BILIRUBIN
ENCEPHALOPATHY
By WILLIAM
J.
WATERS,M.D.,
DANA.
RIGHERT,Pii.D.,
AND HELEN H. RAWSON, M.D.
Syracuse, N.Y.
319
T
HE STRIKiNG correlation betweenjaun-dice, elevated serum bilirubin levels
and brain damage with pigmentation has
been well documented
by
severalinves-tigators in relation to hemolytic disease of the new-born infant.1 In addition this
rather formidable complication has been
re-ported as occurring in other unrelated
dis-ease states,3 but with the apparent
corn-mon factors of hyperbilirubinemia and
“im-maturity” of the individual. The authors
be-came interested in the problem as to whether they could elucidate the nature
of the pigment inyolved in the staining of
the brain. The opportunity to study five
such cases resulting from hernolytic disease
of the newborn infant was presented.
CASE REPORTS
Case 1 : Mother, Type A, Rh-negative,
Gra-vida I, Para 0. No previous history of
transfu-sions or blood derivatives. Anti-Rh titer 1:8.
Male infant, birth weight 2.4 kg., Type A,
Rh-positive, Coomb’s positive. Jaundice noted
shortly after birth with hepatosplenomegaly of
moderate degree and anemia increasing in
se-verity (RBC 2.9 million/cmm.) with
erythro-blastosis (81 nucleated RBC/100 WBC).
Referred at 40 hr. of age for treatment. Total
serum bilirubin level then 33.4 mg./100 cc.
Ex-change transfusion through umbilical vein, 310
cc. Type A, Rh-negative in, 260 cc. out. At 65
hr. of age, developed temperature elevation of
39.2#{176}C. with increasing respiratory difficulty
and hemoptysis but no opisthotonos. Died at 75
hr. of age and postmortem examination
re-vealed yellow pigmentation in the region of the
thalamus, caudate and lentiform nuclei and the
floor of the fourth ventricle.
From the Departments of Pediatrics and
Bio-chemistry of the State University of New York
Medical Center, Syracuse, N.Y.
Presented in part at the Annual Meeting of the
Society for Pediatric Research, May 5, 1953, at
Atlantic City.
(Received for publication Sept. 21, 1953.)
Case 2: Mother, Type A, Rh-negative,
Gra-vida II, Para I. No history of previous
transfu-sions or blood derivatives. Anti-Rh titer 1:1024.
Labor induced 1 mo. early. Male infant,
birth weight 2.8 kg., Type 0, Rh-positive,
Coomb’s positive. Jaundice noted at 3 hr. of age
with hepatosplenomegaly of moderate degree
and anemia (RBC 2.4 million/cmm.) with
erythroblastosis
(
100 nucleated RBC/100WBC). Serum bilirubin not ascertained.
Ex-change transfusion at 5 hr. of age with some
difficulty; 320 cc. Type 0, Rh-negative in, 266
cc. out. At 42 hr. of age had a generalized
con-vulsion and subsequently temperature elevation
to 39.2 and opisthotonos. Died at 52 hr. of age.
Postmortem examination revealed yellow
pig-mentation in the region of the basal ganglia,
thalamus, and some nuclei of the cranial nerves
and cerebellum.
Cases 3 and 4: Twins. Mother, Type AB,
Rh-negative, Gravida V, Para IV, Titer 1:64.
Case 3: Female infant, birth weight 1.3 kg.,
Type A, Rh-positive, Coomb’s positive.
Jaun-dice noted at 8 hr. of age with
hepatospleno-megaly of moderate degree. No significant
anemia or erythroblastosis. At 40 hr. of age
be-gan to have “twitchings.” Subsequently
devel-oped temperature elevation from 35#{176}to 37#{176}C.
Respirations then became irregular and
la-bored. No opisthotonos and Moro was positive
shortly before death at 104 hr. of age. Total
serum bilirubin level at 80 hr. of age was 24.5
mg./100 cc.
Case 4: Female infant, birth weight 1.6 kg.,
Type B, Rh-positive, Coomb’s positive.
Jaun-dice noted at 16 hr. of age but no significant
hepatosplenomegaly, anemia, or
erythroblasto-sis. Serum bilirubin level 22.4 mg./100 cc. at
80 hr. of age. Infant began regurgitating
feed-ings at 84 hr. of age. Developed opisthotonos
with irregular respirations and blood-tinged
vomitus prior to death at 92 hr. of age. No
febrile rise was detected in this patient.
Postmortem examination on both patients re-vealed yellow pigmentation in the region of the
floor of the fourth ventricle, brain stem and
both cerebral hemispheres.
Gra-320
.4
W.
J.
