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Effects of gentamicin on albumin binding of bilirubin
PEDIATRIC
PHARMACOLOGY
THERAPEUTICS
AND
Paul S. Lietman, Editor
Effects of gentamicin on albumin binding of bilirubin The effects of gentamicin on albumin binding of bilirubin and bitirubin transport were studied using Sephadex gel filtration, spectrophotometric analysis, peroxidase assay, and red blood cell uptake, No significant interaction between gentamicin and bilirub~n binding or transport was observed.
Richard P. Wennberg, M.D.,* and L. Fraser Rasmussen, M.S., Seattle, Wash.
G E NT A M I C I N is an important aminoglycoside used in a variety of clinical infections, which include those of the newborn infant due to gram-negative organisms. It has become the drug of choice in treating suspected sepsis in sick neonates in many nurseries in which a high prevalence of kanamycin-resistant organisms has been recognized. Recently, Cukier and associates I reported that gentamicin is a potent displacer of bilirubin from albumin. If true, the potential for kernicterus would weigh heavily in the decision to treat a sick premature infant with this aminoglycoside. The importance of gentamicin in neonatal intensive care together with reports indicating that gentamicin is weakly, if at all, bound to serum proteins 2, 3 prompted us to re-examine the interaction of gentamicin with bilirubinalbumin binding and its effect on the distribution of bilirubin between albumin and cells. MATERIALS
AND METHODS
Purified gentamicin and purified samples of the gentamicin components, C1, Cla , and C2, were supplied by Schering Corp., Bloomfield, N. J. Bilirubin, obtained from Sigma Corp., St. Louis, Mo., had a millimolar extinction co-efficient (1 cm pathlength) of 60.1 in chloroform, contained approximately 10% isomers
From the Department of Pediatries, University of Washington School of Medicine. Supported by United States Public Health Service grant HD-04665 and by United Cerebral Palsy Grant R-265. *Reprint address: Department of Pediatrics, Universityof California, Davis, Calif. 95616.
(thin layer c h r o m a t o g r a p h y ) , and was used without further purification. Model serum was made by dissolving purified human serum albumin (fraction V, Sigma) in phosphate buffer, pH 7.4 or 7.0, ionic strength 0.15. Bilirubin-albumin solutions were prepared by dissolving bilirubin in dilute NaOH (final pH 9.0-9.5) and adding immediately to albumin or plasma. All experiments were performed in subdued light. Bilirubin concentrations in the presence of albumin were determined by direct spectrophotometry assuming a millimolar absorbance of 48.5 at 460 nm. Unbound bilirubin was estimated by difference spectroscopy using a Cary model 16 recording spectrophotometer, and by the peroxidase assay of Jacobsen and Wennberg. 4 Sephadex gel and erythrocytes bind bilirubin in competition with albumin. The influence of gentamicin on a l t e r n a t i v e s u b s t r a t e b i n d i n g was e v a l u a t e d by Sephadex G-25 gel filtration and by the uptake of bilirubin by red blood cells. Sephadex filtration s was performed in 5 cm columns, equilibrated with phosphate buffer, pH 7.4. Fifty microliters of jaundiced model serum (molar ratio 1.2) were applied, and the albumin was completely recovered in a 2.0 ml eluate. Bilirubin absorbed to the gel was removed by a second elution using 50 txl of albumin (3 gm/dl). Red cell uptake of bilirubin was evaluated by the method of Bratlid 6, 7 using fresh heparinized adult blood obtained within two hours of the experiment. Bilirubin, dissolved in dilute NaOH, was added to an equal volume of model serum or plasma after separation from the erythrocytes. One milliliter red blood cells susVol. 86, No. 4, pp. 611-613
612
Wennberg andRasmussen
The Journal of Pediatrics April 1975
Table I. Sephadex gel filtration
Table II. Effects of gentamicin on the unbound bilirubin concentration (peroxidase assay) Bilirubin concentration (mg/dl +_SE)
Unbound bilirubin
[
(nlmol/l) at various bilirubin/ albumin molar ratiost
Initial* Adsorbed (N=3) No gentamicin
Gentamicin powder 10/zg/ml 100/zg/ml Gentamicin injectable (with additives) 10 tzg/ml 100 txg/ml
15.8
2.81 + 0.24
15.8 15.8
2.47 + 0.17 2.57 • 0.17
15.8 15.8
2.83 • 0.10 2.63 • 0A2
*Bilirubin/albumin molar ratio = 1.2,
pended in buffer (pH 7.4, hematocrit 50%) were incubated in a shaking water bath for 15 minutes at 37~ with 3.0 ml buffer containing glucose and gentamiein and 2 ml icteric plasma. The erythrocytes were then w a s h e d three times with buffer, and bilirubin was extracted by a second incubation with 2 ml nonicteric model s e r u m (3 gm/dl fraction V).
