SPECIAL LECTURE

Bilirubin Encephalopathy: Changing Concepts

Leonore Ballowitz, MD

This overview begins with a discussion of clinical approaches to prevent brain damage in jaundiced newborns. Results of longitudinal follow-up studies are given. The main part deals with patho­genesis, whereby bilirubin albumin binding, competition by drugs, bilirubin distribution and equilibration in the body, the increased sensitivity of the infantile nervous system, the mechanism of bilirubin entry into nerve cells and the resulting intracellular damage are emphasized.

Ballowitz L. Bilirubin encephalopathy: changing concepts.

In 1903 Schmor! [1] characterized yellow staining of the basal ganglia of deceased new­born babies as kernicterus, and he had already noticed that the pigment faded on exposure to light. He did some simple diazo tests with ex­tracts and came to the conclusion that the pig­ment was not bilirubin. It was not until the 50s that neurotoxicity of unconjugated, unbound bilirubin became generally accepted as the cause of kernicterus and the new term biliru­bin encephalopathy was suggested. But even today the final mechanism of bilirubin cytoto­xicity is still unknown. There is no obvious explanation for the high sensitivity of certain cerebral regions and for the special susceptibili­ty of newborn and premature babies. Our knowledge of the equilibration between the intra- and extravascular bilirubin pool is insuffi­cient even in a steady state, the more so when the degree of jaundice increases or decreases or

From Children's Hospital, Free University Berlin, Berlin.

Key words: Kernicterus, still persistent sequelae of hyperbilirubinemia, pathogenesis of bilirubin encepha· lopathy, Gunn rats as a model.

Correspondence address: Dr. Leonore Ballowitz, Freie Universitat Berlin, Universitiitsklinikum Charlotten­burg Kinderklinik, Kaiserin Auguste Victoria Haus, Heubnerweg 6-D 1000 Berlin 19, West Germany.

Brain Dev 1980;2:219-27

when the child is under phototherapy. Only very few investigators have compared the pig­ment distribution in fat and collagen of the skin, in the liver, and in the central nervous sys­tem. A clinically applicable method for mea­suring the bilirubin concentration in the ganglia is lacking and will probably never be available for humans. Several problems remain to be solved, despite many years of diligent investi­gation.

On the clinical level much has been done to reduce the gross brain damage and diminish the number of deaths from bilirubin encephalo­pathy. But when the question of kernicterus danger is raised for an individual newborn, laboratory data such as total serum bilirubin concentration, the amount of free bilirubin, reserve binding capacity or saturation of albu­min render sometimes controversial orientation. Often we still rely on the empirically defined critical level of 18-20 mg/1 00 ml serum biliru­bin. But, on the one hand we all know about-a few-mature newborns who survived 30 mg/IOO ml or even higher concentrations without re­markable defects, and on the other hand about premature babies who never had more than 10-12 mg/IOO mI, but nevertheless, autopsy showed kernicterus. Mainly the potential toxi~ city of the "intermediate" serum bilirubin con­centrations (from 10-19 mg/100 ml) has never

o Normal

100 _ Suspect to abnormal

16

t'2- 10

.S 80 u 19 .;:::

6 .... '" E 11 0 60

{3 ~ Po. I'i 0 40

'" 4 E 7 0 u .... 8 20 2

o 0 1 2-3 4-5 ;;'6

No of days indirect bilirubin ~ 15 mg/lOO ml,

p<O.OOl

Fig 1 Duration of exposure to bilirubin versus per­formance on the psychometric examination. Concen­trations of indirect serum bilirubin are corrected to the nearest 0.5 mg/100 mi. A concentration of 15 mg/1 00 ml or more for less than 8 hrs is considered o days of exposure. Such a concentration of bilirubin for 8-31 hrs is considered as 1 day of exposure. 32-56 hrs as 2 days of exposure, and so on [8J.

been clearly defined. Longitudinal follow-up studies-including a

collaborative perinatal project [2] from the USA comprising 27.000 infants-point to a relation between maximum total serum biliru­bin levels and developmental deficits up to the age of one year. Already bilirubin concentra­tions in the range of 10-14 mg/ 100 ml have been discriminated in infants of ~ 36 weeks gestation. The "association of developmental outcome with hyperbilirubinemia was found over and above the variation of maturity" [2] . But, the strongest relationship was revealed in low birth weight/short gestational period infants.

