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The effect of Na benzoate on serum bilirubin of the Gunn rat
May 1975 The Journal o f P E D I A T R I C S
799
The effect of Na benzoate on serum bilirubin of the Gunn rat The administration of Na benzoate to grown and suckling Gunn rats in single doses of 7 and 35 mg/kg failed to significantly alter serum bilirubin concentrations. These doses are comparable to quantities of Na benzoate contained in injectable diazepam used therapeutically for newborn infants. Repeated doses of 7 mg/kg in the grown rat showed no effect, as well. A single dose of 100 or 200 mg/kg of Na benzoate and repeated doses of 35 mg/kg resulted in depressed serum bilirubin concentrations. The higher concentrations o f Na benzoate, however, greatly exceed amounts contained in doses of diazepam recommended for clinical use in the human neonate. The data suggest that the use of injectable diazepam, in appropriate quantities, poses no hazard to the newborn infant in terms o f bilirubin toxicity. The greater affinity for bilirubin o f human albumin, than that of rat albumin, may further minimize the risk.
Gerald Nathenson, M.D.,* Michael I. Cohen, M.D., and Helen McNamara, M.A., Bronx, N.Y.
IN VITRO STUDIES demonstrate that Na benzoate is one of several organic anions which can cause the dissociation of bilirubin from albumin-binding sites. 13 Of considerable concern in clinical situations is the fact that N a benzoate is contained in injectable diazepam, a drug used in the m a n a g e m e n t of various neonatal disorders. In order to further clarify the problem o f the safety of N a benzoate, the G u n n rat, which has been used as an animal model for the study of in vivo effects of drugs on the binding of indirect-reacting bilirubin to albumin,a, 5 was selected for study. The investigation described details the effects of injected Na benzoate, at varying dose levels, on serum bilirubin concentrations in the G u n n rat.
MATERIALS AND METHODS Forty-two grown homozygous G u n n rats, 16 males and 26 females, were used for serial assessments of serum bilirubin concentrations following injections of From the Divisions of Neonatology and Adolescent Medicine, Department o f Pediatrics, Montefiore Hospital and Medical Center, Morrisania City Hospital and the Albert Einstein College of Medicine. *Reprint address:Morrisania CityHospital, 168th St. & Gerard Ave.. Bronx, N. Y. 10452.
N a b e n z o a t e , 0.9% NaCI or sulfisoxazole d i o l a m i n e . Dosages of Na benzoate were 7, 35,100, and 200 mg/kg, respectively, of rat body weight, given intramuscularly or intraperitoneally; and of sulfisoxazole, 200 mg/kg administered subcutaneously. Distilled water was used as the diluent for the benzoate and sulfisoxazole. Injection volumes varied from 0.3 to 0.5 ml. Comparable injection v o l u m e s o f saline were a d m i n i s t e r e d to t h e c o n t r o l animals. The G u n n rats ranged in weight from 86 to 276 gm ( m e d i a n weight: 164 gm) and varied in age from 2 months to 5 months. Bilirubin determinations were perf o r m e d in duplicate b y a m i c r o m o d i f i c a t i o n o f the Malloy-Evelyn technique 6 using a total volume of 0.05 ml of rat serum. Blood was extracted from the tail veins o f animals lightly anesthetized with ether at 0, 10, 20, 45, and 60 minutes, respectively, after single injections of N a benzoate or 0.9% NaC1 and at 0,15, 30, 45, and 60 minutes, respectively, following sulfisoxazole administration. In multiple injection experiments, N a benzoate or 0.9% NaC1 was given at 3 hourly intervals and serum b.ilirubin concentrations were measured at 0, 10, 20, 60, 70, 80, 120, 130, 140, and 180 minutes, respectively. A similar experiment was conducted with t7 suckling homozygous G u n n rats at 14 days of age, weighing from 19 to 29 gm. Single doses of 7 and 35 mg/kg of Na
Vol. 86, No. 5, pp. 799-803
800
Nathenson, Cohen, andMcNamara
The Journal of Pediatrics May1975
BASELINE
"'
BILIRUBIN
CONCENTRATION 117l
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Fig. 1. Single injection series, grown Gunn rats, using Na benzoate, 7 mg/kg, IM H , with a baseline mean bitirubin concentration of 5.0 mg/dl. Na benzoate, 7 mg/kg IP + - - + , with a baseline mean bilirubin concentration of 5.3 mg/dl; 0.9% NaCI, IP (3 (3, with a baseline mean bilirubin concentration of 4.9 mg/dl; sulfisoxazole, 200 mg/kg, SCO . . . . e , with a baseline mean bilirubin concentration of 7.6 mg/dl. The numbers in parentheses represent numbers of animals studied. i,i 7~
