2013

Green Tea and Black Tea Metabolite Concentration Determination through

Proton Nuclear Magnetic Resonance

MICHAEL J. GOETZ CARTHAGE | 2001 Alford Park Drive, Kenosha, WI 53140

Green Tea and Black Tea Metabolite Concentration Determination through Proton Nuclear Magnetic Resonance Abstract Tea has been studied for many years with new findings on their effects on health. While tea still has some unknown effects on the human metabolism, this study aims to determine the effects of green tea and black tea consumption on the human metabolism through metabonomics. Urine samples were studied through proton nuclear magnetic resonance to determine the concentration of metabolites excreted. Subjects who consumed green tea showed a higher concentration of energy metabolites excreted than those who consumed black tea or caffeine alone. This suggests that green tea has a greater positive impact on the human metabolism. As the human metabolism begins to slow down due to aging, daily consumption of green tea may help to combat a slow metabolism. Introduction Tea has been around since the early Chinese empires and assorted tea plants were traded and given as gifts around the world. Recently, tea consumption has increased because of known health benefits tea offers. In the late 1990’s, the health benefits of tea became a popular research topic to determine the benefits of consuming tea.8 Previous research included determining the concentrations of antioxidants in assorted teas, determining the extraction of black tea polyphenols in milk, and if catechins (antioxidants) were acting as a form of antibacterial and an anticarcinogen for the human body.1, 10 Polyphenols and catechins are types of molecules that act as antioxidants in the human body to help remove free radicals in the body10. Free radicals are unpaired electrons that are present in cells due to the cleaving of double bonds of

Page 1 of 18

Green Tea and Black Tea Metabolite Concentration Determination through Proton Nuclear Magnetic Resonance different molecules or any unpaired electrons due to resonance.3 Resonance is the delocalization of electrons in a given molecule or compound.3 The free radicals, if not controlled or removed, cause damage to the cell by breaking down organelles and parts of the cell membrane. A free radical wants to gain another electron (reduced) to pair with the lone electron, the free radical approaches another molecule and takes a valence electron from the molecule.3 The molecule that lost an electron (oxidized), now is a free radical and begins a chain reaction of taking an electron and creating another free radical in the process.2 The breakdown of the organelles and cell membrane causes the cell to “age”, which shortens the life of the cell. In 2006, Ferdi A. Van Dorsten et al, set out to determine if the flavanols that are present in green tea caused a different effect on the human metabolism than the flavanols present in black tea.9 The polyphenols that are present in tea are compounds containing multiple phenol groups, which make up the assorted flavanols. 1 Flavanols are excreted by plants as their metabolites and are present to mediate permeability in plant cells, which also cause the yellow color in tea.1 Caffeine was used as the control because caffeine does not contain any polyphenols and both green tea and black contain natural caffeine, but have different caffeine content. Black tea has a higher natural caffeine content than that of green tea. The effects of different foods and beverages on human metabolism has been a popular area of study due to the fact that as people begin to age, their metabolism processes begin to decline in efficiency as the individual becomes less physically active. If the individual becomes less active, then metabolic pathways do not need to produce or use the same amount of Adenosine

Page 2 of 18

Green Tea and Black Tea Metabolite Concentration Determination through Proton Nuclear Magnetic Resonance Triphosphate (ATP) or to be used to break down glucose molecules.2 Instead a process called glycogenesis will begin, where the excess glucose molecules that are not being used will be added to the long chains of glycogen in the body for storage for a later use.2 Glycogen forms by starting with a glucose molecule and converting it to uridine diphosphate glucose, then is added to the n-terminus of a pre-existing glycogen chain by cleaving the uridine diphosphate from the molecule.2 Long chains of glycogen are stored in the liver for emergency situations with low glucose levels. Oxidation is the process in which a compound or molecule gains an oxygen, or loses a hydrogen atom.3 If the tea leaves are not to be oxidized, the tea leaves are steamed, dried, and then fried to denature the enzymes responsible for oxidation. 7 In contrast, black tea is taken from the same plant and laid out to dry in the sun, completely oxidized, rolled, then is ready to be brewed.7 The oxidation of the tea leaves causes the flavanols in the leaves to begin to breakdown, which will leave the flavanols unable to be used to help rid the body of the free radicals.7 The flavanols in tea are able to reduce the amount of free radicals because the phenol groups of the flavanols are able to donate electrons to the radicals. Although the flavanols are oxidized, the compounds are still stabilized due to the conjugated pi bonds of the phenols. The flavanols that are present in green tea are (-)-Epigallocatechin-3-O-gallate, (-)Epigallocatechin, and (-)-Epicatechin.8 The flavanols that are in black tea are different

