Bilirubin removal by Cibacron Blue F3GA attached nylon-based hydrophilic affinity membrane

Journal of Membrane Science 226 (2003) 9–20

Bilirubin removal by Cibacron Blue F3GA attached nylon-based hydrophilic affinity membrane Baolin Xia, Guoliang Zhang∗ , Fengbao Zhang School of Chemical Engineering & Technology, Tianjin University, Tianjin 300072, China Received 2 April 2003; received in revised form 17 April 2003; accepted 20 August 2003

Abstract Covalent coupling of chitosan to the activated membrane was performed after the reaction of the microporous nylon membrane with formaldehyde. Covalent linkage of chitosan was essential to improve the hydrophilicity and consequently reduced the non-specific interactions of the membranes. Cibacron Blue F3G A (CB F3GA) was then covalently immobilized onto the composite membrane to prepare affinity membrane for bilirubin removal. Different amounts of CB F3GA were attached on the composite membranes by changing the dye-attachment conditions, i.e. initial dye concentration, addition of sodium carbonate, and sodium chloride. The maximum CB F3GA content was obtained at 142.9 ␮mol/g membrane. Dye release in buffers showed a tight covalent bound of CB F3GA on the composite membrane. Bilirubin molecules interacted with the absorbents directly. Non-specific adsorption on the unmodified nylon membrane remains low, and higher bilirubin adsorption capacity, of up to 63.4 mg/g was obtained after CB F3GA immobilization. The effects of the CB F3GA content, temperature, ionic strength, and pH, as well as the adsorption isotherm were investigated in this study. The adsorption capacity increased with increasing the temperature while decreased with increasing the NaCl concentration and a peak at about pH 6.0 was observed during the corresponding experiment. The adsorption isotherm fitted the Freundlich model well. Experiments on regeneration and dynamic adsorption were also performed. © 2003 Elsevier B.V. All rights reserved. Keywords: Bilirubin removal; Cibacron Blue F3GA; Nylon-based affinity membranes; Chitosan; Microporous membranes

1. Introduction Bilirubin is an oxidative product of heme. Normally, it is transported to the liver as a complex with albumin and excreted from hepatocytes into bile mainly as bilirubin glucuronides [1,2]. The free bilirubin is a lipophilic endotoxin and may bind to cellular and mitochondria membranes. High bilirubin concentration may cause cell death in variety of tissues, and hepatic or biliary tract dysfunction; clinically, bilirubin may cause mental retardation, cerebral palsy, deaf∗ Corresponding author. Tel.: +86-22-2740-8778. E-mail address: [email protected] (G. Zhang).

ness, seizures, or death [3,4]. Nowadays researches on experimental animals show that the entry of pigment into the brain is not limited to free or unbound bilirubin only, but the albumin-bound bilirubin, which may enter the brain and further aggravate the risk of bilirubin encephalopathy or kernicterus [5]. There are many studies involving removal bilirubin directly from plasma including hemoperfusion, hemodialysis etc [6,7]. But many disadvantages appear in all these therapeutics. Dialysis membranes only allow hydrophilic small molecules to pass through and it is often not so efficient for bilirubin. Uses of microbeads adsorbents in hemoperfusion, which may cause difficulty in mass transfer, prolong the therapeutic time;

0376-7388/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2003.08.007

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B. Xia et al. / Journal of Membrane Science 226 (2003) 9–20

on the other hand, selectivity of the adsorbents is also a limit in clinical application [8]. Affinity separations rely on the highly specific binding between the counterparts to achieve an efficient separation and purification purpose. And affinity membrane chromatography has become a unique method in separation technology [9]. Microporous membranes have been modified and various affinity ligands have been coupled for use as alternative absorbents for biomedical applications in recent years [10,11]. They have the advantages of large surface area, short diffusion path, and low-pressure drop. As a result of the convective flow of solution through the pores, the mass transfer resistance is tremendously reduced and the binding kinetics dominates the adsorption process. This leads to a rapid processing, and greatly improves the adsorption step [12]. The choice of the membrane material may be difficult, as a compromise must be found regarding the reactivity of the material, stability in polar solvents, pore size, and biocompatibility [13]. The requirements of high selectivity and hydrophilicity, biological, chemical and mechanical stability, fairly large pore size and a narrow pore size distribution, and enough reactive functional groups must be satisfied with the affinity matrices for the clinical application [12,14]. It has been reported that nylon membranes are superior in the structure of pores like uniform pore size and large porosity, so it is used widely. But because of low concentration of primary amino functional groups available in their structure, they have very low ligand density. Hydrolyzing the membranes with acid could solve this problem, and further advantages of hydrophilicity and specificity could be enhanced as well after the treatment. Shang et al. [15] activated the hydrolyzed nylon membrane with dibromide propane. Beeskow et al. [16] activate the nylon membrane with formaldehyde and bisoxirane, and then coupled with hydroxyethyl cellulose. A mixture solution from formic acid, glycerol, and water was introduced by Gholap et al. [17] to prepare nylon membranes. Petsch et al. [18] hydrated the membrane and bind it with polyhydroxyl-containing materials. The requirements of ligand immobilized on the affinity absorbents are their stability, facility for coupling, maintenance of biological activity and biospecificity, inexpensive and non-toxic in use [14]. Interactions between many bio-molecular counterparts including enzymes and substrates, antibodies