RESULTS We observed no difference in the absorption spectrum of bilirubin bound to albumin using molar ratios bilirubin/albumin of 0.75 or 1.6 with either gentamicin or any c o m p o n e n t s . U s i n g difference s p e c t r o s c o p y , Which greatly magnifies very subtle changes in the abSorption spectrum, a very slight decrease in absorbance at 485 n m was observed at gentamicin concentrations in excess of 500/zg/mt. The difference spectrum of unb o u n d bilirubin, however, has a concomitant increase in absorbance at 410 nm, and this was not observed. Spectral changes induced by gentamicin were qualitatively and quantitatively similar at pH 7.0 and 7.4. Sephadex gel filtration can be used to evaluate weakly bound bilirubin which can be competitively adsorbed to the gel. The results of Sephadex filtration are summarized in Table I. Variations in results were negligible. The per0xidase assay can measure very small concentrations of u n b o u n d bilirubin and therefore may be used to analyze both primary and secondary site binding. W e found no evidence of displacement of bilirubin by gentamicin even at very high concentrations of the drug and with high bilirubin/albumin molar ratios (Table II). The uptake of bilirubin from icteric plasma or jaundiced model serum at low molar ratios was not affected b y g e n t a m i c i n (Table III). A slight p o t e n t a t i o n of bilirubin uptake by erythrocytes was suggested at the high bilirubin/albumin molar ratio.
Drug~concentration (ixmol/l)* Gentamicin
0.5
0.75
]
1.70
4.2 3.3 2.4 3,4
15.3 14.1 16.0 17.1
455 418 530 374
4,2 3.4 3.5 3,3
15.3 15.1 19.2 18.2
455 439 426 363
3.7 3.5 3.3 3.6
15.7 16.4 16.2 16.0
563 571 616 548
3.7 4.1 4.0 3.9
15.7 15.5 16.2 --
563 563 548 518
C
0 16 100 500 Gentamicin C~ 0 16 100 500 Gentamicin Cla 0 16 100 500 Gentamicin C2 0 16 100 500
"15 p.molB gentamicin ~ 5 tzgm/ml. ? A l b u m i n concentration = 10/zmol/1, u n b o u n d bilirubin represents the mean of three determinations.
DISCUSSION Our studies indicate minimal, if any, interaction between gentamicin and albumin binding of bilirubin. Although we failed to demonstrate any direct effect of the drug on albumin binding, gentamicin appeared to produce a slight increase in bilirubin uptake by red b l o o d cells at a high b i l i r u b i n / a l b u m i n m o l a r ratio. Under the conditions studied, the u n b o u n d bilirubin concentration was about ten times higher than that observed under clinical situations. 4The increased erythrocyte uptake was far less than Bratlid 6 observed under similar experimental conditions using cloxacillin, tetracycline, novobiocin, sulfisoxazole, or carbenicillin. It is difficult to reconcile our results with reports that g e n t a m i c i n d i s p l a c e s bilirUbin from a l b u m i n . Stern 8 stated that gentamicin increases bilirubin adsorption to Sephadex G-25 columns, but published no supporting data. Kapitulnik and associates 9 reported an infant who had an apparent decrease in binding capacity as measured by Sephadex gel filtration following gentamicin t r e a t m e n t . W e o b s e r v e d n o i n c r e a s e in a d s o r b a b l e bilirubin even with extremely high drug concentrations.