Page 3 of 18

Green Tea and Black Tea Metabolite Concentration Determination through Proton Nuclear Magnetic Resonance than those in green tea due to oxidation, the flavanols are Theaflavin, Theaflavin-3gallate, Theaflavin-3’-gallate, and Theaflavin-3,3’digallate.4 Through this experiment, the potential health benefits could be observed through the study of the metabolite activity after the consumption of the different teas. The different levels of the metabolites being studied correspond to the effects on the different processes including glucose metabolism, protein metabolism, and fatty acid metabolism effected by tea consumption. The metabolites from glucose metabolism studied were glucose, pyruvate, and β-hydroxybutyrate. When glucose is ingested (Figure 1a, Stage 1), the molecules are taken through glycolysis where the glucose molecules are converted into pyruvate.2 The pyruvate is converted into acetyl-CoA by the Pyruvate Dehydrogenase Complex to be fed into the Citric Acid Cycle (Figure 1a Stage 2).2 β-hydroxybutyrate is not part of the normal

Page 4 of 18

Green Tea and Black Tea Metabolite Concentration Determination through Proton Nuclear Magnetic Resonance glucose metabolism because the molecules serve as an emergency back-up for when the brain has low levels of glucose. Ketogenesis is the breakdown of excess acetyl-CoA molecules into acetone, acetoacetate, and β-hydroxybutyrate.2 β-hydroxybutyrate is synthesized by having acetyl-CoA broken down, then fed into the β-hydroxybutyrate dehydrogenase complex.2 The metabolites related to proteins metabolism were oxaloacetate and α-ketoglutarate. α-ketoglutarate is important for protein metabolism because it aids the degradation of amino acids into glutamate (Figure 1b) to be fed into the Urea Cycle.2 Oxaloacetate is necessary for protein metabolism because the molecule is used to aid the production of α-ketoglutarate in the mitochondrial matrix.2 The metabolites studied for fat metabolism were citrate, oxaloacetate, and succinate. Fats that are ingested are broken down into fatty acids, then into acetyl-CoA in a glyoxysome.2 Acetyl-CoA is then fed into the Glyoxylate Cycle which produces succinate, then is taken up by the Citric Acid Cycle in the mitochondrial matrix. 2 After being able to link an effect on metabolite concentration to a type of tea, more research could be done to pinpoint the exact effect tea has on a specific metabolic pathway in the human body. This experiment studied the concentration of numerous metabolites in urine samples from the participants. The different metabolites were categorized into two groups, endogenous non-aromatic metabolites (non-aromatic metabolites that are produced by the individual) and endogenous aromatic metabolites (metabolites with an aromatic ring produced by the individual). The aromatic endogenous metabolites that were studied were hippuric acid, 1, 3-dihydroxyphenyl-2-sulfate, tyrosine, and ten

Page 5 of 18

Green Tea and Black Tea Metabolite Concentration Determination through Proton Nuclear Magnetic Resonance unknown aromatic compounds found in the urine of the participants.9 These aromatic metabolites were studied because hippuric acid is a common metabolite found in human urine.6 The cyclin-dependent protein kinases (CDKs) are the enzymes that contain tyrosine and are at the “checkpoints” of the cell cycle that can determine when the cell is ready to move into the next stage of the cell cycle.2 If there are insufficient amounts of tyrosine in the cell, the regulation of the cell will be minimal and may cause the cell to die prematurely. The list of endogenous metabolites that were studied included six amino acids, seven metabolites used in energy metabolism, betaine, dimethylamine, and N-acetyl (glycoproteins). The different metabolites that were studied were aimed to determine the wide range of effects the consumption of flavanols would have on the body. The molecules were vastly different to attempt to determine the effects on the different processes that include amino acids, energy metabolites, as well as glycoproteins. The aromatic compounds were studied separately because the aromatic compounds came from the breakdown of the flavanols in the gut cells. 1 The study of metabolite concentration were not focused on a single metabolite or a group of metabolites, instead was focused on the overall changes in the metabolite concentrations between black tea and green tea, with respect to caffeine. To analyze the metabolites in the different samples, a technique known as Proton Nuclear Magnetic Resonance (1H NMR) was used to determine the different compounds. 1H NMR is able to detect absorption of specific radio waves emitted by the atomic nuclei in a very strong magnetic field.3 Only the atomic nuclei with a nuclear spin will be able to emit any waves that will be picked up by 1H NMR. The nuclear spin of the

