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Mechanism of creatinine adsorption from physiological solutions onto mordenite
Microporous and Mesoporous Materials 119 (2009) 186–192
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Mechanism of creatinine adsorption from physiological solutions onto mordenite David Bergé-Lefranc a, Hélène Pizzala b, Renaud Denoyel a, Virginie Hornebecq a, Jean-Louis Bergé-Lefranc c, Regis Guieu d, Philippe Brunet e, Habib Ghobarkar a, Oliver Schäf a,* a
MATDIV, Universités d’Aix-Marseille I, II et III-CNRS, UMR 6264, Laboratoire Chimie Provence, Campus St., Jérôme F-13397, Marseille Cedex 20, France Spectrométries Appliquées à la Chimie Structurale, Universités d’Aix-Marseille I, II et III-CNRS, UMR 6264, Laboratoire Chimie Provence, Campus St., Jérôme F-13397, Marseille Cedex 20, France c Service de Biochimie et de Biologie Moléculaire, CHU Conception, Marseille, France d Laboratoire d’Ingénierie des Protéines, FRE 2738, Institut Fédératif de Recherche Jean Roche (IFR 11), Faculté de Médécine Nord, Université de la Méditerranée (Aix-Marseille II), 16 Boulevard Pierre, Dramard 13916, Marseille Cedex 20, France e Laboratoire d’Hématologie-Immunologie, INSERM U608-Université de la Méditerranée (Aix-Marseille II), Faculté de Pharmacie, 13005 Marseille, France b
a r t i c l e
i n f o
Article history: Received 21 May 2008 Received in revised form 13 October 2008 Accepted 21 October 2008 Available online 29 October 2008 Keywords: Zeolite Uremic toxin Mordenite Creatinine Liquid phase adsorption
a b s t r a c t Mordenite framework type zeolites with different Si/Al ratios and H+ as charge compensating cation were found to be specific adsorbents for creatinine. Adsorption isotherms were determined varying the composition of the solvent systematically from pure water to physiological buffered saline solutions containing albumin. The adsorption mechanism is studied using different experiments: by following pH, ionic exchange and adsorption enthalpies during the adsorption process and by analyzing ex situ the chemical form of the adsorbate by 13C MAS-NMR. It is shown that creatinine adsorption, which is the highest when adsorbed from pure water, is reduced when adsorbed from sodium chloride solution or from physiological buffered saline solutions. Once adsorbed, creatinine is present in protonated form within the zeolite, in equilibrium with other cations of the medium. The level of this chemisorption is higher than the one determined in case of carbon type samples. When albumin is added to the physiological buffered saline solutions, adsorption level is only slightly modified, indicating that the weak albumin adsorption observed on the external surface of zeolite particles does not prevent creatinine access to the micropores. Adsorption by zeolites could, thus, be a complementary method for uremic toxins elimination during haemodialysis. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Uremia syndrome is characterized by an accumulation of human metabolism products in blood due to the malfunction of kidneys unable to eliminate them down to a vital level. The artificial removal of these toxins is generally achieved using extra-corporeal detoxification by haemodialysis [1]. During a haemodialysis session, a mesoporous membrane maintains the separation of blood from an isotonic solution. Thus, molecules presenting a molecular weight inferior to 500 g/mol can diffuse from blood through the membrane to the solution [2]. However, removal of some uremic toxins specifically by adsorption during a regular dialysis session may present an interest. For example, protein bound toxins may be insufficiently removed by this well established procedure. Materials efficient for adsorption in such application should possess high surface area, stability under physiological conditions, toxicological safety and some selectivity
* Corresponding author. Tel.: +33 04 91 63 71 22; fax: +33 04 91 63 71 11. E-mail address: [email protected] (O. Schäf). 1387-1811/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.micromeso.2008.10.016
towards the target toxin. Furthermore, their surface chemistry has to allow efficient adsorption not only from aqueous solution, but also under physiological conditions in the presence of salt ions, amino acids, peptides, glucides and proteins. In the case of haemoperfusion [3], a more specific treatment for hepatic coma and certain drug intoxication, blood is already treated by using adsorption onto activated carbons [4]. Nevertheless this type of adsorbent is not specific and adsorbs more or less any molecule that is partially hydrophobic. Moreover, the pore size distribution is generally wide, which prevents their use as molecular sieves. The use of organized porous solids, i.e. solids with a monodispersed pore size and controlled organization, may then be looked for. Recently, silica based mesoporous materials prepared by templating methods have shown interesting properties for biotechnologies. MCM-41 for example presents high adsorption capacities for several small molecules like amino acids [5] but also for larger ones like vitamin B12 [6], cytochrome C, and lysozyme [7]. Nevertheless, their stability as well as their non-toxicity are not yet proved and their pore size is not selective towards small molecules [8]. In contrast, microporous crystallized materials such as zeolites show molecular sieving effects: uremic toxin molecules
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can enter the structure inherent void system (the apertures range between 0.24 and 0.74 nm for aluminosilicate zeolites) if they are small enough. However, adsorption of these molecules only takes place if an interaction with the adsorption sites on the pore walls is established. Zeolites have already been used as vehicles to carry bio-molecules into viable cells [9], a performance which seems to show the non-toxicity of these inorganic solids under suitable conditions. Creatinine (2-amino-1-methyl-5H-imidazol-4-one, C4H7N3O) is representative for small uremic toxins that are not bound to proteins. Creatinine dimensions are found to be 0.39 nm 0.53 nm 0.38 nm for creatinine in the solid-state, as determined by X-ray diffraction [10]. The molecule is important in clinical analytic domain for it is used as a probe molecule to evidence renal failure or muscular dysfunction. Its concentration for a healthy person is in the range of 35–106 lmol/l whereas for a person with uremia this value may increase to concentrations around 2000 lmol/l. Several studies have been devoted to the evaluation of new adsorbents for the elimination of uremic toxins such as activated carbons, carbon nanotubes and synthetic polymers [11–15]. All these materials present interesting creatinine adsorbing properties and will be used as benchmarks in the present study. Nevertheless, no selectivity towards creatinine was really demonstrated excepted in the case of imprinted polymers [16]. Moreover most experiments were performed in aqueous solution or salty solutions far from physiological conditions. Recently, mordenite-type zeolites were identified by our group as potential adsorbents for creatinine [17]. The mordenite framework is constituted of an orthorhombic two-dimensional interconnected channel system in c- and b-directions (Fig. 1). Channel sizes in c- and b-directions are 0.65 0.70 nm and 0.26 0.57 nm, respectively [18]. The aim of the present work is to understand first the adsorption mechanism of creatinine from aqueous solution onto mordenite and second, the influence of solution composition on adsorption properties or on this mechanism. The approach, thus, consists in making the medium progressively more and more complex, starting from pure water, then adding sodium chloride or buffered saline solution to finish with an albumin containing medium. Adsorption isotherms are determined in these various conditions and the adsorption mechanism is studied by following pH variations, ionic exchange and adsorption enthalpies during the adsorption process as well as analyzing ex situ the chemical form of the adsorbate by 13C MAS-NMR.
b
-c Fig. 1. Representation of the two-dimensional channel system of the mordenite structure.
