ANTS Two Research Article Analytical Reviews

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enln2323

Science

Andover Newton Theological School

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The portfolio project is an individual formative assessment all students must complete. Your report should be between 2000-2500 words and follow the guidelines presented.

For this task, you will learn about how to structure and complete your portfolio project. Students must choose two of the research articles provided.

For this task, you will have downloaded and reviewed the slides in Task 3.1.1. You are required to choose from one of the four research papers below;

1.Caffeine and physical performance
The Metabolic and Performance Effects of Caffeine.pdf Click for more options

2. GM tomato to treat cancer

Polyphenolic extract of InsP 5-ptase.pdfClick for more options

You will choose one paper and explain why you have chosen this question (why it interests you) and post this to the discussion board.

This will keep the instructor informed of your topic.

Process

  1. First, you will create a thread
  2. Post the research paper you have chosen
  3. You must explain why you have chosen this question for your project
  4. Submit your post
  5. ------------------------------------------------------------------------
  6. TaskFor this task, you will make sure you have followed the following instructions in developing your portfolio project before submission.1- Identify the research question and rationale posed by the article.2- Provide a hypothesis and null hypothesis that applies to question (and papers associated)3- Discuss the scientific rigor of the articles associated with your question ( ie critically analyze and compare and contrast the articles)4- Analyze the data provided in both papers to determine whether or not this accepts or rejects you null hypothesis (You must explain why and reference the data in this section)5- Generate a conclusive scientific theory from the articles you are provided with and explain the assumptions of the theory identified.6- Provide a conclusion, in which you must relate the findings of the the journal articles and provide areas of future researchProcessStep 1
    1. First, You will make sure you have included all the required parts of the project
    2. Submit a single file in the PDF format.
    3. (I WANT YOU TO DO 2 PROJECTS WITH TWO DIFFERENT SUBJECT ONE FOR ME AND ONE FOR MY FRIEND SAME STEPS BUT DIFFERENT SUBJECT)

