Kernicterus Research and the Basic Sciences: A Prospect for Future Development

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PEDIATRICS (ISSN 0031 4005). Copyright © 1987 by the American Academy of Pediatrics.

154

PEDIATRICS Vol. 79 No. 1 January 1987

EDITORIALS

Kernicterus

Research

and the Basic

Sciences:

A Prospect

for Future

Development

Kernicterus was a major problem of pediatrics in the first half of this century. Important progress has taken place since pediatricians have mastered rhesus disease, introduced exchange transfusion, avoided giving sulfonamides, and have placed at risk infants under blue light. These measures, and above all the general improvements in intensive care in modern neonatology units, have reduced the problem so that kernicterus is now mainly seen as secondary to prematurity, respiratory distress, and severe infections.’ There, it remains a challenge to both clinical and basic science. The mechanism (or mechanisms?) of bilirubin transfer from plasma to brain has not been settled; agreement has not been

reached on laboratory methods for predicting kernicterus; and satisfactory guidance about which drugs to avoid, other than the bilirubin-displacing sulfonamides, remains to be given.

Progress in these fields is steadily achieved through cooperation of clinical and basic scientists. It should be possible to accelerate this development by intensifying the exchange of ideas and know-how, particularly in the phases of planning and experimentation.

A few

examples

will

illustrate

this.

A

prolific area of bilirubin research is concerned with the chemical structure of the bilirubin mole-cule. In the solid state, bilirubin acid can occur as an intramolecularly hydrogen-bonded substance with extremely low solubility in water.2 The circu-lating bilirubin is bound to albumin and is a di-anion, carrying two negative charges and forming soluble salts. Irradiation with blue light results in isomerization, transforming bilirubin into sub-stances that are soluble in water, even in the acid state.3 It is thus possible to understand the effect of phototherapy. The role of acidosis in kernicterus appears to the chemist to be easily explained by precipitation of the insoluble acid. It has indeed been shown that the solubility of biirubin acid and the binding affinity of the dianion to albumin,

measured in vitro, are such that precipitation can take place under the conditions present in infants at risk but not in healthy babies.4 It has further

been shown that such precipitation occurs readily when the icteric and acidotic plasma is in contact with biologic membranes,5 compelling arguments in favor of precipitation of bilirubin acid in kernic-tenis. McDonagh and Lightner have, however, in an interesting experiment demonstrated that bili-rubin can be partitioned from water into olive oil,6 in spite of the fact that the solid bilirubin acid is nearly insoluble in fat and lipid solvents.7 This

experiment calls for renewed investigation of the chemical nature of bilirubin acid. Does a lipophilic modification of bilirubin acid occur? What role, if any, does this substance have in kernicterus?

Another critical issue concerns the choice of suit-able animal models in experimental kernicterus studies. Rats and Gunn rats have been used more than any other animal, and much useful informa-tion has been obtained from them. Experimental work with rats, carried out by two groups,8’9 has demonstrated the importance of the blood-brain barrier for the prevention of bilirubin passage into the brain of these animals. It was found by the latter group9 that hypercarbia opens the barrier, resulting in deposition of bilirubin in the brain, whereas acidosis per so does not. This would

sup-port the concept that acidosis alone may not be a major factor in kernicterus but that acidosis with increased carbon dioxide tension (ie, respiratory acidosis) may increase the permeability of the blood-brain barrier, allowing bilirubin to enter the brain. The suggested applicability of these studies in man raises the crucial importance of the choice of animal models and the difficulty of directly translating fmdings from animal experiments to human infants.

Thus, in vitro studies of bilirubin-binding equi-libria with rat and human serum albumins’#{176} show that rat serum albumin binds bilirubin acid, not the anion, in contrast to the human protein which, as already mentioned, binds the anion, not the acid.

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EDITORIALS

155

Thus, it is understandable that acidosis as such

does not induce precipitation of bilirubin acid in the rat, although seems to do so in human babies.

Another important species difference seems to be

related to the different binding pattern of bilirubin

to rat and human albumin. The main biliary and urinary bilirubin photoisomers excreted by human infants during phototherapy are structural isomers, ie, (EZ)-cyclobilirubin, whereas under similar cir-cumstances geometric isomers, ie, (EZ)- and (ZE)-bilirubins are excreted by Gunn rats.”2 This can be related to species differences observed when bilirubin-albumin is irradiated in vitro.’3 Onishi et al’4 have in recent studies found that the formation of photoisomers ofbilirubin bound to human serum albumin, except for (EZ)-bilirubin, is rapid and

much greater than that for biirubin bound to rat serum albumin. The in vitro findings explain the different composition of the excreted pigments in rats and human infants. It is conceivable that a

difference of bilirubin conformation and

hydrogen-bonding pattern in the complexes with rat and human albumin causes the different photoisomeri-zation kinetics.

Finally, drug-induced bilirubin brain damage is a field in which benefit can be derived from close cooperation between clinical and basic scientists. The well-known outbreak of sulfisoxazole-induced kernicterus, studied by Silverman et al15 in 1956 and explained by Odell16 as displacement of biliru-bin from albumin on competitive binding of the

drug, resulted in a generally accepted avoidance of giving sulfonamides to icteric neonates. As a result of this, it has been suggested that all drugs for use in newborns, and in pregnant and lactating women, should be specifically tested for bilirubin-displacing

properties.’7 In vitro studies of numerous drugs

have shown that few substances in addition to the sulfonamides are sufficiently competitive with bil-irubin to represent a danger, when present in the usual plasma concentrations.’8 On the other hand, one new antibiotic, sodium fusidate (Fucidin, Leo Pharmaceutical Products, Copenhagen), has re-cently been added to the list of potential displacers, thanks to the vigilance of the manufacturers.’9 Most drugs, however, are being put on the market without testing The need for formally required testing persists and a concerted demand raised by both neonatologists and chemists would be timely.