WATERS, D. A. RICHERT AND HELEN H. RAWSON.2
I.0
BLood Serum Prior To Exchange
----o Case A (Moderate)
‘ -, B (Severe)
0.8
0.6
0.4
0.2
ci)
z w
WAVE LENGTH
CHART 1. Absorption spectra of peripheral blood specimens from 2 cases of hemolytic disease of
newborn infant prior to exchange transfusion.
320 360 400 440 480 520 560 600 680
vida X, Para VIII. One stillbirth. No history of previous difficulty and no antepartum titers
were performed. Male infant, birth weight 3.6
kg., Type A, Rh-positive, Coomb’s positive.
Jaundice noted sometime within 1st 24 hr. with
moderately enlarged liver but no apparent splenomegaly. Subsequent anemia (RBC 2.4 million/cmm.) with no significant
erythroblasto-sis. Serum bilirubin level 27.6 mg./100 cc. at
30 hr. At 55 hr. of age developed a
tempera-ture elevation from 36.7#{176}to 38.0#{176}C.,spasticity of the upper extremities, became listless, and
respirations became shallow and irregular at 67
hr. of age. Postmortem examination revealed
yellow pigmentation in the region of the
dor-sum of the pons, around the aqueduct, in the
region of the olivary bodies, pyramids, and
dor-sal nuclei in the medulla.
Clinically the signs associated with
prob-able brain damage have been well
docu-mented in the past.1 In addition the authors
would like to emphasize from the above
case reports that they noted temperature elevation of several degrees from 12 to 24
hours’ duration in 4 out of the 5 cases and
invariably it was associated with
a poor
prognosis.
METHODS OF STUDY
All the absorption spectrum measurements were made with a Beckman spectrophotometer. Peripheral blood specimens from cases of he-molytic disease of the newborn infant (chart 1) and from newborn infants with “physiologic jaundice” (chart 2) were compared spectropho-tometrically. The sera from these bloods were diluted with isotonic saline for the measure-ments. The absorption curves of each showed
a peak at 460, which was found to be typical
for commercial bilirubin dissolved in
chloro-form (chart 3). The pronounced peak in the
410 to 42O.t region exhibited by the sera from
the patients with hemolytic disease indicated
the presence of a hemoglobin compound which
is to be expected as a natural result of
hemoly-S1S.’
At postmortem, tile pigmented areas of the
brains from the above 5 cases were freed of
overlying pial vessels and the tissues were placed in either chloroform or Bloor’s mixture,
macerated, extracted at room temperature for
12 to 24 hr. The extracts were measured spec-trophotometrically and in each instance a curve was obtained showing maximal absorption at
commer-300 340 380 420 460 500 540 580
BILIRUBIN ENCEPHALOPATHY 321
U) z LAJ 0
1.5
L2
.9
.6
.3
CHCL3 Extract Blood Serum
Physiological Jaundice
WAVE LENGTH
620
CHAR! 2. Absorption spectrum of peripheral blood specimen from case of “physiologic jaundice.”
cia! l)ilirubin dissolved in chloroform (chart 3).
A positive van den Bergh was obtained both
ill the 1 mm. and 30 mm. specimens from such
all extract.8 Comparable sections of brain
ob-tamed at postmortem from newborn infants
who did not exhibit jaundice or brain damage
were examined similarly and showed no such
absorption properties at 46O.t (chart 4). In
ad-dition, sections from areas of the medulla and
cerebellum from Case No. 5, which did not
ap-pear grossly stained with pigment, were
ex-tracted with chloroform. The amount of this
tiSSue was equivalent to the pigmented areas
studied. Essentially no bilirubin was detected
spectrophotometrically in the extract (chart 5)
showing that the material with maximal
absorp-tion at 46O.t was concentrated in the pigmented
areas of the brain.
Ill aii atteml)t to make the identification of
the extracted pigment more conclusive, crystals
were obtained from one of the extracts using
the method described by Najjar for the
crystal-lization of bilirubin.#{176} The pigment from a
chloroform extract of brain was re-extracted
from the chloroform by 0.01 molar pyrophos-phate buffer ph 9.5, from which solution the
pigment was crystallized by Najjar’s procedure.
Under the microscope these crystals had a
defi-nite yellow sheen. The crystalline form was
identical with the bilirubin crystals described
by Najjar and Childs in a later
communica-tion.1#{176}Lowry and co-workers have also
de-scribed similar bilirubin crystals)’ When
redis-solved in chloroform, the crystalline material
again showed a typical billirubin absorption
curve as shown in chart 6. Photomicrographs of
the crystals are shown in Fig. 1.