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The Metabolic and Performance Effects of Caffeine Compared to Coffee during Endurance Exercise Adrian B. Hodgson1, Rebecca K. Randell1, Asker E. Jeukendrup1,2* 1 Human Performance Laboratory, School of Sport and Exercise Science, University Of Birmingham, Birmingham, United Kingdom, 2 Gatorade Sport Science Institute, PepsiCo, Barrington, Illinois, United States of America Abstract There is consistent evidence supporting the ergogenic effects of caffeine for endurance based exercise. However, whether caffeine ingested through coffee has the same effects is still subject to debate. The primary aim of the study was to investigate the performance enhancing effects of caffeine and coffee using a time trial performance test, while also investigating the metabolic effects of caffeine and coffee. In a single-blind, crossover, randomised counter-balanced study design, eight trained male cyclists/triathletes (Mean6SD: Age 4167y, Height 1.8060.04 m, Weight 78.964.1 kg, VO2 max 5863 mlNkg21Nmin21) completed 30 min of steady-state (SS) cycling at approximately 55% VO2max followed by a 45 min energy based target time trial (TT). One hour prior to exercise each athlete consumed drinks consisting of caffeine (5 mg CAF/kg BW), instant coffee (5 mg CAF/kg BW), instant decaffeinated coffee or placebo. The set workloads produced similar relative exercise intensities during the SS for all drinks, with no observed difference in carbohydrate or fat oxidation. Performance times during the TT were significantly faster (,5.0%) for both caffeine and coffee when compared to placebo and decaf (38.3561.53, 38.2761.80, 40.2361.98, 40.3161.22 min respectively, p,0.05). The significantly faster performance times were similar for both caffeine and coffee. Average power for caffeine and coffee during the TT was significantly greater when compared to placebo and decaf (294621 W, 291622 W, 277614 W, 276623 W respectively, p,0.05). No significant differences were observed between placebo and decaf during the TT. The present study illustrates that both caffeine (5 mg/kg/BW) and coffee (5 mg/kg/BW) consumed 1 h prior to exercise can improve endurance exercise performance. Citation: Hodgson AB, Randell RK, Jeukendrup AE (2013) The Metabolic and Performance Effects of Caffeine Compared to Coffee during Endurance Exercise. PLoS ONE 8(4): e59561. doi:10.1371/journal.pone.0059561 Editor: Conrad P. Earnest, University of Bath, United Kingdom Received November 26, 2012; Accepted February 15, 2013; Published April 3, 2013 Copyright: ß 2013 Hodgson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no funding or support to report. Competing Interests: AEJ is employed by Pepsi Co. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. * E-mail: a.e.jeukendrup@bham.ac.uk confirmed the ergogenic effects of caffeine using time trial protocols [3–5,7,8], which involves completing an energy based target or set distance in as fast as time possible, thus simulating variable intensities that are likely to occur during competitive events. In most of these studies pure (anhydrous) caffeine was ingested through capsules or dissolved in water. Based on this research it is often assumed that ingesting caffeine in a variety of dietary sources, such as coffee, will result in the same ergogenic effect. Very few studies, however, have shown a positive effect of coffee on exercise performance. Coffee improved performance in some [9,19–21], but not all studies [22–24]. This may seem surprising as reports have shown that coffee is the most concentrated dietary source of caffeine as well as being one of the largest sources of caffeine used by athletes prior to competition [25]. Amongst the current studies, only two investigations have actually used coffee rather than decaffeinated coffee plus anhydrous caffeine [21,22], with only one of these studies showing an ergogenic effect of the coffee [21]. This further identifies the equivocal evidence surrounding the performance effects of coffee. The most cited study is perhaps a study by Graham et al [22], who showed that running time to exhaustion (85% VO2 max) was only improved when runners ingested pure caffeine (4.5 mg CAF/kg BW), prior to exercise, but not when they ingested either regular coffee Introduction Numerous studies to date have shown that caffeine ingested prior to [1–7] and during [8] prolonged sub-maximal and high intensity exercise can improve performance. Since the seminal work by Costill and colleagues [9] it is often cited that caffeine induces its ergogenic effects by an increase in fat oxidation through the sympathetic nervous system, and a sequential sparing of muscle glycogen [2]. However, there is very little support for an increase in fat oxidation [10,11] or an enhancement to the sympathetic nervous system [12] being the principal mechanism of caffeine’s ergogenic effect. Since, recent investigations have elucidated that the principal mechanism of caffeine’s ergogenic effects is through its ability to act as an adenosine receptor antagonist to induce effects on both central and peripheral nervous system [13] to reduce pain and exertion perception [14], improve motor recruitment [13] and excitation-contraction coupling [15– 17]. In the literature to date, the ergogenic effects are well documented with the time to exhaustion test at a fixed power output being the predominant performance measure used [1,2,9– 11]. It was questioned whether assessing endurance capacity in this way would have sufficient ecological validity to translate results to real life events [18]. However since then, a number of studies have PLOS ONE | www.plosone.org 1 April 2013 | Volume 8 | Issue 4 | e59561 Caffeine and Coffee on Exercise Performance trials, each separated by 7 days. Each trial consisted of consuming: caffeine (5 mg CAF/kg BW) (CAF), coffee (5 mg CAF/kg BW) (COF), decaffeinated coffee (DECAF) or placebo (PLA) in the overnight fasted state (8 hrs) 1 h before completing 30-min steady state cycle exercise bout (SS) (55% V_ O2 max). Following this each participant was instructed to complete a time trial lasting approximately 45-min. (4.5 mg CAF/kg BW), decaffeinated coffee plus caffeine (4.5 mg CAF/kg BW), decaffeinated coffee and a placebo control. The authors reported that the difference in performance could not be explained by the caffeine or methylxanthine plasma concentrations 1 h following intake or at end of exercise, as no difference was observed between trials that contained caffeine. Graham et al [22] suggested that other components in coffee known as chlorogenic acids, may have antagonised the physiological responses of caffeine. However, in this study [22] chlorogenic acids in the coffee or in the plasma were not measured. Chlorogenic acids are a group of phenolic compounds that possess a quinic acid ester of hydroxycinnamic acid [26]. The consumption of chlorogenic acids varies significantly in coffee ranging from 20–675 mg per serving [26]. It has previously been shown in vitro that chlorogenic acids antagonize adenosine receptor binding of caffeine [27] and cause blunting to heart rate, blood pressure and cause a dose-dependent relaxation of smooth muscle [28]. For this reason, it is unclear what role chlorogenic acids, found in coffee, will have on the physiological and metabolic effects of coffee and caffeine during exercise in humans. Therefore, due to the large variation of chlorogenic acids between coffee beverages and the unclear performance effects of coffee to date, it is yet to be determined if coffee causes differences in the performance and metabolic effects during exercise when compared to caffeine alone. Therefore the primary aim of the present study was to investigate whether acute intake of coffee (5 mg CAF/kg BW) and anhydrous caffeine (5 mg CAF/kg BW) are ergogenic to cycling performance compared to decaffeinated coffee or placebo beverages when using a validated 45-minute time trial performance test. In addition, completing a steady state exercise bout prior to the time trial performance test is a routine protocol used in our laboratory [18,29]. For this reason it provided any opportunity to also investigate the effect of acute anhydrous caffeine or coffee intake on substrate oxidation and plasma metabolite responses during 30-min steady state exercise (55% VO2 max). The study hypothesis was that despite the previous work by Graham et al [22], 5 mg CAF/kg BW regardless of the form of administration (anhydrous or coffee) would be ergogenic to performance similarly when compared to decaffeinated coffee or placebo, but this effect would not be mediated through changes in fat metabolism. Preliminary Trial Before the experimental trial, participants visited the Human Performance Laboratory at the University of Birmingham on two separate occasions separated by 7 days. During the first visit participants completed an incremental exercise test on an electronically braked cycle ergometer (Lode Excalibur Sport, Groningen, Netherlands) to volitional exhaustion (V_ O2 max test). Prior to beginning the test participants firstly had weight (OHaus, Champ II scales, USA) and height (Seca stadiometer, UK) recorded. Participants mounted the cycle ergometer, which was followed by a 5-min warm up at 75 W, participants then started the test at 95 W for 3-min. The resistance was increased every 3min, in incremental steps of 35 W, until they reached voluntary exhaustion. Wmax was calculated using the following equation: W max ~Woutz½ðt=180Þ|35 Where Wout is the power output of the last stage completed during the test, and t is the time spent, in seconds, in the final stage. Throughout the test respiratory gas measurements (V_ O2 and V_ CO2 ) were collected continuously using an Online Gas Analyser (Oxycon Pro, Jaeger). V_ O2 was considered maximal if 2 out of the 4 following criteria were met: 1) if V_ O2 levelled off even when workload increased 2) a respiratory exchange ratio (RER) of .1.05 3) a heart rate within 10 beats/min of age predicted maximal heart rate 4) a cadence of 50 rpm could not be maintained. Heart rate (HR) was recorded during each stage of the test using a HR monitor (Polar, Warwick, UK). Wmax was used to determine the work load for the steady state exercise bouts throughout all subsequent experimental trials (50% Wmax). Wmax was also used to calculate the total amount of work to be completed during the 45-min time trial and the linear factor, both calculated according to the formula derived by Jeukendrup et al [18]. The bike position was recorded following the test to be replicated in all other trials. Approximately 7 days later participants reported back to the lab, for the second preliminary trial, between 0600 and 0800 having undergone an 8 hr fast. The purpose of the trial was to familiarise each subject to the experimental trial and time trial protocol. All participants completed 30-min SS at 50% Wmax (55% V_ O2 max). Expired breath samples were collected every 10min for measures ofV_ O2 , V_ CO2 , and RER (Oxycon Pro, Jaeger). Immediately following all participants completed a time trial lasting ,45 min. The data collected during the familiarisation trial was not used for any of the final analysis. Materials and Methods Participants Eight trained cyclists/triathletes (Mean 6 SD: Age 4167y, Height 1.8060.04 m, Weight 78.964.1 kg, V_ O2 max 5863 mlNkg21Nmin21) were recruited from local Birmingham cycling and triathlon clubs. Inclusion criteria included participants who trained 3 or more times per week (.90-min/session), had been training for .2 years, and had a low habitual caffeine intake of #300 mg/d (approximately #3 cups coffee/d). Ethics statement All participants were fully informed of the experimental trials and all risks and discomforts associated before providing written informed consent to participate in the study. All procedures and protocols were approved by the Life and Environmental Sciences Ethical Review Committee at the University of Birmingham. Experimental Trial All participants reported to the Human Performance Lab between 0600 and 0800 having completed an 8 h overnight fast. On arrival weight was recorded (Seca Alpha, Hamburg, Germany) and a flexible 20-gauge Teflon catheter (Venflon; Becton Dickinson, Plymouth, United Kingdom) was inserted into an antecubital vein. A 3-way stopcock (Connecta; Becton Dickinson) was attached to the catheter to allow for repeated blood sampling General Study design The study followed a single