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2. Experimental section 2.1. Chemicals Creatinine anhydrous and albumin from bovine serum: (bovine serum albumin, minimum 96%, electrophoresis molecular weight: 67 kDa) were purchased from Sigma–Aldrich. Physiological solution used was the Dulbecco phosphate buffered saline (D-PBS) purchased from Invitrogen. D-PBS composition was: NaCl (8 g L 1), CaCl2 (0.1 g L 1), MgCl2 (0.1 g L 1) KCl (0.2 g L 1), KH2PO4 (0.2 g L 1) and NaH2PO4 (2.16 g L 1). Water was purified by a Millipore milliQ filtration system. Two mordenite zeolite sorbents were purchased from Zeolyst Inc., USA. The first one presents a Si/Al ratio equal to 45 and H+ as charge compensating cation, named H+MOR45 (zeolite formula |H+1.04(H2O)x|[Al1.04Si46.96O96] with x 15). The second one presents a Si/Al ratio equal to 6.5 and Na+ as charge compensating cation and is called Na+MOR6.5 (zeolite formula |Na+6.4(H2O)x|[Al6.4Si41.6O96] with x 24). The water contents were assessed by thermogravimetry. Commercial Lichrosphere silica Si60, provided by Merck, (mesoporous adsorbent with 700 m2/g surface area, without micropores) was used as a reference material [19] to study adsorption on a non-microporous surface. 2.2. Ion exchange procedure The protonated form of MOR6.5 was obtained by treating Na+MOR6.5 with a solution of ammonium chloride (1 M) at 80 °C for 48 h. The solution/solid mass ratio was equal to 30 and the solution renewed twice. During this treatment, NH4+ ions replace Na+ ions in the mordenite channel system. Then, the solid was rinsed with deionised water and calcined at 520 °C for 6 h applying a heating ramp of 12 °C/h. During calcination, ammonium ions are degraded to yield ammonia and H+. These protons remain in the channel system as charge compensating cations. 2.3. Characterization methods The adsorbents were characterized in terms of structure and surface properties. X-ray diffraction (Siemens D5000 Diffractometer, Bragg-Brentano set-up) was used in order to check the phase purity of mordenite sorbents. Morphology and chemical composition of the samples were investigated using Scanning Electron Microscopy (SEM, Philips XL30 ESEM, LaB6 source) and SEM coupled with energy dispersive X-ray analysis (Cambridge S90B with EDAX DX4 detector, tungsten source). Nitrogen adsorption at 77 K measurements with a Micrometrics ASAP 2010 equipment were performed to investigate the porous structure of sorbents. Prior to this measurement samples were outgassed under a pressure lower than 10 2 mBar at 623 K for 12 h. Solid-state NMR spectra were performed on a Bruker Avance DSX 400 NMR at the frequencies of 100.3 and 400.4 MHz for 13C and 1H nuclei, respectively. 13C nuclei were observed by Cross Polarization Magic Angle Spinning (CPMAS) using a standard Bruker broad-band MAS probe. The samples were packed in 4-mm zirconia rotors and spun at 7.5 kHz. 13C CPMAS spectra were acquired with a contact time of 5 ms and a recycle delay of 3 s. To obtain a good signal to noise ratio, 512 transient scans were accumulated for pure creatinine, and 20,000 to 40,000 for adsorbed sample. Chemical shifts were externally referenced to tetramethylsilane (TMS). 2.4. Adsorption experiments form aqueous solutions Adsorption measurements were performed by the solution depletion method in static conditions and in different media: pure
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distilled water, phosphate buffered saline solution (D-PBS) and DPBS solution containing albumin (50 g/l) that is the usual albumin concentration in the human serum. Each sample with a constant liquid/solid ratio (1 mL/20 mg) was stirred at 37 °C until equilibrium is reached, i.e. during 12 h. Then, after centrifugation, creatinine concentration was determined using two different techniques depending on the solvent used. In the case of aqueous and D-PBS solvents, equilibrium concentrations in supernatant were determined by spectrometric measurements. A HPLC set-up (Agilent 1200 series) with diode array detector and Agilent Zorbax SB-C18 column was used. Absorptions were measured at 235 nm which corresponds to the maximum for creatinine and also at 220 nm in order to check the presence of fines particles. The absorbance peaks were next integrated and quantified by comparison with an external calibration (creatinine solutions of well known concentration). Albumin adsorption was followed by the same procedure. In the case of D-PBS containing albumin, creatinine concentrations were determined by the Jaffé reaction [20] performed on an automatic laboratory device: Synchron LX (Beckman Coulter, Fullerton CA). During the Jaffé reaction, the creatinine’s methylene group reacts with sodium picrate in alkaline media to give a orange-red complex measured by photometry at 505 nm [21]. Knowing the concentration variation (initial/equilibrium), the solid mass and the volume of liquid exposed to solid, adsorbed amounts of creatinine were determined for each initial concentration ranging from 0 to 9.5 mM. The isotherm representation gives the specific excess adsorbed amount versus the equilibrium concentration. Adsorption enthalpies were measured by a microcalorimetric method in aqueous solution and in physiological solution D-PBS at 37 °C. A step by step titration of the stirred mordenite suspension by a stock solution of creatinine was performed in a Tian–Calvet type calorimeter. The integral enthalpies were obtained after correction for dilution effects.
