Selivanoff’s Test Reveals Monosaccharide Ketoses in all Trek’s © Engineered Energy Drinks
Monica Narula, Kerri Prediger, Michelle Solomon, Brandon Guelette
LBS 145: Cell and Molecular Biology - Dr. Douglas Luckie
Lab Section Thursday 1 (3-6pm)
Brent Atkinson & Dan Gutteridge
October 12, 2003
ABSTRACT:
A substitute for water, we tested a three-phase energy drink called Trek Engineered Energy in order to determine the validity of the recommended use for each individual step. The three components are Fuel, Nitro, and Recover energy drinks. Fuel, the first energy drink, is designed to prepare and hydrate the body for activity. Nitro is the second stage drink, taken during physical exercise. It is fast-hydrating formula developed to keep one fully hydrated in order to attain maximum endurance. The last phase is a replenishing formula, appropriately called Recover. The energy drinks were tested in a manner of ways, but carbohydrate tests were the most conclusive while trying to characterize the three different liquids. In two of the most relevant tests, stock solutions of each drink were mixed with either Barfoed’s or Selivanoff’s reagent and then treated with heat. The reddish-brown color change of Barfoed’s test infers that all three of Trek’s components contain monosaccharides. At the same time, Selivanoff’s gives evidence that all three contain either monosaccharide aldoses or ketoses. Based on these results the three phases are nearly identical when considering their carbohydrate content. We found through further experimentation that Recover differs from the other components in terms of protein content determined by the Bradford assay. Experimental analysis indicates that there are no significant differences between the Nitro and Fuel energy drinks, implying that there is no need for differentiation. Consumers are being falsely led to believe all three phases are needed for peak performance.
Figure 3-B: Photograph of Tubes after Selivanoff’s Test. The energy drinks in test tubes 1-9 are similar in dark brown color, although they started off as different colors (Figure 3-B). Test tube 10 which contains water has remained clear in color. Glucose, in tube 11 also turned a dark brown similar to all the energy drinks. Test tube 12 which originally contained fructose turned a muddy-brown color. This solution’s color is lighter than the dark brown solutions of the energy drinks and glucose.
DISCUSSION:
The purpose of our analysis of Trek’s Fuel, Nitro, and Recover was to show that there are minimal differences between the three drinks based on their contents and also to confirm our hypothesis that the proposed use of each individual drink is not necessarily valid. Fuel, the drink consumed before exercising, is advertised as being an osmotically-balanced formula with plenty of substrates and carbohydrates (FitnessONE, 2003). Fuel claims to be engineered to fully prepare and hydrate the body before participating in an activity. Nitro, the drink consumed during exercising, is advertised as having a fast-hydrating formula designed to keep one fully hydrated (FitnessONE, 2003). Thus, it is enabling the body for maximum endurance. Recover, the drink consumed after exercising, is advertised as having a replenishing formula (FitnessONE, 2003). This formula is said to be able to “give back” everything lost during activity as well as to prepare one for the next activity (FitnessONE, 2003).
In order to support our hypothesis, several tests were employed to determine the contents of the different drink varieties with respect to their carbohydrate, pigment, and enzyme content. For each test, known solutions were used as positive controls as well as water as a negative control. At the conclusion of our experiments it was our objective to determine if any significant differences exist between the three energy drinks: Fuel, Nitro, and Recover.
Carbohydrate Experiment:
The first experiment consisted of five tests that examined the energy drinks for their carbohydrate compounds. The five tests that were used, to see if variations in carbohydrate content between the three drinks were present, were Benedict’s Test, Barfoed’s Test, Selivanoff’s Test, Bial’s Test and the Iodine Test.
Benedict’s Test determines whether or not a carbohydrate contains a free aldehyde or ketone group. When a red precipitate is formed the test confirms the presence of a free aldehyde or ketone group (Krha, et al. 2003). All three drinks reacted the same way in Benedict’s Test (Table 1). When mixed with Benedict’s reagent the test tubes were a blue color, as can be seen in Figure 4A. Then upon heating, the solutions changed to a brown color and produced a red precipitate. The only minor difference, which is illustrated in Figure 4B, is the shade of brown the solution changed to: Nitro was dark brown, Fuel was a lighter shade of brown, and Recover was an orange/brown. These results lead to the conclusion that all the drinks contain a free aldehyde or ketone group.
Barfoed’s Test was used to determine if there were monosaccharides in the drinks. Only monosaccharides can react fast enough to reduce copper ions and form a red/brown coloration. If there is no change then the solution contains no monosaccharides, disaccharides or polysaccharides (Krha, et al. 2003). A reddish-brown coloration became apparent after boiling solutions of Nitro, Fuel, and Recover for two minutes, implying the presence of a monosaccharide in all three energy drinks (Table 1). Illustrations of before and after the administration of Barfoed’s Test can be seen in Figures 1A and 1B, respectively.