blinded, cross over, randomised counter-balanced study design. Maximal oxygen uptake (V_ O2 max) and power (Wmax) was assessed during a preliminary trial. Following this each participant completed 4 experimental PLOS ONE | www.plosone.org 2 April 2013 | Volume 8 | Issue 4 | e59561 Caffeine and Coffee on Exercise Performance and served in an opaque sports drinks bottle. The exact dose of quinine sulphate was preliminary tested in our lab. A dose of 8 mg was sufficient to prevent the blinded researchers from distinguishing between the placebo and caffeine trial. The coffee and decaffeinated coffee samples were further analysed externally for chlorogenic acids (5-CQA) (Eurofins Scientific, Italy). Based on the analysis, total 5-CQA was 33.91 mg/g and 28.29 mg/g for COF and DECAF respectively. This was then used to calculate the average concentration of total 5-CQA and related isomers for each participants drinks, based on the weight of instant COF and DECAF. The average chlorogenic acid intake in COF and DECAF are presented in Table 1. All of the beverages were prepared in 600 ml of water. This was firstly to avoid any difference in uptake and bioavailability of each of the ingested ingredients, as well as replicating the format of coffee consumption from everyday life. Secondly 600 ml of coffee dissolved in water was not only considered tolerable, based on taste testing by researchers within our laboratory, but also matched similar volumes as used by Graham et al [22]. Once participants received each of the beverages at the beginning of the trial, they had 15 min to consume the entire 600 ml. during the experimental period. An initial 15 ml fasting blood sample was collected (EDTA-containing tubes, BD vacutainers). Following this all participants consumed one of the treatment beverages and rested for 1 h, with further samples taken at 30 min and 60 min (10 ml EDTA). After the rest period, participants then mounted the cycle ergometer, in an identical bike position as recorded during the preliminary trial, and began a 30-min SS at 50% Wmax (55% V_ O2 max). Blood samples (15 ml) and 5-min respiratory breath samples, VO2, VCO2 and RER, (Oxycon Pro, Jaeger) were collected every 10-min during the exercise. The catheter was kept patent during both the rest and exercise period by flushing it with 5 mL isotonic saline (0.9% w/v; B Braun) after every blood sample. In addition, heart rate (HR) was recorded (Polar RS800CX) every 15 min at rest and every 10 min during SS. Ratings of Perceived Exertion (RPE) scale were recorded every 10 min during SS using the 6–20 Borg scale [30]. Upon completion of the SS, the subject was instructed to stop exercising for ,1 min, and the cycle ergometer was set in the linear mode. The participants were instructed to complete an energy-based target amount of work at 70% Wmax in the quickest time possible. The total amount of work (650637 KJ) was calculated for the 45 min time trial. A linear factor, 70% Wmax divided by (90 rpm)2 was entered into the cycle ergometer. The time trial protocol employed has previously been validated and has been shown to be highly reliable [18]. Participants received a countdown prior to starting the time trial and received no verbal or visual feedback regarding performance time or physiological measures throughout the test. No additional measures, blood or respiratory, were taken through the test. Participants received no feedback about their performance until they had completed all 4 experimental trials. Following the completion of the TT, each participant completed a questionnaire to guess the test beverage consumed prior to the commencement of the trial, as well as report any GI distress experienced during the trial. Diet and Exercise Control Participants were instructed to record their food intake the day prior to the preliminary familiarisation trial. Participants had to replicate this diet in the 24 h prior to each experimental trial, as well as refraining from any exercise, consume no alcohol and withdraw from any caffeinated products. Calculations Substrate metabolism was measured during the SS. From the respiratory output measurements of V_ O2 and V_ CO2 (L/min), carbohydrate [1] (CHO) and fat oxidation [2] was calculated every 10 min during the SS. In order to calculate CHO and fat oxidation stoichiometric equations [31] were used, which assume that each of the participants were exercising at a steady state and that protein oxidation was negligible. Treatment Beverages During each visit to the lab, participants ingested one of four treatment beverages. This included caffeine (5 mg CAF/kg BW), regular coffee (5 mg CAF/kg BW), decaffeinated coffee and placebo. Therefore decaffeinated coffee and placebo acted as controls to both of the caffeinated trials. Caffeine (Anhydrous caffeine, 99.8% pure, Blackburn Distributions Ltd, Nelson, United Kingdom) was weighed (394.467.0 mg) prior to the trial, and was immediately dissolved and vortexed for 15 min in 600 ml of water prior to consumption and served in an opaque sports drinks bottle. Coffee was prepared using instant coffee (Nescafe Original). In order to select the correct weight of coffee to equal 5 mg CAF/kg BW, Nescafe states that Nescafe Original instant coffee provides 3.4 g caffeine/100 g of instant coffee. This information was confirmed using a HPLC method (see below), and based on the analysis it was calculated that 0.15 g coffee/kg/BW equalled 5 mg CAF/kg BW. Therefore prior to each trial, coffee was weighed (11.861.0 g) and dissolved in 600 ml hot water (9462uC) and served in a mug. DECAF (Nescafe Original Decaffeinated coffee, ,97% caffeine free) was prepared in an identical fashion, with the same amount of decaffeinated coffee as the COF beverage. Using a HPLC method, decaffeinated coffee provided minimal caffeine throughout each of the prepared beverages (0.17 mg CAF/kg BW or mean intake of 13.4160.70 mg). In order to blind the participants from the taste of the caffeine trial, the placebo trial consisted of 8 mg of Quinine sulphate (Sigma, UK). Quinine sulphate is a food ingredient found in tonic water to give a bitter taste. The quinine sulphate was dissolved in 600 ml of water, vortexed for 15 min, PLOS ONE | www.plosone.org ½1~4:210 V_ CO2 {2:962 V_ O2 ½2~1:65 V_ O2 {1:701 V_ CO2 Blood Analysis Following collection, all tubes were placed in ice until the end of the experimental trial. Following this each tube was centrifuged at 3500 rpm for 15 min at 4uC. Aliquots of plasma were immediately frozen in liquid nitrogen and stored at 280uC for later analysis. Each blood sample taken throughout each experimental trial were analysed for plasma glucose (Glucose Oxidase; Instrumentation Laboratories, England), fatty acids (FA) [NEFA-C; Randox, England], glycerol (Glycerol; Randox, England) and lactate [Lactate, Randox, England] using an ILAB 650 (Instrumentation Laboratory, Cheshire, United Kingdom). Plasma Caffeine and Chlorogenic Acid analysis Plasma caffeine were analysed externally (City Hospital, Dudley, Birmingham) using a reversed-HPLC-UV method. The sample preparation included: 200 mL of plasma were added to 100 mL internal standard (Proxyphylline, Sigma, United Kingdom) before mixing, heating and adding 500 mL of acetic acid. 3 April 2013 | Volume 8 | Issue 4 | e59561 PLOS ONE | www.plosone.org The supernatant was the injected (5 mL) onto a Phenomenex Prodigy 15064.60 mm 5 m Octadecyl Silane (ODS) column using an auto sampler and detected at a UV of 273 nm. Caffeine concentrations were quantified using one point calibration from a calibrator that had previously been internally validated against a 9 point calibration curve (City Hospital, Dudley, Birmingham). With each batch two QC (High and Low) were run, with reference to the internal standard to account for any loses. The caffeine content of coffee and decaffeinated coffee were confirmed at the School of Sport and Exercise Sciences. In brief, coffee and decaf coffee samples were prepared (identical to preparation described above) and cooled before 5 mL of each sample were injected onto a Phenomenex Luna 10 m C18 (2) column using an auto sampler (WPS 3000, Dionex, United Kingdom). The mobile phase consisted of 0.1 M acetic acid in water and 0.1 M acetic acid in acetonitrile. Caffeine concentrations were quantified using a 10-point caffeine calibration curve in water. Coffee and Decaf coffee were analysed for chlorogenic acids. The analysis was conducted externally (Eurofins Scientific, Italy) using a reverse HPLC methodology at 325 nm (Water Symmetry C18, 25064.6 mm, 5 mm) with external 5-QCA standards for quantification on a 3 point calibration (10–250 mg/kg). The mobile phase consisted of aqueous 0.5% formic acid and acetonitrile. Abbreviations: CQA Caffeoylquinic acid, 5-CQA 5-O-Caffeoylquinic acid, 4-CQA 4-O-Caffeoylquinic acid, 5-FQA 5-O-Feruloylquinic acid, 4-FQA 4-O-Feruloylquinic acid, 3,5-diCQA 3,5-O-Dicaffeoylquinic acid, 3,4-diCQA 3,4-ODicaffeoylquinic acid, 4,5-diCQA 4,5-O-Dicaffeoylquinic acid, 4,5-CFQA 4,5-O-Dicaffeoylquinic acid, ml millilitres, mg milligrams, CAF Caffeine, COF Coffee, DECAF Decaffeinated Coffee, PLA Placebo. doi:10.1371/journal.pone.0059561.t001 600 PLA - - - 24% 33% 600 DECAF 13.460.2 328.166.1 23% 7% - 2% 1% 2% 1% - 7% 3% 3% - 2% 3% 7% 8% - 32% 22% - 21% 393.367.3 600 COF 394.467.0 600 CAF 394.467.0 - - 4-FQA 5-FQA 4-CQA 5-CQA CQA % of Total 5-CQA Total 5-CQA (mg/serving) Caffeine content (mg/serving) Serving Volume (ml) Treatment beverage Table 1. Mean caffeine and chlorogenic acid (Total 5-QCA) concentration in each treatment beverage serving. 3,5-diCQA 3,4-diCQA 4,5-diCQA 4,5-CFQA Caffeine and Coffee on Exercise Performance Statistical Analysis Data analysis was performed using SPSS for WINDOWS software (version 17; SPSS Inc, Chicago, IL). Data are expressed as means 6 SEMs, unless otherwise stated. A repeated measure ANOVA was used to assess differences in respiratory, substrate metabolism, plasma metabolite and caffeine concentration as well as time trial performance measurements during each trial. In order to detect differences across time and between treatments a Fisher protected least significant differences post hoc test was used Significance was set at P,0.05. Results Steady State Exercise Whole body respiratory measures, HR and RPE. The selected workload of 50% Wmax during the SS (17167 W) resulted in similar oxygen uptake (V_ O2 ) (2590679, 2595689, 2465679, 2522671 mL/min for CAF, COF, DECAF and PLA respectively P = 0.278). As a result the relative exercise intensity during the SS was similar throughout each trial (5862%, 5862%, 5561% and 5561% for CAF, COF, DECAF and PLA respectively P = 0.337). Energy expended during SS was also shown to be similar (1607649 KJ, 1611654 KJ, 1531650 KJ, 1565643) for CAF, COF, DECAF and PLA respectively P = 0.248). In addition no significant difference was observed in average HR during exercise (11964, 11964, 11964, 12065 bpm for CAF, COF, DECAF and PLA respectively P = 0.281) or RPE values (1060, 1060, 1160 and 1160 for CAF, COF, DECAF and PLA respectively P = 0.091) during SS between trials. Carbohydrate and Fat oxidation. Carbohydrate oxidation rates during SS significantly reduced in all treatments across time (P = 0.001). However there was no significant difference in carbohydrate oxidation between each of the treatments (Figure 1A P = 0.288). Similarly, fat oxidation rates significantly increased during SS in all treatments (P = 0.001). No significant difference in fat oxidation was observed between each of the treatments (Figure 1B P = 0.445). Accordingly the contribution of carbohydrate and fat to total energy expenditure during SS was 4 April 2013 | Volume 8 | Issue 4 | e59561 Caffeine and Coffee on Exercise Performance Figure 1. Carbohydrate oxidation (g/min) (A) and fat oxidation (g/min) (B) rates during 30 min steady state exercise (55% VO2 max) 1 hour following ingestion of caffeine, coffee, decaf or placebo beverages. Data represented seen as Closed circles – Caffeine Open circles – Caffeinated Coffee Closed triangles – Decaffeinated coffee Open triangles – Placebo. Means 6 SE n = 8 doi:10.1371/journal.pone.0059561.g001 not significantly different between any of the treatments (P = 0.463). Plasma metabolite concentrations. Plasma metabolite responses at rest and exercise are displayed in Figure 2 A–D. Plasma glucose concentrations (Figure 2A) were significantly elevated at the end of rest compared to the beginning of rest following CAF and COF (p,0.05 