When Milk Makes You Sick: Genetics of Lactose Intolerance

When Milk Makes you Sick: Genetics of Lactose Intolerance A lab investigation Background: Biochemistry: Enzyme-Substrate The milk produced by human mammary glands contains about the same percentage of fat and less protein than cow's milk, but it has more carbohydrate than that of other mammals. This carbohydrate is in the form of lactose, a disaccharide. Mammalian infants depend on their mother's milk for nourishment and immunological protection. As a fresh, temperature-controlled, uncontaminated food source, the milk from mother's breast is the best at meeting the needs of the infant. Except for certain populations of Western humans, milk consumption stops (or is greatly reduced) with weaning. It also happens that in animals and most humans there is a decline in the level of production of lactase with aging. Lactase is the enzyme that digests milk sugar. The hydrolysis (breakdown) of the lactose sugar by lactase is described below:

Symptoms:

Most human infants produce ample quantities of lactase for milk digestion. However, in the vast majority of adult humans, the gene which specifies production of lactase is turned "off" and these individuals cannot digest lactose - they are lactose intolerant. Symptoms of lactose intolerance include cramps and diarrhea. It goes like this: The lactose molecule, which is large, accumulates in the large intestine and affects the osmotic balance there. Since water moves across semipermeable membranes, such as the intestine, from areas of high concentration to low concentration, the addition of large lactose molecules causes water to enter the intestine. This can result in the very unpleasant experience of watery stool or diarrhea. Since lactose is a sugar, it is an ideal food for the bacteria which normally inhabit our intestine (and are essential to digestion). However, the lactose will be fermented by these same friendly bacteria, and organic acids which are gases, are produced by them. And we all know what discomfort intestinal gas can cause! Even small amounts of dairy products or a single glass of milk can cause extreme discomfort. The onset of symptoms normally occurs within 15 minutes to 3 hours after consuming the offending food. So most folks who are lactose intolerant choose to avoid lactose- containing milk products, or modify the lactose, to avoid the cramps and diarrhea associated with the intolerance syndrome.

Treatment: Several dairy products have been developed to address the problem of lactose intolerance. It is possible to buy lactose-reduced products that have had lactase enzyme added. Most of these remove up to 70% of the lactose in the milk. Persons who still have symptoms after using such products can buy a tablet or liquid to add to milk and remove more of the lactose. The source of the lactase enzyme is a cultured bacteria. In summary, persons who are lactose intolerant have three options regarding dairy product consumption. The first is to consume less than their threshold amount of lactose; a second is to limit usage to fermented forms such as yogurt and hard cheeses; and the third is to add lactase to fluid milk prior to use.

Genetics: The vast majority of the world’s peoples are lactose malabsorbers (lactose intolerant). There is a strong correlation of the incidence of adult lactose absorbers (lactose tolerant) and those whose ancestry included dairying as a means of subsistence. In general, only Northern Europeans, who drink much milk seem to have a high degree of lactose tolerance in the adult population. Many Americans are lactose tolerant, due to mixing of ethnicities - lactose tolerance seems to be genetically transmitted. The speculation is that at some point, a mutation occurred which caused individuals to produce lactase throughout their lives, and for some reason this trait gave these individuals a survival advantage and was selected for during natural selection. In very early human societies, people did not consume milk beyond early childhood, so the lactase gene was "turned off". As people migrated to distant parts of the world and domesticated cattle, in some instances, dairy products provided a food source through adverse winter conditions. Hence, Northern Europeans whose lactase gene remained active could consume milk products without becoming ill, thus providing a survival advantage. This was not an issue in warmer climates where food was cultivated year round and societies were not tied to dairy cattle for sustenance.

Thus, the majority of the world's populations, whose ancestors were not dependent upon dairy products for survival, retained the characteristic of adult lactose malabsorption with no adverse consequences to them.

As most enzymes, lactase is a protein. In humans, the gene for lactase is part of chromosome 2. It turns out that patients that are lactose-intolerant have a normal lactase gene (meaning they can produce the lactase protein) but the gene is being inhibited (down-regulated). This down-regulation starts at the onset of weaning an infant from milk.

Questions: Check your understanding of the background 1. What type of sugar is lactose in milk? What are its subunits?

