[Cells]
The Public Health Epidemic of Diabetes
Lab 3: Molecules in Food Online Only

​Lab 3: Pre-Lab

Your task in Lab 3 will be to construct your own experimental protocol to predict the glycemic index of unknown food items, based on their molecular make-up, using chemical indicators. You may also have some time to continue the analysis you began in Lab 1.

• Introduction
• Do you know enough?
• What will we do in lab?
• LABridge

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1) What is a macromolecule and how can you test for its presence in food?

A marcomolecule (or polymer) is a very large molecule comprised of smaller subunits (or monomers). We tend to recognize four primary types which are highly diverse in atomic make-up and structure and thus,  function. We will focus on the first three (carbs, lipids and proteins) in this lab. You should know the basic subunits of each, their shape, general function and a few examples. You need a basic understanding of dehydration synthesis & hydrolysis (or condensation reactions) which build and breakdown macromolecules. You also need to know which chemical test can be used to indicate each specific macromolecule.

​Please review the slideshow. It provides an overview of macromolecules, the chemical indicators than can detect each in solution, and the affect of each macromolecule on the glycemic index score (an important dietary consideration for diabetics).

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​Please review THIS VIDEO on condensation reactions (<2m).

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What is the glycemic index?

Remember...diabetes is a disease that occurs when blood glucose (i.e., blood sugar) is elevated because the body isn't making enough insulin or doesn’t use insulin well. The glucose then builds up in the blood, and doesn’t reach the cells where it is needed as an energy source. Over time, having too much blood glucose causes the serious and chronic health problems associated with diabetes.

​Please read this quick explanation of the GI from the American Diabetes Association.

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Because the blood glucose levels in diabetics are not well regulated, their diet becomes much more important. Primarily, diabetics have to be concerned with the frequency and types of carbohydrates they consume. Carbohydrates can be identified by their glycemic index (GI), which is a measure of how quickly the carbohydrate is broken down into its component parts to be used by the body. 

• Most simple monosaccharides and disaccharides, like glucose and fructose, are quickly broken down, rapidly increase blood sugar, have high GI scores and are especially detrimental to diabetics.
• Larger, more completed polysaccharides take longer to be broken down, slowly increase blood sugar, have lower GI scores and better for diabetics.

However, keep in mind, diabetics don't just manage their diets based on the GI. They must also select foods that maintain their cholesterol and triglycerides levels, blood pressure, and weight management.

The affect of high vs. low GI foods on blood glucose levels over time.

Lab 3 will proceed in three parts:

1) The pipette challenge: You will learn the basics of measurement, including how to correctly transfer liquids using micropipettes. 

2) Determine the molecular composition of unknown food items: In the lab you will be provided with

• 4 chemical indicators that, when added to a sample, will "indicate" the molecular contents (mono/disaccharide, polysaccharide, or protein). 
• A series of known positive controls for each macromolecule.
• Water to use as a negative control.
• Unknown food samples.
• Common glassware and equipment.

You will then be asked to develop a protocol using the above materials, that might help you predict the GI score for each of the unknown food items. 

3) Predict the GI score of unknown food items. Would any of these foods be good for a diabetic? Can you predict the GI score of a food based on its molecular composition?​ You will analyze your data and predictions vs. the actual GI score of the food items.

If you've never used a micropipette before, try practicing a bit in this virtual lab setup.

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Learn about lab glassware and review the metric system as needed. Make sure you understand WHY scientists use the metric system. 

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Review the importance of a positive and negative control.

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Pipette Virtual Lab. Click to Enter.

​If you feel confident with this material, click the bridge icon and navigate to Blackboard to take the LABridge for Lab 3.
​Be ready to submit your Notebook Entry from last week (Word.doc ONLY) and to be tested on this Pre-Lab material.

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Click here to get to WKU's blackboard to take your LABridge for this week. Be sure your Notebook Entry from last lab is ready to submit!

Lab 1: Protocol

In today's lab you will learn how to use a micropipette. You will set up experiments to try and predict the glycemic index of unknown food items and, if time allows, you can continue analysis of the diabetes data.

​Exercise I. Pipette Challenge
Exercise II. Predict the Glycemic Index
​Exercise III. Continue Analysis 

Lab Objectives: Following today's lab, you should be able to...

• Exercise I
• Exercise II
• Exercise III

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Exercise I. The Pipette Challenge.  

At the beginning of lab, your instructor will demonstrate how to properly use a micropipette.
​Watch carefully and follow along. 

Review the parts of a micropipette: Diagram at right.

1. The Plunger & Volume Dial: If you SLOWLY push the plunger down, you will feel resistance. This is the first stop designed for drawing up fluids. If you continue to push the plunger, you will reach the end or second stop designed to expel fluid. If you draw fluid using the second stop, it will over-draw and your experiment will be compromised. The dial serves as volume adjustment dial. SLOWLY turn the dial to set the volume. DO NOT set a pipette past its listed capacity.
2. Tip Ejector Button: SLOWLY push to eject a tip into the Bio-hazard container.
3. Volume Indicator: Pipettes are labeled with their capacity. 1 - 5mL for larger volumes and 100-1000uL for smaller volumes. 
4. Tips: Disposable tips are provided. Attach a tip by GENTLY pushing the pipette into tip and removing the tip from the box. As part of aseptic technique (methods used to prevent contamination), it is standard practice to dispose of each tips after each transfer and to keep the lid box closed at all times. 

