Chemicals and reagents
Lipopolysaccharide (LPS) from E. coli O55:B5 (≥ 500,000 EU/mg) was purchased from Sigma-Aldrich Co. LLC (Vienna, AT). Polymyxin B- sulfate (PMB), an antibiotic, which is known to bind and inactivate endotoxins, was obtained from AppliChem (Darmstadt, D). Two bentonites (Bentonite 1 – main part smektite), one being a natural sodium-bentonite (Bentonite 2 – main part consist of smectite, feldspar and gypsum), and two clays treated with amines (organophilised) were used: a smectite (Organoclay 1 – fully organophilised: whole surface coated with amines) consisting of montmorillonite and an attapulgite (magnesium phylosillicate) consisting of smectite and palygorskite (Organoclay 2 – part of the surface organophilised).
Reagents used for the Limulus amebocyte lysate (LAL) test were obtained from Charles River Laboratories (CRIVER), Inc. Charleston, US: Limulus amebocyte lysate (Endochrome K; Charge: C4452E), Endotoxin-free (< 0.005 EU/mL) LAL reagent water (LRW; Charge: 99732088) and Endosafe control Standard Endotoxin from E. coli O55:B5 1.000 EU/mL (Charge: EX 14392 and EM11302 – RSE/CSE ratio 10 EU/ng; EX01022 – RSE/CSE ratio 12 EU/ng; EM11302 – RSE/CSE ratio 7 EU/ng). Potassium phosphate monobasic, sodium hydroxide and pancreatin for the preparation of the artificial intestinal fluid were obtained from Merck KGaA (Darmstadt, D).
Materials
All materials used in the experiments were pyrogen free. Glass tubes were obtained from ACILA (16 × 90 mm PYROKONTROL® tubes capped; Weiterstadt, GERD). Material which was reused was heat depyrogenated for 3 h at 230°C. 1.5 mL reaction tubes (Biosphere SafeSeal Tubes 1.5 mL) and Endosafe pipet tips were purchased from Sarstedt (Nümbrecht, D). For the LAL test, 96-well flat bottom microtitre plates (M9005, endotoxin free, Endosafe, CRIVER) were used. For the multipette, combitips were purchased from Eppendorf (Combitips plus, Biopure; Hamburg, D).
Single concentration sorption experiment
A 1000 EU/mL Endosafe control endotoxin standard was diluted in pyrogen free LAL water (LRW) to yield a working solution of 10 endotoxin Units per millilitre (EU/mL). The endotoxin activity in the stock solution was verified by the LAL test. Five milligram of PMB (positive control) and each sorbent were dissolved in 5 mL of the LPS working solution to yield a final concentration of 0.1% w/v sorbent. Reaction mixtures and pure sorbents were shaken at 112 × g for 2 hours on a micro plate shaker at room temperature (22 ± 1°C). Afterwards, the samples were centrifuged at 500 × g for 15 min, supernatant of reaction mixtures and pure sorbents were diluted in LRW water (1:100, 1:10) and endotoxin activity was measured using the LAL assay.
Influence of endotoxin concentration on adsorption efficiency
We used a 250,000 EU/mL stock of LPS of E. coli O55:B5 from Sigma for preparing eight concentrations of endotoxin working solutions, ranging from 2,500-50,000 EU/mL. The endotoxin activity in the stock solution was verified by the LAL test. Each sorbent (5 mg) was dissolved in 5 mL of LRW solution to yield a concentration of 0.1% w/v of sorbent. Incubation was done shaking at 112 × g for 2 hours at 37°C. LPS working solutions and reaction mixtures were diluted and measured as described above. Reaction mixtures had to be diluted up to 1:10,000 before applying on the plate and measured using the LAL assay.
Artificial intestinal fluid sorption experiment
The artificial intestinal fluid (AIF) was prepared according to the “Official Method – Determination of the Disintegration Time of Tablets” by Health Canada (1989). LPS working solutions were prepared from a ≥250,000 EU/mL stock of LPS of E. coli O55:B5 from Sigma to gain concentrations of 25,000 and 120,000 EU/mL. Five milligrams of sorbents and PMB were dissolved in 5 mL AIF to gain a 0.1% w/v solution. Samples were incubated shaking at 112 × g for 2 hours at 37°C. Thereafter, reaction mixtures were treated as described in paragraph “Influence of endotoxin concentration on adsorption efficiency”.