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BILIRUBIN CONCENTRATION ~,o (61
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0
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TIME FOLLOWING INJECTION (MIN) Fig. 2. Single injection series, grown Gunn rats, using Na benzoate, 100 mg/kg, IPQ----O,with a baseline mean bilirubin concentration of 6.5 mg/dl; Na benzoate, 200 mg/kg, IP~) ~ , with a baseline mean bilirubin concentration of 7.2 mg/dl; 0.9% NaCI, IP9 ....O, with a baseline mean bilirubin concentration of 5.5 mg/dl; sulfisoxazole, 200 mg/kg, SCO. . . . O. The numbers in parentheses represent numbers of animals studied. benzoate or 0.9% NaCI were administered intraperitoneally. Bilirubin concentrations in sera were assessed, by the method described, at 0, 15, and 45 minutes after injection. Blood was extracted by direct cardiac puncture in animals lightly a n e s t h e t i z e d with ether. A single suckling rat was used for each determination of serum bilirubin. Mean bilirubin concentrations in the sera of G u n n rats determined at the start of each e x p e r i m e n t and
prior to injection were considered baseline values with which subsequent mean bilirubin concentrations following injection were compared. The differences were expressed as the percent fall from the baseline.
RESULTS Single injection experiments: grown rats. Six control animals were injected with 0.9% NaC1 intraperitoneally in volumes comparable to those given the study rats; 17
Volume 86 Number 5
Effect o f N a benzoate on bilirubin o f Gunn rat
u_J 7 ..._J LJJ
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BASELINE BILIRUBIN CONCENTRATION
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Fig. 3. Multiple injection series, grown Gunn rats, using Na benzoate, 7 mg/kg, IM 9 I , with a baseline bUirubin concentration of 5.4 mg/dl; Na benzoate 7 mg/kg, I P + - - + , with a baseline mean bilirubin concentration of 4.5 mg/dl; Na benzoate, 35 mg/kg, IP ^ /x i with a baseline bilirubin concentration of 4.8 mg/dl; 0.9% NaC19 9 with a baseline mean bilirubin concentration of 4.7 mg/dl; sulfisoxazole, 200 mg/kg, SCO -- --g. The numbers in parentheses represent numbers of animals studied. were given 7 mg/kg of Na benzoate intraperitoneally; four animals received 7 mg/kg of Na benzoate intramuscularly. No significant differences in serum bilirubin concentrations were found among the three groups. F l u c t u a t i o n s from the b a s e l i n e a m o n g the control groups given 0.9% NaC1 were as great as minus 12% (Fig. 1). Sulfisoxazole at 200 mg/kg was administered subcutaneously to two animals. This c o m p o u n d was specifically chosen to demonstrate a significant fall in serum bilirubin concentration because of its ability to uncouple the bilirubin-albumincomplexes. 4,7 Thus, the two a n i m a l s served as " a b n o r m a l c o n t r o l s . " Each manifested a profound decrease in serum bilirubin concentrations after injection. The percents of fall for these two animals are shown in Figs. 1-3 for the purpose of comparison with Na benzoate and saline-treated animals. With the administration of 100 mg/kg of Na benzoate intraperitoneally to two animals, a moderate decrease of serum bilirubin concentrations occurred 45 minutes after injection; the m a x i m u m mean drop was 28% (Fig. 2). At the 200 mg/kg dose level, administered to two animals, a m a x i m u m mean decrease of 47% occurred at the 20 minute mark, approaching that observed in the sulfisoxazole-treated rats (Fig. 2). Though the n u m b e r s of animals studied at these high dosages are too few for statistical analysis, the striking differences observed in comparison with those of the saline-injected control group are in all likelihood significant.
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TIME FOLLOWING INJECTION (MINI Fig. 4. Single injection series, suckling Gunn rats, using Na benzoate, 7 mg/kg IP + +; Na benzoate, 35 mg/kg, IP~--t,; 0.9% NaCI9 9 The numbers in parentheses represent numbers of animals studied. Baseline bilirubin concentration, 9.6 mg/dl.