for both), while no significant difference occurred following DECAF or PLA (P = 0.676 and 0.188 respectively). The elevation in glucose concentrations with CAF following the rest period was significantly higher compared to DECAF only (P,0.05). During exercise, plasma glucose increased over time following CAF and COF, however only COF reached statistical significance (P,0.05). DECAF and PLA glucose concentrations fell during the onset of exercise with a significant increase in both treatments later in exercise (T = 10–30 P,0.05 for both). As a result, CAF had significantly higher glucose concentrations within the first 20 minutes of exercise compared to DECAF and PLA (P,0.05 for both), while at end of exercise CAF and COF had significantly higher glucose concentrations compared to PLA only (P,0.05 for both). Plasma FAs concentration (Figure 2B) were significantly elevated at the end of rest compared to beginning of rest following CAF (P = 0.010), while DECAF and PLA had reduced FA concentration, with only DECAF reaching statistical significance (P = 0.007 and P = 0.072 respectively). Therefore, CAF had significantly higher FAs concentration compared to DECAF and PLA at end of rest period (P,0.05 for both). During the beginning of exercise, CAF continued to have significantly elevated FAs concentration compared to DECAF only (P = 0.037). FA concentration significantly increased during exercise (T = 10–30 P = 0.030) with no significant differences observed between treatments (P = 0.231). Plasma glycerol concentrations (Figure 2C) did not significantly change at rest for CAF (P = 0.066), COF (P = 0.392) and DECAF (P = 0.104) but PLA significantly fell (P = 0.022). DECAF was significantly lower at end of rest compared to CAF, COF and PLA (P,0.05 for all), with no significant differences observed between any other beverage. During exercise there was a significant increase in glycerol concentrations for all treatments over time PLOS ONE | www.plosone.org (P = 0.001), with significantly higher concentrations observed for CAF (P = 0.027) and COF (P = 0.003) at beginning of exercise compared to DECAF only. Plasma lactate concentrations (Figure 2D), were significantly increased following the consumption of CAF and COF only during the rest period compared to DECAF (P = 0.050 and P = 0.003 respectively) and PLA (P = 0.002 and P = 0.001 respectively). In addition DECAF had significantly elevated lactate compared to PLA at end of rest period (P = 0.012). All treatments lactate concentrations significantly increased at the onset of exercise (P = 0.004) with CAF and COF being significantly higher compared to PLA (P = 0.037 and P = 0.010 respectively). CAF and COF had sustained lactate concentrations at the end of exercise, with significantly higher concentrations compared to DECAF (P = 0.008 and P = 0.028 respectively) and PLA (P = 0.050 and P = 0.005 respectively). Plasma caffeine concentrations. The plasma caffeine concentrations following each beverage are displayed in Figure 3. At baseline plasma caffeine concentrations were very low for all treatments (,3 mM), with no significant differences observed (P = 0.478). Plasma caffeine significantly increased following CAF and COF when compared to DECAF (P = 0.000 and P = 0.009 respectively) and PLA (P = 0.000 and P = 0.010 respectively), with peak concentrations observed 60 min after intake (38.262.8 mM and 33.565.0 mM respectively). No significant difference was observed in the plasma caffeine concentrations between CAF or COF (P = 0.156) and DECAF or PLA (P = 0.558) throughout the trials. Time trial performance CAF and COF significantly improved TT finishing times when compared to both DECAF (P,0.05 for both) and PLA (P = 0.007 and P = 0.010) (Figure 4). As a result mean power output during the TT was significantly greater for both CAF and COF compared to DECAF and PLA (29466, 29167, 27667, 27764 W, respectively P,0.05 for both). However no significant differences were seen in average heart rate during the TT between CAF, COF, DECAF and PLA (17063, 16764, 16463, 16564 BPM, respectively P = 0.516). CAF significantly improved TT perfor5 April 2013 | Volume 8 | Issue 4 | e59561 Caffeine and Coffee on Exercise Performance Figure 2. Plasma metabolite responses at rest (t = -60-0) and during 30 min steady state exercise (55% VO2 max) (t = 0–30) following ingestion of caffeine, coffee, decaf or placebo beverages. A Glucose. B Fatty acids (FA). C Glycerol. D Lactate. Data represented seen as Closed circles – Caffeine Open circles – Caffeinated Coffee Closed triangles – Decaffeinated coffee Open triangles – Placebo. a Sig. different between CAF and DECAF (p,0.05) b Sig. different between CAF and PLA (p,0.05) c Sig. different between COF and DECAF (p,0.05) d Sig. different between COF and PLA (p,0.05). Means 6 SE n = 7. doi:10.1371/journal.pone.0059561.g002 acute caffeine ingestion for improving prolonged endurance exercise performance [1–7]. The effects of caffeine on time trial endurance performance (.5 min) have recently been reviewed in a well conducted meta-analysis [7]. The authors concluded that of the 12 studies that investigated caffeine intake (1–6 mg CAF/kg BW), performance was improved by ,3%. Fewer studies have investigated the ergogenic effects of coffee, with results being mixed thus far. In agreement with the literature, the current study found an improvement in performance following caffeine intake of 4.9% and 4.5% when compared to decaf coffee and placebo, respectively (Table 2). Interestingly, the current study also showed that coffee improved performance to the same extent as caffeine when compared to decaf coffee and placebo, 4.7% and 4.3% respectively. Thus, this is the first study to date to demonstrate that coffee consumed 1 h prior to exercise, at a high caffeine dose (5 mg CAF/kg BW), is equally as effective as caffeine at improving endurance exercise performance. Our findings are in line with a number of studies that have shown improvements to performance following coffee intake [9,19–21]. Costill et al [9] were the first to show that decaf coffee plus caffeine (330 mg), improved exercise time to exhaustion (80% mance by 4.9% (95% confidence interval (CI) = 2.3–6.8%) and 4.5% (95% confidence interval (CI) = 2.3–6.2%) compared to PLA and DECAF respectively (p,0.05 for both). Equally, COF significantly improved TT performance by 4.7% (95% confidence interval (CI) = 2.3–6.7%) and 4.3% (95% confidence interval (CI) = 2.5–7.1%) compared to PLA and DECAF respectively (p,0.05 for both) (Table 2). In addition there were no significant differences in TT finishing time between CAF and COF (P = 1.000) or PLA and DECAF (P = 1.000). Following the completion of the TT, 3/8 participants were able to successfully guess the correct order of test beverages consumed prior to the trial. The correct guesses were more consistent for detecting CAF compared to the other drinks, with 6/8 of the participants guessing correctly. None of the participants reported any serious symptoms of GI distress at the end of any of the trials. Discussion The present study examined the effects of acute intake of coffee (5 mg CAF/kg BW) and caffeine (5 mg CAF/kg BW) on time trial cycling performance, as well as substrate utilisation during SS exercise. Numerous studies to date have shown the efficacy of PLOS ONE | www.plosone.org 6 April 2013 | Volume 8 | Issue 4 | e59561 Caffeine and Coffee on Exercise Performance Figure 3. Plasma caffeine concentrations following ingestion of caffeine, coffee, decaf or placebo beverages. a CAF significantly different to DECAF and PLA (p,0.001) b COF significantly different to DECAF and PLA (p,0.05). Data represented seen as Closed circles – Caffeine Open circles – Caffeinated Coffee Closed triangles – Decaffeinated coffee Open triangles – Placebo Means 6 SE n = 7. doi:10.1371/journal.pone.0059561.g003 Figure 4. Time trial finishing time (min) for caffeine, coffee, decaf or placebo beverages a CAF significantly different to DECAF and PLA (p,0.05) b COF significantly different to DECAF and PLA (p,0.05). Data represented seen as Closed bar– Caffeine Open bar – Caffeinated Coffee Dark grey bar– Decaffeinated coffee Light grey bar– Placebo. Means 6 SE n = 8. doi:10.1371/journal.pone.0059561.g004 PLOS ONE | www.plosone.org 7 April 2013 | Volume 8 | Issue 4 | e59561 Caffeine and Coffee on Exercise Performance Table 2. Time trial performance data for each treatment. Improvement compared to PLA % (95% confidence intervals) P value Improvement compared to DECAF % (95% confidence intervals) P value CAF 38.3560.48 a 4.9 (2.326.8) 0.007 4.5 (2.326.2) 0.012 COF 38.2760.57b 4.7 (2.326.7) 0.010 4.3 (2.527.1) 0.012 DECAF 40.2360.63 20.4 (24.023.1) 1.000 - - PLA 40.0660.39 - - 0.3(20.323.9) 1.000 Treatment TT finish time (min) Means 6 SE n = 8 a significantly different to DECAF and PLA (p,0.05) b significantly different to DECAF and PLA (p,0.05) Abbreviations: CAF Caffeine, COF Coffee, DECAF Decaffeinated Coffee, PLA Placebo. doi:10.1371/journal.pone.0059561.t002 bioavailability of plasma caffeine and paraxanthines did not differ to caffeine [22], which is in line with the present study (Figure 4). Further, in vitro studies suggest that chlorogenic acids antagonize adenosine receptor binding of caffeine [27] and cause blunting to heart rate and blood pressure in rats [28]. Yet, in vivo there is no evidence to suggest that chlorogenic acids, especially at the low nanomolar concentration typically observed [32], impact on the mechanisms of action of caffeine that lead to the ergogenic effects. In support of this notion, and in agreement with the current study, regular coffee (1.1 mg/kg/BW) consumed prior to the ingestion of different doses of caffeine (3–7 mg/kg/BW) has been shown not to affect the ergogenic effects of caffeine [21]. The improvement in performance in the current study is unlikely to be explained by alterations to fat oxidation, as no difference during the SS exercise bout was observed (Figure 1B). This is in agreement with a number of investigations that do not support the thesis that caffeine improves exercise performance by augmenting fat metabolism [33,34]. In addition these effects are apparent despite consistent increases in adrenaline though activation of the SNS [33,34] and a subsequent elevation in FA appearance in the circulation following caffeine intake [33,34]. It is evident that the improvement in performance is likely through caffeine’s direct antagonism of adenosine receptors (A1 and A2A) on the skeletal muscle membrane to improve excitation-contraction coupling [13] via a greater release of Ca2+ from the SR [16] and/ or improved Na+/K+ ATPase pump activity [15]. In support of this notion, Mohr et al [12] observed that tetrapelegic patients, who have an impaired sympathoadrenal response [35], showed that caffeine improved exercise performance, while RER did not change during an electrical stimulated cycling test. Further, the authors also observed a significant increase in FA and glycerol at rest and during exercise following caffeine intake, despite a lack of an adrenaline response. This is due to the fact that adenosine has been shown to inhibit lipolysis [36] and enhance insulin stimulated glucose uptake in contracting skeletal muscle in vitro [37]. In support, the current studyobserved a significant increase in plasma glucose, FA and glycerol concentrations following caffeine (Figure 2 A, B, C). In addition, the consistently reported elevation in adrenaline concentrations [33] combined with adenosine receptor antagonism following caffeine intake during exercise may work synergistically to activate glycogenolysis in exercising and non-exercising tissues [34] as well as adipose tissue/skeletal muscle lipolysis [33]. This supports the fact that the current study (Figure 2 D) and others have shown that caffeine increase plasma lactate concentrations at rest and during exercise [33,34]. Though to date there is little supporting evidence that caffeine stimulates exercising skeletal muscle glycogenolysis [33,38], with early studies showing a paradoxical glycogen sparing effect with caffeine [2]. The elevated lactate concentrations are more likely due to a VO2 max) compared with decaffeinated coffee (,18%). More recently, Wiles et al [19] showed that coffee was able to improve 1500 m treadmill running performance when compared to decaffeinated coffee (,3%). However, the current study results are in contrast to a number of other studies [22–24]. For example, the work