Page 6 of 18

Green Tea and Black Tea Metabolite Concentration Determination through Proton Nuclear Magnetic Resonance atomic nuclei allows the nuclei to absorb the energy given off in NMR spectroscopy, then reemits the energy back to the sensor in the NMR spectrometer. The molecules that are being studied absorb the energy, which raises its energy level from its ground state to its excited state. The waves emitted from one nuclei vary from other nuclei depending on the hybridization of the hydrogen emitting the signal.3 Hybridization is the combining of the atomic orbitals of different elements when bonded together. For instance, a hydrogen bonded to a carbon then to another carbon (H-C-C-…) would emit a certain wavelength which is different from a hydrogen that would be bonded to an aromatic compound (H-Ar). The wavelengths that are emitted by different types of hydrogen are able to be differentiated because the spin of the nuclei will generate a magnetic field that will be affected by the electrons that are near the bond, which will weaken the field depending on the location of the electrons in relation to the nuclei. 3 This phenomenon is due to the shielding effect of electrons on nearby protons. 3 The sigma (single) bonded electrons shield the proton, which means that the attractive forces between the proton and electron are decreased due to repulsion force of the electron.3 In contrast, the pi (double) bonds produce a deshielding effect to the proton they are bonded to.3 The deshielding effect is the greater attraction force to the proton from multiple electrons.3 The presence of a hydrogen bonded to a specific atom will generate a peak on the NMR spectra. Depending on which parts of the 1H NMR spectra have the larger areas, the different types of compounds are able to be identified. For instance, the signals that are around 6ppm (parts per million) and 7ppm are known to be the regions of aromatic groups because of the relative amount of energy emitted

Page 7 of 18

Green Tea and Black Tea Metabolite Concentration Determination through Proton Nuclear Magnetic Resonance from the nuclei when the electrons near the hydrogen of the aromatic ring. If there is a large peak and area in that region then the solution that was analyzed likely has an aromatic ring in the compound of interest, the relative concentration of the compound of interest can be determined by the integral of the area of the peak of interest.5 Using 1H NMR, especially when an unknown compound is being analyzed, is very helpful when attempting to identify its structure. The experiment aimed to determine if consumption of green tea or black tea had a larger, positive effect on glycolysis, the Citric Acid Cycle, protein metabolism, and fat metabolism. Although there are differences between the two teas, it is unknown what exactly causes the different effects on the human metabolism. If the relative concentrations of a given compound is too high in the cell, the cell will excrete the excess compounds to reach appropriate amounts for the cell. Assuming that the caffeine content is not the sole cause for the change in metabolism, the breakdown of the different flavanols found in black tea and green tea must be responsible for the differences in metabolite concentration found in the body. Results and Discussion Seventeen males, ages twenty through seventy, were recruited to participate in this trial. The participants were given six portions of one gram tea solids per day, which is equivalent to twelve cups of tea per day. The control of caffeine was distributed as a gelatin capsule of 360 milligrams of caffeine, which equaled that of one tea solid. The participants were kept on a low polyphenol diet two days prior to the beginning of their consumption of tea or caffeine, as well as during their two day substance consumption.

