A comparative study on the extraction of membranebound bilirubin from erythrocyte membranes using various methods

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1 J. Biochem. Biophys. Methods 39 (1999) A comprtive study on the extrction of membrnebound bilirubin from erythrocyte membrnes using vrious methods * Sd Tyyb, Mohmmd Kutub Ali Interdisciplinry Biotechnology Unit, Aligrh Muslim University, Aligrh , U.P., Indi Received 15 September 1998; ccepted 15 November 1998 Abstrct In this study, we used three different methods for the extrction of membrne-bound bilirubin (EMB) from erythrocyte membrnes. Use of 2.5% lbumin, ph 7.4, for elution of EMB resulted in only 34% of the totl EMB which ws estimted fter the solubiliztion of bilirubin-loded erythrocyte membrnes (BLEMs) with 1% SDS. On the other hnd, incubtion of BLEMs with 38 mm sodium crbonte solution contining 5 mm EDTA, ph 11.0, yielded 77% of the totl EMB. Appliction of Fog s rection for the estimtion of EMB directly on the BLEMs resulted in the estimtion of 75% of the totl EMB. These results suggest tht either of the bove methods, i.e. use of lbumin or high ph, or direct Fog s rection cnnot estimte the totl EMB correctly. Increse in ionic strength from 0.15 to 0.45 did not relese ny EMB from erythrocyte membrnes. Therefore, the best method for the estimtion of totl EMB is the solubiliztion of membrne with 1% SDS followed by Fog s rection method Elsevier Science B.V. All rights reserved. Keywords: Bilirubin; Erythrocyte membrne; Sodium dodecyl sulfte; Humn serum lbumin 1. Introduction Studies on the binding of bilirubin to the erythrocyte membrnes hve been mde in order to understnd the mechnism of bilirubin entry into cells which my open the wy * Corresponding uthor. Tel.: ; fx: ; e-mil: btismu@x400.nicgw.nic.in X/ 99/ $ see front mtter PII: S X(98) Elsevier Science B.V. All rights reserved.

2 40 S. Tyyb, M. Kutub Ali / J. Biochem. Biophys. Methods 39 (1999) for developing vrious preventive mesures ginst bilirubin encephlopthy [1,2]. In most of these studies, rdiolbelled bilirubin hs been used [1 3]. Alterntively, unlbelled bilirubin hs lso been used fter its incubtion with the membrnes nd eluting the bound bilirubin with either chloroform or lbumin [1,2,4]. However, both of these methods (chloroform/ lbumin extrction) suffer from serious drwbck of being incpble of removing the totl membrne-bound bilirubin under different conditions (unpublished observtion). Further, modified Jendrssik nd Groff method (Fog s method) [5] hs not been used directly to determine the membrne-bound bilirubin. In view of the bilirubin dissociting potentil of sodium benzote nd cffeine (present in Fog s regent) from the lbumin bilirubin complex [6,7], it seems probble tht these compounds my dissocite bilirubin from the membrnes, thus mking it vilble to dizotized sulfnilic cid. Other fctors such s chnge in ph nd ionic strength my lso dissocite bilirubin from the membrnes. In this pper, we hve compred the eluting potentil of vrious regents for the extrction of bound bilirubin from erythrocyte membrnes. 2. Mterils nd methods 2.1. Mterils Bilirubin, cffeine nhydrous, sulfnilic cid, sodium benzote nd sodium nitrite were purchsed from SD Fine Chemicls (Indi). Sodium potssium trtrte nd sodium hydroxide were obtined from Quligens Fine Chemicls (Indi). Bovine serum lbumin, frction V ws procured from Sigm (USA). Humn serum lbumin ws isolted by the method of Tyyb nd Qsim [8]. Other regents used were of nlyticl grde. Humn blood (in 1.32% sodium citrte nd 1.47% dextrose) ws obtined from the Blood Bnk of J.N. Medicl College, Aligrh Muslim University, Aligrh Methods Protein concentrtion ws determined by the method of Lowry et l. [9] using bovine serum lbumin s the stndrd Preprtion of erythrocyte membrne suspension Humn erythrocytes were collected by centrifugtion of blood t g for 20 min, followed by triple wshings with 50 mm Tris HCl buffer, ph 7.4, contining 100 mm NCl. Erythrocytes were diluted with equl volume of 50 mm Tris HCl buffer, ph 7.4, contining 100 mm NCl to obtin 50% hemtocrit vlue. The membrnes were isolted t 48C from these erythrocytes following the method of Dodge et l. [10]. Finlly, the membrnes were wshed with 50 mm Tris HCl buffer, ph 7.4, nd mixed with the sme volume of the buffer equivlent to the volume of blood used.