Multiple injection experiments: grown rats. Three injections were administered at hourly intervals. Two animals were given 0.9% NaC1, intraperitoneally; four animals Na benzoate, 7 mg/kg, intraperitoneally; one rat, Na benzoate, 7 mg/kg, intramuscularly; and two rats Na benzoate, 35 mg/kg, intraperitoneally. No significant differences in serum bilirubin concentrations
802
Nathenson, Cohen, andMcNamara
were observed when comparing the saline-injected control animals with those receiving 7 mg/kg of N a benzoate, either intraperitoneally or intramuscularly, over the three-hour period of observation. A t the 35 mg/kg dose there were no differences noted during the first hour; b u t moderate depressions of serum bilirubin concentrations following the second and third injections were seen (Fig. 3). S i n g l e injection experiment: suckling rats. T h r e e a n i m a l s were u s e d to e s t a b l i s h the m e a n b a s e l i n e bilirubin concentration. Two control rats were given 0.9% NaC1 at 15 minutes and two at 45 minutes. N a benzoate was administered in doses of 7 and 35 mg/kg to three animals at each dose level at the 15-minute interval and to two animals, respectively, at 45 minutes. The m a x i m u m fall in serum bilirubin concentration at any time was 13%, which is not significantly different from the m a x i m u m fluctuation found in the saline-injected control animals in the grown group of G u n n rats (Fig. 4). DISCUSSION The question of the potential hazard of injectable diazepam in the neonatal period, with regard to the N a benzoate contained in the preparation as a stabilizer, was first raised by Schiff and co-workers, 1 when studying the uncoupling effects of certain drugs on bilirubina l b u m i n b i n d i n g in vitro. I n j e c t a b l e d i a z e p a m has proved therapeutically effective in the human newborn infant for the management of seizure disorders, opiate w i t h d r a w a l , a n d tetanus. 8-1~T h e drug has also b e e n found effective as an adjunct to obstetrical analgesia. 11 B e c a u s e of the u s e f u l n e s s o f d i a z e p a m in p e r i n a t a l medicine N a benzoate was administered to the grown and suckling homozygous G u n n rat in order to evaluate its in vivo effect on serum bilirubin concentrations. Dosages of N a benzoate employed were comparable to those present in diazepam, when used for clinical purposes in the human neonate. A significant decrease in the serum bilirubin concentration of an animal was interpreted as dissociation of bilirubin from albumin complexes and diffusion of free bilirubin from the circulation into extravascular spaces. The administration of 7 mg/kg represents a comparable quantity of N a benzoate present in a 2 mg dose of injectable diazepam for a 3 kg infant,* the highest single dose r e c o m m e n d e d for the m a n a g e m e n t of neonatal narcotic withdrawal. 9 This quantity is close to the amount of benzoate per *Diazepamcontains 5%Na benzoate and benzoic acid(mostly as Na benzoate).A 2 mg dose of diazepamequals0.4 ml and contains 20 mg of Na benzoate.
The Journal of Pediatrics May 1975
dose of diazepam r e c o m m e n d e d for the treatment of neonatal tetanus 1~(11 mg/kg or less of N a benzoate). The dose of 7 mg/kg in the grown and suckling rats appears to have no significant disruptive effect on bilirubin-albumin complexes when administered as a single injection, and no effect in the grown rat after multiple injections as evidenced by failure to appreciate any significant decrease in serum bilirubin concentrations. Na benzoate at 35 mg/kg would be comparable to that amount contained in a 10 mg injection of diazepam, given to a 3 kg infant. This latter concentration falls into the upper range of a therapeutic dose sometimes given in a single i n t r a v e n o u s i n j e c t i o n for the c o n t r o l of protracted neonatal seizures. 