In most reports [3-6] such relations did not show (any more) after the first year of life (at ages 4-7 years). So, that process might be similar to the transient dystonic syndrome first described by Drillien and co-workers [7]. But in a study of Johnson and Boggs [8] statistical­ly significant relations were still found at the age of 4 years when, for instance, the duration of bilirubin exposure, or HBABA binding reserve and the sex of the children were taken

220 Brain & Development, Vol 2, No 3, 1980

t'2-.S u oS '" E 0

..c: u >,

0.. I'i 0

'" E 0 B 8

100 r

80 f- 17

60 f-

40 r-

o Normal _ Suspect to abnormal

5 5

17 r-

':~ 1JL.......L __ _ o

16 r-

BL ,n % ~5O >50 <50 >50

Males p<O.D1

Females NS

Fig 2 Binding level versus performance on the.psy,­chometric examination (males and females). Bmdmg level predicts outcome at the age of 4 yrs in males but not in females in spite of similar degr,ees of exposure to bilirubin. NS = not significant [8].

into account. For more details Figs 1 and 2 are reprinted from their publication. Generally the incidence of suspect ratings was smaller at the age of 7 than 4 years. The difference between the two sexes diminished but still persisted. This may partly at least be due to the fact that maturation in females proceeds somewhat faster during the first years of life than in males. At present, we cannot deny that the concluding comments of Johnson and Boggs [8] may still b~ valid: "We by no means anti­cipated finding such a high incidence of damage among infants whose concentrations of serum bilirubin had been maintained, by and large, within what were then considered safe limits (1965-1966). Damage, we had thought, would be confined to those few infants, whose hyper­bilirubinemia was complicated by other prob­lems such as acidosis and anoxia. And we had look~d forward to finding a sizable number of term infants with uncomplicated hyperbiliru­binemia who would prove not to be at risk in spite of concentrations of serum bilirubin of 18-20 mg/100 ml. Instead we found the dis­tressingly high sequelae rate (and learning disorders associated with minimal brain damage are significant handicaps) at the lower con­centrations reported here." - The final proof

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= :El ~ 15

~ 9 10 ! ~ : Vl I

5 : I I

o 2 4

- Bilirubin level ------ Gantrisin level

5

~ OIl

4 E = ;E

3 e ]

2 E e ~

6 8 10 12 24 Hours

Fig 3 The effect of competition for protein binding of injected sulfisoxazole on the serum bilirubin con­centration in the chronically jaundiced rat (131.

is still lacking that the wide use of photothera­py has decisively changed the panorama. (My personal impression is that the frequency of sequelae was further reduced in the 70s). Recently, Pearlman and coworkers [9] re­viewed post mortem examinations of low birth weight infants who died on the 3rd to 7th days of life. From 1971 through 1976 they failed to reveal any case of kernicterus in infants weighing less than 2,250 g, whereas in 1966/67 the incidence in the same neonatal-premature center was 64%, (a more aggressive policy of exchange transfusion and phototherapy was established in 1970).

As for hearing, speech and learning dis­orders, peripheral (especially high frequency) sensorineural hearing loss is known as a typical defect following bilirubinemia. Audio EEG should therefore be used for screening. Besides this, disorders in central communication func­tions (central auditory perception, memory recall, sound symbolization and so on) have been reported [10] . They may develop in association with but also in the absence of peripheral hearing loss [8]. In Berlin (Giesen M and Keller P, personal communication) evoked brain stem responses to clicks, and 4kHz pips were investigated in infants who had hyperbilirubinemia in the neonatal period_ A latency shift points to myelinization distur­bances of acoustic nerve fibers in the auditory pathway between the nucleus cochlearis and colliculus inferior. Hyperbilirubinemia obvious­ly impairs this part of the central auditory path­way , but it is mostly transitory during the first

months of life . And here too , boys seem to be more frequently affected than girls.

Besides this neurological evaluation of still persistent sequelae of hyperbilirubinemia, fur­ther studies of pathogenesis should enlarge our knowledge of the entity of this syndrome. Therefore, the following paragraphs deal with pathogenesis.

The main bile pigment in the human body is bilirubin IXcx:(Z). In plasma it is firmly bound to albumin with the exception of a small but critical free fraction in equilibrium concentra­tion. Free bilirubin occurs in the plasma as the divalent anion. Its disodium salt is quite soluble in water and in itself it has no 'special affinities for lipid membranes and neither is it cytotoxic as such (for a review see [11]). However, it shows a pronounced tendency to precipitate as a free acid especially at low pH. By the forma­tion of intramolecular hydrogen bonds, the free acid has become nearly insoluble in water. The precipitated hydrophobic-molecule can attach to cells and enter into cells.

