Capillary electrophoresis for the determination of albumin binding capacity and free bilirubin in jaundiced neonates

Capillary Electrophoresis for the Determination of Albumin Binding Capacity and Free Bilirubin in Jaundiced Neonates Chap Y. Yeung, Ying S. Fung, and Dong X. Sun

This study develops a capillary electrophoresis/frontal analysis (CE/FA) method to separate free bilirubln from its albumin bound complex, with only small-sized samples. Under our optimized conditions, CE/FA is proven to be a simple and accurate method in assaying the concentration of free bUirubin in the jaundiced serum. Upon saturation of albumln-binding, the amount of bilirubin added to the serum bears a linear relationship with the absorbance at OD44OnM (R = .9987, P < .00001). Application of this method to study jaundiced neonates has yielded precise data on the residual binding capacity, which may be of significant therapeutic implications. The CE/FA method we have developed appears to be promising and applicable for clinical use as it is higldy reproducible and it only requires a small sample size for tests. Our method should be an invaluable adjunct to the neonatal management of hyperbilirubinaemia. Copyright 9 2001 by W.B. Saunders

Company

inding between albumin and bilirubin is

B clinically important, as u n b o u n d bilirubin

has b e e n shown to damage various tissues especially the brain resulting in kernicterus. 1-a Monitoring both the total bilirubin in the serum and the binding capacity for bilirubin are therefore important parameters in infants with hyperbilirubinemia. Many conditions, such as the presence o f drugs 1,4 and herbs, ~s have demonstrable effects in reducing bilirubin-protein binding; assessing the total serum bilirubin may not provide adequate information for clinical decisions on whether intervention treatment such as exchange transfusions is indicated to avoid brain damage. In the presence o f drugs, which compete for bilirubin binding sites, the level o f bilirubinemia can be deceptive; in fact, low levels of bilirubin have b e e n known to produce brain damage. ~ We adopted a new m e t h o d to assay the residual binding capacity o f serum samples with a novel capillary electrophoresis method. We r e p o r t here the results of our preliminary findings.

Materials and Methods Chemical Agents Bilirubin IX0t and h u m a n serum albumin were obtained from Sigma Chemicals (St Louis, MO). Sodium phosphate buffer was made up of 10 m m o l / L with added ethylenediamine tetraacidic acid (EDTA) 1 m m o l / L to form the running buffer solution before adjusted to p H 7.4. Sodium salicylate was obtained from May and Baker Ltd, England. Doubly Quartz distilled water was used for making up standard solutions and for dilution purposes. Bilirubin stock solution was p r e p a r e d by dissolving 2.5 m g in 200 ~1 of 0 . 1 m o l / L sodium hydroxide, diluting with 780 ~1 distilled water and adding 20 /.d of 0.1 mmol/1 EDTA. T h e stock solution was kept in aliquots in the refrigerator at 4~ and diluted to different concentrations immediately before use. Pooled cord blood serum samples were obtained from newborn babies at Q u e e n Mary Hospital ( H o n g Kong).

Equipment From the Departmentsof Paediatrics and Chemistry, The University of Hong Kong, Hong Kong, China. Address rqrrint requests to Chap E Yeung, FRCP, FRCP (C), FRACP, DCH, Departmentof Paediatrics, The University of Hong Kong, QueenMary Hospita~ Hong Kong. Copyright 9 2001 by W.B. Saunders Company 0146-0005/01/2502-0002535. 00/0 doi:l O.1053/sper.2001.23202

50

Capillary electrophoresis (CE) separation with frontal analysis (FA) to separate u n b o u n d bilirubin from the protein-bound bilirubin was perf o r m e d with the bio-focus CE 2000 capillary electrophoresis system (Bio-Rad Laboratories, Los Angeles, CA). Fused silica capillary columns o f

Seminars in Perinatology, Vol 25, No 2 (April), 2001: pp 50-54

Jaundiced Neonates

36 cm x 50/~m ID (Yong Nian Co; Hebei Province, People's Republic of China) were used for separation studies at 20 kV u n d e r positive polarity in the cartridge maintained at 37~ before spectral p h o t o m e t r y detection at 440 nm.