DISCUSSION
The probability that the pigment
stain-ing the brain in hemolytic disease of the newborn infant is bilirubin was suggested
by the experimental work of Day.12 To the
pig-0.18 _______x Bloor’s Brain Extract No.4
.09
360 380 400 420 440 460 480 500 520
CIIARr 3. Absorption spectra of chloroform and Bloor’s extracts of pigmented Case 4 and commercial bilirubin in chloroform.
- -- CHCL3 Extract of Normal Brain
s -. Bilirubin in CHCL3
brain areas in Case 3 and
>-U)
z
Ui
C)
08
320 340 360 380 400 420 440 460 480 500
CHART 4. Absorption spectra of chloroform extract of normal newborn brain and
commercial bilirubin in chloroform.
322
>- I-U) z
w
C)
0.15
0.12’
.06
.03
2.0
Os
W.
J.
WATERS, D. A. RICHERT AND HELEN H. RA\VSONS
6 “
5’
5’
2
0- .. CHCL3 Brain Extract No. 3
Bilirubin in CHCL3
5’
---o CHCL Extract Non-Pigmented Brain Areas No.5
e #{149}CHCL3 Extract Pigmented Brain Areas No.5
.084
Bilirubin in CHCL3
t s Crystalline Brain Pigment in
.072
.060
360 380 400 420 440 460 480 500
WAVE LENGTH
CHART 6. Absorption spectra of solution of crystals obtained from extract of pigmented
brain areas and commercial bilirubin.
520
0.7
0.6 C
_____
0.5
0.4
>. 5’
0.3
0.2
-0--- -0--- -o--
-a---330 350 370 390 410 430 450 470 490 510
WAVE LENGTH
CUART 5. Absorption spectra of chloroform extracts of pigmented and nonpigmented
brain areas from Case 5.
.048
>-
F-z
Ui
.036
.024
:324
\V. j. WATERS, D. A. RICHERT AND HELEN H. RAWSONFic. 1.
(x
1500) Crystals obtained fromchloro-form extract of pigmented brain areas from case
of hemolytic disease of newborn infant.
ment” in the peripheral blood which might explain why this brain pigmentation occurs
111 selected cases, but it was not possible to
find this by the methods used in this study. In each instance, a spectrophotornetric
curve consistent with bilirubin was obtained
with the blood sera and the extracts of the
pigmented brain areas.
Since in the past it has been well
demon-strated that bilirubin will pass the
cere-brospinal barrier,’35 it would appear
reasonable that this pigment under certain
conditions enters the cell and possibly
in-terferes with its normal metabolic processes.
In the cases so far reported it appears that
pigmentation of the brain as a complication
of jaundice, regardless of etiology, is limited
to the very young infant. The “immaturity” of the total organism may be an essential
factor and may explain why this severe
dis-turbance does not occur in cases of biliary
atresia or in adults with marked jaundice
from various causes. However, this problem
remains for further investigation.
SUMMARY
Peripheral blood specimens from cases of
varying degree of clinical severity of
hemo-lytic disease of the newborn infant have been examined spectrophotometrically and
a curve consistent with the presetc of
bilirubin was found in each case.
Extracts of tile pigmented brain areas
from five cases of hemolytic disease of the
newborn infant, who died with clinical
signs of brain damage, were examined
spectrophotometrically and in each intance
a curve consistent with bilirubin was
ob-tamed. An extract from comparable sections
of brain not appearing grossly pigmented
contained essentially no such pigment when
examined spectrophotonietrically.
Crystals were obtained from a chloroform
extract of pigmented brain areas and when
re-dissolved showed a typical type curve
for bilirubin.
A positive van den Bergh was obtained
on the extract solution.
ACKNOWLEDCMENT
The authors wish to thank Mrs. E. Anita
Schrarnm for her invaluable assistance in
the final preparation of the graphs and
manuscript.
REFERENCES
1. Vaughan, V. C., III, Allen, F. H., Jr., and
Diamond, L. K., Erythroblastosis fetalis.
IV. Further observations 0)1 kernieterus,
PEDIATRICS 6:706, 1950.
2. Hsia, D., Allen, F., Cellis, S., and Diamond,
L. K., Erythroblastosis fetalis. VIII.
Studies of serum bilirubin in relation to
kernicterus, New England
J.
Med. 18:668, 1952.
3. Zuelzer, W. W., and Mudgett, Roxie T.,
Kernicterus : Etiologic study based on
analysis of 55 cases, PEDIATRICS 6:452,
1950.
4. Crigler, j. F., and Najjar, V. A., Congenital
familial nonhemolytic jaundice with
kernicterus, PEDIATRICS 10: 169, 1952.
5. Govan, A. D. T., and Scott,
J.
NI.,Kernic-terus and prematurity, Laneet 1 3 : 6 1 1,
1953.
6. Aidin, R. , and others, Kernicterus and
prematurity, Lancet 1: 1 153, 1950.