Circulatins albumin will simultaneously transport not only the anion of bilirubin IXcx:(Z), fatty acids, hemin, tryptophan, and some other physiological ligands but also sever­al drugs and additives (for a review see [12]). Binding of one ligand interacts with binding of other ligands and changes the blood and tissue distribution of the substances involved.

A model often used to study bilirubin me­tabolism and neurotoxicity is the homozygous Gunn rat_ They have an unconjugated hyperbi­lirubinemia throughout life due to an inherited lack of UDP glucuronyl transferase in the liver­comparable to the Crigler Najjar syndrome in man. In the 50s, Johnson and coworkers [13] decisively contributed to understanding of the deleterious effects of sulfonamides in jaundiced newborn by experimenting with these rats . Fig 3 was taken from one of their publications. It clearly demonstrates competition for albumin binding between sulfisoxazole and bilirubin_ Immediately after the Gantrisin® injection the serum bilirubin concentration sharply declines. Some hours later it rises again reciprocal to the drug elimination. The danger of nerve cell damage is greatest at the time of the lowest serum bilirubin concentration. It is not aston­ishing that injections of albumin influence the distribution of bilirubin between vascular and extravascular spaces in an opposite direction.

Ballowitz : Kernicterus 221

~ SI.2

'" ~ 1.0

o E e 0.8

I':

~ 0.6

:.= :0 0 .4 Q)

.~ ~o.2 "3 E E 8

~ !

40 Serum 30 Bilirubin

mg/lOO ml 20

60 eo Ida f do ,~o ' I~O Time (hours)

Fig 4 The removal of bilirubin by peritoneal dialysis in a 14-year·old patient with familial nonhemolytic hyperbilirubinemia. The diagonal line connecting the X's represents the cummulative removal of bilirubin by continuous dialysis measured on the left-hand ordinate. The patient's serum bilirubin concentrations during the dialyses are represented by the horizontal line plotted from the right·hand ordinate [ 18].

Serum bilirubin increases owing to the in­creased binding capacity [14] .

Remembering the alarming sulfonamide induced neurological sequelae in newborn babies during the 50s-they certainly exceeded the frequency of thalidomide defects-it should be emphasized that a modern drug combina­tion, namely trimethaprime + sulfamethoxazole (Bactrim®, Eusaprim®, Omsat®), has a similar effect. It should also be mentioned that the decrease of serum bilirubin concentration by Bucolome®, a barbiturate mainly used in Japan sometime ago, can probably be explained by the same dangerous mechanism [15] .

It is well known that several other sub­stances, among them solvents, preservatives and stabilizers for injectable preparations, may act as binding competitors. It was quite a striking experience for us [16, 17], when in 1974 we found that 200 mg/kg of a special gentamycin formulation for babies induced lethal kernic­terus in infant Gunn rats., whereas much higher gentamycin doses formulated for adults did not. It turned out that the ampoules for babies contained eight times more benzylalcohol per mg gentamycin than the ampoules for adults, and that the benzylalcohol metabolites caused serum bilirubin decline and kernicterus. The addition of the stabilizer was not declared by the manufacturer at that time. (In the mean­time the critical stabilizer has been removed from the formulations for babies.)

222 Brain & Development, Vol 2, No 3, 1980

Bilirubin distribution and equilibration in plasma, extravascular fluids and tissue cells are important factors . The following data from a newborn of 3,200 g birth weight getting an exchange transfusion may serve as an example. They give an idea of the volume of the extra­vascular bilirubin pool.

Calculated circulating plasma volume ~ISO ml Total serum bilirubin concentration prior

to exchange transfusion 23.8 mg/ I 00 ml Amount of circulating bilirubin prior

to exchange transfusion ~3S mg Amount of bilirubin taken out

dUring exchange transfusion (300 ml plasma at 13 mg/l 00 m!)

Remaining circulating bilirubin after exchange transfusion (9 .S mg/! 00 m!)