conducted by Graham et al [22] showed that coffee (4.5 mg CAF/kg BW), regardless of the format of intake (regular coffee or decaffeinated coffee plus caffeine) did not result in an improvement to running time to exhaustion (75% VO2 max), where as caffeine (4.5 mg CAF/kg BW) significantly improved performance. Therefore it was concluded by Graham et al [22] that the performance effects of coffee may be inferior to caffeine. Despite this evidence, the current study clearly demonstrates that coffee is as effective as caffeine at improving endurance exercise performance. The discrepancy in the performance effects of caffeine and coffee between the present study and Graham et al [22] might be explained by the type of performance test implemented. Time to exhaustion tests have been shown to be highly variable from day to day, with a coefficient of variation (CV) ,27% in one study [18]. It is possible that this large variability may have contributed to the lack of performance effects found by Graham et al [22]. Whereas using a time trial performance measure, as used in the current study, has previously been shown to be highly reproducible (CV,3%) and could detect smaller differences in performance [18]. Also the number of comparisons in the study by Graham et al [22] was greater than in the present study with a similar subject number, indicating that their statistical power was smaller. Perhaps for these reasons, the current study was able to detect similar changes in performance following caffeine and coffee intake (,5%) (Table 2), whereas Graham et al [22] did not. The composition and preparation of coffee in each of the studies [9,19–24] may also explain the discrepancies in the ergogenic effects of coffee. Coffee is ,2% caffeine, with the remainder composed of chlorogenic acids, ferulic acid, caffeic acid, nicotinic acid as well as other unidentifiable compounds [26]. It is evident that the source of coffee beans, roasting, storage and preparation (brewing and filtering) dramatically alters the caffeine and chlorogenic acid content of the coffee [26]. In accordance, recent evidence has shown that the chlorogenic acid content of commercially available espresso coffees range from 24–422 mg/ serving [26]. In support, the current study observes a high chlorogenic acid content in both coffee and decaffeinated coffee samples (Table 1). Graham et al [22] speculated that chlorogenic acids found in coffee may have blunted the physiological effects of caffeine, preventing an improvement in exercise performance. However the authors did not report measurements of chlorogenic acids in coffee or in plasma to support this speculation. Despite the compounds present in coffee, the authors reported that the PLOS ONE | www.plosone.org 8 April 2013 | Volume 8 | Issue 4 | e59561 Caffeine and Coffee on Exercise Performance reduced clearance by the exercising muscle and a greater release by non exercising tissues [33]. Consequently, due to the healthy participants tested in the current study it is likely that the adenosine receptor antagonism by caffeine plays a crucial role in inducing the ergogenic effects of caffeine while regulating the metabolite response synergistically with the SNS. Interestingly, despite coffee producing similar ergogenic effects as caffeine, the metabolite responses were not identical (Figure 2). The current study observed that the significant increase in plasma glucose, FA and glycerol with caffeine was paralleled with an attenuated response for coffee, and a significantly blunted response with decaf coffee when compared to placebo (Figure 2 A, B, C). This is likely due to the compounds in coffee [39] inducing subtle effects on antagonism of adenosine receptors (A1 and A2A) in a variety of exercising and non exercising tissues. In accordance, Graham et al [22] previously showed that coffee resulted in a blunted adrenaline response when compared to caffeine at rest in humans, which was attributed to chlorogenic acids antagonizing adenosine receptor binding of caffeine [27]. In addition nicotinic acid, a fatty acid ester found in coffee known to inhibit lipolysis, has been shown to lower FA concentrations in patients suffering from hyperlipidemia [40]. Chlorogenic acids are also believed to improve glucose uptake at the skeletal muscle when compared to caffeine [41], also by altering the antagonism of adenosine receptors. More recently, caffeic acid has been found to stimulate skeletal muscle glucose transport, independent of insulin, when accompanied with an elevation in AMPK in vitro [42]. Despite the aforementioned evidence, it remains unclear why compounds in coffee appear to modulate the metabolite response but not the ergogenic effects of coffee in the current study. The current study provided a large bolus of caffeine in the form of anhydrous caffeine or coffee one hour prior to exercise (5 mg/ kg BW). The chlorogenic acid content of the coffee beverages was different, which is worth highlighting as a potential limitation of the current study. Previous studies have failed to make comparisons between coffee and decaf coffee and instead have used decaf plus anhydrous caffeine [9,19-21,23,43]. In addition these studies did not examine the chlorogenic acid content of the test beverages. Thus, the novelty of the current study was that the performance effects were investigated between caffeine and coffee, independent of the combined effects of decaffeinated coffee plus caffeine. Adding a decaf plus caffeine trial would have been successful in controlling for chlorogenic acid content of the beverage. However, firstly, investigating the effect of chlorogenic acids on the metabolic and performance effects of caffeine was not the primary aim of the current study. Secondly, and more importantly, the low nanomolar concentration of chlorogenic acids in vivo [32] is unlikely to impact on the mechanisms of action of caffeine when compared to the physiological effects observed in vitro from supra physiological concentrations of chlorogenic acids [27,28]. Yet, as differences in the metabolic effects of caffeine compared to coffee were observed in the current study, it may be important for future studies to control for chlorogenic acid content in coffee beverages or additionally increase the dose of chlorogenic acids to raise the bioavailability in vivo. In turn this will provide further insights into the metabolic differences between caffeine and coffee. In conclusion, the present study showed that caffeine and coffee (5 mg CAF/kg BW) were both able to improve exercise performance to the same extent, when compared to both decaffeinated coffee and placebo. Our data does not support the notion that chlorogenic acids found in coffee impair the ergogenic effects of caffeine. However, the compounds found in coffee may alter the metabolic effects, as the current study observed differences between caffeine and coffee at rest and during exercise. It is yet to be determined if lower doses of caffeine, when ingested as coffee, offer the same ergogenic effects. This would offer a more applicable and realistic nutritional strategy for athletes. Acknowledgments The authors express appreciation to Dr Gareth Wallis for his input and advice in the preparation of this paper. Author Contributions Critically reviewed the paper: ABH RKR AEJ. Conceived and designed the experiments: ABH RKR AEJ. Performed the experiments: ABH RKR. Analyzed the data: ABH AEJ. Wrote the paper: ABH AEJ. References 12. Mohr T, Van Soeren M, Graham TE, Kjaer M (1998) Caffeine ingestion and metabolic responses of tetraplegic humans during electrical cycling. Journal of applied physiology 85: 979–985. 13. Tarnopolsky MA (2008) Effect of caffeine on the neuromuscular system-potential as an ergogenic aid. Applied physiology, nutrition, and metabolism = Physiologie appliquee, nutrition et metabolisme 33: 1284–1289. 14. Doherty M, Smith PM (2005) Effects of caffeine ingestion on rating of perceived exertion during and after exercise: a meta-analysis. Scandinavian journal of medicine & science in sports 15: 69–78. 15. Mohr M, Nielsen JJ, Bangsbo J (2011) Caffeine intake improves intense intermittent exercise performance and reduces muscle interstitial potassium accumulation. Journal of applied physiology 111: 1372–1379. 16. Tallis J, James RS, Cox VM, Duncan MJ (2012) The effect of physiological concentrations of caffeine on the power output of maximally and submaximally stimulated mouse EDL (fast) and soleus (slow) muscle. Journal of applied physiology 112: 64–71. 17. Tarnopolsky M, Cupido C (2000) Caffeine potentiates low frequency skeletal muscle force in habitual and nonhabitual caffeine consumers. Journal of applied physiology 89: 1719–1724. 18. Jeukendrup A, Saris WH, Brouns F, Kester AD (1996) A new validated endurance performance test. Med Sci Sports Exerc 28: 266–270. 19. Wiles JD, Bird SR, Hopkins J, Riley M (1992) Effect of caffeinated coffee on running speed, respiratory factors, blood lactate and perceived exertion during 1500-m treadmill running. Br J Sports Med 26: 116–120. 20. Trice I, Haymes EM (1995) Effects of caffeine ingestion on exercise-induced changes during high-intensity, intermittent exercise. International journal of sport nutrition 5: 37–44. 1. Graham TE, Spriet LL (1995) Metabolic, catecholamine, and exercise performance responses to various doses of caffeine. J Appl Physiol 78: 867–874. 2. Spriet LL, MacLean DA, Dyck DJ, Hultman E, Cederblad G, et al. (1992) Caffeine ingestion and muscle metabolism during prolonged exercise in humans. Am J Physiol 262: E891–898. 3. Jenkins NT, Trilk JL, Singhal A, O’Connor PJ, Cureton KJ (2008) Ergogenic effects of low doses of caffeine on cycling performance. Int J Sport Nutr Exerc Metab 18: 328–342. 4. Irwin C, Desbrow B, Ellis A, O’Keeffe B, Grant G, et al. (2011) Caffeine withdrawal and high-intensity endurance cycling performance. J Sports Sci 29: 509–515. 5. Desbrow B, Biddulph C, Devlin B, Grant GD, Anoopkumar-Dukie S, et al. (2012) The effects of different doses of caffeine on endurance cycling time trial performance. Journal of sports sciences 30: 115–120. 6. Doherty M, Smith PM (2004) Effects of caffeine ingestion on exercise testing: a meta-analysis. Int J Sport Nutr Exerc Metab 14: 626–646. 7. Ganio MS, Klau JF, Casa DJ, Armstrong LE, Maresh CM (2009) Effect of caffeine on sport-specific endurance performance: a systematic review. J Strength Cond Res 23: 315–324. 8. Cox GR, Desbrow B, Montgomery PG, Anderson ME, Bruce CR, et al. (2002) Effect of different protocols of caffeine intake on metabolism and endurance performance. J Appl Physiol 93: 990–999. 9. Costill DL, Dalsky GP, Fink WJ (1978) Effects of caffeine ingestion on metabolism and exercise performance. Med Sci Sports 10: 155–158. 10. Chesley A, Howlett RA, Heigenhauser GJ, Hultman E, Spriet LL (1998) Regulation of muscle glycogenolytic flux during intense aerobic exercise after caffeine ingestion. Am J Physiol 275: R596–603. 11. Graham TE, Spriet LL (1991) Performance and metabolic responses to a high caffeine dose during prolonged exercise. J Appl Physiol 71: 2292–2298. PLOS ONE | www.plosone.org 9 April 2013 | Volume 8 | Issue 4 | e59561 Caffeine and Coffee on Exercise Performance 21. McLellan TM, Bell DG (2004) The impact of prior coffee consumption on the subsequent ergogenic effect of anhydrous caffeine. International journal of sport nutrition and exercise metabolism 14: 698–708. 22. Graham TE, Hibbert E, Sathasivam P (1998) Metabolic and exercise endurance effects of coffee and caffeine ingestion. J Appl Physiol 85: 883–889. 23. Lamina S, Musa DI (2009) Ergogenic effect of varied doses of coffee-caffeine on maximal aerobic power of young African subjects. African health sciences 9: 270–274. 24. Butts N, D C (1985) Effect of caffeine ingestion on cardiorespiratory endurance in men and women. Res Q Exerc Sport 56: 301–305. 25. Desbrow B, Leveritt M (2006) Awareness and use of caffeine by athletes competing at the 2005 Ironman Triathlon World Championships. International journal of sport nutrition and exercise metabolism 16: 545–558. 26. Crozier TW, Stalmach A, Lean ME, Crozier A (2012) Espresso coffees, caffeine and chlorogenic acid intake: potential health implications. Food Funct 3: 30–33. 27. de Paulis T, Schmidt DE, Bruchey AK, Kirby MT, McDonald MP, et al. (2002) Dicinnamoylquinides in roasted coffee inhibit the human adenosine transporter. European journal of pharmacology 442: 215–223. 