Optometry & Vision Science:
The Impact of Tear Film Components on In Vitro Lipid Uptake
Lorentz, Holly*; Heynen, Miriam†; Trieu, Diana; Hagedorn, Sarah J.‡; Jones, Lyndon§
§PhD, FCOptom, FAAO
Centre for Contact Lens Research, School of Optometry, University of Waterloo, Waterloo, Ontario, Canada.
Received November 16, 2011; accepted February 10, 2012.
Holly Lorentz Centre for Contact Lens Research School of Optometry University of Waterloo 200, University Avenue West Waterloo, Ontario, Canada N2L 3G1 e-mail: firstname.lastname@example.org
Purpose. To analyze the influence of various tear film components on in vitro deposition of two lipids (cholesterol and phosphatidylcholine) on three contact lens materials.
Methods. Etafilcon A, balafilcon A, and senofilcon A were incubated in four different incubation solutions for 3 or 14 days: an artificial tear solution containing lipids and proteins, a protein tear solution containing proteins and the lipid of interest, a lipid tear solution containing lipids and no proteins, and a single lipid tear solution containing the lipid of interest only. Each incubation solution contained one of the two radiolabeled lipids: 14C-cholesterol (C) or 14C-phosphatidylcholine (PC). After soaking, lenses were removed from the incubation solution, the lipids were extracted and quantified using a beta counter, and masses of lipid were calculated using standard calibration curves.
Results. This experiment examined several different parameters influencing lipid deposition on contact lenses, including lens material, length of incubation, and the composition of the incubation solution. Overall, lipid deposited differently on different lens materials (p < 0.0005), with the order of deposition most commonly being balafilcon > senofilcon > etafilcon. Incubation solution had a large impact on how much lipid was deposited (p < 0.00001), although cholesterol and phosphatidylcholine demonstrated different deposition patterns. Lipid deposition after 14 days of incubation was consistently greater than after 3 days (p < 0.02).
Conclusions. This in vitro study demonstrates that C and PC deposition are cumulative over time and that silicone hydrogel materials deposit more lipid than group IV conventional hydrogel materials. It also clearly demonstrates that deposition of C and PC is influenced by the composition of the incubation solution and that in vitro models must use more physiologically relevant incubation solutions that mimic the natural tear film if in vitro data is to be extrapolated to the in vivo situation.
In vitro models for examining deposition of tear film components onto contact lens materials have been used for many years.1–10 These models have been used to examine the protein-lipid-mucin interactions that occur on the surface and in the tear film;1–14 to examine the conformation of protein on the surface of a contact lens;5,15 to examine the wettability,16 contact angle, and hydrophobicity of a lens material;16,17 and to analyze the interaction between corneal cells and their interaction with contact lens materials and solutions,18–20 to name just a few. Although in vitro models can never incorporate every element of human contact lens wear, they are indispensable for quickly characterizing and assessing new and commercially available materials and cleaning solutions.
The first in vitro models used were simple.1–6,21 These models included contact lens materials being incubated in a single lipid or protein saline solution for as little as 24 h for an extended wear lens. Earlier analysis techniques11,22 have been updated or replaced with increasingly sensitive technology, including high-performance liquid chromatography, mass spectroscopy, radiochemical assessments, and matrix-assisted laser desorption ionization mass spectroscopy. These techniques are just a few of the methods which are able to assess the interaction of various biological fluids with biomaterials, sometimes without extraction, and can often quantify components in the picogram range.
In the last 20 years, in vitro models of contact lens wear have started to become more complex. In the early 1990s, Mirejovsky et al.10 spear-headed this by using a complex artificial tear solution which incorporated lipids, proteins, and mucin dissolved within a multicomponent saline solution. Since then, many researchers have begun adding more physiological chemicals to their in vitro contact lens interaction models. However, still more research needs to be completed to analyze the influence that different variables have on contact lens deposition, comfort, wettability, and vision.