and antigens, cofactors and acceptors and so on are used in the affinity separation processes. The interactions of the counterparts are highly specific, and the ligands from these counterparts are extremely efficient. However, they are expensive due to high cost of production and/or extensive purification steps. In the process of the preparation of specific sorbents, it is difficult to immobilize certain ligands on the supporting matrix with retention of their original biological activity. Precautions are also required in their use (at sorption and elution steps) and storage [12]. Reactive dyes have been considered as one of general ligands. They are still classified as affinity ligands because they interact with the active sites of many biomolecules by mimicking the structure of the substrates, cofactors, or binding agents for those biological molecules [19]. Generally, they have reactive groups like –Cl, –NH2 , –SO3 H, so that they can be easily immobilized onto the matrixes bearing –OH and –NH2 groups. Dye-ligands are able to bind most types of proteins, especially enzymes, in some case in a remarkably specific manner [20]. Moreover, because of their commercial availability and the advantages vis-a-vis the drawbacks mentioned above, the dye ligands are widely used in affinity separation processes [21–24]. In our work, flat sheet of nylon membrane was activated by formaldehyde. Chitosan was then coupled onto the activated membrane to improve the hydrophilicity. CB F3GA was covalently immobilized onto the composite membrane in alkaline medium as affinity ligand to achieve the affinity membrane. Researches have been taken to character some of the physical or hydraulic properties of the membrane. The modified nylon membrane was then used for bilirubin adsorption. The effects of dye content, temperature, pH value, the regeneration of the membrane, the adsorption of albumin bound bilirubin, and the adsorption isotherm were investigated with batch method. The performance of the membrane stack was also evaluated by dynamic experiments.

2. Experimental section 2.1. Chemicals and apparatus Nylon membranes filters (47 mm in diameter and 0.45 ␮m of the aperture) were purchased from

B. Xia et al. / Journal of Membrane Science 226 (2003) 9–20

Whatman International Ltd., Maidstone, England. Chitosan (Mr ∼ 40,000) was purchased from Fluka, and Cibacron Blue F3GA from Sigma–Aldrich. Bilirubin and BSA were obtained from the chemical reagent company of Beijing and Shanghai, China, respectively. Reagents such as acetic acid, formaldehyde, hydrochloride acid, sodium chloride, sodium hydroxide, sodium carbonate, etc. were reagent grade. The phosphate buffer (0.067 M, formed with potassium dihydrogen phosphate and di-sodium hydrogen phosphate, pH 7.4) was adopted to dispense bilirubin. Normal saline was used as solvent for BSA. A peristaltic pump (Model BT-100, Shanghai, China) was used for the feeding of bilirubin solutions and buffers. The concentration of bilirubin and albumin-conjugated bilirubin were determined using a 752 N UV-Vis spectrophotometer (Shanghai Precision Instruments Co. Ltd., Shanghai, China). The membrane cartridge (donated amicably from the Dalian Chemical and Physical Institute, China) was used to load the membrane stack. 2.2. Membrane preparation 2.2.1. Activation of the unmodified nylon membrane The nylon membrane was firstly activated by formaldehyde [16]. Ten membrane discs were incubated in a solution of 20 ml formaldehyde (>36.5 wt.%) and 0.2 ml phosphoric acid (85 wt.%) for 7 h at 60 ◦ C. The membranes were washed several times with water at 40–50 ◦ C. 2.2.2. Coupling of chitosan Reaction between the membrane and the chitosan took place in a 10 ml chitosan solution (prepared by dissolving 0.15 g chitosan in 10 ml 1 vol.% acetic acid solution) at room temperature for 30 min Then the membrane disc was placed onto a sintered glass filter holder (Autoscience) and the remaining chitosan solution was sucked slowly through the membrane disc by reducing pressure until no further drop was formed at the filtrate side. This was followed by drying the wetted membrane in an oven at 90 ◦ C for 45 min. The subsequent steps involved washing the dried membranes with acetic acid solution (1 vol.%) for several times in 1 h and then with distilled water for 24 h [25].

