1. By: Christian Wawrzonek The Metabolic Enhancer Effect on Microbes

2. Metabolic enhancers can be defined as supplements taken to increase exercise duration and reduce recovery time. GNC is a common distributor of metabolic enhancers. GNC alone sells thousands of products. Some athletic “supplements” are whey protein, muscle milk, energy supplements and different amino acids such as arginine, leucine, glutamine as well as different combinations of these. The catch is, “supplements” are not considered drugs, so the FDA puts little to no regulation on the products. As long as the manufacturer shows that there is no direct toxicity of a product, it is legal. This does not include long term effects and unknown interactions with other drugs. This experiment will target a glutamine supplement and test the effects on yeast. Metabolic Enhancers

3. Glutamine Claims Decreases muscle deterioration during a workout. Used as a fuel for cells. May increase protein metabolism. Can increase hormone production.

4. -One of the 11 nonessential amino acids. -The body produces glutamine from glutamic acid and ammonia. -Found in many protein rich food sources. -Nitrogen carrier in the body. -Precursor for immune cell division. -Important for proper immune system functioning. -When damaged, the body uses glutamine for repair. -Studies show an increase in recovery speed and fewer infections in correlation to glutamine dosage. Glutamine and Uses

5. Saccharomyces cerevisiae Two strains of Saccharomyces cerevisiae were used. Bakers Yeast: best known for alcohol fermentation and bread baking. Laboratory strain: used in many cell/biochemical investigations (obtained from the Jones lab, CMU). Yeast is the most investigated cell in the world. It is used in microbiology due to its ease of manipulation and rapid growth. It is eukaryotic, so it shares similar biochemistry, cell cycle, and genetics with more advanced organisms, including humans.

6. Purpose and Hypothesis Do Metabolic Enhancers really work? To determine if the metabolic enhancer L- Glutamine Powder has any effect on the population growth and cellular respiration of Saccharomyces cerevisiae. Null Hypohtesis: There will be no significant difference between the population growth and cellular respiration of Saccharomyces cerevisiae from the control.

7. Materials Sterile Side Arm Flasks L-Glutamine Powder Saccharomyces cerevisiae (Lab Strain) Sterile Micro Pipettes Sterile Macro pipettes Klett Spectrophotometer YEPD media (0.5% Yeast extract, 2% peptone, 2% glucose) Sterile Deionized water Glutamine Solution (4% [ ]) Rapid Rise Saccharomyces cerevisiae (Cooking Strain), Red Star brand Micro Pipettes Macro Pipettes Balloons (23 cm Unique Industries) 125 ml Erlenmeyer Flasks Deionized Water Sucrose (1% final [ ]) 100mL Graduated Cylinders Plastic Tub Plastic Film Wrap L-Glutamine Powder (GNC Pro Performance) Experiment 1Experiment 2

8. Procedure (Population Growth Exp.) 1.Saccharomyces cerevisiae was grown overnight in sterile YEPD media. 2.A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 3.The culture was placed in an incubator (30°C) until a density of 150 Klett spectrophotometer units was reached. This represents the stock solution of yeast. 4.Glutamine powder was dissolved in sterile water at a concentration of (4%). 5.The glutamine solution, yeast stock, YEPD media, and sterile water were added to sterile side arm flasks as follows: 6.The flasks were swirled to evenly dissolve glutamine solution. 7.Klett spectrophotometer readings were recorded at times: (0,30,60,90,120,165,210,240,285, and 1200 minutes)

9. Table 1Flask A(x5)Flask B (x5)Flask C (x5) Yeast 0.1mL Glutamine Solution 100µl10µl0µl Sterile Water 4mL4.9mL5mL YEPD Media 4.9mL Total 10mL Concentration of Glutamine 1%0.1%0%