3 S. Tyyb, M. Kutub Ali / J. Biochem. Biophys. Methods 39 (1999) Bilirubin binding experiments Bilirubin solution ws prepred by dissolving few crystls of bilirubin in 38 mm sodium crbonte solution contining 5 mm EDTA, ph The concentrtion of bilirubin solution ws determined by Fog s method [5] using clibrtion curve. No difference ws noticed in the clibrtion curve for determining bilirubin concentrtion under ll conditions used for the extrction of membrne-bound bilirubin (EMB). The bilirubin solution ws protected from light nd used within 1 h. All the experiments were crried out under yellow light. The binding of bilirubin to erythrocyte membrnes ws studied by incubting 200 ml of stock bilirubin solution of desired concentrtion with 1.0 ml of erythrocyte membrne suspension nd the finl volume ws djusted to 1.5 ml with 50 mm Tris HCl buffer, ph 7.4. After 30 min incubtion t 378C with intermittent shking, the mixture ws centrifuged in Remi-10 microfuge, t g for 5 min t room temperture nd the superntnt discrded. Membrnes were wshed severl times with 50 mm Tris HCl buffer, ph 7.4, until the lst superntnt ws devoid of yellow color. After finl wshing, the membrne-bound bilirubin ws extrcted nd determined by ech of the following methods Direct Fog s rection Two milliliters of Fog s regent I (contining 30 g sodium benzote, 20 g cffeine nhydrous nd 50 g sodium cette in 400 ml of wter) nd 0.5 ml of regent II (prepred fresh by mixing three drops of 0.5% (w/v) sodium nitrite (regent IIb) in 5.0 ml of regent II (prepred by dissolving 0.5 g of sulfnilic cid in 1.5 ml of concentrted HCl, then diluted with 100 ml of wter)) were directly dded to 1.0 ml of bilirubin-loded erythrocyte membrne (BLEM) suspension. The mixture ws shken well before incubtion. If required, the rection ws crried out with 1.0 ml of diluted BLEM suspension. After 10 min incubtion t room temperture, the mixture ws centrifuged t g for 20 min. To the pink-colored superntnt obtined bove, 1.5 ml of Fog s regent III (contining 30 g sodium hydroxide nd 105 g sodium potssium trtrte in 300 ml of wter) ws dded nd the green-colored lkline zobilirubin ws determined spectrophotometriclly t 600 nm Sodium dodecyl sulfte (SDS) tretment In nother experiment, BLEMs were dissolved in 1% (w/v) SDS (to finl volume of 1.5 ml) by incubting the contents t 608C for 1 h nd Fog s rection ws crried out with 1.0 ml of the solubilized BLEMs to determine bilirubin concentrtion. The turbidity cused by SDS ws removed by centrifugtion t g for 20 min. The cler green-colored superntnt ws collected nd the bsorbnce ws mesured t 600 nm. The precipitte obtined fter centrifugtion ws white nd contined no bilirubin Elution with vrious medi Membrne-bound bilirubin ws lso eluted by incubting BLEMs with 1.0 ml ech of either 3.8% lbumin solution (finl concentrtion, 2.5%) or 38 mm sodium crbonte solution contining 5 mm EDTA, ph 11.0, or with NCl solution of different ionic strengths for 30 min t 378C. The finl volume of the incubtion mixture ws mde up to