Volume 86 Number 4
Effects o f gentam&in on album& binding o f bilirubin
613
Table III. Effect of gentamicin on red blood cell uptake of bilirubin
Protein source Plasma
Bilirubin/albumin molar ratio 0.8
1.6
Albumin solution (fraction V)
1.6
Gentamicin concentration (l~g/ml)
Bilirubin remaining in supernatant (mg/dl) *
RBC uptake bilirubin (ixmol/l)t
0--control 5 500 O-- control 5 500 O--control 5 500
6.1 6.1 6.1 10.8 9.7 9.8 10.9 10.7 10.2
3.11 3.01 3.01 20.0 20.6 21.8 41.9 44.0 43.7
lncrease Uptake (%) --3.0 -3.0 -3.0 9.0 -5.0 4.3
*Albuminconcentration870 mg/dl. tMean concentrationin duplicateextractions(2 ml). Recently, Odell l~and Cukier and associates lreported a more extensive study demonstrating that gentamicin produces a marked shift in the adsorption spectrum of bilirubin-albumin complexes, increases mitochondria uptake of bilirubin, produces a transient decrease in serum bilirubin concentration in G u n n rats, and decreases Plasma bilirubin concentrations in heparinized pre-exchange blood samples. We were unable to demonstrate any significant effect of gentamicin on the absorption spectrum even when using concentrations 100 times higher than those employed by these investigators. In addition, we found only a minimal increase in erythrocyte uptake of bilirubin in the presence of the drug. It is unlikely that the differences in results are due to variations in experimental conditions. Perhaps preparative variation or chemical transformation of the aminogtycoside upon storage is responsible. Our data are consistent with studies demonstrating that gentamicin binds weakly, if at all, to serum proteins. Although it is possible that interaction of the basic drug with cell m e m b r a n e s or b i l i r u b i n could slightly potentiate the uptake of u n b o u n d pigment by tissue, it is unlikely that gentamicin would significantly alter the risk for bilirubin encephalopathy in a sick jaundiced infant.
REFERENCES
1. Cukier JO, Seungdamrong S, Odell JL, and Odell GB. The displacement of albumin-bound bilirubin by gentamicin, Pediatr Res 8:399, 1974. 2. Rift LJ, and Jackson GG: Pharmacology of gentamicin in man, J Infect Dis 124:(Suppl 98), 1971. 3. Gordon RC, Regamey C, and Kirby WMM: Serum protein binding of the aminoglycoside antibiotics, Antimicrob Agents Chemother 2:214, 1972. 4. Jacobsen J, and Wennberg RP: Determination of unbound bilirubin in the serum of newborns, Clin Chem 20:783, 1974. 5. ShiffD, Chan G, and Stern L: Sephadex G-25 quantitative estimation of free bilirubin potential in jaundiced newborn infants' sera: A guide to the prevention of kernicterus, J Lab Clin Med 80:455, 1972. 6. Bratlid D: The effect of antimicrobial agents on bilirubin binding by human erythrocytes, Scand J Clin Lab Invest 30:331, 1972. 7. Bratlid D: Bilirubin binding by human erythrocytes, Scand J Clin Lab Invest 29:91, 1972. 8. Stern L: Drug interactions: II. Drugs, the newborn infant, and the binding of bilirubin to albumin, Pediatrics 49:916, 1972. 9. Kapitulnik J~ Eyal F, and Simcha AJ: Gentamicin and bilirubin-bindingby plasma, Lancet 2:1195, 1972. 10. Odell GB: Influence of binding on the toxicity of bilirubin, Ann NY Acad Sci 226:225, 1973.