B-A

f

51

._,_ono

,~_["-~" Na2 HP04

Procedures Bilirubin binding capacity o f the serum or albumin solution was assayed by adding incremental amounts o f bilirubin to aliquots containing the same a m o u n t of serum or albumin solutions up to levels 2 to 3 times above the expected saturation points. Each aliquot now containing oversaturated bilirubin in serum or albumin solution was then transferred to the silica capillary colu m n for electrophoresis separation (CE/FA) studies ~ u n d e r standardized conditions as listed in Table 1. These oversaturated points o f unb o u n d bilirubin were fitted o n t o the best straight-line, which intersects at the Y-axis where zero absorbance or "no" u n b o u n d bilirubin would be identified. Such intersect represents the 'saturation capacity' of the sample studied. Figure 1 is the diagramatic representation o f the capillary electrophoresis procedure. T h e bilirubin-aibumin (BA) solution was i n t r o d u c e d with Na~HPO4 buffer at 20 kV and 15 psi.second into 1 e n d of the capillary tube. T h e electrophoretic pattern was read at UV 440 n m at the o t h e r end.

Albnmin Binding Capacity Study To each aliquot containing 50 /aL of the same concentration of 61panol/L albumin solution, bilirubin was added to make up the different concentrations as follows: 61 /~mol/L, 122/~mol/L, 153 /~mol/L, 171/~mol/L, 183/~mol/L, 195/~mol/L, 207/~mol/L, 220/~mol/L, 233/~mol/L, and 244 /~mol/L. Each aliquot containing the respective BA solution was introduced with phosphate buffer

Table 1. Optimal Condition for Capillary ElectroPhoresis in our Laboratory

Optimized Conditions

CE/FA Method

Injection (psi.sec) 15 Temperature (~ 37 Buffer pH 7.4 Buffer composition 15 mmol/L sodium phosphate, 1 mmol/L EDTA

Figure 1. The capillary electrophoresis method to determine the albumin binding capacity (or the residual binding capacity of serum) and free bilirubin.

to make up a 400-/zL volume. CE/FA procedures were then performed and the saturated molar ratio of BA complex was calculated by plotting the absorbance of u n b o u n d bilirubin against the total bilirubin concentrations.

Cord Blood Serum Binding Capacity for Bilirubin Pooled cord blood serum samples (100 /~L) were added to the vial and the following bilirubin amounts were a d d e d to each vial: 40/~g, 54 /~g, 68/~g, 80/~g, 100/~g, 108/~g, 120/~g, and 136 p,g. T h e solution was t h e n reconstituted to a volume o f 400 /~L by using phosphate buffer (pH 7.4), and kept at 4~ until CE/FA separation procedures as described earlier were performed. T h e saturation point o f the serum samples o f cord blood was extrapolated from the straight line best fitted onto all the identifiable u n b o u n d bilirubin levels obtained from the capillary electrophoresis. Sodium salicylate was used to test the efficacy o f o u r m e t h o d in identifying the "free bilirubin" generated from an oversaturated BA solution.

Optimal Laboratory Conditions We have optimized o u r experimental conditions to approximate clinical setting as closely as possible by adjusting the samples to the following conditions: cartridge temperature at 37~ phosphate buffer used all t h r o u g h to adjust to p H 7.4 with the r u n n i n g buffer composing of sodium phosphate (15 m m o l / L ) with a d d e d EDTA (1 m m o l / L ) to inhibit oxidation o f bilirubin (Table 1).

Yeung, Fung and Sun

52

Results

3500-

Figure 2 shows the oversaturated albumin solutions generating u n b o u n d bilirubin. T h e straight line j o i n i n g all the u n b o u n d bilirubin levels intersects the X-axis at 0 absorbance, which is the calculated or extrapolated bilirubin concentration that would saturate all the binding sites o f albumin. As n o t e d f r o m Figure 2, the saturated m o l a r ratio for b i l i r u b i n / a l b u m i n binding o b t a i n e d was a r o u n d 2, which is the expected hypothetical b i n d i n g capacity for albumin. x Figure 3 shows the C E / F A results obtained f r o m p o o l e d cord b l o o d serum. As can be noted, the hypothetical bilirubin saturation p o i n t for p o o l e d cord s e r u m was 26 m g / d L , which is the same as r e p o r t e d in the literature. 1,~ By adding sodium salicylate (aspirin) in incremental a o n c e n t r a d o n s f r o m 5, 10, 20, 30, 40 to 50 m g / d L into 100 ~L o f BA solution (containing 3 0 8 / 1 2 3 / z m o l / L respectively), the albumin saturation points were again reassessed. It can be seen f r o m Figure 4 a n d Table 2 that salicylate could significantly displace bilirubin f r o m the albumin binding in a dose-response m a n n e r .

