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Glucose-6-phosphate dehydrogenase deficiency and neonatal jaundice in South African Bantu infants
TROPICAL PEDIATRICS
Glucose-6-pbospbate dehydrogenase deficiency and neonatal jaundice in South African Bantu infants The role o[ G6PD deficiency in the etiology o[ neonatal hyperbilirubinemia has been ~nvestigated in a group o[ South A[rican Bantu in[ants, The incidence o[ this enzyme deject was [ound to be significantly greater in full-term male babies with jaundice with no blood group incompatibility (14 per cent) than in a control group o[ male newborn in[ants (1.3 per cent). Evidence o[ perinatal hypoxia was [ound in 5 o[ 7 G6PD-deficient full-term male jaundiced in[ants with no Rh or ABO incompatibility. It would appear that the combination o[ an increased bilirubin load [rom the shortened red cell life span in G6PD deficiency, together with hypoxic liver damage, is responsible [or icterus in this group.
S. E. Levin, M.B., B.Ch., M.R.C.P.E., D.C.H., ~ R. W. Charlton, M.D., B.Se., M.R.C.P.E., and I. Freiman, M.B., B.Ch., F.C.P. (S.A.) JOHANNESBURG,
REPUBLIC
OF
SOUTt-I
AFRICA
small proportion of cases (10 per cent), and there was a significant number of full-term babies with "physiologic" hyperbilirubinemia. Reports from Malaya, Italy, Nigeria, Greece, and Thailand suggested that deficiency of the enzyme glueose-6-phosphate dehydrogenase (G6PD) in the red cells was an important factor in the etiology of severe neonatal jaundice. 2-~ On the other hand Zinkham 1~ and Hsia 11 could not demonstrate an increased incidence of severe icterus in
I N A previous study of severe neonatal jaundice in South African Bantu infants at this hospital, ~ prematurity was found to be the most frequent cause (70 per cent). ABO or Rh incompatibility was present in only a From the Department of Paediatrics, Baragwanath Hospital, and the Department o[ Medicine, University o[ the Witwatersrand, Johannesburg. ~'Address, Department oJ Paedlatrles, Baragwanath Hospital, Johannesburg, Republic o[ South Africa.
757
758
Levin, CharIton, and Freiman
American Negro neonates with G6PD deficiency compared with normal subjects. Most recently Fessas, Doxiadis, and Valaes, 12 Szeinberg and co-workers, 13 and Zinkham ~4 reported that the enzyme deficiency is not a frequent cause of neonatal jaundice even when the incidence of the trait in the population is high. Nevertheless, these workers concluded that there is an association between the two conditions. In the studies of the first two groups of authors 12' 13 some families with G6PD deficiency developed severe jaundice while others did not. Thus the precise role of this enzyme defect in the etiology of neonatal hyperbilirubinemia has not been finally established. Erythrocyte G6PD deficiency has been demonstrated in South African Bantu male adults, the over-all incidence being 2 to 4 per cent? ~-~s The deficiency is transmitted via a sex-linked gene of intermediate dominance so that marked deficiency is much more common in males than in females? 9-21 In view of the possible relationship between the enzyme deficiency and neonatal hyperbilirubinemia, and the significant incidence of unexplained icterus among newborn. Bantu infants, it was decided to determine the role of G6PD deficiency in the pathogenesis of this condition in babies admitted to Baragwanath Hospital. MATERIAL
AND METHODS
The investigation was divided into two stages. Stage I. In this stage infants were ineluded in the investigation if they became jaundiced within the first 24 hours after birth, or were clinically severely icteric during the first week, or were still jaundiced 14 days after birth. During a 9 month period 159 premature or full-term babies of both sexes were investigated (Group 1 infants). Vitamin K ("Synkavit") in a dose of 10 rag. was given to the mother by intramuscular injection shortly before delivery. Those infants born before arrival at the hospital received an intramuscular injection of 2.5 mg. of the vitamin soon after admission. The mother was asked whether any previous chil-
November 1964
dren had had jaundice. The birth weight and sex of each infant was recorded and a careful watch was kept for signs of kernicterus. Laboratory investigations, carried out on samples of venous blood, included estimation of the hemoglobin and reticulocyte count, 22 blood group determination, and the Coombs test, while the bilirubin concentration was measured by the method of King. 23 Red cell morphology was studied on blood films stained with WHght's stain. Erythrocyte G6PD activity was assessed by the method of Motulsky and Campbell-Kraut, ~4 deficiency of G6PD being confirmed by repeating the test at least once. Blood grouping and tests for abnormal antibodies were also carried out on samples of the mother's blood. Since the criteria for the diagnosis of ABO hemolytic disease have not been established, all Group A or Group B infants born of Group O mothers have been considered to be incompatible. Exchange transfusion was thought to be necessary in 53 infants by the criteria of Shnier and Levin? These babies were given 300,000 units of procaine penicillin by daily intramuscular injection for 4 days after the procedure. No other antibiotic was used. Stage II. In this part of the investigation only full-term male infants with jaundice (as already defined in Stage I) were studied. In the course of the following year an additional 51 babies in this category were admitted (Group 2 infants). Samples of venous blood from these infants and their mothers were analyzed in the same way as in Stage I. In addition, evidence of difficulty with the delivery (such as symphysiotomy, forceps extraction, or cesarean section), or of fetal distress or neonatal asphyxia were noted. As a control, cord blood specimens from 155 serial male full-term hospital births were also tested for G6PD activity. RESULTS
Stage I. The results obtained in the first part of the investigation are summarized in Table I. G6PD deficiency was detected in 5 of the
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G6PD deficiency and jaundice in Bantu in[ants
759
Table I. Analysis of 159 cases of neonatal jaundice
ABO incompatibility Rh incompatibility Unexplained hyperbilirubinemia Total jaundiced infants
Premature Male [ Female 6 13 --
Full-term Male I Female 15" 9
Total 43 1 111
1
51
14
38
8
109 70%
46 30%
155f
175
165
3,3
G6PD deficient Serum bilirubin level 20 mg. per 100 ml. or greater
*I G6PD-deficient infant also had an associated ABO incompatibility. tBirth weights of 4 infants not known. .~Ineludes 1 G6PD-deficient infant.
