A proposal for teaching undergraduate chemistry students carbohydrate biochemistry by problem-based learning activities

Authors

  • Angela C. M. Figueira,

    Corresponding author
    1. Departamento de Química – Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil
    • Address for correpondence to: Departamento de Química – Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil. E-mail: qmcfigueira@gmail.com

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  • Joao B. T. Rocha

    Corresponding author
    1. Departamento de Química – Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil
    • Address for correpondence to: Departamento de Química – Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil. E-mail: qmcfigueira@gmail.com

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Abstract

This article presents a problem-based learning (PBL) approach to teaching elementary biochemistry to undergraduate students. The activity was based on “the foods we eat.” It was used to engage students' curiosity and to initiate learning about a subject that could be used by the future teachers in the high school. The experimental activities (8–12 hours) were related to the questions: (i) what does the Benedict's Reagent detect? and (ii) What is determined by glucose oxidase (GOD)? We also ask the students to compare the results with those obtained with the Lugol reagent, which detects starch. Usually, students inferred that the Benedict reagent detects reducing sugars, while GOD could be used to detect glucose. However, in GOD assay, an open question was left, because the results could be due to contamination of the sugars (particularly galactose) with glucose. Though not stressed, GOD does not oxidize the carbohydrates tested and all the positive results are due to contamination. The activities presented here can be easily done in the high school, because they are simple and non-expensive. Furthermore, in the case of Benedict reaction, it is possible to follow the reduction of Cu (II) “macroscopically” by following the formation of the brick-orange precipitate. The concrete observation of a chemical reaction can motivate and facilitate students understanding about chemistry of life. © 2013 by The International Union of Biochemistry and Molecular Biology, 42(1):81–87, 2014

Abbreviations
PBL

problem-based learning

GOD

glucose oxidase

Introduction

Experimental activities can have an important impact in relation to basic and higher education efficacy. However, the effectiveness of activities of the type “cake recipe” is questionable. In fact, in the “follow-the-steps” of the recipe the student receives protocols with all the steps to be followed and usually the expected results and conclusions are given before or on the spot during the activity. This type of activity leaves few rooms for further discussion and interpretation. Most importantly, it weakens the power of observation, which is the first step to acknowledge or perceive natural phenomena. The high prevalence of this type of well-structured activity during the training of future teachers can give the idea that science is consisted of absolute truths and teleological knowledge, spreading a teaching methodology that is repetitive, and mechanical. This traditional approach normally creates a stereotyped view of the science and does not motivate the students toward science [1-3].

Consequently, the simple use of practical classes does not ensure that students take ownership of knowledge taught, it is necessary that students feel motivated to learn [4]. In an attempting to improve this situation, 10 years ago, we have changed the activities of Experimental Biochemistry discipline to a PBL approach. Since the students of this Chemistry Course will be teachers of the elementary and high-school, we focused all the practical activities in the subject “What we eat and what we drink?” due to its presence in our daily lives and simplicity of obtaining material to work in the classes.

Problem-based learning is an instructional method that challenges students to “learn to learn,” working cooperatively in groups to seek solutions to real world problems [5]. These problems are used to engage students' curiosity and initiate learning of the subject matter. At its most fundamental level, PBL is characterized by the use of “real world” problems as a context for students to learn critical thinking and problem solving skills, and acquire knowledge of the essential concepts of the course [6]. Using PBL, students acquire lifelong learning skills that include the ability to find and use appropriate learning resources [7, 8], according to the same author, “The principal idea behind PBL is that the starting point for learning should be a problem, a query, or a puzzle that the learner wishes to solve.”

The activities reported here have as central theme “What we eat and what we drink?.” The study of carbohydrates is the third topic of the subject during the semester (more details will be presented in the “Material and Methods” section). To put the activities used here in the context of the subject, we now present some explanation about the previous activity developed throughout the semester. The first issue addressed was “The Lambert-Beer's law,” where the students have to determine by eye (and later by normal colorimetric procedures) the quantity of methylene blue in an unknown sample (for details, see [9]). The second topic is “What does the Biuret Reagent detect?,” some aspects about the use of a PBL in teaching protein determination can be found in [10]. Basically, in these classes, students were presented with different kinds of common foods and were asked “What is Biuret detecting?” Normally, based on the results of white egg and some other foods rich in protein (like fish and chicken meat; the cow meat normally gave no clear results because of the interference of the redness of myoglobin), they concluded that Biuret is a protein detector. Here we have tried to address the issue of elementary carbohydrate chemistry starting from the macroscopic chemical behavior of typical monosaccharides found in foods, using the classical Benedict reagent (i.e. Cu [II] reduction by mono- and disaccharides in alkaline medium). Furthermore, we also made studies with Glucose Oxidase (GOD) to introduce questions related to the influence of sugars structure and their interaction with an enzyme.