Please keep the following in mind to increase the likelihood of both accurate and precise measurements in lab:

• Always record the measured value of a liquid at the bottom of the meniscus: the concave or convex surface of a liquid due to cohesion
• You should always use the smallest possible vessel for your measurements. For example, if you need to measure 25mL of solution, you would use 50mL flask, not a 125mL or 250mL.
• When transferring small volumes, you will usually use a micropipette. Occasionally you may be asked to use “drops” from a dropper bottle instead. 
• For liquids: Graduated cylinders are best, second are flasks, never use beakers.

One of our most useful measuring tools is the micropipette, which you will need to use today in Exercise II. However, if this instrument is not used correctly is it neither accurate nor precise, and can be easily damaged. 

Procedure​: Your task is to demonstrate your ability to accurately use a pipette.

Since we have a snow day today a simulation is the best we can do. We will get you all some hand-on practice next week! For now, this will get you ready!

1. Complete the simulation by clicking on the image below or CLICK HERE.
2. Take a screen grab of the final/end screen for your Lab Notebook Guide.

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Metric  Reminder!
​1 mL = 1000uL
​mL = milliliter & ​uL= microliter

Get in the habit of replacing your pipette tip after EVERY use. Contamination is always an issue and will interfere with your results in Exercise II.

Exercise II. Can you use chemical indicators to determine the molecular composition of unknown food items? Can you predict a GI score?

Your task in Exercise II is to develop a series of tests, using chemical indicators, to predict the glycemic index (GI) score of unknown food items. If you have ANY questions about what a macromolecule is, or what the GI is, please refer to the pre-lab above.

Materials. You will be provided a standard lab kit with glassware, test tubes, and a rack. You will also have pipettes, tips and a digital balance. You have solutions of each indicator as well as matching positive controls. You will also have a beaker of distilled water which acts as a negative control for all your tests. A protocol for each chemical indicator in the side bar below.  

Chemical Indicators

Positive Controls

Indicator Protocols

Lab Station Set-Up

Pipettes & Tips

Electric Balance

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Procedure​​

1. Download the Lab 3 Notebook Guide from the sidebar.
2. You won't be completing these activities in lab, but review the chemical indicators and controls as instructed below. 
3. Identify the chemical indicators you would be using for this lab. These four solutions that test positive for for the following macromolecules:
   1. Bendict's solution tests for glucose
   2. Biuret tests for proteins
   3. Lugol's iodine tests for starch
   4. Sudan IV tests for lipids (you may not complete this test, your TA will clarify. ​ 
4. Identify the positive (C+) controls needed for each molecular test. These test to make sure each indicator is working properly.
   1. A 10% glucose solution tests the Benedict's solution
   2. A protein solution (egg albumin) tests the Biuret 
   3. A 1% starch solution tests the Lugol's iodine
   4. An oil based solution tests the Sudan IV
5. You would have used distilled water to serve as the negative control (C-) for each test.
6. Refer to the protocols for each indicator in the sidebar. The protocol will explain exactly how the indicator would have been used and what the testing procedure is. It will also show you how to interpret your results.
7. Your results are posted below.
8. Complete the rest of the Lab Notebook Guide.

Chemical Indicators & Protocols

benedict's

Biuret solution

lugol's iodine

sudan iv

Lab 3 Notebook Guide. Click to download.

All of these tests are time-sensitive!​

A bright yellow/orange result on a Biuret test is common if you wait too long to read it; it is still a NEGATIVE result.

A dark brown test result with Benedict's means it might have stayed in the water bath for too long but is a POSITIVE result. ​

​Exercise III. Would any of these foods be good for a diabetic? How is molecular composition related to GI score?

Procedure

1. If you're not confident with the  Glycemic Index (GI), review the  "Do you know enough?" section of our Pre-Lab.
2. Use your test results for each unknown item to predict what the Glycemic Index score might be, step 5 in lab notebook guide.
3. You should decide on a single number, and a range, and order the unknowns from highest to lowest predicted GI score. Don't forget about the link between GI score and blood glucose levels!
4. Once complete, ask your instructor to provide you with the key, which will give you the name and GI score for each food item.
5. Complete your lab notebook guide.
6. Ensure all members have an electronic copy for reference and be ready to upload your Lab 3 Notebook Entry  in the LABridge before we meet again next week.

If you finish early, work on your report.
LAB 2 Exercise III. has all the details.

Examples of GI scores across food types (from idealnutrition.com)

GI range.

Lab 3 BIOL 120 CONNECTIONS
Section 2.5: Life is Carbon Based
​Chapter 3: Proteins
Chapter 5: Carbohydrates
Big Picture 2: The Chemistry of Life
BioSkills 1: Using the Metric System
​Bioskills 14: Reading Chemical Structures

Faculty Spotlight: Dr. Sigrid Jacobshagen, Emeritus

Email: [email protected]

Dr. Jacobshagen is extremely interested in clocks; not the kind hanging on the wall or on your phone, but biological clocks and circadian rhythms. She explores these internal physiological regulators, like the sleep/wake cycle, using algae as a model organism. Projects in her lab center on particular types of macromolecules called, photoreceptor proteins that are able to reset these circadian clocks. She is an accomplished researcher in the field of biochemistry and teaches our BioChem courses. She is also heavily involved with the Biochemisty Major. Reach out to her if you found Lab 3 particularity interesting or if you're still trying to decide what track you'd like to pursue in our department. 
Research Key Words: Circadian clocks, photoreceptor proteins, gene regulation and mathematical modeling
​Recent Publication:  Chlamydomonas reinhardtii strain CC-124 is highly sensitive to blue light in addition to green and red light in resetting its circadian clock, with the blue-light photoreceptor plant cryptochrome likely acting as negative modulator.