159 babies of Group 1. All 5 enzyme-deficient infants were male. Of the 109 premature babies in this group there were 57 males; ABO incompatibility was present in 6, while of the remaining 51 premature males with no blood group incompatibility one was G6PD deficient. There were 29 males among the 46 fUll-term babies (in 4 cases the birth weights were not recorded). ABO incompatibility was diagnosed in 15 of the 29, one of whom was also G6PD deficient. There were thus 14 full-term males with no blood group incompatibility: in 3 of these G6PD deficiency was found, an incidence of 21 per cent. Rh hemolytic disease was diagnosed in only one infant (a premature female). The total serum bilirubin concentration was between 12 and 19.9 mg. per 100 ml. in 102 (64 per cent) and 20 mg. per 100 ml. or greater in 33 cases (21 per cent). Of the 33 babies with very severe jaundice, 17 were premature and 16 full-term. Two of this group were G6PD deficient, one premature and one full-term. Kernicterus was diagnosed in only 2 cases, in neither of which was G6PD deficiency found. I n both instances signs of kernicterus were already present on admission. Stage II. Five of the 51 male full-term infants with severe jaundice in Group 2 were found to be deficient in G6PD (10 per cent) compared with only 2 of 155 cord blood samples from the control series (1.3 per cent). In 13 of the 51 babies ABO in-
compatibility was demonstrated, one being also deficient in G6PD. Thus 4 of the 38 remaining infants with no blood group incompatibility were G6PD deficient. Obstetrical complications, fetal distress, or neonatal asphyxia were recorded in 5 of the 13 infants with ABO incompatibility (40 per cent), in 17 of the 34 infants with neither blood group incompatibility nor enzyme deficiency (50 per cent), and in 3 of the 4 babies with G6PD deficiency alone, 75 per cent (Table I I ) . Kernicterus was not observed in Group 2. In Table I I I the findings in the 29 fullterm male infants of Group 1 have been combined with those of the 51 babies in Group 2. Of the total of 80 infants, ABO incompatibility was demonstrated in 28, 2 were also G6PD deficient (7 per cent). Seven of the remaining 52 babies with no blood group incompatibility were deficient in G6PD (14 per cent). The difference between this figure and the incidence in the control
Table II. Obstetrical complications, fetal distress, or neonatal asphyxia among 51 icteric full-term male infants
r I,~fa,~t~ I~ypoxi~1% ABO incompatibility No incompatibility (G6PD normal) G6PD deficient (no incompatibility)
13
5*
4~0
34
17
50
4
3
75
Total
51
25
50
*Associated G6PD deficiency in 1 case.
7 6 0
November 1964
L e v i n , CharIton, and F r e i m a n
Table I I I . Incidence of G 6 P D deficiency in combined series of Group 1 and 2 male full-term infants with jaundice i
I ,
ABO incompatibility Unexplained hyperhilirubinemia Total
Group 1 15 (1)t
Group 2 13 (1)
Total 28
G6PD ~ deficient 2 (7 % )
Significance not significant
14 (3) 29
38 (4) 51
52 80
7 (1+%) 9 (11%)
p < 0.01 p < 0.01
~The significance of G 6 P D deficiency has been assessed by comparison with a cord blood incidence of 1.3 per cent. tFigures in parentheses are the number of G6PD-deficient infants in the separate groups.
Table IV. Analysis of 7 full-term male G6PD-deficient infants with unexplained hyperbilirubinemia ftemo-
Reticulocyte
Serum bilirubin level (rag.~1 O0 ml.) ?
Day of li[e
F~x-
Case 1
globin (Gin. 100 m l. ) 16.6
2
13.9
4
4
Normal
2'0.0
Day 4
3 4
16.7 17,0
I 6
12 5
-Normal
11.3 15.6
Day 12 Day 5
5 6
17.2 16.1
1 2
6 8
Normal Normal
19.9 15.4
Day 7 Day 8
7
18.6
2
4
Normal
20.0
Day 4
count
(%)
6
Blood Day of li[e films 3 Normal
group (1.3 per cent) is statistically highly significant (p < 0.01). T h e data relating to the 7 G 6 P D deficient full-term males with no blood group incompatibility are summarized in Table IV. T h e hemoglobin concentrations ranged from 13.9 to 18.6 Gm. per 100 ml. (the child with the hemoglobin concentration of 13.9 Gm. per 100 ml. had a subaponeurotic hemorrhage). Reticulocytes were 1 to 6 per cent, and no morphologic abnormalities were noted in the red cells. T h e serum bilirubin concentrations ranged between t 1.3 and 20.0 rag. per 100 ml. Kernicterus was not observed, but exchange transfusion was performed in 3 instances. Maternal obstetrical complications, fetal distress, or neonatal asphyxia were recorded in 5 of the 7 cases (70 per cent). T h e r e was no family history
change trans[usion -
+ -
+ -
Obstetrical history
Fetal distress Vertex delivery Forceps Subaponeurotic hemorrhage NormaI Cesarean section Disimpacted head District case Cephalopelvic disproportion FetM distress Symphisiotomy Born before arrival at hospitaI Asphyxiated
of severe neonatal jaundice in any of the G 6 P D deficient group. D I S C U S S I O N
O f the 159 cases of neonatal jaundice in the first series, only 5 (all males) were found to be deficient in G6PD. It is thus apparent that deficiency of this enzyme is not a major cause of jaundice in newborn Bantu infants in Johannesburg, One hundred and nine (70 per cent) of the babies in this group were premature, the same proportion as in the previous study reported from this hospital, 1 and it seems clear that hepatic immaturity with inadequate conjugation of bilirubin accounts for most cases of neonatal icterus seen at Baragwanath. There were 14 male babies with no blood group incompatibility a m o n g the 46 full-
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G6PD deficiency and ]aunchce m Bantu infants
term jaundiced infants. Since the incidence of G6PD deficiency in the adult male population in this area is 2 to 4 per cent, 15"1s the demonstration of enzyme deficiency in 3 of these 14 (21 per cent) suggested that it might be an etiologic factor in full-term males. It was in order to examine this possibility that the second part of the study was undertaken. In this stage of the investigation the incidence of G6PD deficiency in jaundiced, male, full-term infants was compared with that in a control group of serial, male, full-term births. After eliminating those with possible ABO or Rh incompatibility, there was a total of 52 infants in the jaundiced group (combining both parts of the study). G6PD deficiency was found in 7, an incidence of 14 per cent. Since only 1.3 per cent of the control group were deficient in the enzyme, it seems reasonable to conclude that G6PD deficiency of the erythrocytes does contribute to the neonatal jaundice of full-term South African Bantu male infants. Investigators working in Greece and in Singapore found evidence of hemolysis in their enzyme-deficient cases, and in addition patients were often admitted with established kernicterusY, a, 7, 2~ In this series the hemoglobin concentrations, reticulocyte counts~ and red cell morphology were not abnormal in the G6PD-deficient infants with no blood group incompatibility (Table IV). (The one child with a low hemoglobin concentration had a subaponeurotic hemorrhage.) Furthermore, none of the 10 G6PDdeficient babies with jaundice in the present investigation were admitted with or developed kernicterus. The differences between the present findings and those of the previous workers may be a reflection of the degree of deficiency of G6PD in the different populations. It has been established that G6PD deficiency is more marked in affected Caucasians than in American Negroes. 26 Certain drugs, such as quinine, quinidine, and chloramphenicol, provoke hemolysis in enzymedeficient Caucasians, but not in affected Negroes. 2~ The pattern of drug sensitivity in G6PD-deficient South African Bantu sub-
