Dialysis, Albumin Binding, and Competitive Binding: A Laboratory

Laboratory Experiment pubs.acs.org/jchemeduc

Dialysis, Albumin Binding, and Competitive Binding: A Laboratory Lesson Relating Three Chemical Concepts to Healthcare Jennifer P. Domingo,† Mohammed Abualia,† Diana Barragan,† Lianne Schroeder,† Donald J. Wink,† Maripat King,‡ and Ginevra A. Clark*,† †

Department of Chemistry, University of Illinois, Chicago, MC-111, 845 W. Taylor Street, Chicago, Illinois 60607, United States College of Nursing, University of Illinois, Chicago, MC-802, 845 S. Damen Street, Chicago, Illinois 60612, United States

‡

S Supporting Information *

ABSTRACT: Introductory Chemistry laboratories must go beyond “cookbook” methods to illustrate how chemistry concepts apply to complex, real-world problems. In our case, we are preparing students to use their chemistry knowledge in the healthcare profession. The experiment described here explicitly models three important chemical concepts: dialysis of small molecules (dye), reversible binding (dye binding to albumin), and competitive binding (dye and a competitor binding to albumin). Moreover, each concept is intimately related to a physiological phenomenon: dialysis is used to treat renal failure, drugs travel in the blood bound to albumin, and competitive albumin binding is a common drug− drug interaction. In the context of this simple series of experiments, students create models, use evidence to validate their models, and finally use their understanding to describe physiological phenomena. This laboratory experiment was implemented in a 100level course for predominantly prenursing majors. Student pre- and postlab models were examined, illustrating an improved conceptual understanding upon performing the lab and use of evidence to improve or support models. This experiment can be performed in 1 h, and can be adapted as a lecture demonstration. KEYWORDS: General Public, First Year Undergraduate/General, High School/Introductory Chemistry, Biochemistry, Laboratory Instruction, Inquiry Based/Discovery Learning, Equilibrium, Membranes, Proteins/Peptides, Drugs/Pharmaceuticals

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INTRODUCTION In transforming the undergraduate curriculum to a more student-centered approach, it is important to consider the goals and interests of the students. Students preparing to enter health-related fields may not understand how their chemistry instruction is relevant to healthcare, or understand foundational chemistry topics well enough to apply them. By developing laboratories that illustrate biological systems, then asking students to develop their own models and understandings, we hope to bridge the gap between chemistry content knowledge and student career interests. This experiment models three concepts that have been identified as foundational in nursing practice: dialysis, reversible binding, and competitive binding. Several studies have surveyed nursing faculty, students, and practitioners to identify concepts that should be taught in chemistry courses.1,2 Dialysis consistently ranks highly, yet few lessons have been developed to clearly illustrate the concepts of dialysis,3 or provide insight for students as to why these concepts are relevant in healthcare. This experiment provides a simple model for dialysis, which is related to kidney function and several other processes in human health. Further, in order for hydrophobic drugs to travel in the bloodstream, they bind to the serum protein albumin.4 In the bound state, albumin acts as a reservoir; in the unbound state, © XXXX American Chemical Society and Division of Chemical Education, Inc.

drugs are able to pass through small pores in the capillary and reach their targets.4 Finally, competitive albumin binding is a common reason for drug−drug incompatibility. For example, sulfonamide antibiotics should not be taken with blood thinners, as competitive binding can alter the effective concentration of either or both drugs in the bloodstream. This can result in high effective concentrations of blood thinner, which can contribute to major bleeding. In this experiment, students perform dialysis of a dye molecule in the presence or absence of albumin and in the presence of albumin with a competitive binder in order to model these three phenomena. This lab is part of a series of laboratories we are developing to teach fundamental concepts in chemistry that meet the needs of prehealth students using the MORE framework.5 The curriculum is being developed with best practices in mind: explicitly stating conceptual goals, including reflective prompts, and allowing students time for discussion.6 MORE, developed by Rickey and co-workers,7 asks students to model a system of interest in both the macroscopic and molecular levels. In lab, Received: February 17, 2017 Revised: May 16, 2017