2. Write the equation that is catalyzed by the enzyme lactase. What is the substrate? What are the products?

3. What happens to a lactose-intolerant person when she drinks milk?

4. What are possible treatments for lactose-intolerance? How does each solution help the patient?

5. Why does lactose intolerance usually shows symptoms only after about 1 year of age?

6. What is the benefit of diagnosing lactose intolerance in newborns, before symptoms occur? 7. Extra credit: Why is lactose-intolerance so common in the world? Why is it relatively more common in Northern Europe? Lactose Intolerance: A Lab Investigation Part 1: Measuring Lactase activity against regular milk Objectives: - Show the activity of a commercial lactase enzyme on milk. - Practice the use of positive and negative controls. Materials: - Regular Milk - Lactose-free milk (“Lactaid”) - Lactase Drops - 12-well plate - Glucose test strips (4 per group. Please do not waste!) - Glucose solution (Diluted Karo Syrup)

Procedure: 1) Fill one well in the plate with regular milk. And another with Lactose-Free milk. 2) Check the two milk types for glucose by dipping a glucose strip and comparing the coloring to the glucose chart on the package. Record the color and the estimated glucose concentration. 3) Add a few drops of Lactase to both types of milk. Shake the plate very gently. - Wait 5 minutes. 4) Check the two milk types for glucose by dipping a new glucose strip and comparing to the glucose chart. NOTE: The glucose strips are very expensive Please use them economically! Results and analysis: Copy the following table to your group report and fill it with your results:

Regular milk Lactose-free milk Optional Color Concentration Color Concentratio n Before Lactase After Lactase

Questions: 1. Compare the glucose concentrations before and after adding lactase to milk: What happened? Explain as detailed as you can.

2. Repeat question 1 for lactose-free milk (“Lactaid”). What can you learn from this result?

3. If there is glucose in the lactase solution, your experiment will be inconclusive (why?). What do you need to do in order to rule out the presence of glucose in the lactase solution?

4. What can you do to be sure the glucose strip is actually working?

5. How can lactose-intolerant patients benefit from your results?

6. In conclusion, write a practical advice to lactose-intolerant patients. Base it on what you have learned in this lab. Lactose Intolerance Investigation Lab Part 2: Measuring Lactase activity in individuals from one family Objective: - Test the lactose digestion by extracted enzyme from patients. - Find out if lactose intolerance is carried by a recessive or dominant allele. Background: Genetic disorders are phenotypes that result from malfunction of mutated alleles. Usually, the defected allele can be either recessive or dominant. If recessive, then both alleles of that particular gene need to be defective in order to show a harmful phenotype. If the disorder is dominant, then just one harmful allele of that particular gene is sufficient to cause a harmful phenotype. It is very important to know if a disorder is recessive or dominant for making predictions about the chances for defects in future generations, and for studying the chemical basis of the symptoms. Often the analysis is based on gathering information from as many people and generations of families with the disorders. As a ‘rule of thumb’ – a disorder (or any other phenotype, for that matter) is identified as recessive if it can ‘skip a generation’ (can you figure out why??).

In this lab, we will try to figure out if lactose intolerance is a recessive or dominant disorder. We will base our analysis on the lactose-tolerance or intolerance phenotypes of three generations in one family (See pedigree chart for the family relations). Instead of interviewing the members of the families, samples of their digestive fluids were collected. We will see if the digestive fluids of these individuals contain lactase activity. Those that will be positive will be called “Lactose Tolerant” (Why?), while those individuals without lactase activity will be called “Lactose intolerant” (Why?).

Materials: - Regular Milk - 12-well plate - Glucose test strips (2 per group. Please do not waste!) - 2 Samples from members of the family in dropper bottles. Procedure: 5) Fill each well that has a sample extract with regular milk. Also fill one well without an extract (What for?). 6) Add a few drops of extract from each family member to the milk. Record the order of the samples in the wells! 7) Shake the plate very gently. - Wait 5-10 minutes. 8) Check the wells for glucose by dipping a glucose strip. Write the estimated concentrations of glucose by comparing to the chart on the package. NOTE: Glucose strips are expensive. Please do not discard strips that did not become darker than light green. 5) Record your results in your own paper, as well as on the class poster-chart. Results and analysis: Fill in the following table: Write both color of the strip and the estimated glucose concentration. (In your notes)

Person’s No Name: Extract Before Lactase After Lactase

Questions: (One per group) 1. List all the family members and next to each one – write the amount of glucose at the end of your measurement. 2. Twice, copy the pedigree chart of the family you are investigating, all three generations. 3. Fill in the symbols that represent patients with no Lactase activity (no glucose after 5 minutes incubation). 4. Analyze the pedigree chart: Dominant or recessive? - In other words, is lack of lactase activity carried by the recessive allele (e) or by the dominant allele (E)? a. Title each of the two pedigree charts to “Suppose it is dominant”, and “Suppose it is recessive”. For each case, write the possible genotypes (EE, Ee, ee) of as many family members as you can based on their phenotypes (filled / empty). b. In one of the inheritance types you will not be able to fit genotypes without changing the phenotypes. And since we do not argue with evidence (phenotypes), this would NOT be the inheritance type you are looking for! c. Conclude: Is lactose intolerance – is it carried by a recessive or dominant allele? How did you get to this conclusion? 5. Jane is about to get married to Juan who tells her that he is lactose intolerant. What are the chances for their future children to develop lactose intolerance after they are weaned form their mother’s breast milk? Explain in detail. You may use a punnet square for this question.