MOR6.5 has a Si/Al ratio small enough to allow the determination of ionic exchange efficiency by EDS. Measurements performed before and after ion exchange (Na+ vs. H+) on Na+MOR6.5 indicated that ion exchange is complete since, no sodium was detected (i.e. Na content below 0.1 mol%). 3.1. Influence of solution composition on adsorption The adsorption isotherms of creatinine onto H+MOR45 are given in Fig. 2 for aqueous- and physiological media (D-PBS). Apparently L-type [23] adsorption isotherms are obtained in both cases, but with different affinities. In aqueous solution high affinity of the molecule towards the adsorbent is found with a maximum adsorption capacity of about 0.39 mmol/g (44 mg/g), a value almost identical for H+MOR6.5. Referred to the crystal structure of mordenite, this value corresponds to 1.12 molecules per unit cell. The observed adsorption capacity is high compared to that observed on activated carbon and carbon nanotubes (about 25 mg/g (0.22 mmol/g), each) [13] or compared to selectively adsorbing imprinted polymers [16] (about 0.1 mmol/g). The small grain size of the mordenites (inducing the observed high specific external surface areas of up to to 15 m2/g) made it necessary to exclude adsorption on the outer surface. Therefore, we determined the adsorption isotherm of creatinine in distilled water onto high surface area silica, Lichrosphere Si60 [19]. This sample has pores much larger than creatinine size [19] (about 6 nm) and should exhibit surface properties similar to that of zeolites external surface (mainly surface silanols of comparable surface density). Creatine adsorption onto Lichrosphere Si60 levels at 0.04 mmol/g, which corresponds to 5.7 10 5 mmol/m2, far from a compact monolayer (around 8.4 10 3 mmol/m2). Finally, even if a compact monolayer is assumed, 0.39 mmol/g of creatinine adsorbed only on the external surface, would need 46 m2/g instead of 15 m2/g found experimentally. These arguments permit to consider that there is mostly no adsorption on the external surface and, thus, that adsorption is mainly taking place in the micropores. In the buffered and isotonic media D-PBS, the initial affinity of creatinine onto mordenite is reduced. The adsorption capacity at high concentrations is around 0.23 mmol/g (26 mg/g) a value that represents 0.66 molecules per unit cell. The two isotherms of Fig. 2 – H+MOR45 in distilled water and in D-PBS – can be related to the corresponding integral enthalpies of adsorption presented in Fig. 3. Again a change of behavior between aqueous- and D-PBS media can be observed. The variation of the enthalpy of adsorption in aqueous solution as a function of the adsorbed amount of creatinine exhibits a constant value characteristic for a single mechanism of adsorption. In the case of D-PBS at low coverage, values of enthalpies of creatinine adsorption are higher than those found in aqueous solution. Differential enthalpies of adsorption then decrease with rising coverage to reach final values lower than those found in aqueous solution. This evolution can be interpreted in terms of successive occupation of energetically different adsorption sites starting from the sites of highest energy to the lowest ones. This decrease of affinity and the evolution of adsorption enthalpies with coverage may indicate that there is a competition for adsorption between creatinine and other components of D-PBS. Zeolites being cation exchanger, the main probability is a competi-
3. Results and discussion Previous screening [17] has revealed that mordenite-type zeolites have a good affinity for creatinine. Starting sorbents used in this study are of commercial origin. Thus, prior to any adsorption experiment, the physical and chemical properties of the samples were analyzed. XRD measurements (not shown) have confirmed the structure of mordenite (IZA code MOR, JCPDS file: 71-1033) for each sample. Mordenite crystallizes in the orthorhombic system (Cmcm) with cell parameters a, b and c equal to 1.81 nm, 2.05 nm and 0.75 nm, respectively [18]. No impurity was detected by XRD. The nitrogen adsorption isotherms at 77 K obtained on H+MOR45 and Na+MOR6.5 are typical for microporous solids [22]. The treatment of these data by the BET equation in the low pressure range (0.05 < p/po < 0.35) allows the determination of an ‘‘equivalent specific surface” area. The internal and external areas as well as the microporous volumes were determined by applying the t-plot method with Harkins–Jura equation. All these parameters are listed in Table 1. The two samples exhibit similar properties but present an internal to external surface area ratio about 10 times smaller than normally expected for zeolites – due to the small grain size.
Table 1 Nitrogen sorption results at 77 K for H+MOR45 and Na+MOR6.5. Solid
Surface area (BET) (m2 g
H+MOR45 Na+MOR6.5
475 399
1
)
Internal area (t-plot) (m2 g 460 391
1
)
External area (t-plot) (m2 g 15 8
1
)
Micropore volume (t-plot) (cm3 g 0.18 0.16
1
)
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Fig. 2. Creatinine adsorption isotherms (adsorbed quantity vs. equilibrium concentration) onto H+MOR45 in aqueous solution and in D-PBS solution.
Fig. 3. Integral adsorption enthalpies (vs. equilibrium concentration) of creatinine onto H+MOR45 in aqueous solution and in D-PBS.
tion with cations. In order to understand this influence of ion presence, adsorption isotherms were determined from sodium chloride solutions, because sodium represents the main cation in physiological solutions. Two concentrations were selected: 2 g/l and 9 g/l, this latter being close to the one found in D-PBS. The results are presented in Fig. 4. The amount of creatinine adsorbed onto H+MOR45 in the presence of sodium chloride is systematically lower than that determined in pure water and the affinity of creatinine towards the porous sorbent decreases with increasing NaCl concentration. Nevertheless, adsorbed amounts are higher than those observed in the presence of D-PBS, indicating the influence either of the other cations constituting D-PBS or of the pH. This latter parameter variation is reported in Fig. 4 in the case of experiments with sodium chloride solutions. The evolution of pH in a buffer free salt solution gives several information: (i) pH strongly decreases with the introduction of H+MOR45, in the absence of creatinine, indicating that H+ ions are partially exchanged for Na+ in the mordenite structure, a fact which is also proven by the EDS analysis (ii) pH increases with rising concentration of creatinine along the adsorption isotherm (see Fig. 4) ranging from 2 two 6.
This indicates that adsorption of creatinine is accompanied by the adsorption of H+ and displacement of Na+. At low pH creatinine is already protonated (pKa of protonated creatinine is 4.4 while it is essentially neutral at pH 7, confirmed by NMR [24]). Its acido–basic equilibrium may be written as:
O H3C-N
O +
N + H NH2
H3C-N + N-H NH2
This points out that at low pH protonated creatinine ‘‘adsorbs” onto mordenite by ion exchange against Na+. Interestingly, the increase of pH from 2 to 6 allows to calculate the amount of proton adsorbed together with creatinine, showing that around one proton is adsorbed with one creatinine molecule. In the case of adsorption from pure water, the pH is stable around 5. In that case neutral creatinine molecules enter the pores and are protonated inside the channels ‘‘in situ”. This picture is confirmed by NMR. Application of 13C Solid-state NMR allows the study of the chemical state of the molecules adsorbed on zeolites [25]. As shown in Fig. 5,
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Fig. 4. Creatinine adsorption isotherms (adsorbed quantity vs. equilibrium concentration) onto H+MOR45 in sodium chloride solution (2 and 9 g/l with corresponding pH) and onto H+MOR45 in D-PBS. The isotherms of H+MOR6.5 and Na+MOR6.5 in D-PBS are given for comparison.
Fig. 5. Solid-state
13
C CPMAS-NMR spectra of polycrystalline (a) and adsorbed creatinine onto mordenite (b).
in the case of adsorbed creatinine both 13C CPMAS-NMR signals attributed to C-2 and C-4 carbons show large high field shifts compared to their chemical shift values in polycrystalline creatinine and neutral D2O solution (not shown). In acidic medium (pH 6 4), similar high field shifts have been described and characterize the protonation of creatinine molecule [26]. This result confirms that creatinine is adsorbed onto mordenite mainly in protonated form. The higher pH, the more predominant is the neutral form of creatinine in solution. It means that under buffer conditions near pH 7, like in pure water, neutral entities should mainly enter the zeo-
lite channel system and get protonated ‘‘in situ”. However, this mechanism is only possible if H+ sites are still present in the zeolite structure despite the presence of other cations. Replacing H+ by other cations in MOR45 consequently reduces the available adsorption sites. By systematic EDS investigations on H+MOR6.5 ion exchanged under equilibrium conditions in D-PBS at 37 °C it was shown that only 1/3 of the H+ ions are exchanged by Na+ and K+ (Na+/K+ ratio of about 2). Other cations present in D-PBS were not detected in the zeolite. Such analysis was not possible with H+MOR45 because of its too small cationic content after exchange. However, ion exchange of H+ against Ag+ in H+MOR45,
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Fig. 6. Creatinine adsorption isotherms (adsorbed quantity vs. equilibrium concentration) onto H+MOR45 in aqueous solution, D-PBS, and protein containing D-PBS (bovine albumin 50 g/l).