A

DOI: 10.1021/acs.jchemed.7b00131 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

students observe the system. On the basis of their observations, students ref lect on their model, using evidence collected in lab to refine their model. The students then explain the model in general terms, or in relation to a similar system. This emphasis on connecting a molecular-level understanding to observation is consistent with many other important curriculum reform efforts. We are using design based research (DBR) to develop these laboratory experiments, and will describe a round of laboratory revision.8 DBR methodologies are based on the design, implementation, and evaluation of learning tools within a specific instructional context. This approach allows us to better describe and understand some learning challenges and opportunities within our efforts. We have previously reported on the importance of in-lab prompts asking students to make specific observations, reflect upon their models, and compare results and models with other groups.5 We will describe how we included similar prompts in this lab.

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Figure 2. Blank vial contains only the buffer solution and is colorless. The other samples show experimental results after incubation with a dialysis tube containing BCG; BCG and albumin; or BCG, albumin, and oleic acid. The dialysis tubing was removed for clearer viewing of results.

METHODS

Box 1. Prelab Question for the First Experimental Setup

Experimental Procedures

Draw a molecular-level and macroscopic picture of what will happen when you put a Bromocresol Green (BCG) solution into a dialysis tube and then place the tube in a buffer solution. What will you observe at the beginning of the experiment? What will you observe over time if your molecular-level picture is correct?

Students perform three experiments and observe the rate at which bromocresol green (BCG) passes through dialysis tubing into an external buffer solution (Figure 1).9,10 BCG is a blue

postlab models. From these models, students are asked to apply their understanding to drug−drug interactions. Description of Implementation

This laboratory was implemented in a one-semester Survey of Organic and Biochemistry course for predominantly prenursing students who have taken one semester of General Chemistry at a large, urban, state university in the midwest United States. The General Chemistry program at this institution has equilibrium content in the first semester. In each week of the 15-week semester of our course, students attend 2.5 h of lecture, 1 h of discussion with a TA, and one 3 h lab with a TA. The population of students at this university is roughly 40% underrepresented minorities, with an average ACT score very close to the US average. In both implementations, students were provided a prelab video describing the relevant content, which was presented by a coauthor who is a nursing faculty member (M. King) using a script codeveloped by the authors.11 The video is approximately 1 min long and is presented by M. King. It describes the important connections between this lab and nursing. The video also presents models of dialysis and albumin binding that may be beneficial for students. Students performed this lab with lab partners. Collection and analysis of student work was performed in accordance with a protocol approved by the university IRB (Internal Review Board). To evaluate learning, we studied outcome measures for which activities students performed correctly in the prelab and postlab. In particular, we determined if macroscopic and molecular-level representations are present and are correct for each of the three experiments. We evaluated student postlab responses for the use of evidence to support their models. We also evaluated student responses to specific prompts regarding reversibility of albumin binding. We present data from two semesters of implementation. In the first semester (Spring of 2016, S16), a total of 32 lab reports

Figure 1. Students observe dye movement of BCG molecules from the dialysis tubing to the external buffer solution.

dye at the pH of this experiment, and diffusion of BCG through the capillary membrane leads to a visible color change in the surrounding buffer solution after about 40 min (Figures 1and 2). For the second experiment, the dialysis tube contains BCG and albumin (Figure 2). Albumin binds reversibly to BCG, and the BCG−albumin complex is too large to pass through the pores of the dialysis tube. Thus, the surrounding buffer solution turns only slightly blue after 40 min. For the final experiment, the dialysis tube contains the BCG, albumin, and oleic acid (Figure 2). Oleic acid competitively binds to albumin, so less BCG is bound to the albumin. This allows BCG to pass through the dialysis tube more rapidly than in the absence of oleic acid. Application of Frameworks and Pedagogy