76 1
jects suggests that the enzyme deficiency resembles the type found in American Negroes rather than in Caucasians. 2s The less severe deficiency of the enzyme, therefore, may be responsible for the milder clinical picture in our patients. Zinkham 14 has recently made a similar suggestion. It has been established that tile life-span of enzyme-deficient cells is about 25 per cent less than normal even in the absence of detectable hemolysisY9 It follows that G6PD deficiency must contribute to neonatal jaundice by increasing the load of unconjugated bilirubin presented to the liver. Not all G6PD-deficient infants become jaundiced, however, so that it is clear that additional factors must operate in the cases which do become icteric. These factors might act in two ways: either the destruction of the defective cells might be accelerated, increasing the load of bilirubin, or else the conjugating capacity of the liver might be depressed. A number of factors are known to hemolyse G6PD-deficient cells. 2~ These include various drugs, naphthalene and favus beans (in the Caucasian type only). Certain viral and bacterial infections, diabetic acidosis, and physiologic hypoglycemia of the newborn enhance the effect of the hemolytic agents. The only recognized hemolytic substance to which the babies in the present study were known to be exposed was vitamin K. However, the dosage employed was that recommended by Zinkham, 8~ and shown recently by the same author 14 not to give rise to significant hyperbilirubinemia in G6PD-deficient Negro babies. In any case, the absence of any hematologic evidence of hemolysis suggests that the additional factor is more likely to be hepatic than hemolytic. The combination of moderate depression of liver function with the 25 per cent greater load of bilirubin in G6PDdeficient infants would be more likely to result in significant jaundice than a similar degree of hepatic dysfunction in babies with normal red cell life spans. Panizon 31 has suggested that fetal hypoxia is commonly associated with severe jaundice in enzymedeficient babies. In the present series situations which would contribute toward the
7 62
Levin, Charlton, and Freiman
development of perinatal hypoxia (such as obstetrical complications, fetal distress, or neonatal asphyxia) were recorded in 5 of the 7 (70 per cent) G6PD-deficient male full-term infants with no ABO incompatibility (Table IV). If hypoxia is the common additional factor in the present series, it seems much more probable that it depresses hepatic function than that it increases erythrocyte breakdown, since defective activity of an enzyme on the oxidative pathway of glucose metabolism would be relieved rather than exaggerated by anaerobic conditions. Evidence of hypoxia was also obtained in 50 per cent of the full-term babies with normal G6PD and no blood group incompatibility (Table I I ) , and this factor alone must be responsible for much of the jaundice in this group of infants. The association between severe neonatal jaundice and hypoxia has been previously described? T M Fessas ar, d colleagues 12 found that severe neonatal jaundice was common in certain families with G6PD deficiency, whereas other G6PD-deficient families did not develop icterus. These workers suggested, therefore, that the additional factor was genetically determined. Szeinberg and coauthors 13 confirmed the familial incidence of jaundice in some of their G6PD-deficient subjects. However, no family history of neonatal jaundice was obtained in any of the G6PD-deficient cases in the present study. If the additional factor in our G6PD-deficient cases is hepatic dysfunction resulting from perinatal hypoxia, it might be expected that the inefficient glucuronide conjugation of prematurity would act in the same way. However, in neither the present study nor that of Doxiadis and associates7 was an increased incidence of the enzyme defect found among jaundiced premature babies. The explanation for this apparent anomaly may lie in the high incidence of jaundice in premature infants, whether G6PD-deficient or not. A survey of the infants in the premature baby unit of this hospital at the present time revealed that more than 50 per cent had developed severe jaundice in the neonatal period. From a statistical point of view,
November 1964
therefore, the possible contribution of G6PD deficiency to the icterus might not be apparent if most premature infants become jaundiced in any case, since the difference between the incidence of the enzyme deficiency in this group and in the general population would not be great. It seems reasonable, however, to postulate that the enzyme deficiency (if present) would aggravate the jaundice of prematurity. It is concluded, therefore, that G6PD deficiency is a factor in the pathogenesis of severe neonatal jaundice in Bantu male fullterm infants. It seems probable that an additional factor is usually present, and that this is commonly perinatal hypoxic damage to the liver. The damaged liver is unable to excrete the increased load of bilirubin resulting from the shortened red cell survival of G6PD deficiency even in the absence of extrinsic hemolytic agents. SUMMARY
The relationship between G6PD deficiency and severe neonatal jaundice was studied in South African Bantu infants admitted to Baragwanath Hospital, Johannesburg. The incidence of the enzyme defect was found to be significantly greater in full-term male jaundiced babies with no blood-group incompatibility (14 per cent) than in a control group (1.3 per cent). In 5 out of 7 G6PDdeficient full-term males with no Rh or ABO incompatibility, evidence of perinatal hypoxia was obtained. It is suggested that the combination of hypoxic liver damage with the increased bilirubin load resulting from G6PD deficiency is responsible for the icterus in this group.
We would like to thank Drs. S. Wayburne, E. Kahn, and H. Stein for allowing us to study cases under their care, and Dr. I. Frack, Superintendent, Baragwanath Hospital, for permission to publish. Dr. M. Shapiro of the South African Blood Transfusion Service kindly arranged the serum bilirubin and blood group determinations and carried out the exchange transfusions, while
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G6PD deficiency and jaundice in Bantu infants
Dr. R. Cassel of the South African Institute for Medical Research performed the hemoglobin and reticulocyte estimations. We are also grateful to Dr. D. Lavery of the Obstetrics Department for the control cord blood samples; to Drs. M. Shnier and L. Taitz for their constructive criticism; and to Mr. C. du Plessis of the National Institute of Personnel Research for the statistical analysis.