Page 8 of 18

Green Tea and Black Tea Metabolite Concentration Determination through Proton Nuclear Magnetic Resonance The samples of urine were collected 24 hours after consumption, then were studied through 1H NMR with the total volume being 0.50mL. The samples were run through the NMR spectrometer at a temperature of 303K (30oC) and had collected 32,000 data points through the analysis. After the urine analysis, blood samples were analyzed via 1H NMR as well. During the analysis of the 1H NMR spectra for urine, the region from 4.3ppm - 6.0ppm was excluded from the analysis due to the resonance of water and the signals that were given off by urea. The energy that is given off by the spinning nuclei of the water and the urea molecule and the electrons that surround the molecule, correlates to the region of 4.3ppm – 6.0ppm. Urea is a compound that is excreted from the body from protein metabolism and is not one of the compounds that was analyzed.6,9 When comparing the chemical shifts between black tea (Figure 2b) and the chemical shifts of green tea (Figure 2c), the peaks from approximately 1.10ppm through about 4.30ppm of green tea are the same peaks found in the black tea and the caffeine NMR spectra (Figure

Page 9 of 18

Green Tea and Black Tea Metabolite Concentration Determination through Proton Nuclear Magnetic Resonance 2a) from 1.10ppm through 4.30ppm. The region from 1.10ppm through 4.30ppm are the metabolites that are common among the three variables. Along with the common metabolites that were identified in the 1H NMR spectra, a compound of interest was at 8.80ppm which is an unknown metabolite that was recorded in both the green tea and black tea NRM spectra. By analyzing the 1H NMR spectra, there are noticeable differences in the different spectra. (Figure 2) For instance, the change in peak area of hippurate and 1,3dihydroxyphenyl-3-O-sulfate (Figure 2c) may shed light on the subtle differences between black tea and green tea. The labeled metabolites (Figure 2) on the NMR spectra correspond to the normal chemical shifts for the types of hydrogens.3 The chemical shift from 1.0ppm-3.0ppm is due to the hydrogen bonded directly to a nitrogen atom, which creatine contains a nitrogen-hydrogen bond.3 The different metabolite concentrations may be due to a few different scenarios. The first being that the metabolites are just due to the breakdown of the flavanols that are present in both teas, but in different concentrations. If the flavanols are present in both teas, then the different concentrations would require different amounts of ATP to be able to breakdown the flavanols in the gut cells. An increased amount for needed ATP would require the Citric Acid Cycle to increase its output of electrons that are available to enter the Electron Transport Chain (Figure 1, Step 3) to produce more ATP. An increase in the needed output in the Citric Acid Cycle would require glycolysis to speed up as well to compensate for the diminished intermediates. The second scenario being that the difference in the metabolites between black and green tea are due to green tea not

Page 10 of 18

Green Tea and Black Tea Metabolite Concentration Determination through Proton Nuclear Magnetic Resonance becoming fully oxidized due to the enzymes in the leaves responsible for oxidizing have not become active. In scenario two, the flavanols of the tea leaves would still be intact and able to be ingested into the body, broken down, and then excreted from the body; which would explain why the 1H NMR of the urine from the individuals who consumed green tea had shown a larger presence of the catechin metabolites than the black tea samples. There is an unknown compound recorded at the peak of 8.80ppm. (Figure 2b/c) This unknown compound peaks could be from the breakdown of the many flavanols that are found in the tea leaves, or a potentially new flavanol to be discovered.

Page 11 of 18

Green Tea and Black Tea Metabolite Concentration Determination through Proton Nuclear Magnetic Resonance The change in the endogenous metabolites after tea consumption served as a way to quantitatively compare differences between black tea and green tea. (Table 1) The metabolites that are listed are labeled with their chemical shifts in the 1H NMR analysis, as well as the direction of change in concentration with respect to the caffeine control (increase, decrease, neutral effect).The direction of change of the concentrations of metabolites versus the different teas may show how different metabolic pathways were altered due to different intake of flavanols. The large increase in concentration of pyruvate in green tea consumption versus an increase in concentration of pyruvate in the consumption of black tea would allow for comparison of differences in effects on human metabolism. Elevated levels of pyruvate in a cell will activate the pyruvate dehydrogenase complex, which will oxidize pyruvate to acetylCoA, then will be used in the citric acid cycle. An active pyruvate dehydrogenase complex will require more pyruvate molecules to be present to remain active, forcing glycolysis to keep running to supply the needed pyruvate; otherwise the complex will build up an excess of acetyl-CoA which is an allosteric inhibitor of the complex which will cause the complex to become inactive. Allosteric inhibition is the inhibition of an enzyme through the binding of a substrate to an allosteric site (not the activation site) of the protein, causing the enzyme to become inactive due to a conformational change. 2 An example of allosteric inhibition would be feedback inhibition which means that the products of an enzyme regulate the activity of the enzyme. The pyruvate substrate acts as a feedback inhibitor to the pyruvate dehydrogenase complex if too much pyruvate is produced. Another explanation may be due to the inhibition of pyruvate being