In alignment with the MORE pedagogy, students were asked to draw prelab macroscopic and molecular-level models for each of the three experimental setups. The prelab prompt for the BCG alone is given in Box 1. Corollary prompts were given for the other two experimental setups. During the lab, students use their observations as evidence to support and/or refine their B




from five different lab sections are analyzed. The coders achieved an inter-rater reliability of 75% or greater for all items. Coding was performed using constant comparison, selective coding. Operational definitions of the codes were defined through discussion between coders, resulting in a detailed rubric. The coding scheme developed for S16 was then applied to analyze 40 lab reports collected in the second semester (Fall of 2016, F16). Coders achieved an inter-rater reliability of 79% or greater on all items. Differences between the remaining items were resolved through discussion between coders for both the S16 and F16 data sets. Differences between the prelab and postlab were evaluated for significance using a Z-test for two population proportions. The laboratories collected in F16 were from six lab sections with four different TAs. All four TAs had previous TA experience, and two of the TAs had previous experience with this course.

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Box 2. Pre/Postlab Prompts Regarding Reversibility Prelab Prompt Drugs are present in the bloodstream either bound to albumin or in the free state. Explain why equilibrium between the bound and unbound state is required for proper drug function. What is the role of the bound state? Unbound state? Postlab Prompt If two drugs compete for albumin binding, how will that impact drug delivery? Can this result in an effective drug concentration being too high? Table 1. Percent of Students with Correct Pre- and Postlabs for Each Experiment BCG Only

RESULTS

Description Molecular-level correct Totally correct

Student Performance and Revision History

Students were able to perform the experiment with few errors. In an earlier implementation, students were not told how long to wait for the dialysis to occur and did not observe any color changes because they would terminate the experiment prematurely. Therefore, we included a data table with observations at 40 and 50 min. In our S16 implementation, we found that students did not successfully observe the relatively subtle change in color for the “BCG and albumin” setup. Further, only 3% of students clearly illustrated the reversibility of BCG−albumin binding in their models, even after the experiment. Students’ descriptions of drug−drug interactions were vague and often taken directly from the lab handout. For some students, a belief emerged that oleic acid was required to displace the BCG from the albumin, and therefore, some students viewed the drug−drug interactions as beneficial in physiological settings. For example, one student wrote: Drugs sometimes bind to albumin in the blood and then the drug will be too large to easily cross the membrane, which would keep the drug/albumin concentration in the bloodstream and not allow it to be absorbed. That is what BCG is doing in this lab; without albumin BCG can cross freely which is what the BCG and buffer showed. But in the presence of albumin you can add [oleic acid] to allow for transport across the membrane. For this reason, we modified the lab for our F16 implementation. We asked students to compare their BCG/ albumin sample to a blank, and directed students to look carefully for minor changes. We further asked students to compare their results with those of another group. We included prompts in the prelab, lab, and postlab asking students to consider the reversibility of binding (see Box 2). In this revision, we also removed a figure from the lab handout, with the aim of encouraging students to prepare models that were more authentic. The figure appears briefly in the prelab video. The laboratory handouts from both implementations are provided in the Supporting Information. Results from S16 are provided in the Supporting Information (Table 1), and results from the F16 refined implementation are provided in Table 1. Note that a lower percentage of students created correct final models in the F16 implementation than in the S16 implementation. For example, 65% of “BCG, albumin, and oleic acid” models were correct in S16, and only 55% were

BCG and Albumin

BCG, Albumin, and Oleic Acid

Pre (%)

Post (%)

Pre (%)

Post (%)

Pre (%)

Post (%)

40

15

20

13

25

10

22

65a

22

55a

15

55a

Statistically significant improvement from pre- to postlab. (p ≤ 0.005), n = 40. a

correct in F16. As stated in the previous paragraph, we removed content from the lab handout to further challenge students in making their models. Students in the F16 class performed better on other learning outcomes, such as incorporating reversibility in their models, as intended by our modification to the lab handout. Differences in student populations and TA assignments are limitations of our study. Different TAs were assigned to the course from S16 to F16, which could account for differences in performance. Also, ACT scores were significantly higher for students in S16 than F16. Learning Outcomes