Selivanoff’s Test is used to differentiate between ketoses and aldoses. Ketoses react quickly in comparison to aldoses. Ketoses react within 1 minute of heating whereas aldoses can require several minutes. Disaccharide sugar compounds containing fructose should react somewhere in between that of fructose alone and any aldose (Krha, et al. 2003). The results of Selivanoff’s Test were nearly uniform amongst the three drinks. After the addition of the reagent, all three drinks turned dark brown in less than a minute, indicating that the monosaccharides, determined to be present in Barfoed’s Test, in each were most likely ketoses. This color change is visible by observing the before picture in Figure 3A and the after picture in Figure 3B.
Bial’s Test is used to identify the presence of furanoses (five-membered rings) within a solution. Pentose furanoses rings react with Bial’s reagent to form a green solution and hexose furanoses react to form an olive/brown solution (Krha, et al. 2003). Again, all three energy drinks reacted similarly and changed from a yellow solution (the color of the solution when Bial’s reagent was added) to an olive/brown solution (Figure 5).
The final test used on the three Trek variants, the Iodine Test, was used to test for the presence of starches (Krha, et al. 2003). Solutions of Nitro, Fuel, and Recover all tested negative for starch content, based on no apparent color change was apparent after the addition of IKI, as shown in Figure 2. Water showed negative results for all five of the conducted tests. The summation of all five tests gives no reason to support the claim that Fuel, Nitro, and Recover have any significant differences.
We chose not to perform the Mucic Acid Test in this experiment. The Mucic Acid test, tests solutions for the presence of galactose (Krha, et al. 2003). Based on our knowledge of Trek’s Engineered Energy Drinks we found no reason to believe galactose would be present in any of the drinks. Furthermore, when the Mucic Acid Test was performed on a set of positive controls it came up negative for all carbohydrates that were hypothesized as possibly being present in Fuel, Nitro and Recover.
Photosynthesis Experiment:
In the second experiment, the drinks were exposed to two tests. The first test was paper chromatography, which tested for pigmentation. As Figure 6 illustrates the chromatogram strip of Fuel, Nitro, and Recover had no observable bands. This led us to believe that there is a lack of pigments within these three drinks. However, if a chromatogram strip had shown a band, indicating that a pigment might be present in the sample, it would have been compared to the chromatogram strip of the positive control (a 50/50 acetone and ethanol solution) in terms of distance and color of the band. This would identify the pigment. Although our experimentation gave no evidence of the presence of any pigmentation, the Recover energy drink lists beta carotene in its ingredients content. However, we were unable to find any verification of this, in the form of experimental research performed by the Trek company themselves, or anyone else. Based on our results and the lack of evidence, we can only assume that if indeed beta carotene or any other pigments are in Fuel, Nitro, or Recover; they are in such scarce quantities that our paper chromatography technique was unable to detect any traces of such pigmentation. Further experimentation would be necessary, involving the concentrating of the stock solution samples, to support the claim that Recover contains beta carotene. However, there is no evidence from the creators of Trek Engineered Energy Drinks. Thus, we remain unconvinced of the presence of any pigments in Fuel, Nitro, or Recover due to the lack of evidence based on our assays.
Beta Carotene is a substance from plants that the body converts into vitamin A. The purpose for beta carotene in the body is to act as an antioxidant and an immune system booster. It is generally found in dark green and orange-yellow vegetables, and additionally in supplements (Gaby, 1991). Water came up negative for all pigments when subjected to the paper chromatography test. However, paper chromatography testing didn’t allow us to confirm that the drinks differ considerably with respect to the presence of pigmentation.
The second test was an absorption spectrum. This test, summarized in Table 3, gives the absorbance values of the three stock solution samples of energy drinks at a variety of wavelengths. Figure 7 visualizes the difference in peaks of the three drinks. Upon graphing the data collected from the absorbance spectrum we found that Fuel increased in absorbency from 400 to 520 nanometers and had a peak between 500 and 550 nanometers with a maximum at approximately 520 nanometers (Figure 7 and Table 2). Conversely, Nitro had a steady increase with a peak from 550 to 700 nanometers, with a maximum around 625 nanometers (Figure 7 and Table 2). Recover had a high absorbency around 1.00 from 400 nanometers to 535 nanometers, and then it was a steady decline to 700 nanometers (Figure 7 and Table 2). Unfortunately, it is difficult to asses this data because of interference in the absorbency readings due to the original colors of the drinks. Fuel being red, Nitro being blue, and Recover being purple could have been significantly dark enough to alter the data collected.