Circulating bilirubin 6 hrs after

~40mg

~14mg

exchange transfusion"rebound" (IS .8mg/!00ml) ~23mg

Another interesting example (Fig 4) is derived from a publication of Blumenschein and coworkers [18]. In a 14-year-old boy with Crigler Najjar-syndrome, 1.2 g of bilirubin was removed by continuous peritoneal dialyses over a period of 5 days. The amount almost equalled one and a half the amount in his entire circulation. But no significant reduction in serum bilirubin concentration could be ob­tained . Obviously , serum albumin concentra­tion did not change during the procedure and the extravascular reservoirs of bilirubin were far in excess of the capacity of plasma albumin to bind the pigment.

The binding 'Capacity of the skin-in fat cells as well as in collagen [19] -is not well defined too. In some sense the skin can be regarded as a comparably safe deposit for bilirubin. In well nourished fat infant Gunn rats kernicterus is, in general, less pronounced than in scrawny ones. The same was discussed as a possible ex­planation for the above cited [8] resistance of girls as compared to boys to the toxic effects of hyperbilirubinemia. Female babies have pro­portionally more fat (less water and a smaller mean size) than male babies of the same gesta­tional age [20, 21) . Fat can provide an effec­tive trap for extravascular bilirubin. This presumption was strengthened by observations of Sato and Semba [22, 23] who compared well suckled infant Gunn rats with starved

littermates. Although unbound bilirubin in plasma was significantly higher in suckled rats (probably due to the high lipid content of rats milk) their bilirubin concentration in the cere­bellum was significantly lower than in starved ones.

Age related differences in bilirubin distribu­tion (partially due to different vascularisation) are likely to be one of the causes for the special susceptibility of immature individuals and of certain areas of the central nervous system to bilirubin neurotoxicity. This also involves the question of the blood-brain-barrier and of tissue "affinities" for bilirubin. With today's know­ledge, we cannot give a generally accepted con­cept for it. It is only possible to quote some relevant observations. Sawasaki and coworkers [24] determined after chloroform extraction the postnatal change in bilirubin content of the brain and the liver in homozygous Gunn rats. In the liver the bilirubin content increased ex­ponentially after birth. In the brain the total (but comparably small) amount of bilirubin in­creased up to the 16th day and declined there­after. But, when the relation between pg bilirubin/g wet weight or ng bilirubin/mg pro­tein was established, the highest "brain bilirubin levels" were measured shortly after birth, with the decline already beginning in the first week.

With sephadex gel filtration , we [25] found out that after sulfadimethoxine application un­bound bilirubin circulated in the plasma of infant Gunn rats for a few minutes only but in adult animals for several hours .

Davis and Yeary [26] followed the distribu­tion of 14C bilirubin in newborn and adult jaun­diced Gunn rats after its displacement from plasma albumin by sulfadimethoxine~ Whereas in the adult animals the unbound bilirubin was primarily taken up by the liver , in the newborn the major portion was distributed to the intes­tine and the liver. The uptake by the brain tissue was not higher in newborn than in adult rats.

In our team, Hanefeld (unpublished data) compared as a first step the development of the cerebellum in infant homozygous Gunn rats, which were under continuous phototherapy during the first 6 weeks of life , with their litter­mates kept in the dark. In the illuminated group, the cerebella remained intact whereas in the dark a marked Purkinje cell loss and a con­secutive weight-reduction of the cerebella occur-

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-30 -20 -10 tJ. 10 20 30 Age Birth Age

Fig 5 Velocity of human brain growth (wet weight) compared with that in other species. Prenatal and postnatal are expressed as follows: human --­in months, guinea pig - - - - - - - - - in days, pig - - - - - in weeks, rat -.-. -. in days [27].

red. As a 2nd step, the 6-week-old (weanling) rats - up to then either illuminated or kept in the dark-were again divided into four groups. Two groups . (one primarily kept in the dark, one primarily illuminated) were placed into dark surroundings, and the two others under the lights. The rats of all four groups were now injected with a high dose of bilirubin. The thus induced hyperbilirubinemia lasted for more than 96 hrs in the dark group and for less than 14 hrs in the phototherapy group . Maximum concentrations markedly surpassed those of the infant rats. At autopsy, the cerebella were unaffected by the 2nd step proceedings. That means, in the 2nd month of life severe hyper­bilirubinemia caused no (new) damage to the nerve cells. This again raises the question of increased sensitivity of the infantile nervous system to bilirubin toxicity.