8 Though the n u m b e r of animals studied at this dosage is small, the data appear to indicate that one injection may be safe in the grown and suckling rat, whereas repeated doses of this concentration at brief intervals could adversely affect the bilirubin-albumin complex. Single doses of 100 and 200 mg/kg clearly diminish serum concentrations of b i ! i r u b i n ; the 200 m g / k g dose p r o d u c e s a d e c r e a s e almost as profound as that observed with sulfisoxazole. But:100 or 200 mg/kg of Na benzoate would be far in excess of the amounts contained in therapeutic doses of d i a z e p a m r e c o m m e n d e d for the n e o n a t a l c o n d i t i o n s described. One might speculate that the rapid conversion in vivo of benzoate to hippurate, a metabolite which does not interfere with bilirubin-binding kinetics, 12 can explain the differences observed in binding ability, when comparing the in vivo model to in vitro systems. But, both the newborn h u m a n infant 13 and suckling rat 14 do not transform benzoate to hippurate as effectively as do older individuals. Therefore, suckling G u n n rats were subjected to concentrations of N a benzoate of 7 and 35 m g / k g in order to e x t e n d our o b s e r v a t i o n s to the younger animal as a subject more representative of the human neonate. Since no significant difference in bilirubin concentrations was observed in the suckling animals, as compared to those in the older G u n n rats, the differing effects of N a benzoate on bilirubin binding in vivo and in vitro remains unclear. Little direct information is available concerning the human newborn infant. Adoni and associates is have shown no adverse effect on bilirubin binding in newborn infants when diazepam, which crosses the placenta, is administered to mothers within minutes to several hours before delivery. In an earlier study we reported r e s e r v e a l b u m i n - b i n d i n g capacities in the sera of several infants treated with diazepam while undergoing narcotic withdrawal. Serum specimens were analyzed
Volume 86 Number 5
by the hydroxyazobenzene benzoic acid and sephadex G-25 techniques. 16 Though it may be difficult to draw precise parallels, the data obtained from these G u n n rat experiments would tend to support the hypothesis that injectable diazepam, containing Na benzoate, is a safe agent when used within the dosage range r e c o m m e n d e d in clinical neonatal medicine. Indeed, a greater margin of safety may in fact exist because of the lesser affinity of rat albumin for bilirubin than of human albumin. 5 The authors would like to thank Dr. Leo Stern for his helpful comments and suggestions. REFERENCES
1. Schiff D, Chan G, and Stern L: Fixed drug combinations and the displacement of bilirubin from albumin, Pediatrics 48:139, 1971. 2. Cohen SN, and Fern LM: The displacement of albuminbound bilirubin by benzoate: A hazard of the use of diazepam in newborn infants (abstr), Pediatr Res 6:404, 1972. 3. Odell GB: The dissociation of bilirubin from albumin and its clinical implications, J PEDIATR55:268, 1959. 4. Johnson L, Sarmiento F, Blanc WA, and Day R: Kernicterius in rats with an inherited deficiency in glucuronyl transferase, Am J Dis Child 97:591, 1959. 5. Schmid R, Diamond I, Hammaker L, and Gundersen CB: Interaction of bilirubin with albumin, Nature 206:1041, 1965.
Effect o f Na benzoate on bilirubin of Gunn rat
803
6. Malloy HT, and Evelyn KA: Determination of bilirubin with photoelectric colorimeter, J Biol Chem 119:48, 1937. 7. Silverman WA, Andersen DH, Blanc WA, and Crozier DN: A difference in mortality rate and incidence of kerniterius among premature infants allotted to two prophylactic antibacterial regimeos, Pediatrics 18:614, 1956. 8. Smith BT, and Masotti RE: Intravenous diazepam in the treatment of prolonged seizure activity in neonates and infants, Dev. Med Child Neurol 13:630, 1971. 9. Nathenson G, Golden GS, and Litt IF: Diazepam in the management of the neonatal narcotic withdrawal syndrome, Pediatrics 48:523, 1971. 10. Hendrickse RG, and Sherman PM: Therapeutic trial of diazepam in tetanus, Lancet 1:737, 1965. 11. Flowers ER, Rudolph AJ, and Desm0nd MM: Diazepam (Valium) as an adjunct to obstetric analgesia, Obstet Gynecol 34:68, 1969. 12. Stern L: Personal communication. 13. Vest MF, and Salzberg R: Conjugation reactions in the newborn infant: the metabolism of paraaminobenzoic acid, Arch Dis Child 40:97, 1964. 14. Brandt IK: The development of the hippuric acid--syn~ thesizing system in the rat, Dev Biol 10:202, 1964. 15. Adoni A, Kapitulnick J, Kaufman NA, Ron M, and Blondheim SH: Effect of maternal administration of diazepam on the bilirubin-binding capacity of cord serum, Am J Obstet Gynecol 115:577, 1973. 16. Nathenson G, Cohen MI, Litt IF, and McNamara H: The effect of maternal heroin addiction on neonatal jaundice, J PEDIATR81:899, 1972.