In both S16 and F16 implementations, student data shows a significant (p ≤ 0.005), improvement in models from pre- to postlab on all three models (Table 1, Supporting Information Table 1), but the remainder of our analysis will focus on results from F16. In F16, we observed that initial models frequently lacked a macroscopic prediction. For example, in the case of “BCG only”, 40% of students correctly illustrated the passage of dye molecules to the external solution, but did not indicate the corresponding color change in the prelab (Table 1). For the “BCG only” experiment, 22% of students’ prelab models were completely correct, while 75% are completely correct in the postlab. Supporting Information Figure 3 provides a sample of student work that lacked a macroscopic description and was corrected in the post lab. In the two experimental setups using albumin, 55% of final models are correct, suggesting that student success decreases as model complexity increases. Interestingly, many student prelab models related to other phenomena that could be reasonably associated with these molecules. In Figure 3, we show work from a student who considered how the difference in osmotic pressure could alter the solution volume inside the dialysis tube. This does occur, but it requires 24 h for an observable difference (data not shown). This student was able to improve their postlab with improved molecular-level representations and the use of evidence to support their model. Other students (e.g., Supporting Information Figure 4) considered proton transfer between BCG and albumin, which does occur at lower pH C




Figure 3. This student initially made no macroscopic predictions in any of their prelab models. Their models become more detailed once they used evidence from the lab. Students’ written descriptions were typed in the figure. Student work reproduced with permission.

values, leading to a color change in the dye.9 Students were exposed to this phenomenon in the prerequisite General Chemistry course, so the inclusion of this idea in prelab models in not surprising.9 In addition to coding model correctness, we coded models to determine the percentage of students who used evidence to support their models and included reversibility in their model for the “BCG and albumin” experimental setup (Table 2). We

have influenced their models. This result can likely be improved with further TA guidance, as some students did not report the use of a blank or comparison with other groups. Students also can be instructed to wait longer if have not yet seen a color change. Further, we coded student responses to determine the percentage of students who correctly answered a prelab prompt regarding reversibility and a postlab prompt regarding competitive binding (Box 2 and Table 2). The pre- and postlab prompts on reversibility and competitive binding are provided in Box 2. We found that 52% of students correctly described reversibility in the prelab, and 45% of students correctly described competitive binding in their postlab (Table 2). Thus, our revision was successful at drawing student attention to the importance of reversibility. For example, one student wrote: Equilibrium between bound and unbound state is required for proper drug function, because the bound state will stick to the albumin. While the unbound state will be free to pass... But once the unbound molecules have passed, some of the bound will be slowly released and will be able to pass through. Despite weekly meetings with the instructor and detailed TA notes, we observed that the lab TA had a significant impact on student performance. As shown in Table 3, the students assigned to TAs A and B significantly outperformed students assigned to TAs C and D. While differences were observed in all areas, Table 3 shows significant TA-related differences in three key areas: use of evidence to support models, correctness

Table 2. Percentage of Students Who Use Evidence or Illustrate Understandings for Various Promptsa Description

Prelab Reversibility

Cite Evidence

Reversibility in Model

Postlab Competitive Binding

% of students

52%

55%

25%

45%

a

n = 40.

found that, in the postlab, 55% of students cite evidence from the experiment to improve or support their model (Table 2). Thus, students are able to use the experimental evidence and corresponding discussions with peers and the TA to improve their models. In our F16 implementation we sought to improve student understanding of the reversibility of dye binding and help students attend to the slow release of dye in the case of “BCG and albumin”. In this implementation, 25% of students incorporated reversible binding into their models (Table 2). We noticed that many students still did not observe the slight color change in the case of “BCG and albumin”, which may D