Materials and Methods

This module for teaching carbohydrates has been applied to 20 classes of courses in Chemistry and Biology of Federal University of Santa Maria in the Experimental Biochemistry discipline, totaling approximately 400 students, which had already attended to theoretical introductory classes of basic biochemistry (60–90 hours) and were attending the last year of Biology or Chemistry course. Here we will give emphasis to the activities carried out in the second half of 2011, with a total of 20 students enrolled. It was a representative semester and the results and activities were similar to those carried out in the previous and subsequent semesters. The activities require two to three classes of four hours each, depending on the particularity of the group and the degree of interference of the teacher.

Reagents Preparation

Benedict Reagent was prepared by dissolving 17.3 g of copper sulfate, 173 g of sodium citrate, and 100 g of anhydrous sodium carbonate in water separately. They were then stirred and the volume was completed to 1000 mL. The solution was filtered and stored in an amber bottle.

Lugol Reagent was prepared by adding 0.5 g of iodine and 1.0 g of potassium iodide to 10 mL of water. This mixture was stirred until the complete dissolution of solutes.

What does the Benedict Reagent Detect?

The first class was divided into two parts. In the first section, we asked: “To what we use the Benedict's reagent?” To answer this question, pair of students received the Benedict's reagent and food samples (soybean oil, wheat flour, lemon soda, cola light, regular cola, beer, corn starch, sugar, milk, common salt, etc). After an initial discussion with the instructor, students were advised to use only 1 mL of Benedict reagent to avoid generating residues and wasting of reagent unnecessarily. After that, students had to decide the quantities of samples and whether or not they should homogenize them. As a rule, the instructor told students to avoid using an excessive amount of food samples and to adapt them to the volume of reagent used. However, students decided by themselves whether or not they should homogenize the dry samples with water before putting the Benedict reagent. In the case of food samples that do not exhibit a good solubility with aqueous solution (for instance, starch or corn or wheat flour), the instructor always asked students about the area of contact and how this could interfere with the results. Then they were asked to keep the tubes at room temperature for 30 min and observe what was going on in each tube. Subsequently the tubes were heated in boiling water bath for about 15 minutes. Students were always and constantly asked to look at the tube to observe any detectable change in the tubes. Typically, most students conclude that Benedict's reagent can be used to detect sugar, due to the fact that the samples containing sugar (biscuit, normal but not light soda, etc) have an orange precipitate. Regarding the sugar (i.e. sucrose), the results varied probably depending on the degree of contamination with glucose or fructose in the samples used. From the pedagogical point of view, teachers should be careful with the use of sucrose, because some commercial sugars did not form the precipitate. In fact, this would be the correct result for pure sucrose, which is not a reducing sugar. For undergraduate students, these technical details can be easily handled during the classes. However, in the elementary level, it can be a confounding factor that will require additional time to be solved. The absence of the reducing power of sucrose molecule can be easily explained by analyzing their Fisher structures in comparison with glucose and fructose (Fig. 1). Therefore, to stimulate students to formulate hypothesis to explain why pure sucrose (which is a dimer of glucose and fructose) does not react with Benedict reagent, whereas glucose or fructose react, they were asked to observe the chemical structures of these sugars. In fact, at this point we always asked the students to search for and observe the Fischer's and Haworth's structures of different sugars.

Figure 1.

Fischer's and Haworth's structures of common sugars.

Figure 2.