Of interest is the composition of the tear film or the artificial tear solution used for in vitro studies. It is known that even though the general composition of lipids found in the human tear film and meibum are similar,22–28 there are still quite a few individual differences between the specific lipids and their concentrations.29–37 Variations in tear film composition have been recorded between people, and an individual's lifestyle can contribute to the tear film's unique composition. A person's diet, work environment, medication, alcohol consumption, and any systemic disease can all greatly affect their tear film composition.29–37 This poses unique challenges when trying to develop an in vitro model and an artificial tear solution (ATS) for that model. In addition, the cost of the components of the ATS may not justify their inclusion if the results are not impeded by their absence. Therefore, some of the main questions that need to be answered before an in vitro contact lens deposition model incorporating an ATS is finalized are “How do the components of the ATS contribute to the deposition profile and how does altering the complexity of the solution change deposition?”
In the mid to late 1990s, Bontempo and Rapp11–14 conducted a set of experiments analyzing lipid deposition and the effect of lipid and protein interactions on conventional hydrogel and rigid gas-permeable contact lens deposition. Their research found that FDA group III contact lens materials deposit the least lipid and group II lenses, the high water non-ionic lenses, deposit the most lipid.11 This finding led them to develop the “pull/push” theory to explain lipid deposition.11,38 In their theory, the “pull” represents the contact lens polymer material attracting the lipids into the matrix and away from the aqueous ATS and the “push” represents the water content of the lens encouraging the lipid to move into the matrix.11,38 Bontempo and Rapp also found that when both lipids and proteins are present in the ATS, the deposition pattern was different than when the contact lenses were exposed to proteins alone or lipids alone. For example, they found that protein deposition onto group IV contact lens material rendered the surface of the lens less hydrophilic, resulting in a subsequent increase in the deposition of lipids. In contrast, when non-polar lipids bound to the contact lens, the surface became more hydrophobic, resulting in decreased protein deposition.12,13 Differences in the deposition of specific lipids were also found, as more polar lipids deposited differently than non-polar lipids depending on the complexity of the ATS.13
The competitive binding research completed by Bontempo and Rapp was a major contribution to the industry's knowledge of deposition and the relationship between lipids and protein in the tear film and on the surface of a contact lens. However, many years have passed and we now have new contact lens materials based on silicone. These materials behave very differently than conventional hydrogel contact lens materials and therefore more work needs to be completed to examine the competitive binding of tear film components on these materials. Even within the group of silicone hydrogel lenses, many differences exist. Some lenses are surface coated, some have internal wetting agents, some have no coatings or wetting agents, some are based on trimethylsiloxy siloxane derivatives, and some are a combination of new siloxane macromers. Due to the uniqueness of each of these new materials, it cannot be assumed that they will behave as the conventional hydrogel lenses or the same as each other.
The purpose of this study was to examine the competitive binding of tear film components onto various contact lens materials by altering the composition of the ATS being used. A radiochemical in vitro model and an ATS, previously optimized by our laboratory, will be used to examine cholesterol and phosphatidylcholine deposition specifically. To examine the role of different tear film components on deposition, four different tear solution compositions will be examined: a protein + lipid ATS, protein only, lipid only, and single lipid. One conventional and two silicone hydrogel lens materials will be examined.
Artificial Tear Solution
A comprehensive protocol outlining the preparation of the ATS that was used in this experiment has been published elsewhere and therefore only a brief overview will be given here.39 Table 1 displays each of the components of the ATS and the final concentrations that were used. Preparing an ATS solution involved several steps. First, all the salt components were dissolved into the desired volume of MilliQ water to prepare the complex saline solution (CSS). To avoid precipitation of some of the components, all the ingredients were dissolved in the order that they are listed in Table 1. Before a lipid tear solution could be made, all the pure lipids required for the specific experiment were first dissolved into a 1:1 hexane:ether solution at high concentrations. This lipid stock solution was prepared previously and stored at −20°C until needed. Once the lipid stock was prepared and was equilibrated to room temperature, it was used to make a lipid tear solution by dispensing an aliquot of the stock into the required volume of CSS. To evaporate the hexane:ether and to ensure that the lipids were fully incorporated, the lipid solution was sonicated at 37°C while being purged with nitrogen gas. Once the odor of solvent had dissipated, the radioactive lipid was then dispensed into the solution and further sonicated for 15 min. The lipid portion of the ATS was now complete and the proteins and mucin were then dissolved into the solution to create the full ATS. In this experiment, a number of different tear incubation solutions were used and therefore not all these steps were required, therefore the individual steps used were adjusted according to the desired incubation solution. The radioactive lipid details can be found in Table 2.