Page 12 of 18

Green Tea and Black Tea Metabolite Concentration Determination through Proton Nuclear Magnetic Resonance synthesized into amino acids (alanine, valine, leucine, and isoleucine).2 The metabolite that was found in urine that was directly involved with the consumption of tea was hippuric acid, which have been linked to the metabolism of the flavanols, and other polyphenols as well.1,6 The increased levels of metabolic intermediates (Table 1) would suggest that consuming tea- green tea especially- may help jump start human metabolism. The concentrations of oxaloacetate, pyruvate, α-ketoglutarate, succinate, and citrate were increased. The metabolites with increased concentrations activate the corresponding dehydrogenase, for example: α-ketoglutarate activate the α-ketoglutarate dehydrogenase in the citric acid cycle.2 The activation of the corresponding dehydrogenase will allow the metabolites to be converted into the next metabolite in the cycle. The increased concentration of pyruvate may suggest that the pyruvate dehydrogenase complex is either inhibited or the conversion of pyruvate to amino acids is inhibited. An increase in citrate concentration may suggest that the Citric Acid Cycle may be inhibited or the Glyoxylate Cycle may be inhibited as well. This may prove to be valid because if the levels of excreted metabolic intermediates increased then the concentration of the intermediates in the body must be in high concentration, where the only way to reach appropriate concentration levels in the cells is to excrete the excess intermediates. The ten unknown metabolites that were found in the urine of the participants may have been due to the breakdown of the different flavanols that are present in green tea, but not black tea. The most common flavanols in green tea are (-)-epicatechin (EG), (-)-

Page 13 of 18

Green Tea and Black Tea Metabolite Concentration Determination through Proton Nuclear Magnetic Resonance epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), and epigallocatechin-3-gallate (EGCG). These compounds are present in green tea because the oxidative enzymes in the tea leaves were never activated. These compounds when excreted are broken down into three different compounds through the gut cells of the body: hippuric acid, 3’methoxy-4’-hydroxyphenylacetic acid, and 3-(3’-hydroxyhenyl) hydracrylic acid.1 Only one of the three products that are excreted after the breakdown of the flavanols were listed in determination of the aromatic metabolites concentrations, which is hippuric acid. All three of the products contain aromatic rings, which could mean that two of the ten unknown aromatic compounds may be 3’-methoxy-4’-hydroxyphenylacetic acid and 3-(3’-hydroxyphenyl) hydracrylic acid. The other six unknown compounds could be hypothesized to be the intermediates of the breakdown of EG, EGC, ECG, and EGCG that were excreted from the body before the compounds were fully broken down.1 Another potential explanation for two of the unknown aromatic compounds is an alternate derivation of the (-)-epicatechin compound. The process by which the EG is derived differently is through microbial degradation in the gut. The compound would be broken down, then would be able to form two different products from the previous stated products. The two products are 5-(4’-hydroxyphenyl)-γ-valeroactone and 3’hydroxyhippuric acid; the 3’-hydroxyhippuric acid derivative is the more stable of the two alternates due to the increased polarity of the molecule, which creates greater intermolecular forces. Polarity of a molecule deals with the uneven distribution of electrons due to greater electronegativity of an atom attracting more electrons towards itself.3

Page 14 of 18

Green Tea and Black Tea Metabolite Concentration Determination through Proton Nuclear Magnetic Resonance Conclusion The observed results showed that the consumption of green tea had a greater, positive impact on the concentration of energy metabolites than that of black tea due to the increased concentrations of glucose, citrate, pyruvate, α-ketoglutarate, and oxaloacetate. The increased levels of the metabolites can then be traced to the corresponding metabolic pathway such as glycolysis, the Citric Acid Cycle, Urea Cycle, and the Glyoxylate Cycle. In the experiment there was an anomaly, which was the distribution of participants. There were an initial total of eighteen participants, all male, ages ranging from twenty years old to seventy years old (the mean age being 63). The outcome of this experiment may have been altered if the demographics of the participants were changed: men and women participants, first trial younger participants, second trial older participants. Different demographics of the tested population may result in different concentrations of excreted metabolites due to the different levels of steroids in the body (estrogen versus testosterone). If the age of the participants was lowered, the metabolism rates may be higher than those of the seventy year old man. An ethical implication that was found in this research was about the sharing of data. On the site of the paper, the 32,000 data points collected from the 1H NMR spectrums were nowhere to be found. This could potentially be scientific misconduct if when prompted for the data, the researchers were unable to come up with the original data.

