45
BILIRUBIN
ENCEPHALOPATHY
Preliminary
Studies
Related
To
Production
By William J. Waters, M.D.,* and Howard A. Britton, M.D.
F
ROM studies previously reported’3 thepigment staining the brain in hemolytic disease of the newborn has been identified
as hilirubin. Additional evidence has been
presented by Claireaux et a!. in support of
these findings.’ It has also been
well-docu-mented that this pigmentation occurs
pri-manly in association with hyperbilirubi-nemia in the young iiifant.5
Buxton and Brookshank reported intense icterus in newborn pigs as a result of
hemo-lytic disease caused by maternal iso-immu-nization.1#{176} They found staining of the brain
ill a high proportion of pigs exhibiting
din-ical icterus. Though the sera contained a
number of different pigments, bilirubin
ap-peared to have been extracted from the
l)raiflS to the exclusion of all others.H Because of the striking clinical incidence
of kernicterils in the human newborn, and
the determination of the pigment as
bili-rubm, we decided to use newborn rats in an attempt to produce staining of the brain with a solution of bilirubin. As controls,
approximately 1-month-old rats were
util-ized.
Previously King’ and others showed that
injection of trypan blue in newborn animals
will produce staining of the brain. In
ad-dition, Frohlich and Mirsky injected rats of
varying ages mntraperitoneally with solutions made from “concentrated bile,” and showed
that ill allimalS ullder 10 days of age they
occasionally obtained staining of the brain; whereas, in the animals over 15 days of age no staining occurred.
From the Department of Pediatrics, State
Uni-versity Medical Center, Syracuse New York.
Read in part by title at the Society for Pediatric Research, May, 1954.
(Received for publication July 16, 1954.)
* ADDRESS: Department of Pediatrics, State
Uni-versity Medical Center, Syracuse, New York.
PROCEDURE
Thirty-six white rats less than 24 hours of
age, and averaging 10 gm. in weight, and 25
1-month-old white rats averaging 1 15 gm. in weight were injected intraperitoneally with a solution of commercial ub#{176} The solution was prepared by dissolving 4 mg. of bilirubill in 1 ml. of a supersaturated solution of Na2CO and
titrating to a pH of between 8 and 9 with iN
HCI. Intraperitoneal injections were given 2 to 3
times daily depending UOfl the degree of
ab-dominal distension observed. The results are
shown in Tables I and II.
DISCUSSION
From Table I it is apparent that tile bili-rubin solution was not well tolerated by
newborn rats since a majority of the rats
died within the first 24 hours. Furthermore
there appeared to be some variability in re-sponse of the newborn group to total dosage
given, as only 5 out of 11 animals that re-ceived a total dose of 5 111g. or more, be-came jaundiced. Four of these showed staining of the brain. None of the rats re-ceiving less than 5 mg. total dose de-veloped visible jaundice or staining of the brain. The single newborn rat that became jaundiced but did not develop staining of the brain, became visibly icteric at 8 days
of age and died at 10 days of age. The lack of staining of the brain may have been due to its failure to develop a critical level of bilirubin early. Thus, it can be seen that
staining of the brain can be produced in
normal newborn rats by the intraperitoneal injection of bilirubin. Although technically it was impossible to obtain serum bilirubin levels in these animals, it was apparent that the staining of the brain could be corre-lated with the degree of jaundice, the latter being, in part, a function of dose.
IT 0
TABLE I
INTIIAPEKITONEAL INJECTION OF BILIRUBIN INTO NEWBORN WHITE RATS,
Av. WT. 10 GM.
o Non-Icteric
Ozcteric Body
#{149}Icteric Body + Brain
#{149}
0#{149} 0 0
5 0 #{149}0 0
4 go 0
0 0
3 oS%o
00
000
2 og
46 WATERS - BILIRUBIN ENCEPHALOPATHY
IS
15
14
0
‘3
0
uJ 2
I’
to
II.-OQ.
wH 8
U)
0
0 .,
-j
4
I-0
I-.
<Id 2d 3d 4d Sd
DAYS OF
In tile adult cOlltrols in Table II it is
ap-parent that jaundice was not as readily pro-duced by an equal dose per gram of body weight. By using much larger doses it was
possible to produce jaundice in a compara-ble number, but staining of the brain was
not observed in these animals.
In the newborn rats studied so far, in
which gross staining of the brain was
pro-Gd Td Sd 9 10
SUR VIVAL
duced, an attempt was made to oi)tain his-tological sections for the purpose of de-termining whether any of the pigment was
TABLE II
INTRAPERITONEAL INJECTION OF BILIRUBIN INTO ONE MONTH OLD WHITE RATS, Av. WT. 115 GM.
0
.0 210
00
200
0
90
180
170
o Non - Icteric
160
O Icteric Body
150 . Lcteric Body + Brain
140
a
130
120
I-C_) 0
w
-) 110 000
100
cr
- 0
_J ‘J
co
U- 80
0
70 #{176}
0
0
_J 60
I-0
50
40 0
30
20 00
0
to
Id 2d 3d 4d Sd 6d i’d 8d 9d tOd ltd I2d 3d 14d 15d ‘t6d
DAYS OF#{149}SURVIVAL
CONCLUSIONS toneal injection of a solution of bilirubin.