REFERENCES 1. Shnier, M. H., and Levin, S. E.: Hyperbilirubinemia and kernicterus in premature and full-term Bantu newborn infants, Brit. M. J. 1: 1004, 1959. 2. Smith, G. D., and Vella, F.: Erythroeyte enzyme deficiency in unexplained kernicterus, Lancet 1: 1133, 1960. 3. Weatherall, D. J.: Enzyme deficiency in haemolytic disease of the newborn, Lancet 2: 835, 1960. 4. Panizon, F.: Erythrocyte enzyme deficiency in unexplained kernicterus, Lancet 2:10931 1960. 5. Gilles, H. M., and Arthur, L. J. H.: Erythroeyte enzyme deficiency in unexplained neonatal jaundice, West African M. J. 9: 266, 1960. 6. Gilles, H. M., and Taylor, B. G.: The existence of the glucose-6-phosphate dehydrogenase deficiency trait in Nigeria and its clinical implications, Ann. Trop. Med. 55: 64, 1961. 7. Doxiadis, S. A., Fessas, Ph., Valaes, T., and Mastrokalos, N.: Glucose-6-phosphate dehydrogenase deficiency. A new aetiologieal factor of severe neonatal jaundice, Lancet 1: 297, 1961. 8. Capps, F. P. A., Gilles, H. M. J., Jolly, H., and Worlledge, S.: Glueose-6-phosphate dehydrogenase deficiency and neonatal jaundice in Nigeria, Lancet 2: 379, 1963. 9. Flatz, G., Sringam, S., and Kornkris, V.: Neonatal jaundice in glucose-6-phosphate dehydrogenase deficiency, Lancet 1: 1382, 1963. 10. Zinkham, W. H.: Yearbook of Pediatrics 1961/1962, Chicago, 1962, Year Book Medical Publishers, p. 295. 11. H~ia, D. Y-Y: Yearbook of Pediatrics, 1961/ 1962, Chicago, 1962, Year Book Medical Publishers, p. 297. 12. Fessas, Ph., Doxiadis, S. A., and Valaes, T.: Neonatal jaundice in glucose-6-phosphate dehydrogenase deficient infants, Brit. M. J. 2: 1359, 1962. 13. 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.
763
14. Zinkham, W. H.: Peripheral blood and bilirubin values ill normal full-term primaquine-sensitive Negro infants: effect of vitamin K, Pediatrics 31: 983, 1963. 15. Charlton, R. W., and Bothwell, T. H.: The incidence of glutathione instability of the red blood cells in the South African Bantu, South African J. M. Sc. 24: 88, 1959. 16. Charlton, R. W., and Bothwell, T. H.: Primaquine-sensitivity of red cells in various races in Southern Africa, Brit. M. J. I: 941, 1961. 17. Bernstein, R. E.: Glutathione assay and metabolism in normal and drug-sensitive red cells, South African M. J. 35; 295, 1961. 18. Bernstein, R. E.: Occurrence and clinical implications of red-cell glucose-6-phosphate dehydrogenase deficiency in South African racial groups, South African M. J. 37: 447, 1963. 19. Childs, B., Zinkham, W. H., Browne, E. A., Kimbro, E. L., and Torbert, J. V.: A genetic study of a defect in glutathione metabolism of the erythrocyte, Bull. Johns Hopkins Hosp. 102: 21, 1958. 20. Adam, A.: Linkage between deficiency of glucose-6-phosphate dehydrogenase and colour-blindness, Nature 189: 686, 1961. 21. Charlton, R. W., Patz, I. M., and Borok, G.: The genetics of glucose-6-phosphate dehydrogenase deficiency, South African M. J. 37" 486, 1963. 22. Dacie, J. V.: Practical haematology, ed. 2, London, 1958, J. & A. Churchill, Ltd., p. 23. 23. King, E. J.: Microanalysis in medical biochemistry, ed. 2, London, 1951, J. & A. Churchill, Ltd., p. 37. 24. Motulsky, A. G., and Campbell-Kraut, J. M.: in Blumberg, B. S., editor: Proceedings of the Conference on Genetic Polymorphisms and Geographic Variations in Disease, London, 1961, Grune & Stratton Company, p. 159. 25. Vella, F.: The incidence of erythrocyte glucose-6-phosphate dehydrogenase deficiency in Singapore, Experientia 17: 181, 1961. 26. Marks, P. A., and Gross, R. T.: Erythrocyte glueose-6-phosphate dehydrogenase deficiency: evidence of differences between Negroes and Caucasians with respect to this genetically determined trait, J. Clin. Invest. 38: 2253, 1959. 27. Tarlov, A. R., Brewer, G. J., Carson, P. E. and Alving, A. S.: Primaquine sensitivity, A. M. A. Arch. Int. Med. 109: 209, 1962. 28. Zail, S. S., Charlton, R. W., and Bothwell, T. H.: The haemolyfic effect of certain drugs in Bantu subjects with a deficiency of glucose6-phosphate dehydrogenase, South African J. M. Se. 27: 95, 1962. 29. Brewer, G. J., Tarlov, A. R., and Kellermeyer, R. W.: The hemolytic effect of primaquine, XII. Shortened erythrocyte life span in primaqulne-sensitive male Negroes in the absence of drug administration, J. Lab. & Clin. Med. 58: 217, 1961.
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Levin, Charlton, and Freiman
30. Zinkham, W. H.: quoted by Tarlov et al. 2~ 3I. Panizon, F.: quoted by Szeinberg et al. 13 32. Crosse, V. M., Meyer, T. C., and Gerrard, J. W.: Kernicterus and prematurity, Arch. Dis. Childhood 30: 501, 1955. 33. Brown, A. K., and Zuelzer, W. W.: Studies in hyperbilirubinemia. I. Hyperbilirubinemia of the newborn unrelated to isoimmunization, A. M. A. J. Dis. Child. 93: 263, 1957.
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34. Miller, C. A., and Reed, H. R.: The relation of serum concentrations of bilirubin to respiratory function of premature infants, Pediatrics 21: 362, 1958. 35. Wood, B. S. B., Culley, P. E., Waterhouse, J. A. H., and Powell, D. J.: Factors influencing neonatal jaundice, Arch. Dis. Childhood 37: 371, 1962.