Several publications of Dobbing (for a re­view see [27]) deal with the development of the brain and its vulnerability during fetal or infantile growth spurt periods. In these periods an extraordinary biochemical activity is going on in the tissue. New structures and functions rapidly emerge . Many components are synthe­sized within the brain itself. Dobbing [27] says : It is erroneously supposed that the phe­nomenon loosely known as the "blood-brain­barrier" develops with increasing age. For many substances (e.g. dyestuffs) it is always closed even in early fetal life; while for others

Ballowitz: Kernicterus 223

(e .g. labelled phosphate) it is only transiently "open" during the brain growth spurt period. Fig 5 demonstrates the brain growth spurt in different species. In humans it runs from mid­pregnancy to about 18 months of postnatal age with a peak around birth. In rats the peak is about 8 -10 days postnatally. Observations from infant rats (or pigs) [28,29] might come closer to the situation in human premature babies than similar tests in guinea pigs. Natural­ly, the transfer of findings from one species to another always remains dubious. But, it is most likely that during the growth spurt period hu­man nerve cells as well as rat nerve cells are especially vulnerable to bilirubin toxicity. In the afore-mentioned investigations by Hanefeld, the severe artificial (2nd step) hyperbilirubin­emia was induced at a time when the growth spurt of the rat brain was over and accordingly it did not harm the cerebella.

.The mechanism of bilirubin entry into cells is not well understood either. But it is obvious­ly influenced by several additional factors such as acidosis, hypoxia or substances which are associated with membrane functions. Acidosis increases the extravascular distribution of bilirubin [30] , the membrane permeability and bilirubin binding to red cells [31], mitochon­dria [32, 33] , fibroblasts [34] and cerebellum tissue [35]. According to the new concepts acidosis may be extremely important since equilibrium of the (non-toxic) free bilirubin IX<x(Z)anion and the cytotoxic free bilirubin acid is inversely proportional to the square of the hydrogen ion concentration. Brodersen [36] calculated that the amount of free biliru­bin acid taken up by the cells, would increase 60 to 100-fold when pH was decreased by 1 (see also Kozuki and coworkers [37]).

Lending and coworkers [38] experimented with puppies under 3 months of age. They were unable to produce yellow staining of the basal ganglia by bilirubin infusions alone . By subjecting the young animals to hypercapnia, hypoxia or prolonged convulsions increased permeability of the blood cerebrospinal fluid barrier could be induced. When both proce­dures were combined, kernicterus rapidly developed in those under 9 weeks of age but not in the older puppies.

Hirata and coworkers [39] produced ex­perimental kernicterus in fetal rats by bilirubin injections: a) after causing hypoxia by clamp-

224 Brain & Development, Vol 2, No 3, 1980

ing the uterine vessels for 3 minutes, and b) by simultaneously applying bilirubin and hyaluro­nidase (which increases vascular permeability). No bilirubin accumulation in the nerve cells occurred by bilirubin injections alone. Finally, the authors were able to prevent the penetration of bilirubin into the brain even after hypoxia and bilirubin injection (condition a) by sub­stances of the adenochrome group (which de­crease vascular permeability).

Goldstein and colleagues [40] studied enzyme activities and stereospecific carrier­mediated transport systems in isolated capil­laries from rat brain. Besides the astrocytic footplates the endothelial cells of the brain capillaries seem involved in regulating the per­meability of the blood brain barrier. And there may be regional variations in the function of brain capillaries. Some endothelial cells may be primarily damaged by unbound bilirubin and only thereafter permit entry of the toxic free bilirubin into nerve cells.

Rasmussen and Wennberg [41] investigated pharmacologic modifications of bilirubin toxic­ity in tissue cultures and found that hydro­cortisone totally protected 929 L-cells from bilirubin toxicity . Prednisolone was slightly less effective. Theophylline and caffeine offered partial protection.

As already mentioned, Pearlman and cowork­ers [42] recently reviewed post mortem exami­nations with regard to kernicterus in 232 new­born babies that died in the years 1971-76. They came to the conclusion that-nowadays under sophisticated intensive care-infections may be the most important predisposing factor in the development of kernicterus. The only cases of proven kernicterus occurred in four near-term infants with antemortem-culture­proven sepsis. Immaturity as a predominant predisposing factor for the development of ker­nicterus was apparently eliminated by aggres­sive management of hyperbilirubinemia.