PHARMACOLOGY
THERAPEUTICS
AND
Paul S. Lietman, Editor
Effects of gentamicin on albumin binding of bilirubin The effects of gentamicin on albumin binding of bilirubin and bitirubin transport were studied using Sephadex gel filtration, spectrophotometric analysis, peroxidase assay, and red blood cell uptake, No significant interaction between gentamicin and bilirub~n binding or transport was observed.
Richard P. Wennberg, M.D.,* and L. Fraser Rasmussen, M.S., Seattle, Wash.
G E NT A M I C I N is an important aminoglycoside used in a variety of clinical infections, which include those of the newborn infant due to gram-negative organisms. It has become the drug of choice in treating suspected sepsis in sick neonates in many nurseries in which a high prevalence of kanamycin-resistant organisms has been recognized. Recently, Cukier and associates I reported that gentamicin is a potent displacer of bilirubin from albumin. If true, the potential for kernicterus would weigh heavily in the decision to treat a sick premature infant with this aminoglycoside. The importance of gentamicin in neonatal intensive care together with reports indicating that gentamicin is weakly, if at all, bound to serum proteins 2, 3 prompted us to re-examine the interaction of gentamicin with bilirubinalbumin binding and its effect on the distribution of bilirubin between albumin and cells. MATERIALS
AND METHODS
Purified gentamicin and purified samples of the gentamicin components, C1, Cla , and C2, were supplied by Schering Corp., Bloomfield, N. J. Bilirubin, obtained from Sigma Corp., St. Louis, Mo., had a millimolar extinction co-efficient (1 cm pathlength) of 60.1 in chloroform, contained approximately 10% isomers
From the Department of Pediatries, University of Washington School of Medicine. Supported by United States Public Health Service grant HD-04665 and by United Cerebral Palsy Grant R-265. *Reprint address: Department of Pediatrics, Universityof California, Davis, Calif. 95616.
(thin layer c h r o m a t o g r a p h y ) , and was used without further purification. Model serum was made by dissolving purified human serum albumin (fraction V, Sigma) in phosphate buffer, pH 7.4 or 7.0, ionic strength 0.15. Bilirubin-albumin solutions were prepared by dissolving bilirubin in dilute NaOH (final pH 9.0-9.5) and adding immediately to albumin or plasma. All experiments were performed in subdued light. Bilirubin concentrations in the presence of albumin were determined by direct spectrophotometry assuming a millimolar absorbance of 48.5 at 460 nm. Unbound bilirubin was estimated by difference spectroscopy using a Cary model 16 recording spectrophotometer, and by the peroxidase assay of Jacobsen and Wennberg. 4 Sephadex gel and erythrocytes bind bilirubin in competition with albumin. The influence of gentamicin on a l t e r n a t i v e s u b s t r a t e b i n d i n g was e v a l u a t e d by Sephadex G-25 gel filtration and by the uptake of bilirubin by red blood cells. Sephadex filtration s was performed in 5 cm columns, equilibrated with phosphate buffer, pH 7.4. Fifty microliters of jaundiced model serum (molar ratio 1.2) were applied, and the albumin was completely recovered in a 2.0 ml eluate. Bilirubin absorbed to the gel was removed by a second elution using 50 txl of albumin (3 gm/dl). Red cell uptake of bilirubin was evaluated by the method of Bratlid 6, 7 using fresh heparinized adult blood obtained within two hours of the experiment. Bilirubin, dissolved in dilute NaOH, was added to an equal volume of model serum or plasma after separation from the erythrocytes. One milliliter red blood cells susVol. 86, No. 4, pp. 611-613
612
Wennberg andRasmussen
The Journal of Pediatrics April 1975
Table I. Sephadex gel filtration
Table II. Effects of gentamicin on the unbound bilirubin concentration (peroxidase assay) Bilirubin concentration (mg/dl +_SE)
Unbound bilirubin
[
(nlmol/l) at various bilirubin/ albumin molar ratiost
Initial* Adsorbed (N=3) No gentamicin
Gentamicin powder 10/zg/ml 100/zg/ml Gentamicin injectable (with additives) 10 tzg/ml 100 txg/ml
15.8
2.81 + 0.24
15.8 15.8
2.47 + 0.17 2.57 • 0.17
15.8 15.8
2.83 • 0.10 2.63 • 0A2
*Bilirubin/albumin molar ratio = 1.2,
pended in buffer (pH 7.4, hematocrit 50%) were incubated in a shaking water bath for 15 minutes at 37~ with 3.0 ml buffer containing glucose and gentamiein and 2 ml icteric plasma. The erythrocytes were then w a s h e d three times with buffer, and bilirubin was extracted by a second incubation with 2 ml nonicteric model s e r u m (3 gm/dl fraction V).
