytic anemia. In these Greek and perhaps also in the Chinese babies the birth process, possibly with its associated hypoxia and hypoglycemia, in some way adversely affects the G-6-P D and results in a temporary increase in the breakdown of red cells. It would be interesting to find out if, later in life, such persons show an abnormal response to the fava bean or other drugs. The Role of Inhibitors Physiologic jaundice in the full-term and particularly in the premature infant is believed to be caused primarily by a functional immaturity of liver glucuronyl transferase. 6 Although it would be attractive to bring about an adaptive increase of this enzyme during the critical first few days of life, repeated efforts to do this have so far been unsuccessful. During recent years increased attention has been turned toward whether the presence of inhibitors might be responsible for the decrease of glucuronyl transferase in the newborn. In 1958 Lathe and Walker22 first reported that serum from pregnant women inhibited the conjugation of bilirubin by liver slices and demonstrated a similar effect with progestational steroids in vitro. Two years later Hsia and his co-workers18 found a similar inhibition upon o-aminophenol conjugation by rat liver homogenate. It was found that serum from pregnant women showed 64.0 -+- 21.8 per cent inhibition, from immediately postpartum women 42.8 -+- 22.5 per cent inhibition, and from newborn infants 48.2 -+- 20.0 per cent inhibition. In contrast, adult males' serum showed only 1.3 -+- 1.0 per cent inhibition, and adult nonpregnant females' serum showed only 26.8 -+- 9.8 per cent inhibition. Pooled serum from pregnant women was purified, and the predominant fraction of the inhibitor substance was found to be pregnanediol. In the meantime two separate clinical syndromes characterized by hyperbilirubinemia in the newborn infant have been described. In 1960
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Lucey, Arias and McKay26 reported on transient familial neonatal hyperbilirubinemia. This condition is manifested by transient nonhemolytic unconjugated hyperbilirubinemia within hours after birth, occurs in siblings and is unrelated to breast feeding. The serum bilirubin level ranged from 25 to 65 mg. per 100 ml. during the first four days of life, and kernicterus was frequently observed. From the second trimester of pregnancy, mothers of infants with this syndrome have in their plasma three to five times higher titers of an inhibitor of glucuronyl transferase than that seen in other pregnant women. Although it would be attractive to postulate that these women have a constitutional or genetic inability to handle pregnanediol or other progestational agents during late pregnancy, no evidence for this has so far emerged. In our own laboratory we have not been able to find any difference in the excretion of pregnanediol in the urine of such women as compared with other women. About the same time a number of reports appeared describing prolonged neonatal jaundice associated with breast feeding. a. 29 In all such instances the removal of human milk and the substitution of cow's Inilk resulted in a rapid disappearance of the hyperbilirubinemia. Milk obtained from the mothers of seven such infants consistently inhibited glucuronyl transferase activity in vitro, while Inilk obtained from other mothers did not demonstrate this inhibition. In contrast, the serum from these women did not inhibit glucuronyl transferase any more than that of other women at the same postpartum stage. From the inhibitory Inilk, pregnane-3a 20 ,a-diol has been consistently isolated, while none has been found in noninhibitory Inilk. It is estimated that 1 mg. of this steroid has been secreted through breast milk by the mother daily. Since pregnanediol in low concentrations acts as a competitive inhibitor of glucuronyl transferase,17 it has been postulated that this material from the milk inhibits an already immature enzyme system and causes increased jaundice due to the inability to conjugate bilirubin. This has been tested in normal full-term infants clinically;12 0.33 mg. per kilogram of pregnane-3a 20 ,a-diol was given to four infants six, eight, 34 and 66 days old and to one adult male. In the two younger infants the serum bilirubin level rose significantly, while no such increase was noted in the two older infants and the adult. It has been suggested that in such situations breast-feeding should be temporarily curtailed for a few days and then resumed. In a survey carried on at several maternity hospitals in our area, we have estimated that this syndrome occurs in about one in 200 to one in 500 births. The Influence of '1atrogenic" Substances A few years ago it was suggested that the majority of instances of unexplained jaundice during the neonatal period were caused by "iatrogenic" substances. As a result, efforts were made to prevent pregnant