When bilirubin has entered a brain cell and its concentration surpasses tolerable levels, membrane alterations 'and mitochondrial dam­age will occur. Bilirubin interactions with lipids [43] especially with gangliosides [44] are dis­cussed as a possible mechanism impairing plasma membranes in the nerve cells. Myelin figures and dense bodies in cytoplasm are con­sidered as pigment lipid complexes [45] origi­nating from this process. It may already be

Fig 6 LDH reaction in the Purkinje cells of the cerebellum in a homozygous Gunn rat given photo­therapy.

effective at bilirubin concentrations which do not disturb the mitochondrial metabolism it­self. When cell metabolism does get involved, several processes-such as oxygen uptake ([28, 29] or for a review [46]), glycolysis [28,29, 47 , 48] and protein synthesis [49] -can be af­fected. But the primary stage is not yet clear. It is unlikely [50] that uncoupling of oxidative phosphorylation is the decisive primary factor­as postulated [51 , 52] -because of the high concentrations necessary for that process in vitro. Nevertheless, Brodersen [36] recently stated that rather high local bilirubin concentra­tions have to be assumed when mitochondria of cerebral neurons become viSibly yellow on light microscopy. This cannot result from bind­ing of discrete amounts of bilirubin to specific receptor proteins. "The pigment must either be dissolved in the lipoid membrane at a very high concentration or be present as aggregates of a crystalline or amorphous nature" (probably as bilirubin IXo:(Z)acid).

In Gunn rats the degree of kernicterus can well be covered by enzyme-histochemical investigations of the Purkinje cells in the cere­bellum [53, 54]. In tissue sections these cells are lined up like pearls in a chain. When injured by bilirubin, staining reactions for dehydro­genases and reductases do not take place any more. An injured cell forms a blank spot in the chain. Figs 6 and 7 give examples with the lac­tic acid dehydrogenase (LDH) reaction. Fig 6 concerns the cerebellum of a homozygous Gunn rat treated with phototherapy. There are only a few blank spots in the nerve cell row (marked with arrows) the enzyme is intact in most of the cells. Fig 7 is a corresponding cere­bellar section. Four days before sulfadimeth­oxine (Madribon~ was administered to the in-

Fig 7 LDH reaction in the Purkinje cells of the cerebellum in a homozygous Gunn rat after sulfadi· methoxine application.

fant rat. The drug obviously displaced bilirubin from albumin bonds and induced penetration of the free pigment into the nerve cells. Only in very few Purkinje cells lactic acid dehydroge­nase is traceable. When such an animal survives, further · growth of its cerebellum will be re­stricted because of the nerve cell loss [55] .

Bilirubin encephalopathy is surely not in the file of medical problems solved. Nevertheless, the prophylactic concept is clear. Kernicterus can be avoided by bilirubin elimination. Ex­change transfusion, phototherapy, glucuronyl­transferase-induction are effective methods. Additional factors affecting neonatal jaundice have to be taken into consideration such as membrane permeability, hypoalbuminemia, acidosis, hypoxia, feeding procedures, drugs , infections and so on.

References 1. Schmorl G. Zur Kenntnis des Ikterus neonato­

rum, insbesondere der dabei auftretenden Gehirn­veranderungen. Verh Dtsch Ges Pathol 1903;6 : 109-15.

2. Scheidt PC, Mellits ED, Hardy JB, Drage JS, Boggs ThR. Toxicity of bilirubin in neonates: infant development during first year in relation to maximum neonatal serum bilirubin concentra­tion. J Pediatr 1977;91 :292-7.

3. Johnston W, Angara Y, Baulmal R, et aI. Erythro­blastosis fetalis and hyperbilirubinemia, a five year follow up with neurological, psychological and audiological evaluation. Pediatrics 1967 ;39: 88-92.

4. Odell G, Storey G, Rosenberg L. Studies in ker­nicterus. III. The saturation of serum proteins with bilirubin during neonatal life and its relation­ship to brain damage at five years. J Pediatr 1970;76 :12-21.

5. Rubin RR, Balow B, Fisch RO. Neonatal serum bilirubin levels related to cognitive development

Ballowitz: Kernicterus 225

at ages 4 through 7 years. J Pediatr 1979 ;94: 601-4.

6. Upadhyay Y. A longitudinal study of full-tenn neonates with hyperbilirubinemia to four years of age. Johns Hopkins Med J 1971 ;128 :265.

7. Drillien CM. Abnonnal neurologic signs in the first year of life in low birth weight infants: possible prognostic significance. Dev Med Child NeuroI1972;14:575-84.

