June 1978
The J o u r n a l o f P E D I A T R I C S
1001
Estimation of unbound serum bilirubin by the peroxidase assay method: Effect of exchange transfusion on unbound bilirubin and serum bindings Unbound and total serum bilirubin concentrations were determined by the peroxidase method in 160 samples from 60 infants. A significant relation existed between unbound and total bilirubin (UB = 0.58 TBR -- 0.24; r = + 0.59," P < 0.001), but the scatter o f values was wide. Serum binding level, the serum concentration o f total bilirubin at which point the unbound bilirubin may be expected to reach 30 nmol/1, was determined in each o f the 60 infants. Binding was related to birth weight (serum binding level = 0.0017 birth weight (gm) + 18. 6," r = + 0.30; P < O.05) and gestational age (serum binding level = 0.45 [gestational age] + 6.67; r = +0.39," P < 0.01), but the scatter o f values was wide. Exchange transfusion reduced high levels o f unbound bilirubin (mean before exchange 14.0 nmol/ l, mean after exchange 4.0 nmol/l," paired analysis P < 0.002). Serum binding level was unaffected or only transiently affected by exchange transfusion.
Howard D. Arkans, M.D., and George Cassady, M.D.,* B i r m i n g h a m , A l a .
EXPERIMENTAL AND CLINICAL DATA indicate that unconjugated, lipid-soluble serum bilirubin, unbound to albumin, relates in a complex and dynamic fashion to risks of neurotoxicity a n d encephalopathy in the newly born infant. 1-5 The concentration of unbound serum bilirubin at which neurotoxic substances reach dangerous levels is often uncertain in the individual patient, in part because of factors which affect albumin binding. Inasmuch as serum from adults binds bilirubin better than that from infants,6, 7 exchange transfusion may favorably influence serum binding properties in the newborn infant. Several methods have been proposed to evaluate albumin binding of bilirubin.8-1~ Unbound bilirubin is directly estimated using the peroxidase method. ~ It is also possible, with this technique, to estimate the total serum bilirubin at which the unbound fraction of bilirubin reaches so-called danger levels. This is done by adding From the Department o f Pediatrics, Division of Perinatal Medicine, University o f Alabama. *Reprint address: Department of Pediatrics, Division of Perinatal Medicine, University of Alabama in Birmingham, University Station, Birmingham, AL 35294.
0022-3476/78/0692-1001500.50/0 9 1978 The C. V. Mosby Co.
aqueous bilirubin to the patient's serum in vitro. 6 While the level at which high affinity binding sites approach saturation has been suggested to be about 30 nmol/1,11 the concentration of bilirubin by this technique which results in dangers of neurotoxicity is not yet known and will require extensive long-term studies. In the present work the peroxidase method is used to explore the relation of total to unbound serum bilirubin, as well as to examine the effect of exchange transfusion on unbound serum bilirubin and serum binding properties. Abbreviations used TBR: total serum bilirubin UB: unbound serum bilirubin ET: exchangetransfusion
MATERIALS AND METHODS Bilirubin studies were performed on 0.2 ml serum samples obtained when clinically required for the care of jaundiced infants. One hundred and sixty studies were performed in 60 patients. Serum was studied a single time in 11 infants, two to three times in 38, and 11 infants had serum studied four to six times. Birth weights ranged from 800 to 4,550 gm; nine infants were less than 1,000 gm,
Vol. 92, No. 6, pp. 1001-1005
1002
Arkans and Cassady
The Journal of Pediatrics June 1978
3C
~20
9
9
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The J o u r n a l o f P E D I A T R I C S
1001
Estimation of unbound serum bilirubin by the peroxidase assay method: Effect of exchange transfusion on unbound bilirubin and serum bindings Unbound and total serum bilirubin concentrations were determined by the peroxidase method in 160 samples from 60 infants. A significant relation existed between unbound and total bilirubin (UB = 0.58 TBR -- 0.24; r = + 0.59," P < 0.001), but the scatter o f values was wide. Serum binding level, the serum concentration o f total bilirubin at which point the unbound bilirubin may be expected to reach 30 nmol/1, was determined in each o f the 60 infants. Binding was related to birth weight (serum binding level = 0.0017 birth weight (gm) + 18. 6," r = + 0.30; P < O.05) and gestational age (serum binding level = 0.45 [gestational age] + 6.67; r = +0.39," P < 0.01), but the scatter o f values was wide. Exchange transfusion reduced high levels o f unbound bilirubin (mean before exchange 14.0 nmol/ l, mean after exchange 4.0 nmol/l," paired analysis P < 0.002). Serum binding level was unaffected or only transiently affected by exchange transfusion.
Howard D. Arkans, M.D., and George Cassady, M.D.,* B i r m i n g h a m , A l a .
EXPERIMENTAL AND CLINICAL DATA indicate that unconjugated, lipid-soluble serum bilirubin, unbound to albumin, relates in a complex and dynamic fashion to risks of neurotoxicity a n d encephalopathy in the newly born infant. 1-5 The concentration of unbound serum bilirubin at which neurotoxic substances reach dangerous levels is often uncertain in the individual patient, in part because of factors which affect albumin binding. Inasmuch as serum from adults binds bilirubin better than that from infants,6, 7 exchange transfusion may favorably influence serum binding properties in the newborn infant. Several methods have been proposed to evaluate albumin binding of bilirubin.8-1~ Unbound bilirubin is directly estimated using the peroxidase method. ~ It is also possible, with this technique, to estimate the total serum bilirubin at which the unbound fraction of bilirubin reaches so-called danger levels. This is done by adding From the Department o f Pediatrics, Division of Perinatal Medicine, University o f Alabama. *Reprint address: Department of Pediatrics, Division of Perinatal Medicine, University of Alabama in Birmingham, University Station, Birmingham, AL 35294.
0022-3476/78/0692-1001500.50/0 9 1978 The C. V. Mosby Co.
aqueous bilirubin to the patient's serum in vitro. 6 While the level at which high affinity binding sites approach saturation has been suggested to be about 30 nmol/1,11 the concentration of bilirubin by this technique which results in dangers of neurotoxicity is not yet known and will require extensive long-term studies. In the present work the peroxidase method is used to explore the relation of total to unbound serum bilirubin, as well as to examine the effect of exchange transfusion on unbound serum bilirubin and serum binding properties. Abbreviations used TBR: total serum bilirubin UB: unbound serum bilirubin ET: exchangetransfusion
MATERIALS AND METHODS Bilirubin studies were performed on 0.2 ml serum samples obtained when clinically required for the care of jaundiced infants. One hundred and sixty studies were performed in 60 patients. Serum was studied a single time in 11 infants, two to three times in 38, and 11 infants had serum studied four to six times. Birth weights ranged from 800 to 4,550 gm; nine infants were less than 1,000 gm,
Vol. 92, No. 6, pp. 1001-1005
1002
Arkans and Cassady
The Journal of Pediatrics June 1978
3C
~20
9
9
%
9
ooo
:D
I0
Oo
I UB(nmol/L)=.58TBR(mg/dl) -.24
9
9 9 0- tto gO 9 9 oo O O~&~ ( ~ I~ 9
,
9
9
I
1~=.o.59 n=160
L
9 o
I
/
9 "
O
9 I0
30
20
TBR (rng/dl) Fig. 1. Total bilirubin vs unbound bilirubin in 160 samples from 60 infants. 40
9
8
"El
~, 30 E
dee 9
20
O0 9 Og$
Se
9 0000
O0
O OLOw 9
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.=6o p=