Protein Experiment:
The third experiment consisted of a test which examined the presence of protein. We used the Protein (Bradford) Assay to determine the concentration of proteins in the three drinks. The assay is a dye-binding assay. It works by CBBG (Coomassie brilliant blue G-250 dye). The dye yields different color responses based on different protein concentrations (Krha, et al. 2003). Demonstrated in figure 8 is the curve of a known protein sample, bovine serum albumin (BSA), which is the protein standard. The Protein (Bradford) Assay was used to determine the concentration of proteins in the three stock solutions. When the calculated protein concentrations of each stock solution were compared to one another, the protein concentration of Fuel was approximately equal to the protein concentration of Nitro (Figure 8 and Table 3). The protein concentration of Recover was approximately double of the protein concentrations of Nitro and Fuel (Figure 8 and Table 3). In order to obtain accurate information from our testing, we found the absorbency with varying ratios of our stock solution to water; we used 20, 30, and 40μl of each stock solution with the correct amounts of water to create 50μl samples. Furthermore, this entire procedure was replicated for another trial. However, due the unforeseen circumstance of the spectrophotometer resetting itself to 500 nanometers, instead of the 595 nanometers we were supposed to be using, the second replication of the experiment gave us very high absorbency values and therefore, these values were not used in creating our graph. The second replication was performed due to an odd value for Nitro 20μl in the first replication, apparently due to experimental error when running the test. Water, the negative control, was also shown to have no protein content.
The protein content was calculated following the procedure on page 132 of the LBS 145 Cell and Molecular Biology Course Packet (Krha, et al., 2003). The linear relationship of micrograms of BSA vs. its absorbency is used to generate a best curve line. When the absorbency of other samples, Trek's Engineered Energy Drinks, is found, we can take this value and extrapolate the BSA data to include what we just found. Essentially, we turn the stock solutions into BSA, and do the process backwards. If we had an absorbency of "y" then we can find what must have been the micrograms of protein in the sample used (Krha, et al., 2003). We are then able to calculate the protein concentration of our sample. In our case, BSA had too great an amount of protein, as compared to our stock solution samples, resulting in too great of a range to properly asses the protein content in our stock solutions. Although, we did find Recover to have approximately twice the amount of protein as Fuel and Nitro, it is risky to accept this analysis because the numbers are almost negligible. However, the trend among Fuel, Nitro, and Recover is relevant, although the calculations result in values that are negative concentrations. Therefore, to properly support this observation, further tests would have to be run, using a protein standard with smaller values, so that the protein standard and the Trek Engineered Energy Drink stock solution samples would be in the same range.
It is our belief, based on what we learned from the Trek Company, that the protein concentration we calculated is mostly based on the concentration of Taurine. Taurine is now added to many infant formulas as a measure of prudence to provide improved nourishment with the same margin of safety for it’s newly identified physiologic functions as that found in human milk (Gaull, 1989). Again this test suggests that Recover has a significantly different composition than Nitro and Fuel. The different composition is in terms of its nutrient replenishing properties (FitnessOne, 2003).
In all trials of all tests within each experiment the different colors of the drinks were taken into consideration. Although when the drinks were inside their container they seem to be very darkly colored, when they were taken out of the bottles and put into test tubes their color was significantly lighter. We found no reason to dilute any of the drinks for any experiment. Our experiments were carried out with extreme precision. But, as with any experiment, human error is always a possible factor. When measuring the correct amount of substance for each test it is possible that the pipette was pressed down too far or not far enough and more or less solution or solvent may have been used in any given experiment. Furthermore, since the stock solutions of drink samples were colored, the subtle color changes that the tests are based on, might not have been apparent to the human eye.
After completing the different experiments on Trek’s three variants, it has become apparent that Fuel and Nitro have no significant differences when considering carbohydrate, pigmentation, and protein content. All tests yielded extremely similar results, with no significant differences. However, the composition of Recover appears to vary slightly from the other two drinks. The higher protein concentration, as revealed by the Protein (Bradford) Assay, in Recover supports the idea that Recover is not identical to Fuel and Nitro.
The test results in all three categories reveal distinct similarities Nitro and Fuel, making them seem virtually identical. On the other hand, based on knowledge discovered from the Trek Company, Fuel contains glycerol, a component that could not be properly tested in the laboratory. Glycerol is released into the bloodstream after the breakdown of fats during fasting and exercise. Thus especially during exercise, when fat breakdown is stimulated, we see elevated plasma glycerol concentrations (Gleeson, 1998).
Based on the results of our experiments, only the proposed use of Recover is valid. Evidence supports our hypothesis only in the case of Fuel and Nitro. Neither Fuel nor Nitro has a clear advantage over the other. It is our findings that Fuel and Nitro can be used interchangeably before and during exercise. On the other hand, using Recover after exercise instead of the other two drinks appears to be a valid proposal.