8. Johnson L, Boggs ThR. Bilirubin-dependent brain damage: incidence and indications for treatment. In: Odell GB, Schaffer G, Simopoulos AP, eds. Phototherapy in the newborn, an overview. Washington DC: Nat Acad Science 1974:122-49.

9. Pearlman MA, Gartner LM, Lee K, Morecki R, Horoupian DS. Absence of kernicterus in low­birth-weight infants from 1971 through 1976: comparison with fmdings in 1966 and 1967. Pediatrics 1978;62:460-4 .

10. Vernon McC V. Rh factor and deafness: the pro­blem, its psychological, physical and educational manifestations. Except Child 1967;34:5-12.

11. Brodersen R. Prevention of kernicterus, based on recent progress in bilirubin chemistry. Acta Paediatr Scand 1977;66 :625-34 .

12. Stern L, Doray B, Chan G, Schiff D. Bilirubin metabolism and the induction of kernicterus. Birth Defects 1976 ;12(2) :255-63.

13. Johnson L, Sarmiento F, Blanc WA, Day R. Ker­nicterus in rats with inherited deficiency of glucuronyl transferase. Amer J Dis Child 1959; 97 :591-608.

14. Odell GB. The distribution and toxicity of bilirubin. Pediatrics 1970;46 : 16-24.

15 . Semba R, Sato H, Yamamura H. Danger of bucolome in infants with hyperbilirubinaemia. Arch Dis Child 1979;53 :503-5 .

16. Ballowitz L, Hanefeld F. Effect of drugs on in­fant Gunn rats under phototherapy. Birth De­fects 1976;12(2):61-80_

17. Ballowitz L, Hanefeld F, Schmid H. The influ­ence of various aminoglycoside preparations on bilirubin/albumin binding. J Perinat Med 1976; 4 :168-83 .

18. Blumenschein SD, Kallen RJ, Storey B, Natzschka JC, Odell GB, Childs B. Familial nonhemolytic jaundice with late onset of neurological damage_ Pediatrics 1968;42:786-92.

19. Kapoor CL. Interaction of bilirubin with recon­stituted collagen fibrils. Biochem J 1975;147: 199-203.

20 . Fomon SJ. Body composition of the male re­ference infant during the fust year of life. Ped­iatrics 1967 ;40:863-74.

21. Owen GM, Jensen RL, Fomon SJ. Sex related difference in total body water and exchangeable chloride during infancy. J Pediatr 1962;60:858-68.

22. Sato H, Semba R. Effect of milk on plasma un­bound-bilirubin concentration in homozygous Gunn rat sucklings. Experientia 1977;33:59-61.

23. Sato H, Semba R. Relationship between plasma unbound-bilirubin concentration and cerebellar bilirubin content in homozygous Gunn rat sucklings. Experientia 1979;34:221.

226 Brain & Development, Vol 2, No 3, 1980

24.

25.

26.

27 .

28.

29.

30.

31.

32.

33.

34.

35.

36 .

37.

38.

39.