6. Reflect on what you have learned, what you liked, and what you suggest to improve, in the two parts of this lab. Lactose Intolerance: A Lab Investigation Teacher-Notes General comments: This mini-unit is designed to help high-school biology students to make the connection between enzyme-catalyzed digestion of carbohydrates to the phenotype (lactose intolerance) and the genotypes of individuals in a family.

Time to allocate: Three full class periods.

Background knowledge students need to know before this unit: - Enzymes and substrates, carbohydrates and proteins, Mendelian genetics.

Materials to prepare: for 10 groups of 3-4 students each.  Glucose strips – I used “Glucose Diastiks” For five class periods I needed 5 packs of 50 strips each. At Walgreen’s I paid $11.00 per pack.. (Prepare in advance! Not every drug store carries them unless you order them. I was also advised next time to buy online, cheaper). - Pour the trips into one larger box, and distribute the packages with the color-scale in various stations. Students will need the boxes to match the color they get with glucose concentrations.  Lactase tablets – one package. Some lactase brands have glucose in them, which adds background to the analysis. It’s not terrible, but if you can afford to buy a few different brands and check, might as well do it. - I dissolved 1 tablet in 50 ml tap water in a conic test tube, and let the insoluble matter sediment before I pour the solution into dropper bottles.  Milk  Lactose-free milk (in any grocery store. Strangely enough – drug stores do not carry it!)  Unknown “samples” in dropper bottles: For a class or 40 students 3 bottles of each ‘individual’. Filled with lactase solution: Eric, Mike, Donna, Jasmine, Fred (“Lactose tolerant”) Filled with water: Lucy, Ben, Jane (“Lactose intolerant”).  Handouts: - Article + questions (to be given as homework before the first lab) - Lab1 directions + questions (colored – pink or other) - Lab2 directions + questions (colored – brown or other)  Power-point slides with journals and charts.  Hand-written poster of the family’s pedigree chart (without the shadings!) – One per class period. I used butcher paper.

For lab part one: Materials per station (I had ten stations):  1 “Lactase” Dropper bottle filled with lactase solution.  Two flasks, one with milk and one with lactose-free milk.  2 glucose strips in each station (not more! See below for a trick to save glucose sticks). I like leaving small things like that in an open Petri dish.  1 12-well plate  Laminated copy of the Lab1 handout.

For lab part two, materials per station:  Two droppers with ‘unknown’ samples. I put one bottle with water and one with lactase, to give an interesting comparison.  12-well plate.  One flask with milk  2 glucose strips  Laminated copy of the lab2 handout Lesson Plan – Part 1: Journal / Warm up: 1. Label the cartoon of the enzyme-catalyzed reaction with lactose, glucose, galactose, carbohydrate, protein, enzyme, lactase, product. 2. Whay are we mentioning enzymes in the genetics unit? 3. What is missing with lactase-intolerant patients? - I let them work independently, and the student / group that figured out all terms first wrote it for the class under the projector. Frontal discussion: Discuss some questions from the homework regarding the symptoms, the causes for the symptoms, and the important of diagnosis in children and adults. Lab Preparation: Explain the procedure, while students are copying the data chart into their notebooks.

Lab run: Within about 10-15 minutes students should be able to complete the testing of milk, before and after adding lactase, and to test the lactose-free milk. NOTE: To save glucose strips, students can re-use a strip that gave a low (blue-light green) result. This enables them to take three measurements with only two strips.

Expected Results: 1) Milk alone gives a blue-green color, which is close to zero glucose. After 5 minutes incubation with 4-5 drips of lactase you get green-brown, which amounts to about ¼ - ½ percent glucose . 2) Lactose free milk shows that it contains a lot of glucose. Ask students how is lactose free milk made, and they easily figure out that lactase was added to the milk in advance (instead of removing lactose from the milk!). Some students may comment that the lactose-free milk is sweeter, which agrees with the glucose content. More points to discuss: 1) It is important to also test for glucose contamination in the lactase solution. As I said the one I bought had plenty, but in the dilution in the milk in the well plate, it gave a low enough result as compared with the product. 2) Positive control, which we didn’t do can be any known glucose solution.