Glucose-6-pbospbate dehydrogenase deficiency and neonatal jaundice in South African Bantu infants The role o[ G6PD deficiency in the etiology o[ neonatal hyperbilirubinemia has been ~nvestigated in a group o[ South A[rican Bantu in[ants, The incidence o[ this enzyme deject was [ound to be significantly greater in full-term male babies with jaundice with no blood group incompatibility (14 per cent) than in a control group o[ male newborn in[ants (1.3 per cent). Evidence o[ perinatal hypoxia was [ound in 5 o[ 7 G6PD-deficient full-term male jaundiced in[ants with no Rh or ABO incompatibility. It would appear that the combination o[ an increased bilirubin load [rom the shortened red cell life span in G6PD deficiency, together with hypoxic liver damage, is responsible [or icterus in this group.
S. E. Levin, M.B., B.Ch., M.R.C.P.E., D.C.H., ~ R. W. Charlton, M.D., B.Se., M.R.C.P.E., and I. Freiman, M.B., B.Ch., F.C.P. (S.A.) JOHANNESBURG,
REPUBLIC
OF
SOUTt-I
AFRICA
small proportion of cases (10 per cent), and there was a significant number of full-term babies with "physiologic" hyperbilirubinemia. Reports from Malaya, Italy, Nigeria, Greece, and Thailand suggested that deficiency of the enzyme glueose-6-phosphate dehydrogenase (G6PD) in the red cells was an important factor in the etiology of severe neonatal jaundice. 2-~ On the other hand Zinkham 1~ and Hsia 11 could not demonstrate an increased incidence of severe icterus in
I N A previous study of severe neonatal jaundice in South African Bantu infants at this hospital, ~ prematurity was found to be the most frequent cause (70 per cent). ABO or Rh incompatibility was present in only a From the Department of Paediatrics, Baragwanath Hospital, and the Department o[ Medicine, University o[ the Witwatersrand, Johannesburg. ~'Address, Department oJ Paedlatrles, Baragwanath Hospital, Johannesburg, Republic o[ South Africa.
757
758
Levin, CharIton, and Freiman
American Negro neonates with G6PD deficiency compared with normal subjects. Most recently Fessas, Doxiadis, and Valaes, 12 Szeinberg and co-workers, 13 and Zinkham ~4 reported that the enzyme deficiency is not a frequent cause of neonatal jaundice even when the incidence of the trait in the population is high. Nevertheless, these workers concluded that there is an association between the two conditions. In the studies of the first two groups of authors 12' 13 some families with G6PD deficiency developed severe jaundice while others did not. Thus the precise role of this enzyme defect in the etiology of neonatal hyperbilirubinemia has not been finally established. Erythrocyte G6PD deficiency has been demonstrated in South African Bantu male adults, the over-all incidence being 2 to 4 per cent? ~-~s The deficiency is transmitted via a sex-linked gene of intermediate dominance so that marked deficiency is much more common in males than in females? 9-21 In view of the possible relationship between the enzyme deficiency and neonatal hyperbilirubinemia, and the significant incidence of unexplained icterus among newborn. Bantu infants, it was decided to determine the role of G6PD deficiency in the pathogenesis of this condition in babies admitted to Baragwanath Hospital. MATERIAL
AND METHODS
The investigation was divided into two stages. Stage I. In this stage infants were ineluded in the investigation if they became jaundiced within the first 24 hours after birth, or were clinically severely icteric during the first week, or were still jaundiced 14 days after birth. During a 9 month period 159 premature or full-term babies of both sexes were investigated (Group 1 infants). Vitamin K ("Synkavit") in a dose of 10 rag. was given to the mother by intramuscular injection shortly before delivery. Those infants born before arrival at the hospital received an intramuscular injection of 2.5 mg. of the vitamin soon after admission. The mother was asked whether any previous chil-
November 1964
dren had had jaundice. The birth weight and sex of each infant was recorded and a careful watch was kept for signs of kernicterus. Laboratory investigations, carried out on samples of venous blood, included estimation of the hemoglobin and reticulocyte count, 22 blood group determination, and the Coombs test, while the bilirubin concentration was measured by the method of King. 23 Red cell morphology was studied on blood films stained with WHght's stain. Erythrocyte G6PD activity was assessed by the method of Motulsky and Campbell-Kraut, ~4 deficiency of G6PD being confirmed by repeating the test at least once. Blood grouping and tests for abnormal antibodies were also carried out on samples of the mother's blood. Since the criteria for the diagnosis of ABO hemolytic disease have not been established, all Group A or Group B infants born of Group O mothers have been considered to be incompatible. Exchange transfusion was thought to be necessary in 53 infants by the criteria of Shnier and Levin? These babies were given 300,000 units of procaine penicillin by daily intramuscular injection for 4 days after the procedure. No other antibiotic was used. Stage II. In this part of the investigation only full-term male infants with jaundice (as already defined in Stage I) were studied. In the course of the following year an additional 51 babies in this category were admitted (Group 2 infants). Samples of venous blood from these infants and their mothers were analyzed in the same way as in Stage I. In addition, evidence of difficulty with the delivery (such as symphysiotomy, forceps extraction, or cesarean section), or of fetal distress or neonatal asphyxia were noted. As a control, cord blood specimens from 155 serial male full-term hospital births were also tested for G6PD activity. RESULTS
Stage I. The results obtained in the first part of the investigation are summarized in Table I. G6PD deficiency was detected in 5 of the
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G6PD deficiency and jaundice in Bantu in[ants
759
Table I. Analysis of 159 cases of neonatal jaundice
ABO incompatibility Rh incompatibility Unexplained hyperbilirubinemia Total jaundiced infants
Premature Male [ Female 6 13 --
Full-term Male I Female 15" 9
Total 43 1 111
1
51
14
38
8
109 70%
46 30%
155f
175
165
3,3
G6PD deficient Serum bilirubin level 20 mg. per 100 ml. or greater
*I G6PD-deficient infant also had an associated ABO incompatibility. tBirth weights of 4 infants not known. .~Ineludes 1 G6PD-deficient infant.