799
The effect of Na benzoate on serum bilirubin of the Gunn rat The administration of Na benzoate to grown and suckling Gunn rats in single doses of 7 and 35 mg/kg failed to significantly alter serum bilirubin concentrations. These doses are comparable to quantities of Na benzoate contained in injectable diazepam used therapeutically for newborn infants. Repeated doses of 7 mg/kg in the grown rat showed no effect, as well. A single dose of 100 or 200 mg/kg of Na benzoate and repeated doses of 35 mg/kg resulted in depressed serum bilirubin concentrations. The higher concentrations o f Na benzoate, however, greatly exceed amounts contained in doses of diazepam recommended for clinical use in the human neonate. The data suggest that the use of injectable diazepam, in appropriate quantities, poses no hazard to the newborn infant in terms o f bilirubin toxicity. The greater affinity for bilirubin o f human albumin, than that of rat albumin, may further minimize the risk.
Gerald Nathenson, M.D.,* Michael I. Cohen, M.D., and Helen McNamara, M.A., Bronx, N.Y.
IN VITRO STUDIES demonstrate that Na benzoate is one of several organic anions which can cause the dissociation of bilirubin from albumin-binding sites. 13 Of considerable concern in clinical situations is the fact that N a benzoate is contained in injectable diazepam, a drug used in the m a n a g e m e n t of various neonatal disorders. In order to further clarify the problem o f the safety of N a benzoate, the G u n n rat, which has been used as an animal model for the study of in vivo effects of drugs on the binding of indirect-reacting bilirubin to albumin,a, 5 was selected for study. The investigation described details the effects of injected Na benzoate, at varying dose levels, on serum bilirubin concentrations in the G u n n rat.
MATERIALS AND METHODS Forty-two grown homozygous G u n n rats, 16 males and 26 females, were used for serial assessments of serum bilirubin concentrations following injections of From the Divisions of Neonatology and Adolescent Medicine, Department o f Pediatrics, Montefiore Hospital and Medical Center, Morrisania City Hospital and the Albert Einstein College of Medicine. *Reprint address:Morrisania CityHospital, 168th St. & Gerard Ave.. Bronx, N. Y. 10452.
N a b e n z o a t e , 0.9% NaCI or sulfisoxazole d i o l a m i n e . Dosages of Na benzoate were 7, 35,100, and 200 mg/kg, respectively, of rat body weight, given intramuscularly or intraperitoneally; and of sulfisoxazole, 200 mg/kg administered subcutaneously. Distilled water was used as the diluent for the benzoate and sulfisoxazole. Injection volumes varied from 0.3 to 0.5 ml. Comparable injection v o l u m e s o f saline were a d m i n i s t e r e d to t h e c o n t r o l animals. The G u n n rats ranged in weight from 86 to 276 gm ( m e d i a n weight: 164 gm) and varied in age from 2 months to 5 months. Bilirubin determinations were perf o r m e d in duplicate b y a m i c r o m o d i f i c a t i o n o f the Malloy-Evelyn technique 6 using a total volume of 0.05 ml of rat serum. Blood was extracted from the tail veins o f animals lightly anesthetized with ether at 0, 10, 20, 45, and 60 minutes, respectively, after single injections of N a benzoate or 0.9% NaC1 and at 0,15, 30, 45, and 60 minutes, respectively, following sulfisoxazole administration. In multiple injection experiments, N a benzoate or 0.9% NaC1 was given at 3 hourly intervals and serum b.ilirubin concentrations were measured at 0, 10, 20, 60, 70, 80, 120, 130, 140, and 180 minutes, respectively. A similar experiment was conducted with t7 suckling homozygous G u n n rats at 14 days of age, weighing from 19 to 29 gm. Single doses of 7 and 35 mg/kg of Na
Vol. 86, No. 5, pp. 799-803
800
Nathenson, Cohen, andMcNamara
The Journal of Pediatrics May1975
BASELINE
"'
BILIRUBIN
CONCENTRATION 117l
~-
20
---!
I.L
120
_1 _J
\
\o\ 40 \ \ 60
CO
(2)
~.