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2.2.3. Immobilization of the Cibacron Blue F3GA CB F3GA was immobilized onto the membranes by the methods of Ruckenstein and Zeng [26]. Two membranes were immersed together into a 20 ml dye solution (containing 10 mg dye per ml solution) for 30 min at 60 ◦ C and 5 ml NaCl aqueous solution (20 wt.%) was added to stimulate the deposition of the dye on the internal surface of the membrane. And in order to accelerate the reaction between the dye and the membranes, 2 ml Na2 CO3 aqueous solution (25 wt.%) was then added after 30 min to adjust the pH-value (to about pH 10) of the solution. The reaction then took place in the mechanical shaker in the following 4 h. Washing with ethanol, 2 M NaCl, 6 M urea, and distilled water successively after the reaction at room temperature, until no dye could be detected. The washed membranes were stored in phosphate solutions (pH 7.0) containing 0.02 wt.% sodium azide at 4 ◦ C. 2.3. Assays about the membrane 2.3.1. Assay of chitosan coupled on nylon membrane The amount of the chitosan coupling onto the nylon membranes was determined by the following method: immerse 30 mg chitosan coupled membrane fragments in 1 ml pure water, and then add 1 ml solution of ninhydrin and DMSO (80 mg ninhydrin/ml DMSO, inert in nitrogen) in, bath on boiling water for 30 min. The colorated solution was then diluted with ethanol aqueous solution (50 vol.%) and detected with a spectrophotometer at the wavelength of 570 nm [25]. 2.3.2. Assay of the dye-ligands density The amount of dye ligands immobilized onto the membrane constitutes an important parameter in the adsorption process. The dye content of the membrane can be determined by the methods of immersing the CB F3GA-attached membrane in an appropriate medium and measuring the absorbency of the solution at the λmax . However, the methods of Ruckenstein et al. [26] and Zeng et al. [10] were not suitable for our chitosan coupled nylon-based dye bound affinity membranes, because of the floccules formed during dilution after hydrolysis. A method developed from Chamber’s [27] was adopted in our study. It keeps the solution at strong acidic surrounding thus avoids flocculation and the bound dye may be released completely in the solution, which could ensure

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the accuracy of the method. The dye content of the present samples was measured by the method of hydrolyzing a certain amount of CB F3GA attached nylon complex membranes in the 12 M hydrochloride acid at 60 ◦ C and then the absorbency of the solution could be read in a spectrophotometer at 505 nm.

2.5. Batch experiments of bilirubin adsorption

2.4. Characterization of the membranes 2.4.1. Hydraulic properties studies Flux of pure water was measured via a membrane filter holder, which can hold one piece of membrane with an effective diameter of 47 mm Pure water was pumped through the membrane holder at 0.2 MPa and 20 ◦ C for 0.5 h The porosity of the composite membranes was measured by determining the amount water adsorbed by the membranes. The average pore size was measured by the permeation rate method which based on the Poiseuille principle. The flux (J), porosity (Pr ) and average pore size (¯rf ) were then calculated by the following equations, respectively: J=

Qm Aeff t

W1 − W 2 100 × Dwater V
8ηH2 O JLd r¯f = Pr p Pr =

spectrophotometer. Three kinds of release media were used: 1 mol/l sodium acetate/HCl buffer solution (pH 2.0), 0.067 M phosphates buffer solution (pH 7.0) and 0.1 mol/l sodium citrate/NaOH buffer solution (pH 12.0).

(1) (2)

(3)

where Qm is the amount of water through the membrane in a given time t, Aeff the efficient membrane area; W1 and W2 the weights of the membrane in wet and dry states, respectively, V the effective volume of the measured membrane in wet state, Dwater the density of pure water at room temperature; Ld the thickness of wet membrane, ηH2 O the viscosity of water and p is the pressure drop across membrane. 2.4.2. Dye-release of the membrane Membranes were incubated in three kinds of media at room temperature to estimate the amount of released CB F3GA. The media were renewed every 24 h and the experiment was continued till no measurable release was observed and the procedure lasted for 10 days. The amount of the dye-release into the medium was measured cumulatively as the absorption band at 610 nm for CB F3GA by a bench-top

Bilirubin adsorption on the membranes was carried out batch wise in a dark room. The amounts of bilirubin removed from the simulation solution were determined with Eq. (4). q=

(ci − ct )Vs m

(4)

where q is the amount of bilirubin adsorbed onto unit mass of the membrane (mg/g); ci and ct are the concentrations of the bilirubin in the initial and in the final solution after adsorption, respectively (mg/l); Vs is the volume of the bilirubin solution; and m is the mass of the membrane (g). The concentration of the solution of the unbound bilirubin was detected by spectrophotometry at the wavelength of 438 nm [5,28] and the ones containing albumin–bilirubin complex, at 460 nm [29]. Some factors that affect the adsorption processes were studied in the present paper. Twenty milligrams of membranes containing different dye amounts were immersed into 10 ml bilirubin solution for 10 h at 25 ◦ C to study the effect of dye content. Thirty milliliters of the bilirubin solution was incubated with 50 mg membrane at different temperatures (i.e. 4, 25 and 37 ◦ C), and then the concentration of the solution was examined at certain time intervals to study the effect of temperature and the equilibrium time at different temperature. The effects of ionic strength and pH value were investigated in the bilirubin solution containing NaCl (the concentrations were 0.05, 0.15, 0.3 and 0.5 M) and of different pH values. 2.6. Regeneration of the membranes The bilirubin-saturated membrane was regenerated with BSA and sodium hydroxide. The bilirubinsaturated membrane was eluted by recirculating the BSA solution. Then the absorbed BSA on the membrane was eluted with 0.5 N NaSCN eluant, and the membranes regenerated successively with 6 M urea,