3000-

Discussion

M a n a g e m e n t o f hyperbilirubinemia in the t e r m infants at o n e p o i n t a p p e a r e d to have b e e n settied. ls,14 Many authors believed that in the absence o f significant sickness a n d hemolytic conditions, t e r m infants should be able to tolerate significantly high levels o f bilirubinemia without r u n n i n g into the risk o f kemicterus, aa,14 More

0.0014 " 9 0.0010 O r-

~ 0.0008

0.0002 0.~ '120

0

i

2000.

."

0.9g~093.8481SE-6 7

G* 140

1;0

180

200

220

2~)

Bilirubin added in i,mol/I (to 61 pmol/I Albumin)

Figure 2. Determination of saturated molar ratio for bilirubin binding to albumin. Note. The extrapolated saturation point at 132 /~mol, which corresponds to BA molar ratio of 2. (BA = 132/~mol/61 /~mol).

Residual Binding Capacity

1500. 1000. 500t

t

9

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t

|

|

r

|

t

e

|

i

t

.10 2o ao 40 so , 0 70 ao ao 100.1.10.120.1a0.140

Bilirubin added (mg/100 mL)

Figure 3. Capillary electrophoresis (CE/FA) on pooled cord serum. Note. Residual binding capacity for bilirubin was around 26 mg/dL. recent o c c u r r e n c e of kernicterus in the t e r m and otherwise healthy looking but j a u n d i c e d infants 15 have revealed that majority o f t h e m did n o t show any features o f hemolysis. These observations have c o n f i r m e d earlier reports o f kernicterus occurring in large n u m b e r s o f nonheo molytic t e r m neonates o f oriental origin. 16,17 Suggestions have b e e n m a d e that the high prevalence rate o f traditional herbal c o n s u m p tion, ~,6A8 m a n y o f which have a d e m o n s t r a b l e effect o f displacing bilirubin f r o m protein binding, is an i m p o r t a n t factor for the o c c u r r e n c e o f kernicterus in these t e r m infants. Recent observation in cell culture m o d e l o f an e n h a n c e m e n t of bilirubin cytotoxicity by e n d o t o x i n a n d t u m o r necrosis factor-a even at subclinical levels has p r o p o s e d yet a n o t h e r i m p o r t a n t factor such as sepsis or subclinical infections also. 19 Table 2. Effect of Salicylate on Binding of Bilirubin and Albumin*

Aspirint Added (mg/dL)

0.0012 "

//

9 2SO0.

0 5 10 20 30 40 50

Free~c Albumin-Bound FreeBilirubin/ Bilirubin Bilirubinw Total Bilirubin (l~mol/L) (izmol/L) (%) 28.5 36.6 40 42.3 49.3 53.7 64.9

280 272 268 266 259 255 243

9.25 11.9 12.9 13.7 16 17.4 21.1

* In 10 mmol/L phosphate buffer, 1 mmol/L EDTA, pH 7.4 with RSD < 3.0% for n = 3. t Sample: 100/~L containing 308 p.mol/L bilirubin and 123 /~mol/L albumin. :[ Determine d by CE/FA. wBy calculation.

Jaundiced Neonates

/l''-t

/1'"

Migration time (min)

Figm'e 4. Capillary electrohoresis on BA solution (with 113/308 gmol/L). Note. Increasing amount of unbound bilirubin generated with increasing quantifies of salicylate added.

I n d e e d failure of the total serum bilirubin level as a prediction of the occurrence o f neurotoxicity or kernicterus in term infants is well acknowledged. 1~176O c c u r r e n c e o f bilirubin encephalopathy with low levels o f serum bilirubin in the low birth weight infants has also b e e n a well-documented p h e n o m e n o n . 9,n,21 O p e n i n g o f the blood brain barrier has b e e n t h o u g h t to be an important p h e n o m e n o n for bilirubin neurotoxicity to o c c u r 3 2 Presence o f excessive a m o u n t o f u n b o u n d bilirubin or reduction of the residual binding capacity for bilirubin in the serum appear to be critical in causing kernicterus also. 11,22,2s Many methods have therefore b e e n proposed to study the serum binding capacity for bilirubin. They have included the sephadex column chromatography, 24 thinqayer chromatography (TLC)35 fluorescent quenching method, 26 high power liquid chromatography (HPLC), or the peroxidase oxidative m e t h o d to assay the u n b o u n d bilirubinW Many o f these methods suffered either from a lack o f reproducibility, long time involved in the testing, or large sample size of serum required for the tests r e n d e r i n g them impractical in clinical setting as monitoring devices. T h e m e t h o d we propose here appears to circumvent all these shortcomings for practical purposes. We only required 3 over saturated study-points to construct the best-fitted straight line to c o m e up with the "residual saturation capacity"; each of the points tested required only 50 izL o f serum and each test requires only minutes to complete. T h e m e t h o d we adopted has b e e n developed to study the interaction between protein and protein. 12 We were delighted to find that apply-