159 babies of Group 1. All 5 enzyme-deficient infants were male. Of the 109 premature babies in this group there were 57 males; ABO incompatibility was present in 6, while of the remaining 51 premature males with no blood group incompatibility one was G6PD deficient. There were 29 males among the 46 fUll-term babies (in 4 cases the birth weights were not recorded). ABO incompatibility was diagnosed in 15 of the 29, one of whom was also G6PD deficient. There were thus 14 full-term males with no blood group incompatibility: in 3 of these G6PD deficiency was found, an incidence of 21 per cent. Rh hemolytic disease was diagnosed in only one infant (a premature female). The total serum bilirubin concentration was between 12 and 19.9 mg. per 100 ml. in 102 (64 per cent) and 20 mg. per 100 ml. or greater in 33 cases (21 per cent). Of the 33 babies with very severe jaundice, 17 were premature and 16 full-term. Two of this group were G6PD deficient, one premature and one full-term. Kernicterus was diagnosed in only 2 cases, in neither of which was G6PD deficiency found. I n both instances signs of kernicterus were already present on admission. Stage II. Five of the 51 male full-term infants with severe jaundice in Group 2 were found to be deficient in G6PD (10 per cent) compared with only 2 of 155 cord blood samples from the control series (1.3 per cent). In 13 of the 51 babies ABO in-
compatibility was demonstrated, one being also deficient in G6PD. Thus 4 of the 38 remaining infants with no blood group incompatibility were G6PD deficient. Obstetrical complications, fetal distress, or neonatal asphyxia were recorded in 5 of the 13 infants with ABO incompatibility (40 per cent), in 17 of the 34 infants with neither blood group incompatibility nor enzyme deficiency (50 per cent), and in 3 of the 4 babies with G6PD deficiency alone, 75 per cent (Table I I ) . Kernicterus was not observed in Group 2. In Table I I I the findings in the 29 fullterm male infants of Group 1 have been combined with those of the 51 babies in Group 2. Of the total of 80 infants, ABO incompatibility was demonstrated in 28, 2 were also G6PD deficient (7 per cent). Seven of the remaining 52 babies with no blood group incompatibility were deficient in G6PD (14 per cent). The difference between this figure and the incidence in the control
Table II. Obstetrical complications, fetal distress, or neonatal asphyxia among 51 icteric full-term male infants
r I,~fa,~t~ I~ypoxi~1% ABO incompatibility No incompatibility (G6PD normal) G6PD deficient (no incompatibility)
13
5*
4~0
34
17
50
4
3
75
Total
51
25
50
*Associated G6PD deficiency in 1 case.
7 6 0
November 1964
L e v i n , CharIton, and F r e i m a n
Table I I I . Incidence of G 6 P D deficiency in combined series of Group 1 and 2 male full-term infants with jaundice i
I ,
ABO incompatibility Unexplained hyperhilirubinemia Total
Group 1 15 (1)t
Group 2 13 (1)
Total 28
G6PD ~ deficient 2 (7 % )
Significance not significant
14 (3) 29
38 (4) 51
52 80
7 (1+%) 9 (11%)
p < 0.01 p < 0.01
~The significance of G 6 P D deficiency has been assessed by comparison with a cord blood incidence of 1.3 per cent. tFigures in parentheses are the number of G6PD-deficient infants in the separate groups.
Table IV. Analysis of 7 full-term male G6PD-deficient infants with unexplained hyperbilirubinemia ftemo-
Reticulocyte
Serum bilirubin level (rag.~1 O0 ml.) ?
Day of li[e
F~x-
Case 1
globin (Gin. 100 m l. ) 16.6
2
13.9
4
4
Normal
2'0.0
Day 4
3 4
16.7 17,0
I 6
12 5
-Normal
11.3 15.6
Day 12 Day 5
5 6
17.2 16.1
1 2
6 8
Normal Normal
19.9 15.4
Day 7 Day 8
7
18.6
2
4
Normal
20.0
Day 4
count
(%)
6
Blood Day of li[e films 3 Normal
group (1.3 per cent) is statistically highly significant (p < 0.01). T h e data relating to the 7 G 6 P D deficient full-term males with no blood group incompatibility are summarized in Table IV. T h e hemoglobin concentrations ranged from 13.9 to 18.6 Gm. per 100 ml. (the child with the hemoglobin concentration of 13.9 Gm. per 100 ml. had a subaponeurotic hemorrhage). Reticulocytes were 1 to 6 per cent, and no morphologic abnormalities were noted in the red cells. T h e serum bilirubin concentrations ranged between t 1.3 and 20.0 rag. per 100 ml. Kernicterus was not observed, but exchange transfusion was performed in 3 instances. Maternal obstetrical complications, fetal distress, or neonatal asphyxia were recorded in 5 of the 7 cases (70 per cent). T h e r e was no family history
change trans[usion -
+ -
+ -
Obstetrical history
Fetal distress Vertex delivery Forceps Subaponeurotic hemorrhage NormaI Cesarean section Disimpacted head District case Cephalopelvic disproportion FetM distress Symphisiotomy Born before arrival at hospitaI Asphyxiated
of severe neonatal jaundice in any of the G 6 P D deficient group. D I S C U S S I O N
O f the 159 cases of neonatal jaundice in the first series, only 5 (all males) were found to be deficient in G6PD. It is thus apparent that deficiency of this enzyme is not a major cause of jaundice in newborn Bantu infants in Johannesburg, One hundred and nine (70 per cent) of the babies in this group were premature, the same proportion as in the previous study reported from this hospital, 1 and it seems clear that hepatic immaturity with inadequate conjugation of bilirubin accounts for most cases of neonatal icterus seen at Baragwanath. There were 14 male babies with no blood group incompatibility a m o n g the 46 full-
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G6PD deficiency and ]aunchce m Bantu infants
term jaundiced infants. Since the incidence of G6PD deficiency in the adult male population in this area is 2 to 4 per cent, 15"1s the demonstration of enzyme deficiency in 3 of these 14 (21 per cent) suggested that it might be an etiologic factor in full-term males. It was in order to examine this possibility that the second part of the study was undertaken. In this stage of the investigation the incidence of G6PD deficiency in jaundiced, male, full-term infants was compared with that in a control group of serial, male, full-term births. After eliminating those with possible ABO or Rh incompatibility, there was a total of 52 infants in the jaundiced group (combining both parts of the study). G6PD deficiency was found in 7, an incidence of 14 per cent. Since only 1.3 per cent of the control group were deficient in the enzyme, it seems reasonable to conclude that G6PD deficiency of the erythrocytes does contribute to the neonatal jaundice of full-term South African Bantu male infants. Investigators working in Greece and in Singapore found evidence of hemolysis in their enzyme-deficient cases, and in addition patients were often admitted with established kernicterusY, a, 7, 2~ In this series the hemoglobin concentrations, reticulocyte counts~ and red cell morphology were not abnormal in the G6PD-deficient infants with no blood group incompatibility (Table IV). (The one child with a low hemoglobin concentration had a subaponeurotic hemorrhage.) Furthermore, none of the 10 G6PDdeficient babies with jaundice in the present investigation were admitted with or developed kernicterus. The differences between the present findings and those of the previous workers may be a reflection of the degree of deficiency of G6PD in the different populations. It has been established that G6PD deficiency is more marked in affected Caucasians than in American Negroes. 26 Certain drugs, such as quinine, quinidine, and chloramphenicol, provoke hemolysis in enzymedeficient Caucasians, but not in affected Negroes. 2~ The pattern of drug sensitivity in G6PD-deficient South African Bantu sub-