28. Tse SY (1992) Cholinomimetic compound distinct from caffeine contained in coffee. II: Muscarinic actions. Journal of pharmaceutical sciences 81: 449–452. 29. Currell K, Jeukendrup AE (2008) Validity, reliability and sensitivity of measures of sporting performance. Sports medicine 38: 297–316. 30. Borg G (1982) Psychophysical bases of perceived exertion. Med Sci Sports 14: 377–381. 31. Jeukendrup AE, Wallis GA (2005) Measurement of substrate oxidation during exercise by means of gas exchange measurements. Int J Sports Med 26 Suppl 1: S28–37. 32. Stalmach A (2012) Bioavailability of Coffee Chlorogenic Acids, in Coffee: Emerging Health Effects and Disease Prevention (ed Y.-F. Chu). WileyBlackwell, Oxford, UK 33. Graham TE, Helge JW, MacLean DA, Kiens B, Richter EA (2000) Caffeine ingestion does not alter carbohydrate or fat metabolism in human skeletal muscle during exercise. J Physiol 529 Pt 3: 837–847. PLOS ONE | www.plosone.org 34. Raguso CA, Coggan AR, Sidossis LS, Gastaldelli A, Wolfe RR (1996) Effect of theophylline on substrate metabolism during exercise. Metabolism: clinical and experimental 45: 1153–1160. 35. Van Soeren M, Mohr T, Kjaer M, Graham TE (1996) Acute effects of caffeine ingestion at rest in humans with impaired epinephrine responses. Journal of applied physiology 80: 999–1005. 36. Kather H, Bieger W, Michel G, Aktories K, Jakobs KH (1985) Human fat cell lipolysis is primarily regulated by inhibitory modulators acting through distinct mechanisms. The Journal of clinical investigation 76: 1559–1565. 37. Vergauwen L, Hespel P, Richter EA (1994) Adenosine receptors mediate synergistic stimulation of glucose uptake and transport by insulin and by contractions in rat skeletal muscle. J Clin Invest 93: 974–981. 38. Graham TE, Battram DS, Dela F, El-Sohemy A, Thong FS (2008) Does caffeine alter muscle carbohydrate and fat metabolism during exercise? Applied physiology, nutrition, and metabolism = Physiologie appliquee, nutrition et metabolisme 33: 1311–1318. 39. Beaudoin MS, Graham TE (2011) Methylxanthines and human health: epidemiological and experimental evidence. Handbook of experimental pharmacology: 509–548. 40. Wahlberg G, Walldius G (1992) Effects of nicotinic acid treatment on fatty acid composition of plasma lipids and adipose tissue in hyperlipidaemia. Scandinavian journal of clinical and laboratory investigation 52: 547–553. 41. Ong KW, Hsu A, Tan BK (2012) Chlorogenic acid stimulates glucose transport in skeletal muscle via AMPK activation: a contributor to the beneficial effects of coffee on diabetes. PloS one 7: e32718. 42. Tsuda S, Egawa T, Ma X, Oshima R, Kurogi E, et al. (2012) Coffee polyphenol caffeic acid but not chlorogenic acid increases 5’AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal muscle. The Journal of nutritional biochemistry. 43. Butts N, Crowell D (1985) Effect of caffeine ingestion on cardiorespiratory endurance in men and women. Res Q Exerc Sport 56: 301–305. 10 April 2013 | Volume 8 | Issue 4 | e59561 RESEARCH ARTICLE Polyphenolic extract of InsP 5-ptase expressing tomato plants reduce the proliferation of MCF-7 breast cancer cells Mohammad Alimohammadi1☯, Mohamed Hassen Lahiani1☯, Diamond McGehee1☯, Mariya Khodakovskaya1,2* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Biology, University of Arkansas at Little Rock, Little Rock, Arkansas, United States of America, 2 Institute of Biology and Soil Sciences, Far-Eastern Branch of Russian Academy of Sciences, Vladivostok, Russia ☯ These authors contributed equally to this work. * mvkhodakovsk@ualr.edu Abstract OPEN ACCESS Citation: Alimohammadi M, Lahiani MH, McGehee D, Khodakovskaya M (2017) Polyphenolic extract of InsP 5-ptase expressing tomato plants reduce the proliferation of MCF-7 breast cancer cells. PLoS ONE 12(4): e0175778. https://doi.org/ 10.1371/journal.pone.0175778 Editor: Rajeev Samant, University of Alabama at Birmingham, UNITED STATES Received: October 30, 2016 Accepted: March 9, 2017 Published: April 27, 2017 Copyright: © 2017 Alimohammadi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. In recent years, by extensive achievements in understanding the mechanisms and the pathways affected by cancer, the focus of cancer research is shifting from developing new chemotherapy methods to using natural compounds with therapeutic properties to reduce the adverse effects of synthetic drugs on human health. We used fruit extracts from previously generated human type I InsP 5-ptase gene expressing transgenic tomato plants for assessment of the anti-cancer activity of established genetically modified tomato lines. Cellular assays (MTT, Fluorescent microscopy, Flow Cytometry analysis) were used to confirm that InsP 5-ptase fruit extract was more effective for reducing the proliferation of breast cancer cells compared to wild-type tomato fruit extract. Metabolome analysis of InsP 5-ptase expressing tomato fruits performed by LC-MS identified tomato metabolites that may play a key role in the increased anti-cancer activity observed for the transgenic fruits. Total transcriptome analysis of cancer cells (MCF-7 line) exposed to an extract of transgenic fruits revealed a number of differently regulated genes in the cells treated with transgenic extract compared to untreated cells or cells treated with wild-type tomato extract. Together, this data demonstrate the potential role of the plant derived metabolites in suppressing cell viability of cancer cells and further prove the potential application of plant genetic engineering in the cancer research and drug discovery. Data Availability Statement: All relevant data are within the manuscript and the Supporting Information files. Introduction Funding: Authors are grateful to Arkansas Space Grant Consortium for providing a stipend to Diamond McGehee. This project was supported by National Space Grant College Fellowship Program (NNXISAR7H) through Research Infrastructure Award provided by Arkansas Space Grant Consortium (award to MK). The funders had no role in study design, data collection and analysis, Cancer is one of the leading causes of death in humans. Scientific advances in recent years and the use of chemoprevention therapy has led to a significant reduction in death rates for different types of cancers [1–3]. Recently, natural compounds with cancer preventive properties have been more widely used in cancer therapy [4]. Natural compounds with antioxidant activity can be categorized into three major groups: compounds that can directly inhibit cell proliferation, compounds that affect tissues outside the cancer cells, and immune-stimulating compounds [5]. Epidemiological studies have shown a positive correlation between the long- PLOS ONE | https://doi.org/10.1371/journal.pone.0175778 April 27, 2017 1 / 21 Polyphenolic extract of InsP 5-ptas tomato plants reduce the proliferation of cancer cells decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. term consumption of fruits and vegetables containing naturally occurring antioxidants with a reduced risk of several types of cancer [6–10]. One of such naturally occurring antioxidants are polyphenols that can be found in various amounts in many types of fruits and vegetables [11–13]. They can be classified into two main groups according to their chemical structure: flavonoids and non-flavonoid compounds. These compounds are particularly valuable because of their high antioxidant activity [14–16]. Several clinical studies indicate that dietary intake of flavonoids and some other phenolic compounds such as caffeic acid and chlorogenic acid can significantly reduce the risk of multiple types of cancer including breast, lung, prostate, and pancreatic cancers [17–20]. Studies have also shown that the use of dietary phenolic compounds can have better preventive and therapeutic results compared to the common synthetic drugs used for cancer treatment since these natural compounds demonstrate less toxicity compared to synthetic chemo-preventive medicines [21]. Dietary flavonoids and other important phenylpropanoids naturally exist in plants. A good example of the commonly used crop plants with a high content of phenolic compounds is tomato (Solanum lycopersicum) [22]. Consumption of tomato has preventive and therapeutic effects on several types of diseases, including cancer [23]. The observed anti-cancer effects of tomato are mainly related to the properties of phenolic compounds that allow them to bind to or interact with a wide range of molecules, affect cell signaling processes, or even serve as a signaling molecule [24–27]. Several attempts have been made to improve the level of health promoting compounds in tomato through conventional breeding techniques as well as genetic engineering tools [28,29]. We recently generated transgenic tomato lines with increased biosynthesis of antioxidants such as lycopene, vitamin C and several flavonoids [30]. Particularly, the transgenic lines were generated by overexpression of InsP 5-ptase gene which affects the phosphoinositol stress signaling pathway through changes in the metabolism of InsP3, the key metabolite of the phosphoinositol pathway [31]. We also reported that the increase in metabolism of InsP3 in transgenic plants positively affects the biosynthesis of several flavonoids, such as chlorogenic acid and rutin, by changing the expression level of the main components of the light-signaling pathway that is linked to secondary metabolism in plants [32]. The observed increase in biosynthesis of phenolics and other secondary metabolites with antioxidant properties in InsP 5-ptase overexpressing transgenic plants suggest an increase in health beneficial properties of these transgenic tomato plants. Despite the obvious potential of the genetically enhanced crop plants with enhanced nutraceutical value, general concerns regarding consumption of food products containing genetically modified (GM) ingredients significantly limits the use of GM crops in medicine. In such circumstances, the extraction of desirable pharmaceuticals from GM crops can serve as an alternative approach to the direct consumption of GM crops [33– 35]. These compounds can then be purified and used in medicine as drugs or supplements. Here, we tested the anti-cancer activity of the total metabolite extract containing flavonoids and other phenolic compounds from InsP 5-ptase expressing tomato fruits in vitro. Anti-proliferative effects of extracts obtained from transgenic fruits on breast cancer cell line (MCF-7) were documented by a number of standard assays including cell viability assay, cell morphological analysis, and flow cytometry. Total transcriptome analysis of cancer cells treated with a mix of metabolites extracted from InsP 5-ptase fruits suggested possible pathways involved in anti-cancer effects of applied extracts. To identify metabolites that may play a role in the antiproliferative activity of InsP 5-ptase fruit extracts, we analyzed and compared extracts from wild-type tomato fruits (control) and extracts from transgenic fruits using LC-MS as a powerful and modern metabolomics tool. LC-MS data confirmed the up-regulation of a number of phenolic compounds with strong anti-proliferative potential in InsP 5-ptase fruit extracts. The design of our study is shown in Fig 1. PLOS ONE | https://doi.org/10.1371/journal.pone.0175778 April 27, 2017 2 / 21 Polyphenolic extract of InsP 5-ptas tomato plants reduce the proliferation of cancer cells Materials and methods Plant growth conditions InsP 5-ptase tomato lines were generated and described in details in our previous publication (30). Tomato (cv. Micro-Tom) seeds of control lines (wild-type, empty vector control, and transgenic lines (lines 6 and 7) were germinated in pots containing a combination of 75% Sun Gro Redi-earth ‘Plug and Seedling’ Mix (Sun Gro Horticulture, Bellevue, WA) and 25% sand. The seeds were germinated in a growth chamber under high-light conditions (800 μmol m-2 s-1) with intervals of 16 h light (25˚C) and 8 h dark (22˚C). The red fruits were collected between 6 to 8 weeks of growth under controlled environment and exposure to high-light. The red tomato fruits were immediately frozen in liquid nitrogen after harvest and stored at -80˚C or immediately used in the experiment. For phenolic extraction, fruit samples were immediately lyophilized and stored in the dark environment at room temperature before being used in the experiment. Total phenolic compounds extraction and quantification Total phenolics were extracted based on the method described by Ainsworth and Gillespie (2007) [36]. The colorimetric assay works based on the transfer of electrons in alkaline medium from phenolic compounds to phosphomolybdic/ phosphotungstic acid complexes. A three step sequential aqueous/methanol extractions method was used to extract Polyphenols, hydroxycinnamates, flavonoids, and their glycosides from lyophilized red tomato fruits. One milliliter of the methanol/water (2:1) solution was mixed with 100 mg of each fruit sample after which the samples were vortexed for 30 minutes. Next, the sample extracts were centrifuged at 10, 000 g for 5 minutes at 4˚C and the supernatant was collected. One milliliter of fresh extraction solution was added to the pellet and the above extraction process was repeated twice and at the end of each extraction, the supernatant was collected. It is important to protect the samples from light throughout the extraction process. Folin-Ciocalteau micro method as described by Slinkard et al. (1977) was used to measure the concentration of the phenolic compounds in the extraction solution [37]. Twenty microliters of the tomato extract were diluted in 1.58 ml of H2O and 100 μl of the Folin-Ciocalteau reagent was added to the solution. The mixture was vortexed for 5 minutes after which 300 microliters of sodium carbonate solution (250 μg/ml) were added to the reaction, mixed and incubated for 30 minutes at 4˚C. Following the incubation period, absorbance was read at 765 nm against the blank. A galic acid Fig 1. Overview of applied research strategy. Total metabolites of transgenic tomato fruits were analyzed for their potential anti-cancer properties by application of the total metabolite mixture to MCF-7 cancer cells followed by cell viability assays and transcriptome analysis of the cancer cells. The total tomato wild-type and transgenic fruit extracts were scanned for the potential metabolite with antioxidant and anti-cancer properties by using LC-MS. https://doi.org/10.1371/journal.pone.0175778.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0175778 April 27, 2017 3 / 21 Polyphenolic extract of InsP 5-ptas tomato plants reduce the proliferation of cancer cells calibration curve (0–1 g/L) was used as standard and the flavonoid concentrations were expressed as galic acid equivalents. Fruit tissue preparation for LC-MS analysis Whole, intact tomatoes were pulverized using a mortar and pestle with liquid nitrogen to keep tissue frozen. Approximately 20 mg of frozen tissue powder was then extracted using 80% methanol by using a bead beater for each of the three technical replicates for each line. Samples were then vortexed, sonicated, and centrifuged. The supernatant was filtered through 13 mm syringe filters into microcentrifuge tubes and dried overnight in a 45˚C vacufuge. Samples were then stored at -80˚C for future use. Extraction solution (80% methanol) was used to reconstitute samples to equal volumes. Samples were vortexed and sonicated to ensure all residues had dissolved before they were centrifuged. The supernatant was transferred to a labeled autosampler vial and analyzed immediately. Chromatography and mass spectrometry Samples were analyzed using a Grace C-18 (Grace Davison Discovery Sciences, USA) reverse phase column on an Agilent 1100 series LCMS (Agilent Technologies, Waldbronn, Germany) equipped with a G1379A degasser, G1312A binary pump, G1329A autosampler, G1316A column oven, G13158B diode array detector, and G2445C MS. The aqueous phase was acidified HPLC-grade water (0.05% formic acid), and the organic phase was HPLC-grade methanol. Ten microliter injections were pumped at 0.6 mL/min with the following elution gradient: 0–2 min, 5% B; 2–22 min, 75% B; 22–27 min, 75% B; 27–28 min, 5% B; 28–32 min. A fiveminute wash was included after each sample run. Negative-mode electrospray ionization was used to detect the metabolites by utilizing a trap mass spectrometer scanning of 100–1500 m/z. The target was set at 10,000 and maximum accumulation time at 100.00 ms with two averages. Chemstation software (http://www.agilent.com/en-us/products/software-informatics/ massspec-workstations/lc-ms-chemstation-software), provided with the Agilent machine used to analyze samples and collect data was used to convert files to netCDF format. Further conversion to mzXML format was completed with msConvert (http://proteowizard.sourceforge.net/ tools.shtml) [38]. Files were then loaded into MZmine software (http://mzmine.github.io/) and processed [39]. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database, available at http://www.genome.jp/kegg/tool/map_pathway1.html, was used for tentative online compound identification and was completed through MZmine using the gap-filled peak list [40]. Further statistical analysis was carried out by uploading the identified peak list to Metaboanalyst (http:// www.metaboanalyst.ca/) for analysis and by comparison to publications [41–43]. More information regarding data structure can be found in S4 Fig. Cell culture conditions MCF-7 breast cancer cells (ATCC) were seeded in T-75 culture flasks (Thermo Scientific) and maintained in Dulbecco’s modified Eagle‘s medium (DMEM) media, supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 U/ml streptomycin. The culture plates were maintained at 37˚C with 5% carbon dioxide. The medium was changed every two days, and the cells were passaged at 80% confluency before the experiment. MTT assay Cells were divided into six groups: blank group (no cells), control group (no treatment) and four experimental groups (WT, EV, L6 and L7 lines extract treatments). Cells were seeded in PLOS ONE | https://doi.org/10.1371/journal.pone.0175778 April 27, 2017 4 / 21 Polyphenolic extract of InsP 5-ptas tomato plants reduce the proliferation of cancer cells 96-well plates 24 hours prior the experiment at the density of 104 cells/well. The next day, the medium was changed and metabolite extract (34 μg/μl was supplemented to the fresh medium. The cells were incubated 24 hours with the medium containing the metabolite extract. After the incubation period, 3-(4,5-dim ethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was then added to each well at the concentration of 5 mg/ml. The cells were incubated for 4 hours, after which the supernatant was replaced with 200 μL of dimethyl sulfoxide (DMSO). The absorption was measured at 570 nm using a micro-plate reader. The results were presented as OD 570–620 using the following formula: MTT OD 570–620 = (Mean A 570–560)—(Mean A of Blank) / (Mean A Negative Control)—(Mean A of Blank). The results are based on two independent experiment with each experiment consisting of 3 technical replicates. Double immuno-staining and microscopy MCF-7 were seeded into each well of Lab-Tek 2 chamber slide (Thermo Scientific Nunc. NY) and incubated for 4 hours at 37˚C (5x105 cells in each chamber) in a humidified, 5% carbon dioxide atmosphere to attach. Cells were divided into five groups: control group (no treatment) and four experimental groups (WT, EV, L6 and L7 lines extract treatments). Total metabolite extract (34 μg/μl) was then added to fresh DMEM medium and applied to the wells and incubated for 24 hours. After incubation, cells were washed once with phosphate buffer saline (PBS) and stained with 1% Acridine orange/Ethidium bromide solution in PBS for 1 minute. Chambers were then washed two times with PBS after which slides were detached from the chamber and air dried. Images were then taken by fluorescence microscopy. A Nikon Eclipse 90i microscope equipped with a 12V-100W halogen lamp, external transformer, flyeye lens built-in and NCB11, ND8, ND32 filters, was used to visualize the stained samples. The following Nikon filters were used: barrier filter BP365, reflector filter FT 395 and exciter filter LP395. Live/dead cells were counted using Image J software. Live cells fluoresce green (FITC/ green) and dead cells fluoresce red/orange (Texas Red/red). Flow cytometry analysis Cells of MCF-7 breast cancer cell line were seeded in 6-well plates at a density of 5 x105 cells per well and incubated for 24 hours at 37˚C in an incubator with 5% carbon dioxide to attach. After the initial seeding, the cells were incubated with fresh medium containing 34 micrograms per microliters of extract and were incubated for 24 hours at 37˚C. Next day, cells were trypsinized and collected in 2 ml tubes. Samples were centrifuged for 15 minutes at 1,300 rpm at 37˚C. The supernatant was discarded and the cells were resuspended by addition of cold PBS. Samples were briefly vortexed, transferred to Flow Cytometry tubes, and then placed on ice. Components of YO-PRO kit (Life Technologies) were used for labeling the samples. One sample was kept as a control (without label). One microliter of the YO-PRO -1 stock solution (Component A) was added directly to the mixture in each tube followed by one microliter of the propidium iodide (PI) stock solution (Component B). Labeled tubes were incubated on ice for 30 minutes. Cells were analyzed by flow cytometry using BD LSRFortessa Cell Analyzer (BD biosciences, CA) (to detect green (YO-PRO-1) and red (propidium iodide) signals. Gene expression analysis using microarray (Affymetrix platform) Breast cancer cell line MCF-7 cells were seeded in 6 well plates at a concentration of 106 cells/ well, 24 hours prior to the experiment. Afterwards, cells were washed two times with warm media and were incubated for 24 hours with fresh medium containing tomato extracts from different plant lines (WT, EV, L6, L7) at a concentration of 34 μg/μl. RNeasy Mini Kit (Qiagen Sciences, Maryland, USA) was used to isolate RNA samples with modification of standard PLOS ONE | https://doi.org/10.1371/journal.pone.0175778 April 27, 2017 5 / 21 Polyphenolic extract of InsP 5-ptas tomato plants reduce the proliferation of cancer cells Qiagen protocol. Cells were disrupted briefly by using Trizol reagent (Ambion, Grand Island, NY), and total RNA was extracted by using chloroform extraction method. After the RNA purification, on-column DNA digestion using the RNase-free DNase Kit (Qiagen Inc. Valencia, CA) was used to remove the residual DNA. The purity of RNA samples was confirmed by electrophoresis and the concentration was quantified by using Nanodrop spectrophotometer (Thermo Scientific. Wilmington, DE). Affymetrix Human Genome Arrays were used as the microarray platform. Biotinylated cRNA targets were synthesized by Affymetrix IVT Express target labeling assay as specified in the Affymetrix GeneChip Expression Analysis Technical Manual. Hybridization reactions to the Affymetrix Human GeneChips were carried out by Expression Analysis, Inc. (Durham, NC). Statistical analysis The cell viability data were analyzed by Tukey’s test and expressed as mean ±S.D. by which the significant differences (P value < 0.05) between groups were determined. To analyze the microarray raw data, column-wise normalization using a reference sample (control)was applied. The resulting data was then visualized using Multi Experiment viewer (Mev). The data was further analyzed by ANOVA and Tukey test with a p-value of 0.001 while assuming variance between variables are equal. The genes with significantly different expression were clustered by hierarchical clustering. In addition, all the known and unknown genes with changes in expression were functionally analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 and PANTHER for gene classification. Microarray data were deposited in GEO database (GEO number: GSE94548). Results Effect of InsP 5-ptase overexpression in tomatoes on total phenolic content in transgenic fruits InsP 5-ptase overexpressing (two independent transgenic lines) and control (two control lines) tomato fruits were tested for their total content of metabolites with phenolic nature. Results of the experiment demonstrated that the fruits of transgenic tomato lines contain more phenolic compounds (37% for L6, 50% for L7) compared to control lines (Fig 2). These results confirm Fig 2. Total phenolic content in mature (red) InsP 5-ptase expressing and wild-type tomato fruits. (A) Standard curve using galic acid (0–1000 mg) as a standard reagent. (B) Total phenolic content in the mature tomato fruits of InsP 5-ptase transgenic lines (L6 and L7) and control lines (WT and EV). The total phenolic content is expressed as mg/g galic acid equivalent. The different letters (a,b) means statistically different groups (p
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Running Head: RESEARCH ARTICLE ANALYSIS