Contact Lens Materials
Three contact lens materials were examined (n = 4) including Acuvue 2 (etafilcon A; Vistakon), Acuvue® OASYS (senofilcon A; Vistakon), and PureVision (balafilcon A; Bausch & Lomb). The material characteristics of all contact lens materials can be found in Table 3. As can be seen from Table 3, each of these three materials have very different polymer compositions, are grouped into different categories according to their FDA classifications, and have different methods of surface modification, if any. Due to their varied specifications and current popularity in the contact lens market, they were chosen as the three contact lenses of interest for this study.
A flow chart outlining the experimental setup is shown in Fig. 1. 6 ml glass incubation vials (Wheaton, VWR, Mississauga, ON, Canada) were all incubated in non-radioactive ATS for at least 4 days at 37°C before lens incubation. This process allowed lipid and protein to coat the interior surface of the vial. Previous work in our laboratory has found that pretreatment of the vials is a necessary step to ensure that the contact lens incubation solution components do not preferentially adsorb to the vial and are therefore available to deposit onto the lens. This experiment used several different solution formulations for the incubation of lenses. The vial pretreatment solution that was used was matched with the specific incubation solution that was to be used. After pretreatment, the solution was removed, saline was used to rinse the vial, and 6.0 ml of the prepared radioactive solution was inserted.
All contact lens materials were removed from their packaging solutions and then soaked in 12-well plates filled with CSS for at least 24 h to remove components of the individual blister pack solutions. After each lens was soaked, they were rinsed twice in CSS, blotted on lens paper, and placed into their incubation vials that had been filled with one of four different incubation solution compositions: an ATS containing the full complement of lipids and proteins (ATS), a lipid-only tear solution (LTS), a protein-only tear solution (PrTS), and a single lipid tear solution (SLTS). Each of these solutions was tested with each radioactive lipid independently. The details of each of these solutions are outlined in Fig. 1. The vials were capped, sealed with Parafilm, and incubated at 37°C with shaking for 3 or 14 days. Four replicates of each lens material with each of the radioactive solutions were tested.
At the end of incubation times (3 or 14 days), each lens was rinsed twice in CSS to remove loosely bound lipid. For the 14C-cholesterol samples, the lenses were extracted in 20 ml glass scintillation vials each with 2 ml of 2:1 chloroform:methanol extraction solvent and incubated at 37°C for 3 h while shaking at a speed of 60 RPM. Each lens was extracted twice and the extracts were pooled together for processing. For the 14C-phosphatidylcholine samples, the lenses were extracted in 20 ml glass scintillation vials with 2 ml of 60:50:1:4 chloroform:methanol:acetic acid:water extraction solvent and were incubated at 37°C for 3 h while shaking at the same speed. Once again, two extractions were completed and pooled together.
The contact lens extracts were then dried under a steam of nitrogen gas, resuspended in 1 ml of chloroform, sonicated for 1 min, and 10 ml of Ultima Gold F scintillation fluor was dispensed into the vials. The lens extract samples and prepared standard radioactive counts were counted for their radioactive signals using a Beckman-Coulter L6500 liquid scintillation counter. In the experiment, the radioactive lipid was used as a probe, and the ratio of radioactive lipid to non-radioactive lipid in the incubating ATS was kept constant. Therefore, quantification of the total amount of cholesterol and phosphatidylcholine deposited was extrapolated and calculated using standard radioactive lipid calibration curves. Statistical analysis was performed with Statistica 9, using repeated measures analysis of variance (ANOVA) and Tukey's post hoc analysis when required.
The results for cholesterol deposition on each of the three contact lens materials and for each of the four incubation solutions tested are found in Fig. 2 and Table 4. Overall, after 14 days, balafilcon A and senofilcon A lenses deposited the most cholesterol when incubated in the PrTS (5754.73 ± 245.23 and 6353.08 ± 255.19 ng/lens, respectively) and the least in the lipid-only LTS solution (770.19 ± 68.98 and 423.73 ± 84.57 ng/lens, respectively). Etafilcon A deposited the least amount of cholesterol for all incubation solutions.
The results of the repeated measures ANOVA are seen in Table 5. The repeated measures ANOVA results found statistically significant differences between all the variables tested (lens materials, incubation time, and incubation solution) and the interactions between these variables as well.