B. Xia et al. / Journal of Membrane Science 226 (2003) 9–20

1% Tween 80 and distilled water. The elution process by the alkaline solution included immersing the bilirubin absorbed membranes in 0.1 N NaOH aqueous solution, followed by the procedure of washing with large volume of distilled water and phosphate buffer (pH 7.4). The regenerated membranes were then reused for bilirubin equilibrium adsorption. 2.7. Dynamic experiments of the membrane stack The dynamic experiments were carried out in the cartridge to investigate the breakthrough performance. It was impelled by peristaltic pump with two different flow rate (i.e. 2 and 4 ml/min) to flow through the membrane stack (containing eight overlapped membranes). Then the amounts of bilirubin through the membrane cartridge were measured in succession with a spectrophotometer.

3. Results and discussion 3.1. Characteristics of CB F3GA dyed nylon-based hydrophilic membranes The reactive groups (–CH2 OH) on the nylon membranes were multiplied after activation with the solution of formaldehyde and phosphoric acid. Since it is difficult to dissolve in the water, the chitosan was dissolved in the 1 vol.% acetic acid solution (pH value was adjusted to 2.0 by 2 M hydrochloride acid). The reaction took place in the acidic medium. It was observed that the percolation and drying operations, which were reaction related, were quite important for the membrane preparation process, hence, they must be treated carefully. The average amount of chitosan coupled on membrane was 84.2 mg/g unmodified nylon membrane. Chitosan has a structure similar to that of cellulose and possesses a large number of reactive groups of –CH2 OH and –NH2 . In this paper, dye ligand CB F3GA was immobilized on the chitosan coupled membranes, via the reaction between the chlorine group on the triazine ring and the –CH2 OH and –NH2 groups of the chitosan under alkaline conditions in a relatively higher temperature range from 60 to 80 ◦ C. The dye contents reached 80–142.9 ␮mol/g membrane (Table 1). This result was close to that from Ruckenstein and Zeng [26] in the entire chitosan membrane.

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The release of dye molecules in three different kinds of media showed that there was nearly no release of dye molecules in the acidic medium (pH 2.0), and in the neutral and alkaline medium, the dye release was 0.07 and 0.78%, respectively. This result was lower than that of CB F3GA attached polyamide hollow fibers from the experiment of Senel ¸ et al. [12], and this suggested a further application. The release in the strongly alkaline might due to the existence of strong ionic interactions. The release in the neutral medium might just be the physically occluded dye molecules along with any weakly/physically-bound dye [12]. Fortunately, these interactions are not so significant for biomedical application. It also indicated a fairly strong covalent bound between CB F3GA and the composite membrane. The permeation water flux of the modified membrane is 4.7 cm/(min atm), the reduction of the pure water flux is only 5.8% comparing to the unmodified nylon membrane, which means that the pores in the membrane could be kept at micron level during the modification process. Porosity of the membrane was calculated through Eq. (1) by measuring the related parameters in it. Membranes were stacked with some glue at the rim and compacted in the cartridge device. Some of the characteristics of the membrane stack were listed in Table 1. 3.2. Effects of CB F3GA loading on bilirubin adsorption Dye content is one of the several factors that affect the adsorption characteristics of affinity membranes. Fig. 1 showed the relationship between the bilirubin adsorption capacity and the CB F3GA content in the membranes. The adsorption capacity increases rapidly at relatively low dye contents (<53.3 ␮mol/g), but much slower at higher dye content (only about 7 mg bilirubin was increased while the dye content increased from 53.3 to 142.9 ␮mol/g), and it reached almost a constant value. This may be explained as follows: When the dye content increases, the attached amount of hydrophobic groups on the membrane surface, which would interact with the bilirubin will increase, leading to higher bilirubin adsorption, while it gets to a high dye content, some of the dye molecules might become inaccessible to bilirubin.

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Table 1 The characteristics of the CB F3GA attached membrane stack Items

Property

Membrane material Configuration Average pore size of the membrane Porosity of the membrane Thickness of a membrane Effective surface area of the membrane Chitosan density Ligand (CB F3GA) content Hydraulic permeability of pure water

Nylon, chitosan and Cibacron Blue F3GA A stack of eight flat membranes 0.35 ␮m 49.1% 0.2 mm 13.9 cm2 84.2 mg/g nylon membrane 124.5 ␮mol/g complex membrane 4.7 cm/(min atm)

3.3. Effects of bilirubin initial concentration

illustrated a Freundlich isotherm (Fig. 3). For the Freundlich model, a plot of ln q∗ versus ln c∗ should yield a straight line (linear least square regression analysis) with slope of m and with an intercept of ln κ.