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mothers and their newborn babies from receiving drugs and other substances. Although this is a good idea in principle, up until the present only three groups of drugs has been convincingly shown either to alter the bilirubin level or to increase the susceptibility to development of kernicterus. The role of vitamin K analogues in hyperbilirubinemia is well known. Several groups5. 28 have presented clear evidence that the use of large doses of vitamin K will result in increased levels. of indirect bilirubin in the serum, particularly in the premature infant. Surprisingly, the mechanism for this increased hemolysis in non-G-6-P D-deficient erythrocytes is not properly understood. Zinkham 44 suggested that Synkayvite might lower the glucose level of newborn erythrocytes more rapidly than adult erythrocytes and that this would cause increased hemolysis. The role of Gantrisin in the development of kernicterus among premature infants is also well known. In 1956 Silverman and his coworkers 37 carried out a controlled study on the relative effectiveness of two prophylactic antibacterial regimens. Quite unexpectedly, they found 29 examples of kernicterus among 141 necropsies. Of these, 27 instances occurred among infants receiving penicillin and sulfisoxazole and only two instances among infants receiving oxytetracycline. The subsequent studies of Ode1l30 have shown that this occurs because the sulfonamide prevents bilirubin from attaching to serum proteins. The free bilirubin can thus diffuse through protein-impermeable membranes and cause kernicterus. This may occur even if the circulating serum bilirubin levels are not excessively high. In 1961 Sutherland and Keller39 showed that novobiocin can cause hyperbilirubinemia in the newborn infant. They found that jaundice developed in 60 out of 688 infants (9 per cent) who received 50 mg. of novobiocin three times daily, while the incidence was only 50 out of 2044 (2 per cent) in the preceding six months and 67 out of 1809 (3 per cent) in the subsequent six months when novobiocin was not administered. When novobiocin was injected into newborn rats, they became jaundiced, and there was also a decreased excretion of bilirubin in rabbits after the administration of novobiocin. The excessive jaundice caused by novobiocin occurs because this antibiotic is an inhibitor of glucuronyl transferase. l4 • 25 Although a large number of other compounds have been implicated as "iatrogenic" substances in hyperbilirubinemia of the newborn, they have not stood up to rigid control trials. For example, many clinical observations have been made showing that babies born of mothers receiving Demerol or Phenergan are more jaundiced than those not receiving these drugs. At least three controlled trials have been carried out showing that there is no difference in the serum bilirubin levels between
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those recelvmg and those not recelvmg the drugs. 4. 21.34 These data emphasize the need of using rigid trial conditions for documenting clinical observations.
THE TREATMENT OF NEONATAL JAUNDICE
The primary objective of therapy is to prevent the formation of indirect-reacting bilirubin and to promote its removal from the circulation. For the former, we have discussed the prevention of red cell breakdown, the removal of inhibitors, and the exclusion of "iatrogenic" substances. Despite these measures, some infants will still accumulate excessive amounts of indirect-reacting bilirubin. This section will discuss the current thinking on methods of removal. Hydration In 1962 Hubbell and his co-workers 19 observed that infants of diabetic mothers who were not fed for 48 hours become more significantly jaundiced than those who were fed shortly after birth. Haworth and Medovy15 did not find this in a group of healthy premature infants. They found that a group of infants fed 15 ml. of 5 per cent glucose water per kilogram per day, starting at four hours of age and fed every three hours, showed the same bilirubin level as a similar group of infants receiving no feedings for the first 36 hours. Although obviously more data are needed on this important point, it would appear logical that the bilirubin levels in the serum should be lower in the better hydrated infants. Whether this decrease is meaningful or not in terms of the development of kernicterus remains unknown. Chelating Agents In 1962 Lester, Hammaker and Schmid23 found that when Gunn rats with a genetic deficiency of glucuronyl transferase were given cholestyramine resin, there was a decrease of serum bilirubin levels and an increase of bilirubin-l-C14 clearance. It was felt that this compound, which exhibits a high affinity for bilirubins, irreversibly binds intestinal bilirubin and prevents its reabsorption. In this way the efficiency of pigmentation disposition would be increased and the plasma level would be reduced to a new, lower plateau. According to this same reasoning, cholestyramine was given to a group of premature infants 24 hours after birth. There was little difference between the mean 48-hour bilirubin levels of the resin and the control groups. More recently Ulstrom and Eisenklam41 have performed similar studies using activated charcoal (N. F. Mallinckrodt) in full-term infants. They found that if the charcoal was administered at 12 hours of age, it had no effect on the serum bilirubin levels. But if the charcoal was given at four hours of age, the treated group showed a peak serum