4 42 S. Tyyb, M. Kutub Ali / J. Biochem. Biophys. Methods 39 (1999) ml with the buffer. Then, the mixture ws centrifuged t g for 5 min nd 1.0 ml of the superntnt contining eluted bilirubin ws subjected to Fog s rection for the determintion of bilirubin concentrtion in the eluent. Necessry volume corrections were mde in determining the bilirubin concentrtion. The pellet left ws wshed gin to remove the eluted bilirubin nd then dissolved in 1% (w/v) SDS (to finl volume of 1.5 ml) nd the mount of bilirubin present in the finl pellet ws determined by Fog s method [5]. 3. Results nd discussion Tble 1 shows the comprison of vrious eluting medi for their bility to relese bilirubin from BLEMs with the mount of bilirubin relesed fter solubilizing the BLEMs in 1% (w/v) SDS nd mesuring the bilirubin directly by Fog s method. It should be noted tht the mount of bilirubin relesed fter the solubiliztion of BLEMs with 1% (w/v) SDS represented the totl mount of EMB. As cn be seen from Tble 1 t given bilirubin lod in the incubte (72 mm), the mount of bilirubin relesed fter SDS solubiliztion of BLEMs ws much higher thn tht eluted with the other medi tested. Use of 2.5% lbumin, ph 7.4, solution eluted only 34% of totl EMB. These results suggest tht 2.5% lbumin solution which is reported to elute bout 95% of bound bilirubin from intct erythrocytes [11], cnnot be used s bilirubin eluting medium from erythrocyte membrnes. In view of the higher mount of bilirubin bound with lysed erythrocyte membrnes compred to the seled membrnes [3] nd dul nture of the binding of bilirubin to erythrocyte membrnes (i.e. bilirubin dinion to polr heds of phospholipids nd bilirubin cid binding to lipophilic regions of bilyer) [12], it seems tht lbumin removes tht frction of EMB which is bound to polr heds. This view is lso supported by the observtion tht lbumin could not remove ggregted bilirubin cid from membrne [13]. Use of 38 mm sodium crbonte solution contining 5 mm EDTA, ph 11.0, Tble 1 Estimtion of membrne-bound bilirubin fter its extrction from erythrocyte membrnes using vrious methods Eluting medium Estimted bilirubin (mm) Totl estimted bilirubin (mm) In elute EMB left fter elution 1% SDS % Albumin M NCl mm N2CO31 5 mm EDTA, ph Fog s regents I nd II The totl bilirubin concentrtion in the incubte ws 72 mm. Ech vlue represents men of three observtions from three independent experiments. Bilirubin ws estimted by Fog s method [5].

5 S. Tyyb, M. Kutub Ali / J. Biochem. Biophys. Methods 39 (1999) commonly used bilirubin solvent, resulted in the elution of bout 76.4% of totl EMB which ws much higher thn the lbumin eluted bilirubin from BLEMs. These results were in greement with the previous reports on the ph-dependent binding of bilirubin to erythrocyte membrnes, i.e. decresed binding of bilirubin to erythrocyte membrnes with the increse in ph of the incubtion medium [1] nd reversibility of bilirubin ggregte t high ph [13]. Since 24% of totl EMB still remined bound with the membrnes s mesured by SDS tretment it cn be sid tht increse in ph cnnot dislodge ll the bound bilirubin from erythrocyte membrnes. Therefore, use of 38 mm sodium crbonte solution contining 5 mm EDTA, ph 11.0, cnnot be successful tretment for the estimtion of totl EMB. Incubtion of BLEMs with solutions of different ionic strengths, i.e M NCl, did not relese ny mount of bilirubin from the membrnes s no bilirubin ws detected in the eluent. These results suggest tht the binding forces between bilirubin nd membrnes cnnot be wekened by the increse in ionic strength. This ws in greement with n erlier observtion tht binding between bilirubin nd membrnes is hydrophobic [14]. Incubtion of BLEMs with Fog s regents I nd II (contining sulfnilic cid, cffeine nd sodium benzote) lso resulted in the removl of bound bilirubin in the form of zobilirubin. However, the mount of bilirubin estimted from the direct rection of Fog s regents with BLEMs ws found to be 72% of the totl EMB. The vrition in either the incubtion time of Fog s regents I nd II with BLEMs from 10 to 120 min (Tble 2) or ddition of Fog s regent III directly into the rection mixture did not result in ny significnt chnge in the mount of bilirubin estimted. In ll the bove tretments, the frction of EMB left unttcked by eluting medium ws relesed by SDS tretment nd the totl sum of the mount of bilirubin relesed by vrious eluting medi nd SDS tretment ws found to be within the rnge of bilirubin directly estimted fter SDS solubiliztion of BLEMs (see Tble 1). These results strongly suggest tht the estimtion of totl EMB by Fog s method is most relible when the estimtion is crried out directly fter solubiliztion of BLEMs with 1% (w/v) SDS. In yet nother experiment, erythrocyte membrnes were incubted with incresing concentrtions of bilirubin ( mm) nd the mount of EMB ws estimted either by direct rection of Fog s regents with BLEMs or with Fog s rection fter SDS solubiliztion of the BLEMs. As cn be seen from Fig. 1, in both cses, the mount of Tble 2 Effect of time on the direct Fog s rection with BLEMs in the determintion of EMB Time (min) Time of incubtion of BLEMs with Fog s regents I nd II. b Ech vlue represents men of two observtions from two different experiments. Estimted EMB (mm) b