Fig. 2 showed the specific adsorption of bilirubin onto the membranes. The specific bilirubin adsorption on the membrane is quite low, and the amount was about 0.6 mg bilirubin/g unmodified membrane only. While much higher binding capacity, up to 63.4 mg bilirubin/g membrane was obtained after hydrophilic process in case of the CB F3GA immobilization. The specific bilirubin adsorption increased with the increasing of bilirubin initial concentration at the given concentration range. Batch experiment provides the equilibrium parameters of the membranes for the bilirubin adsorption. The bilirubin adsorption on the modified membrane

ln q∗ = ln κ + m ln c∗

(5)

where q∗ is the amount of bilirubin adsorbed on the solid phase (the CB F3GA attached chitosan-coupled nylon-based membrane, mg/g) at equilibrium, c∗ is equilibrium concentration of bilirubin in the liquid phase mg/l). And κ and m are empirical parameters of the Freundlich model. The correlation coefficient (r) of the isotherm was 0.9983, indicating that the data generally fits the model well. The Freundlich isotherm is a purely empirical relationship, the values of the

Adsorption capacity (mg/g membrane)

70

60

50

40

30

20 20

40

60

80

100

120

140

160

CB F3GA content (µmol/g membrane) Fig. 1. Effect of CB F3GA content on bilirubin adsorption. Bilirubin concentration: 200 mg/l; medium: phosphate buffer (pH 7.4, 0.067 mol/l); temperature: 25 ◦ C and equilibrium time: 10 h.

B. Xia et al. / Journal of Membrane Science 226 (2003) 9–20

15

Adsorption capacity (mg/g membrane)

50 original nylon membrane modified membrane

40

30

20

10

0 0

40

80

120

160

200

Bilirubin initial concentration (mg/L) Fig. 2. Effect of bilirubin initial concentration on adsorption. Dye content 124.5 ␮mol/g; temperature: 25 ◦ C; medium: phosphate buffer (pH 7.4, 0.067 mol/l); equlibrium time: 10 h.

4.0 3.5

2.5

*

ln(q /[M])

3.0

2.0 1.5 1.0 0.5 1.0

1.5

2.0

2.5

3.0

3.5

4.0

ln(Ct/[C]) Fig. 3. The adsorption isotherm of bilirubin on CB F3GA attached nylon composite membrane. The same adsorption conditions with the legend of Fig. 2.

empirical parameters are κ = 0.758 and m = 1.005 in our study. 3.4. Effects of temperature The effect of temperature was studied under various temperatures, shown in Fig. 4. The adsorption capac-

ity increases with the increasing of temperature. The result was similar to that of other related experiments [8,12,25,30]. The hypothesis for these phenomena is that a conformational change takes place in the bilirubin molecule. The bilirubin molecule changed from a cis configuration to a trans configuration with increasing temperature. This would allow for lessened

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B. Xia et al. / Journal of Membrane Science 226 (2003) 9–20

Adsorption capacity (mg bilirubin/g membrane)

70 60 50 40 30 o

20

37 C o 25 C o 4C

10 0 0

50

100

150

200

250

300

350

400

Time (min)

Fig. 4. Effects of temperature on bilirubin adsorption. Dye content: 124.5 ␮mol/g; bilirubin concentration: 100 mg/l; medium: phosphate buffer (pH 7.4, 0.067 mol/l).

steric hindrance in the binding of bilirubin to the attached CB F3GA molecules [12]. The adsorption rate also increases with the increasing of temperature at the incipient stage (from the beginning to about 50 min). The reasonable explanation for this case is that bilirubin molecules move more quickly at a higher temperature. Data from batch experiments also showed an equilibrium time of approximate 2.5 h. 3.5. Effects of ionic strength It was presented in Fig. 5 that the effect of the ionic strength on bilirubin adsorption. As seen in the figure, the adsorption capacity decreases with increasing the concentration of NaCl in the binding buffer (0.067 M, phosphate, pH 7.4). When the salt concentration changes from 0.05 to 0.5 M, the adsorption capacity decreases by about 5%, and it inclines to have a notable influence in high salinity solutions. One of the possible explanations was given by Lu et al. [31] that the negative carboxyl ion of bilirubin in the experimental conditions could attract antiparticles around it to form an “ionic atmosphere”. The increasing of the salinity of the solution intensified the course. On the other hand, CB F3GA attached membranes were also negatively charged in the solution, which were also sources for “ionic atmosphere”, because of their numerous –SO3 H groups. Thus, the interactions between