t,l
W Q_
0
lO' 30 2"0 ' ' 45 ' TIME FOLLOWING INJECTION (MIN)
60 '
Fig. 1. Single injection series, grown Gunn rats, using Na benzoate, 7 mg/kg, IM H , with a baseline mean bitirubin concentration of 5.0 mg/dl. Na benzoate, 7 mg/kg IP + - - + , with a baseline mean bilirubin concentration of 5.3 mg/dl; 0.9% NaCI, IP (3 (3, with a baseline mean bilirubin concentration of 4.9 mg/dl; sulfisoxazole, 200 mg/kg, SCO . . . . e , with a baseline mean bilirubin concentration of 7.6 mg/dl. The numbers in parentheses represent numbers of animals studied. i,i 7~
"J
BASELINE
BILIRUBIN CONCENTRATION ~,o (61
z_ '~ ~"' ~ 20 rn
_j
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~,:t,
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60
~ - ~ - e (21
0
I
I
I
I
I
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20
30
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TIME FOLLOWING INJECTION (MIN) Fig. 2. Single injection series, grown Gunn rats, using Na benzoate, 100 mg/kg, IPQ----O,with a baseline mean bilirubin concentration of 6.5 mg/dl; Na benzoate, 200 mg/kg, IP~) ~ , with a baseline mean bilirubin concentration of 7.2 mg/dl; 0.9% NaCI, IP9 ....O, with a baseline mean bilirubin concentration of 5.5 mg/dl; sulfisoxazole, 200 mg/kg, SCO. . . . O. The numbers in parentheses represent numbers of animals studied. benzoate or 0.9% NaCI were administered intraperitoneally. Bilirubin concentrations in sera were assessed, by the method described, at 0, 15, and 45 minutes after injection. Blood was extracted by direct cardiac puncture in animals lightly a n e s t h e t i z e d with ether. A single suckling rat was used for each determination of serum bilirubin. Mean bilirubin concentrations in the sera of G u n n rats determined at the start of each e x p e r i m e n t and
prior to injection were considered baseline values with which subsequent mean bilirubin concentrations following injection were compared. The differences were expressed as the percent fall from the baseline.
RESULTS Single injection experiments: grown rats. Six control animals were injected with 0.9% NaC1 intraperitoneally in volumes comparable to those given the study rats; 17
Volume 86 Number 5
Effect o f N a benzoate on bilirubin o f Gunn rat
u_J 7 ..._J LJJ
801
BASELINE BILIRUBIN CONCENTRATION
0
< z
20
\ 40
\
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Q..
~ o 121
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20
40
6
TIME
inj. inj. FOLLOWING INJECTIONS (MIN)
inj.
8
lO0
120
140
160
Fig. 3. Multiple injection series, grown Gunn rats, using Na benzoate, 7 mg/kg, IM 9 I , with a baseline bUirubin concentration of 5.4 mg/dl; Na benzoate 7 mg/kg, I P + - - + , with a baseline mean bilirubin concentration of 4.5 mg/dl; Na benzoate, 35 mg/kg, IP ^ /x i with a baseline bilirubin concentration of 4.8 mg/dl; 0.9% NaC19 9 with a baseline mean bilirubin concentration of 4.7 mg/dl; sulfisoxazole, 200 mg/kg, SCO -- --g. The numbers in parentheses represent numbers of animals studied. were given 7 mg/kg of Na benzoate intraperitoneally; four animals received 7 mg/kg of Na benzoate intramuscularly. No significant differences in serum bilirubin concentrations were found among the three groups. F l u c t u a t i o n s from the b a s e l i n e a m o n g the control groups given 0.9% NaC1 were as great as minus 12% (Fig. 1). Sulfisoxazole at 200 mg/kg was administered subcutaneously to two animals. This c o m p o u n d was specifically chosen to demonstrate a significant fall in serum bilirubin concentration because of its ability to uncouple the bilirubin-albumincomplexes. 4,7 Thus, the two a n i m a l s served as " a b n o r m a l c o n t r o l s . " Each manifested a profound decrease in serum bilirubin concentrations after injection. The percents of fall for these two animals are shown in Figs. 1-3 for the purpose of comparison with Na benzoate and saline-treated animals. With the administration of 100 mg/kg of Na benzoate intraperitoneally to two animals, a moderate decrease of serum bilirubin concentrations occurred 45 minutes after injection; the m a x i m u m mean drop was 28% (Fig. 2). At the 200 mg/kg dose level, administered to two animals, a m a x i m u m mean decrease of 47% occurred at the 20 minute mark, approaching that observed in the sulfisoxazole-treated rats (Fig. 2). Though the n u m b e r s of animals studied at these high dosages are too few for statistical analysis, the striking differences observed in comparison with those of the saline-injected control group are in all likelihood significant.
BASELINE BILIRUBIN CONCENTRATION 0 uJ ~ _J ILl :7"
+
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(2)
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~: 6o U.J l.u
J
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TIME FOLLOWING INJECTION (MINI Fig. 4. Single injection series, suckling Gunn rats, using Na benzoate, 7 mg/kg IP + +; Na benzoate, 35 mg/kg, IP~--t,; 0.9% NaCI9 9 The numbers in parentheses represent numbers of animals studied. Baseline bilirubin concentration, 9.6 mg/dl.