The above examples indicate that the effective development of kernicterus research is closely linked to the proper positioning of the basic

scien-tist in such research groups. Examples of excellent

cooperation exist, but the chemist interested in

these topics is more often confined to reading the

clinician’s paper with 2 years’ delay and giving his

own contributions as a ten-minute presentation at meetings where pressure of time and nervousness admittedly may result in over-heated and useless exchanges. Otherwise, the basic scientist is called in as a referee when the clinician’s paper has been finished and submitted. He is then too late to make a positive contribution and often has the feeling that earlier contact could have prevented a disas-trous waste of effort and dashed hope for the inves-tigator.

Is it conceivable that this practice could be re-versed? The basic scientist should be involved in the early stages of research planning as well as during the experimental work and analysis of

re-sults. In return, the clinical investigator ought to

be reassured that his paper will be acceptable for publication once it has been endorsed by the same specialists who would otherwise serve as referees for the journal to which it is ultimately submitted. By such means, not only would the scientific basis of such studies be markedly enhanced but the din-ical applicability of the information acquired may therefore be more relevant and, because of this, more useful.

REFERENCES

LEO STERN, MD Brown University Providence, RI

ROLF BRODERSEN, PHD

Institute of Medical Biochemistry University of Aarhus

Aarhus, Denmark

1. Gartner LM, Snyder RN, Chabon RS, et al: Kernicterus: High incidence in premature infants with low serum

biliru-bin concentrations. Pediatrics 1970;45:906

2. Bonnett R, Davies JE, Hursthouae MB, et al: The structure

of bilirubin. Proc R Soc London BiOL 1978;202:249

3. Zenone EA, Stoll MS, Ostrow JD: Mechanism of excretion of unconjugated bilirubin (UCB) during phototherapy. Gas-troentemlogy 1977;72:1180

4. Cashore WJ, Oh W, Brodersen R: Reserve albumin and bilirubin toxicity index in infant serum. ActaPaediatr Scand

1983;72:415

5. Eriksen EF, Danielson H, Brodersen R: Bilirubin-liposome interaction: Binding ofbilirubin dianion, protonization, and

aggregation of bilirubm acid. J Biol Chem 1981;256:4269

6. McDonagh AF, Lightner DA: ‘Like a shrivelled blood or-ange’-Bilirubin, jaundice, and phototherapy. Pediatrics 1985;75:443-455

7. Brodersen R: Bilirubin solubility and interaction with al-bumin and phospholipid. J BiOL Chem 1979;254:2364

8. Levine RL, Fredericks WR, Rapoport SI: Entry of bilirubin into the brain due to opening of the blood-brain barrier. Pediatrics 1982;69:255-259

9. Bratlid D, Cashore WJ, Oh W: Effect ofacidosis on bilirubin deposition in rat brain. Pediatrics 1984;73:431-434

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156 KERNICTERUS

RESEARCH

10. Frandsen PC, Brodersen R: Bilirubin-rat serum albumin

interaction. Acta Chem Scand 1985;40B:55

11. Onishi 5, Miura I, Isobe K, et al: Structure and thermal interconversion of cyclobilirubin IXa. Biochem J 1984;218:667

12. Onishi S, Ogino T, Yokoyama T, et al: Biliary and urinary excretion rates and serum concentration changes of four

bilirubin photoproducts in Gunn rats during total darkness and low or high illumination. Biochem J 1984;221:717 13. McDonagh AF, Palma LA, Lightner DA: Phototherapy for

neonatal jaundice: Stereospecific and regioselective

photo-isomerization of bilirubin bound to human serum albumin

and NMR characterization ofintramolecularly cycized

pho-toproducts. J Am Chem Soc 1982;104:6867

14. Onishi S, Itoh 5, Yamakawa T, et al: Comparison of kinetic

study of the photochemical changes of (ZZ)-bilirubin IXa

bound to human serum albumin with that bound to rat serum albumin. Biochem J 1985;230:561

15. Silverman WA, Andersen DH, Blanc WA, et al: A difference

in mortality rate and incidence of kernicterus among pre-mature infants allotted to two prophylactic antibacterial regimens. Pediatrics 1956;18:614

16. Odell GB: The dissociation of bilirubin from albumin and

its clinical implications. J Pediatr 1959;55:268

17. Stern L: Drug interactions: Part II. Drugs, the newborn infant, and the binding of bilirubin to albumin. Pediatrics 1972;49:916

18. Brodersen R, Friis-Hansen B, Stern L: Drug-induced

dis-placement of bilirubin from albumin in the newborn. Dev

Pharmacol Ther 1983;6:217

19. Brodersen R Fusidic acid binding to serum albumin and interaction with binding of bilirubin. Acta Paediatr Scand

1985;78:874

UNEXAMINED PEER REVIEW PROCESS

. . . that data on the performance of the reviewing system are lacking is all

the more astounding in view of the momentous influence the system exerts on

the lives of those who write biomedical articles.

Submitted by Student

From Ingelfinger RJK: Peer review in biomedical publication. Am J Med 1974;56:686-692.

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1987;79;154

Pediatrics

LEO STERN and ROLF BRODERSEN