bilirubin and the absorbent were interfered which led to a decreasing of bilirubin adsorption on the membranes. 3.6. Effects of pH Fig. 6 provided the effects of pH value on bilirubin adsorption. The adsorption capacity increases firstly at acidic conditions and then decreases with the increasing of pH (<11) at alkaline environments. And at stronger alkaline solution, a peak appears at about pH 6.0, which identifies a suitable solution pH range for adsorption. This might be explained as follows: Intact inner hydrogen bond makes bilirubin molecule itself wrapped with hydrophobic groups. Thus the adsorption process needs a dissolution and a successively diffusion procedure from the bulk solution to the solid phase. With the increasing of pH, the hydrogen bonds in the molecular structure were destroyed gradually and the solubility of bilirubin increased little by little, and this resulted in the increasing of binding capacity on the membranes. But as the dissociation of the carboxyl of bilirubin to form anions and the transformation in the structure of bilirubin in higher pH surroundings, it becomes more and more disadvantageous for the adsorption (pKa for bilirubin is about 4.2–4.9 [32]). Additionally, since sulfogroups (pKa ∼ 0.8) are readily dissociated and the primary and

B. Xia et al. / Journal of Membrane Science 226 (2003) 9–20

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Adsorption capacity (mg/g membrane)

36 35 34 33 32 31 30 0.0

0.1

0.2

0.3

0.4

0.5

Concentration of NaCl (mol/L) Fig. 5. Effects of ionic strength on bilirubin adsorption. Dye content: 124.5 ␮mol/g; bilirubin concentration: 100 mg/l; temperature: 25 ◦ C; medium: phosphate buffer (pH 7.4, 0.067 mol/l); equilibrium time: 10 h.

3.7. Adsorption of BSA bound bilirubin

secondary amines are comparatively slightly dissociated in CB F3GA molecules in the solution, the dye ligands tend to be negatively charged, which distribute a positive function in acidic solution and a negative influence on bilirubin adsorption to the membranes. Therefore, the adsorption curve vs. pH value behaves as the phenomena described above.

It is generally known that bilirubin exists in three forms in serum as free bilirubin, conjugated bilirubin (in form of monoglucuronide and diglucuronide) and delta bilirubin (covalently bound with albumin with one or two molecules) [33–35]. Here, we adopted a

Adsorption capacity(mg/g membrane)

42

40

38

36

34

32

30

28 3

4

5

6

7

8

9

10

pH value Fig. 6. Effects of pH on bilirubin adsorption. Dye content: 124.5 ␮mol/g; bilirubin concentration: 100 mg/l; medium: phosphate buffer (pH 7.4, 0.067 mol/l); temperature: 25 ◦ C; equilibrium time: 10 h.

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B. Xia et al. / Journal of Membrane Science 226 (2003) 9–20

1:1 molar ratio of BSA bound bilirubin to observe the adsorption performance of the membranes. The clearance ratio (bilirubin adsorbed on the membrane versus the overall bilirubin in the initial solution) of the BSA conjugated bilirubin is high up to 58.8% (the absolute amount is about 17.8 ␮mol bilirubin per unit mass of membrane) in presence, and the loss of BSA, which defined as the ratio of BSA adsorbed by membranes versus to original BSA in the bulk solution, is only about 23%, this occurs because it still exist the binding capacity of BSA in the CB F3GA attached membranes. The results indicate a tight binding between BSA and bilirubin, and they also imply a considerable competitive ability of CB F3GA attached membranes to combine bilirubin with BSA. BSA and human serum albumin (HSA) both bind one or two molecules of bilirubin with high affinity, the binding constant being 107 M−1 [5,36]. Results of the adsorption in BSA conjugated bilirubin proved the possibility of the membrane in biomedical use.

Table 2 Regeneration of the membrane and reutilization for bilirubin adsorption

Recovery of eluant (%) Adsorption capacity for bilirubin (mg/g membrane) Reduction ratio of the adsorption capacity (%)

BSA regenerated membranes

Caustic alkaline regenerated membranes

83.1 45.1

– 48.7

19.5

13.1

Membranes were obtained from the equilibrium adsorbed membranes at 25 ◦ C in the batch experiments for effect of temperature; the adsorption temperature was 25 ◦ C for the regenerated membranes.

attached membrane (Table 2). The bilirubin adsorption was still remaining a relatively high level. And the physical character of the membrane keeps nearly unchanged. 3.9. Breakthrough of the membrane stack

3.8. Regeneration and reuse of the membranes The membranes have very preferable mechanical and chemical properties. Both BSA and NaOH were used to regenerate the membrane, and the regenerated membrane seems to have a comparative performance of adsorption capacity approximate to the CB F3GA

Breakthrough of the membrane stack was investigated under two flow rates. Fig. 7 gives the breakthrough curve of the membrane stack in a membrane container. Higher velocity of the bilirubin solution makes out a rapid breakthrough and the lower one shows a laggard penetration.