Multiple injection experiments: grown rats. Three injections were administered at hourly intervals. Two animals were given 0.9% NaC1, intraperitoneally; four animals Na benzoate, 7 mg/kg, intraperitoneally; one rat, Na benzoate, 7 mg/kg, intramuscularly; and two rats Na benzoate, 35 mg/kg, intraperitoneally. No significant differences in serum bilirubin concentrations
802
Nathenson, Cohen, andMcNamara
were observed when comparing the saline-injected control animals with those receiving 7 mg/kg of N a benzoate, either intraperitoneally or intramuscularly, over the three-hour period of observation. A t the 35 mg/kg dose there were no differences noted during the first hour; b u t moderate depressions of serum bilirubin concentrations following the second and third injections were seen (Fig. 3). S i n g l e injection experiment: suckling rats. T h r e e a n i m a l s were u s e d to e s t a b l i s h the m e a n b a s e l i n e bilirubin concentration. Two control rats were given 0.9% NaC1 at 15 minutes and two at 45 minutes. N a benzoate was administered in doses of 7 and 35 mg/kg to three animals at each dose level at the 15-minute interval and to two animals, respectively, at 45 minutes. The m a x i m u m fall in serum bilirubin concentration at any time was 13%, which is not significantly different from the m a x i m u m fluctuation found in the saline-injected control animals in the grown group of G u n n rats (Fig. 4). DISCUSSION The question of the potential hazard of injectable diazepam in the neonatal period, with regard to the N a benzoate contained in the preparation as a stabilizer, was first raised by Schiff and co-workers, 1 when studying the uncoupling effects of certain drugs on bilirubina l b u m i n b i n d i n g in vitro. I n j e c t a b l e d i a z e p a m has proved therapeutically effective in the human newborn infant for the management of seizure disorders, opiate w i t h d r a w a l , a n d tetanus. 8-1~T h e drug has also b e e n found effective as an adjunct to obstetrical analgesia. 11 B e c a u s e of the u s e f u l n e s s o f d i a z e p a m in p e r i n a t a l medicine N a benzoate was administered to the grown and suckling homozygous G u n n rat in order to evaluate its in vivo effect on serum bilirubin concentrations. Dosages of N a benzoate employed were comparable to those present in diazepam, when used for clinical purposes in the human neonate. A significant decrease in the serum bilirubin concentration of an animal was interpreted as dissociation of bilirubin from albumin complexes and diffusion of free bilirubin from the circulation into extravascular spaces. The administration of 7 mg/kg represents a comparable quantity of N a benzoate present in a 2 mg dose of injectable diazepam for a 3 kg infant,* the highest single dose r e c o m m e n d e d for the m a n a g e m e n t of neonatal narcotic withdrawal. 9 This quantity is close to the amount of benzoate per *Diazepamcontains 5%Na benzoate and benzoic acid(mostly as Na benzoate).A 2 mg dose of diazepamequals0.4 ml and contains 20 mg of Na benzoate.