(3) Ribeiro, I. A. C.; Faustino, C. M. C.; Guedes, R. C.; Alfaia, A. J. I.; Ribeiro, M. H. L. Exploring drug diffusion through a membrane: A physical chemistry experiment for health and life sciences undergraduate students. J. Chem. Educ. 2015, 92 (5), 924−927. (4) Lehne, R. Pharmacology for Nursing Care; Elsevier: St. Louis, MO, 2003. (5) Abualia, M.; Schroeder, L.; Garcia, M.; Daubenmire, P. L.; Wink, D. J.; Clark, G. A. Connecting Protein Structure to Intermolecular Interactions: A Computer Modeling Laboratory. J. Chem. Educ. 2016, 93 (8), 1353−1363. (6) DeKorver, B. K.; Towns, M. H. General Chemistry Students’ Goals for Chemistry Laboratory Coursework. J. Chem. Educ. 2015, 92 (12), 2031−2037. (7) Rickey, D.; Teichert, M. A.; Tien, L. T.; Pienta, N. J.; Cooper, M. M.; Greenbowe, T. J. Model-Observe-Reflect-Explain (MORE) Thinking Frame Instruction: Promoting Reflective Laboratory Experiences to Improve Understanding of Chemistry. In Chemists Guide to Effective Teaching, Vol II; Pearson Prentice Hall: Upper Saddle River, NJ, 2008; Vol. 2. (8) The Design-Based Research, C.. Design-Based Research: An Emerging Paradigm for Educational Inquiry. Educ. Res. 2003, 32 (1), 5−8. (9) Sohl, J. L.; Splittgerber, A. G. The binding of coomassie brilliant blue to bovine serum albumin - A physical biochemistry experiment. J. Chem. Educ. 1991, 68 (3), 262. (10) Suzuki, M. Applications and analogies; The movement of molecules and heat energy two demonstrative experiments. J. Chem. Educ. 1993, 70 (10), 821−822. (11) Clark, G. A., 2016. vimeo.com/162659020. (12) Velasco, J. B.; Knedeisen, A.; Xue, D.; Vickrey, T. L.; Abebe, M.; Stains, M. Characterizing instructional practices in the laboratory: The laboratory observation protocol for undergraduate STEM. J. Chem. Educ. 2016, 93 (7), 1191−1203. (13) Luft, J. A.; Kurdziel, J. P.; Roehrig, G. H.; Turner, J. Growing a Garden without Water: Graduate Teaching Assistants in Introductory Science Laboratories at a Doctoral/Research University. J. Res. Sci. Teach. 2004, 41 (3), 211−233.

Table 3. Comparison of TA Effects: Percentage of Students Who Cite Evidence in Their Postlab, Correctly Model BCG and Albumin, and Include Reversibility in Their Model of BCG and Albumin, Grouped by TA, F16 Description TAs A and Ba TAs C and Db

Cite Evidence

BCG and Albumin Model Correct

Reversibility in Model

77%c

77%c

41%d

27%

27%

5%

a

n = 22. bn = 18. cStatistically significant difference between TA groupings, F16 (p ≤ 0.005). dp = 0.010.

of the model for “BCG and albumin”, and the inclusion of reversibility in the model for “BCG and albumin”. This result illustrates both an opportunity and potential pitfall, as careful implementation can improve students’ outcomes. Further examination of the role of the TA is beyond the scope of this study.12,13

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CONCLUSIONS Using the MORE framework, students were able to use evidence to not only refine their models but also relate models to drug distribution. With lab refinement, students displayed an understanding of the concepts of reversible binding and competitive binding. Several ideas are discussed to improve implementation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.7b00131. Table of student outcomes form S16, three additional examples of student work, teaching notes, laboratory prep sheet, and laboratory handouts from F16 and S16 (PDF, DOCX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Ginevra A. Clark: 0000-0003-3065-2627 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The material is based on work supported by the National Science Foundation under Grant DUE-1431926. The authors would like to thank the teaching assistants who have participated in this study for their insights and efforts. The authors would further like to thank the students who have performed this lab.

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REFERENCES

(1) Brown, C. E.; Henry, M. L. M.; Barbera, J.; Hyslop, R. M. A Bridge between Two Cultures: Uncovering the Chemistry Concepts Relevant to the Nursing Clinical Practice. J. Chem. Educ. 2012, 89 (9), 1114−1121. (2) Walhout, J. S.; Heinschel, J. Views of nursing professionals on chemistry course content for nursing education. J. Chem. Educ. 1992, 69 (6), 483−487. E


Dialysis, Albumin Binding, and Competitive Binding: A Laboratory

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