Cyclization of planar structure (Fisher structure) of glucose and fructose forming the Hemiacetal Intermediate of Glucose (glucopyranose) and fructose (fructofuranose). Fischer and Haworth structures are shown in the center and on the right, respectively. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

After the initial open activity (where the students were allowed to select the majority of the food to be tested), students were then asked to repeat the previous procedure, but with the following samples: fructose, starch, alanine, aspartic acid, sucrose, galactose, glucose, lactose, and maltose. After doing experiment with these samples, the majority of students realized that sugars (other than sucrose) are reducing agents. Moreover, some students were able to observe that fructose reacts with the reagent of Benedict even without heating the solution (15–30 minutes). Indeed, depending on the quantity of fructose put in a given tube, the orange precipitate can be observed easily. These results can be used to discuss the functional groups present in sugars and the reactivity of aldehyde (glucose, galactose, lactose) compared with ketone group found in fructose.

The general conclusion reached by students was that a reducing sugar is any sugar that has either an open-chain form with a free aldehyde, a free hemiacetal or a free ketone group (as in the case of fructose; see Fig. 1 and 2).

Accordingly, the aldehyde functional group of simple carbohydrates gives to molecule the reducing power in alkaline and hot medium. All monosaccharides and some disaccharides (e.g. maltose) are reducing sugars. One of the most simple tests for detecting reducing sugars is the Benedict's test [11], where reducing sugars are heated with an alkaline solution of copper (II) sulfate forming an insoluble precipitate of copper (I) oxide. The color of the precipitate changes from green to yellow, orange, brown or strong red depending on the quantity of reducing sugar present.

Testing Lugol's Solution

Another important carbohydrate present in our diet is the starch, a polysaccharide that is found in many parts of a plant in the form of small granules or grains, for example, starch grain in chloroplasts. Especially large amount occur in seeds and storage organs such as potato tubes. Starch is easily detected by the Lugol's reagent [12]. In this reaction, the transparent and slight brown to yellow color of iodine present in the potassium iodide solution turns into a blue-black color after interacting with starch molecules. The changes in color of the Lugol solution are due to the absorption of iodine molecules in the centre of the helix of starch polymer molecules. It is important to carry out this reaction at room temperature, because at high temperatures the starch helix can unwind, releasing the iodine. Consequently, the dark-blue color of Lugol returns to its transparent yellow-to-brown coloration seen in the absence of starch.

Considering the importance of starch in our diet, we usually complement the studies on sugars by asking students to repeat the tests performed earlier with Lugol reagent. The students are asked to put foods in test tubes as they did before for Benedict reaction and to add 1 mL of Lugol solution per tube. We then inform the students that it is not necessary to keep the tubes in a water bath and ask them to compare results obtained with Lugol to those of reaction with Benedict.

What can be Detected with Glucose Oxidase (GOD)?

Aiming to encourage observation of formulas and also to introduce the issue of three-dimensionality in biomolecules, we introduce students to Glucose Oxidase (GOD) and ask: “What is the purpose of GOD?” Students are asked to choose some foods, including different types of soda (normal, i.e. with sugar and light or diet with no added sugar). Food samples were placed in a test tube and 0.5 mL of GOD solution was added. The GOD solution consisted of 30 mmol/L phosphate buffer (pH 7.5); 1 mol/L phenol; glucose oxidase (12.5 U/mL), peroxidase (0.8 U/mL); 0.29 mmol/L 4-aminoantipyrine, and 7.5 mmol/L sodium azide. It is important to note that the color of samples may interfere with visualization of the results, for example, in cola soda, its dark color masks the positive test for glucose. This type of problem does not occur with lemon soda, which is colorless. In this case, if teacher wants to do a more directed activity, only colorless soda must be used. The general conclusion of the reaction of foods in the GOD test is that GOD detects sugars. The differences between normal and light or diet soft drinks are usually well perceived by the students. After this activity, students received samples of glucose, galactose, lactose, sucrose, and fructose, which should be added to the test tubes and added to 0.5 mL of GOD. The time course of color development was asked to be observed and recorded (and to rank the sugars by color intensity of reaction), first at room temperature and then in a water bath for about 15 minutes at 37°C. Furthermore, at the end of the class students were also asked to check the color of the tubes. Occasionally, they left the tubes all the time at room temperature to follow the reaction by eye as a function of time. Here the teachers must be aware of the influence of room temperature on the velocity of color appearance with glucose (positive control), because in the winter it took more time to see the appearance of the pink color than in the summer.