RESULTS We observed no difference in the absorption spectrum of bilirubin bound to albumin using molar ratios bilirubin/albumin of 0.75 or 1.6 with either gentamicin or any c o m p o n e n t s . U s i n g difference s p e c t r o s c o p y , Which greatly magnifies very subtle changes in the abSorption spectrum, a very slight decrease in absorbance at 485 n m was observed at gentamicin concentrations in excess of 500/zg/mt. The difference spectrum of unb o u n d bilirubin, however, has a concomitant increase in absorbance at 410 nm, and this was not observed. Spectral changes induced by gentamicin were qualitatively and quantitatively similar at pH 7.0 and 7.4. Sephadex gel filtration can be used to evaluate weakly bound bilirubin which can be competitively adsorbed to the gel. The results of Sephadex filtration are summarized in Table I. Variations in results were negligible. The per0xidase assay can measure very small concentrations of u n b o u n d bilirubin and therefore may be used to analyze both primary and secondary site binding. W e found no evidence of displacement of bilirubin by gentamicin even at very high concentrations of the drug and with high bilirubin/albumin molar ratios (Table II). The uptake of bilirubin from icteric plasma or jaundiced model serum at low molar ratios was not affected b y g e n t a m i c i n (Table III). A slight p o t e n t a t i o n of bilirubin uptake by erythrocytes was suggested at the high bilirubin/albumin molar ratio.
Drug~concentration (ixmol/l)* Gentamicin
0.5
0.75
]
1.70
4.2 3.3 2.4 3,4
15.3 14.1 16.0 17.1
455 418 530 374
4,2 3.4 3.5 3,3
15.3 15.1 19.2 18.2
455 439 426 363
3.7 3.5 3.3 3.6
15.7 16.4 16.2 16.0
563 571 616 548
3.7 4.1 4.0 3.9
15.7 15.5 16.2 --
563 563 548 518
C
0 16 100 500 Gentamicin C~ 0 16 100 500 Gentamicin Cla 0 16 100 500 Gentamicin C2 0 16 100 500
"15 p.molB gentamicin ~ 5 tzgm/ml. ? A l b u m i n concentration = 10/zmol/1, u n b o u n d bilirubin represents the mean of three determinations.
DISCUSSION Our studies indicate minimal, if any, interaction between gentamicin and albumin binding of bilirubin. Although we failed to demonstrate any direct effect of the drug on albumin binding, gentamicin appeared to produce a slight increase in bilirubin uptake by red b l o o d cells at a high b i l i r u b i n / a l b u m i n m o l a r ratio. Under the conditions studied, the u n b o u n d bilirubin concentration was about ten times higher than that observed under clinical situations. 4The increased erythrocyte uptake was far less than Bratlid 6 observed under similar experimental conditions using cloxacillin, tetracycline, novobiocin, sulfisoxazole, or carbenicillin. It is difficult to reconcile our results with reports that g e n t a m i c i n d i s p l a c e s bilirUbin from a l b u m i n . Stern 8 stated that gentamicin increases bilirubin adsorption to Sephadex G-25 columns, but published no supporting data. Kapitulnik and associates 9 reported an infant who had an apparent decrease in binding capacity as measured by Sephadex gel filtration following gentamicin t r e a t m e n t . W e o b s e r v e d n o i n c r e a s e in a d s o r b a b l e bilirubin even with extremely high drug concentrations.