which is much more sensitive in EDS analysis, confirmed that at maximum only 45% of H+ are exchangeable. The amounts of creatinine adsorbed in H+MOR45 and H+MOR6.5 samples in distilled water and equilibrated with D-PBS are of the same level (about 1.12 creatinine/unit cell at the plateau in water (results for H+MOR6.5 not shown) and 0.66 creatinine/unit cell at the plateau in D-PBS – see Fig. 4). This indicates that H+MOR45 also has enough protonated sites in the channels despite the exchanges with cations of D-PBS. In the absence of H+ sites, i.e. pure Na+MOR6.5 in D-PBS, no creatinine is adsorbed (see also Fig. 4). As indicated above, H+MOR45 cannot completely be converted to Na+MOR45 as only 1/3 of the H+ are exchangeable. Comparing adsorption isotherms of H+MOR45 and H+MOR6.5 after equilibration with D-PBS, but also values obtained in distilled water (H+MOR45 and H+MOR6.5 show the same saturation level), it is finally surprising that they are not very different, although the number of H+ sites inside H+MOR6.5 is largely higher than that in H+MOR45 (6 against 1). After ion exchange, these values are at least 4 against less than 1. It means that the level of H+ adsorption on H+MOR6.5 in distilled water and the reduction of creatinine adsorption level in D-PBS in the case of H+MOR6.5 is due to an additional factor independent from H+ reduction. The principal MOR channel system has the following dimensions: 0.65 0.7 nm [18]. About 1.12 creatinine molecules are found per unit cell in pure H+MOR45 with Si/Al = 45 (corresponding to 1.04 H+ per unit cell). This represents almost the maximum possible amount adsorbed under the chosen experimental conditions because it already corresponds to 71% of maximum filling, calculated on pure geometrical considerations. At lower Si/Al ratios, i.e. Si/Al = 6.5, there are much more H+ adsorption sites (6.4 per unit cell) but these available additional sites are not accessible for sterical reasons. During the partial ion exchange 1/3 of H+ are replaced by other cations, notably Na+ and K+. While this leads to a 1/3 reduction of available adsorption sites in MOR45, replacing 1/3 of them in MOR6.5 still leaves more available adsorption sites than initially present in H+MOR45. As for both H+MOR45 and H+MOR6.5 adsorption levels of creatinine in pure water are almost the same, it can be concluded that a big part of the adsorption sites in H+MOR6.5 have been sterically blocked due to the lack of space. 3.2. Influence of albumin on the adsorption In this last part of the study, the complexity of the medium is again increased: a protein, the bovine serum albumin, is added to the D-PBS solution. This was done in order to approach in a closer
manner biomedical conditions. The amino acids sequence of serum albumin is highly conserved in mammals. For example human and bovine serum albumin shares more than 85% similarities. In humans, serum albumin account for more than 50–55% of total serum proteins and its average concentration in adults is 35–50 g/l. In such biological samples molecules like creatinine are known to be free solute, i.e. not bound to albumin. However, interactions between proteins and solid have to be taken into account [27]. Recently it was shown that Ovalbumin (egg protein) is adsorbed at the surface of zeolite [9]. Therefore, in a first step the amount of protein adsorbed onto the external surface of mordenite particles was determined. It is found that albumin is adsorbed at a constant level (in the 1–50 g/l concentration range) around (0.020+/ 0.005) mg/g corresponding to about 1 lg albumin per square meter of zeolites external surface. This corresponds only to a small fraction of a monolayer (about 2 mg/g) [28]. In Fig. 6 the adsorption isotherms of creatinine onto H+MOR45 performed in water, D-PBS solution without – and with albumin are presented. The shape of the adsorption isotherm in the presence of albumin is very similar to the one obtained in D-PBS. However, the capacity of adsorption is slightly lower: 17 mg/g against 22 mg/g. This corresponds to 0.43 creatinine molecules per mordenite unit cell. Creatinine adsorption quantities onto H+MOR45 in different media are summarized in Table 2. This decrease of the adsorbed amount of creatinine onto mordenite is small and close to the error range of the measurements. It can be explained by several phenomenon. Small amino acids constituting the primary structure of albumin are certainly still present (purity of albumin sample is 96%) and they may be adsorbed into zeolite [29] channels, thus, entering in competitive adsorption with creatinine. Channel sizes of the mordenite structure are too small to allow proteins to enter in (internal surface) but albumin can be adsorbed on the external surface [30]. This adsorption of albumin onto the external surface of particles can partially block accesses to a certain fraction of channels, but due to its conformation and quantity, it cannot block all pores.
Table 2 Summary of creatinine adsorption levels onto H+MOR45 in different media. Media
Aqueous
DPBS
DPBS and albumin
0.38 1.12
0.23 0.66
0.17 0.43
Adsorption capacity mmol/g molecule/unit cell
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4. Summary and conclusions This study shows that mordenite framework type zeolites with different Si/Al ratios and H+ as charge compensating cation are good adsorbents for creatinine. The crystallinity of these porous solids gives them a good thermodynamic stability necessary in biological environments. Two mordenite-type zeolites have been modified and the adsorption results compared in various conditions in order to understand the adsorption mechanism. Results, notably pH variation and NMR, show that creatinine is adsorbed on mordenite in its protonated form. When adsorbed from pure water on the H+ form of the mordenite, there is protonation of the creatinine inside the channels. There is a unique adsorption mechanism as evidenced by microcalorimetry, i.e. constant adsorption enthalpy versus coverage. When adsorption is done on partially exchanged forms of mordenite, the adsorption mechanism may also include a cationic exchange between the protonated creatinine and other cations. In that case there is a composite mechanism: adsorption enthalpies decrease with coverage. A more detailed analysis also shows that steric hindrance occurs when H+ concentration in MOR is too high (H+MOR6.5: more adsorption sites available than sterically accessible) but also when sodium or other cations are present in the channels of mordenite. Removal of uremic toxin molecules such as creatinine by adsorption is not a competitive method to haemodialysis but an alternative process to eliminate one particular toxin molecule specifically. As shown here in the case of albumin, microporosity prohibits the access of proteins to the internal reactive surface. These results are a first promising step in the improvement of dialysis procedure based on a specific adsorption process. References [1] T. Murphy, S. Robinson, Anesth. Intens. Care. Med. 7 (2006) 247. [2] R. Vanholder, R. De Smet, G. Glorieux, A. Argiles, U. Baurmeister, Ph. Brunet, W. Clark, G. Cohen, P.P. De Deyn, R. Deppish, B. Descamps-Latscha, T. Henle, A.