The Journal of Pediatrics May 1975
dose of diazepam r e c o m m e n d e d for the treatment of neonatal tetanus 1~(11 mg/kg or less of N a benzoate). The dose of 7 mg/kg in the grown and suckling rats appears to have no significant disruptive effect on bilirubin-albumin complexes when administered as a single injection, and no effect in the grown rat after multiple injections as evidenced by failure to appreciate any significant decrease in serum bilirubin concentrations. Na benzoate at 35 mg/kg would be comparable to that amount contained in a 10 mg injection of diazepam, given to a 3 kg infant. This latter concentration falls into the upper range of a therapeutic dose sometimes given in a single i n t r a v e n o u s i n j e c t i o n for the c o n t r o l of protracted neonatal seizures. 8 Though the n u m b e r of animals studied at this dosage is small, the data appear to indicate that one injection may be safe in the grown and suckling rat, whereas repeated doses of this concentration at brief intervals could adversely affect the bilirubin-albumin complex. Single doses of 100 and 200 mg/kg clearly diminish serum concentrations of b i ! i r u b i n ; the 200 m g / k g dose p r o d u c e s a d e c r e a s e almost as profound as that observed with sulfisoxazole. But:100 or 200 mg/kg of Na benzoate would be far in excess of the amounts contained in therapeutic doses of d i a z e p a m r e c o m m e n d e d for the n e o n a t a l c o n d i t i o n s described. One might speculate that the rapid conversion in vivo of benzoate to hippurate, a metabolite which does not interfere with bilirubin-binding kinetics, 12 can explain the differences observed in binding ability, when comparing the in vivo model to in vitro systems. But, both the newborn h u m a n infant 13 and suckling rat 14 do not transform benzoate to hippurate as effectively as do older individuals. Therefore, suckling G u n n rats were subjected to concentrations of N a benzoate of 7 and 35 m g / k g in order to e x t e n d our o b s e r v a t i o n s to the younger animal as a subject more representative of the human neonate. Since no significant difference in bilirubin concentrations was observed in the suckling animals, as compared to those in the older G u n n rats, the differing effects of N a benzoate on bilirubin binding in vivo and in vitro remains unclear. Little direct information is available concerning the human newborn infant. Adoni and associates is have shown no adverse effect on bilirubin binding in newborn infants when diazepam, which crosses the placenta, is administered to mothers within minutes to several hours before delivery. In an earlier study we reported r e s e r v e a l b u m i n - b i n d i n g capacities in the sera of several infants treated with diazepam while undergoing narcotic withdrawal. Serum specimens were analyzed
Volume 86 Number 5
by the hydroxyazobenzene benzoic acid and sephadex G-25 techniques. 16 Though it may be difficult to draw precise parallels, the data obtained from these G u n n rat experiments would tend to support the hypothesis that injectable diazepam, containing Na benzoate, is a safe agent when used within the dosage range r e c o m m e n d e d in clinical neonatal medicine. Indeed, a greater margin of safety may in fact exist because of the lesser affinity of rat albumin for bilirubin than of human albumin. 5 The authors would like to thank Dr. Leo Stern for his helpful comments and suggestions. REFERENCES
1. Schiff D, Chan G, and Stern L: Fixed drug combinations and the displacement of bilirubin from albumin, Pediatrics 48:139, 1971. 2. Cohen SN, and Fern LM: The displacement of albuminbound bilirubin by benzoate: A hazard of the use of diazepam in newborn infants (abstr), Pediatr Res 6:404, 1972. 3. Odell GB: The dissociation of bilirubin from albumin and its clinical implications, J PEDIATR55:268, 1959. 4. Johnson L, Sarmiento F, Blanc WA, and Day R: Kernicterius in rats with an inherited deficiency in glucuronyl transferase, Am J Dis Child 97:591, 1959. 5. Schmid R, Diamond I, Hammaker L, and Gundersen CB: Interaction of bilirubin with albumin, Nature 206:1041, 1965.
Effect o f Na benzoate on bilirubin of Gunn rat
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6. Malloy HT, and Evelyn KA: Determination of bilirubin with photoelectric colorimeter, J Biol Chem 119:48, 1937. 7. Silverman WA, Andersen DH, Blanc WA, and Crozier DN: A difference in mortality rate and incidence of kerniterius among premature infants allotted to two prophylactic antibacterial regimeos, Pediatrics 18:614, 1956. 8. Smith BT, and Masotti RE: Intravenous diazepam in the treatment of prolonged seizure activity in neonates and infants, Dev. Med Child Neurol 13:630, 1971. 9. Nathenson G, Golden GS, and Litt IF: Diazepam in the management of the neonatal narcotic withdrawal syndrome, Pediatrics 48:523, 1971. 10. Hendrickse RG, and Sherman PM: Therapeutic trial of diazepam in tetanus, Lancet 1:737, 1965. 11. Flowers ER, Rudolph AJ, and Desm0nd MM: Diazepam (Valium) as an adjunct to obstetric analgesia, Obstet Gynecol 34:68, 1969. 12. Stern L: Personal communication. 13. Vest MF, and Salzberg R: Conjugation reactions in the newborn infant: the metabolism of paraaminobenzoic acid, Arch Dis Child 40:97, 1964. 14. Brandt IK: The development of the hippuric acid--syn~ thesizing system in the rat, Dev Biol 10:202, 1964. 15. Adoni A, Kapitulnick J, Kaufman NA, Ron M, and Blondheim SH: Effect of maternal administration of diazepam on the bilirubin-binding capacity of cord serum, Am J Obstet Gynecol 115:577, 1973. 16. Nathenson G, Cohen MI, Litt IF, and McNamara H: The effect of maternal heroin addiction on neonatal jaundice, J PEDIATR81:899, 1972.