At the end of each lesson students have to present their results to the whole class as well as to do written reports in their laboratory notebooks, which are evaluated at the end of the semester and used to give the students grades. It is important to highlight that no lectures or protocols to be followed are given to students. All reagents were previously prepared by the laboratory technicians.

The Glucose Oxidase

Glucose oxidase is the most widely employed analytical enzymatic method for detecting glucose in clinical analysis laboratories. The GOD catalyzes the oxidation of glucose according to the following reaction [13]:

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The hydrogen peroxide formed reacts with 4-aminoantipyrine, under catalytic action of peroxidase, forming an antipirilquinonimine (oxidezed form of 4-aminoantipyrine), a pink to red substance which intensity is proportional to the concentration of glucose in samples.

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The use of this method is interesting because in addition to the experimental work, it is possible to discuss theoretical aspects related to enzyme chemistry and catalysis. In fact, questions about the tridimensionality of the active center and also the question of enzyme specificity can be discussed here, comparing the activity of GOD with the structures of the sugars tested. The question of galactose contamination can be discussed in terms on how we can do provisional interpretation of our experimental data. In fact, at the end of the semesters where we did not have ultrapure galactose, we concluded and emphasized that we can speculate that GOD might be oxidizing aldehyde groups located in sugars which has a difference in the spatial distribution of [BOND]OH of a given carbon. In the specific case of galactose, it is carbon 4. However, since the purity of the analytical reagent is about 97–99%, the color observed could be due to the presence of glucose as a major contaminant of galactose. This discussion was done in the cases where we have used the ultrapure reagent. With the use of ultrapure galactose, it became clear that the false positive reaction with GOD was due to glucose contamination of common galactose. Based on these questions, we are now planning, for the next academic semesters, to ask students to look at the review articles of [14] where they cite the literature showing that GOD only oxidizes glucose to create the habit in the students to confront their results with the data of the literature. Although this activity can seem trivial, students do not have the habit of reading papers in English and possibly only few (if any) has habit of confront experimental observations done in a practical activities with the literature data.

Results and Discussion

The results presented here were transcribed from the laboratory notebooks used by students during the semester, which were collected by the teacher at the end of the semester for evaluation. In the first part of these classes the majority of students obtained the results shown in Table 1.

Table 1. Reaction of common foods with Benedict and Lugol Reagents
SampleWith Benedict reagent (Room temperature)With Benedict reagent (After heating in boiling water)With Lugol reagent
BlankNegativeNegativeNegative
Soybean oilNegativeNegativeNegative
Wheat flourNegativeNegativePositive
Stuffed cookieNegativePositivePositive
Lemon soft drinkNegativePositiveNegative
Cola lightNegativeNegativeNegative
Cola NormalNegativePositiveNegative
StarchNegativeNegative/PositivePositive
BeerNegativePositivePositive
Cane sugarNegativePositiveNegative
Cooking saltNegativeNegativeNegative
PastaNegativeNegativePositive
BananaNegativePositivePositive
AppleNegativePositivePositive
OnionNegativePositiveNegative
LemonNegativePositiveNegative
Sweet potatoNegativePositivePositive
EggwhiteNegativeNegativeNegative
Egg yolkNegativeNegativeNegative
HoneyNegativePositiveNegative
White chocolateNegativePositiveNegative
MilkNegativePositiveNegative

Based on the observation of the results, students inferred that there is some substance in the stuffed cookie, lemon soft drink, regular cola, beer, starch, cane sugar, banana, apple, lemon, sweet potato, honey, and white chocolate that made Benedict's reagent to change color blue to orange (brick color). However, the results obtained with cane sugar and starch varied both depending on the degree of contamination with glucose and/or fructose as well as a function of the amount of food placed in the test tubes.

After performing the second part of the classes, most of the students realize that only fructose reacted with Benedict at room temperature. When substances were heated, they usually observed the reaction of fructose, galactose, lactose, glucose, maltose, and starch. Thus, the students concluded that Benedict's reagent is used to identify reducing sugars, this conclusion was reinforced by the observation of Fisher or planar structures of the sugars. It was also realized that the samples of pure sucrose did not react, while impure samples could give positive results depending on the quantity of sample added to the tubes. These results are shown in Table 2.