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Mechanism of creatinine adsorption from physiological solutions onto mordenite David Bergé-Lefranc a, Hélène Pizzala b, Renaud Denoyel a, Virginie Hornebecq a, Jean-Louis Bergé-Lefranc c, Regis Guieu d, Philippe Brunet e, Habib Ghobarkar a, Oliver Schäf a,* a
MATDIV, Universités d’Aix-Marseille I, II et III-CNRS, UMR 6264, Laboratoire Chimie Provence, Campus St., Jérôme F-13397, Marseille Cedex 20, France Spectrométries Appliquées à la Chimie Structurale, Universités d’Aix-Marseille I, II et III-CNRS, UMR 6264, Laboratoire Chimie Provence, Campus St., Jérôme F-13397, Marseille Cedex 20, France c Service de Biochimie et de Biologie Moléculaire, CHU Conception, Marseille, France d Laboratoire d’Ingénierie des Protéines, FRE 2738, Institut Fédératif de Recherche Jean Roche (IFR 11), Faculté de Médécine Nord, Université de la Méditerranée (Aix-Marseille II), 16 Boulevard Pierre, Dramard 13916, Marseille Cedex 20, France e Laboratoire d’Hématologie-Immunologie, INSERM U608-Université de la Méditerranée (Aix-Marseille II), Faculté de Pharmacie, 13005 Marseille, France b
a r t i c l e
i n f o
Article history: Received 21 May 2008 Received in revised form 13 October 2008 Accepted 21 October 2008 Available online 29 October 2008 Keywords: Zeolite Uremic toxin Mordenite Creatinine Liquid phase adsorption
a b s t r a c t Mordenite framework type zeolites with different Si/Al ratios and H+ as charge compensating cation were found to be specific adsorbents for creatinine. Adsorption isotherms were determined varying the composition of the solvent systematically from pure water to physiological buffered saline solutions containing albumin. The adsorption mechanism is studied using different experiments: by following pH, ionic exchange and adsorption enthalpies during the adsorption process and by analyzing ex situ the chemical form of the adsorbate by 13C MAS-NMR. It is shown that creatinine adsorption, which is the highest when adsorbed from pure water, is reduced when adsorbed from sodium chloride solution or from physiological buffered saline solutions. Once adsorbed, creatinine is present in protonated form within the zeolite, in equilibrium with other cations of the medium. The level of this chemisorption is higher than the one determined in case of carbon type samples. When albumin is added to the physiological buffered saline solutions, adsorption level is only slightly modified, indicating that the weak albumin adsorption observed on the external surface of zeolite particles does not prevent creatinine access to the micropores. Adsorption by zeolites could, thus, be a complementary method for uremic toxins elimination during haemodialysis. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Uremia syndrome is characterized by an accumulation of human metabolism products in blood due to the malfunction of kidneys unable to eliminate them down to a vital level. The artificial removal of these toxins is generally achieved using extra-corporeal detoxification by haemodialysis [1]. During a haemodialysis session, a mesoporous membrane maintains the separation of blood from an isotonic solution. Thus, molecules presenting a molecular weight inferior to 500 g/mol can diffuse from blood through the membrane to the solution [2]. However, removal of some uremic toxins specifically by adsorption during a regular dialysis session may present an interest. For example, protein bound toxins may be insufficiently removed by this well established procedure. Materials efficient for adsorption in such application should possess high surface area, stability under physiological conditions, toxicological safety and some selectivity
* Corresponding author. Tel.: +33 04 91 63 71 22; fax: +33 04 91 63 71 11. E-mail address: [email protected] (O. Schäf). 1387-1811/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.micromeso.2008.10.016
towards the target toxin. Furthermore, their surface chemistry has to allow efficient adsorption not only from aqueous solution, but also under physiological conditions in the presence of salt ions, amino acids, peptides, glucides and proteins. In the case of haemoperfusion [3], a more specific treatment for hepatic coma and certain drug intoxication, blood is already treated by using adsorption onto activated carbons [4]. Nevertheless this type of adsorbent is not specific and adsorbs more or less any molecule that is partially hydrophobic. Moreover, the pore size distribution is generally wide, which prevents their use as molecular sieves. The use of organized porous solids, i.e. solids with a monodispersed pore size and controlled organization, may then be looked for. Recently, silica based mesoporous materials prepared by templating methods have shown interesting properties for biotechnologies. MCM-41 for example presents high adsorption capacities for several small molecules like amino acids [5] but also for larger ones like vitamin B12 [6], cytochrome C, and lysozyme [7]. Nevertheless, their stability as well as their non-toxicity are not yet proved and their pore size is not selective towards small molecules [8]. In contrast, microporous crystallized materials such as zeolites show molecular sieving effects: uremic toxin molecules
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can enter the structure inherent void system (the apertures range between 0.24 and 0.74 nm for aluminosilicate zeolites) if they are small enough. However, adsorption of these molecules only takes place if an interaction with the adsorption sites on the pore walls is established. Zeolites have already been used as vehicles to carry bio-molecules into viable cells [9], a performance which seems to show the non-toxicity of these inorganic solids under suitable conditions. Creatinine (2-amino-1-methyl-5H-imidazol-4-one, C4H7N3O) is representative for small uremic toxins that are not bound to proteins. Creatinine dimensions are found to be 0.39 nm 0.53 nm 0.38 nm for creatinine in the solid-state, as determined by X-ray diffraction [10]. The molecule is important in clinical analytic domain for it is used as a probe molecule to evidence renal failure or muscular dysfunction. Its concentration for a healthy person is in the range of 35–106 lmol/l whereas for a person with uremia this value may increase to concentrations around 2000 lmol/l. Several studies have been devoted to the evaluation of new adsorbents for the elimination of uremic toxins such as activated carbons, carbon nanotubes and synthetic polymers [11–15]. All these materials present interesting creatinine adsorbing properties and will be used as benchmarks in the present study. Nevertheless, no selectivity towards creatinine was really demonstrated excepted in the case of imprinted polymers [16]. Moreover most experiments were performed in aqueous solution or salty solutions far from physiological conditions. Recently, mordenite-type zeolites were identified by our group as potential adsorbents for creatinine [17]. The mordenite framework is constituted of an orthorhombic two-dimensional interconnected channel system in c- and b-directions (Fig. 1). Channel sizes in c- and b-directions are 0.65 0.70 nm and 0.26 0.57 nm, respectively [18]. The aim of the present work is to understand first the adsorption mechanism of creatinine from aqueous solution onto mordenite and second, the influence of solution composition on adsorption properties or on this mechanism. The approach, thus, consists in making the medium progressively more and more complex, starting from pure water, then adding sodium chloride or buffered saline solution to finish with an albumin containing medium. Adsorption isotherms are determined in these various conditions and the adsorption mechanism is studied by following pH variations, ionic exchange and adsorption enthalpies during the adsorption process as well as analyzing ex situ the chemical form of the adsorbate by 13C MAS-NMR.
b
-c Fig. 1. Representation of the two-dimensional channel system of the mordenite structure.