(1) Staining of the brain can be produced (2) Under similar conditions it was not
48 WATERS - BILIRUBIN ENCEPHALOPATHY
a comparable series of normal adult rats. (3) This study further emphasizes the im-portance of the immaturity of the organism
in the production of hyperbilirubinemia and
its consequent passage through the
“blood-brain barrier” (Bilirubin Encephalopathy).
(4) This adds to the increasing evidence
that immaturity of biochemical and physio-logical processes determines the pathologi-cal states which appear to be unique to the newly born organism.
REFERENCES
1. Waters, W.
J.,
Richert, D., Rawson, H.:Bilirubin encephalopathy. Tr. Soc.
Pediat. Research, Am.
J.
Dis. Children, 86:483, 1953.2. Waters, W.
J.,
Richert, D., Rawson, H.:Bilirubin encephalopathy. PEDIAi’mcs, 13:319, 1954.
3. Waters, W.
J.,
Vincent, W., Britton, H.:Bilirubin encephalopathy. Further stud-ies related to identification and
produc-tion. Read by title. Soc. Ped. Research,
May, 1954.
4. Claireaux, A. E., Cole, P. G., Lathe, G. H.: Icterus of the brain in the newborn.
Lancet, 11:1226, 1953.
5. Hsia, David, Allen, F. H., Jr., Gelliss, S. S., and Diamond, L. K.: Erythroblastosis
fetalis. VIII. Studies of serum bilirubin in relation to kernicterus. New England
J.
Med., 247:668, 1952.6. Zuelzer, W. W., and Mudgett, Roxie T.:
Kernicterus : etiological study based on analysis of fifty-five cases. PEDIATRICS,
6:452, 1950.
7. Crigler,
J.
F. and Najaar, V. A. : Congenital familial non-hemolytic jaundice withkernicterus. PEDIATRICS, 10: 169, 1952.
8. Govan, A. D. T., and Scott,
J.
M. :Kern-icterus and prematurity. Lancet, 1:611,
1953.
9. Aidin, R., et a!. Kernicterus and
pre-maturity. Lancet, I : 1 153, 1950.
10. Buxton,
J.
C., and Brooksbank, N. H.:Hemolytic disease of newborn pigs
caused by iso-immunization of
preg-nancy. Nature, 172:355, 1953. 11. Buxton,
J.
C.: Personal communication.12. King, L. S. : Research Publications of the
Association for Research in Nervous and
Mental Disease, Vol. 18, 1938, Chap. 6. 13. Frohlich, A., and Mirsky, I. A. :
Suscepta-bility to convulsions in relation to age.
II. Influences of bile in rats. Soc. Exper.
Biol. & Med. Proc., 50:25, 1942.
SPANISH ABSTRACT
Encefalopatla por Bilirrubina Estudios Preliminares en Relaci#{243}n a su
Producci#{243}n
Se ha identificado ya a la bilirrubina como
el pigmento que se fija en el cerebro en Ia enfermedad hemoiltica del reci#{233}nnacido, por
un proceso asociado fundamentalmente a la
hiperbilirrubinemia debido
a
su frecuenciacimnica en el reci#{233}nnacido humano, los
auto-res presentan un estudio experimental en ratas
tomando en consideraci#{243}n tambi#{233}n que otros
mnvestigadores ya habIan encontrado fijaci#{243}nde Ia bilirrubina en el cerebro de estos animales
menores de diez dIas en tanto que no se observaba en ratas mayores de quince dIas
de edad. Se emplearon 36 ratas blancas dentro de las primeras 24 horas de nacidas, con un
peso de 10 gramos como promedio, ye como
control 25 ratas de un mes de edad y un
peso de 115 gramos; a ambos grupos se les
inyect#{243}una soluci#{243}ncommercial de bilirrubina
por via intraperitoneal.
La mayorIa de las ratas reci#{233}nnacidas
fallecieron durante las siguientes 24 horas;
Ia respuesta variO en las dem#{225}s de acuerdo
con Ia dosis total de bilirrubina inyectada:
cmnco de once ratas que recibieron 5 miligramos
6 m#{225}spresentaron ictericia; de ellas, cuatro mostraron pigmentaci#{243}n cerebral; en cambio
ninguna de las ratas que recibi#{243}menos de 5 miligramos ni desarroll#{243} ictericia ni mucho menos pigmentaci#{243}n cerebral. Por razones t#{233}cnicasno se pudieron obtener secciones his-t#{243}logicas de los cerebros pigmentados. En el
grupo control no se produjo ictericia tan
facil-mente con dosis iguales por gramo de peso corporal; con dosis mucho mayores se observ#{243} ictericia pero sin que ninguna de las ratas
mostrara adem#{225}s pigmentaci#{243}n cerebral.
De acuerdo con su experiencia los autores prueban Ia pigmentaci#{243}n cerebral con
bilir-rubina cuando se aplica por via intraperitoneal a ratas reci#{233}nnacidas en contraste con ratas
adultas. De aqul recalcan Ia importancia de Ia inmadurez org#{225}nica en la producci#{243}n de hiperbilirrubinemia con su consecuente paso
por la barrera hematoencef#{225}lica; asImismo, presentan una prueba m#{225}sdel significado que
tiene Ia inmadurez de los procesos bioqulmicos
y fisiolOgicos en determinar condiciones