1.0

0.8

C/Co

0.6

0.4

4.0ml/min 2.0ml/min

0.2

0.0 0

100

200

300

400

500

600

Time (s) Fig. 7. Breakthrough curves for bilirubin with a stack of eight CB F3GA attached membranes. Dye content: 124.5 ␮mol/g; bilirubin concentration: 100 mg/l; temperature: 25 ◦ C; medium: phosphate buffer (pH 7.4, 0.067 M).

B. Xia et al. / Journal of Membrane Science 226 (2003) 9–20

It also shows a high rate for bilirubin removal in dynamic experiments. Because the mass transfer in dynamic experiment is convective mode, the resistance is greatly reduced. As a result, the operation for bilirubin removal could be speeded up, and the ligands on the membranes could be made the most usage by recirculating the plasma with certain flow rates.

4. Conclusions Nylon modified microporous hydrophilic membranes were prepared. It involved activating the primal nylon membrane, which containing little active groups, with formaldehyde, then the well hydrophilic material namely chitosan was coupled on it, and finally the reactive dye (CB F3GA) was immobilized on the composite membranes. The membrane had good hidrophilicity and high ligand content, which were benefit for bilirubin adsorption. Batch experiments revealed a high bilirubin adsorption capacity. Optimal adsorptions could be achieved in a suitable pH range, ionic strength and appropriate temperature. The adsorption process showed a Freundlich isotherm at the given range of the bilirubin concentration. The high capacity under suitable adsorption environment (pH 6.0, 37 ◦ C) and a preferable higher competitive ability to albumin implied a prospect for clinical application. Regeneration of the membrane suggested good mechanical and chemical stability. Dynamic experiments indicate a higher bilirubin removal rate compared to the batch-wise experiments. Higher efficiency of bilirubin removal in the future application should lie in the circulating process. References [1] T. Kamisako, Y. Kobayashi, K. Takeuchi, T. Ishihara, K. Higuchi, Y. Tanaka, E.C. Gabazza, Y. Adachi, Recent advances in bilirubin metabolism research: the molecular mechanism of hepatocyte bilirubin transport and its clinical relevance, J. Gastroenterol. 35 (2000) 659. [2] Y. Andreu, M. Ostra, C. Ubide, J. Galbán, S. de Marcos, J.R. Castillo, Study of a fluorometric-enzymatic method for bilirubin based on chemically modified bilirubin-oxidase and multivariate calibration, Talanta 57 (2002) 343. [3] A. Lavin, C. Sung, A.M. Klibanov, R. Langer, Enzymatic removal of bilirubin from blood: a potential treatment for neonatal jaundice, Science 230 (1985) 543.

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[4] P.D. Berk, A computer simulation study relating to treatment of fulminant hepatic failure by hemoperfusion, Proc. Soc. Exp. Biol. Med. 155 (1977) 535. [5] M.M. Khan, S. Tayyab, Understanding the role of internal lysine residues of serum albumins in conformational stability and bilirubin binding, Biochim. Biophys. Acta 1545 (2001) 263. [6] K. Morishita, H. Yokoyama, S. Inoue, T. Koshino, Y. Tamiya, T. Abe, Selective visceral and renal perfusion in thoracoabdominal aneurysm repair, Eur. J. Cardio-thoracic Surg. 15 (1999) 502. [7] S. Sideman, L. Mor, M. Mihich, S. Lupovich, J.M. Brandes, M. Zeltzer, Resin hemoperfusion for unconjugated bilirubin removal, Contrib. Nephrol. 29 (1982) 90. [8] L. Jiang, G. Zhang, F. Zhang, W. Shi, Chitosan membranes used for bilirubin removal, J. Chem. Eng. Chin. Univ. 17 (2003) 128. [9] W. Guo, Z. Shang, Y. Yu, L. Zhou, Membrane affinity chromatography of alkaline phosphatase, J. Chromatogr. A 685 (1994) 344. [10] X. Zeng, E. Ruckenstein, Supported dye-affinity membranes and their protein adsorption properties, J. Membr. Sci. 117 (1996) 271. [11] A. Kassab, H. Yavuz, M. Odabasi, A. Denizli, Human serum albumin chromatography by Cibacron Blue F3GA-derived microporouse poly amide hollow fiber affinity membranes, J. Chromatogr. B 746 (2000) 123. [12] S. Senel, ¸ F. Denizli, H. Yavuz, A. Denizli, Bilirubin removal from human plasma by dye affinity membrane microporous hollow fibers, Separation Sci. Technol. 37 (2002) 1989. [13] K. Kugel, A. Moseley, G.B. Harding, E. Klein, Microporous poly (caprolactom) hollow fibers for therapeutic affinity adsorption, J. Membr. Sci. 74 (1992) 115. [14] S.R. Narayanan, Preparative affinity chromatography of proteins, J. Chromatogr. A 658 (1994) 237. [15] Z. Shang, D. Zhou, W. Guo, Y. Yu, L. Zhou, K. Zhou, Preparation and characteristics of column and membrane affinity matrixes used for endotoxin removal, Anal. Chem. (China) 25 (1997) 1010. [16] T.C. Beeskow, W. Kushayoto, F.B. Anspach, K.H. Kroner, W.D. Deckwer, Surface modification of microporouse polyamide membranes with hydroxyrthyl cellulose and their application as affinity membranes, J. Chromatogr. A 715 (1995) 49. [17] S.G. Gholap, D.A. Musale, S.S. Kulkarni, S.K. Karode, U.K. Kharu, Protein and buffer transport through anionically grafted nylon membranes, J. Membr. Sci. 183 (2001) 89. [18] D. Petsch, T.C. Beeskow, F.B. Anspach, W.D. Deckwer, Membrane adsorbers for selective removal of bacterial endotoxin, J. Chromatogr. B 693 (1997) 79. [19] A. Denizli, E. Pigskin, Dye-ligand affinity systems, J. Biochem. Biophys. Meth. 49 (2001) 391. [20] C.R. Lowe, D.A.P. Small, A. Atkinson, Some preparative and analytical applications of triazine dyes, Int. J. Biochem. 13 (1981) 33. [21] M.Y. Arica, C. Halýcýgil, G. Alaeddino˘glu, A. Denizli, Affinity interaction of hydroxypyruvate reductase from