53

ing this technology to study the interaction between bilirubin and protein also works. We have tested the reproducibility o f the results of single sample o n the same and different days o f the week. O u r results indicated that within the same week the results were highly reproducible. As can be seen in Figure 2, there was a narrow range of standard deviations (SD) when the same sample of albumin solution was tested with varying bilirubin-additions over a period o f 7 days u n d e r our optimized experimental conditions. O u r study o n commercially available h u m a n albumin (Sigma) has shown the binding capacity o f albumin for bilirubin is 2, ie, BA molar ratio = 2 (Fig 2), which is similar if not identical to those expected from o t h e r reports. 1-3 O u r findings on p o o l e d cord serum (Fig 3) from local infants showed a residual binding capacity for bilirubin of 26 m g / d L , which is also nearly identical to those r e p o r t e d in other studies. 1,2 Such highly comparable results obtained by our m e t h o d have indicated that this novel m e t h o d as proposed here should be a most appropriate and applicable test for clinical use. We have also f o u n d sodium salicylate, which is well known for its competitive binding to albumin, to be highly effective in displacing bilirubin in a dose-response manner. Even in dosages well below the usual therapeutic level of 20 m g / d L , significant amounts o f free bilirubin was generated. This observation has further attested to the usefulness o f o u r m e t h o d as an adjunct in assessing the risk of kernicterus in the j a u n d i c e d infant. Because the residual binding capacity of the serum in the sick infants is m u c h lower than their healthy counterparts, 27 the measurements of the total serum bilirubin alone does not predict the risk of bilirubin brain toxicity. It is particularly relevant and i m p o r t a n t in ethnic groups like the Chinese in whom there is a high prevalence rate o f self-medication and herbal consumption. 5,6 Unfortunately serf-medication and herb usage may n o t be readily revealed in the medical history, although many may displace bilirubin from protein binding. We do not consider measuring the ' u n b o u n d bilirubin' in the serum o f the j a u n d i c e d infant of significant predictive value for bilirubin brain damage, as the water-insoluble 'freed bilirubin' would be precipitated out o f the circulation. Therefore, it is important that accurate, micro-scale methods

54

Yeung, Fung and Sun

like o u r s , w h i c h c a n i d e n t i f y t h e " r e s i d u a l b i n d ing capacity" of the neonatal serum, be available as a n a d d i t i o n a l a s s e s s m e n t t o o l f o r t h e h y p e r bilirubinemic neonates. The recent resurgence of kemicterus occurr i n g in t h e t e r m i n f a n t s 15 h a v e n o t o n l y r e k i n dled intense interest to improve the understanding of the mechanism of producing kernicterus, b u t also i n c r e a s e d t h e q u e s t f o r a s e a r c h o f a c c u r a t e a n d p r a c t i c a b l e m e t h o d s to assess t h e risk o f b r a i n d a m a g e o f t h e j a u n d i c e d i n f a n t s . S u c h tests a r e n o l o n g e r u n i q u e l y n e e d e d o n l y f o r t h e l o w b i r t h w e i g h t , as p r e v i o u s l y b e l i e v e d , b u t also n e e d e d f o r t h e t e r m i n f a n t s w h o a r e discharged early and are breast-feeding but othe r w i s e a p p e a r r e l a t i v e l y h e a l t h y . 15 T h e m e t h o d we p r o p o s e d h e r e h o p e f u l l y s h o u l d a d d a n e w d i m e n s i o n to a s s e s s i n g t h e risk o f b i l i r u b i n n e u r o t o x i c i ~ i n t h e j a u n d i c e d n e o n a t e . 2s