BILIRUBIN METABOLISM
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bilirubin level of 6.2 -+- 3.1 mg. per 100 ml., while the control group showed a peak level of 8.9 -+- 3.6 mg. per 100 ml., a difference which was significant (p = 0.036). Although we are not at the stage where these agents can readily be used therapeutically in the treatment of hyperbilirubinemia, they nevertheless offer a fresh approach to the problem. Exchange Transfusion This remains the only effective means of reducing serum bilirubin levels and preventing kernicterus. During the past few years there has been continued controversy as to what should be regarded as the critical serum bilirubin level for exchange transfusions in full-term and premature infants. Although there is no magic in the level of 20 mg. per 100 ml. of indirect bilirubin, charts to aid in the interpretation of bilirubin levels at various ages are available! and provide a rough guide as to when an initial exchange transfusion should be performed. More important is the need for establishing uniformity in the measurement of serum bilirubin levels in different hospitals. For example, Lucey and his co-workers27 found that in the same community over the same period, one hospital had performed exchange transfusions on 44 out of 160 newborn infants, while the other had performed only four in 156 newborn infants, even though the same criteria had been used as guidelines for both hospitals. A careful investigation showed that the bilirubin measurements in the two hospitals were very different. Similarly, Westphal, Viergiver and Roth43 reported that 13 different teaching hospitals had an average discrepancy of 3.6 mg. per 100 ml. Because of this wide variability, the Committee on the Fetus and Newborn of the American Academy of Pediatrics 33 has recommended that an acceptable primary bilirubin standard must give a i-cm. molar absorptivity at 453 millimicrons in chloroform at 25°C. between 59,100 and 62,300. Only in this way can the results from one hospital be effectively compared with those of another hospital. Odell and Cohen3 ! have suggested that the removal of bilirubin from infants by exchange transfusion might be augmented if albumin was given before the exchange transfusion. An average of 41 per cent more bilirubin was removed in the first exchange transfusion from jaundiced infants when they were primed with 1 gm. per kilogram of birth weight of albumin one to two hours before exchange transfusion. Although this approach was proposed nearly five years ago, no definitive data are available on whether it is of any significant value in reducing the incidence of kernicterus. Sequelae of Hyperbilirubinemia Although considerable information is available on the neurologic sequelae of infants with kernicterus, much less is known about the effects of hyperbilirubinemia upon those who fail to show gross abnormalities
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during the neonatal period. In a study undertaken before the era of widespread use of exchange transfusions, Gerver and D ay l3 reported that for a group of 68 children who recovered from erythroblastosis fetalis without suffering obvious motor nerve damage, the mean intelligence quotient was 11.8 points lower than that of their unaffected older brothers and sisters. When these same studies were carried out in an equal group of similar children after the introduction of exchange transfusions, Day and Haines 8 showed that the difference was only 5.7 points. But when the data were analyzed further, they found that the most anemic infants with hemoglobin values of less than 15 gm. per 100 m!. were 8.9 points lower, the infants who appeared most jaundiced were 14.5 points lower, and those infants who were both most anemic and most jaundiced were 23.1 points lower. Recently, data have also been accumulated on infants of low birth weight without erythroblastosis fetalis. Heimer and her co-workers l6 compared the peak bilirubin levels and the Gesell Gross Motor Schedule at one year in 68 males and 94 females of low birth weight. They found a negative coefficient of correlation of -0.34 (significant at 0.10 level) for males and -0.11 (not significant) for females. Schiller and Silverman 35 undertook a three-year follow-up of a group of 110 infants of low birth weight from whom serum bilirubin levels were obtained during the neonatal period. They were unable to detect any significant difference in either the neurologic or mental status between these infants with peak serum bilirubin levels which were over and under 18 mg. per 100 m!. Vuckovich and his co-workers 42 in a four-year follow-up on a group of 61 infants of low birth weight could not find any striking correlation between the peak serum bilirubin levels and the psychologic and neurologic tests carried out at six months, one year, three years and four years. These findings would lead us to believe that kernicterus appears in general to be an all-or-none phenomenon. If the infant escapes gross neurologic damage in the neonatal period, he is likely to show both motor and mental development very similar to that of other infants of the same gestation length and birth weight.