187
2. Experimental section 2.1. Chemicals Creatinine anhydrous and albumin from bovine serum: (bovine serum albumin, minimum 96%, electrophoresis molecular weight: 67 kDa) were purchased from Sigma–Aldrich. Physiological solution used was the Dulbecco phosphate buffered saline (D-PBS) purchased from Invitrogen. D-PBS composition was: NaCl (8 g L 1), CaCl2 (0.1 g L 1), MgCl2 (0.1 g L 1) KCl (0.2 g L 1), KH2PO4 (0.2 g L 1) and NaH2PO4 (2.16 g L 1). Water was purified by a Millipore milliQ filtration system. Two mordenite zeolite sorbents were purchased from Zeolyst Inc., USA. The first one presents a Si/Al ratio equal to 45 and H+ as charge compensating cation, named H+MOR45 (zeolite formula |H+1.04(H2O)x|[Al1.04Si46.96O96] with x 15). The second one presents a Si/Al ratio equal to 6.5 and Na+ as charge compensating cation and is called Na+MOR6.5 (zeolite formula |Na+6.4(H2O)x|[Al6.4Si41.6O96] with x 24). The water contents were assessed by thermogravimetry. Commercial Lichrosphere silica Si60, provided by Merck, (mesoporous adsorbent with 700 m2/g surface area, without micropores) was used as a reference material [19] to study adsorption on a non-microporous surface. 2.2. Ion exchange procedure The protonated form of MOR6.5 was obtained by treating Na+MOR6.5 with a solution of ammonium chloride (1 M) at 80 °C for 48 h. The solution/solid mass ratio was equal to 30 and the solution renewed twice. During this treatment, NH4+ ions replace Na+ ions in the mordenite channel system. Then, the solid was rinsed with deionised water and calcined at 520 °C for 6 h applying a heating ramp of 12 °C/h. During calcination, ammonium ions are degraded to yield ammonia and H+. These protons remain in the channel system as charge compensating cations. 2.3. Characterization methods The adsorbents were characterized in terms of structure and surface properties. X-ray diffraction (Siemens D5000 Diffractometer, Bragg-Brentano set-up) was used in order to check the phase purity of mordenite sorbents. Morphology and chemical composition of the samples were investigated using Scanning Electron Microscopy (SEM, Philips XL30 ESEM, LaB6 source) and SEM coupled with energy dispersive X-ray analysis (Cambridge S90B with EDAX DX4 detector, tungsten source). Nitrogen adsorption at 77 K measurements with a Micrometrics ASAP 2010 equipment were performed to investigate the porous structure of sorbents. Prior to this measurement samples were outgassed under a pressure lower than 10 2 mBar at 623 K for 12 h. Solid-state NMR spectra were performed on a Bruker Avance DSX 400 NMR at the frequencies of 100.3 and 400.4 MHz for 13C and 1H nuclei, respectively. 13C nuclei were observed by Cross Polarization Magic Angle Spinning (CPMAS) using a standard Bruker broad-band MAS probe. The samples were packed in 4-mm zirconia rotors and spun at 7.5 kHz. 13C CPMAS spectra were acquired with a contact time of 5 ms and a recycle delay of 3 s. To obtain a good signal to noise ratio, 512 transient scans were accumulated for pure creatinine, and 20,000 to 40,000 for adsorbed sample. Chemical shifts were externally referenced to tetramethylsilane (TMS). 2.4. Adsorption experiments form aqueous solutions Adsorption measurements were performed by the solution depletion method in static conditions and in different media: pure
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distilled water, phosphate buffered saline solution (D-PBS) and DPBS solution containing albumin (50 g/l) that is the usual albumin concentration in the human serum. Each sample with a constant liquid/solid ratio (1 mL/20 mg) was stirred at 37 °C until equilibrium is reached, i.e. during 12 h. Then, after centrifugation, creatinine concentration was determined using two different techniques depending on the solvent used. In the case of aqueous and D-PBS solvents, equilibrium concentrations in supernatant were determined by spectrometric measurements. A HPLC set-up (Agilent 1200 series) with diode array detector and Agilent Zorbax SB-C18 column was used. Absorptions were measured at 235 nm which corresponds to the maximum for creatinine and also at 220 nm in order to check the presence of fines particles. The absorbance peaks were next integrated and quantified by comparison with an external calibration (creatinine solutions of well known concentration). Albumin adsorption was followed by the same procedure. In the case of D-PBS containing albumin, creatinine concentrations were determined by the Jaffé reaction [20] performed on an automatic laboratory device: Synchron LX (Beckman Coulter, Fullerton CA). During the Jaffé reaction, the creatinine’s methylene group reacts with sodium picrate in alkaline media to give a orange-red complex measured by photometry at 505 nm [21]. Knowing the concentration variation (initial/equilibrium), the solid mass and the volume of liquid exposed to solid, adsorbed amounts of creatinine were determined for each initial concentration ranging from 0 to 9.5 mM. The isotherm representation gives the specific excess adsorbed amount versus the equilibrium concentration. Adsorption enthalpies were measured by a microcalorimetric method in aqueous solution and in physiological solution D-PBS at 37 °C. A step by step titration of the stirred mordenite suspension by a stock solution of creatinine was performed in a Tian–Calvet type calorimeter. The integral enthalpies were obtained after correction for dilution effects.