Lesson Plan – Part 1: Journal / Warm up: Show a pedigree chart with 4 generations and several people with names. Then ask them to identify the relationships between pair of individual sin the family. Lecture/exercise: Pedigree analysis (unless you already taught this): - Have students copy a small pedigree of some disorder twice. - Label each diagram “Dominant?” and “Recessive?”. - If the pedigree is of a recessive disorder start with the dominant and show that the genotypes are coming to a contradiction with the phenotypes. - Move to the correct inheritance type and show genotypes of all family members that agree with the phenotypical data. Lab Preparation: - Introduce the “family of the lab”. You can ask each class period to make up last names. They LOVE it. Lab Run: Within 10 minutes students should be able to complete their measurements. When they finally sit down at their desks, collect the data from the entire class on a transparency.

Expected Results:

Lesson 3: Summary and lab report Journal/warm-up: 1. If Jane’s sample resulted with a blue glucose strip, does this mean that she is lactose tolerant or lactose intolerant? 2. Copy the family’s pedigree andanalyze – recessive or dominant.

ANSWER: 1. A brown strip indicates glucose presence that developed from the mixing of enzyme (sample) and substrate (lactose in the milk). So a brown strip indicates for the presence of lactase, which makes the patient lactose-tolerant. If the glucose strip remains blue, it means that the person lacks lactase and is lactose intolerant (“Shaded circle/square in the pedigree). You can use the cartoon they labeled in the journal of the first day to clarify the parts of the system. 2. Lactose intolerance is carried by a recessive allele. * An interesting question you can ask: can the inheritance type be different in a different family? They mistakenly answer yes, and it is of course NO. Group work on two lab reports, one for each day.

Lesson 4: In the evolution unit – What is normal: lactose tolerant or intolerant? Why is lactose-intolerance more common in European countries? (Not prepared yet..)

From “Genome” / Matt Ridley p.192-193 Ref: Holden, C., and Mace, R. (1997) Phylogenetic analysis of the evolution of lactose digestion in adults. Human Biology 69: 605-28

“…We are all born with this (lactase) gene switched on in our digestive system, but inmost mammals – and therefore in most people – it switches off during infancy. This makes sense: milk is something you drink in infancy and it is a waste of energy making the enzyme after that. But some few thousand years ago, human beings hit on the underhand trick of stealing the milk from domestic animals for themselves, and so was born the dairy tradition. This was fine for the infants, but for adults, the milk proved difficult to digest in the absence of lactase. One way around the problem is to let bacteria digest the lactose and turn the milk into cheese. Cheese, being low in lactose, is easily digestible for adults and children. Occasionally, however, the control gene which switches off the lactase gene must suffer a mutation and the lactase production fails to cease at the end of infancy. This mutation allows its carrier to drink and digest milk all through life. Fortunately for the makes of Corn Flakes and Weetabix, most western Europeans by decent can drink milk as adults, compared with less than thirty per cent of people from parts of Africa, eastern and south-eastern Asia and Oceania. The frequency of this mutation varies fro people to people and place to place in a fine and detailed pattern, so much so that it enables us to pose and answer a question about the reason people took up milk drinking in the first place. There are three hypotheses to consider. First and most obvious, people took up milk drinking to provide a convenient and sustainable supply of food from herds of pastoral animals. Second, they took up milk drinking in places where there is too little sunlight and there is therefore a need for an extra source of vitamin D, a substance usually made with the help of sunlight. Milk is rich in vitamin D. This hypothesis was sparked by the observation that northern Europeans traditionally drink raw milk, whereas Mediterranean people eat cheese. Third, perhaps milk drinking began in dry places where water is scarce, and was principally an extra source of water for desert dwellers. Bedouin and Tuareg nomads of the Saharan and Arabian deserts are keen milk drinkers, for example. By looking at sixty-two separate cultures, two biologists were able to decide between these theories. They found no good correlation between the ability to drink milk and high latitudes, and no good correlation with arid landscapes. This weakens the 2nd and 3rd hypotheses. But they did find evidence that the people with the highest frequency of milk- digestion ability were ones with a history of pastoralism. The Tutsi of central Africa, the Fulani of western Africa, the Bedouin, Tuareg and Beja of the desert, the Irish, Czech and Spanish people – this list of people has almost nothing in common except that all have a history of herding sheep, goats or cattle. They are the champion milk digesters of the human race. This evidence suggests that such people took up a pastoral way of life first, and developed milk-digesting ability later in response to it. It was not the case that they took up a pastoral way because they found themselves genetically equipped for it. This is a significant discovery. It provides an example of a cultural change leading to an evolutionary, biological change. The genes can be induced to change by voluntary, free-willed, conscious action. By taking up t4he sensible lifestyle of dairy herdsmen, human beings created their own evolutionary pressures. It almost sounds like the great Lamarckian heresy that bedeviled the study of evolution for so long: the notion that a blacksmith, having acquired beefy arms in his lifetime, then had children with beefy arms. It is not that, but it is an example of how conscious, willed action can alter the evolutionary pressures on a species – on our species in particular.