REFERENCES 1. Allen, F. H., Jr., and Diamond, L. K.: Erythroblastosis FetaUs. Boston, Little, Brown & Company, 1958. 2. Arias, I. M.: The Chemical Basis of Kernicterus. Adv. Clin. Chern., 3:35, 1960. 3. 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 20j3-diol in Maternal Milk That Inhibits Glucuronide Formation in Vitro. J. CUn. Invest., 43:2037, 1964. 4. Boggs, T. R., Lucine, A. A., and Needleman, H. L.: Promethazine Hydrochloride and Neonatal Bilirubin Concentrations. ]. Pediat., 62: 160, 1963. 5. Bound, J., and Telfer, T.: Effect of Vitamin K Dosage on Plasma Bilirubin Levels in Prematures. Lancet, 1:720, 1956.
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6. Brown, A. K., and Zuelzer, W. W.: Studies on the Neonatal Development of the Glucuronide Conjugating System. J. Clin. Invest., 37:332,1958. 7. Capps, F. P. A., Gilles, H. M., Jolly, H., and Worlledge, S. M.: Glucose-6-Phosphate Dehydrogenase Deficiency and Neonatal Jaundice in Nigeria. Lancet, 2:379, 1963. 8. Day, R., and Haines, M. D.: Intelligence Quotients of Children Recovered from Erythroblastosis Fetalis since the Introduction of Exchange Transfusion. Pediatrics, 13:333, 1954. 9. Doxiadis, S. A., Fessas, P., and Valaes, T.: Glucose-6-Phosphate Dehydrogenase Deficiency. A New Aetiological Factor of Severe Neonatal Jaundice. Lancet, 1 :297, 1961. 10. Doxiadis, S. A., Valaes, T., Karaklis, A., and Stavrakakis, D.: Risk of Severe Jaundice in Glucose-6-Phosphate Deficient Newborns. Differences in Population Groups. Lancet, 2:1210,1964. 11. Fessas, P., Doxiadis, S. A., and Valaes, T.: Neonatal Jaundice in Glucose-6-Phosphat Dehydrogenase Deficient Infants. Brit. M.J., 2: 1359, 1962. 12. Gartner, L. M., and Arias, 1. M.: Production of Unconjugated Hyperbilirubinemia in Full-Term Newborn Infants Following Administration of Pregnane-3a 20/3diol. J. Pediat., 65:1045,1964. 13. Gerver, J. M., and Day, R.: Intelligence Quotient of Children Who Have Recovered from Erythroblastosis Fetalis. ]. Pediat., 36:342, 1950. 14. Hargreaves, T., and Holton, J. B.: Jaundice of the Newborn Due to Novobiocin. Lancet, 1:839, 1962. 15. Haworth, J. C., and Medovy, H.: The Effect of Early and Late Feeding and Glucagon upon Blood Sugar and Serum Bilirubin Levels of Premature Infants. J. Pediat., 63:498,1963. . 16. Heimer, C. B., and others: The Sequelae of Neonatal Hyperbilirubinemia of Prematurity at the Age of One Year. Am. J. Dis. Child., 100:495, 1960. 17. Hsia, D. Y.-Y., Riabov, S., and Dowben, R. M.: Inhibition of Glucuronosyl Transferase by Steroid Hormones. Arch Biochem. & Biophys., 103:181, 1963. 18. Hsia, D. Y.-Y., Dowben, R., Shaw, R., and Grossman, A.: Inhibition of Glucuronyl Transferase by Progestational Agents from Serum of Pregnant Women. Nature, 187:693,1960. 19. Hubbell, J. P., and others: "Early" versus "Late" Feeding of Infants of Diabetic Mothers. New England]. Med., 265:835, 1961. 20. Kirkman, H. N., Doxiadis, S. A., Valaes, T., Tassopoulos, N., and Brinson, A. G.: Diverse Characteristics of Glucose-6-Phosphate Dehydrogenase from Greek Children. ]. Lab. & Clin. Med., 65:212, 1965. 21. de Lamerens, S., Tuttle, A. H., and Aballi, A. J.: Neonatal Bilirubin Levels after Use of Phenothiazine Derivatives for Obstetrical Analgesia. J. Pediat., 65:925, 1964. 22. Lathe, G. H., and Walker, M.: Inhibition of Bilirubin Conjugation in Rat Liver Slices by Human Pregnancy and Neonatal Serum and Steroids. Quart. J.. Exp. Physiol., 43:257, 1958. . 23. Lester, R., Hammaker, L., and Schmid, R.: A New Therapeutic Approach to Unconjugated Hyperbilirubinaemia. Lancet, 2:1257, 1962. 24. Levin, S. E., Charlton, R. W., and Freiman, I.: Glucose-6-Phosphate Dehydrogenase Deficiency and Neonatal Jaundice in South African Bantu Infants. J. Pediat., 65:757, 1964. 25. Lokietz, H., Dowben, R. M., and Hsia, D. Y.-Y.: Studies on the Effect of Novobiocin on Glucuronyl Transferase. Pediatrics, 32:47, 1963. 26. Lucey, J., Arias, I., and McKay, R.: Transient Familial Neonatal Hyperbilirubinemia. Am. J. Dis. Child., 100:787, 1960. 27. Lucey, J. F., Phillips, C. L., Utterback, J. G., and McKay, R. J.: A Difference in the Incidence of Hyperbilirubinemia among Premature Infants in Two Hospitals. Pediatrics, 30:3, 1962. 28. Meyer, T. C., and Angus, J.: The Effect of Large Doses of "Synkavite" in the Newborn. Arch Dis. Childhood, 31:212, 1956. 29. Newman, A. J., and Gross, S.: Hyperbilirubinemia in Breast-Fed Infants. Pediatrics, 32:995, 1963.