When examining the specific solutions in which the lenses were incubated, it was found that cholesterol deposition on balafilcon A and senofilcon A followed the order PrTS > SLTS > ATS > LTS after 3 days and PrTS > ATS > SLTS > LTS after 14 days of incubation. However, etafilcon A deposited the most cholesterol when incubated in PrTS > SLTS > LTS > ATS after 3 days and SLTS > PrTS > LTS > ATS after 14 days. Etafilcon A cholesterol deposition was lower and more variable between the time points and solutions. The orders listed above represent the ranking for the overall deposition amounts. However, when the statistical differences between the individual solutions are examined using a Tukey's post hoc analysis (Table 6), it is found that not all solution comparisons were statistically different.
The results for phosphatidylcholine (PC) deposition on each of the three contact lens materials and for each of the four incubation solutions tested are found in Fig. 3 and Table 7. Overall, after 14 days, balafilcon A and senofilcon A lenses deposited the most PC when incubated in the SLTS (581.39 ± 85.32 and 366.95 ± 62.17 ng/lens, respectively) and the least in the lipid-only LTS solution (107.89 ± 34.75 and 114.25 ± 15.03 ng/lens, respectively). Etafilcon A deposited the least amount of PC for all incubation solutions.
The results of the repeated measures ANOVA are seen in Table 8. The repeated measures ANOVA results found statistically significant differences between all the variables tested (lens materials, incubation time, and incubation solution) and the interactions between these variables as well.
When examining the specific solutions in which the lenses were incubated, it was found that PC deposition on balafilcon A and senofilcon A followed the order SLTS > ATS > PrTS > LTS after 3 and 14 days of incubation. However, etafilcon A deposited the most PC when incubated in SLTS > LTS > PrTS > ATS after 3 days and SLTS > ATS > LTS > PrTS after 14 days. Etafilcon A PC deposition was lower and more variable between the time points and solutions. The orders listed above represent the ranking for the overall deposition amounts. However, when the statistical differences between the individual solutions are examined using a Tukey's post hoc analysis (Table 9), it is found that not all solution comparisons are statistically different.
This study was designed to examine lipid binding to contact lenses in the presence or absence of other macromolecules. Specifically, the contact lens competitive binding profiles for cholesterol and phosphatidylcholine were examined by varying the components and complexity of the incubation solutions used through use of a radiochemical carbon-14 in vitro model. The simpler incubation solutions used in this study (SLTS, LTS, and PrTS) were used to represent the complexity and composition of previously used simple incubation solutions dominating in vitro experiments in the literature over the past 20 years,1,6,40–42 whereas the composition and complexity of the more complex ATS was more similar to that of human tear fluid. Many other researchers are now beginning to incorporate a more complex artificial tear fluid into their in vitro models for material, deposition, and solution testing.
The results from this study show quite clearly that experiments performed with simple, moderately complex, or complex incubation solutions will exhibit different deposition results. Cholesterol and phosphatidylcholine behaved differently in their deposition profiles between the four different incubation solutions and between silicone hydrogel and conventional hydrogel contact lens materials. For the two silicone hydrogel contact lens materials, cholesterol deposition was highest when the lenses were incubated in the PrTS followed by the ATS and then SLTS. The lowest deposition was found with the LTS incubation. This implies that the cholesterol is outcompeted for binding sites when it is in the presence of other lipids, but when protein is present in the solution and most likely depositing on the material, protein deposition increases cholesterol deposition, as is seen with the deposition profile in the PrTS and ATS. This is likely occurring because protein deposition and denaturation is making the lens surface more hydrophobic during binding, thus providing additional binding sites for cholesterol. Protein denaturation is thought to be more prevalent on silicone hydrogels than on conventional hydrogel materials, where a much higher percentage of the protein remains in its native state.5,43,44 This trend of increasing non-polar lipid deposition was also seen by Bontempo and Rapp12–14 in their solution composition studies with conventional hydrogel lenses.
When examining the cholesterol deposition on etafilcon A, a different trend was seen. In this case, when incubating in the SLTS solution for 14 days, the greatest amount of lipid was deposited followed closely by the PrTS. Incubation in the LTS deposited significantly less cholesterol, and the ATS incubation deposited only minute amounts of cholesterol. However, the only statistical differences in cholesterol deposition on etafilcon A were found between the SLTS solution and the other three. Therefore, these results imply that cholesterol is easily outcompeted for the hydrophobic binding sites by other non-polar lipids on this material and that protein deposition does more to encourage deposition than other lipids.