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Research Article Analysis
Institute:
Name:
Date:

RESEARCH ARTICLE ANALYSIS

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INTRODUCTION
Cancer is among the leading cause of human deaths all over the world. Recently,
advancement in scientific technology, together with chemoprevention therapy, has
significantly reduced death rates caused by the various types of cancer. There is an increased
interest in fruits and vegetables with a high concentration of antioxidants due to their
reported chemotherapeutic potential. The natural compounds containing antioxidant activity
are broadly placed in three categories: compounds that inhibit cell proliferation directly,
compounds affecting tissues surrounding cancer cells, and those that stimulate the immune
system. Polyphenols are antioxidants that occur naturally and are found in varying amounts
in fruits and vegetables. This paper analyses two articles on the use of polyphenols in Cancer
prevention, identifying their rationale, hypothesis, scientific rigor, data provided, and a
conclusive scientific theory of the articles.
The first article is: Polyphenolic extract of InsP 5-ptase expressing tomato plants
reduces MCF-7 breast cancer cells proliferation. In this article, extracts of fruits from human
type I InsP 5-ptase gene expressing transgenic tomato plants generated earlier were used to
assess the anticanceranticancer activity of established genetically modified tomato lines. The
research question in this article was “Does polyphenolic extract of InsP 5-ptase expressing
tomato plants reduce the proliferation of MCF-7 breast cancer cells?”. This article's rationale
was the recently growing interest in the use of naturally occurring compounds with
antioxidant activity in cancer therapy and treatment. This was based on the epidemiological
studies showing a positive connection between consumption of vegetables and fruits
containing antioxidants with lowered risk of cancer occurrence in the long run.

RESEARCH ARTICLE ANALYSIS

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This article hypothesizes that there is a statistically significant relationship between
the use of polyphenolic extracts of InsP 5-ptase conveying tomato plants and the reduction of
proliferation of MCF-7 breast cancer cells. According to previous epidemiological studies,
there is a positive correlation between consuming vegetables and fruits that contain
antioxidants and reduced cancer risk. This article states that tomato (Solanum Lycopersicum)
contains phenolic properties that provide preventative and therapeutic effects on cancer. The
null hypothesis states that there lacks a positive correlation between the use of the
polyphenolic extracts of InsP 5-ptase in tomato plants and the reduction of proliferation of
MCF-7 breast cancer cells. This would mean that tomatoes' phenolic compounds do not
inhibit cancer disease development. This is the hypothesis that was tested in this research
paper.
The second article is: Potential Effects of Pomegranate Polyphenols in Cancer
Prevention and Therapy. In contrast to the first research article, a review article provides an
inclusive examination of known marks and mechanisms and a critical assessment of
polyphenols extracted from pomegranate fruit as future anticancer agents. This article is
similar to the first one in that it also looks into the use of naturally occurring antioxidants in
fruits in cancer therapy and treatment. According to this article, pomegranate fruit contains
anti-invasive and anti-proliferative effects. The flavonoids in this fruit are also involved in
inducing apoptosis by modifying Bcl-2 proteins, regulating p21 and p27, and downregulating
the cyclin-CDK system. Some experimental studies on pomegranate use established a
tendency of efficacy in increasing the prostate cancer antigens and, consequently, prolonging
prostate cancer patients' lives.

RESEARCH ARTICLE ANALYSIS

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In the first article, the cell viability results established that cancer cells' viability was
reduced up to 70% when incubated with phenolic extracts from control fruits. When the
cancer cells were incubated with InsP 5-ptase extract, their viability reduced even more, with
values as low as 30-50%. This shows that there is a statistically significant relationship
between the use of the polyphenolic extract of InsP 5-ptase expressing tomato plants and the
reduction of proliferation of MCF-7 cells involved in breast cancer. The alternative
hypothesis can therefore be accepted, and the null hypothesis rejected. The second article
relied solely on previous studies on pomegranate use in cancer therapy and treatment. Several
researches, both in vivo and in vitro, proved the efficacy of polyphenols in pomegranate in
hindering cancer pathogenesis and progression. A study by Patrick et al. conducted on
patients with recurrent prostate cancer showed a statistically significant PSA extension;
increasing time from 15 to 54 months following the usage was established. A recent research
examining the possibility of a genotoxic outcome of pomegranate extract was conducted. The
fruit extract prompted mutation of points, sister chromatid exchanges, and chromosome
aberrations. This study showed a significant genotoxic impact on mammary cancer cells.
This indicates that the polyphenols in pomegranate extract contain an anticancer effect.
Therefore, the null hypothesis in this article was rejected, and the alternative hypothesis was
accepted.
These two articles prove that fruits such as tomatoes and pomegranate contain
naturally occurring antioxidants with chemo-preventative and chemotherapeutic potential.
Many clinical investigations show that long-term consumption of phenolic compounds can
decrease the risk of many cancer types such as prostate, lung, pancreatic, and breast cancers.
The two articles acknowledge that using naturally occurring polyphenols can have enhanced
preventative and healing outcomes compared to conventional cancer treatment drugs. This s
because the natural compounds show less toxicity than synthetic cancer prevention drugs.
This theory assumes that there is a known concentration of antioxidants that is effective in
destroying cancer cells.
Conclusively, naturally occurring anticancer compounds exists in high amounts in
some fruits. Research on understanding the cancer mechanisms should therefore shift from
the development of new methods of chemotherapy to the use of natural compounds; this will

RESEARCH ARTICLE ANALYSIS

reduce the adverse effects of synthetic drugs on human health. These two articles
demonstrate the success of polyphenols both in cancer therapy and cancer treatment. There,
however, lacks a standardized method of assessing the concentration of polyphenols that are
ideal for cancer treatment and prevention. This is an area that needs further research to
enhance the success of naturally occurring compounds in overcoming the frustrating effects
of this disease.

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RESEARCH ARTICLE ANALYSIS

References:
Turrini, E., Ferruzzi, L., & Fimognari, C. (2015). Potential effects of pomegranate
polyphenols in cancer prevention and therapy. Oxidative medicine and cellular
longevity, 2015.
Alimohammadi, M., Lahiani, M. H., McGehee, D., & Khodakovskaya, M. (2017).
Polyphenolic extract of InsP 5-ptase expressing tomato plants reduces the
proliferation of MCF-7 breast cancer cells. PloS one, 12(4), e0175778.

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Running Head: RESEARCH ARTICLE ANALYSIS

Research Article Analysis Caffeine and Impacts on Exercise
Institute:
Name:
Date:

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Running Head: RESEARCH ARTICLE ANALYSIS

Research question:
Article one: The research question is what the performance-enhancing effects of caffeine in
coffee are in a time trial performance test. Second, to figure out the metabolic effects of caffeine
and coffee.
The research question is does caffeine positively improve exercise in adults?
Hypothesis:
The hypothesis is that caffeine leads to an improvement in exercise function in adults.
The null hypothesis is that caffeine does not lead to an improvement in exercise function in
adults.
Scientific Rigor and Compare and contrast articles

The scientific rigor of this article, "The Metabolic and Performance Effects of Caffeine
Compared to Coffee during Endurance Exercise.” is strong. The literature review provided by
the paper discusses multiples studies that have shown how caffeine leads to "prolonged submaximal and high-intensity exercise can improve performance". Second, that the mechanism of
action is n increase in fat oxidation through the sympathetic nervous system, and a sequential
sparing of muscle glycogen. Despite this, there is contradicting information on the fat oxidation
is the mechanism of action of caffeine's impact. Other theories show that caffeine's ergogenic
effects are through its ability to act as an adenosine receptor antagonist to induce effects on both
central and peripheral nervous system to reduce pain and exertion perception [, improve motor

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Running Head: RESEARCH ARTICLE ANALYSIS

recruitment and excitation-contraction coupling (Hogson, et al., 2013 p1). The article "The
Metabolic and Performance Effects of Caffeine Compared to Coffee during Endurance
Exercise." Is an experimental trial to assess if the caffeine in coffee leads to improved
performance in timed trials. The article has much rigor because it is a randomized clinical trial.
The sample size was low with only 8 cyclists or triathletes used.
The Hodgson study would have benefited from an increase in the number of participants.
Further, the inclusion-exclusion criteria included participants who had trained 3 or more times a
week and had a low caffeine intake to ensure that the participants had similar traits. The ethical
standard of this paper was high because participants were informed of risks and discomforts
before signing informed consent. Further, this study was a "single-blinded, cross over,
randomized counter-balanced study design” (Hodgson, et al., p2). One area of concern is in the
design, decaffeinated coffee and placebo were both used as controls. Decaffeinated coffee still
contains caffeine and could have skewed results.
The second article is styled after a systematic review article. It provides background information
on caffeine and its impacts on exercise. This is a strong article because it has been reviewed by
multiple authors. Further, the article addresses if there were any ethical problems which there
were none. Second, that there were no funding resources and any conflict of interests. While the
systematic article lacks a method section on how the resources were picked and verified
decreases the validity of this study. Adding a method section with inclusion and exclusion
criteria would have greatly benefited this research article especially since it is styled as a
systematic review.

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Running Head: RESEARCH ARTICLE ANALYSIS

The second article is strong for background information on previous studies that have been
conducted on caffeine and its impact on exercise. For example, "Several meta-analyses
examining the effects of caffeine ingestion on exercise performance have been conducted,
exploring the effects of caffeine on a broad array of exercise tests, including 1 repetition
maximum (1 RM) strength, isokinetic peak torque, vertical jump height, power output across
different exercise types, aerobic endurance performance, and muscular endurance.” (Pickering,
C., & Grgic, J.,2019 p 1).

Data:
The data in the Hodgson study was conducted by using 8 cyclists or triathletes. The design was a
single-blinded, cross-over, randomized counter-balanced study design. During the preliminary...

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