Sawasaki Y, Yamada N, Nakajima H. Studies on kernicterus. III . Developmental change in brain bilirubin level in Gunn rats. Proc Japan Acad 1976 ;52:41-4. Ballowitz L, Hanefeld F, Jarofke R, Keller P, Korn J, Schweitzer U. Sephadex-Gel filtration (SGF) in infant and adult Gunn rats. BioI Neonate 1978;33:13-7. Davis DR, Yeary RA. Effect of sulfadimethoxine on tissue distribution of [14 C] bilirubin in the newborn and adult hyperbilirubinemic Gunn rat. Pediatr. Res 1975 ;9 :846-50. Dobbing J. The later development of the brain and its valnerability. In: Davis JA, Dobbing J , eds. Scientific foundations of paediatrics. London: William Heinemann Medical Books Ltd, 1974:565-77. Abe T, Sugiyama J, Osawa A, Baba K. Expect­mental kernicterus and the change of cerebral metabolic rate. XIII Internat Congr Pediat (Vienna) 1971;9:157-67_ Baba K, Okada T, Ariizumi M, et al . Chemical injuries of the brain in neonatal period. Nihon Univ J Med 1967 ;9 : 157-67. Diamond I, Schnid R. Experimental bilirubin en­cephalopathy: the mode of entry of bilirubin-14C into the central nervous system. J Clin Invest 1966 ;45 :678-89 . Bratlid D. The effect of pH on bilirubin binding by humap erythrocytes. Scand J Clin Lab Invest 1972;29:453-7. Odell GB. The influence of pH on distribu tion of bilirubin between albumin and mitochondria. Proc Soc Exp BioI Med 1965;120:352. Odell GB , Cohen SN, Kelly PC. Studies in ker­nicterus II. The detennination of saturation of serum albumin with bilirubin J Pediatr 1969;74 : 214-30. Nelson T, Jacobsen J , Wennberg RP. Effect of pH on the interaction of bilirubin with albumin and tissue culture cells. Pediatr Res 1974 ;8 : 963-7 . Silberberg DH, Johnson L, Ritter L. Factors in­fluencing toxicity of bilirubin on cerebellum tis­sue culture. J Pediatr 1970;77:386-96 . Brodersen R. 'Free bilirubin in blood plasma of the newborn : effects of albumin, fatty acids, pH, displacing drugs and phototherapy. In: Stern L, Oh W, Friis-Hansen B, eds. Intensive care of the newborn II. New York: Masson Publ, 1979:331-45 . Kozuki K, Oh W, Widness J, Cashore, WJ. In­crease in bilirubin binding to albumin with cor-rection of neonatal acidosis. Acta Paediatr Scand 1979 ;68 :213-7 . Lending M, Slobody LB, Mestern J. The relation­ship of hypercapnia to the production of ker­nicterus. Dev Med Child Neuro/1967;9 : 145-51. Hirata Y, Matsuo T, Shibata M, Takatera Y, Nakamura K. Experimental studies on the de-velopment of kernicterus. BioI Neonate 1968; 12:371-7 .

40. Goldstein GW, Wolinsky JS, Csejtey G, Diamond S. Isolation of metabolically active capillaries from rat brain. J Neurochem 1975;25:715-7 .

41. Rasmussen LF, Wennberg RP. Pharmacologic modification of bilirubin toxicity in tissue cul­ture. Res Commun Chern Pathol Pharmacol 1972;3:567-78.

42. Pearlman MA, Gartner LM, Lee K, Eidelman AI, Morecki R, Horoupian SO. The association of kernicterus with bacterial infection in the new­born. Soc Ped Res Meeting New York 1978 April.

43. Mustafa MG, King TE. Binding of bilirubin with lipid. J BioI Chern 1970;245:1084-9.

44. Weil ML, Menkes JH. Bilirubin interaction with ganglioside: possible mechanism in kernicterus. Pediatr Res 1975;9:791-3.

45. Luse SA. The neuron. In: Minkler, Jed. Patho­logy of the nervous system. Vol. I. New York: McGraw-Hill Book Co, 1968:572.

46. Cowger ML. Bilirubin encephalopathy. In: Gaull EG, ed. Biology of brain dysfunction. Vol II. New York· London: Plenum Press, 1973 :265-93.

47. Katoh R, Kashiwamata S, Niwa F. Studies on cellular toxicity of bilirubin: effect on the car­bohydrate metabolism in the young rat brain. Brain Res 1975;83 :81-92 .

48. Katoh-Semba R. Studies on cellular toxicity of bilirubin: effect on brain glycolysis in the

young rat. Brain Res 1976;113:33948. 49. Greenfield S, Majumdar APN. Bilirubin encepha­

lopathy: effect on protein synthesis in the brain of the Gunn rat. J Neurol Sci 1974;22:83-9.

50. Diamond I. Studies on the neurotoxicity of bilirubin and the distribution of its derivates. Birth Defects 1970;6(2):124-7.

51. Ernster L. The mode of action of bilirubin on mitochondria. In : Sass-Kortsak A, ed. Kernic­terus. Toronto : Univ. of Toronto Press, 1961: 174-92.

52. Schenker S, McCandless OW, Zollman PE. Studies on cellular toxicity of unconjugated bilirubin in kernicteric brain. J Clin Invest 1966; 45:1213-20.

53. Hanefeld F, Natzschka J. Histochemical studies in infant Gunn rats with kernicterus. Neuropaed­iatrie 1971 ;4:428-38.

54. Schutta HS, Johnson L. Bilirubinencephalopathy in the Glinn rat : a fine structure study of the cerebellar cortex. J Neuropathol Exp Neurol 1967;26:377-96.

55. Hanefeld F, Ballowitz L. Influence of hyper­bilirubinaemia on growth of the cerebellum·. Neuropaediatrie 1977 ;8:521-2 .

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