References 1. Odell GB: Neonatal hyperbilirubinemia. New York, NY, Grune and SLratton, 1980 2. Odell GB: Studies in kernicterus III. The Saturation of serum proteins with bilirnbin during neonatal life and its relationship to brain damage at five years. J Pediatr 76:12-21, 1970 3. OstrowJD: Bile pigment and jaundice: Molecular, metabolic and medical aspect. New York, NY, Marcel Decca & Corp, 1980 4. Walker PC: Neonatal bilirnbin toxicity. A review of kernicterus and the implications of drug-induced bilirnbin displacement. Clin Pharmacokinet 13:26-50, 1987 5. Yeung CY: Neonatal hyperbilirubinaemia in Chinese, Trop Geogr Med 25:151-157, 1973 6. Yeung CY: Changing pattern of neonatal jaundice and kernicterns in Chinese neonates. Chinese Med J 110: 448-454, 1997 7. Yeung CY, Leung CS, Chen YZ, et al: Effect of a poplular Chinese herb on neoantal bilirnbin protein binding. Biology Neonates 58:98-103, 1990 8. Yeung CY, et al: An old traditional herbal remedy for neonatal jaundice with a newly identified risk. J Paediatr Child Health 29:292-294, 1993 9. Pledger DR, Scott JM, Belfield A: Kernicterns at low levels of serum bilirubin: The impact of bilirubin albumin-binding capacity. A 10-year retrospective survey. Biol Neonate 1982; 41:3844, 1982 10. Kim MH, Yoon.~, SherJ, et al: Lack of predictive indices in kernicterns: A comparison of clinical and pathologic factors. Pediatrics 66:852-858, 1980

11. Cashore WJ, Oh W: Unbound bilirubin and kernicterus in low birth weight infants. Pediatrics 69:481-485, 1982 12. Chen N, Wang L, Zhang YK: Electrophoresis selectivity as a function of operating parameters in free-solution capillary electrophoretic separation of dipeptides. J Liq Chromatogr 16:3609-3622, 1993 13. WatchkoJF, Oski F: Bilirnbin 20mg/dl = Vigintiphobia. Pediatrics 71:660-665, 1983 14. Newman TB, Maisels JM: Evaluation and treatment of jaundice in the term newborn: A kinder, gender approach. Pediatrics 91:1218-1219, 1993 15. Johnson L, Brown AK: A pilot registry for acute and chronic kernicterus in term and near-term infants, pediatrics 103:736, 1999 (suppl) 16. Yeung CY: Kernicterus in term infants. Aust Paediatr J 21:273-274, 1985 17. Wong HB: Singapore kernicterns. Singapore Med J 29: 556-558, 1980 18. Yeung CY: The role of native herbs in neonatal jaundice. J Sing Paediatr Soc 36:7-8, 1994 19. Ngai KC, Yeung CY: Additive effects of TNFa and endotoxin on bilirubin cytotoxicity. Pediatric Res 45:526-540, 1999 20. Robertson AF, Karp WB, Davis HC, et al: Predicting the need for exchange transfusion in newborn infants. A comparison of five methods. Cain Pediatr 22:533-536, 1983 21. Silverman WA, Anderson D, Blanc W, et al: A difference in mortality rate and incidence of kernicterus among premature infants allotted two prophylactic antibiotic agents. Pediatrics 18:614-615, 1956 22. Wennberg RP, Hance AJ: Experimental bilirubin encephalopathy: Importance of total bilirnbin, protein binding and blood-brain barrier. Pediatr Res 20:789-792, 1986 23. Cashore WJ: Free bilirubin concentrations and bilirubinbinding affinity in term and preterm infants.J Pediatrics 96:521-527, 1980 24. Schiff D, Chan G, Stern L: Sephadex G25 quantitative estimation of free bilirubin in jaundiced newborn infants sera: A guide to the prevention of kernicterns. Lab Clin Med 80:455-458, 1972 25. Trivin F, Odievre M, Lemonnier A: Faster estimation of reserve bilirubin binding capacity of serum from the neonate by thin-layer-chromatography on sephadex. Clin Chem 23:541-545, 1977 26. Lee K, Gartner LM, Zaraf UL: Fluorescent dye method for determination of the bilirnbin- binding capacity of serum albumin. J Ped 86:280-285, 1975 27. Jacobson J, Wennberg RP: Determination of unbound bilirnbin in the serum of newborns. Clin Chem 20:783784, 1974 28. Cashore WJ, Oh W, Blumberg WE, et al: Rapid fluorometric assay of bilirubin and bilirubin-binding capacity in blood of jaundiced neonates: Comparisons with other methods. Pediatrics 66:411-416, 1980