76 1
jects suggests that the enzyme deficiency resembles the type found in American Negroes rather than in Caucasians. 2s The less severe deficiency of the enzyme, therefore, may be responsible for the milder clinical picture in our patients. Zinkham 14 has recently made a similar suggestion. It has been established that tile life-span of enzyme-deficient cells is about 25 per cent less than normal even in the absence of detectable hemolysisY9 It follows that G6PD deficiency must contribute to neonatal jaundice by increasing the load of unconjugated bilirubin presented to the liver. Not all G6PD-deficient infants become jaundiced, however, so that it is clear that additional factors must operate in the cases which do become icteric. These factors might act in two ways: either the destruction of the defective cells might be accelerated, increasing the load of bilirubin, or else the conjugating capacity of the liver might be depressed. A number of factors are known to hemolyse G6PD-deficient cells. 2~ These include various drugs, naphthalene and favus beans (in the Caucasian type only). Certain viral and bacterial infections, diabetic acidosis, and physiologic hypoglycemia of the newborn enhance the effect of the hemolytic agents. The only recognized hemolytic substance to which the babies in the present study were known to be exposed was vitamin K. However, the dosage employed was that recommended by Zinkham, 8~ and shown recently by the same author 14 not to give rise to significant hyperbilirubinemia in G6PD-deficient Negro babies. In any case, the absence of any hematologic evidence of hemolysis suggests that the additional factor is more likely to be hepatic than hemolytic. The combination of moderate depression of liver function with the 25 per cent greater load of bilirubin in G6PDdeficient infants would be more likely to result in significant jaundice than a similar degree of hepatic dysfunction in babies with normal red cell life spans. Panizon 31 has suggested that fetal hypoxia is commonly associated with severe jaundice in enzymedeficient babies. In the present series situations which would contribute toward the
7 62
Levin, Charlton, and Freiman
development of perinatal hypoxia (such as obstetrical complications, fetal distress, or neonatal asphyxia) were recorded in 5 of the 7 (70 per cent) G6PD-deficient male full-term infants with no ABO incompatibility (Table IV). If hypoxia is the common additional factor in the present series, it seems much more probable that it depresses hepatic function than that it increases erythrocyte breakdown, since defective activity of an enzyme on the oxidative pathway of glucose metabolism would be relieved rather than exaggerated by anaerobic conditions. Evidence of hypoxia was also obtained in 50 per cent of the full-term babies with normal G6PD and no blood group incompatibility (Table I I ) , and this factor alone must be responsible for much of the jaundice in this group of infants. The association between severe neonatal jaundice and hypoxia has been previously described? T M Fessas ar, d colleagues 12 found that severe neonatal jaundice was common in certain families with G6PD deficiency, whereas other G6PD-deficient families did not develop icterus. These workers suggested, therefore, that the additional factor was genetically determined. Szeinberg and coauthors 13 confirmed the familial incidence of jaundice in some of their G6PD-deficient subjects. However, no family history of neonatal jaundice was obtained in any of the G6PD-deficient cases in the present study. If the additional factor in our G6PD-deficient cases is hepatic dysfunction resulting from perinatal hypoxia, it might be expected that the inefficient glucuronide conjugation of prematurity would act in the same way. However, in neither the present study nor that of Doxiadis and associates7 was an increased incidence of the enzyme defect found among jaundiced premature babies. The explanation for this apparent anomaly may lie in the high incidence of jaundice in premature infants, whether G6PD-deficient or not. A survey of the infants in the premature baby unit of this hospital at the present time revealed that more than 50 per cent had developed severe jaundice in the neonatal period. From a statistical point of view,
November 1964
therefore, the possible contribution of G6PD deficiency to the icterus might not be apparent if most premature infants become jaundiced in any case, since the difference between the incidence of the enzyme deficiency in this group and in the general population would not be great. It seems reasonable, however, to postulate that the enzyme deficiency (if present) would aggravate the jaundice of prematurity. It is concluded, therefore, that G6PD deficiency is a factor in the pathogenesis of severe neonatal jaundice in Bantu male fullterm infants. It seems probable that an additional factor is usually present, and that this is commonly perinatal hypoxic damage to the liver. The damaged liver is unable to excrete the increased load of bilirubin resulting from the shortened red cell survival of G6PD deficiency even in the absence of extrinsic hemolytic agents. SUMMARY
The relationship between G6PD deficiency and severe neonatal jaundice was studied in South African Bantu infants admitted to Baragwanath Hospital, Johannesburg. The incidence of the enzyme defect was found to be significantly greater in full-term male jaundiced babies with no blood-group incompatibility (14 per cent) than in a control group (1.3 per cent). In 5 out of 7 G6PDdeficient full-term males with no Rh or ABO incompatibility, evidence of perinatal hypoxia was obtained. It is suggested that the combination of hypoxic liver damage with the increased bilirubin load resulting from G6PD deficiency is responsible for the icterus in this group.
We would like to thank Drs. S. Wayburne, E. Kahn, and H. Stein for allowing us to study cases under their care, and Dr. I. Frack, Superintendent, Baragwanath Hospital, for permission to publish. Dr. M. Shapiro of the South African Blood Transfusion Service kindly arranged the serum bilirubin and blood group determinations and carried out the exchange transfusions, while
Volume 65 Number 5
G6PD deficiency and jaundice in Bantu infants
Dr. R. Cassel of the South African Institute for Medical Research performed the hemoglobin and reticulocyte estimations. We are also grateful to Dr. D. Lavery of the Obstetrics Department for the control cord blood samples; to Drs. M. Shnier and L. Taitz for their constructive criticism; and to Mr. C. du Plessis of the National Institute of Personnel Research for the statistical analysis.