MOR6.5 has a Si/Al ratio small enough to allow the determination of ionic exchange efficiency by EDS. Measurements performed before and after ion exchange (Na+ vs. H+) on Na+MOR6.5 indicated that ion exchange is complete since, no sodium was detected (i.e. Na content below 0.1 mol%). 3.1. Influence of solution composition on adsorption The adsorption isotherms of creatinine onto H+MOR45 are given in Fig. 2 for aqueous- and physiological media (D-PBS). Apparently L-type [23] adsorption isotherms are obtained in both cases, but with different affinities. In aqueous solution high affinity of the molecule towards the adsorbent is found with a maximum adsorption capacity of about 0.39 mmol/g (44 mg/g), a value almost identical for H+MOR6.5. Referred to the crystal structure of mordenite, this value corresponds to 1.12 molecules per unit cell. The observed adsorption capacity is high compared to that observed on activated carbon and carbon nanotubes (about 25 mg/g (0.22 mmol/g), each) [13] or compared to selectively adsorbing imprinted polymers [16] (about 0.1 mmol/g). The small grain size of the mordenites (inducing the observed high specific external surface areas of up to to 15 m2/g) made it necessary to exclude adsorption on the outer surface. Therefore, we determined the adsorption isotherm of creatinine in distilled water onto high surface area silica, Lichrosphere Si60 [19]. This sample has pores much larger than creatinine size [19] (about 6 nm) and should exhibit surface properties similar to that of zeolites external surface (mainly surface silanols of comparable surface density). Creatine adsorption onto Lichrosphere Si60 levels at 0.04 mmol/g, which corresponds to 5.7 10 5 mmol/m2, far from a compact monolayer (around 8.4 10 3 mmol/m2). Finally, even if a compact monolayer is assumed, 0.39 mmol/g of creatinine adsorbed only on the external surface, would need 46 m2/g instead of 15 m2/g found experimentally. These arguments permit to consider that there is mostly no adsorption on the external surface and, thus, that adsorption is mainly taking place in the micropores. In the buffered and isotonic media D-PBS, the initial affinity of creatinine onto mordenite is reduced. The adsorption capacity at high concentrations is around 0.23 mmol/g (26 mg/g) a value that represents 0.66 molecules per unit cell. The two isotherms of Fig. 2 – H+MOR45 in distilled water and in D-PBS – can be related to the corresponding integral enthalpies of adsorption presented in Fig. 3. Again a change of behavior between aqueous- and D-PBS media can be observed. The variation of the enthalpy of adsorption in aqueous solution as a function of the adsorbed amount of creatinine exhibits a constant value characteristic for a single mechanism of adsorption. In the case of D-PBS at low coverage, values of enthalpies of creatinine adsorption are higher than those found in aqueous solution. Differential enthalpies of adsorption then decrease with rising coverage to reach final values lower than those found in aqueous solution. This evolution can be interpreted in terms of successive occupation of energetically different adsorption sites starting from the sites of highest energy to the lowest ones. This decrease of affinity and the evolution of adsorption enthalpies with coverage may indicate that there is a competition for adsorption between creatinine and other components of D-PBS. Zeolites being cation exchanger, the main probability is a competi-
3. Results and discussion Previous screening [17] has revealed that mordenite-type zeolites have a good affinity for creatinine. Starting sorbents used in this study are of commercial origin. Thus, prior to any adsorption experiment, the physical and chemical properties of the samples were analyzed. XRD measurements (not shown) have confirmed the structure of mordenite (IZA code MOR, JCPDS file: 71-1033) for each sample. Mordenite crystallizes in the orthorhombic system (Cmcm) with cell parameters a, b and c equal to 1.81 nm, 2.05 nm and 0.75 nm, respectively [18]. No impurity was detected by XRD. The nitrogen adsorption isotherms at 77 K obtained on H+MOR45 and Na+MOR6.5 are typical for microporous solids [22]. The treatment of these data by the BET equation in the low pressure range (0.05 < p/po < 0.35) allows the determination of an ‘‘equivalent specific surface” area. The internal and external areas as well as the microporous volumes were determined by applying the t-plot method with Harkins–Jura equation. All these parameters are listed in Table 1. The two samples exhibit similar properties but present an internal to external surface area ratio about 10 times smaller than normally expected for zeolites – due to the small grain size.
Table 1 Nitrogen sorption results at 77 K for H+MOR45 and Na+MOR6.5. Solid
Surface area (BET) (m2 g
H+MOR45 Na+MOR6.5
475 399
1
)
Internal area (t-plot) (m2 g 460 391
1
)
External area (t-plot) (m2 g 15 8
1
)
Micropore volume (t-plot) (cm3 g 0.18 0.16
1
)
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Fig. 2. Creatinine adsorption isotherms (adsorbed quantity vs. equilibrium concentration) onto H+MOR45 in aqueous solution and in D-PBS solution.
Fig. 3. Integral adsorption enthalpies (vs. equilibrium concentration) of creatinine onto H+MOR45 in aqueous solution and in D-PBS.
tion with cations. In order to understand this influence of ion presence, adsorption isotherms were determined from sodium chloride solutions, because sodium represents the main cation in physiological solutions. Two concentrations were selected: 2 g/l and 9 g/l, this latter being close to the one found in D-PBS. The results are presented in Fig. 4. The amount of creatinine adsorbed onto H+MOR45 in the presence of sodium chloride is systematically lower than that determined in pure water and the affinity of creatinine towards the porous sorbent decreases with increasing NaCl concentration. Nevertheless, adsorbed amounts are higher than those observed in the presence of D-PBS, indicating the influence either of the other cations constituting D-PBS or of the pH. This latter parameter variation is reported in Fig. 4 in the case of experiments with sodium chloride solutions. The evolution of pH in a buffer free salt solution gives several information: (i) pH strongly decreases with the introduction of H+MOR45, in the absence of creatinine, indicating that H+ ions are partially exchanged for Na+ in the mordenite structure, a fact which is also proven by the EDS analysis (ii) pH increases with rising concentration of creatinine along the adsorption isotherm (see Fig. 4) ranging from 2 two 6.
This indicates that adsorption of creatinine is accompanied by the adsorption of H+ and displacement of Na+. At low pH creatinine is already protonated (pKa of protonated creatinine is 4.4 while it is essentially neutral at pH 7, confirmed by NMR [24]). Its acido–basic equilibrium may be written as:
O H3C-N
O +
N + H NH2
H3C-N + N-H NH2
This points out that at low pH protonated creatinine ‘‘adsorbs” onto mordenite by ion exchange against Na+. Interestingly, the increase of pH from 2 to 6 allows to calculate the amount of proton adsorbed together with creatinine, showing that around one proton is adsorbed with one creatinine molecule. In the case of adsorption from pure water, the pH is stable around 5. In that case neutral creatinine molecules enter the pores and are protonated inside the channels ‘‘in situ”. This picture is confirmed by NMR. Application of 13C Solid-state NMR allows the study of the chemical state of the molecules adsorbed on zeolites [25]. As shown in Fig. 5,
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Fig. 4. Creatinine adsorption isotherms (adsorbed quantity vs. equilibrium concentration) onto H+MOR45 in sodium chloride solution (2 and 9 g/l with corresponding pH) and onto H+MOR45 in D-PBS. The isotherms of H+MOR6.5 and Na+MOR6.5 in D-PBS are given for comparison.
Fig. 5. Solid-state
13
C CPMAS-NMR spectra of polycrystalline (a) and adsorbed creatinine onto mordenite (b).
in the case of adsorbed creatinine both 13C CPMAS-NMR signals attributed to C-2 and C-4 carbons show large high field shifts compared to their chemical shift values in polycrystalline creatinine and neutral D2O solution (not shown). In acidic medium (pH 6 4), similar high field shifts have been described and characterize the protonation of creatinine molecule [26]. This result confirms that creatinine is adsorbed onto mordenite mainly in protonated form. The higher pH, the more predominant is the neutral form of creatinine in solution. It means that under buffer conditions near pH 7, like in pure water, neutral entities should mainly enter the zeo-
lite channel system and get protonated ‘‘in situ”. However, this mechanism is only possible if H+ sites are still present in the zeolite structure despite the presence of other cations. Replacing H+ by other cations in MOR45 consequently reduces the available adsorption sites. By systematic EDS investigations on H+MOR6.5 ion exchanged under equilibrium conditions in D-PBS at 37 °C it was shown that only 1/3 of the H+ ions are exchanged by Na+ and K+ (Na+/K+ ratio of about 2). Other cations present in D-PBS were not detected in the zeolite. Such analysis was not possible with H+MOR45 because of its too small cationic content after exchange. However, ion exchange of H+ against Ag+ in H+MOR45,
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Fig. 6. Creatinine adsorption isotherms (adsorbed quantity vs. equilibrium concentration) onto H+MOR45 in aqueous solution, D-PBS, and protein containing D-PBS (bovine albumin 50 g/l).