When examining the phosphatidylcholine deposition, a different trend of deposition was seen. The highest amount of PC was deposited on all materials using the SLTS solution followed by ATS, PrTS, and the LTS depositing the least amount of PC. This order of deposition was statistically significant for both silicone hydrogel materials, but this was not the case for etafilcon A. For the two silicone hydrogel lens materials, phosphatidylcholine was deposited in the highest masses when it did not have to compete with any other tear film constituents, proteins or lipids alike. PC is in fairly low concentration in the tear film and does not have a strong attraction toward these hydrophobic materials and thus is easily outcompeted by more prevalent, hydrophobic, and attractive lipids and proteins. This is evident when incubating in the LTS, as the other lipids available in the solution restrict the deposition of PC. When PC is surrounded with proteins, as is the case when incubating in the PrTS and ATS, PC deposition decreases when compared with SLTS deposition levels but increases significantly when compared with the LTS. This shows that protein deposition alters the lens surface chemistry so that it is less hydrophilic and thus creates more sites for PC to bind.
Etafilcon A results show a similar trend, with higher deposition of PC occurring when it is the only lipid present; however, no significant differences were seen in deposition masses for the other three solutions, p > 0.05. Therefore, these results show that phosphatidylcholine has little affinity for etafilcon A, especially with other tear film components present.
When the overall deposition relating to lens material is analyzed, it is seen that balafilcon A usually accumulates the most lipid for both cholesterol and PC; however, senofilcon A deposited the most cholesterol when incubating in the PrTS. Balafilcon A's propensity to deposit higher masses of lipid has also been found by other researchers and has been attributed to its polymer composition, polymeric structure, and its plasma oxidation process.44–46 As seen in Table 3, one of the monomeric constituents of balafilcon A is N-vinyl pyrrolidone (NVP),47–50 a monomer known to be lipophilic and a cause of increased lipid deposition for FDA group II conventional hydrogel lens materials12,13,51,52 which incorporate it. In addition to NVP, the incorporation of silicone, a very hydrophobic molecule, also increases the lens' lipophilic nature. To mask the hydrophobic matrix of balafilcon A, the lens is subjected to a plasma oxidation process which converts the surface into silicate.53 However, this does not create a continuous silicate surface but creates silicate “glassy” islands across the entire surface of the lens (Fig. 4A).53 Therefore, there are portions of the polymer surface that have not been converted into silicate and are therefore still relatively hydrophobic. Finally, the balafilcon A material also contains a vast number of pores (Fig. 4B).48,54–57 These pores are classified as macropores and are much larger in size than what is common in other silicone hydrogels as network pores.48,55,56 These macropores are thought to be continuous from the anterior to the posterior surface of the lens material and therefore are another area for lipid to deposit.48,55,56 For a lipid such as cholesterol, which is non-polar, balafilcon A still provides many available hydrophobic sites and thus results in higher levels of cholesterol deposition. When analyzing phosphatidylcholine deposition, balafilcon A is also an ideal deposition matrix. Phosphatidylcholine is an amphiphilic molecule that contains both a hydrophilic “water loving” head group (in this case choline) and a hydrophobic “water hating” tail group which contains two fatty acid chains, one saturated and one unsaturated. This dual nature of PC allows it to then deposit not only on the hydrophobic portions of the surface but also on the more hydrophilic silicate islands.
Senofilcon A is a second-generation silicone hydrogel lens and unlike balafilcon A does not have a silicate surface coating. However, it does have an internal wetting agent incorporated into its polymeric structure which is designed to aid with surface wettability. This wetting agent is a high-molecular-weight molecule of polyvinylpyrrolidone.58 Polyvinylpyrrolidone is a polymer of the monomer NVP which is known to be lipophilic in nature (as discussed with balafilcon A), and thus senofilcon A may also deposit increased amounts of cholesterol and phosphatidylcholine when compared with a conventional hydrogel such as etafilcon A.