REFERENCES 1. Shnier, M. H., and Levin, S. E.: Hyperbilirubinemia and kernicterus in premature and full-term Bantu newborn infants, Brit. M. J. 1: 1004, 1959. 2. Smith, G. D., and Vella, F.: Erythroeyte enzyme deficiency in unexplained kernicterus, Lancet 1: 1133, 1960. 3. Weatherall, D. J.: Enzyme deficiency in haemolytic disease of the newborn, Lancet 2: 835, 1960. 4. Panizon, F.: Erythrocyte enzyme deficiency in unexplained kernicterus, Lancet 2:10931 1960. 5. Gilles, H. M., and Arthur, L. J. H.: Erythroeyte enzyme deficiency in unexplained neonatal jaundice, West African M. J. 9: 266, 1960. 6. Gilles, H. M., and Taylor, B. G.: The existence of the glucose-6-phosphate dehydrogenase deficiency trait in Nigeria and its clinical implications, Ann. Trop. Med. 55: 64, 1961. 7. Doxiadis, S. A., Fessas, Ph., Valaes, T., and Mastrokalos, N.: Glucose-6-phosphate dehydrogenase deficiency. A new aetiologieal factor of severe neonatal jaundice, Lancet 1: 297, 1961. 8. Capps, F. P. A., Gilles, H. M. J., Jolly, H., and Worlledge, S.: Glueose-6-phosphate dehydrogenase deficiency and neonatal jaundice in Nigeria, Lancet 2: 379, 1963. 9. Flatz, G., Sringam, S., and Kornkris, V.: Neonatal jaundice in glucose-6-phosphate dehydrogenase deficiency, Lancet 1: 1382, 1963. 10. Zinkham, W. H.: Yearbook of Pediatrics 1961/1962, Chicago, 1962, Year Book Medical Publishers, p. 295. 11. H~ia, D. Y-Y: Yearbook of Pediatrics, 1961/ 1962, Chicago, 1962, Year Book Medical Publishers, p. 297. 12. Fessas, Ph., Doxiadis, S. A., and Valaes, T.: Neonatal jaundice in glucose-6-phosphate dehydrogenase deficient infants, Brit. M. J. 2: 1359, 1962. 13. 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.
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14. Zinkham, W. H.: Peripheral blood and bilirubin values ill normal full-term primaquine-sensitive Negro infants: effect of vitamin K, Pediatrics 31: 983, 1963. 15. Charlton, R. W., and Bothwell, T. H.: The incidence of glutathione instability of the red blood cells in the South African Bantu, South African J. M. Sc. 24: 88, 1959. 16. Charlton, R. W., and Bothwell, T. H.: Primaquine-sensitivity of red cells in various races in Southern Africa, Brit. M. J. I: 941, 1961. 17. Bernstein, R. E.: Glutathione assay and metabolism in normal and drug-sensitive red cells, South African M. J. 35; 295, 1961. 18. Bernstein, R. E.: Occurrence and clinical implications of red-cell glucose-6-phosphate dehydrogenase deficiency in South African racial groups, South African M. J. 37: 447, 1963. 19. Childs, B., Zinkham, W. H., Browne, E. A., Kimbro, E. L., and Torbert, J. V.: A genetic study of a defect in glutathione metabolism of the erythrocyte, Bull. Johns Hopkins Hosp. 102: 21, 1958. 20. Adam, A.: Linkage between deficiency of glucose-6-phosphate dehydrogenase and colour-blindness, Nature 189: 686, 1961. 21. Charlton, R. W., Patz, I. M., and Borok, G.: The genetics of glucose-6-phosphate dehydrogenase deficiency, South African M. J. 37" 486, 1963. 22. Dacie, J. V.: Practical haematology, ed. 2, London, 1958, J. & A. Churchill, Ltd., p. 23. 23. King, E. J.: Microanalysis in medical biochemistry, ed. 2, London, 1951, J. & A. Churchill, Ltd., p. 37. 24. Motulsky, A. G., and Campbell-Kraut, J. M.: in Blumberg, B. S., editor: Proceedings of the Conference on Genetic Polymorphisms and Geographic Variations in Disease, London, 1961, Grune & Stratton Company, p. 159. 25. Vella, F.: The incidence of erythrocyte glucose-6-phosphate dehydrogenase deficiency in Singapore, Experientia 17: 181, 1961. 26. Marks, P. A., and Gross, R. T.: Erythrocyte glueose-6-phosphate dehydrogenase deficiency: evidence of differences between Negroes and Caucasians with respect to this genetically determined trait, J. Clin. Invest. 38: 2253, 1959. 27. Tarlov, A. R., Brewer, G. J., Carson, P. E. and Alving, A. S.: Primaquine sensitivity, A. M. A. Arch. Int. Med. 109: 209, 1962. 28. Zail, S. S., Charlton, R. W., and Bothwell, T. H.: The haemolyfic effect of certain drugs in Bantu subjects with a deficiency of glucose6-phosphate dehydrogenase, South African J. M. Se. 27: 95, 1962. 29. Brewer, G. J., Tarlov, A. R., and Kellermeyer, R. W.: The hemolytic effect of primaquine, XII. Shortened erythrocyte life span in primaqulne-sensitive male Negroes in the absence of drug administration, J. Lab. & Clin. Med. 58: 217, 1961.
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30. Zinkham, W. H.: quoted by Tarlov et al. 2~ 3I. Panizon, F.: quoted by Szeinberg et al. 13 32. Crosse, V. M., Meyer, T. C., and Gerrard, J. W.: Kernicterus and prematurity, Arch. Dis. Childhood 30: 501, 1955. 33. Brown, A. K., and Zuelzer, W. W.: Studies in hyperbilirubinemia. I. Hyperbilirubinemia of the newborn unrelated to isoimmunization, A. M. A. J. Dis. Child. 93: 263, 1957.
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34. Miller, C. A., and Reed, H. R.: The relation of serum concentrations of bilirubin to respiratory function of premature infants, Pediatrics 21: 362, 1958. 35. Wood, B. S. B., Culley, P. E., Waterhouse, J. A. H., and Powell, D. J.: Factors influencing neonatal jaundice, Arch. Dis. Childhood 37: 371, 1962.