7. Lembert, R., and Legge, j. W., Hematin
compounds and bile pigments, New
York, Interscience Publishers, Inc., 1949.
8. Hsia, D., Hsia, H. H., and Gellis, S.,
Micro-method for serum bilirubin,
J.
Lab. &Clin. Med. 40:610, 1952.
9. Najjar, V. A., Metabolism of bilirubin,
BILIRUBIN ENCEPHALOPATHY 325
10. Najjar, V. A., and Childs, B.,
Crystalliza-tioti and properties of serum bilirubin,
J.
Biol. Chem. 204:359, 1953.
1 1. Lowry, P. , Bessenmaier, I., and Watson,
C.
J.,
Isolation of bilirubin from feces,J.
Biol. Chem. 202:305, 1953.12. Day, R., Kernicterus problem:
Experi-mental in vivo and in vitro staining of
brain tissue with bilirubin, Am.
J.
Dis. Child. 73:241, 1947.13. Roberts, M. H., Relation of pigment
con-tent in serum and spinal fluid of
new-born infants, South. M.
J.
6:460, 1928. 14. Cantarow, A., and Trumper, M., ClinicalBiochemistry, Philadelphia, W. B.
Saun-ders Company, 1950, p. 409.
15. Amatuzio, D. S., Weber, L.
J.,
and Nesbitt,S., Bilirubin and protein in C. S. F. of
jaundiced patients with severe liver
dis-ease,
J.
Lab. & Clin. Med. 41:615, 1953.SPANISH ABSTRACT
Encefalopatla
por
Bilirrubina
La enfermedad hemolItica del reci#{233}nnacido
COIl frecuencia se acompafla de ictericia,
hiper-bilirrubinemia acentuada y lesion cerebral; Ia
lesion cerebral tambi#{233}n se describe en otros
padecimientos no relacionados a Ia enfermedad
hemolItica del reci#{233}n nacido, pero como en
ella, con factores comunes de
hiperbilirrubi-nemia e “inmadurez” del individuo. El estudio de 5 casos de enfermedad hemolItica del reci#{233}n
nacido les di#{243}oportunidad a los presentes
autores para tratar de elucidar la naturaleza
del pigmento qiie al fijarse en la
c#{233}lulanervi-osa, Ia lesiona.
A los signos cinicos de lesion cerebral, cuidadosamente estudiados por otros investi-gadores, Waters y sus colaboradores agregan
otro que ellos observaron en su serie: alza de Ia
temperatura en varios grados, con duraciOn de
12 a 24 horas en 4 de los cinco casos, que se
asociO invariablemente a un pronOstico fatal.
El espectro de absorciOn se midiO con 1111
espectrofotOmetro de Beckman; se utilizO
sangre periferica de los reci#{233}n nacidos con
en-fermedad hemolItica, de grado variable en severidad cilnica, compar#{225}ndose los datos con Ia sangre de reci#{233}nnacidos con ictericia fisio-lOgica; con los datos se traz#{243}una curva de
bilimibinemia en cada caso. La bilirrubina se
demostrO adem#{225}s en Ia c#{233}lulanerviosa: las areas cerebrales pigmentadas de los 5 casos
objeto de este estudio, se examinaron espectro-fotom#{233}tricamente y se determinO una curva
tIpica del pigmento; sirvieron de control
see-ciones cerebrales comparables pero no
pigmen-tadas macroscOpicamente, de reci#{233}nnacidos sin
ictericia ni daflo cerebral que no mostraron
tam-poco pigmento a la espectrofotometria. Por #{241}ltimose obtuvieron curvas de bilirrubina con los cristales logrados de las areas cerebrales pig-mentadas tratadas eon cloroformo; con este mismo extracto Ia reacci#{243}nde Van Den Bergh fue positiva.
Los trabajos experimentales de Day sugirie-ron la probabilidad de que el pigmento fijado
al cerebro en Ia enfermedad hemoIltica del
reci#{233}nnacido fuera bilirrubina; se ha demo-strado que #{233}stapasa f#{225}cilmente Ia barrera cere-broespinal, con lo que era razonable suponer que bajo determinadas condiciones penetrara a las c#{233}lulasnerviosas e interviniera en sus pro-cesos metabOlicos normales. En los casos hasta
ahora reportados se encuentra que Ia
pigmen-taciOn del cerebro, como complicaciOn de las ictericias independientemente de su etiologla, se limita a los lactantes muy peque#{241}os; Ia “in-madurez” general del organismo puede ser un factor esencial y explicar por qu#{233}este trastorno no se presenta en casos de atresia biliar o en adultos con ictericia asociada de varias causas. Esta aseveraciOn, dicen los autores, requiere
mayor investigaciOn.