Page 15 of 18

Green Tea and Black Tea Metabolite Concentration Determination through Proton Nuclear Magnetic Resonance Looking forward, the next step that this type of research can go into is the determination of which flavanols in tea are responsible for the different effects on metabolism. The experiment setup would include the isolated flavanols from green tea and to have the different groups consume one flavanol to determine if a single flavanol is responsible for the effects on metabolism. After being able to link an effect on metabolite concentration to a specific flavanol in a tea, further research could be explored to pinpoint the exact effect a specific tea has on a metabolic pathway in the human body. A potential application of this research would be to have a specific tea synthesized for an individual with a metabolic pathway ailment, and to be able to reactivate the specific pathway to work as new. To further research, researchers would want to change the demographics to include males and females to determine if the effects are the same between sexes. This research may be a stepping stone to a large possible breakthrough in the medical field. Recently, research has discovered that tea compounds are able to act as an anticarcinogen to assorted cancerous cells.11 With the small steps that research is taking, potentially tea may be able to cure cancer in the future.

Page 16 of 18

Green Tea and Black Tea Metabolite Concentration Determination through Proton Nuclear Magnetic Resonance References 1. Alan Crozier, M. N. C., and Daniele Del Rio, Ed. (2012). Bioavailability of Dietary Monomeric and Polymeric Flavan-3-ols. Flavonoids and Related Compounds. Boca Raton, FL, CRC Press. 2. David L. Nelson, M. M. C. (2009). Principles of Biochemistry. New York, NY, W.H. Freeman and Company. 3. Eckert, D. T. (2011). Principles of Organic Chemistry. Kenosha, Wi, Dr. Timothy Eckert. 4. N. Subramanian, P. V., Shovan Ganguli, Vilas P. Sinkar (1999). "Role of Polyphenol Oxidase and Peroxidase in the Generation of Black Tea Theaflavins." Journal of Agricultural and Food Chemistry(47): 8. 5. Peter Atkins, J. D. P. (2010). Atkin's Physical Chemistry. New York, NY, W.H. Freeman and Company. 6. Putnam, D. F. (1971). "Composition and Concentrative Properties of Human Urine." National Aeronautics and Space Administration: 112. 7. Shii, T., et al. (2011). "Polyphenol Composition of a Functional Fermented Tea Obtained by Tea-Rolling Processing of Green Tea and Loquat Leaves." Journal of Agricultural and Food Chemistry 59(13): 7253-7260. 8. Toyoda, M., et al. (1997). "Profiles of Potentially Antiallergic Flavonoids in 27 Kinds of Health Tea and Green Tea Infusions." Journal of Agricultural and Food Chemistry 45(7): 2561-2564.

Page 17 of 18

Green Tea and Black Tea Metabolite Concentration Determination through Proton Nuclear Magnetic Resonance 9. Van Dorsten, F. A., et al. (2006). "Metabonomics Approach To Determine Metabolic Differences between Green Tea and Black Tea Consumption." Journal of Agricultural and Food Chemistry 54(18): 6929-6938. 10. Yoshiaki Miyake, K. S., Shigenori Kumazawa, Kanefumi Yamamoto, Naohide Kinae, Toshihiko Osawa (2000). "Identification and Antioxidant Activity of Flavonoid Metabolites in Plasma and Urine of Eriocitrin-Treated Rats." Journal of Agricultural and Food Chemistry(48): 8. 11. Mendel Friedman, B. E. M., Hyun-Jeong Kim, In-Seon Lee, Kap-Rang Lee, Seung-Un Lee, Etsuko Kozukue, and Nobuyuki Kozukue (2007). "StructureActivity Relationships of Tea Compounds against Human Cancer Cells." Journal of Agricultural and Food Chemistry(55): 11.

Page 18 of 18