which is much more sensitive in EDS analysis, confirmed that at maximum only 45% of H+ are exchangeable. The amounts of creatinine adsorbed in H+MOR45 and H+MOR6.5 samples in distilled water and equilibrated with D-PBS are of the same level (about 1.12 creatinine/unit cell at the plateau in water (results for H+MOR6.5 not shown) and 0.66 creatinine/unit cell at the plateau in D-PBS – see Fig. 4). This indicates that H+MOR45 also has enough protonated sites in the channels despite the exchanges with cations of D-PBS. In the absence of H+ sites, i.e. pure Na+MOR6.5 in D-PBS, no creatinine is adsorbed (see also Fig. 4). As indicated above, H+MOR45 cannot completely be converted to Na+MOR45 as only 1/3 of the H+ are exchangeable. Comparing adsorption isotherms of H+MOR45 and H+MOR6.5 after equilibration with D-PBS, but also values obtained in distilled water (H+MOR45 and H+MOR6.5 show the same saturation level), it is finally surprising that they are not very different, although the number of H+ sites inside H+MOR6.5 is largely higher than that in H+MOR45 (6 against 1). After ion exchange, these values are at least 4 against less than 1. It means that the level of H+ adsorption on H+MOR6.5 in distilled water and the reduction of creatinine adsorption level in D-PBS in the case of H+MOR6.5 is due to an additional factor independent from H+ reduction. The principal MOR channel system has the following dimensions: 0.65 0.7 nm [18]. About 1.12 creatinine molecules are found per unit cell in pure H+MOR45 with Si/Al = 45 (corresponding to 1.04 H+ per unit cell). This represents almost the maximum possible amount adsorbed under the chosen experimental conditions because it already corresponds to 71% of maximum filling, calculated on pure geometrical considerations. At lower Si/Al ratios, i.e. Si/Al = 6.5, there are much more H+ adsorption sites (6.4 per unit cell) but these available additional sites are not accessible for sterical reasons. During the partial ion exchange 1/3 of H+ are replaced by other cations, notably Na+ and K+. While this leads to a 1/3 reduction of available adsorption sites in MOR45, replacing 1/3 of them in MOR6.5 still leaves more available adsorption sites than initially present in H+MOR45. As for both H+MOR45 and H+MOR6.5 adsorption levels of creatinine in pure water are almost the same, it can be concluded that a big part of the adsorption sites in H+MOR6.5 have been sterically blocked due to the lack of space. 3.2. Influence of albumin on the adsorption In this last part of the study, the complexity of the medium is again increased: a protein, the bovine serum albumin, is added to the D-PBS solution. This was done in order to approach in a closer
manner biomedical conditions. The amino acids sequence of serum albumin is highly conserved in mammals. For example human and bovine serum albumin shares more than 85% similarities. In humans, serum albumin account for more than 50–55% of total serum proteins and its average concentration in adults is 35–50 g/l. In such biological samples molecules like creatinine are known to be free solute, i.e. not bound to albumin. However, interactions between proteins and solid have to be taken into account [27]. Recently it was shown that Ovalbumin (egg protein) is adsorbed at the surface of zeolite [9]. Therefore, in a first step the amount of protein adsorbed onto the external surface of mordenite particles was determined. It is found that albumin is adsorbed at a constant level (in the 1–50 g/l concentration range) around (0.020+/ 0.005) mg/g corresponding to about 1 lg albumin per square meter of zeolites external surface. This corresponds only to a small fraction of a monolayer (about 2 mg/g) [28]. In Fig. 6 the adsorption isotherms of creatinine onto H+MOR45 performed in water, D-PBS solution without – and with albumin are presented. The shape of the adsorption isotherm in the presence of albumin is very similar to the one obtained in D-PBS. However, the capacity of adsorption is slightly lower: 17 mg/g against 22 mg/g. This corresponds to 0.43 creatinine molecules per mordenite unit cell. Creatinine adsorption quantities onto H+MOR45 in different media are summarized in Table 2. This decrease of the adsorbed amount of creatinine onto mordenite is small and close to the error range of the measurements. It can be explained by several phenomenon. Small amino acids constituting the primary structure of albumin are certainly still present (purity of albumin sample is 96%) and they may be adsorbed into zeolite [29] channels, thus, entering in competitive adsorption with creatinine. Channel sizes of the mordenite structure are too small to allow proteins to enter in (internal surface) but albumin can be adsorbed on the external surface [30]. This adsorption of albumin onto the external surface of particles can partially block accesses to a certain fraction of channels, but due to its conformation and quantity, it cannot block all pores.
Table 2 Summary of creatinine adsorption levels onto H+MOR45 in different media. Media
Aqueous
DPBS
DPBS and albumin
0.38 1.12
0.23 0.66
0.17 0.43
Adsorption capacity mmol/g molecule/unit cell
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4. Summary and conclusions This study shows that mordenite framework type zeolites with different Si/Al ratios and H+ as charge compensating cation are good adsorbents for creatinine. The crystallinity of these porous solids gives them a good thermodynamic stability necessary in biological environments. Two mordenite-type zeolites have been modified and the adsorption results compared in various conditions in order to understand the adsorption mechanism. Results, notably pH variation and NMR, show that creatinine is adsorbed on mordenite in its protonated form. When adsorbed from pure water on the H+ form of the mordenite, there is protonation of the creatinine inside the channels. There is a unique adsorption mechanism as evidenced by microcalorimetry, i.e. constant adsorption enthalpy versus coverage. When adsorption is done on partially exchanged forms of mordenite, the adsorption mechanism may also include a cationic exchange between the protonated creatinine and other cations. In that case there is a composite mechanism: adsorption enthalpies decrease with coverage. A more detailed analysis also shows that steric hindrance occurs when H+ concentration in MOR is too high (H+MOR6.5: more adsorption sites available than sterically accessible) but also when sodium or other cations are present in the channels of mordenite. Removal of uremic toxin molecules such as creatinine by adsorption is not a competitive method to haemodialysis but an alternative process to eliminate one particular toxin molecule specifically. As shown here in the case of albumin, microporosity prohibits the access of proteins to the internal reactive surface. These results are a first promising step in the improvement of dialysis procedure based on a specific adsorption process. References [1] T. Murphy, S. Robinson, Anesth. Intens. Care. Med. 7 (2006) 247. [2] R. Vanholder, R. De Smet, G. Glorieux, A. Argiles, U. Baurmeister, Ph. Brunet, W. Clark, G. Cohen, P.P. De Deyn, R. Deppish, B. Descamps-Latscha, T. Henle, A.
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