Volume 86 Number 4
Effects o f gentam&in on album& binding o f bilirubin
613
Table III. Effect of gentamicin on red blood cell uptake of bilirubin
Protein source Plasma
Bilirubin/albumin molar ratio 0.8
1.6
Albumin solution (fraction V)
1.6
Gentamicin concentration (l~g/ml)
Bilirubin remaining in supernatant (mg/dl) *
RBC uptake bilirubin (ixmol/l)t
0--control 5 500 O-- control 5 500 O--control 5 500
6.1 6.1 6.1 10.8 9.7 9.8 10.9 10.7 10.2
3.11 3.01 3.01 20.0 20.6 21.8 41.9 44.0 43.7
lncrease Uptake (%) --3.0 -3.0 -3.0 9.0 -5.0 4.3
*Albuminconcentration870 mg/dl. tMean concentrationin duplicateextractions(2 ml). Recently, Odell l~and Cukier and associates lreported a more extensive study demonstrating that gentamicin produces a marked shift in the adsorption spectrum of bilirubin-albumin complexes, increases mitochondria uptake of bilirubin, produces a transient decrease in serum bilirubin concentration in G u n n rats, and decreases Plasma bilirubin concentrations in heparinized pre-exchange blood samples. We were unable to demonstrate any significant effect of gentamicin on the absorption spectrum even when using concentrations 100 times higher than those employed by these investigators. In addition, we found only a minimal increase in erythrocyte uptake of bilirubin in the presence of the drug. It is unlikely that the differences in results are due to variations in experimental conditions. Perhaps preparative variation or chemical transformation of the aminogtycoside upon storage is responsible. Our data are consistent with studies demonstrating that gentamicin binds weakly, if at all, to serum proteins. Although it is possible that interaction of the basic drug with cell m e m b r a n e s or b i l i r u b i n could slightly potentiate the uptake of u n b o u n d pigment by tissue, it is unlikely that gentamicin would significantly alter the risk for bilirubin encephalopathy in a sick jaundiced infant.
REFERENCES
1. Cukier JO, Seungdamrong S, Odell JL, and Odell GB. The displacement of albumin-bound bilirubin by gentamicin, Pediatr Res 8:399, 1974. 2. Rift LJ, and Jackson GG: Pharmacology of gentamicin in man, J Infect Dis 124:(Suppl 98), 1971. 3. Gordon RC, Regamey C, and Kirby WMM: Serum protein binding of the aminoglycoside antibiotics, Antimicrob Agents Chemother 2:214, 1972. 4. Jacobsen J, and Wennberg RP: Determination of unbound bilirubin in the serum of newborns, Clin Chem 20:783, 1974. 5. ShiffD, Chan G, and Stern L: Sephadex G-25 quantitative estimation of free bilirubin potential in jaundiced newborn infants' sera: A guide to the prevention of kernicterus, J Lab Clin Med 80:455, 1972. 6. Bratlid D: The effect of antimicrobial agents on bilirubin binding by human erythrocytes, Scand J Clin Lab Invest 30:331, 1972. 7. Bratlid D: Bilirubin binding by human erythrocytes, Scand J Clin Lab Invest 29:91, 1972. 8. Stern L: Drug interactions: II. Drugs, the newborn infant, and the binding of bilirubin to albumin, Pediatrics 49:916, 1972. 9. Kapitulnik J~ Eyal F, and Simcha AJ: Gentamicin and bilirubin-bindingby plasma, Lancet 2:1195, 1972. 10. Odell GB: Influence of binding on the toxicity of bilirubin, Ann NY Acad Sci 226:225, 1973.