6 44 S. Tyyb, M. Kutub Ali / J. Biochem. Biophys. Methods 39 (1999) Fig. 1. Comprison of the mount of EMB estimted by direct Fog s rection with BLEMs (d) nd fter 1% SDS solubiliztion of BLEMs (s). EMB estimted incresed with the increse in the concentrtion of bilirubin in the incubte. However, within the rnge of bilirubin concentrtion used in this study, the mount of EMB estimted by direct Fog s rection with intct BLEMs ws lwys less thn tht of bilirubin estimted by the sme method fter the solubiliztion of BLEMs with 1% (w/v) SDS. From these results, there seems to be competition between EMB nd sulfte-binding proteins nd lipids of membrnes for dizotized sulfnilic cid, s dizotized sulfnilic cid is lso known to bind membrne sulfte-binding proteins nd lipids [15]. Further, sodium benzote nd cffeine (ccelertors in Fog s rection) with known potentil of displcing bilirubin from the bilirubin lbumin complex [6,7], were found to be ineffective in dissociting bilirubin from the bilirubin membrne complex. This my ccount for the difference in the estimted bilirubin by two different methods. In ddition to the bove, ineffectiveness of dizotized sulfnilic cid to enter into the bilyer membrnes [15] might prevent the rection of dizotized sulfnilic cid with some of the bound bilirubin which ws reported to be hydrophobiclly inserted within the membrne bilyer [12]. On the other hnd, the solubiliztion of BLEMs with SDS fvored the estimtion of totl EMB s ll the EMB ws vilble for dizotized sulfnilic cid to rect. From these results, it ppers tht SDS solubiliztion of membrnes nd estimtion of EMB by Fog s method cn be pplied successfully in the study of bilirubin binding to erythrocyte membrnes.

7 Acknowledgements S. Tyyb, M. Kutub Ali / J. Biochem. Biophys. Methods 39 (1999) This work ws finncilly supported by reserch grnt from the Deprtment of Science nd Technology, New Delhi, Indi. Fcilities provided by Aligrh Muslim University nd finncil ssistnce to one of us (MKA) in the form of Senior Reserch Fellowship of University Grnts Commission, New Delhi, Indi re grtefully cknowledged. We re lso grteful to the members of Distributed Informtion Sub-Centre, Aligrh Muslim University for providing us with reprogrphic fcilities. References [1] Sto H, Kshiwmt S. Interction of bilirubin with humn erythrocyte membrnes. Biochem J 1983;210: [2] Sto H, Aono S, Semb R, Kshiwmt S. Interction of bilirubin with humn erythrocyte membrnes. Biochem J 1987;248:21 6. [3] Krp WB, Subrmnym SB, Ho CK, Robertson AF. Drugs ffecting bilirubin uptke by humn erythrocyte ghosts. Am J Med Sci 1985;289: [4] Hyer M, Piv M-T, Sieso V, de Bornier BM. Experimentl studies on unconjugted bilirubin binding by humn erythrocytes. Clin Chim Act 1989;186: [5] Fog J. Determintion of bilirubin in serum s lkline zobilirubin. Scnd J Clin Lb Invest 1958;10: [6] Bessrd G, Chourqui JP, Remy C, Rmbud P. Effect of sodium benzote on the cutneous bilirubin content of the dult Gunn rt. Biol Neonte 1983;44: [7] Frnzini C, Cttozzo G. Cffeine-splitting of bilirubin/ lbumin complex its relevnce to the spectrophotometry of bilirubin in serum. Clin Chem 1987;33: [8] Tyyb S, Qsim MA. Purifiction nd properties of bufflo serum lbumin. Biochem Int 1990;20: [9] Lowry OH, Rosebrough NJ, Frr AL, Rndll RJ. Protein mesurement with the Folin phenol regent. J Biol Chem 1951;193: [10] Dodge JT, Mitchell C, Hnhn DJ. The preprtion nd chemicl chrcteristics of hemoglobin-free ghosts of humn erythrocytes. Arch Biochem Biophys 1963;100: [11] Brtlid D. Bilirubin binding by humn erythrocytes. Scnd J Clin Lb Invest 1972;29:91 7. [12] Cestro B, Cervto G, Ferrri S, Di Silvestro G, Monti D, Mnitto P. Interction of bilirubin with smll unilmellr vesicles of diplmitoylphosphtidylcholine. Itl J Biochem 1983;32: [13] Vzquez J, Grci-Clvo M, Vldivieso F, Myor F, Myor Jr. F. Interction of bilirubin with the synptosoml plsm membrne. J Biol Chem 1988;263: [14] Kirschner-Zilber I, Rbizdeh E, Shkli N. The interction of hemin nd bilirubin with the humn red cell membrne. Biochim Biophys Act 1982;690: [15] Berg HC. Sulphnilic cid dizonium slt: A lbel for the outside of the humn erythrocyte membrne. Biochim Biophys Act 1969;183:65 78.