Limulus amebocyte lysate test (LAL)
The test was performed using a 96-well microtitre plate and optical density was read at 405 nm. Reaction was measured over 70 minutes. For each clay mineral, 100 μL of the diluted adsorbent alone and the sample LPS mixture was applied in duplicate onto the 96-well-plate. Spiking experiments were carried out in order to determine the LPS recovery for identifying interferences of endotoxins, reagent and used materials. For that purpose, a spiking solution with a concentration of 10 EU/mL was prepared. Subsequently, 50 μL of the spiking solution was added to the supernatant of the reaction mixtures. Each sample and the respective spiked sample were diluted to meet the calibration line and applied in duplicate. Preparation of a four-point calibration line (0.05, 0.5, 5, 50 EU/mL), application of 100 μL reaction reagent (Endochrome K as a chromogenic substrate) and the measurements were performed according to European Pharmacopoeia 5.0 2005 (chapter 2.6.14., Method D, pp. 161–168). For all experiments done in LRW solution, calibration line was done with LRW. For experiments in AIF solution, calibration line was assayed in AIF buffer. Data were calculated by the EndoScanV 9.1 software (CRIVER).
Performance standards of the LAL test
Performance characteristics of the LAL test were defined according to manufacturer’s specifications: The LPS recovery of the spiked sample had to be 50% to 200%, and the coefficient of variation of the sample, analysed in duplicate, had to be lower than 10%. Additionally, the coefficient of correlation of the calibration line had to be equal or better than 0.980. Each plate included a negative control (endotoxin free test water, LRW) and the related spiked sample. Invalid recoveries of spiked samples show false negative or false positive readings therefore, they can be excluded. Data which did not fulfil the performance standards of the test were not considered in our calculations.
Endotoxin removal efficiency (E)
The endotoxin removal efficiency was calculated for experiments in LRW and different increasing endotoxin concentrations and experiments in AIF by equation (1), where E was the removal efficiency in percentage (%), and C0 and C were the endotoxin concentrations measured in the stock solution and in sample supernatant, respectively.
(1)
Endotoxin adsorption capacity (q) and isothermal equations
In comparison to PMB, two sorbents were chosen to calculate the amount of endotoxin adsorbed (q) and to fit them to isothermal equations. Therefore, the endotoxin activity (EU/mL) was converted into endotoxin EU/mg sorbent. This referred to the potency of the used endotoxin standard, which was defined by the certificate of analysis from the producer, to be between 7–12 EU/ng (depending on the certificate of the standard/charge).
To calculate the adsorbed amount of endotoxin on the control and the used sorbents (q), the following equation (2) was used
C0 and C were the concentrations of endotoxin in the initial endotoxin solution and in the supernatant after adsorption. V was the volume of the solution (mL) and Wm was the amount of adsorbent used (mg).
The data were fitted to different isothermal models (linear and Freundlich isotherm). The equation used for the linear model (3) was
and the equation used for the Freundlich model (4) was
For both equations, q corresponded with the concentration of endotoxin bound (EU/mg). C was the concentration of endotoxin in solution (EU/mL). Kd represented the dimensionless distribution coefficient. KF corresponded to the Freundlich coefficient. The Freundlich exponent n described the deviation of the isotherm from a linear correlation and was a measure for the sorption intension. If n = 1, a linear isotherm is described and the distribution between the two phases is independent of the concentration of the sorbent. 1/n < 1 is a reference for a normal Langmuir isotherm and 1/n > 1 indicates a cooperative sorption.
Calculations and diagrams
All calculations for the isotherm models were done with Table Curve 2D v5.0. Diagrams and tables were prepared with Microsoft Excel 2010. For statistical comparison of mean binding efficiencies and to demonstrate significant decrease of endotoxin activity when sorbents used, a one-sided T-test (equal variances not assumed; Welch correction) was performed.