Table 2. Results of the reaction of purified carbohydrates with Benedict reagent
SampleWith Benedict reagent (room temperature)With Benedict reagent (after heating)
FructosePositivePositive
StarchNegativePositive/Negative
AlanineNegativeNegative
Aspartic acidNegativeNegative
SucroseNegativePositive/Negative
GalactoseNegativePositive
GlucoseNegativePositive
LactoseNegativePositive
MaltoseNegativePositive

Since several foods tested here are expected or believed to have no sugar, we have also asked the students to compare the results of Benedict with that Lugol (which is very simple method to detect starch). The positive reaction of Lugol indicates the presence “of hidden sugar” in a given food. Here the instructor has the possibility to discuss why starch occasionally can give positive reactions.

Subsequently, students were asked to find out what is the function of Glucose oxidase reagent. The results generally obtained with GOD can be seen in Table 3. Based on the own observations, students infer that GOD is a reagent which detects the presence of sugars.

Table 3. Reaction of common foods with Glucose Oxidase
SampleWith GOD (after incubating at 37°C)
ColaPositive
Cola lightNegative
Cola dietNegative
LemonPositive
Lemon lightNegative
Lemon dietNegative
Cooking saltNegative
Stuffed cookiePositive
HoneyPositive
Cane sugarPositive
White chocolatePositive
Sweet potatoPositive
Soybean oilNegative

For the second test with Glucose oxidase, we ask students to present their results as shown in Table 4, where a comparison is made of the intensities of the colors resulting from the reaction with different foods or sugars.

Table 4. Relative subjective color of food samples after reaction with GOD reagent
Sugar (10 mM)Intensity
  1. a

    Indicates that can vary depending on the purity of source of the sugar used.

Glucose10 (very intense pink)
Galactose3 (weak to moderate pink)a
Lactose2 (weak pink)a
Sucrose2 (weak pink)a
Fructose1 (very weak pink)a
Water0

The above table shows the color intensity observed by eye of the reaction of glucose oxidase with common foods. It can be observed that the staining intensity is very strong for glucose (represented by 10), while galactose was rated between 1 and 5 (with a median of 3). For the case of lactose (2), sucrose (2) and fructose (1), the pink intensity is very weak. Note that the results marked with an asterisk can vary depending on the purity of samples (contamination by glucose).

Conclusions

The use of this problem based teaching method provides the opportunity of integrating aspects of organic chemistry (biologically relevant organic functions) with the study of physiologically relevant molecules, using simple macroscopic tests (Benedict, Lugol and Glucose Oxidase). We also intend with these activities to instigate the students, who will be future teachers, to formulate hypotheses to explain simple chemical phenomena of biological significance.

The major problem we have been facing with the application of this type of activities is the complaints of a good proportion of the students about the absence of well defined protocols to be followed (the recipe). As rule, at the beginning of the activities the students stated they were disoriented regarding on how to do the experiments and, what is even more worrying is the complaints that students always ask for “what we must observe” in the sense that they ask for the “correct answer” even after we have just sad “you must do your own observations and conclusions.” This fact is related to behavioral difficulties to break away their passive learning habits, because in all their classes, students always receive protocols that must be followed to the letter and that generate a previously known response. In contrast to the traditional methodology, the activities reported here lead frequently to errors or alternative results. We understand that the presence of errors or inconclusive experiments is of outmost importance to counteract the general belief that science does not contain errors [1]. In fact, the teaching of science with the follow-the-recipe approach certainly contributes to the stereotyped view of science and to the notion that all the scientific knowledge is known a priori.

Another important point is the discussion of the final results from the class, which requires an active student, unlike the passivity of the lectures regularly attended by the undergraduate students. Indeed, it is possible to observe a high level of motivation among students during classes proposed here. Finally, we hope that this proposal can help future teachers (undergraduate students) as well as in service teachers to propose activities to their students that encourage the observation, perception, interpretation, and curiosity. Though these skills can be considered intuitive and trivial in science students, our empirical observations along the years of teaching biochemistry have indicated a tremendous deficiency of students in getting, interpreting and formulating a simple task such as to follow macroscopically a chemical reaction.

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