20

[22]

[23]

[24]

[25]

[26]

[27]

[28]

B. Xia et al. / Journal of Membrane Science 226 (2003) 9–20 Methylophilus spp. with Cibacron blue F3GA-derived poly (HEMA EGDMA) microspheres: partial purification and characterization, Proc. Biochem. 34 (1999) 375. J.A. López-Mas, S.A. Streitenberger, F. Garc´ıa-Carmona, Á. Sánchez-Ferrer, Cyclodextrin biospecific-like displacement in dye-affinity chromatography, J. Chromatogr. A 911 (2001) 47. N.E. Labrou, Y.D. Clonis, Biomemetic-dye affinity chromatography for the purification of mitochondrial l-malate dehydrogenase from bovine heart, J. Biotechnol. 45 (1996) 185. F.J. Wolman, M. Grasselli, E.E. Smolko, O. Cascone, Preparation and characterization of Cibacron Blue F3G-A poly (ethylene) hollow-fiber membranes, Biotechnol. Lett. 22 (2000) 1407. W. Shi, F. Zhang, G. Zhang, S. Wang, et al. Polylysineimmobilized affinity nylon membrane used for bilirubin adsorption, Mol. Simul. (2003), in press. E. Ruckenstein, X. Zeng, Albumin separation with Cibacron Blue carrying macroporous chitosan and chitin affinity membranes, J. Membr. Sci. 142 (1998) 13. G.K. Chambers, Determination of Cibacron Blue F3GA substitution in Blue Sephadex and Blue Dextran-Sepharose, Anal. Biochem. 83 (1977) 551. M. Bouvier, G.R. Brown, L.E. St-Pierre, The binding of bilirubin by immobilized oligopeptides containing lysine, Can. J. Chem. 67 (1989) 596–602.

[29] A.F. McDonagh, A. Giovanni, D.A. Lightner, Induction of wavelength-dependent photochemistry in bilirubins by serum albumin, Monatshefte für Chemie 129 (1998) 649. [30] A. Denizli, M. Kocakulak, E. Pi¸skin, Bilirubin removal from human plasma in a packed-bed column system with dye-affinity microbeads, J. Chromatogr. B 707 (1998) 25. [31] L. Lu, Z. Yuan, K. Shi, B. He, Studies on the adsorption of bilirubin by macroporous poly(2-hydroxyethyl methacrylate) adsorbents, Ion. Exchange. Adsorp. (China) 18 (2002) 105. [32] A.F. McDonagh, A. Phimister, S.E. Boiadjiev, D.A. Lightner, Dissociation constants of carboxylic acids by 13 C-NMR in DMSO/water, Tetrahedron Lett. 40 (1999) 8515. [33] K. Kurosaka, S. Senba, H. Tsubota, H. Kondo, A new enmzymatic assay for selectively measuring conjugated bilirubin concentration in serum with use of bilirubin oxidase, Clinica Chim. Acta 269 (1998) 125. [34] S. Shiomi, D. Habu, T. Kuroki, S. Ishida, N. Taisumi, Clinical usefulness of conjugated bilirubin levels in patients with acute liver diseases, J. Gastroenterol. 34 (1999) 88. [35] Y. Andreu, J. Galban, S. Marcos, J.R. Castillo, Determination of direct-bilirubin by a fluorimetric-enzymatic method based on bilirubin oxidase, Fresenius J. Anal. Chem. 368 (2000) 516. [36] T. Færch, J. Jacobsen, Kinetics of the binding of bilirubin to human serum albumin studied by stopped-flow technique, Arch. Biochem. Biophys. 184 (1977) 282.