10. Procedure (Respiration Exp.) 1.Glutamine powder was dissolved in sterile water at a concentration of (4%). 2.Sugar, water, and glutamine solution were added to 12 flasks in the ratios as follows: 3. 7 grams of Red Star rapid rise Cooking Yeast was added to each flask and balloons were immediately affixed to each flask. 4.The flasks were transferred to a warm (30 C) water bath. 5.After 90 minutes of incubation, each balloon was removed from the flask (careful to prevent any leakage of gas; each balloon was pinched at the neck and twisted off). 6.A plastic tub was filled with water. 7.Each graduated cylinder was filled with water to the brim and sealed with plastic wrap. 8.The cylinder was inverted and immersed into the water and the plastic wrap removed. 9.The balloon was placed into the water with the mouth placed into the cylinder. 10.The mouth was slowly released and the air was pumped into the graduated cylinder. The volume of gas was then recorded.

12. Table 2Flask A (x3) Flask B (x3) Flask C (x3) Flask D (x3) Yeast 7 grams Sucrose (10%) 4mL Distilled Water 31mL35mL35.9mL36mL Glutamine Solution (4%) 5mL1mL.1mL0mL Total Volume 40mL [Glutamine] 0.5%0.1%.01%0%

14. Sig. P>.05

15. ANOVAs TestF valueF criticalP valueInterp. Respiration Experiment 14.95873.86250.000764 Significant Growth Experiment Growth at 120 Min. 19.2274.2560.000563 Significant 240 Min.12.8454.2560.002307 Significant 285 Min.4.5154.2560.043851 Significant 1200 Min. 0.8584.2560.455861 Insignificant

16. TimeVariable Compared t value Interpretation 120 Minutes1% vs. c.3.88Significant 120 Minutes0.1% vs. c.6.1Significant 240 Minutes1% vs. c.0.038Insignificant 240 Minutes0.1% vs. c.4.51Significant 285 Minutes1% vs. c.0.861Insignificant 285 Minutes0.1% vs. c.2.12Insignificant Dunnett's Tests#1 t critical = 3.22 α = 0.05

17. Dunnett's Tests#2 Variable Compared t value Interpretation 0% [stock] to 0.01% [stock] 4.41Significant 0.1% vs C5.08Significant 0% [stock] to 1% [stock] 7Significant α = 0.05 t critical = 3.73

18. Conclusions Exp. 1: Hypothesis Accepted: Glutamine powder yielded a significant decrease in rate of population growth of Saccharomyces cerevisiae at 120,240, and 285 minutes. - Failed to reject the Null Hypothesis at the time of 1200 minutes. - Dunnett’s test was significant for both concentrations at 120 and 240 minutes. Exp. 2: Accepted Hypothesis: Glutamine supplementation significantly increased cell respiration (as measured by CO2 evolved) at all concentrations (0.5%, 0.1%, 0.01%). - Null Hypothesis Rejected - Dunnett’s test showed all concentrations significant.

19. Limitations and Extensions Much larger sample sizes, Might require a team of researchers. Slight variations in timing of readings for growth experiment (Only one Klett Spec available) Possible slight variations in optical properties of side arm flasks. Variation in the balloon elasticity (trap the gas in more quantitative manner). Balloons placed awkwardly on flask limiting CO2 intake. Properly place balloons.

20. References http://www.online-medical- dictionary.org/L+Glutamine.asp?q=L+Glutamine http://mental-health.emedtv.com/glutamine/does- glutamine-work.html http://www.vitamins-supplements.org/amino- acids/glutamine.php http://en.wikipedia.org/wiki/Glutamine http://genome.wellcome.ac.uk/doc_wtd020808.html http://biochemie.web.med.uni- muenchen.de/Yeast_Biol/03%20Yeast%20Metabolism.pdf http://www.bodybuilding.com/store/glutpep.html http://mental- health.emedtv.com/glutamine/glutamine.html

21. Need paragraph on m.e. in general Need paragraph on just glutamine Find more info (list of other m.e.) Add slide bout scientific thoughts Review to make sure there are no mistakes