Etafilcon A is a FDA group IV conventional hydrogel material composed of poly-2-hydroxyethyl methacrylate (HEMA) and methacrylic acid (MA). Conventional hydrogel materials based on HEMA have a long history of proving to be relatively low lipid depositors, especially when compared with silicone hydrogels. However, ionic materials such etafilcon A tend to deposit large amounts of proteins, specifically lysozyme. This is because the MA in etafilcon A gives the lens material a net negative charge and therefore attracts positively charged lysozyme through electrostatic interactions.3,59,60 These electrostatic interactions cause heavy deposits of lysozyme onto these materials, thus allowing little lipid to deposit in comparison.
Some of the other newly published in vitro lipid articles have quantified higher masses of phospholipids and cholesterol depositing on these materials. For example, in vitro work from Pucker et al.6 and Carney et al.1 both published higher lipid deposition masses than the work presented in this article. However, the experimental procedures in these other in vitro studies had at least one of these variations: altered concentrations of lipids in the ATS, different incubation durations and volumes, the use of different incubation solution compositions, and replenishment of the ATS throughout incubation.1,6,9 Therefore, these alterations in method may help to explain the differences in lipid deposition found.
When the cholesterol and phosphatidylcholine deposition results found in this in vitro experiment are compared with recent ex vivo data, it is seen that the results tend to fall within the same range of deposition. However, direct comparisons should not be made as the wear time of the ex vivo lenses are much longer than the incubation time of this experiment, ex vivo lenses are cleaned nightly, and it would be incorrect to extrapolate the data.46,61 In addition, many of the published ex vivo and in vitro studies on lipid deposition have quantified different lipid species and examined different contact lens materials than those in this study.
The use of a more complex ATS for in vitro contact lens studies has been a major topic of discussion for many years, specifically at contact lens conferences over the past year or so. Many researchers believe a more complex ATS better represents the composition of the natural tear film and will better predict and model lipid deposition during an in vitro experiment. However, the cost of creating a more complex tear fluid can be high and therefore the question becomes “Is the increase in ATS complexity worth the cost and will it impact the in vitro deposition of the tear film components?”
The experiment designed and described in this article examined the effect that the ATS composition had on the resulting deposition of two different model lipids. The four different tear solution compositions that were examined in this study were meant to represent the main solution compositions and complexities often utilized in previous literature (simple, moderate, and complex) and have allowed us to examine the impact that various individual components have on deposition.
The results from this experiment show that the composition of the incubation solution, the lipids under examination, length of incubation, and the lenses used will all have an influence on the overall deposition profile. The interactions between the components of the tear film and the contact lens surface will dictate the deposition that occurs. If in vitro models are really meant to mimic in vivo conditions then it is imperative that more complex models are utilized and that every attempt is made to make the in vitro conditions as similar as possible to human contact lens wear. It is only by completing these types of experiments that we can improve an in vitro model's usefulness and systematically explore the relationships that are occurring during human contact lens wear and then test and incorporate them into the in vitro models.
This in vitro study demonstrates that cholesterol and phosphatidylcholine deposition is cumulative over time and that silicone hydrogel materials deposit more lipid than a conventional hydrogel FDA group IV material. It also clearly demonstrates that deposition of cholesterol and phosphatidylcholine is influenced by the composition of the incubation medium. Specifically, cholesterol exhibited significant increases in deposition with protein-rich incubation solutions; however, significant competition with other lipids decreased deposition in the lipid-rich LTS and ATS solutions. Phosphatidylcholine deposited extremely well when it was the only component in the incubation solution, only moderately well with a protein-rich ATS and ATS solutions, and very poorly when it competes with other lipids in the LTS.
These results prove that in vitro models must use more physiologically relevant incubation solutions that mimic the natural tear film if in vitro data is to be extrapolated to the in vivo situation.
Centre for Contact Lens Research
School of Optometry
University of Waterloo
200, University Avenue West
Waterloo, Ontario, Canada N2L 3G1
The authors thank Dr. Zoya Leonenko for the use of AFM and Liz Drolle for assistance with imaging. Lyndon Jones has received funding over the past 3 years from the following companies which are either directly involved in products used in this manuscript or involved in the manufacture of competing products: Alcon, AMO, B&L, CIBA Vision, CooperVision, and Johnson & Johnson.
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lipid; deposition; contact lens; tear film; artificial tear solution
© 2012 American Academy of Optometry
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