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30. Odell, G. B.: Studies in Kernicterus. I. The Protein Binding of Bilirubin. J. Clin. Invest., 38:823, 1959. 31. Odell, G. B., and Cohen, S.: Albumin Priming in the Management of Hyperbilirubinemia by Exchange Transfusion. Am. J. Dis. Child., 102:699, 1961. 32. O'Flynn, M. E. D., and Hsia, D. Y.-Y.: Serum Bilirubin Levels and Glucose-6Phosphate Dehydrogenase Deficiency in Newborn American Negroes. J. Pediat., 63:160,1963. 33. Recommendations on a Uniform Bilirubin Standard. Pediatrics, 31:878, 1963. 34. Rowley, W. F., Tannrikulu, 0., Grossman, A., and Hsia, D. Y.-Y.: A Controlled Study on Effect of Promethazine Hydrochloride and Meperidine Hydrochloride upon Serum Bilirubin Levels in the Newborn Infant. J. Pediat., 62:934, 1963. 35. Schiller, J. G., and Silverman, W. A.: Uncomplicated Hyperbilirubinemia of Prematurity. Am.]. Dis. Child., 101:587, 1961. 36. Schmid, R., Forbes, A., Rosenthal, I. M., and Lester, R.: Lack of Effect of Cholestyramine Resin on Hyperbilirubinaemia of Premature Infants. Lancet, 2:938, 1963. 37. Silverman, W. A., Andersen, D. H., Blanc, W. A., and Crozier, D. N.: A Difference in Mortality Rate and Incidence of Kernicterus among Premature Infants Allotted to Two Prophylactic Antibacterial Regimens. Pediatrics, 18:614, 1956. 38. Smith, G. D., and Vella, F.: Erythrocyte Enzyme Deficiency in Unexplained Kernicterus. Lancet, 1:1133, 1960. 39. Sutherland, J. M., and Keller, W. H.: Novobiocin and Neonatal Hyperbilirubinemia. Am. J. Dis. Child., 101:447, 1961. 40. Szeinberg, A., Oliver, M., Schmidt, R., Adam, A., and Sheba, C.: Glucose-6-Phosphate Dehydrogenase Deficiency and Haemolytic Disease of the Newborn in Israel. Arch. Dis. Childhood, 38:23, 1963. 41. Ulstrom, R. A., and Eisenklam, E.: The Enterohepatic Shunting of Bilirubin in the Newborn Infant. ]. Pediat., 65:27, 1964. 42. Vuckovich, D. M., Haimowitz, N., Bowers, N. D., Cosbey, J., and Hsia, D. Y.-Y.: The Influence of Serum Bilirubin Levels upon the Ultimate Development of Low Birthweight Infants. ]. Ment. Def. Res., to be published. 43. Westphal, M., Viergiver, E., and Roth, R.: Analysis of a Bilirubin Survey. Pediatrics, 30: 12, 1962. 44. Zinkham, W. H.: An in-Vitro Abnormality of Glutathione Metabolism in Erythrocytes from Normal Newborns: Mechanisms and Clinical Significance. Pediatrics, 23: 18, 1959. 45. Idem: Peripheral Blood and Bilirubin Values in Normal Full-Term PrimaquineSensitive Negro Infants: Effect of Vitamin K. Pediatrics, 31:983, 1963. 46. Zuelzer, W. W., and Brown, A. K.: Neonatal Jaundice. Am. J. Dis. Child., 101:87, 1961. Children's Memorial Hospital 707 Fullerton Avenue Chicago, Ill. 60614